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Logo of neuroncolAboutAuthor GuidelinesEditorial BoardNeuro-Oncology
Neuro-oncol. 2006 October; 8(4): 391–500.
PMCID: PMC1871956

Abstracts for the Eleventh Annual Meeting of the Society for Neuro-Oncology

November 16–19, 2006



Shideng Bao, Qiulian Wu, Sith Sathornsumetee, Yueling Hao, Zhizhong Li, Anita B. Hjelmeland, Qing Shi, Roger E. McLendon, Darell D. Bigner, and Jeremy N. Rich; Departments of Surgery, Pathology, Medicine, and Neurobiology, Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA

Malignant gliomas are highly lethal cancers that depend on angiogenesis for malignant progression. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of these glioma cancer stem cells to support tumor angiogenesis. Glioma cancer stem cells isolated from human glioblastoma biopsy specimens and xenografts expressed neural stem cell markers, formed neurospheres, and differentiated along multiple nervous system lineages. Glioma cancer stem cells derived from multiple gliomas potently generated tumors when they were implanted into the brains of immunocompromised mice, while glioma nonstem tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from glioma cancer stem cells were morphologically distinguishable from nonglioma cancer stem cell tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for glioma cancer stem cells in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by glioma cancer stem cells. In comparison to the matched glioma nonstem tumor cell population, glioma cancer stem cells consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, glioma cancer stem cells conditioned media significantly increased endothelial cell migration and tube formation compared with glioma nonstem cell tumor cell conditioned media. The pro-angiogenic effects of glioma cancer stem cells on endothelial cells were specifically abolished by the anti-VEGF-neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Parallel results were detected in in vivo animal studies in which bevacizumab treatment blocked the angiogenic effects of the cancer stem cells. These data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting pro-angiogenic factors from stem cell-like tumor populations may be critical for patient therapy. This study was supported in part by funds from the Pediatric Brain Tumor Foundation of the United States, Accelerate Brain Cancer Cure, Childhood Brain Tumor Foundation (J.N.R.), and Southeastern Brain Tumor Foundation (A.B.H.). This work was also supported by NIH grants NS047409, NS054276 and 1 P50 CA 108786 (J.N.R.). A.B.H. is a Paul Brazen/American Brain Tumor Association Fellow. J.N.R. is a Damon Runyon-Lilly Clinical Investigator supported by the Damon Runyon Cancer Research Foundation and a Sidney Kimmel Cancer Foundation Translational Scholar.


Mark N. Jabbour,1,2 Weijun Wang,1 Ligaya Pen,2 Thomas C. Chen,1,2 and Florence M. Hofman2; Departments of 1Neurological Surgery and 2Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

The functional role of the vasculature in tumor growth remains an important issue in cancer biology. In this study, we examined the effects of human brain endothelial cells (hBEC) on tumor growth, using the human metastatic breast cancer cell line (MDA-435) in an in vivo co-culture system. We hypothesize that endothelial cells enhance the growth of metastatic breast cancers. The human in vivo co-culture assay was performed by combining the metastatic breast cancer cell line MDA-435 with cultures of non-malignant, primary human brain-derived endothelial cells. This mixture of cells was injected subcutaneously or stereotaxically and intracranially into nude mice. HBECs were labeled with a green fluorescent dye (CFD-SE). Antibodies to CD31 and vonWillebrand Factor (vWF), used to identify both mouse and human blood vessels, were used in the standard immunostaining protocol. The average microvascular density (MVD) was calculated as “hot spots” = areas demonstrating a relative increase in vascularity at 200× magnification. In the subcutaneous model, the tumor volume of co-cultured cells (MDA-435 plus hBEC) was approximately 2 times larger than the volume of tumor cells alone (P < 0.05) at 74 days postinjection. The survival time of mice with intracranial tumors was less for the co-culture groups compared with tumor cells alone but was not statistically significant. However, a histopathologic examination of these intracranial tumor specimens revealed increased microvessel and tumor cell density and apparent invasion of breast cancer cells toward the peripheral skull region. HBECs, found only within the tumor sample, were distinctly fluorescent green, staining positively for CD31, vWF, and forming capillary-like structures. The total number of hot spots present in animals with co-cultures was significantly greater than that of tumor cells alone, with a 1.8–fold increase in the vascularity in co-cultures compared with tumor cells alone (21 +/− 4 vs. 12 +/− 1 microvessels/0.25 mm2). Normal mouse brain had an average MVD of approximately 8 microvessels/0.25 mm2. We have established an in vivo co-culture model that will enable us to examine the contribution of endothelial cells to tumor growth both in the subcutaneous and orthotopic model. Using this model, we have shown that human brain endothelial cells enhance tumor growth, and this finding may be a reflection of significantly increased microvessel density in these tumors.


Ok-Hee Lee, Juan Fueyo, Jing Xu, Gregory Fuller, Kenneth Aldape, Howard Colman, Joy Gumin, Frederick Lang, W.K. Alfred Yung, and Candelaria Gomez-Manzano; Departments of Neuro-Oncology, Neuropathology, and Neurosurgery, Brain Tumor Center, University of Texas, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Tie2 receptor tyrosine kinase was considered to be a specific endothelial cell receptor, but recently Tie2 expression has also been found in hematopoietic stem cells as a mediator of adhesion in the niche, and it is subsequently protected from myelosuppressive stress. We have previously reported the expression of Tie2 in the glial compartment of malignant gliomas. Our current research is focused on the elucidation of the function of the Ang1/Tie2 pathway in the neoplastic astrocytes and its relation to the brain tumor stem cell population. Interestingly, cultures of neurospheres from surgical glioblastoma multiforme (GBM) specimens co-expressed Tie2 and several stem cell markers, including the recently identified brain tumor stem cell marker CD133, and showed proliferating and renewal properties. Double immunofluorescence of human GBM tissues using anti-Tie2 and anti-Factor VIII antibodies showed that Tie2+ nonendothelial cells were located in vessel-rich regions that resemble a stem cell niche, where cancer and endothelial cell cross-talk could occur. In this regard, we have gathered data showing the existence of a functional Ang1/Tie2 pathway in glioma cells that is involved in the neoplastic phenotype of gliomas. We found that Ang1 significantly enhanced adhesion of Tie2–expressing glioma cells to several components of the basement membrane, as collagen I and IV, by upregulating integrin β1 and activating the focal adhesion kinase. Consistent with this data, we observed changes in the cytoskeleton organization with the formation of focal adhesions, as assessed by immunolocalization of paxillin. Using a system of isogenic glioma cell lines, we determined the role of Ang1/Tie2 in the production of glioma cell-to-endothelial cell adhesion and migration that would justify the perivascular localization of Tie2+ cells the in human GBM specimens we have previously described. Our data showed that Tie2 expression in the glial compartment of malignant gliomas co-exists with several stem cell-like markers. Moreover, we have identified a functional Ang1/Tie2 network involved in the neoplastic glial phenotype, specifically in adhesion to vascular structures and migration. The Tie2 present in the membrane of glioma cells could serve as a target for novel therapies for gliomas.


Lily Pal,1 Shaleen Kumar,2 and Sanjay Behari3; Departments of 1Pathology, 2Radiotherapy and 3Neurosurgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India

Oligodendrogliomas, diffusely infiltrating glial neoplasms, encompass a spectrum of well differentiated to frankly malignant tumors. As these tumors have lesser growth potential than the corresponding grade of astrocytomas, patients usually present with a long history of neurological symptoms. However, these tumors do have an inherent tendency to recur and undergo malignant transformation. Microvascular proliferation is an important phenomenon in the progression of low- to high-grade astrocytic tumors. Its role in oligodendroglioma is uncertain. The characteristic quiescent-looking delicate angulated vessels of low-grade oligodendroglioma often disappear during its anaplastic progression. A diffusible angiogenic factor, such as VEGF, is one of the potential targets for therapeutic strategies. Hence, the aim of our study was to (1) study the pattern of VEGF expression in oligodendrogliomas; (2) quantitate angiogenesis (MVD/mm2); and (3) correlate VEGF expression and MVD with tumor grade. After reviewing the histologic parameters, 35 cases were considered for analysis. Immunohistochemistry was performed using antibodies against CD-34 and VEGF. The MVD count/mm2 was done at 20× magnification in 9 fields using graticule for the mean MVD/mm2. Subjective VEGF expression was assessed as 3+ (strong signal at multiple sites), 2+ (easily detected at multiple sites), 1+ (focal positivity), and 0 (no detectable stain). The average MVD/mm2 in grade II was 84.058 and was 137.583 in anaplastic oligodendrogliomas. For glioblastomas (GBM) and anaplastic astrocytomas (control cases) the average MVD/mm2 was 40.5 and 30.66, respectively. In anaplastic oligodendrogliomas small vascular buds were prominent as opposed to glomeruloid morphology in GBM and delicate vessels in grade II oligodendrogliomas. Regarding VEGF, 6.66% of grade II showed 3+, 20% had 2+, 60% had 1+, and 2 were negative. In the anaplastic variant, 28.57% showed 3+, 42.85% had 2+, and 28.5% had 1+ positivity. GBM and gemistocytic astrocytomas showed 3+ positivity. VEGF expressed predominantly in tumor cells. Medulloblastoma, a high-grade primitive tumor, did not show any increase in MVD or VEGF expression. It appears that in anaplastic oligodendrogliomas, MVD is markedly increased compared with its low-grade counterpart and GBM. The morphology of the vessels also changes with higher grade. Regarding VEGF, only 28.57 % had 3+ and 42.85% showed 2+ in anaplastic oligodendroglioma, whereas VEGF expression was 3+ in gemistocytic astrocytomas and in GBM. It is possible that microvascular proliferation may not be totally dependent on VEGF production by the tumor cells, and VEGF may be involved in tumor progression irrespective of its role in microvascular proliferation.


Jenilyn Virrey,1 Ligaya Pen,1 Christiana Charalambous,2 Thomas Chen,1,3 and Florence Hofman1,3; Departments of 1Pathology, 2Molecular Microbiology and Immunology, and 3Neurosurgery, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA

No effective treatment is currently available for the estimated 30% of breast cancer patients who have metastases to the brain. Antiangiogenic therapy is being recognized as an emerging treatment for targeting cancer growth. Breast cancer brain metastases flourish under the highly vascularized microenvironment of the brain, which provides nutrients and oxygen to the tumor. The tumor vasculature is very different from normal blood vessels: the tumor-associated blood vessels are hemorrhagic, tortuous, and form a chaotic network of vessels. The purpose of this study was to analyze the phenotypic and functional properties of endothelial cells derived from metastatic breast cancer to the brain (BBEC). The BBEC were isolated from brain tissue specimens of a patient with advanced breast cancer brain metastases. Primary cultures of human BBEC were purified using flow cytometry and characterized as endothelial cells based on positive immunostaining for Factor VIII, CD31, and CD105 antibodies. The BBECs are morphologically larger and more flattened than normal brain endothelial cells (BEC). The BBECs proliferate at a significantly slower rate than the BECs. Furthermore, the BBECs migrate faster and do not respond to interleukin (IL)-8 stimulation in the migration assay as do the control BECs. A functional analysis of BBECs demonstrates that these cells proliferate more slowly than do BECs and exhibit a significantly higher expression of the pro-angiogenic growth factors: vascular endothelial growth factor (VEGF), endothelin-1 (Et-1), and IL-8 as compared with the control BECs. These characteristics of BBECs appear to be more similar to primary cultures of human endothelial cells derived from glioma tissue (TuBEC) than normal, control brain endothelial cells. In conclusion, the BBECs are phenotypically and functionally distinct from normal endothelial cells and similar to glioma-derived endothelial cells. The nature of BBECs must first be examined to appropriately target the tumor vasculature for antiangiogenic therapy.


Anna Yanai, Carolyn Lutzko, Vazgen Khankaldyyan, Maya Otto-Duessel, Ignacio Gonzalez-Gomez, Donald B. Kohn, Mike Rosol, Anat Erdreich-Epstein, and Walter E. Laug; Childrens Center for Cancer and Blood Diseases, Childrens Hospital Los Angeles, Los Angeles, CA, USA

High vascularization of glioblastoma typically predicts poor prognosis. Local sprouting of existing brain capillaries (angiogenesis) is known to provide tumors with new blood vessels. We hypothesize that bone marrow (BM)-derived endothelial progenitor cells (EPC) also participate in the process of neovascularization of malignant brain tumors. BM progenitor cells from nude mice were transduced in vitro with a lentiviral vector-encoding enhanced green fluorescent protein (EGFP) and infused into syngeneic mice (5 × 106 cells per animal). Engraftment was documented by FACS analysis of blood for EGFP-expressing cells. Eight weeks after bone marrow transplantation (BMT), the animals were orthotopically injected with 105 U87MG glioblastoma cells. Tumor growth was monitored by MRI scans. Mice were sacrificed 8–10 weeks after tumor xenotransplant and the brains were fixed with 4% paraformaldehyde. Tumors were examined by H&E staining and immunohistochemistry was used to test for tumor vascularity (CD31 staining) and the presence of BM-derived cells (EGFP-labeled). Co-localization was done by confocal microscopy. All 8 mice that underwent BMT showed engraftment. Circulating EGFP-positive cells were only 32.6% in 1 mouse and over 80% in the other 7 mice. All mice had successful tumor xenotransplant (MRI scanning and autopsy). EGFP-positive cells were found in all tumors. CD31 and EGFP were co-localized in 36.58% of the vessel area covered by CD31 and analyzed in 10–20 randomly selected fields in the brains of 4 mice. CD45-EGFP double-positive cells were abundant in the perivascular area of the tumor but not in the normal brain. Our data demonstrate that BM-derived progenitor cells contribute significantly to endothelial coverage in brain tumor neovascularization. BM cells also localize to perivascular sites in the tumor. Whether these BM-derived endothelial cells behave biologically similarly to those derived by sprouting angiogenesis remains to be seen. Furthermore, these data raise the issue of antiangioblast therapy for brain tumors.


David Zagzag, Yevgeniy Lukyanov, Li Lan, M. Aktar Ali, Herman Yee, Evelyn Voura, and Elizabeth W. Newcomb; Microvascular and Molecular Neuro-Oncology Laboratory, Department of Pathology, Division of Neuropathology, Department of Neurosurgery and New York University Cancer Institute, New York University School of Medicine, New York, NY, USA

From our previous studies examining the role of hypoxia and HIF-1α in glioblastoma multiforme (GBM) and given the knowledge that CXCR4 can respond to HIF-1α and is involved in angiogenesis, we hypothesized that CXCR4 would be regulated by hypoxia in GBMs. CXCR4 is a specific chemokine receptor for stromal cell-derived factor-1 (SDF-1) α, also known as CXCL12. First, we investigated the expression of HIF-1α and CXCR4 in GBM tumor tissues. CXCR4 was consistently found in pseudopalisading glioma cells around areas of necrosis where it co-localized with HIF-1α expression. We found that CXCR4 levels correlated with the level of HIF-1α in these cells. In addition, angiogenic vessels were strongly positive for CXCR4. To understand these results, we tested the in vitro effect of hypoxia and VEGF on the expression of CXCR4 in glioma cells and in human brain microvascular endothelial cells (HBMECs). We showed that significant CXCR4 and HIF-1α expression was induced in glioma cells after exposure to hypoxia and that the level of CXCR4 expression increased in HBMECs after exposure to exogenous VEGF. To further assess the role of HIF-1α in CXCR4 expression, we transfected HIF-1α into U87MG glioma cells. We observed that CXCR4 was up-regulated in HIF-1α expressing normoxic U87MG-transfected glioma cells. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at 2 levels: first through the VEGF-stimulated angiogenic response in HBMECs and, second, by HIF-1α in the pseudopalisading tumor cells themselves. We believe this knowledge may lead to a potentially important 2–pronged therapy against GBM progression using chemotherapy targeting CXCR4.


Abdessamad Zerrouqi,1,2 Beata Pyrzynska,1,2 Daniel Brat,1,4 and Erwin G. Van Meir1,2,3,5; 1Laboratory of Molecular Neuro-Oncology, Departments of 2Neurosurgery, 3Hematology/Oncology, 4Pathology and 5Winship Cancer Institute, Emory University School of Medicine, GA, USA

The Ink4a/Arf locus on chromosome 9p21 is frequently inactivated in the progression of malignant astrocytoma, which suggests a role in tumor suppression. This locus encodes 2 tumor suppressor proteins: p16INK4A and the alternative reading frame product, p14ARF. Recent studies have indicated that tumor vascularity is enhanced by the loss of the Ink4a/Arf locus in gliomas and inhibited by the restoration of p16 in colon cancer; however, the specific role of p14ARF in the regulation of angiogenesis has not been addressed to date. Here, we examined whether p14ARF, independent of p16, regulates angiogenesis in gliomas. To evaluate the effect of ARF gene replacement on angiogenesis in glioma, we engineered human glioma cells deficient in the INK4A/ARF locus to conditionally express a human ARF cDNA. We used cells that carried either a transcriptionally active p53 (LN229) or null allele (LNZ308). Our results show that the conditioned media of cells expressing p14ARF inhibits endothelial cell migration/invasion through gelatin-coated filters in vitro. Furthermore, the restoration of ARF expression in LN229 cells inhibits vessel formation in matrigel plugs in vivo. These effects are independent upon the expression levels of the known glioma pro-angiogenic factors VEGF, angiopoietin, and IL-8. Using microarray analyses, we found that induction of p14ARF upregulates the expression level of the tissue inhibitor of metalloproteinase-3 (TIMP-3), both in p53-positive (LN229) and p53-negative (LNZ308) glioma cells. The silencing of TIMP3 expression abolishes the p14ARF inhibitory effect of conditioned media on endothelial cell migration, which suggests that secreted TIMP3 has a role inregulating endothelial cell migration mediated by ARF. We investigated the mechanism of TIMP-3 upregulation by ARF and found that it was dependent on SP1 transcription factor activity. The silencing of SP-1 (but not p53) inhibited the induction of TIMP3 by p14ARF. These findings indicate that p14ARF has an additional activity in tumor suppression, independent of p53 via the negative regulation of angiogenesis. This activity is mediated by TIMP3 induction, emphasizing the importance of both p14ARF and regulators of extracellular matrix remodeling in suppression of angiogenesis.


Ping-Pin Zheng,1 Marcel van der Weiden,1 Martin J. van den Bent,2 Peter A.E. Sillevis Smitt,2 Theodorus M. Luider,2 and Johan M. Kros1; Departments of 1Pathology and 2Neurology, Erasmus Medical Center, Rotterdam, The Netherlands

Neovascularization (vasculogenesis and angiogenesis) is essential for tumor growth and invasion. Most endothelial progenitor cells (EPCs) reside in bone marrow and are mobilized and enter the circulation by cytokines or angiogenic growth factors triggered by various physiological or pathological stimuli. In experimental models, it has been shown that EPCs enter the interstitial space where they promote de novo vessel formation by integrating into vessels or take part in the formation of entirely new vessels (vasculogenesis). Whether this scenario actually takes place in patients with glial tumors is unknown. So far, the recruitment of EPCs and their incorporation into tumor tissues has been investigated only in animal models and in ex vivo experiments with exogenously transported EPCs. In this study, we investigated the presence of activated EPCs in biopsy specimens and peripheral blood samples of patients with glial tumors. The EPCs were found predominantly as solitarily deposited cells and cell chains throughout the tumors or as constituents of hyperplastic vessels. We were able to identify increased numbers of circulating EPCs in blood samples taken preoperatively from the glioma patients. We matched the percentages of these EPCs with those present in the tissue samples of the tumors by double- and triple-labeling experiments. The findings provide evidence that EPCs contribute to glioma neovascularization in vivo. The presence of the EPC population may become a diagnostic parameter for glioma progression or serve as a potential target for antiangiogenic therapy.


James C. Yao,1 Jun Zhang,1 Zhiliang Jia,1 and Keping Xie1,2; Departments of 1Gastrointestinal Medical Oncology and 2Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Vascular endothelial growth factor (VEGF) is a critical proangiogenic factor in almost all solid tumors. However, its expression and role in human carcinoid development and progression remain unclear. Using immunohistochemical analysis, we investigated VEGF expression patterns in 50 cases of human carcinoid with various clinicopathologic characteristics. We found that strong VEGF expression was detected in tumor cells, whereas no or very weak VEGF expression was detected in stromal cells surrounding or within the tumors. The levels of VEGF expression directly correlated with the expression levels of Sp1 and microvessel density and were associated with a short period of progression-free survival. VEGF expression was also associated with metastasis. Using in vitro and in vivo models, we treated human carcinoid cell lines with bevacizumab, a monoclonal antibody targeting VEGF. Bevacizumab did not inhibit the growth of carcinoid cells in vitro but significantly reduced tumor angiogenesis and impaired tumor growth in animals. Our data suggest that the overexpression of VEGF promotes the growth of human carcinoid, in part through the upregulation of angiogenesis.



Etty (Tika) Benveniste, Emily Brantley, L. Burton Nabors, G. Yancey Gillespie, and Susan Nozell; University of Alabama at Birmingham, Birmingham, AL, USA

Inflammatory and immune responses are mediated by the STAT and NF-κB families of transcription factors, which regulate the expression of genes that facilitate cell invasion, adhesion, and angiogenesis. Constitutively activated STATs, particularly STAT-3, have been detected in a wide variety of primary tumors, and NF-κB is constitutively activated in many cancers. This suggests that in cancer, mechanisms that regulate STAT-3 and NF-κB activity have failed, enabling STAT-3 and NF-κB to function as tumor promoters. Our preliminary results demonstrated that STAT-3 and NF-κB were constitutively activated in most glioma specimens compared with control brain specimens. STAT-3 was constitutively phosphorylated on both tyrosine and serine residues, indicative of an activated state. Furthermore, NF-κB levels were elevated and phosphorylated on serine residues 276 and 536, again indicative of NF-κB activation. Another parameter reflective of NF-κB activation is the phosphorylation of IκBα, which was observed in most glioma specimens but not in controls. The protein inhibitors of activated STATs (PIAS) proteins negatively regulate activated STAT proteins. PIAS3 specifically inhibits activated STAT-3, suppressing its transcriptional activity. PIAS3 has recently been shown to inhibit NF-κB transcriptional activity. We made the striking observation that the PIAS3 protein is either absent or expressed at low levels in glioma tissue samples compared with control brain tissue. Given the inhibitory effect of PIAS3 on both STAT-3– and NF-κB-mediated transcriptional activity, we hypothesize that the loss of PIAS3 expression in gliomas may be responsible, in part, for constitutively activated STAT-3 and NF-κB. Interestingly, in glioma cell lines cultured in vitro, the PIAS3 protein was abundantly expressed, suggesting an influence of the CNS microenvironment on PIAS3 expression in vivo. We generated human glioma cell lines that inducibly regulated endogenous PIAS3 expression through the use of inducible siRNA. These cell lines are currently under analysis to determine the influence of the absence or presence of PIAS3 on STAT-3– and NF-κB-mediated gene expression (VEGF, survivin, IL-8, IL-6, MMP-9, and iNOS) and aspects of apoptosis and proliferation. These studies will determine the functional involvement of PIAS3, STAT-3, and NF-κB in gliomagenesis.


Hetal Bhanushali, Sharon L. Longo and Gregory W. Canute; Department of Neurosurgery, SUNY Upstate Medical University, Syracuse, NY, USA

We determined the effect of the irreversible epidermal growth factor receptor (EGFR)/erbB2 tyrosine kinase inhibitor, EKB-569, on glioblastoma multiforme (GBM) with differential EGFR expression. EKB-569 inhibits EGF-induced phosphorylation of EGFR and the growth of tumors that overexpress EGFR, but we need to better understand the biologic and clinical criteria for patient selection and how to best use the available EGFR inhibitors. Cell lines that mimic the molecular status of the primary tumor are needed to analyze these agents before they can be used clinically. The purpose of this study was to compare GBM cell lines, which naturally overexpress wild-type (wt) EGFR, with an artificially transfected wtEGFR line and GBM with low levels of EGFR. A flow cytometry analysis was used to determine EGFR levels. Both the naturally occurring wtEGFR line and the transfected wtEGFR line demonstrated high levels of receptor expression; the unamplified GBM line had low levels of EGFR. When we measured cytotoxicity using an MTT assay only, the cell line with naturally overexpressing wtEGFR was sensitive to EKB-569. We analyzed the cell cycle after exposing the cells to EKB-569 and found that the transfected wtEGFR and unamplified lines demonstrated G2M arrest at high drug concentrations, whereas the naturally overexpressing wtEGFR underwent apoptosis at much lower concentrations. A preliminary analysis of these cell lines demonstrated diverse molecular profiles that could contribute to their differential responses to EGFR inhibition. Natural wtEGFR cells behaved differently from artificially transfected wtEGFR cells. Evaluating the preclinical response of EGFR inhibitors using cell lines with artificially transfected wtEGFR may result in inaccurate predictions of clinical outcome.


Atreyi Dasgupta,1 Baisakhi Raychaudhuri,2,3 Talat Haqqi,2,3 Erwin G. Van Meir,4 Michael Vogelbaum,2,3 and S. Jaharul Haque1,3; 1Department of Cancer Biology, 2Department of Neurosurgery, 3Brain Tumor Institute, Cleveland Clinic, Cleveland, OH, USA, 4Department of Neurosurgery, Hematology/Oncology and Winship Cancer Institute, Emory University, Atlanta, GA, USA

In a previous study, we found that > 90% of primary GBM tissues and all GBM cell lines examined contained persistently activated Stat3 that promoted the growth of GBM cells in vitro. This was, in part, mediated by the suppression of spontaneous apoptosis due to an induction of the bcl-2 family of pro-survival genes (bcl-2, bcl-x, and mcl-1). To understand the role of activated Stat3 in the growth of GBM in vivo, we generated U87–derived stable cell lines that express varying levels of a dominant-negative mutant (DN)-Stat3 protein in an inducible fashion. To determine tumor-specific expression, we used an expression vector (V6L) that is activated under hypoxia, a physiologic property of GBM and other solid tumors. Hypoxia and associated cell necrosis and angiogenesis are cardinal features of GBM. We placed DN-Stat3 cDNA under the control of the hypoxia-inducible factor-responsive promoter found in V6L (Post, D. and Van Meir, E.G., Gene Ther. 8: 1801–7, 2001) and derived stable clones in U87 cells. Two clones, designated N714 and N716, exhibited tight regulation of DN-Stat3 expression under hypoxia in vitro and were selected for subcutaneous mouse tumorigenicity studies. Both clones formed tumors in 2 weeks, but the tumors were markedly smaller than vector-control tumors. Moreover, they did not grow further, whereas the volume of the control tumors was ~3 times larger in 4 weeks. These data indicate that activated Stat3 may play a key role in the tumorigenesis of malignant gliomas. Further analyses of the expression profile of DN-Stat3 and the levels of persistently activated Stat3 in the hypoxic regions of harvested tumors are being conducted. We anticipate that reduced tumor growth will correlate with reduced Stat3 activation in the hypoxic regions of the tumors. This study was supported by National Institutes of Health grant R01 CA095006 to S.J.H. and CA87830 to E.G.V.M.


Atreyi Dasgupta,1 Pankaj Sharma,1 and S. Jaharul Haque1, 2; 1Department of Cancer Biology, Lerner Research Institute and 2Brain Tumor Institute, Cleveland Clinic, Cleveland, OH, USA

The brain tumor stem cell hypothesis predicts that differentiated GBM cells are not capable of supporting tumor formation. Consistent with this prediction, U251 cells that express the glial differentiation marker GFAP form > 100 times smaller tumors in rats than U87 cells that do not express GFAP. Activated Stat3 is persistent in U251 cells, which binds to the GFAP promoter. We previously showed that Stat3 also binds to the promoters of bcl-2, bcl-x, and mcl-1. Stat3 signaling is required for both glial differentiation and GFAP expression. To understand the role of activated Stat3 in chromatin remodeling during the differentiation of GBM cells, we used the chromatin immunoprecipitation (ChIP) assay. ChIP is an indispensable tool for studying chromatin remodeling during the expression and silencing of genes. A conventional ChIP assay using antibodies that are specific for a given transcription factor is designed to pull down all chromatin fragments that are associated with the transcription factor. This is a major drawback of this assay in addressing promoter-specific epigenetic alterations. To circumvent this problem, we developed a novel method that allows us to immunoprecipitate chromatin fragments that encompass the promoter of the gene of interest. This method uses two vectors: (1) pFA-CMV (Strata-gene) expressing the DNA-binding domain of yeast GAL4 protein and (2) pChIP, which we constructed using pcDNA3.1/Hygro+ vector (Invitrogen) as the backbone. pChIP contains the open reading frame (ORF) of green fluorescent protein (GFP), upstream of which are multiple cloning sites (MCS) to subclone the promoter of interest. At the 5′-end of the MCS, 5 copies of the yeast upstream activating sequence (UAS) that binds to GAL4 are inserted, which are flanked at the 5′-end by an antisense ORF of DsRed-Express protein to function as a stuffer region. When these 2 vectors are co-expressed in mammalian cells, in principle, GAL4 DBD would bind to the UAS located upstream to the promoter of interest, and chromatin fragments of desired lengths containing the promoter could be immunoprecipitated using anti-GAL4–DBD antibodies. To prove this principle, we transiently transfected the ChIP vector containing a 2.02–Kb human mcl-1 promoter with or without pFA-CMV in 293T cells and demonstrated that the exogenous mcl-1 promoter is chromatinized and immunoprecipitated with anti-GAL4 DBD monoclonal antibody but not with 2 isotype-matched control antibodies. These data strongly suggest that this method can be used in dissecting promoter-specific chromatin remodeling during proliferation, differentiation, and de-differentiation of normal and tumor cells, including malignant glioma cells. This study was supported by National Institutes of Health grant R01 CA095006 to S.J.H.


Sarah Dunlap, J. Matthew McDonald, David Cogdell, Valerie Dunmire, Qingyi Wei, Anna Starzinski-Powitz, Raymond Sawaya, Janet Bruner, Gregory N. Fuller, Kenneth Aldape, and Wei Zhang; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The allelic loss of the short arm of chromosome 1 has been frequently observed in oligodendroglioma (60%–80%). We evaluated 177 oligodendroglial tumor samples and defined a consensus region of deletion of approximately 630 kb. This region contains a single gene, SHREW1, which encodes a novel transmembrane protein in adherens junctions. A mutation was not detected in the coding region of the SHREW1 gene in oligodendrogliomas, but expression of the gene was consistently decreased in oligodendrogliomas relative to normal brain tissues. The restoration of SHREW1 expression resulted in suppression of cell adhesion and migration. The SHREW1 protein was shown to be localized in adherens junctions and filament-like protrusions in SHREW1–GFP-transfected stable clones. Thus, SHREW1 may play an important role in cell-cell communication and cell-stromal interaction, inactivation of which may play a role in the development of oligodendroglial tumors.


Sarah M. Dunlap,1 Joseph Celestino,1 Rongcai Jiang,1 Hua Wang,1 Eric C. Holland,2 Gregory N. Fuller,1 and Wei Zhang1; 1The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Insulin-like growth factor-binding protein 2 (IGFBP2) has been shown to be overexpressed in 80% of the most advanced type of diffuse glioma, glioblastoma multiforme (GBM), by combined cDNA microarray and tissue microarray approaches. Furthermore, overexpressed IGFBP2 correlates with poor survival in diffuse gliomas. In vitro functional studies have provided evidence that IGFBP2 increases glioma cell migration and invasion. We therefore hypothesized that IGFBP2 is a key regulator of glioma progression. We tested our hypothesis using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific cell lineages. In this system, avian virus receptor is expressed exclusively in glial cells via linkage to the neuroglial-specific nestin promoter. Genes of interest are cloned into an avian RCAS vector, and viral particles are expanded in DF1 avian fibroblasts. When injected into the neonatal mouse brain, the virions infect only glial cells, and the genes of interest borne by the virions are thus only expressed in glial cells. For these experiments, the study genes were IGFBP2, platelet-derived growth factor (PDGF), K-Ras, and Akt, which were delivered separately and in combination. IIp45 (invasion inhibitory protein at 45 kDa), which was discovered and characterized by our laboratory, was also co-delivered. IIp45 has been shown to bind IGFBP2 and diminish its ability to increase invasion and migration in vitro. Our results show that PDGF signaling leads exclusively to the formation of low-grade (World Health Organization grade II) oligodendrogliomas. PDGF delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (World Health Organization grade III). These higher grade tumors are characterized by increased cellular density, vascular proliferation, and poor survival. IIp45 injected in combination with PDGF and IGFBP2 ablated IGFBP2-induced tumor progression and resulted in the formation of low-grade oligodendrogliomas. Combined K-Ras and Akt led to the formation of astrocytomas; K-Ras alone or Akt alone did not result in tumor formation. IGFBP2 in combination with K-Ras produces astrocytomas, which are histologically similar to the gliomas resulting from K-Ras/Akt stimulation. No tumors resulted from the simultaneous delivery of Akt and IGFBP2, suggesting that IGFBP2 and Akt likely lie in the same pathway or in converging pathways. The present studies show that: (1) IGFBP2 is associated with progression from low-grade oligodendroglioma to high-grade anaplastic oligodendroglioma in gliomas initiated by PDGFb overexpression in vivo, (2) IGFBP2-induced tumor progression can be ablated by IIp45, and (3) IGFBP2 can synergize with the Ras pathway to produce diffuse gliomas in vivo. Collectively, our data demonstrate that IGFBP2 actively contributes to diffuse glioma initiation and progression. Studies are ongoing to further elucidate the signaling pathways of IGFBP2-induced gliomagenesis.


Xing Fan, William Matsui, Leila Khaki, Duncan Stearns, Jiong Chun, Yue-Ming Li, and Charles G. Eberhart; Departments of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD; Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

The Notch signaling pathway is required in both nonneoplastic neural stem cells and embryonal brain tumors, such as medulloblastoma, that are derived from such cells. We investigated the effects of Notch pathway inhibition on medulloblastoma growth using pharmacologic inhibitors of gamma-secretase. Notch blockade suppressed expression of the pathway target Hes1 and caused cell cycle exit, apoptosis, and differentiation in medulloblastoma cell lines. Interestingly, viable populations of better-differentiated cells continued to grow when Notch activation was inhibited but were unable to efficiently form soft agar colonies or tumor xenografts, suggesting a cell fraction required for tumor initiation had been depleted. It was recently hypothesized that a small population of stem-like cells within brain tumors is required for the long-term propagation of neoplastic growth and that CD133 expression and Hoechst dye exclusion (side population) can be used to prospectively identify such tumor-initiating cells. We found that Notch blockade reduced the CD133-positive cell fraction almost five-fold and totally abolished the side population, suggesting that the loss of tumor-initiating capacity could be due to the depletion of stem-like cells. Notch signaling levels were higher in the stem-like cell fraction, suggesting that they are more sensitive to inhibition of this pathway. We also observed that apoptotic rates after Notch blockade were 10-fold higher in primitive nestin-positive cells compared with nestin-negative ones. Stem cell-like cells in brain tumors thus appear to be selectively vulnerable to agents that inhibit the Notch pathway.


Sven Hanson,1,2 Kira Erber,1,2 Kiran Todkar,1,2 Nadine Pettkus,1,2 Alf Giese,1,2 and Ella Kim1,2; 1Laboratory of Neuro-Oncology, Department of Neurosurgery, University of Schleswig-Holstein, Campus Luebeck; 2Translational Neuro-Oncology Research Group, Department of Neurosurgery, Georg-August University of Goettingen, Germany

Tumor suppressor p53 elicits distinct cellular responses, such as survival or programmed cell death. The intricate balance between the survival-promoting and apoptotic activities of p53 can be influenced by alternative splicing; several splice isoforms of p53 have recently been identified in human cells. p53β is a transcriptionally inactive isoform with unknown functions. Identified originally in blood screenings, p53β expression was thought to be a hallmark of quiescent cells until recently, when it was detected in cancer cells. There is inconsistency in the medical literature concerning the functions and expression patterns of p53β. It has been proposed that p53β expression is tissue-specific and does not occur in the brain. We analyzed p53β expression in brain tissues and cultured cells derived from brain tumors (GBMs). p53β expression was detected in normal astrocytes, malignant brain tissues, and cultured glioma cells, as ascertained by RT-PCR and immunochemical analyses using anti-p53β antibody. Furthermore, we were able to clone p53β from normal human astrocytes and assessed its functions in vitro and in vivo. We show, for the first time, that p53β is an anti-apoptotic factor that protects cells from apoptosis by localizing to mitochondria. Inhibition of p53β by iRNA sensitized cells to spontaneous and camthothecin-induced apoptosis, supporting the role of p53β as a survival-promoting factor. Considering that p53β is expressed in glioma cells with transcriptionally impaired mutant p53, the retention of anti-apoptotic p53β activity in conjunction with the loss of p53 activities may confer a growth advantage to glioma cells with the mutated TP53 gene.


Chu-Cheng Kan,1 Dattatreyudu Nori,1 Gloria C. Li,2 Angel Aguso-Sacido,3 and John A. Boockvar3; 1The Frank Randazzo Jr. Cell Biology Laboratory, Integrated Department of Radiation Oncology, Weill Cornell and NYHQ, USA; 2 Laboratory of Hyperthermia and Radiobiology, Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, NY, USA; and 3Neurosurgical Laboratory for Translational Stem Cell Research, Department of Neurological Surgery, Weill Medical College, USA

Statins are inhibitors of HMG-CoA reductase and have shown anti-cancer efficacy in preclinical trials. Epidermal growth factor receptor- target therapies have been shown to potentiate statin-induced cytotoxicity in cancer cells. In this study, we determined the cytoxicity of glioblastoma cell lines with varied levels of EGFR signaling to treatment with simvastin and lovastatin. U87MG glioblastoma cells were transfected with wild-type EGFR (WT-EGFR), constitutively active EGFR (EGFRvIII), and empty vector controls. These stable cell lines were then cultured for 24 hours, followed by treatment with either simvastatin or lovastatin at 75 microM in 0.5% FBS. An MTT assay was performed at day 3 after treatment to assess cell viability. Data were analyzed by the paired Student’s t-test or one-way ANOVA. The proliferation of U87 WT-EGFR, U87 EGFRvIII, and U87MG cells was abolished by simvastin (mean = 5% viability; P < 0.05). The cytotoxic effect of lovastatin was much less significant than that of simvastatin in U87 MG, U87WT-EGFR, and U87EGFRvIII cells (mean = 50% viability; P < 0.05). Mevalonic acid lactone antagonized the lovastatin effect but not the simvastatin effect. The effect of simvastatin on U87EGFRvIII cells (87% cytotoxicity) was less robust than on WTEGFR and control cells (97%; P < 0.05). Our results indicate that statins show reactivity against glioblastoma cells, even with activated levels of EGFR signals. These preliminary studies also suggest that simvastin has more activity against GBM than does lovastatin. Activated EGFR signals, such as the presence of EGFRvIII, may render GBM cells less responsive to statins. Tyrosine kinase inhibitors may, therefore, demonstrate additive cytotoxic effects in combination with statins in GBM.


Gurpreet S. Kapoor and Donald M. O’Rourke; Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA

EGFR is an important therapeutic target in many human cancers, particularly de novo glioblastomas (GBM). We previously showed that kinase-inactive erbB2 mutants that interfere with EGFR signaling inhibited the transformation of wild-type p53 glioblastoma cells, suggesting that disabled erbB receptor complexes may cooperate with p53 to confer growth inhibitory effects. Mutations involving the TP53 gene are frequently identified in up to 50% of all human tumors, including glioblastomas. An analysis of expression patterns of TP53 in glioblastomas showed that it is mainly mutated in secondary glioblastomas that are derived from lower grade tumors and is less common in primary GBMs that arise de novo. Moreover, the prognostic significance of TP53 loss of function in astrocytomas has always been controversial. To further evaluate whether p53 is required for the inhibition of glial cell transformation, we used mutant p53 GBM cells to determine the effects of non-functional EGFR/erbB2 receptor assembly on glioblastoma cell transformation and signaling in the absence of functional p53. The ectopic expression of erbB2 mutants in U373MG cells (mutant p53) resulted in slower growth and more flattened and untransformed morphologic characteristics. Importantly, disabled erbB receptors inhibited transformation in soft agar assays and tumor formation in nude mice. A biochemical analysis showed reduced Akt and GSK-3α/β but not p42/44MAPK phosphorylation in mutant erbB2–expressing cells compared with parental controls. A cell cycle analysis showed reduced cyclin D1 and CDK6 and increased phospho-Cdc-2 (Tyr15) and p15INK4B in erbB2–inhibited cells. These observations suggest that non-functional EGFR/erbB2 receptor complexes exert their inhibitory effects via the PI3–K/Akt pathway and affect different cell cycle regulatory proteins to block the progression of tumor cells at the G2/M phase. Collectively, our data provide the basis for receptor-based therapies that disable erbB receptors, such as activated EGFR. Non-functional erbB2–mediated strategies appear to inhibit EGFR signals independently of p53 and may represent an alternative approach to EGFR-specific targeting of human cancer cells, including glioblastoma cells.


Tomohiro Kawaguchi,1 Yoji Yamashita,1 Masayuki Kanamori,1 Krystof S. Bankiewicz,1 Suzanne J. Baker,2 Gabriele Bergers,1 and Russell O. Pieper1; 1Brain Tumor Research Center, Department of Neurological Surgery and the University of California–San Francisco Comprehensive Cancer Center, University of California–San Francisco, San Francisco, CA, USA; and 2 Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA

The collagen type IV cleavage fragment tumstatin and its active sub-fragments bind to integrin αVβ3, and in endothelial cells (but not tumor cells) inhibit the activation of FAK, PI3K, Akt, and mTOR. The resultant endothelial cell apoptosis accounts for the ability of tumstatin to function as an endogenous inhibitor of angiogenesis and an indirect suppressor of tumor growth. We hypothesized that the inability of tumstatin to directly suppress tumor cell growth might be the result of the constitutive activation of the Akt/mTOR pathway commonly seen in tumors. Consistent with this idea, several integrin αVβ3-expressing glioma cell lines with PTEN mutations and high levels of pAkt were unaffected by exposure to an active fragment of tumstatin (T3), whereas αVβ3-expressing glioma cell lines with a functional PTEN and low levels of pAkt exhibited T3-induced growth suppression that could be bypassed by siRNA-mediated suppression of PTEN, introduction of a constitutively expressed Akt, or introduction of the Akt and mTOR target eIF4E. The direct tumor-suppressive actions of T3 were further demonstrated in an αVβ3-deficient in vivo mouse model in which T3 was unable to alter the tumstatin-insensitive vasculature contributed by the αVβ3-deficient host but nonetheless suppressed the growth and proliferative index of intracranially implanted αVβ3-expressing PTEN-proficient glioma cells. These results show that tumstatin, previously considered to be only an endogenous inhibitor of angiogenesis, also directly inhibits the growth of tumors in a manner dependent on Akt/mTOR activation. The PTEN-proficient subset of GBM may, therefore, be an especially good target for therapy using tumstatin or other endogenous inhibitors of angiogenesis.


John J.P. Kelly, Andrew Chojnacki, Xueqing Lun, Donna Senger, Peter Forsyth, Ian F. Parney, and Samuel Weiss; The University of Calgary, Calgary, Alberta, Canada

The relationship between neural stem cells (NSCs) and brain tumors presents an opportunity to improve our understanding of the cellular origin of gliomas. Gliomas have heterogeneous histologic features and biologic behavior within and between lesions of the same pathologic grade. We hypothesized that gliomas arise from cells at different stages along neural stem cells to astrocyte lineage and consequently demonstrate heterogeneity when cultured using the neurosphere assay system. We sought to determine whether gliomas are heterogeneous with respect to growth factor responsiveness, brain tumor neurosphere formation, differentiation potential, self-renewal capacity, and ability to initiate tumors in immunocompromised mice. The neurosphere assay system was used to culture 30 fresh human brain tumor specimens. Ten glioblastoma (GBM) specimens, which formed tumor neurospheres, were analyzed in detail. The differentiation potential of primary and secondary brain tumor spheres was determined by immunocytochemical analysis. The self-renewal of tumor spheres under various growth factor conditions was assessed using single-cell dissociation assays. Karyotyping confirmed the isolation of genetically abnormal cells. Tumor sphere cells were implanted intracranially into immunocompromised mice to demonstrate tumorigenicity. Eighteen of 30 glial tumors cultured generated tumor spheres. Grade IV gliomas were most amenable to culture; 14 of 18 cultures formed spheres. Sphere formation occurred not only in response to epidermal growth factor and fibroblast growth factor but also to platelet-derived growth factor; most importantly, it also occurred in the absence of additional growth factors. Oligodendrogliomas did not form tumor spheres. Of 10 GBM specimens characterized in detail, all demonstrated properties of normal stem cells and cancer cells. Nine of 10 GBMs displayed multilineage differentiation, producing astrocytes (GFAP+), oligodendrocytes (O4+), or neurons (MAP2+). Abnormal differentiation was evident by retention of nestin and CD133 expression. Nine of 10 GBMs demonstrated self-renewal under various growth factor conditions. Brain tumor sphere-forming cells from all growth conditions were karyotypically abnormal and formed tumors upon intracranial implantation in immunocompromised mice. Both Cd133+ and Cd133− cells, determined by flow cytometry, gave rise to infiltrating tumors. Tumors that formed in mice were much different from those formed by the U87 cell line. GBM sphere cells displayed aggressive behavior, forming tumors that infiltrated white matter tracts and, in some cases, honed to the subventricular zone. Glioma sphere-forming cells displayed properties of both normal neural stem cells and cancer cells, which proliferated independently, without exogenous growth factor stimulation. This is the first demonstration of varied and abnormal responses to growth factor stimulation of human glioma stem cells. Gliomas demonstrate heterogeneity with respect to brain tumor sphere formation, self-renewal, and multilineage differentiation. The cellular origin of gliomas remains uncertain. Glial tumors likely arise from cells at different stages along the NSC to astrocyte lineage pathway.


E. Kim,1, 2 R. Wüstenberg,1 J. Leppert,1 Sven Hanson,1,2 E. Pawlak,1 N. Pettkus,1,2 and A. Giese2; 1Laboratory of Neuro-Oncology, Department of Neurosurgery, University of Schleswig-Holstein, Campus Lübeck and 2Translational Neuro-Oncology Research Group, Department of Neurosurgery, Georg-August University of Goettingen, Germany

The tumor-suppressing functions of p53 rely on its ability to regulate transcription. A remarkable feature of p53 is its functional versatility, with its still-growing pool of p53 target genes involved in the regulation of cell survival, DNA repair, or apoptosis. Although the loss of the p53 function by mutations in the TP53 gene is frequent in brain tumors, nearly half of human gliomas have wild-type p53. The search for agents that would be effective in (re)activating the p53 transcriptional response in tumor cells has been an actively developing area of cancer research. We found that DNA-intercalating agent chloroquine was a potent activator of the p53 DNA-binding and transcriptional activity in glioma cells that express wild-type p53. Cloroquine inhibited the growth of glioma cells, induced cell death by apoptosis, and sensitized glioma cells to gamma irradiation and BCNU. The mechanism of p53 activation by chloroquine was distinct from the one underlying activation of p53 by gamma irradiation in that it bypassed ATM-dependent phosphorylation of the p53 protein. The results of our ChIP analyses indicate that chloroquine may act by facilitating p53 binding to the promoters of apoptotic genes. Its effectiveness in killing glioma cells and its long history of safe clinical use make chloroquine an attractive candidate drug that may be used to complement existing glioma therapies.


Roger A. Kroes, Nigel J. Otto, Mary Schmidt, Verne Hulce, E. Malcolm Field, and Joseph R. Moskal; The Falk Center for Molecular Therapeutics, Department of Biomedical Engineering, Northwestern University, Evanston, IL, and Field Neurosciences Institute, Saginaw, MI, USA

Aberrant cell surface glycosylation patterns are present on virtually all tumors and have been linked to oncogenic transformation, tumor progression, metastasis, and invasion. Northern blot analyses of glycosyltransferase and glycosylhydrolase gene expression, the enzymes that regulate cell surface glycosylation, in high-grade glioma specimens demonstrated a wide variety of quantitative and qualitative differences in expression among tumors. Of particular interest was the significant overexpression of α2,3 sialyltransferase (α2,3ST) mRNA found in most tumors and the complete lack of measurable α2,6 sialyltransferase (α2,6ST) in all tumors examined. The α2,3ST, together with α2,6ST, are the 2 enzymes responsible for effectively all of the terminal sialylation of N-linked glycoproteins oligosaccharides. To test the hypothesis that modifying glycogene expression would alter the cell-surface carbohydrate patterns found in gliomas and inhibit their invasive potential, stable α2,6ST transfectants were created and characterized. Studies with these cells showed that, indeed, expressing the α2,6ST gene in the human glioma cell line, U373MG, inhibited invasion in vitro without affecting cell proliferation. Further studies revealed that these effects were mediated by the observed alterations in α3β1 integrin sialylation and focal adhesion kinsase (p125fak) signal transduction. An adenoviral vector containing the α2,6ST gene was constructed and used to transiently infect U373MG cells. When these cells were stereotactically implanted directly into the brains of SCID mice, no tumor formation was observed. We created an oligonucleotide microarray platform representing all the cloned human glycosidases, glycosyltransferases, polysaccharide lyases, carbohydrate esterases, and carbohydrate-binding proteins and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Of the many significant changes in glycogene expression observed, of particular interest was the observation that another sialyltransferase, SIAT7E, was expressed at low levels in all glioma and glioma cell lines examined compared with normal brain. These results were corroborated by quantitative PCR (qRT-PCR). SIAT7E catalyzes the formation α2,6 sialic acid linkages only in gangliosides. Similar experiments to those reported here using other models of glioma invasivity will be important, but the results reported here strongly suggest that this approach has significant therapeutic potential.


Khatri Latha,1 Yeo-Hyeon Huang,1 Robert Dejournett,1 Jian Kuang,2 and Oliver Bogler1; 1Department of Neurosurgery, Brain Tumor Center; and 2Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

EGFR overexpression and mutation drive glioblastoma cell proliferation and survival, and EGFR inhibition is an important area of translational research. Direct targeting of the receptor is an area of active clinical investigation, but inhibition of downstream effectors of EGFR may be a necessary complimentary strategy. Signals that promote cell survival make particularly attractive targets. We have identified a new link between EGFR and apoptosis and are investigating its potential as a new therapeutic target. In this pathway, adaptor proteins SETA/CIN85 and Alix, which are associated with EGFR endocytosis, were shown to modulate Bax-dependent mitochondrial apoptosis via a member of the endophilin family, Bif-1. Bax insertion into the mitochondrial membrane is dependent on Bif-1 via an undefined mechanism. In normal and transformed cells of mesenchymal origin, increased apoptosis in response to Bif-1 overexpression is dependent on a cooperating apoptotic insult. In contrast, our data showed that all 5 glioma cell lines tested underwent apoptosis in response to elevated Bif-1 alone, suggesting that this pro-apoptotic signal is primed in this tumor type. Furthermore, elevated levels of SETA/CIN85 and Alix potentiated Bif-1–mediated glioma cell apoptosis, and this depended on their ability to interact with Bif-1. We are testing the role of this new pathway in the EGFR survival signal that countermands glioma cell apoptosis in response to DNA damage. In addition to determining the dependence of EGFR-mediated survival on SETA/CIN85, Alix, and Bif-1, we are testing whether this pathway is independent of PI3K-Akt signaling.


Iris Lavon,1,2 Dana Fuchs,1,2 Daniel Zrihan,1,2 Yakov Fellig,3 Bracha Zelikovitsh,1,2 Tali Siegal1, 2; 1Gaffin Center for Neuro-Oncology and Departments of 2Neurology and 3Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel

The activation of nuclear factor (NF)-κB in response to alkylating agent-induced DNA damage has been described previously, related mainly to its role in cell survival pathways, which allows normal cell cycles in cases of limited DNA damage. It was demonstrated that inhibition of NF-κB potentiates the anti-tumor activity of alkylating agent. Therefore, NF-κB may play a crucial role in the development of resistance to chemotherapy. Recently, tumor necrosis factor-α-induced protein 3 (TNFAIP3) was identified as a component of a putative cytoplasmic signaling cascade that mediates NF-αB activation in response to alkylating agents. However, the specific NF-αB target gene involved in chemoresistance to alkylating agents is yet unknown. MGMT is the only known critical DNA damage repair enzyme involved in the direct reversal of the biologic effects of O6-methylguanine. Therefore, a tumor’s resistance to alkylating agents often correlates with the extent of MGMT expression. MGMT induction after a variety of DNA-damaging treatments is regulated at the transcriptional level. The function of the transcription factors SP-1 glucocorticoid-responsive elements and AP-1 in MGMT regulation has been described before. Besides the previously identified binding sites, we have found 2 putative NF-αB sites within the MGMT promoter region that suggest that NF-αB induces drug resistance by as yet unknown mechanisms. We demonstrated, by an electrophoretic mobility shift assay, a specific and direct interaction between NF-αB and each of the NF-αB-binding sites. Moreover, we showed that transfection of the NF-αB subunit P65 (NF-αB/p65) to HEK 293 cells induced a 90–fold increase in the MGMT mRNA transcription level. The addition of the NF-αB superrepressor ΔNI-κB completely abrogated this induction. We also found a significant correlation between the extent of NF-κB activation and the MGMT expression level in both glioma cell lines and human glial tumors. These findings are of potential clinical significance, as we showed that cell lines with either forced expression of p65 or high constitutive activity of NF-κB are less sensitive to nitro-sourea (e.g., BCNU) treatment. Our findings strongly suggest that NF-κB plays a major role in MGMT regulation. Together, these observations shed light on a novel role of NF-κB in the regulation of DNA damage repair mechanisms and the emergence of chemoresistance.


Yunqing Li, Lauren Fuller, Fadila Guessous, David Schiff, and Roger Abounader; Departments of Neurology and Microbiology, University of Virginia, Charlottesville, VA, USA

Overexpression of the growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor c-Met and loss of the tumor suppressor PTEN are frequent occurrences in human glioblastoma. Both occurrences have been shown to significantly contribute to glioblastoma malignancy. PTEN interacts with and can regulate c-Met-dependent pathways at various levels of cell signaling. In the present study, we determined the relative contribution of PTEN loss and restoration to c-Met-induced glioblastoma malignancy and associated molecular events. We restored PTEN or PTEN lipid-phosphatase mutant (G129E) or phosphatase dead mutant (C124A) to PTEN-null glioblastoma cells (U87 and A172) using adenovirus-based transfections. We subsequently treated the cells with or without 10 ng/mL SF/HGF and analyzed them for proliferation by cell counting, for cell cycle progression by flow cytometry, and for anchorage-independent growth by clonogenicity in soft agar. We also analyzed the cells for the expression and activation levels of various regulators of cell proliferation and cell death by immunoblotting. We found that PTEN expression inhibited basal cell proliferation, basal cell cycle progression, and basal anchorage-independent growth. PTEN restoration also completely inhibited SF/HGF-induced cell cycle progression and anchorage-independent growth and partially inhibited SF/HGF-induced cell proliferation. Neither PTEN mutants G129E and C124A nor the pharmacologic PI3K inhibitors wortmannin and LY294002 reproduced the inhibitory effects of PTEN on the SF/HGF-induced malignant end points described above, indicating that PTEN modulates c-Met-dependent effects via lipid-phosphatase-dependent and independent mechanisms. PTEN reconstitution inhibited basal and SF/HGF-mediated activation/inactivation of cell cycle regulatory proteins p27, cyclin E, and E2F-1 and cell death-regulatory proteins AKT, mTOR, GSK-3, JNK, and BAD as well as SF/HGF-induced c-Myc oncoprotein expression levels. We also determined the effects of PTEN restoration on total cell protein tyrosine phosphorylation and found that PTEN strongly inhibited the SF/HGF-induced tyrosine phosphorylation of several proteins. The identity of these proteins is currently being investigated. Together, these findings show that c-Met-induced glioblastoma malignancy is strongly amplified by PTEN loss. These findings indicate that experimental therapeutic approaches that combine inhibition of SF/HGF:c-Met with reconstitution of PTEN could lead to improved anti-tumor effects. Such experimental therapeutic approaches are currently being tested in our laboratory. This study was supported by National Institutes of Health grant R01 NS045209 (R.A.).


Juinn-Lin Liu,1 Ta Jen Liu,1 Kenneth D. Aldape,2 Zhenyu Mao,1 Tiffany A. LaFortune,1 and W.K. Alfred Yung1; 1Brain Tumor Center, Department of Neuro-Oncology; and 2Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The phosphoinositide 3-kinase (PI3K)/AKT/mTOR/p70S6K signaling cascade is prevalently activated in GBMs as a result of deletion/mutation of the PTEN tumor suppressor gene or activation/amplification of PI3K-signaling components. PTEN plays differential growth-regulatory roles in the cytoplasm versus the nucleus. In the cytoplasm, it has intrinsic lipid phosphatase activity that specifically antagonizes the PI3K pathway. In the nucleus, PTEN displays Akt-independent growth-suppressing activities. Previously, we demonstrated that PTEN’s cell cycle–dependent nuclear export is triggered by the PI3K cascade, particularly p70S6K, during G1/S transition. The exported PTEN is not only released from its nuclear growth-suppressing activities but can also dephosphorylate cytoplasmic PIP3 to prevent the constitutive activation of Akt-mediated signaling pathways. This scenario exemplifies a reciprocal regulation between PI3K and PTEN. To identify the downstream target genes specifically modulated by nuclear PTEN, we developed an ecdysone-inducible expression system in U251–HF cells. An Affymetrix oligonucleotide microarray analysis identified several nuclear PTEN-specific genes that may contribute to cell growth suppression, including topoisomerase IIα, cdk1, cyclin A2, and cyclin B1. We also verified their expression at the protein level. In congruence with the findings in other advanced human tumors, our immunohistochemical analysis results showed that PTEN was predominantly expressed in the cytoplasm if it was not deleted in GBM tumors. Conversely, S6 is constitutively phosphorylated regardless of PTEN status. This is further supported by our observation of preferential cytoplasmic localization of PTEN in several GBM cell lines, including LN229. We next evaluated the potential of using nuclear PTEN as a therapeutic molecule in GBMs expressing cytoplasmic wild-type PTEN. Our preliminary data showed that nuclear PTEN, but not wild-type PTEN, suppressed the anchorage-independent growth of LN229 cells. Our long-term goal is to establish the profile of PTEN’s subcellular localization in relation to the activation states of the PI3K-signaling cascade to improve the molecular diagnosis, prognosis, prediction, and individualized targeted therapy for GBM.


Tapati Mazumdar,1 Pankaj Sharma,1 Phyllis Harbor,1 Rikhia Chakraborty,1 Baisakhi Raychaudhuri,2,3 Hamid Daneshvar,2,3 Andrew Kanner,2,3 Olga Chernova,2,3 Gene Barnett,2,3 Robert Miller,3,4 Abhijit Guha,5 Michael Vogelbaum,2,3 and S. Jaharul Haque1,3; 1Department of Cancer Biology, 2Department of Neurosurgery, 3Brain Tumor Institute, Cleveland Clinic, Cleveland, Ohio, USA; 4Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio, USA; and 5Arthur and Sonia Labatts Brain Tumor Center, Hospital for Sick Children, University of Toronto, Toronto, Canada

Tumors such as glioblastoma multiforme (GBM) are composed of heterogeneous populations of neoplastic cells. In accordance with the emerging cancer stem cell hypothesis, a minor population of transformed cells that exhibit somatic stem cell-like properties are thought to maintain neoplastic clones within cancer tissue. It was recently reported that both normal and transformed neural stem cells expressed CD133, a transmembrane protein originally identified as a marker of hematopoietic stem cells. In correlating the expression of CD133 and the tumor-initiating potential of primary cultures of GBM, we found that of 4 primary cultures of GBM, CCF-310 and CCF-334, which form neurospheres, a cardinal property of neural stem cells, contained 50.0% and 16.5% CD133-expressing cells, respectively. By contrast, the primary GBM cultures CCF-247 and CCF-252 did not contain CD133-expressing cells. Although lacking CD133-expressing cells, CCF-252 cells were capable of forming neurospheres. The tumor-forming potential of these primary GBM cultures is being studied; we have found that U87 and U251, established cell lines that form tumors in rodents, did not express CD133. Furthermore, U87 cells, which form markedly larger (> 100 times) tumors than U251 cells in rodents, did not express glial fibrillary acidic protein (GFAP) but did express c-Myc. In contrast, U251 cells did not express c-Myc at detectable levels but expressed GFAP. Importantly, it has been demonstrated that activation of Stat3 is required for glial differentiation and GFAP expression. Consistent with these observations, we found that expression of constitutively activated Stat3 downregulated the c-Myc expression in U87 cells and significantly reduced (> 5-fold) the volume of U87-derived tumors in nude mice. These data suggest that the regulated expression of c-Myc and loss (or lack) of expression of GFAP are critical determinants of initiation and progression of tumor growth, whereas expression of CD133 on the cell surface is a bystander in these processes.


K.N. Mendes, W. S. Song, G.N. Fuller, and W. Zhang; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Glioblastoma multiforme (GBM) has a high mortality rate that is attributed to GMB’s high resistance to chemotherapy and radiotherapy and its invasiveness. The invasion-inhibitory gene IIp45, a putative tumor suppressor, was identified in our lab as a binding partner for the insulin-like growth factor-binding protein 2 (IGFBP2), a protein that is overexpressed in high-grade gliomas. In addition to inhibiting cell motility, recent observations have indicated a proapoptotic function for IIp45. A microarray analysis of IIp45-induced genes showed an induction of the BH3-only protein PUMA, suggesting that IIp45 is involved in a mitochondria-dependent apoptotic pathway. Mitochondrial isolation followed by immunoblot analysis indicated an increase in PUMA in the mitochondria of IIp45-expressing cells prior to drug treatment, confirming the microarray data. A colony-formation assay revealed no significant differences between IIp45-expressing cells and control cells, indicating that the IIp45-dependent PUMA increase was not directly responsible for cell apoptosis. These data, along with our ability to generate IIp45-stable lines, led us to determine the effect of IIp45 on the manipulation of the threshold of cells to apoptotic stimuli. Transfection of IIp45 reduced cell viability (as analyzed by an MTS assay) in LN229 cells in response to treatment with the DNA-damaging chemotherapeutic drugs BCNU and etoposide. Similar results were obtained when the LN229 cells transfected with IIp45 were exposed to radiation treatment. A flow cytometric cell cycle analysis after treatment of IIp45-expressing cells revealed an increase in G2/M arrest at lower doses of the drugs, supported by an increase in the p21 protein levels in IIp45-expressing cells. TUNEL staining revealed that IIp45 also increased the apoptotic cell population after treatment with BCNU or etoposide. These results indicate that IIp45 plays a role in DNA damage-induced cell death by sensitizing the cells to cell-cycle arrest and apoptosis. Elucidation of the pathways regulated by IIp45 may lead to new approaches, such as an effective combination strategy using IIp45 and traditional therapy to overcome the problems related to the high resistance of GBM tumors.


J. Mukherjee, A. Wolf, A. Pandita, and A. Guha; Arthur & Sonia Labatts Brain Tumor Center, Hospital for Sick Children’s Research Institute, University of Toronto, Toronto, Canada

GBMs are characterized by pathologic heterogeneity at a cellular and regional level, likely reflecting the molecular heterogeneity in response to the tumor microenvironment. We investigated this cellular and regional molecular heterogeneity, with a focus on molecular regulators of angiogenesis. We obtained 22 paired flash-frozen and paraffin-embedded sections from the “center” and “periphery” of GBMs, on the basis of stereotactic MRI with subsequent neuropathologic verification, along with 5 nonneoplastic control brain specimens. Laser capture microdissection (LCM) on the frozen sections was used to isolate tumor and endothelial cells. Micro-quantities of LCM-isolated RNA were checked for quality and specificity by RT-PCR. A quantitative real-time PCR (QRT-PCR) analysis for known angiogenesis modulators (VEGF, VEGFRs, angiopoietin 1 and 2, Tie1, Tie2, and neuropilin 1 [NRP1]) was undertaken. The expression levels were semi-quantitatively evaluated by immunohistochemical analysis. The functional significance of differential expression of Ang-1, Ang-2, and NRP1 were analyzed by co-culture assay and siRNA-mediated downregulation. QRT-PCR from the center demonstrated increased VEGF and VEGFRs in both the TC/EC compartments vs. the periphery. In contrast, angiopoietin expression was higher in the periphery invading edge, suggesting its role in neovascularization and invasion. NRP1 was highly expressed in tumor cells of the periphery, suggesting it has a role in conjunction with VEGF in regulating endothelial cell motility. Results from the functional assay clearly showed that endothelial tubule formation was regulated and maintained when the Ang-1 level was higher than the Ang-2 level, whereas high Ang-2 expression clearly diminished the tubular structure. Moreover, downregulation of NRP1 in a glioma cell line by siRNA and a neutralizing antibody decreased invasion and resistance to apoptosis. This cellular characterization of known angiogenic modulators in 2 regional compartments of GBMs supports our thesis that the tumor microenvironment differentially influences regulators of angiogenesis, which underlie the regional pathologic differences in GBM angiogenesis.


J. Mukherjee, A. Wolf, and A. Guha; Arthur & Sonia Labatts Brain Tumor Center, Hospital for Sick Children’s Research Institute, University of Toronto, Toronto, Canada

The analysis of expression of natural caspase inhibitors, collectively termed inhibitors of apoptosis (IAPs), has not been undertaken thoroughly in gliomas and is important for our understanding of glioma biologic characteristics and potential therapy and resistance. We evaluated the expression levels of 4 IAP family members (cIAP1, cIAP2, XIAP, and survivin) in low- and high-grade human glioma and any regional differences in their expression between the “center” and “periphery” of human GBMs. Surgically resected flash-frozen sections of low-grade astrocytoma and paired center and periphery regions of GBMs with corresponding paraffin- embedded sections were obtained. Tumor cells were isolated by laser capture microdissection (LCM) from frozen sections, and the isolated micro-RNA was purified. Quantitative real-time PCR was used to determine the expression levels of IAPs. Results were validated by immunohistochemical analysis in the corresponding paraffin-embedded sections. cIAP1 expression was the only IAP member of the 4 we tested that had increased expression in low-grade astrocytomas vs. normal brain. cIAP2 expression was increased (4×) in the periphery of GBMs compared with the center. In contrast, XIAP expression was increased in the center vs. the periphery of GBMs. Similarly, survivin expression was also increased in the center (6.5×) compared with the periphery of GBMs. Resistance to apoptosis in low-grade astrocytomas seemed to be mediated by overexpression of cIAP1, whereas in GBMs, the expression of IAPs was characterized by regional differences, likely representing influences of the tissue micro-environment. In the relatively nutrient-deprived hypercellular center of GBMs, there was increased expression of XIAP and survivin, which are known to be regulated by hypoxia, whereas the periphery of GBMs express cIAP2. Our current study is directed towards understanding the differences in IAP expression profiles in low-grade astrocytomas and the noted regional differences in GBMs. We hypothesize that the differences in IAP expression profiles also play an important role in resistance and recurrence, the understanding of which may translate to better glioma therapy. This study was supported by National Institutes of Health grant R01 CA095006 to S.J.H.


Tatsunori Okamura, Timothy Haystead, Darell D. Bigner, and Francis Ali-Osman; Duke Comprehensive Cancer Center, Departments of Surgery and Pathology and the Preston Robert Tisch Brain Tumor Center, Duke University, Durham, NC, USA

Akt (protein kinase [PK] B), a serine/threonine kinase involved in cell growth and survival, contains a kinase domain that is structurally similar to those of PKC and PKA. Akt is upregulated in a number of human cancers, notably glioblastoma multiforme (GBM), in which lack of the tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a negative regulator of Akt activation, is frequently observed. Akt plays crucial roles in the neoplastic biology of these tumors by phosphory-lating many critical downstream targets, such as mTOR, GSK3, and the proapoptotic BAD proteins. An ever-increasing list of Akt substrates has defined the multiple functions of this kinase family in normal and disease states. We report here that the human glutathione S-transferase P1 (GSTP1) protein, a major drug/carcinogen-metabolizing and stress response-signaling protein, is a heretofore unidentified downstream target of Akt. In cell-free systems, we showed that human recombinant full-length active Akt1 phosphorylates human recombinant GSTP1–1 protein when in its physiologic glutathione (GSH)-bound state. Because human Akt has 3 isozymes, Akt1, Akt2, and Akt3, all of which have emerged as critical mediators of the PI3K-Akt pathway, each Akt kinase was applied to the GSTP1 phosphorylation assay. Interestingly, the Akt2 (ΔPH,S474D) protein had the strongest catalytic activity for GSTP1 phosphorylation of the 3 isozymes. After the phosphatidylinositol 3–kinase (PI3K)/Akt signaling pathway had been activated, endogenous GSTP1 phosphorylation in human GBM cells was examined using PTEN-negative U-87MG and MGR3 cells and PTEN-positive LN-229 cells. In human GBM cells, endogenous GSTP1 was phosphorylated, and its phosphorylation was enhanced after activation of PI3K/Akt pathway, especially in PTEN-positive LN-229 cells. Acid hydrolysis and phosphoamino acid-mapping of Akt-phosphorylated GSTP1 using thin-layer chromatography confirmed both serine and threonine as the phospho-acceptor residues; we are conducting a phosphorylation site analysis by mass spectrometry. Preliminary data on the effects of the Akt phosphorylation on GSTP1 protein function will be presented. These findings, the first-ever demonstration of GSTP1 as a downstream target of Akt, are significant in the context of malignant gliomas in that the genetic background of these tumors, particularly de novo GBM, includes mutations of the PTEN gene that encode a phosphatase whose action results in activation of the PI3K/Akt pathway. This, coupled with the overexpression and high activity of GSTP1 and the high levels of GSH in GBM cells, suggest that interaction of these 2 genetic characteristics could contribute to the drug resistance and aggression of GBMs. This study was supported by grants R01 CA91438, R01 CA79644, and P20–CA096890 from the National Institutes of Health (USA).


Akihiro Otsuki, Ankita Patel, Suzanne Camilli, Kazue Kasai, Sigeru Tanaka, E. Antonio Chiocca, and Yoshinaga Saeki; Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA

Glioblastoma is among the most devastating malignancies in humans and is often rapidly fatal despite aggressive modern treatments. Recent evidence suggests that neoplastic tumors are initiated and maintained exclusively by a rare fraction of cancer cells with stem cell properties (cancer stem cells). Several groups of investigators have reported the presence of stem cell-like cells in human brain tumor specimens, and Dirks and his colleagues elegantly demonstrated that only the CD133-positive fraction of human brain tumor cells has stem-like properties, including self-renewal ability, multipotency, and the potential to develop brain tumors in SCID mice. Normal human neural stem/progenitor cells express high levels of anti-apoptotic proteins, DNA repair enzymes, and ATP-binding cassette (ABC) transporters, which is thought to be the basis for the resistance of these cells to a number of chemotherapeutic drugs and irradiation. According to the cancer stem cell theory, brain tumor therapy should target CD133-positive, stem cell-like cells in brain tumors; they may upregulate several of the same genes as normal neural stem/progenitor cells and thus have similar resistance to most conventional cancer therapies. Oncolytic HSV mutant rQNestin34.5, which lacks viral ribonucleotide reductase (RR) activity and expresses the viral virulence protein ICP34.5 under the control of a nestin/hsp68 chimeric promoter, was recently demonstrated to have enhanced oncolytic activity in cultured glioma cells and in a mouse brain tumor model in vivo. We hypothesized that the upregulated expression of anti-apoptotic and DNA-repair proteins, together with the higher transcriptional activity of the nestin/hsp68 promoter in CD133-positive brain tumor stem cells, would enhance the replication and cytotoxicity of rQnestin34.5. To characterize gene expression profiles of CD133-positive brain tumor stem cells, we prepared human glioma cells from subcutaneous xenograft tumors maintained in nude mice, enriching the CD133-positive and -negative fractions of cells using an immunoaffinity-based cell separation method. We prepared total RNA from these cells and determined the expression levels of stem cell markers, ABC transporters, and anti-apoptotic genes. The propagation and cytotoxicity of rQNestin34.5 and rHSVQ1, a control virus lacking both RR and ICP34.5, were evaluated in CD133-positive and -negative cells. rQNestin34.5 resulted in higher propagation and cytotoxicity in CD133-positive brain tumor stem cells than did rHSVQ1. These results indicate that rQNestin34.5 has great potential in treating brain tumors because of its enhanced oncolytic activity against brain tumor stem cells.


Amith Panner,1,2,3 Jean L. Nakamura,3 Andrew T. Parsa,1,2,3 Pablo Rodriguez-Viciana,3 Mitchel S. Berger,1,2,3 David Stokoe,1,2,3 and Russell O. Pieper1,2,3; 1Dept. of Neurological Surgery, 2The Brain Tumor Research Center, and 3University of California-San Francisco Comprehensive Cancer Center, University of California-San Francisco, San Francisco, CA, USA

RalA, a member of the Ral GTPase family of proteins, has been identified as an important mediator in Ras-induced oncogenic growth and morphologic transformation. The mechanism by which RalA contributes to Ras signaling is thought to involve mTOR and enhanced translation of proteins critical for proliferation and transformation. In addition to its effects on proliferation and transformation, RalA regulates sensitivity to death-inducing ligands, although the means by which this occurs is unknown. In this study, we showed that RalA sensitized cells to the death-inducing ligand TRAIL through an mTOR-independent mechanism. This mechanism involves RalA acting through its binding partner, RalBP1, to suppress the cdc42-mediated activation of S6 kinase and ultimately reduce translation of the anti-apoptotic protein FLIPS. These results suggest that in addition to the mTOR-dependent component of the RalA-signaling pathway involved in proliferation and transformation, an mTOR-independent component of RalA signaling controls extrinsic cell death pathways. The mTOR-independent component can, in turn, be exploited to sensitize gliomas to TRAIL-based therapies.


Peter Pyrko,1 Florence M. Hofman,1 Axel H. Schönthal,2,5 Amy S. Lee,4,5 and Thomas C. Chen1,3; Departments of 1Pathology, 2Molecular Microbiology and Immunology, 3Neurosurgery, 4Biochemistry and Molecular Biology, 5Norris Cancer Center, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) stress protein, that is elevated by hypoxia, hypoglycemia, and reductive stress. It was previously demonstrated that increased GRP78 had protective effects against etoposide and doxorubicin, two DNA-targeting drugs. Glioblastoma multiforme (GBM), the most malignant type of glioma, is characterized by a necrotic, hypoxic, hypoglycemic microenvironment; it is highly resistant to both radiation and chemotherapy. We hypothesized that GBM would have elevated levels of GRP78 and that reduction of GRP78 would increase its chemosensitivity and radiation sensitivity. We examined the tissue sections of patients with GBM and demonstrated that GRP78 was increased in these tumors via immunohistochemical analysis and in tumor cell lysates via Western blot analysis. In malignant glioma cell lines (A-172, U-87, T-98G, U-251, LN-18, LN-229, and U-138), increased expression of GRP78 was demonstrated via immunohistochemical analysis and Western blot analysis in all cell lines except U-138. Moreover, treatment of U-87 and A-172 cell lines with temozolomide (12–1000 μM) increased GRP78 expression 2–fold. The same effect was found with radiation (1–7 Gy) on LN229 cells. Downregulation of GRP78 levels by GRP78 siRNA increased chemosensitivity to temozolomide in U87 cells. Transfection of GRP78 siRNA alone decreased the number of colonies in the colony-forming assay (CFA) by 20%. The addition of TMZ (200 μM) alone decreased colony formation by 50%; the combination of GRP78 siRNA with TMZ (200 μM) decreased colony formation by an additional 30% compared with TMZ alone. Similar effects were observed in U251 and LN229 glioblastoma cells. We also evaluated the effect of downregulating GRP78 on the radiosensitivity of glioblastoma. In U251 cells, we found that the cells in which GRP78 was downregulated were at least 30% more sensitive to radiation than the cells transfected with control siRNA. Our results demonstrate that GRP78 plays an important role in conferring resistance to chemotherapy and radiation in glioblastomas. Because radiation and temozolomide are the 2 pillars of glioblastoma treatment, the reduction of GRP78 may be an important step in increasing chemosensitivity and radiosensitivity in this previously untreatable cancer.


Y. Rong, D.L. Durden, E.G. Van Meir, and D.J. Brat; Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA

Glioblastoma (GBM) is the highest grade astrocytoma and is characterized by aggressive biologic properties and rapid clinical progression. The development of both hypoxia and pseudopalisading necrosis is central to angiogenesis and the accelerated growth of GBM. Intravascular thrombosis and subsequent vaso-occlusion could initiate or propagate this cascade of events. Tissue factor (TF), the main cellular initiator of blood coagulation, is overexpressed in GBMs and likely favors thrombosis. Amplified EGFR genes are found in 40%–50% of GBMs, either in wild-type or mutant forms. Gene amplification is accompanied by overexpression of the wild-type EGFR (wtEGFR) or mutant EGFR (vIII). Upregulated cell signaling through these receptors could contribute to TF expression, thrombosis, and the aggressive course of GBM. In the current study, we investigated the role of wtEGFR and vIII in the regulation of TF expression by GBM cells under normoxia and hypoxia (1% O2). Human GBM cells (U87MG) that had been stably transfected with either wtEGFR (U87-wtEGFR) or EGFRvIII (U87-vIII) showed strong basal upregulation of TF expression by Western blot analysis compared with parental U87MG cells, which had little EGFR expression. U87-wtEGFR showed a dose-dependent increase in TF expression in response to exogenous EGF stimulation, whereas U87-vIII cells had constitutive upregulation of TF that did not respond to EGF. pAkt levels were mildly increased under basal conditions in U87-vIII, whereas pErk1/2 levels were markedly increased in U87-wtEGFR cells compared with parental U87MG cells. We found that TF expression was significantly inhibited by the PI-3K inhibitor LY294002 and the mTOR inhibitor rapamycin under normoxia for both cell lines. The MEK/Erk1/2 inhibitor U0126 caused decreased expression of TF in U87–vIII cells but not in U87–wtEGFR cells, suggesting potentially different signaling pathways in TF activation between wt and mutant EGFR. To determine how these signaling events affect TF expression, we investigated TF gene activation. The TF promoter contains binding sites for Egr-1, Sp1, AP1, and NF-κB. Luciferase reporter assays in U87–wtEGFR cells treated with EGF showed increased Egr-1– and c-Jun/AP1–mediated transcriptional activities but not NF-κB. Hypoxia strongly upregulated TF expression in both U87–wtEGFR and U87–vIII cells. This appeared to be dependent on Egr-1 and AP1 but not NF-κB transcriptional activities. Under hypoxia, only LY294002 caused decreased TF expression by U87–wtEGFR, whereas only U0126 moderately inhibited hypoxia-induced TF expression in U87–vIII cells. In conclusion, both wtEGFR and EGFRvIII caused upregulation of TF expression in GBM cells, and this upregulation was even higher under hypoxia. Differential signaling was involved in the regulation of TF expression by wtEGFR and EGFRvIII under normoxia and hypoxia. Both the EGFR-mediated and hypoxia-mediated TF depended mostly on Egr-1 and AP1 transcriptional activities. Upregulated TF expression by wtEGFR and EGFRvIII, both under normoxia and hypoxia, could be responsible for the prothrombotic events that occur in the progression of GBM.


Qing Shi,1 Shideng Bao,1 Anita B. Hjelmeland,1 Darell D. Bigner,2 and Jeremy N. Rich1,3,4; Departments of 1Surgery, 2Pathology, 3Medicine, and 4Neurobiology, Duke University Medical Center, Durham, NC, USA

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein frequently expressed in several solid cancers upon adoption of metastatic or invasive behaviors. SPARC expression in malignant glioma cell lines induces tumor cell invasion and promotes tumor cell survival upon serum withdrawal; the latter process is dependent on SPARC activation of AKT. To determine the intracellular mediators of SPARC that activate AKT, we examined the effects of SPARC on the activation state of 2 non-receptor tyrosine kinases involved in tumor invasion, focal adhesion kinase (FAK) and integrin-linked kinase (ILK). We selected FAK and ILK for study as they are commonly activated in glioma samples and function to activate AKT. Treatment with exogenous SPARC protein or constitutive overexpression of SPARC activated both FAK and ILK in glioma cells previously characterized as responsive to SPARC. Targeting the expression of either FAK or ILK by small interfering ribonucleic acid (siRNA) transfection inhibited SPARC-mediated AKT phosphorylation. Targeting both FAK and ILK expression with siRNA blocked SPARC-mediated AKT activation more potently than targeting either FAK or ILK alone. This decrease in SPARC-mediated AKT activation correlated with a reduction in SPARC-dependent invasion and survival upon the downregulation of FAK or ILK expression. In addition, SPARC facilitated a novel molecular interaction between FAK and ILK, as either treatment with exogenous SPARC protein or overexpression of SPARC induced a physical association between FAK and ILK. These data further confirm the function of SPARC in glioma tumor progression through the activation of specific intracellular kinases that may provide novel therapeutic targets for advanced cancers. This study was supported in part by funds from the Pediatric Brain Tumor Foundation of the United States, Accelerate Brain Cancer Cure, Childhood Brain Tumor Foundation (J.N.R.), and the Southeastern Brain Tumor Foundation (A.B.H.). This work was also supported by National Institutes of Health grants NS047409, NS054276, and 1 P50 CA108786 (J.N.R.). A.B.H. is a Paul Brazen/American Brain Tumor Association Fellow. J.N.R. is a Damon Runyon-Lilly Clinical Investigator supported by the Damon Runyon Cancer Research Foundation and a Sidney Kimmel Cancer Foundation Translational Scholar.


Liliana Soroceanu,1,7 Samir Kharbanda,1 Ruihuan Chen,1 Robert Soriano,2 Jiping Zha,4 Anjan Misra,6 Ken Aldape,5 William Forrest,3 Janice M. Nigro,6 Zora Modrusan,2 Burt Feuerstein,6 and Heidi S. Phillips1; 1Department of Tumor Biology and Angiogenesis, 2Department of Molecular Biology, 3Department of Biostatistics, 4Department of Pathology, Genentech, Inc., South San Francisco, CA, USA; 5Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 6Brain Tumor Research Center, University of California San Francisco, San Francisco, CA, USA; 7 Current affiliation: California Pacific Medical Center Research Institute, San Francisco, CA, USA

Gene amplification and overexpression of growth factor receptors are frequently found in high-grade gliomas. Expression profiling of 165 high-grade glioma cases revealed that insulin-like growth factor 2 (IGF2) overexpression was a distinct feature of a subset of glioblastomas (GBM) that lack amplification or overexpression of epidermal growth factor receptor (EGFR). Thirteen percent of grade IV astrocytomas and 5% of grade III astrocytomas showed IGF2 mRNA levels that exceeded by 50-fold the median value of all tumors. The mutually exclusive pattern of overexpression between IGF2 and EGFR was validated by Taqman and histologic approaches. Further analyses indicated that IGF2-overexpressing tumors frequently had PTEN deletion, were highly proliferative, displayed intense phospho-Akt staining, and belonged to a subclass of GBMs with poor outcome. Using a serum-free culture system, we showed that IGF2 could substitute for EGF in promoting the growth of GBM-derived neurospheres. We further demonstrated that within GBM cells, IGF2–induced growth was mediated by the insulin-like growth factor receptor 1 (IGF1R) and that IGF1R recruited PIK3R3, a PI3-kinase regulatory subunit found to show genomic gains and overexpression in some highly proliferative GBMs. Stable PIK3R3 knock-down inhibited neurosphere growth induced by IGF2 and, to a lesser extent, growth supported by EGF. These data provide evidence that the IGF2–IGF1R-PIK3R3 signaling pathway is involved in supporting the growth of a subclass of aggressive GBMs that lack EGFR amplification and suggest that this axis is an alternate mechanism to EGFR amplification for promoting tumorigenesis. Furthermore, our results highlight the similarities that exist between signaling pathways alterations that characterize primary human gliomas and factors that promote the in vitro growth of tumor-derived stem cell-like cells.


Liliana Soroceanu,1 Matthias Kraus,2 Scott Denham,2 Wenyue Zhang,2 William Britt,2 Russ Pieper,3 and Charles Cobbs1; 1California Pacific Medical Center Research Institute, San Francisco, CA, USA; 2University of Alabama School of Medicine, Birmingham, AL, USA and 3Department of Neurosurgery, University of California, San Francisco, CA, USA

Infection caused by human cytomegalovirus (HCMV) is ubiquitous, and its persistent latent presence has been documented in several organ systems, including the central nervous system (CNS). Recent immunohistochemical and in situ hybridization data from several laboratories have identified the presence of HCMV in human glioblastoma (GBM) patient tissue, raising the interesting possibility that reactivation of a latent HCMV infection may contribute to the neoplastic process in some high-grade gliomas. To test this hypothesis, glioma, immortalized astroglial, and human embryonic lung (HEL) cells, either mock-infected or infected with the Towne strain of HCMV, were used to determine changes in the tyrosine phosphorylation of several key intracellular signaling molecules. HCMV infection caused rapid phosphorylation of Akt in all cell types and induced recruitment of phosphatidylinositol–3 kinase (PI3K) and phospholipase C (PLCγ) in human fibroblasts. In response to HCMV stimulation, the p85 regulatory subunit of the PI3K was found to co-immunoprecipitate with a high-molecular weight receptor tyrosine kinase, the identity of which we are currently investigating. Using epidermal growth factor receptor (EGFR)-null cells, we demonstrated that activation of cellular tyrosine kinase signaling by HCMV did not require a functional EGFR. Furthermore, HCMV treatment enhanced extracellular matrix-dependent migration of human glioma cells (but not normal astrocytes) associated with tyrosine phosphorylation of endogenous focal adhesion kinases (FAK). Stable expression of the HCMV immediate early gene IE1, which is essential for viral infection, revealed that this gene product induced either proliferation or growth arrest of various glioma cell lines by regulating the steady-state activity of cell cycle-controlling proteins, such as Rb and p53, and by chronic stimulation of Akt. In summary, our results demonstrate that HCMV infection and IE1 expression can trigger rapid intracellular phosphorylation of several components of the signaling pathways regulating tumor cell survival (e.g., PI3K-Akt), motility (e.g., FAK), and cell cycle progression (e.g., p53 and pRb). Taken together, these data suggest a possible role for HCMV in the development and progression of some high-grade gliomas.


Charles B. Stevenson,* Larry A. Pierce,*, Moneeb Ehtesham, Kyle D. Weaver; Department of Neurological Surgery, Vanderbilt University Medical Center, Nashville, TN, USA

Malignant gliomas are highly infiltrative tumors with neoplastic cells that invade extensively through distant and functional brain. Because of this, and despite aggressive surgical and chemotherapeutic intervention, tumor recurrence or progression is almost universal. The development of an effective therapy requires a better understanding of the specific processes that govern glioma growth and invasion. Recent work suggests that the CXCR4/CXCL12 chemokine signaling axis plays a prominent role in delineating an infiltrative phenotype in gliomas, with overexpression of the CXCR4 receptor promoting proliferation and invasion. However, the mechanisms of CXCR4 gene regulation remain unclear. DNA hypermethylation within promoter CpG islands of multiple cancer-related genes and their resultant epigenetic silencing has been implicated in gliomagenesis and progression. The methylation status of the CXCR4 promoter in normal brain tissue and glioma has yet to be reported. Genomic DNA was extracted from 21 gliomas (2 World Health Organization grade I, 3 grade II, 2 grade III, and 14 grade IV) and 2 normal brain specimens using organic techniques. Purified DNA was bisulfite-treated and subjected to methylation-specific polymerase chain reaction (MSP) using both methylated and unmethylated primer sets. MSP products were then visualized on a standard agarose gel and assayed in a semiquantitative fashion. Methylation was detected in a CpG island within the CXCR4 promoter in all 21 gliomas and normal brain specimens. No detectable methylation was present in normal human leukocyte control samples; leukocytes are known to robustly express CXCR4 protein. We observed differential promoter methylation patterns between tumor grades and extensive variability among high-grade tumors; however, these findings were not statistically significant. To our knowledge, this study is the first to demonstrate near-ubiquitous methylation of the CXCR4 promoter in both normal brain tissue and glioma. The variability of CXCR4 promoter methylation in the 14 glioblastomas studied may correspond to the previously characterized differential expression pattern of CXCR4 in GBM. Of note, 2 GBM specimens were largely unmethylated, in contrast to the predominantly methylated status of normal brain tissue and lower-grade gliomas. Given the known pro-invasive role of CXCR4 in glioma, the finding of decreased methylation of the CXCR4 promoter in high-grade tumors suggests a role for epigenetic dysregulation of CXCR4 in the progression and invasion of malignant glioma. Functional studies using quantitative MSP, reverse-transcriptase PCR, and inhibitors of methylation are planned to further understand this interaction.


K. Todkar,1 S. Hanson,1,2 S. Schlaffer,1 N. Pettkus,1,2 E. Pawlak,1 V. Tronnier,1 E. Kim,1,2 and A. Giese1,2; 1Laboratory of Neuro-Oncology, Department of Neurosurgery, University of Schleswig-Holstein, Campus Lübeck, Germany; 2Translational Neuro-Oncology Research Group, Department of Neurosurgery, Georg-August University of Goettingen, Germany

A high potential for invasion and resistance to apoptosis are hallmarks of glioblastoma multiforme (GBM), the most aggressive type of intrinsic brain tumor. The oncogenic factor ets-1 is associated with glioma invasion and is frequently overexpressed in GBMs. We have previously reported that the oncogenic functions of ets-1 are negatively controlled by tumor suppressor p53, which interacts with the ets-1 protein and inhibits transcriptional activation of anti-apoptotic and invasion genes by ets-1. Our new results further reveal that the antagonistic relationship between the 2 factors is reciprocal — ets-1 acts as a potent negative regulator of p53 transcriptional activity. Our results demonstrate, for the first time, that ets-1 inhibits p53 by affecting the stability of the p53 protein. Considering that the TP53 gene is intact in more than 50% of gliomas, our findings indicate that overexpression of ets-1 might be a mechanism that abrogates p53 functions in the absence of inactivating TP53 mutations. The reciprocal and antagonistic relationship between ets-1 and p53 thus comprises a feedback loop that may be deregulated in glioma cells with overexpressed ets-1.


S. Tye, A.G. Gilg, J. Knapp, L. Olson, J.R. Bethard, C.A. Welsh, Z. Rumbolt, I. Takacs, B.P. Toole, and B.L. Maria; Charles P. Darby Children’s Research Institute, Medical University of South Carolina, Charleston, SC, USA

The multicopper oxidase enzyme Ceruloplasmin was recently shown to be secreted by a desmoplastic infantile ganglioglioma that probably arises from multipotent progenitor cells (Knapp J., et al, 2005). The purpose of this study was to determine whether Ceruloplasmin is regulated by hyaluronan (HA), a large polysaccharide that promotes anti-apoptosis, invasion, and drug resistance in malignant cells. Through FACS analysis, we isolated a side population (0.2%–0.72%) of cells from the rat C6 glioma cell line (C6SP) that expressed ABCG2 (BCRP) and were highly drug-resistant by virtue of their BCRP efflux of chemotherapy; in addition, we isolated BCRP-positive neurospheres from the U87 human glioma cell line. C6SP cells cultured on matrigel for 10 days expressed both neuronal (MAP2 and Tuj1) and glial (GFAP) markers. Western blotting showed that C6SP cells and U87 neurospheres contained significantly more Ceruloplasmin than their respective parent lines. Antagonizing hyaluronan/CD44 interactions by treating C6SP and U87 neurospheres with hyaluronan oligomers decreased Ceruloplasmin production. IL-1 beta increased Ceruloplasmin and HIF-1 alpha production in C6 cells more than in IL-6, and IL-6 increased Ceruloplasmin and HIF-1 alpha production more in C6SP. C6 and C6SP cells engrafted into the rodent central nervous system both expressed abundant Ceruloplasmin. Taken together, these results suggest that (1) glioma cells and their progenitor subpopulations express Ceruloplasmin in vitro and in vivo, (2) Ceruloplasmin production in glioma progenitors is heavily dependent on hyaluronan/CD44 interactions, and (3) downstream from hyaluronan/CD44 interactions, inflammatory mediators modulate Ceruloplasmin production differently in glioma progenitors. Ongoing studies will determine how hyaluronan-mediated Ceruloplasmin production contributes to anti-apoptosis, invasion, and drug resistance in glioma progenitor cells.


Timothy Van Meter, Anil Kumar, Catherine Dumur, William C. Broaddus, and Gary Tye; Departments of Neurosurgery, Anatomy and Neurobiology, and Pathology, School of Medicine, Virginia Commonwealth University Health Systems, VA, USA

Previous studies from our laboratories reported characterization of AKT isotype expression and activity in PNET and medulloblastoma cell lines an enhanced sensitivity to cisplatin-induced cell death in the presence of micromolar doses of PH-domain-directed AKT inhibitors. We determined the growth-suppressive effects of AKT inhibitors that have been characterized for their isotype selectivity. We demonstrated the sensitivity profiles of pediatric tumor cells to each inhibitor class using paired luminescent viability and apoptosis assays and IP kinase assays. Oligonucleotide microarray studies suggested novel candidate proteins involved in the cell cycle in medulloblastoma that are diminished by AKT inhibition during the administration of sublethal doses of inhibitors. We have further characterized and validated these candidates by quantitative PCR in cell lines and clinical samples. AKT2 siRNA treatment of model cell lines sensitized DAOY and VC312 cells to cisplatin. Confocal immunofluorescence microscopy studies revealed an association between active AKT and specific stages of mitosis. Our data suggest that the success of the therapeutic inhibition of AKT as a means of enhancing chemotherapeutic response depends on activity toward the AKT2 isotype and that AKT inhibition may also have sustained cytostatic effects that can be exploited in vivo.


Nicole M. Warrington,1 B. Mark Woerner,1 Arie Perry,3 Girish C. Daginakette,2 David H. Gutmann,2 Joshua B. Rubin1,2,4; Departments of 1Pediatrics, 2Neurology, 3Pathology, 4Anatomy and Neurobiology, Washington University School of Medicine, St Louis, MO, USA

Astrocytoma (glioma) formation in neurofibromatosis type 1 (NF1) occurs preferentially along the optic pathway during the first decade of life. The molecular basis of this unique pattern of gliomagenesis is unknown but appears to be dependent on factors derived from the surrounding brain microenvironment. We previously demonstrated that the chemokine CXCL12 (SDF-1α) is expressed in a temporal and anatomic pattern that correlates with the growth of NF1–associated gliomas and that CXCL12 uniquely promotes the growth of Nf1/−, but not wild-type, astrocytes. These data suggest that CXCL12 is a key factor present in the evolving tumor microenvironment that regulates the expansion of Nf1–deficient astrocytes and facilitates NF1–associated glioma growth. To further evaluate this hypothesis, we determined the molecular basis of the difference between wild-type and Nf1/− astrocyte responses to CXCL12 and found that Nf1 loss promoted growth responses to CXCL12 by inhibiting the desensitization of the CXCL12 G protein-coupled receptor (CXCR4). Receptor desensitization is a negative regulatory process that limits the strength and duration of receptor signaling. Desensitization is initiated by ligand-induced, G protein receptor kinase (GRK)-mediated, receptor phosphorylation. In this regard, CXCL12 treatment of wild-type astrocytes induced a 3–fold increase in CXCR4 phosphorylation (desensitization), which resulted in a brief suppression of intracellular cAMP levels. As cAMP suppression is necessary for CXCL12–induced growth responses in astrocytes and gliomas, we observed no effect of CXCL12 on wild-type astrocyte growth. In contrast, loss of Nf1 expression was associated with an MEK-dependent increase in the inactive phosphorylated form of GRK2 and a near total loss of CXCL12–induced, GRK2–mediated CXCR4 phosphorylation. The loss of CXCR4 phosphorylation (desensitization) in Nf1–deficient astrocytes correlated with sustained suppression of intracellular cAMP levels and increased astrocyte growth. Together, these studies provide the first evidence of a novel form of ligand-dependent glioma growth regulation in NF1 and indicate that genetic alterations in tumor cells can transform normal homeostatic microenvironmental signals into potent tumor growth stimuli. The identification of cAMP suppression as a growth stimulus in NF1–associated gliomas supports the evaluation of cAMP-elevating agents, such as phosphodiesterase inhibitors, in the treatment of patients with NF1–associated gliomas.


Todd A. Whitwam, Matthew W. VanBrocklin, and Sheri L. Holmen; Van Andel Research Institute, Grand Rapids, MI, USA

The poor efficacy of current treatments makes glioblastoma multiforme (GBM) tumors ideal candidates for molecularly targeted therapies. Recent advances in molecular biology and proteomics have made it possible to identify characteristic molecular profiles of GBM, and several genetic alterations have been identified, but it remains unclear which of these gene products is required for tumor maintenance and progression. In human GBM, signaling through the phosphatidylinositol 3-kinase (PI3K)/AKT- and RAS/mitogen-activated protein kinase (MAPK) pathways is thought to originate from abnormally activated receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). The loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression also results in increased AKT signaling. RAS is activated in almost all cases of GBM1, and AKT is activated in ~70% of GBM tumors. Once these kinases are active, they signal several downstream pathways, including multiple survival- and cell death-signaling cascades. Understanding which of these signaling cascades is required for tumor progression and maintenance has been a major challenge in glioma oncology. To determine the signaling cascades that drive glioblastoma maintenance, we used pharmacologic inhibitors to target these pathways in human glioblastoma cell lines and assessed the consequences of target inhibition on tumor-free survival using a mouse model of human GBM based on the avian RCAS/TVA system. We observed that inhibition of RAS signaling in glioblastoma tumors induced by expression of activated RAS and AKT resulted in cell death by apoptosis, indicating the importance of this pathway in glioblastoma maintenance in this context. Likewise, inhibition of downstream AKT signaling by the molecular target of rapamycin (mTOR) blockade resulted in apoptosis in a subset of glioblastoma tumor cells. Studies have demonstrated that blocking only 1 pathway often leads to a transient response (e.g., delayed time to progression), but tumors eventually progressed; therefore, the more effective therapies are likely to be those that inhibit more than 1 target. For these reasons, we are currently testing whether inhibition of both the RAS/MAPK and AKT/mTOR pathways in combination results in a synergistic increase in apoptosis in glioblastoma cells. The treatment of glioblastoma cells with an MAPK inhibitor and an mTOR inhibitor in combination has not previously been reported and therefore represents a new approach in the field. Moreover, using compounds that are already being studied in clinical trials for other cancers should allow us to translate the results of these experiments into clinical practice as quickly as possible.


Jill Wykosky and Waldemar Debinski; Wake Forest University School of Medicine, Brain Tumor Center of Excellence, Winston-Salem, NC, USA

We have found that the EphA2 receptor is overexpressed and associated with malignant features in glioblastoma multiforme (GBM) (MCR 3:541–551, 2005; cover). The ligand for EphA2, ephrinA1, is expressed at low levels when the receptor is elevated. Furthermore, a soluble, recombinant homodimer, ephrinA1–Fc, activates EphA2 in GBM and other tumor cells, profoundly affecting their morphologic and malignant features. Hence, we hypothesized that ephrinA1 is a tumor suppressor in several solid tumors. However, the prevailing notion has been that ephrinA1 is a membrane-bound protein, a characteristic that facilitates the formation of stable oligomeric complexes necessary to activate EphA2 on neighboring cells. To determine what form of ephrinA1 fulfills a tumor suppressor function, we transfected U-251 MG GBM cells with ephrinA1 and observed diminished EphA2 levels in confluent cells by Western blotting and immunofluorescence; this supported the membrane-anchored presence of ephrinA1 and cell-to-cell contact responsible for EphA2 activation and ensuing degradation. Unexpectedly, when cells were not in contact, we found the same decrease in EphA2, suggesting the presence of a full-length, functional monomeric ephrinA1 that was not anchored to the cell membrane. Next, we detected a monomer of ephrinA1 in the media of the U-251–ephrinA1(+) cells but not in the media of control cells. We also found that U-251–ephrinA1(+) media caused downregulation of EphA2 in control U-251 MG cells, confirming the specific activation of the receptor. Moreover, U-251–ephrinA1(+) media induced profound, dose-dependent morphologic changes in GBM cells and suppressed the expression of phosphorylated-ERK protein, both of which we have observed previously to result from treatment with ephrinA1-Fc. In a direct investigation of the functional capability of a soluble monomer of ephrinA1, we reduced the recombinant ephrinA1-Fc homodimer and permanently blocked sulfhydryl groups to obtain monomeric ephrinA1-Fc. This monomer had a similar potency to the homodimer in activating EphA2 and causing morphologic changes in GBM cells. We then used SK-BR-3 breast cancer cells of known high levels of endogenous ephrinA1 and found phenomena similar to U-251 MG-ephrinA1(+) cells: (1) EphA2 was suppressed even when cells were not in contact, (2) the media contained a monomer of ephrinA1, and (3) the media had EphA2 level-suppressing activity. Thus, cell-to-cell interaction is not necessary for the activation and downregulation of EphA2 in solid tumors. This is because ephrinA1 can, indeed, function in a paracrine manner and is not entirely dependent on juxtacrine interactions. These findings are important for our understanding of the role of ephrinA1 and EphA2 in GBM pathogenesis. They also have direct implications for the design of therapies against solid tumors such as GBM that exploit the ephrinA1/EphA2 system because soluble monomeric ephrinA1 is a tumor-suppressing factor.


Jill Wykosky and Waldemar Debinski; Wake Forest University School of Medicine, Brain Tumor Center of Excellence, Winston-Salem, NC, USA

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in the oncogenesis of many solid tumors. We have found that the EphA2 receptor is overexpressed in glioblastoma multiforme (GBM) in an inactivated, oncogenic form. Of interest, ephA2 belongs to a small group of genes that are regulated by epidermal growth factor receptor (EGFR)-vIII variant in GBM. We have also found that the ephrinA1 ligand is expressed at low levels, which likely contributes to the lack of EphA2 activation (phosphorylation) and ligand-induced receptor degradation in GBM. Importantly, we have shown that exogenous ephrinA1 has a profound anti-oncogenic effect on GBM cells overexpressing EphA2. The purpose of this study was to determine the role of ephrinA1 as a tumor-suppressing factor in GBM. The primary focus was on the effect of ephrinA1 on the EphA2 oncoprotein and the associated changes in cell morphologic characteristics, migration, and intracellular signaling that result. A Western blot analysis revealed that U-251 MG GBM cells treated with soluble, recombinant ephrinA1–Fc or stably expressing an ephrinA1 transgene exhibited significant downregulation of EphA2. In addition, treatment of U-251 MG cells with ephrinA1–Fc resulted in a rapid and dramatic change in cell morphologic characteristics and cytoskeletal architecture, as revealed by time-lapse microscopy and staining of cells with phalloidin, respectively. Within 5 min, most of the cellular processes were retracted or lost, and cells became strikingly rounded. This phenomenon was reversible, with cells regaining their original shape within 8 hr after stimulation. We also investigated the changes in intracellular signaling mediated by EphA2. After treating U-251 cells with ephrinA1, we performed Western blotting for phospho-ERK and total-ERK protein and EphA2 immunoprecipitation and immunoblotting for the tyrosine-phosphorylated protein. We observed rapid, transient phosphorylation of EphA2 by ephrinA1, followed by a significant decrease in the level of phosphorylated ERK, but not total ERK, that persisted for at least 24 hr. In addition, the treatment of U-251 MG cells with ephrinA1 had a prominent effect on cell migration. In the presence of ephrinA1, these cells exhibited an impaired ability both in migration towards laminin in a trans-well migration assay and in wound closure in a wound-healing assay. Thus, the ephrinA1 ligand, which is present at low levels in GBM, has the potential to downregulate the EphA2 oncoprotein, with ensuing changes in the malignant behavior of GBM cells. This potential tumor-suppressing role can be mediated, at least in part, through suppression of the RAS/MAPK pathway and is not fulfilled in GBM. Therefore, the ephrinA1/EphA2 system may play a dual role in GBM, with ephrinA1 as a tumor suppressor acting through the EphA2 oncoprotein. This scenario will be exploited for the specific therapeutic targeting of GBM.


Shuli Xia,1 Eliot M Rosen,2 John Laterra1; 1The Kennedy-Krieger Institute, Johns Hopkins School of Medicine, Baltimore, MD; and 2Department of Oncology, Lombardi Cancer Center, Washington, DC, USA

A prominent feature of glioblastoma is its resistance to death-receptor-mediated cell apoptosis. In this study, we explored the possibility of modulating Fas-induced cell death with the strong c-Jun NH2 terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and causing “ribotoxic stress.” Anisomycin alone induced cell cycle arrest in U87 glioblastoma cells. We found that anisomycin together with agonistic anti-Fas antibody CH-11 induced synergistic cell death in human glioblastoma cells as anisomycin reduced the IC50 of agonistic anti-Fas antibody CH-11 more than 20-fold (from 500 ng/mL to 25 ng/mL) in U87 and U373 cells. In U87 cells, cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P < 0.001), respectively. Cell death occurred predominantly through apoptosis involving both extrinsic and intrinsic pathways, as evidenced by the cleavage of caspases-3, -8, and -9 PARP and Bid. The expression of Fas, FasL, Flip, and FADD was not changed after treatment with anisomycin + CH-11. JNK was activated 6-fold by the ribotoxic stress caused by anisomycin in U87 cells. Inhibiting JNK activation with the specific JNKK inhibitor CEP-11004 or with dominant-negative MEKK2 significantly prevented cell death induced by the combination of anisomycin + CH-11. On the other hand, activation of JNK with transgenic JNKK1, JNKK2, and MEKK2 did not potentiate the cell death induced by CH-11. We further found that aniso-mycin upregulated the pro-apoptotic protein Bim and CH-11 enhanced this response ~2–fold. Inhibiting Bim expression with siRNA desensitized U87 cells to anisomycin + CH-11. These findings demonstrate that ribotoxic stress sensitizes glioblastoma cells to Fas-induced apoptosis via a mechanism requiring JNK activation and Bim.


Jing Zhang, Yan Zhou, Rwena Cua, and V. Wee Yong; Departments of Clinical Neurosciences and Oncology, University of Calgary, Calgary, AB, Canada

Gliomas in situ have a high density of microglia, suggesting the possibility that a glioma-microglia interaction facilitates the growth of tumors. As chemokines are important mediators of cell-cell communication, we first determined the expression of several chemokines in 16 glioma lines to identify those that were commonly expressed. CCL2 (macrophage chemoat-tractant protein-1, MCP-1) was expressed in 11 glioma cell lines, but no expression of its receptor, CCR2, was detected. Instead, CCR2 was found on microglia and monocytes, suggesting that the tumor-secreted MCP-1 protein interacts with these monocytoid cells to affect glioma growth. We next overexpressed MCP-1 in human cell line U87, which has low baseline level of MCP-1. Stable clones with 8–10-fold more MCP-1 levels had a similar growth rate and invasive capacity as vector controls when cultured by themselves. However, when MCP-1 overexpression clones were co-cultured with human microglia in a 3D collagen gel matrix, the invasiveness of both MCP-1 clones and microglia increased. Superarray analyses demonstrated that specific growth factors, including glial-derived neurotrophic factor, keratinocyte growth factor, fibroblast growth factor-14, and interleukin-6 were consistently increased in the co-culture. These studies revealed new roles and mechanisms for MCP-1 in glioma biology. Targeting MCP-1 or downstream factors may lead to new therapies for currently incurable malignant gliomas.


S. Zhang,1 A. Perry,2 F. Xue,1 E. Henson,1 S.B. Gibson,1 D.D. Eisenstat1; 1Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, MB, Canada; 2Department of Pathology, Washington University, St. Louis, MO, USA

Oligodendroglial tumors are classified as oligodendrogliomas ([OD] World Health Organization grade II) or anaplastic oligodendrogliomas ([AO] grade III). A lack of response to therapy may be due to tumor hypoxia, which facilitates resistance to radiation and chemotherapy. The BCL2 19 kilodalton-interacting protein (BNIP3) is a BCL-2 family member that is upregulated in hypoxic regions in many tumors. BNIP3 is directly activated by the transcription factor HIF1α and mediates cell death in a caspase-independent manner through the interaction of its transmembrane (TM) domain with mitochondria. We have determined that BNIP3 is upregulated in oligodendroglial tumors compared with normal brain tissue. This increased BNIP3 expression was correlated with increased HIF1α and glut-1, indicating hypoxic regions within these tumors. In 17% of primary (de novo) GBMs, we detected mutations in the PEST domain of BNIP3 that targets BNIP3 to the proteosome. These BNIP3 mutations result in a truncated protein that lacks a functional TM domain. We sequenced the BNIP3 gene in 45 oligodendroglial tumors (21 ODs and 24 AOs) and found mutations in 10 (22%). In 21 of 24 AO tumors examined, we found predominantly nuclear expression of BNIP3. Overexpression of BNIP3 in glioma cells induced cell death, whereas treatment with mutant BNIP3 blocked hypoxia-induced cell death. This blockage of cell death was caused by the failure of BNIP3 to localize to the mitochondria and inhibition of BNIP3–mediated mitochondrial dysfunction. Our discovery suggests that BNIP3 function may be abrogated in OD tumors, either by sequestration in the nucleus or inactivating mutations. This could explain why treatments for ODs are often ineffective in hypoxic regions of these tumors.


Yanhua Zheng, Dexing Fang, Yan Xia, and Zhimin Lu; Department of Neuro-Oncology and Molecular Genetics, The University of Texas M.D. Anderson Cancer, Houston, TX, USA

Brain metastases from breast cancer are common and cause significant morbidity and mortality, but their underlying mechanism is still largely unknown. We found that brain metastatic breast cancer cells had higher Ras activity, corresponding to a reduced FAK phosphorylation level than that found in parental breast cancer cells. Dephosphorylation and inhibition of FAK was dependent on Ras activity and mediated by Cdc42 but not other Ras downstream-signaling molecules, such as Raf, PI-3K, Ral, and Rac. Furthermore, overexpression of the wild-type tyrosine phosphatase PTP-PEST, but not its catalytic domain mutant, dephosphorylated FAK. Constitutive activation of FAK in Ras-transformed cells blocked cell migration and invasion. These results strongly suggest that the dephosphorylation and inhibition of FAK resulting from PTP PEST, which is downstream from the activation of Ras and Cdc42, contribute to the spread of breast cancer cells to the brain.


Amotz Ziv-Av,1 Cunli Xiang,2 Wei Lu,2 Simona Cazacu,2 Cathie G. Miller,2 Ronit Sarid, 1 and Chaya Brodie1,2; 1The Everard and Mina Goodman Faculty of Life Sciences, Faculty of Life-Sciences, Bar-Ilan University, Ramat-Gan Israel; and 2Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA

RTVP-1 is a novel gene that was initially cloned from human glioblastoma cell lines and was identified as GLIPR (glioma pathogenesis-related protein) or RTVP-1 (related to testes-specific, vespid, and pathogenesis protein). RTVP-1 was reported to be expressed at high levels in gliomas and glioma cell lines, whereas no expression was observed in other cells or tumors of the central nervous system. In addition, RTVP-1 has also been implicated as a marker of myelomonocytic differentiation in macrophages and has been reported to act as a tumor-suppressor gene in prostate cancer. In this study, we characterized the expression of RTVP-1 in various astrocytic tumors and studied its functions in glioma cells. We found that RTVP-1 was expressed in high levels in glioblastomas, whereas its expression in low-grade astrocytomas, oligodendrogliomas, and normal brain tissues was low. The transfection of glioma cells with siRNAs targeting RTVP-1 decreased cell proliferation in all cell lines examined and induced cell apoptosis in some of them. Overexpression of RTVP-1 increased the anchorage-independent growth of the cells and rendered the cells more resistant to the apoptotic effect of TRAIL and serum-deprivation. To delineate the molecular mechanisms involved in the survival effects of RTVP-1, we determined the expression and phosphorylation of various apoptosis-related proteins. We found that overexpression of RTVP-1 increased the expression of Bcl2 and decreased the phosphorylation of JNK, whereas the expression of Bax and the expression and phosphorylation of AKT were not altered. Silencing of Bcl2 partially abolished the protective effect of RTVP-1 against cell apoptosis. Finally, we found that RTVP-1 regulated the invasion of glioma cells, as evidenced by their enhanced migration through Matrigel and by their increased invasion in the spheroid confrontation assay. The increased invasive potential of the RTVP-1 overexpressers was also demonstrated by the increased activity of MMP2 in these cells. Our results suggest that the expression of RTVP-1 correlates with the degree of malignancy of astrocytic tumors and that RTVP-1 is involved in the regulation of the growth, survival, and invasion of glioma cells. Collectively, these findings suggest that RTVP-1 is a potential diagnostic marker and a therapeutic target in gliomas.


Bingbing Dai, Shin-Hyuk Kang, Frederick F. Lang, Christopher E. Pelloski, Kenneth D. Aldape, Raymond Sawaya, and Suyun Huang; Department of Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The transcription factor Forkhead box Ml (FoxMl) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found that FoxMlB was the predominant FoxMl isoform expressed in human glioma but not in normal brain tissue. The level of FoxMl protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxMl protein expression in human GBM tissues was inversely correlated with patient survival. Enforced FoxMlB expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse models. Inhibition of FoxMl expression in GBM U-87MG cells suppressed their tumorigenicity in vivo. Furthermore, we found that FoxMl regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27Kipl. Finally, we found that FoxMl overexpression increased the invasiveness of glioma cells, whereas inhibition of FoxMl expression significantly suppressed the invasiveness of GBM cells. These results demonstrate that FoxMl is overexpressed in human glioblastomas and contributes to glioma tumorigenicity and invasion. Therefore, FoxMl might be a new potential therapeutic target in human malignant gliomas.



Fred G. Barker, Bob S. Carter, and William T. Curry; Neurosurgical Service, Massachusetts General Hospital, Boston, MA, USA

Racially based disparities in American health care are well documented. We previously reported disparities in outcomes (in-hospital mortality and discharge disposition) after 40,101 craniotomies for brain tumors (primary tumors, metastases, meningiomas, and acoustic neuromas) in the United States from 1988 to 2000. Here, we extend these results by examining racially based differential access to high-volume care, severity of disease at time of admission, and trends in disparities over time. The data source used was the Nationwide Inpatient Sample (HCUP, AHRQ, Rockville, MD). Analyses (multivariate proportional-odds ordinal logistic regression corrected for clustering by hospital and random-effects meta-analysis with bootstrapped heterogeneity estimation) were adjusted for age, sex, primary payer for care, income in ZIP code of residence, geographic region, admission type and source, medical comorbidity, year of treatment, and hospital volume of care, as well as disease-specific factors (metastases: lung cancer diagnosis, systemic metastases). We previously reported that in-hospital mortality and adverse discharge disposition were both more likely in black patients than in others for all tumor types. Pooled odds ratios for blacks were 1.70 for in-hospital craniotomy mortality (95% CI, 1.4–2.1; P < 0.001) and 1.41 for adverse discharge disposition (95% CI, 1.3–1.6; P < 0.001). Blacks tended to present with markers of higher disease severity: more likely emergency admission (P < 0.05 for all four tumor types; pooled OR, 2.1), more likely hydrocephalus for acoustics (P = 0.04), more likely hemiparesis/hemiplegia for primary tumors and meningiomas (P < 0.001), and more likely lung cancer diagnosis for metastases (P < 0.001). Surgeon annual case volume was lower for blacks in all four tumor types, and hospital volume was lower for three of the tumor types. Trend analyses comparing the second to the first half of the study period showed no significant reduction in outcome disparities over time. Black patients were significantly more likely to suffer death or adverse discharge disposition after tumor craniotomy in the United States from 1988 to 2000. Blacks had more severe disease at presentation and lower-volume surgical providers (hospital and surgeon), but disparities persisted after adjustment for these factors. There was no evidence that these disparities lessened over time during the 1990s.


S. Erdal,1 J. Mendes,2 D.D. Bigner,3 and F. Davis2; 1Environmental and Occupational Health Sciences, School of Public Health, University of Illinois at Chicago, Chicago, IL, USA; 2Epidemiology and Biostatistics, School of Public Health, University of Illinois at Chicago, Chicago, IL, USA; 3Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, NC, USA

Epidemiological evidence for brain cancer has so far been inconclusive and/or failed to demonstrate associations or causal links between exposure to chemical agent(s) and brain tumors. We hypothesized that this may be due to inadequate exposure data underlying the epidemological analysis and/or exposure data nonspecific to chemicals that have been demonstrated to cause neurocarcinogenicity in animal models. However, multimedia and multipathway human exposure analyses for known and suspected animal neurocarcinogens (ANCs) have not been attempted previously in an epidemiological investigation of brain cancer. We developed such an exposure framework and model specific to chemical and environmental fate and transport properties of ANCs and used these new human exposure assessment tools and methods in a case-control study of brain tumors. We identified 27 known and 15 suspected ANCs based on our review of original toxicity literature of brain cancer, along with consultation with experts. We developed an environmental and occupational exposure questionnaire specific to these ANCs using their respective physical, chemical, toxicological, and environmental fate and transport information along with information on their use in commerce and commercial products. This fundamental information specific to each ANC guided us in creating an exposure matrix informing the analyst of exposure media and pathway(s) of concern for each ANC. In addition, a weighted scoring system was developed, which takes exposure frequency and exposure duration into account and then enables development of relative exposure score distibutions for a given pathway (e.g., inhalation), a given exposure medium (e.g., air), and/or a given ANC. We also have the ability to separately analyze the exposure scores pertaining to each question in the questionnaire. We have currently obtained data on approximately 200 cases from brain tumor clinics and 180 friend and 180 sibling controls. Variability in the exposure scores seen in the preliminary results suggest that this exposure assessment methodology is effective and useful in cancer epidemiological investigations. In addition, preliminary data indicate that exposure to selected ANCs in the breathing zone may increase the risk of brain tumors. Specific environmental exposure scenarios associated with higher risk are also identified. These data, combined with biomarkers of exposure, will eventually provide a robust data set for understanding exposure to ANCs externally and internally and critical exposure characterization data to better understand the etiology of brain cancer.


N. Esmen,1 S. Lacey,1 K. Kennedy,1 and R. Hancock,1 G. Marsh,2 J. Buchanich,2 F. Lieberman,2 A. Youk,2 and Z. Bornemann2; 1University of Illinois at Chicago, Chicago, IL, USA; 2University of Pittsburgh, Pittsburgh, PA, USA

An exposure reconstruction is under way at the University of Illinois at Chicago (UIC) as part of a collaborative effort with the University of Pittsburgh (UPitt) on their occupational epidemiology study of glioblastoma multiforme (GBM) mortality among a cohort of jet-engine manufacturing workers. The 7-year study began in 2002, when concern over a suspected cluster of brain cancer arose in one of a company’s several manufacturing plants. The primary goal of the UPitt study is to determine whether the cohort has an increased risk of mortality from GBM, and the goal of the UIC study is to examine possible occupational risks for this outcome. Exposures will be reconstructed for workers employed at one or more of eight plants from 1952, when the plant where the concern arose opened, through 2001, the designated cohort end date. An abundance of data exist for the study period and have been assembled into a time-dependent spatial database. These data include company job descriptions with task information, manufacturing process records, machine procurement records, plant layouts, and annual space allocation records. Examination of manufacturing operations and materials used, along with their carcinogenic plausibility, has generated a list of agents for which exposure will be determined individually. In addition to exposure to specific agents, an approach is being used to examine exposure to parts manufactured and the processes to produce them. A job dictionary of exposure to parts, processes, and specific agents by job class over time is being constructed and will be used by UPitt epidemiology collaborators to establish a unique exposure profile for each worker and link those profiles to total and cause-specific mortality with emphasis on brain cancer. This presentation describes the data available for the reconstruction of occupational exposures and the general methodology used to inform the UPitt epidemiology study of GBM mortality.


S.J. Hepworth,1 J.M. Birch,2 P.A. McKinney,1 and the UKCCS investigators; 1Paediatric Epidemiology Group, University of Leeds, Leeds, UK; 2Cancer Research UK Paediatric and Familial Cancer Research Group, University of Manchester, UK

Little is known about the etiology of childhood CNS tumors, but recent epidemiological evidence has suggested that environmental and social factors related to a potential infectious etiology are one area where further investigation is warranted. Immune response and infectious exposure are areas of increasing interest in the investigation of the etiology of CNS tumors in both adults and children. A proxy measure for exposure to common infections in early life is the level and diversity of social contact. Children who attend daycare, and therefore have increased exposure to common infections, appear to be at a reduced risk of developing acute lymphoblastic leukemia in childhood. Thus, lack of exposure increases risk (the “hygiene hypothesis”). It has been hypothesized that infections play a similar role in the development of CNS tumors. Patterns of exposure to infections are likely to critically influence the developing immune system, particularly during the first year of life. We aimed to test the hypothesis that attendance at daycare and social contact in the first year of life and early infancy (as a proxy for infectious exposure) protects against the development of subtype-specific CNS tumors. The UK Childhood Cancer Study is a national population-based case-control study carried out between 1991 and 1996 in 10 regions of the UK to investigate environmental, medical, and familial factors and possible associations with childhood cancer. The study covered all diagnostic groups and included 577 brain tumor cases (aged 2–14 years at diagnosis) and 6,305 healthy, individually matched controls. Information was collected during a personal interview on aspects of social contact, including daycare attendance, social activity with other children, birth order, number of siblings (including those who were attending school), residential history, and sharing of a bedroom with family members. These are all considered possible proxies for infectious exposure. Among the controls, 2,980 (47%) reported attending a preschool group or activity (day-care, swimming, clubs, etc.) in the first year of life, with 736 (12%) reporting formal daycare (defined as any attendance at a day nursery or nursery school, at least two half-day sessions a week at a playgroup or mother-and-toddler group, or at least two half-day sessions a week at a child minder with a minimum of four children attending). We present results from case-control analyses using logistic regression of different aspects of social contact in the first 12 months of life and their association with CNS tumor risk for each specific diagnostic subtype in children aged 2–14 years.


K.A. Jaeckle, A.F. Furth, W. Wu, K.V. Ballman, P.J. Flynn, P.D. Brown, E. Galanis, and J.C. Buckner; Mayo Clinic, Jacksonville, FL, USA; Mayo Clinic, Rochester, MN, USA; Metro MN, USA; and St. Louis Park, MN, USA

Approximately 70%–80% of patients accrued to NCCTG phase II and III glioma trials are from the community, with the remainder being from major referral centers (primarily Mayo Clinic facilities). However, formal comparisons of patient outcomes based on location of treatment have seldom been performed. We analyzed six prospective NCCTG trials of 1,307 newly diagnosed glioma patients (156 from Mayo facilities and 1,151 from non-Mayo facilities) and 330 patients from 14 trials for disease recurrence (78 Mayo and 252 non-Mayo). Demographic factors, prognostic variables, and outcome endpoints were compared between groups. Quality-of-life (QOL) assessments (LASA and FACT-Br) were available for comparison in two trials. Multivariable analysis was performed using Cox proportional hazards models (time-to-event data, such as overall survival), logistic regression (binary outcome data, such as progression-free survival [PFS]), and linear regression (continuous data, such as QOL scores) to examine for differences between Mayo and community patients after adjusting for known prognostic factors. For newly diagnosed patients, there were no significant differences in demographic or prognostic variables (age, ECOG performance status, baseline MMSE, and steroid use at entry) between groups, except for the extent of resection (P = 0.001), with fewer community patients having biopsy (18% vs. 32%) and more having subtotal resection (63% vs. 48%). After adjusting for demographic and prognostic factors in a multivariable model, there were no statistically significant differences between Mayo and community members in outcome (response rate, immediate progression, overall survival, PFS, PFS at 6 months, or time to progression). For patients with recurrent disease, there were no significant univariate differences between community and Mayo patients in demographic or prognostic variables except for age (median age, 55 vs. 50; P = 0.047) and primary resection (biopsy, 13% vs. 27%; P = 0.042). There were also no differences in response rate, time to progression, 6-month PFS, or overall survival in our multivariable models. There were no differences in QOL, as measured by FACT-Br or LASA, or in the change in scores from baseline to end of cycle 1 for these instruments between Mayo and non-Mayo sites. Preliminary analysis indicates no significant differences in the proportion of grade 3 or higher non-hematologic toxicities between groups in a multivariable model for newly diagnosed patients. These data demonstrate that there is an excellent concordance between demographic features, prognostic variables, outcome measures, and QOL for patients treated on NCCTG clinical trials at either community sites or at an academic medical center (Mayo Clinic). This analysis also suggests that outcomes of patients treated within the context of clinical trials are independent of treatment location. Since the majority of patients in these trials were treated in community practices, these outcomes should reflect those of the general population.


G. Marsh,1 J. Buchanich,1 F. Lieberman,1 A. Youk,1 Z. Bornemann,1 N. Esmen,2 S. Lacey,2 K. Kennedy,2 and R. Hancock2; 1University of Pittsburgh, Pittsburgh, PA, USA; 2University of Illinois at Chicago, Chicago, IL, USA

In 2002, the University of Pittsburgh undertook a 7-year exploratory historical cohort study and nested case-control study to investigate a suspected cluster of malignant brain cancer at a jet-engine manufacturing plant in North Haven, CT. A preliminary comparative cancer incidence analysis conducted by the CT Department of Health was inconclusive. Our cohort includes more than 250,000 former and current employees with work experience since 1952 in one or more of eight manufacturing facilities in the Hartford, CT, area. The total and cause-specific mortality experience of the cohort will be examined from 1952 to 2004 and compared with the standard populations of the US, the state of CT, and the CT regional county area. The benign and malignant brain cancer experience of the cohort will also be evaluated from 1976 to 2004 and compared to national and regional brain cancer incidence rates. Each identified case of benign and malignant brain cancer will be matched on age, race, sex, and year of birth to a cohort member without brain cancer at the diagnosis date of the case. The case-control study will enable the collection of data on potential risk factors for brain cancer and co-exposures unavailable from existing record sources. If the participation rate is adequate, the study will also will afford a comprehensive and focused evaluation of brain cancer occurrence in relation to demographic, work history, and occupational exposures while controlling for potential confounding factors. Case-control data collection includes a structured telephone interview and the acquisition of medical records and tissue specimens from cases of malignant glioblastoma multiforme. We plan to use microdissection-based genotyping of the tumor specimens obtained with the appropriate consent from subjects to determine whether these tumors have similar or different mutational profiles than sporadic high-grade glioblastoma multiforme. The target number for our pilot genetic study is 20 subjects who were younger than 70 years at the time of diagnosis. Our investigation is complemented by a companion exposure assessment project at the University of Illinois at Chicago that will characterize the historical work practices and exposures that occurred in each study plant. We will use the work history and exposure data in our cohort and case-control studies to examine the relationship between brain cancer mortality and incidence and the past working environment of the plants. Our study is the largest and most comprehensive study of its kind ever conducted to investigate environmental agents possibly causally related to development of benign and malignant brain cancers. This paper will present and discuss the background, objectives, and methodologies used in our study.


B.J. McCarthy,1,2 C. Xu,1 J.M. Propp,1,2 T. Valyi-Nagy,1 and J.L. Villano1; 1University of Illinois at Chicago, Chicago, IL, USA; and the Central Brain Tumor Registry of the United States (CBTRUS), Chicago, IL, USA

Olfactory tumors, especially olfactory neuroblastomas and carcinomas with neuroendocrine differentiation, are extremely rare. Little information is available and is frequently not population based. The objective of this study was to more fully describe selected olfactory tumor groupings from the nasal cavity (C30.0 ICD-O-3 site code) and accessory sinuses (C31.0-C31.9 site codes) diagnosed between 1973 and 2002 using nine registries of the population-based Surveillance, Epidemiology and End Results (SEER) November 2004 public-use data set. Frequencies, incidence rates, and relative survival rates for olfactory neuroblastomas (ON; ICD-O-3 histology codes 9364, 9473, 9500, 9520, 9521, 9522, and 9523) and carcinomas with neuroendocrine differentiation (CND; ICD-O-3 histology codes 8041, 8240, and 8246) were estimated using SEER*Stat 5.2 software. Squamous-cell carcinomas (SCC; ICD-O-3 histology codes 8070–8072) were used as a comparison group. Incidence rates were age-adjusted to the 2000 US standard population. Of the 5,084 tumors found in the nasal cavity and accessory sinuses, 283 were ON, 62 were CND, and 2,241 were SCC. The incidence of ON was 0.044/100,000 person-years (p-y), while the incidence of CND was 0.009/100,000 p-y and the incidence of SCC was 0.353/100,000 p-y. The incidence of ON was higher in the nasal cavity, while the incidence of CND and SCC were both higher in the accessory sinuses. The incidence was highest in the ‘other’ racial category for all three histology groupings (0.054, 0.017, and 0.420, respectively), although this was not significantly different from the incidences in whites (0.044, 0.009, and 0.341, respectively) or blacks (0.024, sample size too small, and 0.392, respectively). Although there were slightly more males than females diagnosed in all three histology groupings, there were no statistically significant differences in incidence by gender. Median age at diagnosis was 53 years for ON, 57 years for CND, and 66 years for SCC, although patterns by age at diagnosis differed between the three histology groupings. The 1- and 5-year relative survival estimates were 90.2% and 71.1%, respectively, for ON; 78.6% and 52.1% for SCC; and 81.0% and 36.7% for CND. Additional incidence and survival estimates will be presented. This descriptive study will provide a detailed analysis of selected olfactory tumor groupings, which are often excluded from epidemiological studies because of their rarity.


B.J. McCarthy,1,2 J.M. Propp,1,2 and F.G. Davis1; 1University of Illinois at Chicago, Chicago, IL, USA; and the 2Central Brain Tumor Registry of the United States, Chicago, IL, USA

Five-year survival is the standard measure of outcome for many cancer diagnoses. In adults with a primary malignant brain tumor, 5-year survival rates are dismally low. However, for those who have survived some length of time post-diagnosis, the probability of survival increases dramatically. The objective of this study was to provide estimates of the conditional probability of survival by histology and age at diagnosis for adults with primary malignant brain and CNS tumors who have already survived for a specific period of time post-diagnosis. Adults (aged 20 and older at the time of diagnosis) with primary malignant brain and CNS tumors (ICD-O-3 site codes C70.0–C72.9 & C75.1–C75.3) diagnosed between 1990 and 2002 were identified from nine regions (Atlanta, Connecticut, Detroit, Hawaii, Iowa, New Mexico, San Francisco-Oakland, Seattle-Puget Sound, and Utah) of the Surveillance, Epidemiology, and End Results (SEER) public-use November 2004 dataset. Relative survival in 1-month intervals was estimated using the actuarial method adjusted for heterogeneity in withdrawal using the SEER-Stat software. The conditional probabilities of surviving for various time periods (n-year survival conditional on select lengths of time; e.g., 2-year survival given survival to 6 months) were calculated. Between 1990 and 2002, 17,989 adults were diagnosed with a primary malignant brain or CNS tumor. Two- and 5-year relative survival rates were 33.0% and 25.0%, respectively, for all primary malignant brain tumors. Survival estimates were inversely associated with age at diagnosis and varied significantly by histology. Conditional probabilities of survival also varied by histology and age at diagnosis. Five-year survival of all patients with primary brain and CNS tumors conditional on 2–year survival was 82.7% for those aged 20–29, 77.3% for those aged 30–39, 72.9% for those aged 40–49, 64.4% for those aged 50–59, 58.6% for those aged 60–69, and 64.6% for those aged 70+. For those with glioblastoma, the 5-year conditional probability of survival increased from 2.8% at the time of diagnosis to 9.6% for those surviving 1 year post-diagnosis, 34.4% for those surviving 2 years post-diagnosis, and 62.1% for those surviving 3 years post-diagnosis. For those with oligodendroglioma, the 5-year conditional probability of survival increased from 76.3% at the time of diagnosis to 82.5% for those surviving 1 year post-diagnosis, 86.6% for those surviving 2 years post-diagnosis, and 90.5% for those surviving 3 years post-diagnosis. Conditional survival for other time periods and intervals will be presented. Conditional survival provides more useful and encouraging information for brain tumor patients who have survived for some specified time after diagnosis, compared to 5-year survival estimates made at the time of diagnosis.


S.M. Sawrie,1 B.L. Guthrie,2 S.A. Spencer,1 R.A. Nordal,1 R.F. Meredith,1 J.M. Markert,2 and J.B. Fiveash1; University of Alabama School of Medicine, Departments of 1Radiation Oncology and 2Neurosurgery, AL, USA

The objective of this study was to identify a subgroup at low risk for distant brain failure (DBF, metachronous brain metastasis) using three previously identified predictors in patients treated initially with stereotactic radiosurgery (SRS) alone for newly diagnosed brain metastases. Records were reviewed from 100 patients treated at our gamma-knife facility from 2003 to 2005. All patients were newly diagnosed with brain metastases and underwent SRS as the initial treatment for their CNS disease. The primary endpoint was DBF. The three previously identified predictors of DBF were number of metastases, extent of extracranial disease, and histology. The median number of metastases was 2 (range, 1–6), with a median total tumor volume of 6.1 cm3 (range, 0.3–54.4 cm3). Forty-seven percent of the patients had non-small-cell lung cancer; melanoma (21%), breast cancer (17%), renal cell (8%), and other histologies (7%) comprised the remaining sample. Extracranial disease was either absent (28%), stable (22%), or not controlled (50%). Forty-eight percent developed DBF during the follow-up period. The 1-year actuarial risk of DBF was 61% for all patients. Three distinct risk groups were identified using combinations of the three predictor variables noted above. The low-risk group included those with [less-than-or-eq, slant] 3 metastases, no extracranial disease, and non-melanoma histology (N = 17). Three patients in this subgroup developed DBF during our follow-up period. This group had a median time to DBF of 90 weeks. One-year actuarial freedom from DBF was 92%. The high-risk group (N = 21) included all patients with a melanoma histology, regardless of number of metastases or status of extracranial disease. These patients had a median time to DBF of only 18 weeks. All other patients comprised a distinct intermediate-risk group, with a median time to DBF of 37 weeks. The Kaplan-Meier curves for these three groups were statistically distinct (log-rank = 26.34; P < 0.0001). Significant heterogeneity exists in time to DBF among patients with newly diagnosed brain metastases treated initially with SRS alone. Patients with [less-than-or-eq, slant]3 lesions, no extracranial disease, and non-melanoma histology appear to have a low risk of DBF. These patients may be candidates for initial localized treatment, reserving whole-brain radiotherapy (WBRT) for salvage. Patients in the intermediate or high-risk groups may be candidates for initial WBRT or should be considered for clinical trials. Clinical trials not using WBRT should stratify for these risk factors.


M.E. Scheurer,1 M. Wrensch,2 R.A. El-Zein,1 M. Moghadassi,2 R. Miike,2 K.D. Aldape,3 and M.L. Bondy1; Departments of 1Epidemiology and 3Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; and 2Department of Epidemiology and Biostatistics, The University of California, San Francisco, San Francisco, CA, USA

First-generation antihistamines cross the blood-brain barrier with high affinity and have significant sedative effects. In addition, histamine (H-1) receptors affected by these drugs are found on glial cells and appear to have an effect on the neural inflammatory response. We found an increased risk for anaplastic astrocytoma (AA) and low-grade glioma (LGG) among cases reporting the use of antihistamines. We also confirmed the protective effect of anti-inflammatory drug use previously reported for glioblastoma (GBM) but did not find similar effects for AA or LGG. Data collected as part of the Harris County Adult Glioma Study and the Bay Area Adult Glioma Study were pooled for the purpose of this analysis. This allowed the inclusion of 830 brain tumor cases and 831 frequency-matched controls. Because of differences in reporting, proxy-reported cases (N = 197) were excluded from this analysis. Additional cases (N = 23) with missing or ambiguous histology were also excluded. Of the remaining cases, there were 339 cases of GBM, 117 cases of AA, and 154 cases of LGG. Logistic regression models were built comparing each histologic grade to all controls. All models were adjusted for the matching variables (age, sex, and race) and study series. Antihistamine use was associated with an increased risk for AA (OR, 2.73; 95% CI, 1.60–4.65) and LGG (OR, 1.86; 95% CI, 1.11–3.11) but not for GBM. Anti-inflammatory use was associated with a reduced risk for GBM (OR, 0.69; 95% CI, 0.51–0.94) but not for other histologic grades. A history of asthma or allergies was protective for all histologic types (GBM OR, 0.65; 95% CI, 0.46–0.92; AA OR, 0.49; 95% CI, 0.28–0.85; LGG OR, 0.64; 95% CI, 0.40–1.02). A history of chicken pox was protective for GBM (OR, 0.60; 95% CI, 0.42–0.87) and AA (OR, 0.42; 95% CI, 0.25–0.72) but not for LGG (OR, 0.78; 95% CI, 0.45–1.37). While it is apparent that inflammatory processes play a role in the development of gliomas, these data point to potentially different pathways for GBM compared to AA and LGG. This report is the first analysis to examine the effects of antihistamines on the development of gliomas. It also confirms prior reports of a protective effect of anti-inflammatory use for GBM, but it is the first to show that this protection is not extended to AA and LGG. Since AA and LGG arise by molecular pathways distinct from GBM, it is possible that these causative factors are related to specific molecular subtypes of glioma. The relationship of these pathways with neural inflammation deserves further investigation.


M.L. Siker,1 B. Berkey,2 K. Porter,3 D. Nelson,4 W. Curran,5 J. Michalski,6 L. Souhami,7 A. Chakravarti,8 W. Yung,9 J. DelRowe,10 C. Coughlin,11 and M.P. Mehta1; 1University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 2Radiation Therapy Oncology Group (RTOG), Philadelphia, PA, USA; 3American College of Surgeons (ACoS), Commission on Cancer, Chicago, IL, USA; 4Mayo Clinic, Rochester, MN, USA; 5Thomas Jefferson University, Philadelphia, PA, USA; 6Washington University Medical School, St. Louis, MO, USA; 7McGill University, Montreal, PQ, Canada; 8Harvard Medical School, Boston, MA, USA; 9The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 10Montefiore Medical Center, Bronx, NY, USA; 11Dartmouth-Hitchcock Medical Center, Lebanon, NH,USA

Grade IV glioblastoma (GBM) is rare in early adulthood, and little data are available on this subgroup. In a retrospective comparison of two large databases, we investigated whether very young age has an independent effect on survival. In a recently completed RTOG database analysis, age was found to be an independent predictor of survival. To determine whether this phenomenon is also present in the non-clinical trial patient population, we compared these results to the ACoS National Cancer Data Base (NCDB). In the RTOG evaluation, we analyzed all eligible GBM cases from all treatment arms of 17 RTOG studies from 1974 to 2002. All patients with GBM during the years 1985–1998 in the NCDB were included for comparison. Patients were divided into three cohorts: ages 18–30, 31–49, and 50 or greater. Overall survival as a function of age (discrete and continuous) was assessed. In the RTOG review of 3,136 patients, 112 cases (3.6%) were 18–30 years old, 780 (24.9%) were 31–49, and 2,244 (71.6%) were 50 or older. The median survival times of the three groups were 21, 13.5, and 9.1 months, respectively (P < 0.0001). Significant improvement in survival for younger patients was demonstrated, with adjustment for recursive partitioning analysis (RPA) class. Of the 37,260 patients analyzed in the NCDB, 796 cases (2.1%) were 18–30 years old, 5,711 (15.3%) were 31–49, and 30,753 (82.5%) were 50 or older. The median survival times of the three groups were 18, 12.8, and 6.3 months, respectively (P < 0.0001). Data were not available for RPA class. GBM is very rare in young adulthood (age [less-than-or-eq, slant] 30 years), comprising 2.1%–3.6% of patients in our study; these patients have superior survival even when adjusted for RPA class in the RTOG database. Investigations on the unique biologic characteristics of tumors in this population are the subject of future research.


E.P. Zeitler, M. Goodman, P.A.S. Johnstone, N. Esiashvili; Emory University, Atlanta, GA, USA

Data from the SEER database were analyzed to better understand trends in the incidence and survival of infants less than 1 year old with CNS malignancies. Earlier analyses pointed to an increasing incidence in CNS malignancies in that age group, but data now available through the SEER database expand the ability to thoroughly investigate possible trends. Population-based data from 1973 to 2002 in the SEER program were used to examine the frequency, incidence, and survival of patients younger than 1 year with CNS malignancies. Incidence was analyzed for the entire group and separately for patients with different demographic characteristics and histologic types of CNS tumors. Actuarial survival rates were examined in 1-, 5-, and 10-year intervals to detect changes in survival over time. Overall incidence of CNS tumors in infants was slightly increased over time, from 15 to 30 cases per million from 1973 to 1987, with stabilization of rate for the following decade (average, 28.7 cases per million). There was predominance in the rate of CNS tumors among whites (32.1 per million) versus blacks (18.6 per million). The most common diagnosis was astrocytoma, followed by primitive neuroectodermal tumors and ependymoma, respectively. When 5-year observed survival was examined for the entire group by 5-year intervals, there was no significant improvement over the past three decades, with an average of 45% survival at 5 years. There was no difference in survival between sexes or between blacks and whites. Among tumor types, the best survival was seen for astrocytoma. There has been little change in the frequency, survival, and distribution of CNS tumors in the infant population over the past 30 years. There is a clear need to improve our understanding of tumor biology and develop better management strategies to optimize the outcome of these very challenging cases.


John Wiencke,1 Margaret Wrensch,1 Joe Wiemels,1 Rei Miike,1 Joe Patoka,1 Michelle Moghadassi,1 Alex McMillan,2 Karl T. Kelsey,3 Kenneth Aldape,4 Kathleen R. Lamborn,1 Andrew Parsa,1 Jennette D. Sison,1 and Michael D. Prados1; 1Department of Neurological Surgery and 2Comprehensive Cancer Center Biostatistics Core, University of California San Francisco, San Francisco, CA, USA; 3Department of Genetics and Complex Diseases, Harvard School of Public Health, Harvard University, Boston, MA, USA; and 4Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

In a population-based study of glioma patients, we examined survival in relation to potentially pertinent constitutive polymorphisms, serologic factors, and tumor genetic and protein alterations in epidermal growth factor receptor (EGFR), MDM2, and TP53. Subjects were newly diagnosed adults residing in the San Francisco Bay Surveillance Epidemiology and End Results Area during 1991 to 1994 and 1997 to 1999 with central neuropathology review (n = 873). Subjects provided blood for serologic studies of IgE and IgG to four herpes viruses and constitutive specimens for genotyping 22 polymorphisms in 13 genes (n = 471). We obtained 595 of 697 astrocytic tumors for marker studies. We determined treatments, vital status, and other factors using data from registries, interviews, medical records, and active follow-up. Cox regressions for survival were adjusted for age, gender, ethnicity, study series, resection versus biopsy only, radiation, and chemotherapy. Using a stringent P < 0.001, glioma survival was associated with ERCC1 C8092A (hazard ratio [HR], 0.72; 95% confidence limits [95% CL], 0.60–0.86; P = 0.0004) and GSTT1 deletion (HR, 1.64; 95% CL, 1.25–2.16; P = 0.0004); glioblastoma patients with elevated IgE had 9 months longer survival than those with normal or borderline IgE levels (HR, 0.62; 95% CL, 0.47–0.82; P = 0.0007), and EGFR expression in anaplastic astrocytoma was associated with nearly threefold poorer survival (HR, 2.97; 95% CL, 1.70–5.19; P = 0.0001). Based on our and others’ findings, we recommend further studies to (1) understand the relationships of elevated IgE levels and other immunologic factors with improved glioblastoma survival, which are potentially relevant to immunologic therapies, and (2) determine which inherited ERCC1 variants or other variants in the 19q13.3 region influence survival. We also suggest that tumor EGFR expression be incorporated into the clinical evaluation of patients with anaplastic astrocytoma.



Marta M. Alonso, Candelaria Gomez-Manzano, Hong Jiang, OK-Hee Lee, Yuji Piao, Frederick Lang, W.K. Alfred Yung, and Juan Fueyo; Brain Tumor Center, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Novel therapies are needed for gliomas, and the combination of oncolytic vectors and chemotherapy offers hope for the treatment of this malignancy. In addition, this combination may allow for lower virus doses to achieve an anticancer effect, thus resulting in lower undesirable toxicities due to viral proteins. Mathematical modeling shows that the balance between the rate of tumor cell growth and virus spread is a critical determinant of the outcome of oncolytic virus infection. In this work, we determined whether a combination of the oncolytic adenovirus Delta-24-RGD and temozolomide (TMZ) or RAD001 resulted in an enhanced anti-glioma effect in vivo. We determined the in vitro cytotoxic effects and replication properties of Delta-24-RGD alone and in combination with TMZ or RAD001 in the U87 MG glioma cell line by MTT and TCID50, respectively. Athymic mice bearing glioma xenografts (5 × 105 U87 MG cells/animal) received 3 intratumoral injections (days 3, 5, and 7 after implantation) of Delta-24-RGD (108 p.f.u./animal). TMZ was administered over 5 days at a dose 7.5 mg/kg, and RAD001 was given in a regimen of 5.0 mg/kg/day for 5 days until the end of the experiment. Survival was analyzed by the Kaplan-Meier method and the log-rank test. Pathologic examination and adenoviral protein immunostaining were used to assess the anti-glioma effect and the in vivo replication of Delta-24-RGD. Our data showed that in vitro treatment with TMZ or RAD001 not only did not interfere with adenovirus replication but enhanced its oncolytic properties. The combination of Delta-24-RGD and TMZ or RAD001 resulted in a potent anti-glioma effect, and 80% of animals were still alive after 100 days. Pathologic analyses of the animals showed marked areas of necrosis and expression of late adenoviral genes, indicating in vivo replication. The combination of Delta-24-RGD and TMZ or RAD001 significantly increased survival in vivo and generated a high percentage of animals that were asymptomatic for a long time. The results of this study suggest that this combination of treatments should be tested in a clinical trial of patients with glioblastoma multiforme.


Joshua C. Anderson,1 J. Robert Grammer,1 Wenquan Wang,2 L. Burton Nabors,3 Jerry E. Stewart Jr.,1 and Candece L. Gladson1; Department of 1Pathology, Division of Neuropathology, 2Medicine Hematology-Oncology Division (Biostatistics Section), and 3Neurology (Neuro-Oncology), University of Alabama at Birmingham, Birmingham, AL, USA

Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models, and a modified TSR peptide, ABT-510, has now entered Phase II clinical efficacy trials in non-glioma tumors. Because microvascular endothelial cells (MvEC) exhibit heterogeneity, we determined the ability of the modified TSR peptide (NAcSarGlyValDallo-IleThrNvaIleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and induce apoptosis of brain microvessel endothelial cells (MvECs) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19) completely inhibited the growth of human malignant astrocytoma tumors established in the brains of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvECs was significantly higher (3-fold) in the tumors of ABT-510–treated animals. Similar results were found in which an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvECs propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8–dependent mechanism. It also inhibited tubular morphogenesis of MvECs propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that is blocked by an antibody against the TSP-1 receptor (CD36) on MvECs. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.


Hiroshi Aoki,1 Eiji Iwado,1 Mark S. Eller,2 Yasuko Kondo,1 Keishi Fujiwara,1 Guang-Zhi Li,2 Kenneth R. Hess,3 Doris R. Siwak,4 Gordon B. Mills,4 Barbara A. Gilchrest,2 and Seiji Kondo1,5,6; 1Department of Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Department of Dermatology, Boston University School of Medicine, Boston, MA, USA; Departments of 3Biostatistics and Applied Mathematics and 4Molecular Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 5The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA; and 6Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA

Telomere 3 overhang-specific DNA oligonucleotides (T-oligos) induce cell death in cancer cells by mimicking telomere loop disruption. Therefore, T-oligos may be useful as a new therapeutic strategy for various cancers. The purpose of this study was to elucidate how T-oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T-oligos inhibited the proliferation of malignant glioma cells through induction of non-apoptotic cell death and mitochondria hyperpo-larization, whereas normal astrocytes were resistant to T-oligos. Tumor cells treated with T-oligos showed autophagic features, with development of autophagic vacuoles and conversion of an autophagy-related protein, microtubule-associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reverse-phase protein microarray analysis revealed that treatment with T-oligos inhibited the Akt/mammalian target of rapamycin (mTOR) pathway that is associated with regulation of autophagy. Moreover, pretreatment with T-oligos significantly prolonged the survival time of the animals intracranially inoculated by malignant glioma cells compared with the untreated and control oligonucleotides (C-oligos)-treated groups (P = 0.0065 and P = 0.043, respectively). These results indicate that T-oligos are a promising agent to treat malignant gliomas by stimulating the induction of non-apoptotic autophagic cell death.


Vladimir E. Belozerov,1 Taku Narita,1 Jiyoung Mun,2 Rita Noronha,3 Hui Mao,2 Saroja N Devi,1 Mark Goodman,2 Kyriakos C. Nicolaou,3 Ruiwen Zhang,4 and Erwin G. Van Meir1; 1Department of Neurosurgery & Hematology/Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA; 2Department of Radiology, School of Medicine, Emory University, Atlanta, GA, USA; 3Department of Chemistry, Scripps Research Institute, La Jolla, CA, USA; and 4Cancer Pharmacology Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA

Hypoxia is a common feature of brain tumors that develop as the tumor mass outgrows the existing vascular supply. Intratumoral hypoxia strongly contributes to the malignant phenotype of brain tumors by enhancing angiogenesis, migration, and clonal selection of mutations in anti-apoptotic genes (e.g., in TP53) and maintaining an undifferentiated state of cancer stem cells. Further, hypoxic areas of the tumor are resistant to traditional chemotherapies and radiotherapies; therefore, they represent an important therapeutic target. HIF-1 is a key transcription factor that orchestrates a range of adaptive molecular responses, allowing cancer cells to survive and proliferate in a hypoxic environment. Our laboratory and others have shown that the reduction of HIF-1 levels in cancer cells significantly slows down tumor growth in vivo. These findings are the basis for our efforts to characterize and develop specific small molecule inhibitors of HIF-1. Recently, we screened a combinatorial library of natural product-like compounds using a cell-based assay for HIF activity and discovered a new class of selective HIF-1 inhibitors. Our studies suggest that the lead inhibitor of this class, KCN1 (IC50 ~ 4 μM), potently reduces hypoxic levels of HIF-1α in glioma cell lines while exerting minimal effects on other short-lived proteins, HIF-1β, and control proteins. Further, the activity of KCN1 appeared to be independent of the activation state of major signal transduction pathways. Evidence addressing the molecular mechanism of KCN1 will be presented. Our initial animal experiments suggest that KCN1 inhibits HIF-1 signaling in tumor xenografts and accumulates in orthotopic brain tumors, providing the basis for further in vivo studies. Preliminary data on the systemic administration of KCN1 in LN229 glioma xenograft-bearing mice suggest that intratumoral levels of HIF-1α are reduced compared with controls, and this reduction correlates with increased necrosis in treated tumors. We are currently determining the pharmacokinetic characteristics of KCN1 to define the best administration route and dosing to evaluate its anti-tumor effects in glioma models.


Vaibhav Chumbalkar,1 Yeo-Hyeon Huang,1 Ho-Shin Gwak,1 Wei Zhang,2 Yasuko Kondo,1 Nicholas P. Farrell,3 and Oliver Bogler1; Departments of 1Neurosurgery, Brain Tumor Center and 2Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; and 3Department of Chemistry, Virginia Commonwealth University, Richmond, VA, USA

Polynuclear platinum chemotherapeutics (PPCs) BBR3464, BBR3571, and BBR3610 are potent agents against glioma cells in cultures and animal models; thus, we determined their mechanism of action at the cellular level. BBR3610, the most potent compound, had an IC90 dose 250 times lower than cisplatin for glioma cells and significantly extended survival in mice with U87 intracranial tumors. An analysis of apoptosis and cell cycle distribution showed that PPCs induced G2/M arrest in the absence of cell death, whereas cisplatin predominantly induced apoptosis. The cell cycle arrest induced by PPCs was accompanied by hallmarks of autophagy, including vacuole acidification and LC3 clustering and processing. Both PPCs and cisplatin induced ERK1/2 phosphorylation, and inhibition of this pathway at the level of MEK antagonized the induction of G2/M arrest or apoptosis, respectively. An analysis of Chk1, Chk2, and survivin did not reveal what underlies the different response. This prompted a broader molecular analysis, undertaken at the mRNA and protein levels. Using Agilent microarray-based gene expression analysis in glioma cells, we identified genes that were specifically suppressed by treatment with cisplatin but not BBR3610. Knockdown by siRNA of these genes is being evaluated as a strategy to enhance the apoptosis seen in response to PPCs. We have also used 2–dimensional liquid chromatography on the PF2D platform to profile the proteome in PPC-treated cells. This approach led to the isolation of 39 peaks specifically associated with BBR3610 treatment, and we are identifying the proteins using LC-MS/MS to elucidate PPC’s mechanism of action and develop biomarkers indicative of response.


John F. de Groot,1 Li Lu,1 Ta-Jen Liu,1 Gregory Fuller,2 W.K. Alfred Yung1; Brain Tumor Center, Departments of 1Neuro-Oncology and 2Neuropathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

High-grade gliomas release excitotoxic concentrations of glutamate, which has been shown to enhance tumor proliferation and migration. A pharmacologic blockade of glutamate receptors decreases tumor viability and improves the therapeutic efficacy of cytotoxic chemotherapy in vitro. Despite ongoing therapeutic trials aimed at targeting glutamate receptors for the treatment of glioma, there remains an incomplete understanding of the mechanisms by which glutamate enhances tumor growth and invasion. We examined AMPA-mediated signaling in glioma cell lines and used shRNA to stably knockdown the GluR1 subunit of the a-amino-3–hydroxy-5–methylisoxazole-4–propionic acid (AMPA) receptor to evaluate its effects on signaling and tumor proliferation and tumorigenicity, both in vitro and in vivo. The mRNA and protein expression of GluR1–4 of AMPA subunits was evaluated in a panel of glioma cell lines. GluR1, the most abundantly expressed subunit, was further examined using an immunohistochemical analysis in a tissue microarray of 108 high- and low-grade human astrocyte tumor samples. There was a statistically significant increase in GluR1 expression in glioblastoma samples compared with anaplastic astrocytoma and low-grade tumors (P < 0.0004). Furthermore, areas of vascular proliferation in glioblastoma samples had intense GluR1 expression. In vitro, we observed a time- and dose-dependent increase in MAPK phosphorylation after exposure to AMPA, independent of the presence of calcium-containing medium, which was blocked with AMPA receptor antagonists and the MEK1 inhibitor PD98059. The retroviral delivery of shRNA reduced GluR1 expression in U251 and U87 glioma cell lines, as assessed by real-time PCR and Western blot analysis. Knockdown of GluR1 inhibited AMPA-mediated increases in MAPK phosphorylation and decreased glioma proliferation in vitro, as determined by direct cell counting and colony-forming assays. To test the tumorigenicity of GluR1 knockdown cells, we implanted U251 and U87 shGluR1 cell lines into the flanks of nude mice and measured tumor growth over time. Compared with the non-silencing shRNA control, both GluR1 knockdown caused cell lines to grow significantly more slowly and, in U251, caused almost complete inhibition of tumor growth in vivo. Taken together, these results suggest that AMPA receptors are abundantly expressed in high-grade gliomas and gene silencing of the GluR1 AMPA receptor subunit results in abrogation of AMPA-mediated signaling and tumor growth.


D. Dinger,1 A. Block,3 H. Wulff,3 T. Ries,2 K. Lamszus,1 M. Westphal,1 and N.O. Schmidt1; 1Klinik und Poliklinik fuer Neurochirurgie, 2Radiologische Klinik, and 3Klinik fuer Innere Medizin, Universitaetsklinikum HH-Eppendorf, Hamburg, Germany

Interleukin-12 (IL-12) has shown potent anti-tumor activity through stimulation of the glioma-suppressed cytotoxic T-cell system. However, systemic therapy is limited by severe side effects. Local adenoviral-mediated gene transfer has the potential to overcome this obstacle, but vector systems are needed that result in high and controllable transgene expression with minimized viral toxicity. The therapeutic efficiency of local IL-12 gene therapy was evaluated by intratumoral injection of adenoviral vectors in the well-established syngenic orthotopic glioma xenograft model (GL261–C56/BL6). The adenoviral IL-12 transgene was under the control of a bidirectional promoter and a tetracycline-suppressible transactivator that was cloned into the E1 region of an E1–deleted recombinant adenoviral vector (Ad.3rmIL12). One week after intratumoral injection of Ad.3rmIL12, glioma growth was inhibited by 73% (P < 0.05), as assessed by T1–Gd-enhanced MRI compared with tumors injected with NaCl or control adenovirus (n = 12). Survival was significantly prolonged in Ad.3rmIL12–treated animals, and a CD3 immunohistochemical analysis demonstrated a significantly higher intratumoral T-cell infiltration (>4-fold, p < 0.001). The treatment appeared to be safe, as no side effects were observed. Transgene expression in vivo was localized in close vicinity to the viral injection site, as demonstrated by eGFP staining. The in vitro expression of IL-12 by GL261 glioma cells was significantly higher after infection with Ad.3rmIL-12 compared with an adenoviral vector with transgene expression under the control of a conventional CMV-promoter (Ad.CMV-IL-12). IL-12 expression after infection with Ad.3rmIL-12 was significantly suppressed in the presence of doxycycline. Local in vivo gene therapy with Ad.3rmIL-12 resulted in a higher reduction of tumor burden compared with Ad.CMV-IL-12 at the same dosage (6 × 107 pfu) (n = 5, not significant). Here, we demonstrated that local IL-12 gene therapy counterbalanced glioma-induced immunosuppression by inducing intratumoral T-cell infiltration, which resulted in a significant reduction of tumor growth. This new adenoviral vector system may contribute to the safety of viral gene therapy by resulting in more controllable and higher transgene expression than is obtained with conventional CMV-promoter driven vectors.


Heather G. Gatcombe, Chi-Ming Chang, and Hui-Kuo G. Shu; Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA

Epidermal growth factor receptor (EGFR) is overexpressed or amplified in roughly 50% of glioblastoma multiformes (GBMs), and many of these tumors with amplified EGFR express a mutant, constitutively active form of this receptor, termed EGFRvIII. The role of specific small molecule inhibitors of EGFR in the treatment of GBMs has been explored. To date, clinical responses have been disappointing. Previously, we showed that these tumors may display resistance to first-generation quinazoline-based EGFR inhibitors. Agents such as PD153035 and ZD1839 (Iressa) can inhibit EGF-dependent receptor autophosphorylation with high efficiency in LN229/EGFR, a model glioma cell line with overexpressed wild-type EGFR (wtEGFR), but much higher levels of drug were needed to inhibit downstream signaling to PI-3K/AKT and MEK/ERK. We hypothesized that the persistent EGF-dependent downstream signaling seen with EGFR inhibition was due to signaling through erbB2, which is not efficiently inhibited by these agents. After ligand binding, wtEGFR has been reported to form homodimers and heterodimers with itself and other family members, which initiates the signaling cascade. Through in vivo crosslinking studies, erbB2 has been shown to comprise a significant portion of the activated EGFR complex seen after ligand stimulation. We reasoned that if erbB2 is important in resistance to these inhibitors, concomitant inhibition of erbB2 would increase the efficiency at which EGF-dependent signaling is suppressed. Later-generation EGFR inhibitors that inhibit erbB2 with equal efficiency were tested and found to suppress receptor autophosphorylation and EGF-dependent downstream signaling at similar drug concentrations. To further support our hypothesis, erbB2 expression was knocked down in LN229/EGFR by RNA interference, resulting in inhibition of EGF-dependent signaling to PI-3K/AKT with reduced levels of the EGFR inhibitor PD153035. Responses of malignant gliomas to first-generation EGFR inhibitors in clinical trials have been poor overall, but recent studies suggest that a subset of tumors that express EGFRvIII may be more sensitive to these inhibitors. Therefore, another LN229 line was generated that overexpressed EGFRvIII (LN229/vIII), and its response to EGFR inhibition was tested. Compared with inhibition of signaling originating from wtEGFR, EGFRvIII-dependent signaling was inhibited with lower levels of the drug. EGFRvIII is believed to behave similarly to an EGF-bound form of wtEGFR, but our data suggest that it behaves differently, and this may account for its increased sensitivity to EGFR inhibition. Receptor crosslinking on LN229/vIII cells revealed that erbB2 is not a significant component of the activated receptor complex, unlike what was previously found with wtEGFR in LN229/EGFR. Thus, EGFRvIII-dependent signaling likely arises predominantly from EGFRvIII homodimers, which accounts for the efficient inhibition of this mutant receptor and may explain why responses to Iressa and Tarceva have mainly been seen in GBMs expressing EGFRvIII. Because wtEGFR is more efficiently suppressed with concomitant inhibition of erbB2, strategies targeting both kinases may ultimately prove superior to targeting EGFR alone.


Martin R. Graf,1 Ross S. Johnson,3 and Roger M. Loria2; 1Department of Neurosurgery and 2Departments of Microbiology and Immunology, Pathology and Emergency Medicine, Virginia Commonwealth University Medical Center, Richmond, VA, USA; and 3Department of Biology, Virginia State University, Petersburg, VA, USA

The androstene neuro-steroids 3β androstene 17α diol (α-AED); 3β androstene 17β diol (β-AED); 3β androstene 7α, 17β triol (α-AET); and 3β androstene 7β, 17β triol (β-AET) are metabolites of the common steroid dehydroepiandrosterone and are produced in neuro-ectodermal tissue. The androstenediols (α-AED and β-AED) are epimers of each other, as are the androstenetriols (α-AET and β-AET). α-AED and β-AED are chemically identical, except for the orientation of a hydroxyl group on 17th carbon, but the 2 molecules have significantly different biologic actions. In this regard, α-AED can inhibit proliferation and induce apoptosis in human myeloid tumor cells and human breast cancer cells. In contrast, β-AED is an enhancer of immune regulation, and its metabolite, β-AET, is more potent as an immune modifier. In the current study, we determined the anti-tumor activity of these 4 androstene neuro-steroids on glioma cells. In these studies, a 1:1 mixture of PEG400/ethanol was used as the excipient. The results of proliferation studies using the human T98 glioma cell line showed that α-AED was the most potent inhibitor of the neuro-steroids and that the concentration required to inhibit 50% of cellular proliferation (IC50)compared with sham-treated cultures was 8.5 μM. The IC50s for α-AET and β-AET in treated T98 glioma cells were 180 and 380 μM, respectively. Although β-AED marginally suppressed T98 glioma cell proliferation at high concentrations, an IC50 was not achievable. Further studies showed that α-AED can effectively inhibit the proliferation of several different human (LN18, LN-229, LN-Z308, U251, and U87), rat (F98, RT-2, and T9), and murine (GL261) glioma cell lines. The IC50 for α-AED in these gliomas ranged from 10–30 μM. The presence of inhibitors specific for the ERK1/2, JNK, or p38 mitogen-activated protein (MAP) kinase cascades did not block α-AED-mediated growth inhibition of T98 glioma cells and, in some instances, increased the suppressive action of α-AED. Additional studies with inhibitors specific for the phosphatidylinositol 3-kinase (PI3K) or NF-κB pathways also failed to block the inhibitory function of α-AED in T98 glioma cells. These findings demonstrate that (1) androstene neuro-steroids can suppress the proliferation of glioma cells and the effectiveness is dependent upon the number of hydroxyls and their position on the androstene molecule; (2) the α-AED neuro-steroid is the most potent androstene inhibitor of glioma cell growth and is effective on human and rodent gliomas; and (3) α-AED inhibition of glioma cell proliferation may involve cell signaling pathways other than the MAP kinase, PI3K/AKT, and NF-κB pathways.


Christopher W. Gregory, Masha Kononov, James L. Barbee, Carol Giamario, and Eric S. Werdin; Voyager Pharmaceutical Corporation, Raleigh, NC, USA

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults in the United States. The median survival of patients with GBM is 9–15 months, and most die within 2 years. Surgical resection, radiotherapy, and chemotherapy are the currently used strategies to treat patients with GBM, although treatment often fails because tumors are resistant to cytotoxic chemotherapy and radiotherapy. The development of new targeted agents is warranted. Our laboratory has found a signaling mechanism in several glioblastoma cell lines that recapitulates the hypothalamic-pituitary-gonadal (HPG) axis, an endocrine system that is essential for reproductive function. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to demonstrate the expression of HPG axis genes, including gonadotropin-releasing hormone I (gnrhI), gnrh I receptor (gnrhI-r), luteinizing hormone β (βlh), lh receptor (lh-r), follicle-stimulating hormone β (βfsh), and fsh receptor (fsh-r) in glioblastoma cell lines LN229, U118–MG, U87–MG, and CCF-SttG1. An immunoblot analysis demonstrated protein expression of the hormones GnRHI, LH, and FSH, as well as expression of the cognate receptors GnRH-RI, LH-R, and FSH-R. On the basis of these expression profiles, studies were performed to determine the effect of the GnRH agonist leuprolide acetate on the growth of cultured glioblastoma cell lines. The dose response and time course cell proliferation assays were performed using three doses of leuprolide acetate (10−11, 10−8, and 10−5M) for up to 5 days. At the highest dose of 10−5 M, leuprolide acetate significantly inhibited cell proliferation on days 3 and 5 compared with untreated cells (% inhibition for each cell line: 15% for LN229, 10% for U118–MG, 40% for U87–MG, and 60% for CCF-SttG1). On the basis of in vitro data, the cell lines were used as subcutaneous xenograft tumor models to determine the in vivo effects of sustained leuprolide acetate exposure on tumor growth. Leuprolide acetate was administered as a polymer-based subcutaneous implant. LN229, U118–MG, and U87–MG cell lines were injected in Matrigel. LN229 tumors were growth-inhibited by up to 88% by leuprolide acetate treatment for more than 3 months compared with tumors in placebo-treated mice. Leuprolide acetate treatment of mice bearing U118–MG tumors caused up to 90% growth inhibition compared with placebo for 144 days. U87–MG tumors were growth-inhibited by leuprolide acetate by up to 68% for approximately 60 days. To our knowledge, there have been no published reports that leuprolide acetate has growth-inhibitory effects in GBM. Clinical trials are planned to determine the efficacy of leuprolide acetate in extending disease-free survival after tumor resection in GBM patients.


Vinay Gupta,1 Yuzhuang S. Su,2 Christian G. Samuelson,3 Florence M. Hofman,1,2 Axel H. Schönthal,4 and Thomas C. Chen1,2,5,6; 1Departments of Pathology, 2Neurosurgery, 4Molecular Microbiology and Immunology, 5K. Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA; and 3Temple University School of Medicine, Temple University, Philadelphia, PA, USA

Although most meningiomas are treated surgically, atypical or malignant meningiomas and surgically inaccessible meningiomas may not be removed completely and tend to recur in most cases. We determined the effects of the topoisomerase I inhibitor, CPT-11 (Camptostar, Irinotecan), on primary meningioma cultures and a malignant meningioma cell line in vitro and in vivo. The effects of CPT-11 on cellular proliferation in primary meningioma cultures and the IOMM-Lee malignant meningioma cell line were measured by an MTT assay and flow cytometry analysis. Apoptosis after drug treatment was evaluated by TUNEL and the DNA-laddering assay. The effects of CPT-11 in vivo in a meningioma model were determined with a subcutaneous murine tumor model using the IOMM-Lee cell line. CPT-11 induced a dose-dependent anti-proliferative effect, with subsequent apoptosis in primary meningioma cultures (up to 100 μM) and the IOMM-Lee human malignant meningioma cell line (up to 20 μM). In our animal model, CPT-11 treatment led to a statistically significant decrease in tumor growth. An HPLC analysis demonstrated conversion of CPT-11 to the active metabolite SN-38 in tumor specimens. Treatment was accompanied by a decrease in Bcl-2 and survivin levels and an increase in apoptotic cell death. CPT-11 inhibited meningioma growth both in vitro and in vivo. CPT-11 was much more effective against the malignant meningioma cell line than against primary meningioma cultures. Therefore, this drug may have an important therapeutic role in the treatment of atypical or malignant meningiomas and should be evaluated further for this purpose.


Vinay Gupta,1 Yuzhuang S. Su,2 Christian G. Samuelson,3 Florence M. Hofman,1,2 Axel H. Schönthal,4 and Thomas C. Chen1,2,5,6; 1Departments of Pathology, 2Neurosurgery, 4Molecular Microbiology and Immunology, 5K. Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA; and 3Temple University School of Medicine, Temple University, Philadelphia, PA, USA

There is currently no effective chemotherapy for meningioma. Overexpression of platelet-derived growth factor (PDGF) and its receptors (PDGFRs) have been previously demonstrated in meningiomas. We hypothesized that Gleevec (imatinib mesylate) would inhibit meningioma growth by interrupting PDGFR phosphorylation. Primary meningioma cell cultures and a malignant meningioma cell line were treated in vitro and in vivo with Gleevec. The effects of Gleevec on cellular proliferation in primary meningioma cultures and the IOMM-Lee malignant meningioma cell line were measured by an MTT assay and flow cytometry analysis. Apoptosis after drug treatment was evaluated by the TUNEL and the DNA laddering assay. The effects of Gleevec in an in vivo meningioma model were determined with a subcutaneous murine tumor model using the IOMM-Lee cell line. Gleevec induced a dose-dependent anti-proliferative effect, with subsequent apoptosis in the primary meningioma cultures and the IOMM-Lee human malignant meningioma cell line. In our animal model, Gleevec treatment induced up to a 70% decrease in tumor size. Further experiments entail assessment of proliferation, the apoptotic index, and Bcl-2, Bax, and survivin protein levels in treatment versus control groups. Because Gleevec demonstrated growth-inhibitory effects in meningiomas, both in vitro and in vivo, this drug may have an important therapeutic role in the treatment of meningiomas and should be evaluated further for this purpose.


Anita B. Hjelmeland,1 Katie Lattimore,1 Brian Fee,1 Sarah Wickman,1 Stephen T. Keir,1 Mark D. Hjelmeland,1 David Batt,2 Heidi Lane,2 Darell D. Bigner,1,3 Henry S. Friedman,1,3,4 and Jeremy N. Rich1,5,6; Departments of 1Surgery, 3Pathology, 4Pediatrics, 5Medicine, and 6Neurobiology, Duke University Medical Center, Durham, NC, USA; 2Novartis Institutes for Biomedical Research, Cambridge, MA, USA; Novartis Pharmaceuticals Oncology, East Hanover, NJ, USA

Monotherapies have proven largely ineffective for the treatment of glioma, suggesting that increased patient benefits may be achieved by combining therapies. Two pro-tumorigenic pathways active in brain cancers include Raf and target of rapamycin (TOR). Ras activity results in the phosphorylation and activation of Raf to control the transcription of multiple target genes. TOR is a central regulator of nutrient sensing and protein translation. We determined the efficacy of a combination of novel low-molecular-weight inhibitors targeting these 2 pathways. LBT613 (Novartis) is a low-molecular-weight inhibitor of Raf. RAD001/everolimus is a rapamycin derivative that inhibits TOR activities. LBT613 decreased phosphorylation of Erk1 and Erk2, downstream effectors of Raf, in the human glioma cell line D54MG. RAD001 resulted in decreased phosphorylation of the TOR effector S6. Thus, LBT613 and RAD001 inhibit the phosphorylation and activation of signals downstream of Raf and TOR, respectively. To determine whether the decrease in Raf and TOR activity resulted in decreased pro-tumorigenic glioma cellular behavior, we evaluated the abilities of LBT613 and RAD001 to affect the proliferation, migration, and invasion of human glioma cells. Treatment with 0.5 uM LBT613 alone or 0.05 uM RAD001 alone significantly decreased the proliferation of D54MG, U87MG, U251MG, and U373MG cells. Furthermore, LBT613 and RAD001 in combination blocked cell proliferation to a greater degree than either drug alone in all tested lines. In U87MG cells, the decrease in glioma cell proliferation was associated with a G1 cell-cycle arrest. Glioma invasion is a critical contributor to tumor malignancy. The combination of LBT613 and RAD001 inhibited the invasion of D54MG and U251MG cells through Matrigel to a greater degree than treatment with either drug alone. These data suggest that the combination of LBT613 and RAD001 may reduce glioma cell proliferation and invasion, resulting in decreased tumor growth in vivo. Indeed, orally administered LBT613 (100 mg/kg qd for 14 days 2 hr before RAD001) and RAD001 (5 mg/kg tiw) delayed the growth of subcutaneous human glioma xenografts grown in immunocompromised mice (6.7 days delay, P < 0.001). The combination also resulted in regressions in 3 of 8 animals. Together, these results suggest that the combination of Raf and mTOR inhibitors should be studied in the treatment of glioblastoma patients.


Grace H. Huynh,1 Tomoko Ozawa,2 Dennis F. Deen,2 and Francis C. Szoka, Jr.1,3; 1Joint Graduate Group in Bioengineering, University of California, San Francisco, CA, USA and University of California, Berkeley, San Francisco, CA, USA; 2 Brain Tumor Research Center, Department of Neurological Surgery, University of California, San Francisco, CA, USA; and 3Departments of Pharmaceutical Chemistry and Biopharmaceutical Sciences, University of California, San Francisco, CA, USA

A retroconvection-enhanced delivery (R-CED) method has been developed to improve the delivery of intravenously administered therapeutics within solid brain tumors. R-CED uses an osmotic gradient to withdraw brain interstitial fluid (ISF) through a microdialysis membrane in a controlled manner via an implanted probe. Withdrawal of ISF increases the local tissue specific gravity in normal brain tissue. The application of R-CED doubled the extravasation of intravenous Evans blue-albumin in both normal brain tissue and orthotopic 9L tumor tissue. R-CED also increased the extravasation of 67 nm fluorescent liposomes nearly 5-fold in tumor tissue, demonstrating that substances in the blood can be readily transferred into the tissue parenchyma. After probe removal, the magnitude of the R-CED effect on EB-albumin extravasation decreased to control values within 1.5 hours in normal brain tissue; however, the effect persisted beyond 6 hours in tumor tissue. There was no evidence of histologic damage to the neurons. Our findings establish the feasibility of applying R-CED to increase the distribution of systemically administered drugs to both the normal tissue-tumor margin and the central tumor core, which may lead to improved antitumor drug efficacy.


Hong Jiang, Ok-Hee Lee, Hiroshi Aoki, Seiji Kondo, Fredrick F. Lang, Yasuko Kondo, W.K. Alfred Yung, and Juan Fueyo; Brain Tumor Center, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Accumulating evidence suggests that only the population of cancer cells within a glioma, identified as cancer stem cells (CSC), have the capacity to initiate the formation of new tumors. Because of their resistance to conventional chemotherapy and radiotherapy, CSCs are considered to be the cause of tumor recurrence. We hypothesized that oncolytic adenoviruses can be used to overcome the hurdle caused by brain tumor stem cells (BTSCs) in glioma therapy. Thus, we evaluated the oncolytic effect of Delta-24 and Delta-24–RGD in 8 brain tumor stem cell lines that were established from fresh tumor specimens from patients with GBM. First, we examined the adenoviral receptors on the cell surface by flow cytometry analysis. We showed that BTSCs expressed higher levels of the native viral receptor Coxsackie and adenovirus receptor (CAR) but lower levels of αvβ3 integrin than glioma cell lines; BTSCs also had variable levels of αvβ5 integrin. A flow cytometric analysis of GFP expression in the BTSCs infected with AdGFP and AdGFP-RGD indicated that RGD modification increased adenoviral infectivity in BTSCs because the RGD motif in the HI loop of fiber protein enhances adenoviral tropism to αvβ3 and αvβ5 integrins in addition to CAR binding. Thus, Delta-24–RGD was more potent than Delta-24 in inducing the cytopathic effect in BTSCs, as assayed by MTS. Consistently, titration of the progenies of the viruses in infected BTSCs through TCID50 assays showed that the replication efficiency of Delta-24–RGD was 10– to 1000–fold higher than Delta-24. Interestingly, further studies demonstrated that Delta-24–RGD induced cell death in BTSCs through nonapoptotic autophagy, which is characterized by acidic vesicular organelles that can be identified by acridine orange staining. This is the first report of the autophagic effect of an oncolytic adenovirus on cancer stem cells. Our data indicate that autophagy is part of the mechanism of the adenovirus-mediated antiglioma effect. Combined with our previous report on Delta-24–RGD’s potency in glioma cells lack of CAR expression, our data further support the argument that Delta-24–RGD is a promising choice for efficient glioma therapy in the clinical setting.


Rongcai Jiang, Woonyoung Choi, W.K. Alfred Yung, Eugene Gerner, Stanley R. Hamilton, and Wei Zhang; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; Arizona Cancer Center, Tucson, AZ, USA

N1, N11–diethylnorspermine (DENSPM), a spermine analog that activates polyamine catabolism, was recently shown to have therapeutic effects in a number of tumor types in preclinical studies and thus is being tested in clinical trials. However, the molecular mechanism of the cell death induced by DENSPM still remains obscure, and there have been no reports of its application in glioma. In our in vitro experiments, DENSPM alone resulted in sub-G1 aggregation in 2 different GBM cell lines, U87 and LN229. Treated cells were detached from the culture plates resembling anoikis. DENSPM treatment led to a decrease in polyamine levels, an increase in acetylated polyamine levels, generation of hydrogen peroxide, a reduction of mitochondrial membrane potential, an elevation of spermidine/spermine N1–acetyltransferase mRNA, and DNA degradation. The mRNA expression level of SMO/PAO, which regulates H2O2 production, was also induced. An inhibitor of SMO/PAO attenuated the production of H2O2, as well as DNSPM’s inhibitory effect on both cell lines. To understand the mechanism through which this cell death occurs, we evaluated the protein expression of a number of apoptosis regulators and cell adhesion molecules. Surprisingly, we did not observe cytochrome C release from mitochondria in the treated cells, suggesting a new mechanism that is independent of cytochrome C. We observed a marked decrease in many proteins, including pro-apoptotic proteins and cell adhesion proteins. As a control, temozolomide induced marked increasing expression of several pro-apoptotic proteins, including p53 and cleaved Caspase 9. We did not detect an increase in ubiquitination in DENSPM-treated cells. Furthermore, the pre-incubation of MG132, an inhibitor of proteosome degradation pathway, did not alter the cellular toxicity of DENSPM or prevent a protein decrease. This observation led to a further examination of the mTOR-related protein initiation pathway. We found a decrease in proteins downstream of mTOR, including phosphorylated mTOR, mTOR, p70S6K, phosphorylated p70S6K, 4E-BP1, phosphorylated 4E-BP1, and eIF4B. The mTOR upstream proteins, Akt and PI3K, remained stable after DENSPM treatment. An m7GTP-Sepharose pulldown assay showed that more 4E-BP1 bound to the complex, prohibiting formation of the active translation initiation complex for 5′-cap-dependent initiation of protein translation. In a xenograft experiment, we gave mice DENSPM at a dose of 0.15 g/kg/day intraperitoneally for 6 days (PBS as control) 2 weeks after GBM cells were transplanted into their brains. DENSPM extended the life of U87 xenograft mice. In summary, this study provides preclinical evidence that DENSPM targets the mTOR-controlled protein initiation pathway, induces anoikis in GBM cells, and extends the life of glioma xenograft mice. DENSPM may be useful in the clinical therapy of GBM.


Adel Kardosh,1 Nathaniel Soriano,2 Peter Pyrko,2 Jasim Uddin,3 Nicos A. Petasis,3 Florence M. Hofman,2 Axel H. Schönthal,1,5 and Thomas C. Chen2,4; Departments of 1Molecular Microbiology and Immunology, 2Pathology, 3Chemistry, 4Neurosurgery, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA

Multiple myeloma is a systemic hematologic malignancy that is characterized by monoclonal proliferation of plasma cells. Disseminated disease with multiple osteolytic lesions or infiltration of the bone marrow is seen in most cases, whereas a solitary lesion may be present in 5–10% of cases. Because of the distribution of hematopoietic cells, the spine is one of the most commonly affected sites. Patients with multiple myeloma frequently develop drug-resistant disease and ultimately die. 2,5–Dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex). In contrast to celecoxib, however, DMC lacks the COX-2–inhibitory function (i.e., it is not a coxib). Nevertheless, DMC is able to potently mimic the anti-tumor effects of celecoxib in vitro and in vivo. In this study, we found that DMC and celecoxib inhibited the proliferation of various multiple myeloma cell lines, including several multi-drug-resistant variants. Growth inhibition in drug-sensitive and drug-resistant cells was mediated via multiple drug effects, which included diminished signal transducer and activator of transcription 3 (STAT-3) and mitogen-activated protein (MAP) kinase kinase (MEK) activity. In addition, DMC and celecoxib reduced the expression of survivin, an anti-apoptotic protein that is highly expressed in tumor cells and known to confer resistance of such cells to anticancer treatments. The downregulation of survivin was closely correlated with drug-induced growth inhibition and apoptosis. Remarkably, these effects were not restricted to in vitro conditions but also took place in xenografted tumors of drug-treated animals, where both drugs similarly repressed survivin, induced apoptosis, and inhibited tumor growth in vivo. Thus, our study demonstrates that inhibition of proliferation and induction of apoptosis by DMC and celecoxib can be accomplished even in highly drug-resistant multiple myeloma cells and that this effect is achieved via the blockage of multiple targets that are critical for multiple myeloma cell growth and survival in vitro and in vivo. In consideration of the life-threatening side effects that have recently emerged with the use of coxibs (which are a class effect and due to the inhibition of COX-2 and the resulting imbalance of prostanoid levels), our results further indicate that DMC might be a better alternative for the treatment of cancer; because DMC does not inhibit COX-2, those side effects that have been related to altered prostanoid levels are not expected to occur with this drug.


Makoto Katayama, Mitchel S. Berger, and Russell O. Pieper; Dept. of Neurological Surgery and The Brain Tumor Research Center, University of California-San Francisco, CA, USA

Although autophagy enhances cell survival in nutrient-deprived cells by increasing ATP production, it remains unclear whether autophagy functions similarly in cells treated with cytotoxic chemotherapy agents. To address this issue, we measured both the ability of DNA damaging agents (temozolomide and etoposide) to induce autophagy-dependent production of ATP and the effects of modulation of autophagy on drug-induced cell death. Both drugs induced an autophagy-associated increase in ATP production in multiple glioma cell lines. The drug-induced ATP surge was not blocked by glucose starvation but was blocked by pre-incubation with the autophagy inhibitor 3-methyladenine or the mitochondrial inhibitor oligomycin. Inhibition of autophagy-induced ATP production increased nonapoptotic cell death associated with micronucleation, whereas restoration of the 3-methyladenine–inhibited ATP surge by addition of pyruvate suppressed cell death. These results show that DNA-damaging agents induce an autophagy-associated ATP surge that protects cells and may contribute to drug resistance.


J. Gaspar Kitange, Brett L. Carlson, Eduard Dinca, Jeff LaMont, Mark Schroeder, C. David James, Jann N. Sarkaria; Mayo Clinic & Foundation, Rochester, MN, USA

O6–methyguanine-DNA-methyltransferase (MGMT) plays a critical role in tumor resistance against the alkylating agent temozolomide (TMZ). Because silencing of MGMT by aberrant promoter methylation is associated with a TMZ response in glioblastoma patients, we investigated this association using an intracranial glioblastoma xenograft model. MGMT promoter methylation was observed in 10 of 23 xenografts (44%), and among these cases there was a strong but not absolute association between MGMT promoter methylation and the expression of MGMT protein: 5 of 6 (83%) tumors with MGMT promoter methylation showed minimal or no expression of MGMT protein, whereas 10 of 12 (83%) tumors without methylation expressed much higher levels of this protein. Of these GBMs, 14 (8 MGMT non-methylated and 6 MGMT methylated) have been tested for TMZ response in vivo using a clinically relevant dosing regimen (200 mg/m2 PO daily × 5). The MGMT non-methylated tumors showed varying responses to TMZ, ranging from complete resistance to relative sensitivity (relative survival prolongation ranging from −7% to 191%). In contrast, all xenografts with methylated MGMT promoter were sensitive to TMZ (relative survival prolongation of 71% to 290%). Interestingly, one of the xenografts that is sensitive to TMZ showed high levels of MGMT and a lack of MGMT promoter methylation (GBM14). The treatment of sensitive GBM14 flank tumors with a 5-day course of TMZ resulted in downregulation of MGMT, whereas the same treatment in resistant GBM43 flank tumors resulted in upregulation of MGMT levels. A similar upregulation in MGMT levels after 100 mM TMZ treatment was observed in vitro in the resistant GBM10, 43, and 44 xenograft lines, whereas MGMT was suppressed or not expressed after TMZ treatment in the sensitive GBM12 and 14 lines. Thus, our results confirm that MGMT methylation is an important biomarker of TMZ sensitivity, and TMZ-induced MGMT induction may be an important factor that contributes to TMZ resistance.


Dimpy Koul,1 Ruijun Shen,1 Jennifer Edge,1 TJ Liu,1 Garth Powis,1 D. Lynn Kirkpatrick,2 and W.K. Alfred Yung1; 1The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA and 2ProlX Pharmaceuticals, Tucson, AZ, USA

Glioblastoma, the most malignant form of primary glioma, is refractory to conventional therapies, making the development of new, rational, targeted therapies an urgent necessity. The phosphatidylinositol-3-kinase (PI-3-kinase)/PTEN/Akt pathway is a survival-signaling pathway that is activated in many types of human cancer, including glioblastomas, where it leads to increased proliferation and inhibition of apoptosis. The PTEN tumor suppressor antagonizes PI-3-kinase signaling by dephosphorylating PI-3-phosphates (PI-3P). Abnormalities in PTEN sequence, expression, or function combined with activation of EGFR leads to constitutive activation of the PI3K pathway in glioma, rendering the PI3K/PTEN pathway an attractive target for therapeutic development. Small molecules targeting various signaling nodes along receptor tyrosine kinases (RTKs) and the PTEN-PI3K-Akt pathway have been developed and are currently in different stages of testing. We demonstrated that PX866, a newly developed irreversible PI3K inhibitor, with selectivity for p110α (ProlX Pharmaceuticals), effectively inhibited signaling through the PI3K/Akt cascade in U87, U251, and LN229 glioma cells. PX866 blocked both basal and EGF-induced phosphorylation of Akt and downstream targets, including p70S6K1, tuberin, and pS6. Treatment of all 3 glioma cell lines with PX866 showed 50% growth inhibition at 4–8μM concentrations at the 48-h time point. The decrease in cell growth was directly correlated with G1 cell cycle arrest. In addition, PX866 induced autophagy-programmed cell death type-2 in U87 glioma cells in a dose-dependent manner, as shown by the development of acidic vesicular organelles and the autophagosome membrane association of the microtubule-associated protein light chain 3, which are characteristic of autophagy, in malignant glioma cells; apoptotic cell death was not observed. An in vivo experiment with U87 SC xenografts demonstrated an 84% growth inhibition after 4 weeks of treatment at an oral dose of 2.5mg/kg on a qod schedule. PX-866 increased the survival of animals with i.c. U87 tumors from 31 to 38 days. Taken together, these data demonstrate that PI3K inhibitor PX866 has significant activity in signal inhibition, cell cycle arrest, growth inhibition, and autophagy in human glioblastoma in vitro and in vivo, affirming that the PI3K/Akt pathway is a highly specific molecular target for molecular therapeutics development for glioblastoma and other cancers with aberrant PTEN/PI3K expression.


Kazuhiko Kurozumi,1 Jennifer Cutter,1 Jayson Hardcastle,1 Ming Yang,2 Gregory Christoforidis,2 William Carson,3 E. Antonio Chiocca,1 and Balveen Kaur1; Departments of 1Neurological Surgery, 2Radiology and 3Surgery, The Ohio State University Medical Center, OH, USA

Oncolytic viruses (OVs) are being investigated as treatment modalities for many cancers because of their ability to selectively replicate and lyse in tumors. The results from recent clinical trials with OV have revealed the safety of this approach, yet evidence for efficacy remains elusive. We believe changes elicited in the tumor microenvironment affect the therapeutic efficacy of OV treatment. We are using a syngeneic rat glioma model to uncover these alterations. Seven days after intracerebral tumor implantation (D74HveC rat glioma cells), rats were treated with direct intratumoral injection of hrR3 or PBS. The effect of OV on tumor vasculature was evaluated by a tail vein injection of FITC-conjugated dextran. Quantification of leaked fluorescent dye within tumor tissue revealed a 4-fold increase in vascular leakage upon OV infection compared with the PBS-injected control. This observation was confirmed on ultra high-field, high resolution magnetic resonance (MR) imaging performed at 8.0-Tesla using an ultra-small particles of iron oxide (USPIO)-enhanced gradient echo sequence. An analysis of OV-induced changes in gene expression revealed significant induction of iNOS but not VEGF, suggesting that increased vessel permeability could be the result of endothelial cell activation in response to inflammation induced by OV infection of the tumor. Additionally, tumor-bearing rats treated with OV or PBS were killed, and an evaluation of vessel density in size-matched tumors revealed a 5-fold increase in angiogenesis in OV-treated tumors compared with PBS-injected controls (OV: 101 ± 21.6 vs. PBS: 19.8 ± 10) (P = 0.0037). Vascular normalization achieved by pre-treatment with a single dose of cyclic RGD peptide (cRGD; a known anti-angiogenic agent and antagonist of integrins αvβ3) significantly increased the survival of rats compared with rats treated with a single agent (median survival: PBS: d13; cRGD: d13; OV: d17; and cRGD + OV: d21) (P = 0.0003 between OV and cRGD + OV). In conclusion, vessel normalization achieved prior to OV infection enhanced the therapeutic efficacy of OV.


Kazuhiko Kurozumi,1 Jayson Hardcastle,1 Giulia Fulci,2 Hirokazu Kambara,1 E. Antonio Chiocca,1 and Balveen Kaur1; 1Department of Neurological Surgery, Ohio State University Medical Center, OH, USA; and 2Department of Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA

Oncolytic virus (OV) treatment of brain tumors uses genetically engineered viruses designed to selectively infect or replicate in tumor cells but not in normal cells. Despite the exciting preclinical results obtained from animal models, OV therapy has not yielded encouraging results for patients with malignant gliomas. We have observed that, consistent with published findings, transient suppression of innate immunity by cyclophosphamide (CPA) increases the efficiency of OV-mediated oncolysis in the brain. To elucidate these mechanisms, we investigated the time course of transcriptional profiling of the brain in response to viral infection by DNA chip analysis. D74HveC glioma cells were implanted orthotopically as intracerebral brain tumors in rats. Five days after tumor implantation, the rats were infected intratumorally with the hrR3 OV (hrR3) with or without systemic CPA treatment. Total RNA were extracted 12 or 72 hours after the OV injection from CPA-treated or -untreated animals. The presence of hrR3 was detected by RT-PCR for lacZ (encoded by hrR3) from the RNA. CPA pretreatment led to a marginal increase in the amount of hrR3 12 hrs after virus infection. Moreover, CPA pretreatment led to a dramatic increase in the amount of hrR3 72 hrs after OV treatment of brain tumors in rats. The RNA were also hybridized to Affymetrix rat genome array 230 2.0, which represents 28,000 rat cDNAs. Global changes in gene expression were analyzed by cluster analysis and expression analysis of systematic explorer (EASE). 33 and 303 genes were dysregulated by CPA 12 hrs and 72 hrs after injection of the virus, respectively. An EASE analysis for microclusters of dysregulated genes by CPA after 12 and 72 hrs revealed that genes involved in immune response, defense response, and response to biotic stimuli were overrepresented. EASE is a powerful tool for rapidly converting the results of functional genomics studies from genes to ‘themes’. The results of real-time quantitative PCR confirmed that the observed differences in expression were real and significant. Future experiments would elucidate the role of each of these functions in suppressing the host response to viral infection, and suppression of these functions may lead to ways to improve the therapeutic efficacy of OV infection.


Bachchu Lal,1 David E. Gerber,1 Jin Kim,2 and John Laterra1; 1Johns Hopkins University and Kennedy Krieger Institute, Baltimore, MD, and 2Galaxy Biotech, Inc, Mountain View, CA, USA

Most glioblastomas express hepatocyte growth factor (HGF), and all express its transmembranous receptor tyrosine kinase c-Met. Expression levels of HGF and c-Met are associated with malignant progression in glioma and in medulloblastoma. Experimental evidence indicates that c-Met activation contributes to glioma malignancy by enhancing tumor mitogenicity, motogenicity, angiogenesis, and resistance to cytotoxic therapies. We showed that inhibition of expression levels of HGF and c-Met are associated with tumor cell growth inhibition in vivo and in vitro. c-Met activation by HGF activates multiple cell signaling pathways that mechanistically contribute to the biologic response to receptor activation. PI3K-AKT and RAS-MAPK are 2 pathways potently activated by c-Met. We recently reported that systemic delivery (twice/week, 5 mg/kg, I.P.) of mL2G7, a murine monoclonal antibody that inhibits HGF binding to c-Met, inhibited the growth of established intracranial U87MG xenografts and prolonged animal survival. We now report that inhibition of intracranial U87MG glioma growth via systemic delivery of mL2G7 is dose dependent, with anti-tumor activity using doses as low as 0.625 mg/kg body weight delivered I.P. twice/week. The systemic delivery of mL2G7 (twice/week, 5 mg/kg, I.P.) also had modest anti-tumor activity against pre-established subcutaneous primary human glioblastoma xenografts that express ~30-fold less HGF than the U87MG glioma model. Humanized anti-HGF antibody (hL2G7), administered by I.P. injections (1.25 mg/kg) biweekly to scid mice bearing pre-established intracranial U87MG glioma xenografts, also showed significant tumor growth inhibition compared with animals treated with control IgG. We determined whether mL2G7 therapy alters the downstream targets of c-Met activation, phospho-MAPK and phospho-AKT, concurrent with tumor growth inhibition. Mice bearing pre-established subcutaneous U87MG glioma xenografts (~300 mm3) were treated with I.P. injections of mL2G7 (5 mg/kg) every alternate day for 3 days. Tumors regressed by ~70% over this brief treatment period. Using a semiquantitative immunoblot analysis, we found inhibited phosphorylation of phospho/total AKT (2.5-fold) and phospho/total MAPK (1.8-fold) in mL2G7-treated tumors. These findings suggest that systemic neutralizing anti-HGF monoclonal antibody inhibited the growth of HGF-expressing gliomas by downregulating AKT- and MAPK-dependent signal transduction pathways downstream of c-Met.


Michael Lim, Yi-Shan Yang, Leroy Sims, Steven Choi, Yingyun Wang, and Samira Guccione; Stanford University, Stanford, CA, USA

We have developed an anti-angiogenic approach that kills tumor neovasculature (NPTx). This approach is an integrin-targeted delivery system that can carry chemotherapy, radiation, or genes to the tumor vasculature. In this presentation, a gene was delivered to the tumor endothelium that causes VEGF- or FGF-activated endothelial cells to apoptose. We tested the efficacy of NPTx on a variety of tumor models, including the RT2 primary brain tumor model in rats. Tumors were initiated by stereotactic injection of the RT2 cells into the striatum of F344 rats. After confirming the presence of the tumor mass with MRI, we treated rats with our integrin-targeted therapeutic (NPTx). Treatment was fractionated over 3 days with intravenous injection of NPTx. FDG-PET and MRI scans were used to follow up the treated and untreated animals. All control animals died of mass effects in less than 28 days. Treated animals survived beyond 1 year after treatment, with no signs of recurrence, as determined by FDG-PET, MRI, or histologic evaluation. Temporal evaluation of the course of treatment using functional imaging provides an efficient method for early detection of response to therapy. The vascular delivery platform we have developed combines effective targeting of therapeutics in the tumor and potent, selective destruction of the tumor endothelium. The resulting combined effect of this approach causes significant tumor mass reduction. Recurrence or drug resistance has not been observed with this approach thus far. Anti-angiogenic therapies that destroy the tumor neovasculature can be an effective therapeutic approach for vascular tumors such as GBM. We are currently conducting toxicity studies to evaluate NPTx for clinical translation; some of these toxicity results will be presented here.


Ta-Jen Liu,1 Tiffany LaFortune,1 Toshiyuki Honda,2 Osamu Ohmori,2 Dowdy Jackson,2 John de Groot,1 and W.K. Alfred Yung1; 1Brain Tumor Center, Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Oncology-Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

Multiple genetic aberrations in human gliomas contribute to their highly infiltrative and rapid growth characteristics. Focal adhesion kinase (FAK) regulates tumor migration and invasion. Insulin-like growth factor-I receptor (IGF-IR), whose expression correlates with tumor grade, is involved in proliferation and survival. We hypothesized that inhibiting the phosphorylation of FAK and IGF-IR by NVP-TAE226 (hereafter called TAE226), a novel dual tyrosine kinase inhibitor of FAK and IGF-IR, would suppress the growth and invasion of glioma cells. In culture, TAE226 inhibited extracellular matrix-induced autophosphorylation of FAK (Tyr397). TAE226 also inhibited IGF-I-induced phosphorylation of IGF-IR and the activity of its downstream target genes, such as mitogen-activated protein kinase (MAPK) and Akt. TAE226 retarded tumor cell growth as assessed by a cell viability assay and attenuated G2/M cell cycle progression associated with a decrease in cyclin B1 and phosphorylated cdc2 (Tyr15) protein expression. TAE226 treatment inhibited tumor cell invasion by at least 50% compared with the control in an in vitro matrigel invasion assay. Interestingly, TAE226 treatment of tumor cells containing wild-type p53 (wt-p53) mainly exhibited G2/M arrest, whereas tumor cells bearing mutant p53 underwent apoptosis. Induction of apoptosis by TAE226 was substantiated by detection of caspase-3/7 activation and poly (ADP)-ribose polymerase (PARP) cleavage and by an annexin V apoptosis assay. More importantly, TAE226 treatment significantly increased the survival rate of animals in 2 intracranial glioma xenografts models (U87: P = 0.042 and LN229: P = 0.0031). Collectively, these data show that blocking the signaling pathways of FAK and IGF-IR with TAE226 has the potential to be an efficacious treatment for human gliomas.


Ta-Jen Liu, Tiffany A. LaFortune, John F. de Groot, and W.K. Alfred Yung; Brain Tumor Center, Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Therapies using small-molecule inhibitors targeting a single signaling pathway for glioma are likely to be ineffective because of the action of compensatory or collateral pathways that overcome the effect of single-target inhibition. For example, phase I/II trials for various cancers with RAD001, an mTOR inhibitor, yielded little or no clinical response. Studies have demonstrated that mTOR inhibition elicits positive feedback of the IGF-IR pathway, leading to a reactivation of Akt. Under these circumstances, one could envision that combining the antagonist to IGF-IR signaling with RAD001 would be a rational therapy for glioma. We tested combinations of the RAD001 and TAE226, a dual kinase inhibitor of FAK and IGF-1R, on tumor proliferation, signal transduction, and survival of mice implanted with U87 xenografts. The combination effect of RAD001 and TAE226 on glioma cell proliferation was assessed using the sulforhodamine B (SRB) assay. Isobologram plots were generated, and the combination index (CI) (Calcusyn; Biosoft, UK) was used to determine the optimal synergistic (CI < 1) combinations of these inhibitors. The dose-response experiments using multiple drug combinations indicated a synergistic anti-proliferative effect of RAD001 and TAE226 on 5 glioma cell lines with diverse genetic profiles. Synergistic combinations resulted in alterations of cell cycle distribution compared with single agents alone. For example, there was a 20% increase in the subG1 population of LN229 cells. Additionally, inhibition of downstream markers of the mTOR/Akt pathway, such as phospho-Akt (Ser473) and phospho-S6 ribosomal protein in U87 and LN229 cell lines, as well as an increase in cleaved caspase-3 levels in LN229 cells, were noted by Western blot analysis. In vivo studies were performed on U87 tumor xenografts administered intracranially to nude mice. When treated for 6 weeks (5/7 schedule), animals receiving a combination of RAD001 and TAE226 had a significant increase in median survival time over the single-agent controls (P < 0.0001). Taken together, this study demonstrates an additive benefit of the combination of these 2 small molecules on the inhibition of tumor proliferation in both in vitro and in vivo glioma models. Furthermore, our study suggests that the combined inhibition of the mTOR and IGF-IR pathways have significant therapeutic potential in the treatment of glioma patients.


A.B. Madhankumar, Becky Slagle-Webb, Jonas M.Sheehan, and James R. Connor; Department of Neurosurgery, Pennsylvania State University College of Medicine, Hershey, PA, USA

Most of the chemotherapeutic agents for glioma therapy are associated with either toxic side effects or poor response rates. To improve the drug delivery and uptake by tumor cells, we developed targeted nanovesicles using IL13Rα2, a receptor for interleukin-13 (IL-13), which is selectively expressed in high-grade astrocytomas. Previously, we demonstrated the selective targeting ability of IL-13-conjugated liposomes to high-grade astrocytomas in cell culture models and the ability of these liposomes to cross the blood-brain barrier under in vitro conditions. In this investigation, we determined the efficacy of targeted liposomes carrying doxorubicin (DXR) in cell culture models and a glioma tumor-bearing mouse model. Most of the brain tumors conferred drug resistance to glioma cells because of the expression of P-glycoprotein (Pgp). The uptake and accumulation of IL-13-conjugated liposomes in glioma cells was analyzed using fluorescence microscopy and FACS analysis. The effects of the liposomes encapsulating pharmaceutical agents were compared with unencapsulated cytotoxins or non-targeted liposomal cytotoxins in U251 glioma cells. For our animal experiments, U251 glioma cells were implanted subcutaneously in female nude mice. Two weeks after implantation, when the tumors reached 21 mm2, we injected the IL-13-conjugated liposomes encapsulated with DXR intraperitoneally once a week. The size of the tumors was measured weekly. The results of the in vitro cell culture model indicated that the cytotoxicity of IL-13-conjugated liposomal DXR was higher than that of unconjugated liposomal DXR. The level of DXR detected in the cells after delivery via IL-13-conjugated liposomes was even higher than that seen with free DXR and cyclosporine A, a Pgp inhibitor. In the in vivo model, the tumor size decreased by 66% after weekly injections of 15 mg/kg body weight) of DXR (group 1) but not 7.5 mg/kg body weight of DXR over a 2-week time period. To date, only 1 of 5 animals in group 1 has died. The tumors in animals injected with PBS alone have continued to grow. The in vivo studies are ongoing.


Y.J. Piao, H. Jiang, J. Xu, Y.J. Ji, M.M. Alonso, O-K. Lee, C. Conrad, C. Gomez-Manzano, and J. Fueyo; Brain Tumor Center, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Malignant gliomas are characterized by infiltrative growth that causes progressive neurologic dysfunction and, almost invariably, death. Currently there is no effective treatment for glioblastoma; however, recent advances in our understanding of brain tumor biology suggest that treatment strategies that target the fundamental molecular defects of brain tumors may provide effective and selective therapies. Oncolytic adenoviruses are a promising therapy for the treatment of gliomas. Previously, we studied the antiglioma activity of the tumor-selective Delta-24 adenovirus, which encompasses an early 1A adenoviral deletion in the retinoblastoma protein-binding region. However, the paucity of Coxsackie-adenovirus receptor on tumor cells is a major stumbling block for adenovirus-based treatment. In this study, we enhanced the tropism of Delta-24 through genetic incorporation of cancer receptor-binding peptides to improve antiglioma efficiency and maintain replication selectivity for cancer cells. The mutant variant III of EGFR constitutes the prototype of the cancer-specific receptor. We hypothesize that redirection of Delta-24 infectivity to mutant EGFR would result in the targeting of the cancer cells responsible for the advanced neoplastic phenotype of gliomas. Using isogenic cell lines, we first confirmed that the peptide PEPCH1 binds preferentially to the membrane of cells overexpressing EGFRvIII (U87-Delta-EGFR and 293.EGFRvIII). Then we constructed an adenovirus with retargeted infectivity via EGFR (Delta-24-RIVER) by inserting the PEPCH1–coding sequence in the HI loop of a Delta-24-detargeted (unable to bind CAR) adenovirus. Delta-24-RIVER infected and replicated efficiently in EGFR-expressing A549 cells, allowing for adenoviral production. Importantly, Delta-24-RIVER showed selective cytotoxicity in cancer cells expressing high levels of EGFR. Thus, infection with similar doses of Delta-24-RIVER induced a cytopathic effect in A549 cells but not in 293 cells (EGFR negative). We evaluated the EGFR expression level in several cancer cell lines and normal cells, including MDA-MB-468 breast cancer cells, A549 non-small cell lung cancer cells, MRC-5 human lung fibroblasts, U2OS and Saos-2 sarcoma cells, and normal human astrocytes. MDA-MB-468 and A549 cells showed high EGFR expression levels. Conversely, U2OS, Saos-2, and MRC-5 were characterized by low EGFR expression. We found that Delta-24-RIVER acquired a replication phenotype, as assessed by expression of late genes (fiber), and induced significant cytolysis in a dose-dependent manner in cancer cells with high levels of EGFR but did not replicate in normal fibroblasts and astrocytes. To further prove the EGFR-dependent infectivity and cytolysis of the Delta-24-RIVER construct, we tested Delta-24-RIVER and Delta-24 in the U87 MG isogenic system. Delta-24-RIVER induced potent cytolysis in the U87-Delta-EGFR cell line but not in the U87 parental line. In vivo experiments using U87MG and U87-Delta-EGFR xenografts implanted in nude mice treated intratumorally with a dose of 10e8 pfu/animal of Delta-24-RIVER are in progress. Taken together, these data constitute proof of principle for the direct target of a cancer-specific receptor using replication competent tumor-selective adenoviruses.


Dawn E. Post,1,2,6 Eric M. Sandberg,1,2 Narra Sarojini Devi,1,2 Daniel J. Brat,4,6 Zhiheng Xu,5,6 Mourad Tighiouart,5,6 and Erwin G. Van Meir1,2,3,6; 1Laboratory of Molecular Neuro-Oncology, Departments of 2Neurosurgery, 3Hematology/Oncology and 4Pathology, 5Biostatistics Research and Informatics, 6Winship Cancer Institute, Emory University, Atlanta, GA, USA

There are no approved therapies for hypoxic/HIF-active tumor cells, which are associated with tumor resistance to therapy, and have therefore become an important therapeutic target. We previously developed an oncolytic adenovirus (HYPR-Ad) for the specific killing of hypoxic/HIF-active tumor cells. Here, we present a second-generation HYPR-Ad that has been armed with an interleukin-4 gene (HYPR-IL-4-Ad). The IL-4 cytokine possesses strong anti-tumor activity, including the induction of a host immune response against the tumor and inhibition of tumor angiogenesis. A bidirectional hypoxia/HIF-responsive promoter was used to conditionally regulate the expression of the Ad E1A viral replication and IL-4 genes within HYPR-IL-4-Ad. HYPR-IL-4-Ad displays hypoxia-dependent E1A and IL-4 protein expression. It induces viral replication and conditional cytolysis of hypoxic but not normoxic cells. HYPR-IL-4-Ad treatment of established human tumor xenografts by intratumoral injection resulted in a rapid regression in tumor size that was maintained long-term. Importantly, the antitumor activity of this virus was as potent as that of the wild-type dl309-Ad. HYPR-IL-4-Ad–treated tumors displayed extensive necrosis, fibrosis, and leukocyte infiltrates and widespread viral replication and hypoxia. The use of an oncolytic Ad that locally delivers IL-4 to tumors is novel, and we expect that HYPR-IL-4-Ad will have broad therapeutic use for all solid tumors that have hypoxia or active HIF, regardless of tissue origin or genetic alterations.


Vinay K. Puduvalli, Jihong Xu, Deepa Sampath and Yuanfang Liu; Departments of Neuro-Oncology and Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Vorinostat is a histone deacetylase inhibitor with promising activity against malignancies in preclinical and early clinical studies. We determined the activity of vorinostat against gliomas in vitro as a single agent or in combination with cytotoxic and cytostatic agents. Glioma cells (D54, U87, and U373) were exposed to vorinostat, and the effect on proliferation was determined using a WST-1 assay. Induction of apoptosis and effects on cell cycle were determined using a flow cytometric analysis. Anchorage-independent growth was assessed using a soft agar clonogenic assay. The cells were treated concurrently with vorinostat and cytotoxic agents or isotretinoin to assess the effects of the combination. Vorinostat induced a dose- and time-dependent decrease in proliferation in glioma cells and induced apoptosis as seen by phenotypic, functional, and biochemical changes. Treatment with vorinostat in a soft agar clonogenic assay resulted in inhibition of anchorage-independent growth of glioma cells. Vorinostat-treated cells exhibited hyperacetylation of the histones H3 and H4, increased levels of p21, and reduced levels of cyclin B1. These biochemical changes were accompanied by a G2-M cell cycle arrest that was followed by induction of apoptosis, suggesting that vorinostat-induced cell death was a result of the failure of cells to cross the G2-M checkpoint. Vorinostat did not activate markers of the DNA damage signaling pathway, such as H2AX, Chk 1, or Chk 2; in addition, a decrease in level of p53 was seen in D54 cells, suggesting that the changes seen in response to the agent were independent of p53 or DNA damage. Concurrent treatment with vorinostat and etoposide (a topoisomerase inhibitor) or cisplatin (a DNA cross-linking agent) resulted in synergistic effects on apoptosis. Combined treatment with vorinostat and isotretinoin also exhibited a synergistic effect in inducing apoptosis, suggesting a cooperative interaction between these 2 agents. Vorinostat exhibited activity against gliomas in vitro, both alone and in combination with cytotoxic agents. The effects were mediated by modulation of cell cycle-related proteins and induction of G2-M arrest, leading to apoptosis. Of particular interest, vorinostat showed synergistic interaction with cytotoxic agents and isotretinoin showed a resultant increase in apoptosis. These findings suggest a therapeutic potential for HDAC inhibitors against gliomas, both as single agents and in combination with existing therapies.


Sith Sathornsumetee,1 Anita B. Hjelmeland,1 Stephen T. Keir,1 Roger E. McLendon,2 David Batt,3 Timothy Ramsey,3 Naeem Yusuff,3 B.K. Ahmed Rasheed,2 Mark W. Kieran,4 Andrea Laforme,4 Darell D. Bigner,1,2 Henry S. Friedman,1,2,5 and Jeremy N. Rich1,6,7; Departments of 1Surgery, 2Pathology, 5Pediatrics, 6Medicine, and 7Neurobiology, Duke University Medical Center, Durham, NC, USA; 3Novartis Institutes for Biomedical Research, Cambridge, MA, USA; 4Department of Pediatrics, Dana-Farber Cancer Institute, Boston, MA, USA

Malignant gliomas are highly proliferative, and angiogenic cancers are resistant to conventional therapies. Although RAS and RAF mutations are uncommon in gliomas, RAS activity is increased in gliomas. Additionally, vascular endothelial growth factor (VEGF) and its cognate receptors are highly expressed in gliomas. We now report that AAL881, a novel low-molecular weight inhibitor of the kinase activities associated with B-RAF, C-RAF (RAF-1), and VEGFR-2, demonstrated activity against glioma cell lines and xenografts. In culture, AAL881 inhibited downstream effectors of RAF in a concentration-dependent manner, with inhibition of proliferation associated with G1 cell cycle arrest, induction of apoptosis, and decreased colony formation. AAL881 decreased proliferation of bovine aortic endothelial cells and tumor cell secretion of VEGF and inhibited invasion of glioma cells through an artificial extracellular matrix. Orally administered AAL881 was well tolerated, with minimal weight loss in non-tumor bearing mice. Established subcutaneous human malignant glioma xenografts grown in immunocompromised mice treated with a 10–day course of oral AAL881 exhibited growth delays relative to control tumors, frequently resulting in long-term complete regressions. AAL881 treatment extended the survival of immunocompromised mice bearing orthotopic glioma xenografts compared with placebo controls. The intraparenchymal portions of orthotopic AAL881 treated tumors underwent widespread necrosis consistent with vascular disruption compared with the subarachnoid elements. These effects are distinct from our prior experience with VEGFR-2 inhibitors, suggesting that targeting RAF itself or in combination with VEGFR-2 induces profound tumor responses in gliomas and may serve as a novel therapeutic approach in patients with malignant gliomas. This study was supported in part by funds from the Pediatric Brain Tumor Foundation of the United States, Accelerate Brain Cancer Cure, and Southeastern Brain Tumor Foundation (J.N.R.). This work was also supported by National Institutes of Health grants NS047409, NS054276, and 1 P50 CA108786 (J.N.R.). A.B.H. is a Paul Brazen/American Brain Tumor Association Fellow. J.N.R. is a Damon Runyon-Lilly Clinical Investigator supported by the Damon Runyon Cancer Research Foundation and a Sidney Kimmel Cancer Foundation Scholar.


C. Sauvageot,1 J. Barnes,2 J. Weatherbee,1 N. Ramakrishna,3 S. Kesari,4 C. Stiles,1 M. Kieran,2 and P. Wen4; Departments of 1Cancer Biology, 2Pediatric Oncology, 3Radiation Oncology, 4Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA

The malignancy and progression manifested by glioblastoma multiforme (GBM) tumor cells arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Research to date has revealed that inhibitors of single targets are only modestly effective at attenuating the growth and malignancy of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a well-characterized group of client proteins, many of which are deregulated in GBM. 17-allylamino–17-demethoxygeldanamycin (17-AAG) is an HSP90 inhibitor that has been shown to promote growth inhibition in multiple tumor cell lines and anti-tumor activity in in vivo and preclinical models. Given that 17-AAG targets many of the aberrant signal transduction pathways in GBM, we were interested in assessing the ability of 17-AAG to inhibit the growth of glioma cells both in vitro and in vivo. In addition, we assessed whether 17-AAG would synergize with radiation or temozolomide, which are the best treatment modalities currently available for GBM. Our results reveal that 17-AAG inhibits the growth of numerous glioma cell lines in vitro, targets the appropriate proteins within these cells, inhibits the growth of intracranial tumors, and synergizes with radiation, both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide. We found that 17-AAG is a promising compound for the treatment of GBM.


Amish C. Shah,1 Jacqueline N. Parker,2 G. Yancey Gillespie,3 James M. Markert,1, 2, 3 and Kevin A. Cassady2; Departments of 1Physiology and Biophysics, 2Pediatrics Infectious Diseases, and 3Surgery — Neurosurgery, University of Alabama at Birmingham, Birmingham, AL, USA

The oncolytic herpes simplex virus (HSV)-1 γ134.5–deletion mutant is a promising agent for the treatment of malignant glioma and other tumors. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We sought to determine whether substitution of γ134.5 with human cytomegalovirus (HCMV) genes that allow late viral protein synthesis in infected cells would improve HSV replication and anti-tumor efficacy without restoring neurovirulence. C130 and C134 are Δγ134.5 HSV vectors expressing the HCMV PKR-evasion genes TRS1 and IRS1, respectively. Various human glioma cell lines were infected in vitro to determine whether the viruses could overcome the PKR-mediated inhibition of protein translation and replicate more efficiently. These viruses were subsequently tested in vivo to determine neurovirulence and anti-tumor efficacy in 2 brain tumor models. The HCMV/HSV-1 chimeric viruses (1) maintained late viral protein synthesis in the human malignant glioma cells tested (U87, U251, and D54); (2) replicated to wild-type levels in these cells; (3) were aneurovirulent, with LD50 measurements of 4 to more than 6 logs higher than that of wild-type HSV-1; and (4) improved survival in 2 brain tumor models: a human malignant glioma in severe combined immunedeficient (SCID) mice and a syngeneic immunocompetent murine neuroblastoma model. These findings suggest that replication of oncolytic HSV-1 vectors in partially restrictive tumor cells due to anti-viral PKR responses can be significantly improved by encoding PKR-evasion genes from a related herpesvirus. The chimeric HSV exhibit advantageous therapeutic ratios by demonstrating superior anti-tumor efficacy and low neurovirulence.


Qing Shi,1 Anita B. Hjelmeland,1 Stephen T. Keir,1 Sarah K. Wickman,1 Guanghong Wu,1 Dowdy Jackson,2 Osamu Ohmori,2 Darell D. Bigner,1,3 Henry S. Friedman,1,3,4 and Jeremy N. Rich1,5,6; Departments of 1Surgery, 3Pathology, 4Pediatrics, 5Medicine, and 6Neurobiology, Duke University Medical Center, Durham, NC, USA; 2Novartis Institutes for Biomedical Research, Cambridge, MA, USA; Novartis Pharmaceuticals Oncology, East Hanover, NJ, USA.

Glioblastomas are highly lethal cancers that resist current therapies. Glioblastomas frequently overexpress the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), which contributes to tumor malignancy by increasing cellular migration, invasion, and proliferation. We evaluated the efficacy of a novel low-molecular-weight inhibitor of FAK, TAE226, against human glioma cell lines and xenografts. TAE226 inhibited the phosphorylation of FAK and the downstream effectors AKT, extracellular signal-related kinase (ERK), and S6 ribosomal protein. TAE226 demonstrated a concentration-dependent decrease in cellular proliferation with an associated G2 cell cycle arrest in multiple glioma cell lines. TAE226 also induced apoptosis while decreasing cellular adhesion, migration, and invasion in vitro, demonstrating the potential benefit of TAE226 in glioma therapy. In vivo, orally administered TAE226 (100 mg/kg qd on a 5 day on/2 day off/5 day on schedule) induced a modest growth delay of subcutaneous human glioma xenografts grown in immunocompromised mice (3 to 4 days delay, P < 0.001). TAE226 also increased the survival (3.5 days, P = 0.078) of immunocompromised mice bearing orthotopic intracranial human glioma xenografts until neurologic deficits occurred. As glioma xenografts rely primarily on proliferative expansion rather than invasion for tumor growth and FAK plays a major role in tumor invasion, these results may underestimate the efficacy of TAE226 in humans. Therefore, further evaluation of TAE226 and similar FAK inhibitors alone and in combination with other established therapies for the treatment of glioblastoma patients is warranted. This study was supported in part by funds from the Pediatric Brain Tumor Foundation of the United States (J.N.R.), Accelerate Brain Cancer Cure (J.N.R.), Childhood Brain Tumor Foundation (J.N.R.), and Southeastern Brain Tumor Foundation (A.B.H.). This work was also supported by National Institutes of Health grants NS047409, NS054276, and 1 P50 CA108786 (J.N.R.). A.B.H. is a Paul Brazen/American Brain Tumor Association Fellow. J.N.R. is a Damon Runyon-Lilly Clinical Investigator supported by the Damon Runyon Cancer Research Foundation and a Sidney Kimmel Cancer Foundation Scholar.


Atsushi Suzuki,1 Shinya Yamada,1 Vazgen Khankaldyyan,1 Anat Erdreich-Epstein,1 Fred Dorey,1 Ignacio Gonzalez-Gomez,1 Simon L. Goodman,2 Michael Meyring,3 and Walter E. Laug1; 1Childrens Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles, Los Angeles, CA, USA; 2Oncology Research, Merck KGaA, Darmstadt; and 3 Institute of Drug Metabolism and Pharmacokinetics, Merck KGaA, Grafing, Germany

EMD 121974 (Cilengitide) is an αv-integrin antagonist peptide that inhibits neo-angiogenesis. Specifically, it suppresses orthotopic glioma growth in nude mice and encouragingly, has shown promising results in a phase I trial in adult glioblastoma patients. In the present study, we investigated the optimal dose and interval of drug administration in the orthotopic U87MG brain tumor model. Daily intraperitoneal administration of 5 mg/kg and 10 mg/kg EMD 121974 suppressed glioblastoma growth by 93% ± 13 SD (n = 12) and 82% ± 27 (n = 15), P < 0.0001 for both. Surprisingly, higher doses (30 mg/kg and 90 mg/kg, n = 8 for each) did not inhibit tumor growth (P > 0.12). Maximal inhibition of tumor growth and maximal survival were seen with daily dosing, most likely because of the short half-life of the peptide in the circulation (T½ = ~20 min). Pharmacokinetic data indicated that EMD 121974 was rapidly and well absorbed from the peritoneum, with peak levels attained at 5 min. Mean peak plasma concentrations on day 7 in mice treated daily with 200 μg EMD 121974 were 14.7 μg/mL ± 3.5 (SD, n = 3; ~25 μM). The mean peak level in the brains of the same mice were 0.46 μg/g ± 0.13 (n = 3). Administration of EMD 121974 (90 μg/mouse/day) by continuous infusion using a subcutaneous Alzet pump did not affect tumor growth. Although our PK data indicate that the drug was well absorbed using Alzet pumps, the mean steady-state plasma level (91.6 ng/mL ± 14.4, n = 9; ~150 nM) was considerably lower than the peak levels attained with daily i.p. administration. This may explain the lack of efficacy we found when using Alzet pumps. Overall, these data show that in the orthotopic mouse model, optimal suppression of glioma growth is obtained when EMD 121974 is given as a daily i.p. bolus at a dose of 5–10 mg/kg. Moreover, it underscores the need for effective surrogate markers to determine the optimal biologic dosing of EMD 121974. These data may help guide the treatment of glioma patients in upcoming phase II trials.


Ilya V. Ulasov, Matthew Tyler, Zeng B. Zhu, David T. Curiel, and Maciej S. Lesniak; The University of Chicago, Section of Neurosurgery, Chicago, IL, USA and University of Alabama at Birmingham, Gene Therapy Center, Birmingham, AL, USA

Improved gene therapy approaches for the treatment of malignant glioma requires a reliable in vivo gene transfer method. We recently developed a replication-competent vector containing a tumor-specific survivin promoter that drives E1A viral replication and demonstrated oncolytic effects against human glioma in vitro and in vivo. To enhance the observed oncolytic effect, we subsequently created a new vector, CRAd-S-pk7, containing a wild-type backbone with a polysine chain incorporated into the adenovirus fiber protein. We hypothesized that a vector containing tumor-specific survivin promoter and a polylysine fiber modification would allow a higher level of specificity and efficacy of glioma killing. To test the efficacy of our vector, we performed infections of human glioma cells in vitro and in vivo. After infection with CRAd-S-pk7, crystal violet staining demonstrated a higher rate of glioma cell killing, with a higher level of E1A protein expression. Moreover, E4 copy quantification assays indicated that CRAd-S-pk7 has enhanced glioma-specific replication. U87MG human glioma xenografts were subsequently established in nude mice and infected with the new vector. CRAd-S-pk7 significantly inhibited tumor growth by 60%, whereas the tumor infected with wild-type virus or mock virus infected showed little or no growth inhibition. These results were confirmed with an immunohistochemical analysis using Ki-67 along with anti-hexon staining. A biochemical analysis using anti-human caspase 3 antibodies confirmed the proapoptotic character mediated by the CRAd-S-pk7 vector. The efficiency of the CRAd-S-pk7 gene transfer and enhanced oncolytic capacity warrants further exploration of this novel oncolytic vector for testing in clinical trials of malignant brain tumors.


Mahmud Uzzaman, Ron Benveniste, Gordon Keller, and Isabelle Germano; Department of Neurosurgery and Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY, USA

The treatment of malignant gliomas with current protocols remains a challenge in neuro-oncology. Most of the tumor recurs after aggressive surgical and medical treatment. The aim of this study was to generate transgene-expressing cells that can be implanted in situ and deliver genes under external control. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been shown to induce apoptosis in a variety of tumor cells, including gliomas. Recently, we developed an expression system using embryonic stem cells (ESCs) differentiated into astrocytes. This system can express transgene (ESC) under doxycycline control. The aim of this study was to assess the pro-apoptotic effects of transgene-expressing ES-derived astrocytes on malignant gliomas in vivo. Malignant glioma A172 cells were used to induce tumors in nude mice. ESC-derived astrocytes expressing TRAIL were injected into the tumors. Doxycycline was administered to the experimental group. Tumor growth was measured daily. Tissue was assayed by histologic analysis, TUNEL, and PCR. Mice in the experimental group (n = 6) were injected 7 days after tumor induction with ESC-derived astrocytes expressing TRAIL and then treated with doxycycline. The control groups (n = 3 each) included: 1) no ESC-derived astrocyte injection, 2) no ESC-derived astrocyte injection plus doxycycline, and 3) injection of ESC-derived astrocyte without doxycycline. A 30% reduction in tumor size occurred 48 hrs after injection of ESC-derived transgenic astrocytes in the experimental group. This was not observed in the control group. TUNEL revealed abundant apoptotic tumor cells in the experimental group. PCR data expressing TRAIL after doxycycline induction will be presented. Our data suggest that ESC-derived astrocytes expressing TRAIL can be used as vectors to deliver gene therapy for malignant gliomas. The tight control under tet promoter, previously reported in vitro, is maintained in vivo, corroborating the safety of this system. Similar experiments with U87 cells are underway.


Weijun Wang,1 Adel Kardosh,2 Axel Schonthal,2 and Thomas C. Chen1,3; Departments of 1Neurosurgery, 2Molecular Microbiology & Immunology, and 3Pathology, University of Southern California, Los Angeles, CA, USA

It is well known that the prognosis of patients with central nervous system (CNS) lymphoma is extremely poor, with a 5–year survival rate of 34% for primary CNS lymphoma and 33% for metastatic CNS lymphoma. Because lymphomas have a dramatic cytotoxic response to steroids, we hypothesized that similar effects would be seen with non-steroidal anti-inflammatory drugs (NSAIDS) such as Celebrex, which has been demonstrated to increase tumor cell sensitivity to radiation and chemotherapy. To understand whether the inhibitory properties of Celebrex are secondary to inhibition of COX-2, we used a close structural analog of Celebrex, dimethyl-Celebrex (DMC), which lacks the COX-2 inhibitory function but maintains the ability to induce apoptosis. An intracranial CNS lymphoma animal model was formed in athymic nude mice. B-cell Raji lymphoma cells (5 × 104) were injected subdurally into mice brains and 1000 ppm DMC and Celebrex were administered orally to the animals the day after tumor cell implantation. The therapeutic effect was monitored by animal survival. The animals treated with Celebrex had significantly improved survival compared with those treated with DMC and controls. There was no significant difference between DMC-treated mice and controls. Celebrex is a promising therapeutic agent in the treatment of CNS lymphomas. The combination of Celebrex and radiation is being investigated.


Shinya Yamada,1,5 Xing-Yao Bu,2 Vazgen Khankaldyyan,2 Stefan Bluml,3,5 Ignacio Gonzales-Gomez,4 J. Gordon McComb,1,5 Anat Erdreich-Epstein,2 and Walter E. Laug2; Departments of 1Neurological Surgery, 2Pediatrics, 3Radiology and 4Pathology, Keck School of Medicine, University of Southern California, Los Angeles and Children’s Hospital Los Angeles, Los Angeles, CA, USA; and 5The Rudi Schulte Research Institute, Santa Barbara, CA, USA

The objective of this study was to determine the effect of the angiogenesis inhibitor Cilengitide (EMD121974) on glioblastoma growth and associated angiogenesis in the brains of nude mice. U87MG human glioblastoma cells (105) in 1–μL medium were stereotactically injected over 20 min into the caudate/putamen of nude mice. Mice were injected daily IP with Cilengitide or solvent (control) starting 5 days after tumor implantation. Mice were killed 1 hr to 63 days after tumor implantation and examined for tumor size, vascularity, apoptosis, and tumor cell proliferation. This injection technique resulted in highly reproducible, localized, spherical tumor cell placement in the parenchyma, without reflux into the subarachnoid space or penetration into the ventricle. Serial brain sections showed stable tumor size (1 to 2 mm3) for the first 30–40 days. Thereafter, control tumors showed exponential growth to a maximal volume of 120 mm3, leading to signs of distress that required us to kill the mice at 8–9 weeks. Serial staining for Ki-67, a marker for cell proliferation and CD31, an endothelial cell marker, demonstrated that tumor cell proliferation and tumor angiogenesis increased more than the increase in tumor volume. In contrast, in Cilengitide-treated mice, the tumor volume remained stable at 1–2 mm3, and staining for Ki-67 and CD31 remained low throughout the 9 weeks. This standardized brain tumor model is highly reproducible and useful for testing new treatment regimens. Cilengitide is highly effective at suppressing blood vessel growth and tumor cell proliferation, thereby controlling the orthotopic growth of this glioblastoma cell line. It remains to be determined whether the effect of Cilengitide is due to a combination of inhibition of angiogenesis and direct inhibition of tumor cell proliferation or only direct inhibition of angiogenesis with secondary inhibition of tumor cell proliferation.


Lihua Yang,1 Erin Jackson,2 B. Mark Woerner,1 Arie Perry,3 David Piwnica-Worms,2,4 and Joshua B. Rubin1,5,6; Departments of 1Pediatrics, 2Molecular Imaging Center, Mallinckrodt Institute of Radiology, 3Pathology, 4Molecular Biology and Pharmacology, 5Anatomy and Neurobiology, 6Neurology, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, MO, USA

CXCL12 and its receptor CXCR4 are important regulators of malignant brain tumor growth, and targeting this pathway has become a therapeutic goal. However, the molecular basis for CXCL12-induced tumor growth remains unclear, and the optimal approach to inhibiting CXCR4 function in cancer is unknown. We investigated the mechanism of CXCR4-mediated brain tumor growth in in vitro and in vivo models. We found that CXCL12 (0.1 μg/mL) increased both the U87 glioblastoma and Daoy medulloblastoma cell numbers by approximately 300% and that this growth effect was dependent on the sustained suppression of intracellular cAMP to 30% of baseline. We further found that the anti-tumor activity of the CXCR4 antagonist AMD 3465 was associated with blocking CXCL12–induced cAMP suppression. The importance of sustained cAMP suppression in tumor cell growth was also demonstrated by the growth inhibitory properties of the adenylyl cyclase activator forskolin and the phosphodiesterase inhibitor Rolipram. Both of these drugs elevated intracellular cAMP and completely blocked CXCL12 growth effects. In corollary fashion, overexpression of phosphodiesterase 4A reduced intracellular cAMP levels, stimulated tumor cell growth to a similar degree to CXCL12 treatment, and abrogated any additional growth effect of CXCL12. To evaluate the importance of cAMP suppression to in vivo tumor growth, we treated intracranial xenografts of U87 and Daoy cells with AMD 3465 (50 μg/day by continuous subcutaneous infusion) or Rolipram (100 μg/day in water) and found that Rolipram had equivalent activity to that of AMD 3465, inhibiting intracranial U87 growth by 90% and Daoy medulloblastoma growth by 70%. The anti-tumor effects of both AMD 3465 and Rolipram were correlated with in vivo increases in tumor cAMP levels from 45.5 pmol/mg protein to 62.3 and 79.7 pmol/mg protein, respectively. These data demonstrate that CXCR4-mediated tumor growth is dependent on the suppression of intracellular cAMP and that directly elevating cAMP has a comparable anti-tumor effect to that of CXCR4 antagonists. The current clinical availability of phosphodiesterase inhibitors support the evaluation of these agents in patients with brain tumors.


Qingyu Zhou, Ping Guo, Xiaoming Wang, and James M Gallo; Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, PA, USA

Metronomic dosed (MD) chemotherapy, as opposed to conventional dosed (CD) chemotherapy of standard cytotoxic anticancer drugs, is considered an alternate strategy to target endothelial cells and angiogenesis. There is interest in defining the optimal biologic dose of MD drugs, but there has not been any attempt to integrate PK with PD measurements, which would assist in defining optimal metronomic dosing regimens. The main objective of the present study was to compare the PK and PD of temozolomide (TMZ) using MD and CD regimens. Nude rats bearing subcutaneous gliomas overexpressing VEGF were entered into either a CD (18 mg/kg/d IV for 5 days) or MD (3.24 mg/kg/d IV for 28 days) treatment schedule. TMZ and its metabolite, AIC, were measured by LC/MS/MS in plasma and tumors by microdialysis on the first and last days of dosing. PD measurements collected through the study periods consisted of gene and protein expression of various angiogenic markers such as VEGF, Ang1, Ang2, Tie2, TSP1, HIF-1α, tumor pH, and interstitial fluid pressure. In addition to PK/PD measurements, tumor size was monitored throughout the 28-day period. A comparison of the PK parameters of total clearance and volume of distribution revealed no significant changes between the MD and CD groups. Although the tumor:plasma AUC ratio was initially higher on day 1 for the MD group compared with the CD group, the final day comparisons indicated an analogous tumor exposure of slightly higher than 1. The PD assessment is ongoing, and both the MD and CD groups caused a significant (P < 0.05) decrease in IFP and tumor size compared with vehicle control treatment. The PK results confirmed that TMZ exhibited linear pharmacokinetics, and based on the MD and CD regimens employed, do not yield a significantly different pattern of drug accumulation in the tumor. The reduction in interstitial fluid pressure in the TMZ groups may contribute to the accumulation of TMZ into the tumor. Thus far, there does not seem to be any contrasting effects on the PD parameters between the MD and CD regimens, suggesting that additional low-dose regimens should be examined.



K. Aldape, L. Zhang, H. Phillips, C. Wei, C. Nutt, D. Louis, J.G. Cairncross, B.G. Feuerstein, R. Wang, R.B. Jenkins, and H. Colman; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; Genentech Inc., South San Francisco, CA, USA; Massachusetts General Hospital, MA, USA; University of California-San Francisco, San Francisco, CA, USA; and Mayo Clinic, Rochester, MN, USA

The use of expression microarray data to distinguish molecular subtypes of tumors associated with distinct clinical outcomes has the potential for both identification of novel therapeutic targets and individualization of treatment based on molecular profile. However, a significant limitation in the use of microarray data from an individual study to prospectively identify robust predictors of outcome is that the high number of genes investigated, combined with a relatively low number of samples, results in a high false-discovery rate. This leads to a corresponding low likelihood that the top survival genes observed in one study will predict outcome in an independent set of samples. To overcome this problem, we conducted a meta-analysis by combining Affymetrix expression array data from four different institutions comprising 110 cases of newly diagnosed glioblastoma (GBM). Algorithms were developed to merge data from different Affymetrix chips (U133A and U95A), remove institutional bias, normalize data, and identify samples having significant contamination of normal brain tissue. We identified the top 200 survival genes from each of the four datasets individually using the fold-change between the typical GBM survivor group (less than 2 years) and the long-term survivor group (2 years or greater). We identified the most robust consensus set by identifying the top survival genes common to all 4 datasets. This analysis identified 38 genes that were ranked in the top 200 in data from all four institutions, a result found to be highly unlikely to be due to chance. A composite survival index derived from these 38 genes predicted survival in all four datasets and will be further refined and validated in independent sample sets. These findings provide proof of concept that gene expression profiles derived from one GBM dataset can predict survival in an independent dataset and that a consensus multigene survival classifier for GBM can be identified. Preliminary RT-PCR analysis on independent samples indicates that a subset of these genes predict outcome. Refinement and validation of this classifier using additional independent sample sets from uniformly treated patients is planned, with the goal of designing a clinical test to be used for treatment response prediction in GBM.


Francis Ali-Osman; Departments of Surgery and Pathology and the Preston Robert Tisch Brain Tumor Center, Duke University, Durham, NC, USA

The gene encoding EGF, the ligand for the receptor tyrosine kinase EGFR, harbors a single nucleotide polymorphism resulting from an A-to-G transition at position +61 in its 5′-untranslated region. It has been suggested that the 61G EGF promoter is transcriptionally more active than is the 61A. The polymorphism has been associated with increased risk for melanoma and a more aggressive disease in malignant gliomas. In this study, we developed a TaqMan allele discrimination assay for the 61A and 61G EGF alleles and used it to determine the EGF genotypes of 332 glioma patients, using genomic DNA isolated from their peripheral blood lymphocytes. Patient survival data and histological diagnoses were obtained from patient hospital records and used in the statistical analyses. The results showed that among all patients, regardless of histology, 48% were heterozygous A/G, 33% homozygous A/A, and 19% homozygous GG. Interestingly, the frequency of the GG genotype decreased with increasing grade of astrocytoma, being 37%, 27%, 13%, and 14% for low-grade astrocytoma, astrocytoma, anaplastic astrocytoma, and GBM, respectively; the relationship approached statistical significance (P = 0.075). In contrast, no statistically significant relationship was observed between patient age and EGF genotype (P = 0.285). The results of Kaplan-Meier survival analysis showed that the EGF A61G polymorphism was highly associated with patient survival. The log-rank test comparing the survival distribution of the three EGF genotypes was statistically significant (P = 0.021), with the G/G genotype having a better survival than the A/A group. The Cox proportional hazards model showed the number (0, 1, or 2) of A61 alleles to be a statistically significant (P = 0.0053) predictor of patient survival. Our results confirm previous findings of an association between EGF polymorphism and clinical outcome in malignant gliomas (Bhowmick et al., Cancer Res 64, 2004). Our data, however, contrast with those findings in that we found the presence of the A/A genotype to be associated with a shorter disease-free survival than is the G/G genotype. To establish the significance of the polymorphisms in the transcriptional activity of the EGF promoter, we cloned each of the EGF promoter variants into phagemid vector constructs to drive luciferase gene expression and then used them to demonstrate the differential transcriptional activity of the two promoters. The discrepancy between our results and those of the only other published study of EGF polymorphism in gliomas indicates the need for more investigations on the association of this genetic polymorphism and of EGF expression in the biological behavior and clinical outcome of human gliomas. Supported by grants RO1 CA 91438, P50 CA108786, and 5P30 CA 114236 from the National Cancer Institute.


J.W. Barnes,1, 3 D. Cervi,3 K.L. Ligon, L.H. Tseng,4 J.A. Longtine,4 D.M. Chaponis,1,3 P.Y. Wen,2 S. Kesari,2 J. Drappatz,2 P.M. Black,5 and M.W. Kieran1, 3; Divisions of 1Pediatric Oncology and 2Adult Neuro-Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 3Vascular Biology Program, Children’s Hospital, Boston, MA, USA; Departments of 4Neuropathology and 5Neurosurgery, Brigham and Women’s Hospital, Boston, MA, USA

Primary brain tumors are typically highly aggressive and refractory to current treatments. A number of chemotherapeutic agents, such as carmustine (BCNU), lomustine (CCNU), procarbazine, and temozolomide, target the O6 position on guanine, forming DNA adducts and cross-links that induce cytotoxicity. The DNA repair enzyme O6–alkylguanine DNA alkyltransferase, encoded by the gene O6–methylguanine-DNA methyltransferase (MGMT), repairs alkylation at this site and is responsible for protecting both tumor and normal cells from environmental insults and chemotherapeutic agents. Recent clinical studies have demonstrated the importance of this resistance pathway in the treatment and prognosis of malignant gliomas of the CNS. Current methods to assess MGMT activity rely on indirect valuation of promoter methylation, which is very susceptible to tissue handling and sample preparation. In addition, there are other ways of inhibiting protein expression that would not be detected by this method. We now report the development of a high-throughput proteomic technique that directly evaluates MGMT protein in clinical samples from brain tumors. Fresh frozen tissue was obtained at the time of surgical resection. Total protein was extracted and directly applied to proteomic chips without additional fractionation or treatment. All samples were compared to purified MGMT as well as cancer-cell lysates with known expression of MGMT. In addition, promoter methylation status was obtained in a blinded fashion for all samples. Treatment/outcome was available for the patients providing these samples. Using tissue extracts, a clear quantitative molecular signature of MGMT was obtained at picogram concentrations of total protein using the SELDI-TOF platform. Signatures were validated using previously characterized expression from cell lines and tissue samples. Samples were also validated using Western blotting analysis and micro-sequencing. This new approach was compared to standard immunohistochemical and promoter methylation status and was able to identify MGMT expression in some samples that were classified as negative by these other techniques. A detailed description of the expression pattern for adult and pediatric tumor samples is discussed. In an effort to make direct MGMT protein analysis reliable and feasible on a large scale, we developed a highly sensitive and specific proteomic approach for MGMT detection from tumor specimens. Evaluating the levels of MGMT in human tumors may provide information that clinicians can use to formulate effective treatment combinations, leading to better clinical outcomes.


Daniel P. Cahill, Kymberly K. Levine, Candice A. Romany, Michelle G. Rooks, Jennifer E. Roy, Tracy T. Batchelor, and David N. Louis; Molecular Pathology Unit, Neurosurgical Service, Brain Tumor Center and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

Glioblastomas are devastating primary brain tumors, with expected survival of 1–3 years from diagnosis. Surgical resection followed by a combination of radiotherapy and the alkylating chemotherapeutic agent temozolomide has become the standard of care for treatment of this disease. However, virtually all patients have recurrent progressive tumors despite this treatment regimen. Both O-6 methylguanine methyltransferase (MGMT) levels and somatic mutation of the mismatch repair gene hMSH6 have been linked to alkylator resistance in experimental models and in patients treated with temozolomide. We sought to comparatively evaluate the roles of these resistance pathways in human glioblastomas. We directly sequenced the coding frame of hMSH6 in pre-treatment and recurrent tumors. hMSH6 mutation was not observed in any pre-treatment glioblastoma, although a subgroup of recurrent cases had somatic mutations in the gene, indicating that hMSH6 mutation was associated with recurrence. To expedite further MSH6 analysis and confirm these findings in an independent set of patients, we optimized an immunohistochemical assay for MSH6 and analyzed both MSH6 and MGMT status in a well-characterized panel of matched pre- and post-treatment glioblastomas derived from patients undergoing adjuvant therapy. MSH6 protein expression was detectable in all pre-treatment samples analyzed. Notably, a substantial fraction of post-treatment samples displayed loss of MSH6 expression. These data suggest that MSH6 deficiency represents a clinically relevant mechanism of alkylator-chemoresistant tumor-cell escape and may have important implications for the development of new chemotherapeutic regimens using temozolomide in combination with other agents.


Andrew M. Donson, B.K. Kleinschmidt-DeMasters, Steven O. Addo-Yobo, and Nicholas K. Foreman; The Children’s Hospital, Denver and The University of Colorado at Denver and Health Sciences Center, Aurora, CO, USA

Radiation-induced glioblastomas (XRT-GBMs) comprise a significant proportion of GBMs seen in children and young adults. Little is known about their underlying biology, and studies thus far have suggested no unique histological or cytogenetic characteristics. Files were searched to ascertain cases, and XRT-GBMs were investigated using Affymetrix gene-expression microarray profiling. GeneSpring data analysis was used to create a list of the 100 most overexpressed genes in pooled gene-expression profiles of three pediatric XRT-GBMs versus a surrogate control panel of non-astrocytic tumors (5 AT/RT, 6 MED, 2 CPP, and 16 EPN). To examine whether XRT-GBM constitutes a distinct molecular subset, we contrasted this list of genes with similar lists obtained using spontaneous GBM (N = 5) and pilocytic astrocytoma (PA; N = 5). Eight of 24 and 2 of 38 pediatric and young-adult GBM patients, respectively, had XRT-GBMs. All patients with XRT-GBMs were male, whereas patient with sporadic GBMs manifested more equitable gender ratios. Pediatric patients with XRT-GBMs had very short survival (4.5 months; range, 1–17 months) compared to patients with sporadic pediatric GBMs (9.5 months; range, 1– 70 months). One of the young-adult patients with XRT-GBMs showed prolonged survival of 4+ years. Using Venn diagram analysis to assess similarity between the lists of 100 most overexpressed genes, we found that XRT-GBM had a much stronger overlap with PA than with sporadic pediatric GBM. Thirty-seven percent of XRT-GBM genes were exclusively shared with PA, in contrast with only 5% of XRT-GBM genes exclusively shared with sporadic GBMs. Both Sox10 and ErbB3 were exclusively overexpressed in both XRT-GBM and PA. In addition, XRT-GBM gene-expression profiles were more conserved than in sporadic GBMs, which exhibited heterogeneous gene-expression profiles. XRT-GBMs represent a unique molecular subset of gliomas by gene analyses, despite their lack of distinguishing histological or cytogenetic features. Surprisingly, the XRT-GBM molecular subset shows a strong similarity with PA but not with sporadic GBM. As previously documented in PA by our group, overexpression of Sox10 and ErbB3 may be responsible for driving growth in XRT-GBM. XRT-GBM may therefore be amenable to therapies that target ErbB3.


P.J. French,1 J. Peeters,2 S. Horsman,2 E. Duijm,1 M.J. van den Bent,1 J.M. Kros,3 P. van der Spek,2 and P. Sillevis Smitt1; Departments of 1Neurology, 2Bioinformatics and 3Pathology, Erasmus MC, Rotterdam, The Netherlands

There is strong evidence that aberrant splice isoforms are involved in tumor initiation and/or progression of gliomas. For example, glioblastomas frequently express EGFRvIII, a tumor-specific, ligand-independent, constitutively active isoform of the epidermal growth factor receptor that lacks exons 2–7. Such aberrant splice variants may serve as targets for novel treatment modalities. We therefore set out to perform a genome-wide screen of expressed splice variants in gliomas. Our screen was performed by profiling the expression levels of virtually all known and predicted exons that are present in the human genome. Profiling was performed on 26 glioblastomas (GBM), 18 of which had amplification of the EGFR locus; 22 oligoden-drogliomas (OD) with loss of heterozygosity on 1p and 19q; and 6 control brain samples. Unsupervised clustering analysis indicates that exon-level expression profiling identifies subgroups based on their histological appearance (OD, GBM, and control). In addition, our screen identified approximately 700 exons that are not present in public-domain databases but are expressed in normal brain and/or glial brain tumors. We next used pattern-based correlation (PAC) as an algorithm to identify splice variants that are differentially regulated between GBM and OD. PAC calculates the level of splicing of a given exon in relation to expression of its transcript. Transcript levels were calculated using only constitutive exons; that is, exons that are always incorporated in the transcript. PAC identified approximately 400 exons that are significantly differentially spliced between oligodendroglial tumors and glioblastomas. RT-PCR confirmed the differential expression of 9 of 19 alternatively spliced exons. Our data therefore indicate that exon arrays can identify brain-tumor subgroups based on their histological appearance and that exon arrays can identify novel, differentially expressed splice variants.


S.G. Gregory,1 N.V. Johnson,1 J.J. Connelly,1 J. Virgadamo,1 R.E. McLendon,2 J.M. Vance,1 and D.D. Bigner2; 1Duke Center for Human Genetics and 2Duke Comprehensive Cancer Center, Durham, NC, USA

Primary brain tumors account for 1% of new cancer cases in the United States. Given the mortality of 4.1% per 100,000 persons, primary brain tumors accounted for 13,100 deaths in 2001 alone. These tumors have a broad histopathology and variable sensitivity to treatment and, therefore, have unpredictable progression and survival times. In general, the molecular mechanisms underlying all these variables are poorly understood. For many years it has been contended that genetic instability leads to cancer development via non-random chromosome losses and gains that contribute to tumor malignancy. To elucidate these underlying molecular mechanisms, we have generated high-resolution comparative genomic hybridization (hrCGH) data using our whole-genome tilepath microarrays to identify chromosomal rearrangements associated from 110 oligodendroglioma (OD) tumors. We have generated hrCGH data at 100–kb resolution from 45 tumors histopathologically determined to be well-differentiated OD, 40 anaplastic OD, and 25 with only an OD designation. Analysis of the entire tumor set identified characteristic loss of 1p and 19q within 72% of the tumors analyzed. In addition to ‘gross’ chromosome rearrangements, amplifications, or deletions (>4 Mb) throughout other regions of the genome, we observed a number of single-clone deletions and amplifications within our tumor set. The novel data associated with our analysis therefore represents either novel copy number polymorphisms or, more likely, genomic loci that contain genes associated with the development and progression of OD. Additionally, we observed an accumulation of genomic rearrangements between stages II and III. We will present a detailed analysis of genomic intervals that define minimally deleted and amplified regions between all OD tumors. These regions now form the focus of ongoing candidate gene analysis.


Shawn L.A. Hervey-Jumper, M. Oskar Nowicki, E. Antonio Chiocca, and Sean E. Lawler; The Dardinger Laboratory for Neuro-Oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University Medical Center, Columbus, OH, USA

The Wnt family of secreted glycoproteins is involved in various differentiation events during embryonic development. Inappropriate and aberrant activation of Wnt genes leads to tumorigenesis and is known to be particularly important in certain malignancies such as colon cancer. Although there have been numerous reports on over- and underexpression of Wnt genes in human cancers, there are little data on Wnt expression in gliomas. We conducted an analysis of gene expression levels of Wnt ligands, receptors, and intracellular signaling components by quantitative RT-PCR. We found that, in comparison with normal brain (temporal lobe) RNA, many pro-Wnt signaling molecules are downregulated in patient glioblastoma multiforme samples. In contrast, we observed a trend toward upregulation of negative regulators of Wnt signaling. We found significant upregulation of Vangl1 and Vangl2, two closely related genes known to be involved in the establishment of planar cell polarity and Wnt pathway signaling via their interaction with Dishevelled proteins. Vangl overexpression is associated with several solid and epithelial-origin tumors. Its interaction with the C terminus of KAI1 (a known metastasis suppressor) promotes adhesion and increased invasiveness of colon cancer cells. In addition, Vangl1 was upregulated in migrating U87 cells in vitro, suggesting a possible functional role for these genes in glioma invasion.


Michael J. Jarzynka,1 Bo Hu,1 Weisong Gu,2 Ifat Bar-Joseph,1 Ping Guo,1 Russel O. Pieper,3 Xiao Zeng,4 Ray Blanchard,4 and Shi-Yuan Cheng1; 1University of Pittsburgh Cancer Institute, Department of Pathology, Hillman Cancer Center, Pittsburgh, PA, USA; 2Ohio Supercomputer Center Springfield, Springfield, OH, USA; 3Brain Tumor Center, Department of Neurosurgery & Cancer Center, University of California, San Francisco, CA, USA; 4SuperArray Biosciences Corporation, Frederick, MD, USA

High-grade gliomas characteristically invade adjacent brain parenchyma, leading to a poor clinical prognosis even with combined treatments including surgery, radiation, chemotherapy, and immunotherapy. We previously reported that overexpression of angiopoietin-2 (Ang2) leads to the acquisition of invasiveness in engineered U87MG intracranial gliomas in mice through the activation of MMP-2. However, U87MG and other established glioma cell lines contain genetic alterations in pathways such as p53 and PTEN, rendering the precise determination of the molecular mechanisms of invasion difficult. We have therefore developed a minimally genetically altered human astrocyte cell line that stably expresses Ang2 and forms invasive tumors resembling human grade III anaplastic astrocytomas in the murine brain. To begin to explore the mechanisms of Ang2–induced glioma invasion, we first used the Affymetrix GeneChip HG-U133A to identify genes differentially expressed in both U87MG and the constructed astrocytic model systems. Similar data were obtained using a Human Genome Oligonucleotide Version 2.1.2 microarray from Operon. The data generated from these two platforms were cross-compared, and shared alterations and unique differences in gene expression were ascertained using both cell model systems. Initial analyses revealed 261 genes that are associated with cell migration/motility, growth, and survival pathways. To further characterize differences in gene expression, we designed an Oligo GEArray Human Custom Microarray (SuperArray Biosciences Corporation, Frederick, MD) containing these genes of interest. Additional validation was performed using quantitative real-time PCR and Western blotting. These thorough analyses have led to the discovery of novel functions of genes and pathways involved in human glioma cell motility and invasion. Such an understanding of the genes and pathways involved in glioma invasion may lead to the development of novel therapies for the treatment of these deadly brain tumors.


Rongcai Jiang, Cristian Mircean, Ilya Shmulevich, David Cogdell, Yu Jia, Ioan Tabus, Kenneth Aldape, Raymond Sawaya, Janet M. Bruner, Gregory N. Fuller, and Wei Zhang; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; Institute of Signal Processing, Tampere University of Technology, Tampere, Finland; Institute for Systems Biology, Seattle, WA, USA

The progression of gliomas has been extensively studied at the genomic level using cDNA microarrays. However, systematic examinations at the protein translational and posttranslational levels are far more limited. We constructed a glioma protein lysate array from 82 different primary glioma tissues and surveyed the expression and phosphorylation of 46 different proteins involved in signaling pathways for cell proliferation, cell survival, apoptosis, angiogenesis, and cell invasion. An analysis algorithm was used to robustly estimate protein expression in these samples. When ranked by their discriminating power to separate 37 high-grade gliomas from 45 lower-grade gliomas, the following 12 proteins were identified as the most powerful discriminators: IκBα, EGFRpTyr845, AKTpThr308, PI3K, BadpSer136, IGFBP2, IGFBP5, MMP9, VEGF, pRB, Bcl-2, and c-Abl. Clustering analysis showed a close link between PI3K and AKTpThr308, IGFBP5, and IGFBP2, and between IκBα and EGFRpTyr845. Another cluster includes MMP9, Bcl-2, VEGF, and pRB. These clustering patterns may suggest functional relationships that warrant further investigation. The marked association of phosphorylation of AKT at Thr308, but not Ser473, with glioblastoma suggests a specific event of PI3K pathway activation in glioma progression.


F.W. Khwaja,1 M. Reed,2 J.S. Duke-Cohan,5 D.J. Brat,3 B.J. Schmotzer,4 J.J. Olson,1 G.Y. Gillespie,6 A. Guha,7 M.D. Groves,8 J. Pohl,2 and E.G. VanMeir1; 1Laboratory of Molecular Neuro-Oncology, Departments of Neurosurgery, Hematology/Oncology and Winship Cancer Institute, 2Emory University Microchemical and Proteomics Facility, 3Department of Pathology and Laboratory Medicine, and 4General Clinical Research Center, School of Public Health, Emory University School of Medicine, Atlanta, GA, USA; 5Dana-Farber Cancer Institute, Department of Medical Oncology, Harvard School of Medicine, Boston, MA, USA; 6University of Alabama at Birmingham, Birmingham, AL, USA; 7Arthur and Sonia Labatts Brain Tumor Center, Hospital for Sick Children, University of Toronto, Department of Pathology, Division of Neurosurgery, Toronto, Ontario, Canada; and 8The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Measurements of alterations in protein composition of the CSF are sensitive indicators of CNS pathology, yet their systematic application to analysis of CNS neoplasia has been limited. In this report, we used two proteomic techniques, two-dimensional gel electrophoresis and cleavable isotope-coded affinity tag, to compare the CSF proteomes and identify tumor- and grade-specific biomarkers in patients bearing brain tumors of differing histologies and grades. Analyses were performed on 60 samples derived from WHO grades II, III, and IV astrocytomas, schwannomas, metastatic brain tumors, inflammatory samples, and non-neoplastic controls. We identified 103 tumor-specific markers, of which 20 were high grade and astrocytoma specific. Among the high-grade markers, attractin was found to be the principal mediator of a glioma cell-motility activity discovered in the CSF, a property critical to the invasive nature of diffusely infiltrating astrocytic neoplasms. We confirmed that attractin was highly upregulated in 97% of high-grade astrocytomas in an independent series of 100 tumor samples by both Western and immunohistochemical analyses and showed that it is produced and secreted by the tumor cells. These investigations allowed us to identify a spectrum of signature proteins defining specific astrocytoma grades, which may represent new diagnostic and prognostic markers alone or in combination. These candidate biomarkers, like attractin, may also have functional properties that play a critical role in the development and malignant progression of human astrocytomas, thus representing novel therapeutic targets for this highly lethal disease.


A. Law1 and R.E. Brown2; 1Division of Oncology and 2Division of Pathology, Geisinger Medical Center, Danville, PA, USA

GBM is uniformly fatal. The identification of signaling-protein circuitry in individual cases of GBM can have therapeutic implications. We systemically studied immunohistochemical expression of the major signal transduction pathways in GBM. A mini-microarray was constructed from paraffin-embedded blocks of five cases of GBM using a triple-core technique. Immunohistochemical procedures were carried out for the detection of protein analytes: epidermal growth factor receptor; platelet-derived growth factor receptor (PDGFR)-α; PDGFR-β; protein kinase C-α (cPKC- α); α-subunit common to farnesyl and geranylgeranyl transferase (FT/GGTα); N-ras; Ser2448–phosphorylated mammalian target of rapamycin (p-mTOR); Thr389p-p70S6K; Thr202/Tyr204p-extracellular signal-regulated kinase-1/2 (p-ERK1/2); Thr180/Tyr182p-p38 mitogen-activated protein kinase (p-p38MAPK); Ser536p-nuclear factor-kappaB p65 (p-NF-κBp65); estrogen receptor (ER)-α; ER-β; peroxisome proliferator-activated receptor-gamma; bcl-2; and S phase-associated protein kinase-2. The intensity of the chromogenic signal was scored from 0 to 3+ using bright-field microscopy and noting subcellular compartmentalization. Moderate to strong (2+ or 3+) expression in all five cases, accompanied by constitutive activation as evidenced by phosphorylation or compartmental translocation, was noted for the following: p-mTOR; nuclear p-p70S6K, p-ERK1/2, p-p38MAPK, and p-NF-κBp65; and plasmalemmal cPKC-α. Moderate nuclear expression of ER-β and correlative cytoplasmic and plasmalemmal expression of FT/GGTα and N-ras, respectively, were common to all cases. Morphoproteomic analysis utilizing phosphospecific probes, cellular compartmentalization, and correlative expression of protein analytes in our case series confirmed the constitutive activation of cPKC-α, mTOR, ras/Raf kinase, NF-κB tumorigenic pathways, and the potentially anti-tumorigenic pathway involving ER-β and p-p38MAPK. This represents a promising tool for identification of therapeutic targets in individual cases.


Sang Y. Lee, Ramaz Geguchadze, Becky Slagle-Webb, Jonas M. Sheehan, and James R. Connor; Penn State College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA

Mutants of HFE, a gene that regulates cellular iron status, have been associated with hepatocarcinoma and neurodegenerative diseases. In this study, we characterize the expression and effect of the most common HFE mutations, H63D and C282Y, on brain tumor cells and solid tumors to begin to understand the role of the HFE in brain tumor carcinogenesis. We determined the expression level of HFE in brain tumor tissue and found that meningiomas and the majority of high-grade astrocytoma tumors expressed HFE protein. We generated stable human neuroblastoma SH-SY5Y cells carrying the wild-type (WT) and mutant HFE genes to determine the effect of the HFE mutation on cells that do not normally express the HFE protein. The expression of the C282Y, but not the H63D, mutation was associated with a rapid proliferation of cells. The C282Y cells failed to differentiate when exposed to 13–cis retinoic acid or dibutyryl cAMP, and although these cells were responsive to the chemotherapeutic agent BCNU, Temodar was not cytotoxic to these cells. However, when H63D and WT HFE cells were treated with identical concentrations of these agents, normal differentiation or toxicity was observed. In addition, the C282Y cells were resistant to gamma radiation, while the WT and H63D cells were sensitive. We also obtained and genotyped a number of astrocytoma cell lines from the American Type Culture Collection. Resistance to radiation was found in a C282Y-heterozygous astrocytoma cell line (CCF-STTG1) but not in WT or H63D-heterozygous (T98G) astrocytoma cell lines. To identify the molecular footprint of the mutant form of HFE on the cells, we performed gene expression profiling in stably transfected SH-SY5Y cells using target-specific gene arrays. C282Y-transfected cells had altered expression of cancer and angiogenesis genes, such as angiopoietin 1, p16ink4, COX-2, and cyclin D1, compared to WT HFE-transfected cells. Furthermore, protein profiling by two-dimensional gel analysis also showed different protein expression patterns between HFE cells. In conclusion, HFE protein is strongly expressed in brain tumor cells, and cellular data indicate that the C282Y mutation significantly alters gene and protein expression and cell proliferation, differentiation, and response to gamma radiation and anti-tumor drugs. If the rates of C282Y mutations in brain tumor populations are similar or higher than those in than the general population, almost 10% of the brain tumor population may be less responsive to existing therapeutic strategies.


K. McDonald,1 J. Parkinson,1,5 H. Wheeler,2 M. O’Sullivan,3 G. Stone,3 J. Brewer,4 R. Cook,5 M. Biggs,5 N. Little,5 C. Teo,6 and B. Robinson1; 1Cancer Genetics, Kolling Institute of Medical Research, 2Northern Cancer Institute, North Shore Private Hospital, 3Mathematical and Information Sciences, CSIRO, Departments of 4Anatomical Pathology and 5Neurosurgery, RNSH, St Leonards, and 6Center of Minimally Invasive Neurosurgery, Randwick, NSW, Australia

Histologically, gliomas are separated into groups based on their presumed cell of origin. The two most common groups are astrocytoma and oligodendroglioma. Glial tumors are then graded pathologically, on the basis of the most malignant area identified, according to the WHO system. This system uses presence of nuclear atypia, mitosis, microvascular proliferation, and necrosis as indicators of increasing tumor aggressiveness. Our understanding of the molecular genetics of gliomas has advanced significantly in recent years, but we are still far from understanding the complex mechanisms that underlie tumor initiation and progression. Microarray analysis offers unbiased, quantitative, and reproducible evaluation of tumor specimens by parallel monitoring of expression levels of thousands of genes. However, its translation into clinical practice has been limited. It has been difficult to find individual gene/protein assays for use both at a diagnostic level and as a target for future treatment development. We conducted a series of microarray experiments to identify genes that could be used as robust diagnostic markers to discriminate between astrocytic and oligodendroglial tumors as well as gene markers that could discriminate between the different grades of tumors. We used an analysis program called GeneRaVE that is able to find small sets of genes with better predictive accuracy than the usually much larger sets found by existing technology. We identified two novel gene sets with highly significant discriminatory power when we separated tumors according to the presence or absence of necrosis, microvascular proliferation, mitoses (more than 6 per 10 HPF), and a Ki67 percentage greater than 20%. These genes were further validated at the mRNA level using real-time PCR and at the protein level using immunohistochemistry. These gene/protein sets may serve as new histological markers for high-grade tumors. However, unlike the histological stains used now, these gene/protein sets may help our understanding of tumor progression and potentially become new targets for pharmacological intervention.


Michael Mitchell, Laura L. Eggink, and Adrienne C. Scheck; Neuro-Oncology and Neurosurgery Research, Barrow Neurological Institute of SJHMC, Phoenix, AZ, USA

Malignant gliomas are typically treated with surgery, radiation, and chemotherapy. However, recurrence is common, and the median survival following diagnosis continues to be less than a year. It is thought that recurrent tumors may arise, in part, from a subpopulation of cells present in the primary tumor that possess genetic and/or epigenetic attributes that confer resistance to radiation and chemotherapy. In addition, the treatments themselves cause DNA damage, which can lead to genetic changes in the cells that survive. Many studies have investigated changes in DNA and RNA expression that lead to therapy resistance, but few studies have been done to study the global protein changes that occur between the primary tumor and recurrence following therapy. An understanding of the changes that occur in the cell populations of primary tumors leading to the formation of recurrent tumors may point to additional targets for the design of novel therapies. We have used two-dimensional polyacrylamide gel electrophoresis to analyze proteins isolated from cells from primary and recurrent tumor pairs with or without in vitro selection for drug resistance. The proteins were then identified by MALDI-TOF analysis of a tryptic digest. To date, we have analyzed total protein samples from three primary/recurrent tumor pairs prior to and following selection for resistance to 1,3–bis(2–chloroethyl)-1–nitrosourea (BCNU). Fifty-two differentially expressed proteins were consistently detected, ranging in size from 28 to 65 kDa and in pI from 4.6 to 6.2. Sixteen proteins have been identified to date. Nine were upregulated in BCNU-resistant cells relative to mock-treated cells. These include hexo-kinase-2, heat-shock protein apg-1, La-binding protein-1, and the hypothetical protein FLJ20364. Seven proteins were downregulated relative to mock-treated cells, and subsets of proteins were found to be differentially expressed in primary versus recurrent tumor cells. Further protein identification is in progress, as are analyses of additional cell and tissue samples from primary and recurrent tumors. Global proteomic analysis of primary and recurrent malignant gliomas will provide critically needed additional data for the identification of new therapeutic targets for the treatment of recurrent tumors. This work was supported by The Brain Tumor Society and the Barrow Neurological Foundation.


T. Nagasaka,1 M. Gunji,2 N. Hosokai,3 K. Hayashi,3 H. Ikeda,1 M. Ito,2 and S. Inao1; Departments of 1Neurosurgery and 2Department of Pathology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan; 3Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo, Japan

Glioblastoma is the most malignant and frequent of the gliomas. A minor fraction of glioblastomas may contain areas showing oligodendroglioma-like tumor cell differentiation. Several authors have described such tumors as “glioblastoma with oligodendroglial component (GBMO).” GBMO may represent the ultimate level of malignancy in the oligodendroglial lineage. Presence of an oligodendroglial component and combined loss of chromosomal arms 1p and 19q in glioblastoma indicate increased survival. In our study, we analyzed 1p and 19q status in a series of 12 glioblastoma and 8 oligodendroglial tumors using fluorescence in situ hybridization (FISH) on paraffin-embedded tissues. In each case, hybridization status was classified as deletion, imbalance, polysomy, amplification, or normal pattern. Other genetic alterations, such as CDKN2A (p16), RB, and EGFR, were also assessed. On histological review, 2 (17%) of 12 glioblastomas were classified as GBMO. Chromosome 1p/19q deletion was detected in 3 (25%) of 12 glioblastomas. In contrast, all eight oligodendroglial tumors showed 1p/19q deletion. All GBMOs had 19q deletion with imbalance, while 1 (10%) of 10 ordinary glioblastomas had 19q deletion with imbalance. All but 1 (90%) ordinary glioblastoma showed CDKN2A (p16) deletion, and no GBMOs displayed this alteration. Our results indicate that GBMO may be a distinct subtype of glioblastoma harboring a characteristic molecular profile. FISH on paraffin-embedded specimens is a useful method for sub-classification of glioblastoma.


A. Pandita,1 L. Bereskin,1 J. Wiley,1 M.Z. Karim,1 P. Shannon,2 and A. Guha1,2; 1Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, Canada; 2The Toronto Western Hospital, Toronto, Canada

Patients with neurofibromatosis type 1 (NF1) harbor several subtypes of neurofibromas, with dermal nfibs (DNFs) remaining benign and plexiform nfibs (PNFs) having a 10–15% risk of transformation to a malignant peripheral nerve sheath tumor (PNST). The genetic alterations leading to the initiation and malignant transformation of these PNSTs are largely unknown. This is the first study to successfully perform arrayCGH analysis on LCM-isolated Schwann-cell DNA from various PNSTs. Gains and losses were observed in the DNA sequences of three of five DNFs, seven of eight PNFs, and all eight malignant PNSTs. The DNFs revealed very few non-random alterations (10.5 per tumor), with losses being mainly prominent on chromosomal regions 7p14, 7q11.2, and 9q34 and gains on 8q11, 8q21, and 18q21–22. In contrast, the PNFs and malignant PNSTs uncovered a higher number of alterations (17.5 and 18.5 per tumor, respectively), with losses being more common in the benign tumors and gains in their malignant counterparts. Loss of regions on chromosomes 1q, 2q, 6q13–15, 11q12–13, 13q13–21, 15q23–25, and 17q11.2 were common to both benign and malignant tumors, likely harboring genes involved early in tumor initiation. Gains on chromosomal arms 4q22–34, 5p14–15.2, 6q22–24, 8q, 13q22–33, 17q22–25, and 20q11.2–13.2 were found only in malignant PNSTs and are expected to be involved in the malignant progression of PNFs. High-copy-number gains were observed in malignant PNSTs on chromosomes 5p14–15.3, 17q22–25 (62.5%), 20q12–13 (50%), and 12q12–13 (37%). Some of these results, including loss of NF1, were confirmed by FISH analysis. The distinct pattern of genetic alterations uncovered in each PNST subtype by the current study reflects alterations that are responsible for the varying clinical and biological behavior of these PNSTs. These chromosomal regions provide the basis for molecular identification of novel oncogenes and tumor suppressor genes of pathogenetic relevance in PNSTs.


A. Pandita,1 A. Balasubramanium,1 R. Perrin,2 and A. Guha1,2; 1Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, Canada; 2The Toronto Western Hospital, Toronto, Canada

Gangliogliomas are generally benign, indolent primary brain tumors that contain both transformed neuronal and glial elements, with rare malignant progression of the glial elements. In the current study, a patient specimen with both benign and malignant ganglioglioma was used to address two interesting issues: first, deduction of the genetic alterations associated with initiation and progression of gangliogliomas, and second, whether the malignant component arises from clonal progression of the benign component. Conventional and arrayCGH were used to examine genetic alterations, while the HUMARA assay was used to examine clonality. The high-resolution genetic alteration map revealed losses to be predominant in the benign portion, while gains were more prevalent in the malignant regions of the ganglioglioma. Losses in the benign region, suggestive of genetic alterations leading to initiation of the ganglioglioma, involved chromosomal regions 1p35–36, 2p16–15, 3q13.1–13.3, 3q24–25.3, 6, 9p, Xp11.3–11.22, and Xq22.1–22.3. Gains in the malignant region were clustered around chromosomal regions 1p35–34.2, 2q24.1–32.3, 3q13.1–13.3, 6q13–16.2, 7q11.2–31.3, 8q21.1–23, 11q12–31, and 12q13.2–21.3, indicative of genes involved in progression. In addition, immunohistochemical and DNA mutational analysis revealed alterations involving p53, p16, and p19 to be involved in malignant transformation. HUMARA assay revealed the benign and malignant components to be clonal, suggestive of the tumor likely developing from a single precursor cell. This work is the first to undertake a detailed molecular study of a ganglioglioma that has progressed towards malignancy. Genes residing in these chromosomal regions are of current interest in our understanding of these rare brain tumors and the more common gliomas.


Larry Panyon,1 Emmanuelle J. Meuillet,4 Raymond Schilling,3 Christopher Biggs,3 Gabriel Martinez,1 and Adrienne C. Scheck1,2; 1Neuro-Oncology Research, 2Neurosurgery Research, 3Radiation Oncology, Barrow Neurological Institute of SJHMC, Phoenix, AZ, USA; and 4University of Arizona, Tucson, AZ, USA

Malignant gliomas are typically treated with surgery, radiation, and chemotherapy. Despite this, these tumors recur and are resistant to additional treatment. Studies in our laboratory using cells cultured from primary and recurrent tumors from the same patient revealed the presence of three specific translocations between chromosomes 11 and 22 [t(11;22)] in cells from the recurrent tumors but not in cells from the primary tumor. Furthermore, t(11;22) has been found in paraffin-embedded tissue (in vivo) from recurrent tumors but not from the primary tumor from the same patient. In vitro, the percentage of cells with a translocation increased when cells from the recurrent tumor were selected for resistance to 1,3–bis(2–chloroethyl)-1–nitrosourea (BCNU), whereas we could not cause this translocation in cells from primary tumors using in vitro selection for BCNU resistance. Cells from recurrent tumors are often more radioresistant than cells from primary tumors. To determine whether t(11;22) could be caused by radiation, we treated cells from a primary tumor with repeated radiation at doses of 4, 8, and 16 Gy. t(11;22) was detectable in cells from this tumor after three doses of 4 Gy. Bacterial artificial chromosomes (BACs) and long-range PCR demonstrated that the chromosome 11 breakpoint is within BAC CTC 824H1, mapped to 11q22.3. There was one predicted (GeneScan) mRNA mapped to this region. The chromosome 22 breakpoint is within the distal 50,000 bp of BAC CTD 2570M18 (22q11.23). Genes mapped to this vicinity include D-dopachrome tautomerase, a gene related to migration-inhibitory factor-1; glutathione S-transferase theta-1 (GSTT1) and GSTT2, genes thought to play a role in carcinogenesis; and calcineurin-binding protein-1, a gene thought to be involved in calcineurin-mediated signal transduction. Our results suggest a role for t(11;22) in therapy resistance and/or tumor recurrence in glioma. Further fine mapping of the breakpoints and gene expression studies are in progress. This work was supported by The Brain Tumor Society and the Barrow Neurological Foundation.


Vinay R. Raj,1 Sarah Griffin,1 Yue Teng,1 Kenneth R. Hess,2 W.K. Alfred Yung,2 Mark E. Linskey,3 and Yi-Hong Zhou1; 1University of Arkansas for Medical Sciences Arkansas Cancer Center, Little Rock, AR, USA; 2The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3The University of California, Irvine, CA, USA

Glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA) are malignant astrocytic gliomas with variable survival periods. The objective of this study was to assess the prognostic significance of genes involved in regulation of apoptosis and cell cycle arrest and their functions in prognosticating outcome of patients with malignant astrocytic gliomas. We have established a comprehensive prognostic model using gene expression and clinical variables (Zhou et al., Neuro-Oncology, 2005). The new gene variables in this study include mutations and expression of the gene encoding tumor suppressor p53 (TP53) and expression of the genes encoding p53–binding protein-2 (ASPP2), BRCA2, CDKN1A-interacting protein (BCCIP), and baculoviral IAP repeat-containing-5 (BIRC5). The gene expression levels were measured by real-time quantitative RT-PCR. The p53 mutations were identified by direct sequencing following PCR amplification. Wilcoxon rank-sum test and Spearman rank correlation analysis were applied to study the correlation of gene expression and association with p53 mutation and tumor grade. p53 mutations were identified in 68% of AA (N = 34) but only in 28% of GBM (N = 40). The distribution of the mutations in AA was different from that in GBM, which is in accordance with the fact that the majority of GBMs are derived de novo. ASPP2 expression was significantly lower in GBM than in AA (P < 0.0001). This lower expression of ASPP2 was significantly associated with wild-type p53. BCCIP expression was significantly lower in GBM than in AA (P < 0.0001). ASPP2 and BCCIP were significantly positively correlated (R = 0.63; P < 0.0001). Both ASPP2 and BCCIP expression exhibited a significant negative correlation with genes encoding vascular endothelial growth factor (VEGF) and insulin-like growth factor binding protein-2 (IGFBP2) from our previous study. The expression of TP53 and BIRC5 did not correlate with either grade of the tumor. Since ASPP2 regulates p53 function in apoptosis, our study suggests that although most GBMs have wild-type p53, p53 function may be impaired by low expression of ASPP2. BCCIP, an important cofactor for BRCA2 in tumor suppression, delays the G1-to-S progression. The low expression of BCCIP is consistent with the malignant nature of GBM. Our study suggests that ASPP2 and BCCIP expression may be significant prognostic factors for gliomas.


N.O. Schmidt,1 F.J. Müller,2 M. Messing,1 U. Kehler,1,3 J.F. Loring,4 F. Zeigler,5 K. Lamszus,1 and M. Westphal1; 1Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum HH-Eppendorf, Hamburg, Germany; 2Zentrum für Integrative Psychiatrie, Universität Schleswig-Holstein Campus Kiel, Germany; 3Neurochirurgische Klinik, Allgemeines Krankenhaus Altona, Hamburg, Germany; 4The Burnham Institute, La Jolla, CA, USA; 5Orion Biosolution Inc., Vista, CA, USA

Previous reports have demonstrated the extensive migratory and tumor-targeting capabilities of neural stem cells derived from fetal tissue. However, ethical and logistical difficulties are major obstacles to obtaining human tissue suitable for the isolation of neural progenitor cultures. The aim of this study was to establish well-characterized cultures of neural progenitor cells from human adult brain tissue and to explore their potential as drug delivery vehicles for targeting invasive glioma cells. We modified previously reported protocols for establishing neural progenitor cultures from fetal and postmortem brain tissue. By using Neurobasal/B27 medium and various combinations of growth factors, we obtained highly proliferative neuroec-todermal cells using surgical specimens from amygdalohippocampectomies (N = 8) and brain tissue adherent to resected arteriovenous malformations (N = 2). We isolated and propagated highly proliferative neural progenitor cultures from human cortex, hippocampus, and amygdala and characterized them by immunohistochemistry, RT-PCR, and FACS for the expression of stem cell and lineage markers. Our cultures grew as homogenous monolayers of progenitor cells expressing markers such as Nestin, A2B5, SOX2, BMI1, and, infrequently, CD133 and Musashi, while expressing markers of neuronal and oligodendroglial lineage. Using Illumina Sentrix whole-genome microarrays, we found the global gene expression pattern to be different from well-characterized long-term fetal neural stem cell cultures. Established neural progenitor cell cultures displayed a directed, transcal-losal migration toward intracerebral glioma when transplanted in the contralateral hemisphere of nude mice. Our data indicate that the human adult brain still contains highly motile populations of progenitor cells. Human adult brain tissue derived from surgical procedures contains neural progenitor cells that can be isolated, expanded, and used for further exploration of their therapeutic potential in a variety of neurological disorders.


Mukesh K. Sharma, David B. Mansur, Guido Reifenberger, Arie Perry, Kenneth D. Aldape, Simon Loeser, Jeffrey Leonard, Mark A. Watson, Rakesh Nagarajan, and David H. Gutmann; Departments of Neurology, Radiation Oncology and Pathology and Immunology, Washington University School of Medicine, St. Louis MO, USA; Department of Neuropathology, Heinrich-Heine-University, Düsseldorf, Germany; Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Pilocytic astrocytomas (PAs) are the most common glioma in children. These tumors are glial fibrillary acidic protein (GFAP)-positive tumors, which are classified by the WHO as grade I astrocytomas. While many PAs are slow growing or clinically indolent, others exhibit more aggressive features with tumor recurrence and death. Compared with other pediatric brain tumors, little is known about the molecular genetic changes associated with PA formation and progression. To identify genetic signatures that might predict PA clinical behavior and divide PA into clinically or biologically relevant subgroups, we performed microarray-based gene expression profiling on 41 primary PAs arising sporadically. No expression signature was found that could discriminate (1) clinically aggressive or recurrent tumors from more indolent cases, (2) tumors in male patients from those in female patients, and (3) tumors arising in patients under or over age 18. Significance analysis of microarrays identified a gene expression signature that stratified PAs by location (supratentorial versus posterior fossa). We validated select genes by real-time RT-PCR (qRT-PCR) and by immunohistochemistry on an independent series of supratentorial and posterior fossa PAs. We also identified this genetic signature in the neocortex and cerebellum of mice at postnatal days 1 and 30. In addition, qRT-PCR identified this molecular signature in GFAP-positive astrocytes from these distinct brain regions. Moreover, microarray-based gene expression profiling and immunohistochemical analyses confirmed the gene expression pattern in another glial tumor (ependymoma) arising supratentorially compared to those originating in the posterior fossa. Our findings provide evidence for molecular heterogeneity among glial tumors based on brain region and suggest that that these brain region-specific genes might be involved in the pathogenesis and progression of glial cell tumors.


Kristen L. Templeton,1 Gaspar J. Kitange,1 Thomas M. Kollmeyer,1 Mark E. Law,1 Hilary E. Blair,1 Bruce W. Morlan,2 Karla V. Ballman,2 and Robert B. Jenkins1; 1Division of Laboratory Genetics and 2Biostatistics, Mayo Clinic and Foundation, Rochester, MN, USA

Oligodendrogliomas commonly lose both the chromosome 19q and 1p arms. Tumors with this deletion have a better prognosis and response to treatment than those without the deletion. To identify the target 19q gene(s), we previously mapped 19q deletions in a series of glioma cell lines. Glioma cell lines with deletions of chromosome 19q were complemented with normal human chromosome 19 by microcell-mediated transfer and maintained under selection with G418. The hybrid cell lines had different growth characteristics than the parental lines, with slower proliferation rates and lower migratory potential than the parental cell lines. The gene expression profile of the cell lines was examined by Affymetrix U133 Plus 2.0 Gene Chip analysis. Significant differences in expression were noted in the genes from the commonly deleted regions (19q13.3) in the glioma cell lines. All probes (N = 10) found to be significantly overexpressed for seven potential candidate genes (APOE, FLJ10781, RCN3, SAE1, BCL2L12, CCDC8, and STRN4) when compared to the parental cell lines in all hybrid cell lines examined (N = 7; P < 0.01). Changes in expression were confirmed by qRT-PCR. Candidates are being evaluated by RT-PCR in a panel of tumors to examine the expression difference in tumors with the deletion versus those without the deletion. The genes are also being further evaluated by siRNA analysis of the chromosome 19 hybrids to assess their effects on cell line phenotype. Our results suggest that one or more genes in 19q13.3 are the target of 19q deletion in oligodendrogliomas.


Wei Zhang and Ilya Shmulevich; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; The Institute for Systems Biology, Seattle, WA, USA

High-throughput genomic and proteomic studies of clinical samples have generated large amounts of data but very little information and much less wisdom. We understand that transcripts and proteins are linked, but it is a major challenge to develop appropriate mathematical models that reveal the logical and physical relationships among the components of biological systems. We submit that a key modeling criterion is that the model must be data driven; that is, it must be able to take in biological data and produce experimentally testable diagrams or networks. Only when this correlation is repeatedly demonstrated can we reach the conclusion that a biologically appropriate mathematical model has been created. We have developed a mathematical model, termed the Probabilistic Boolean Network (PBN), considering the uncertainties and probabilistic nature of biological systems. We applied this PBN model to a set of microarray data generated from 25 glioma tissues from different stages of cancer development. We then generated two subnetworks focusing on two genes important for glioma development and progression: vascular endothelial growth factor (VEGF) and insulin-like growth factor binding protein-2 (IGFBP2). VEGF is required for angiogenesis, which is critical for providing nutrients for tumor growth. The VEGF subnetwork revealed a number of relationships that are supported by the literature. IGFBP2 is overexpressed in 80% of cases of the most advanced glioma, glioblastoma multiforme (GBM), and contributes to glioma cell migration and invasion. Mathematical modeling with glioma gene expression profiling data suggested that IGFBP2 is linked to the integrin pathway. This notion was subsequently validated by the demonstration that IGFBP2 interacts with integrin through an RGD domain. We hypothesized that IGFBP2 is a key regulator of glioma progression. We tested our hypothesis using a glial-specific somatic gene transfer mouse model called the RCAS-tva model. Our results showed that IGFBP2 actively contributes to tumor initiation and progression in two lineages of gliomas. Through these functional genomic and mathematical modeling studies, we believe we have gained important insight into the systems biology of gliomas.



D. Bassiri,1 N. Luther,1 I.J. Dunkel,2 M.A. Edgar,2 N.K. Cheung,2 Q.C. Wang,3 I. Pastan,3 P.H. Gutin,2 and M.M. Souweidane1,2; 1The Weill Medical College of Cornell University, New York, NY, USA; 2Memorial Sloan-Kettering Cancer Center, New York, NY, USA; and 3National Cancer Institute, Bethesda, MD, USA

Interstitial infusion of tumor-directed immunotoxins has recently been introduced into clinical trials. 8H9 is a murine monoclonal antibody (mAb) that is highly selective against human neuroectodermal-derived tumors. When genetically engineered as a single chain Fv fragment (scFv) and fused to a mutant form of Pseudomonas exotoxin (PE38), it becomes a tumor-selective immunotoxin (8H9scFv-PE38). This agent has yet to be tested with respect to toxicity or efficacy following interstitial delivery. Naïve Sprague-Dawley rats underwent interstitial infusion of escalating doses (N = 5/dose level; 0.5 μg, 0.75 μg, 1.0 μg, 2.0 μg, and 4.0 μg) of 8H9scFv-PE38 (conc. 1.4 μg/μL). To investigate potential toxicity, the animals were observed and serially imaged using MRI. Histopathologic sections were assessed for microscopic evidence of injury or reactive changes. The maximum tolerated dose (MTD) was then used to treat athymic animals bearing an immuno-reactive human glioma (U87). Radiographic response, clinical assessment, survival, and histologic changes were assessed. All animals tolerated the stereotactic infusion of 8H9scFv-PE38 without surgical morbidity. During the period of observation, infrequent behavioral changes (1/5) were witnessed at the 1.0 μg dose level, whereas both physiologic and behavioral changes were consistently seen at the 2.0 μg dose level. Nonspecific histologic changes at the site of 8H9scFv-PE38 were observed but without clinical correlates in animals infused with 0.75 μg of 8H9scFv-PE38, thus establishing this as the MTD. When animals bearing the U87 xenografts were treated with 0.75 μg of 8H9scFv-PE38 by interstitial delivery, MRI-defined tumor responses were observed and survival was prolonged. Local toxicity following interstitial delivery of the immunotoxin 8H9scFv-PE38 in the rat striatum was dose dependent. Treatment of U87 xenografts at the MTD (0.75 μg) of 8H9scFv-PE38 resulted in both radiographic response and prolonged survival with minimal toxicity.


N.L. Bryant,1 C. Suarez-Cuervo,2 B. Gehrs,2 G.Y. Gillespie,3 L.B. Nabors,4 and L.S. Lamb2; Divisions of 1Pediatric Hematology and Oncology, 2Hematology and Oncology, 3Neurosurgery, and 4Neuro-Oncology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA

Glioblastoma multiforme (GBM) is an almost universally fatal disease despite numerous advances made in chemotherapy, surgery, and radiotherapy. Classical systemic immunotherapy approaches have shown little effect, making GBM ideal for local therapy. We have previously shown that γδ T cells are cytotoxic to GBM cells and do not damage normal astrocytes. Unlike standard cytotoxic lymphocyte-based therapies, γδT cells are part of the innate immune system. They act directly and immediately via stress-associated antigens and do not require MHC antigen recognition. To further define the role of γδ T cells in patients with GBM, we examined the γδ T cell number and function at specified time points during GBM therapy: at diagnosis, 1–14 days postresection, and 7–14 weeks postresection. Peripheral blood was obtained from patients with GBM and from healthy volunteers. Lymphocyte phenotype, γδT cell population, and vδ1 and vδ2 subclasses were examined using flow cytometry. The mitogenic potential of γδ T cells was determined using standard methods. Primary GBM tumor samples from these patients were also embedded in paraffin, sectioned, and then labeled with antibodies to CD3 and TCR-γδ to determine the extent to which these cells invaded the tumor parenchyma. Cytotoxicity of expanded/activated γδ T cells from allogeneic healthy controls was evaluated against established GBM cell lines and primary GBM tumors. Patients showed a preoperative decrease in the CD4+ T cell population compared with healthy controls but did not differ substantially in γδ T cell profile and mitogenic response. In the immediate postoperative period, CD4+ T cell counts recovered, but CD8+ and Vδ2+ counts fell substantially. A global decrease in all T cell subsets occurs 7–14 weeks after surgery with the exception of a slight recovery of Vδ2+ cells in patients receiving local radiation therapy. Immunohistochemical staining showed some perivascular cuffing of lymphocytes but no infiltration into the brain parenchyma and no presence of γδ T cells. Expanded γδ T cells kill GBM cell lines D54, U373, and U251, as well as primary GBMs, without damaging healthy astrocytes. There is no evidence for an effective γδ T cell response prior to resection of the tumor even though the γδ T cell phenotype and function are no different from healthy controls. The substantial decline in the Vδ2 T cell population in the immediate postoperative period suggests that a loss of this component of innate immune function postoperatively concurrent with the removal of the tumor that will require further investigation to define. Allogeneic γδ T cells effectively kill GBM cell lines and primary tumors and can be effectively expanded in healthy controls and preoperatively in patients with GBM, thus opening the possibility for either autologous or allogeneic local (intracavitary) therapy in the immediate postoperative period following steroid taper.


Michael J. Ciesielski,1,2 Carla Castro,1 Tara Barone,1 and Robert A. Fenstermaker1, 2; Department of Neurosurgery, 1Roswell Park Cancer Institute, and 2State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY, USA

Survivin represents a tumor-specific target for cancer immunotherapy. It is possible to produce an MHC-I-restricted cellular immunologic attack in response to a survivin vaccine. We have previously reported that bone marrow dendritic cells (DC) that express survivin induce effective anti-tumor responses to GL261 glioma. Using a defined dendritic cell line as a vaccine vehicle, we can induce an immune response with peptide epitopes of the survivin protein sequence. In this study, we have set out to define the most immunogenic epitopes in our model system. Several potential peptide epitopes of survivin were selected, using SYFPEITHI and BIMASS algorithms, as being likely to be presented by MHC-I (H-2Kb) molecules. Two peptides (svn57/CFFCFKEL and svn82/SGCAFLSV) scored sufficiently high so that they were chosen for further study. DCs loaded with survivin peptides were as effective as the entire survivin protein at stimulating anti-tumor immune responses. Following intracerebral implantation of GL261 glioma cells, serial peptide-loaded DC vaccination resulted in the following median survival times: 20 days (range, 18–23 days) for control peptide; 29 days (range, 22–35 days) with svn57 peptide (P < 0.0049 vs. control); 27 days (range, 20–32 days) with svn82 peptide (P < 0.0092 vs. control); and 40 days (range, 23–94+ days) with a combination of svn57 and svn82 peptides (P < 0.0016 vs. control). Specific CD8+ T-cells for peptides svn57 and svn82 were detected in mice that were initially vaccinated with the full-length survivin protein, which indicates the immunodominance of these 2 peptides in vivo (svn82-specific CD8+ cells = 1.78% of total splenocytes and svn57-specific CD8+ cells = 0.52% of total splenocytes). Studies utilizing peptide-specific CTL showed lytic activity against GL261 cells in cytotoxicity assays. We are actively developing these peptide vaccines and characterizing their efficacy in mouse brain tumor models. The results of this study could aid in the development of a clinical trial of survivin peptide-loaded DC vaccines in glioma patients.


Guillermo R. DeAngulo,1 Hernan Vasquez,1 Nadezhda V. Koshkina,1 Wei Sun,2 S. Farzana Hussain,2 Eugenie S. Kleinerman,1 Johannes Wolff,1 Raymond Sawaya,2 and Amy B. Heimberger2; 1Children’s Cancer Hospital and the 2Brain Tumor Center, Department of Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Glioblastoma multiforme (GBM) has marked cellular heterogeneity; thus, combination therapy will likely become the standard. Recent strides have been made in prolonging survival in GBM patients with both chemotherapy and immunotherapy. Conventional opinion has been that administration of chemotherapy would mitigate the efficacy of immunotherapy; however, this opinion may be erroneous. The expression of Fas/CD95 on tumors can render them susceptible to CD8+ cytotoxic T cell killing. The malignant glioma cell line U-87 was treated with titrated physiological doses of topotecan, temozolomide, gemcitabine, and cisplatin over time to determine expression of Fas with flow analysis cytometry. Topotecan demonstrated a 65% increase of FAS expression, as did cisplatin (33%) to a lesser degree, in contrast to temozolomide (< 5%) and gemcitabine (13%). Administration of soluble Fas ligand (SFL) in MTT cell proliferation assays demonstrated a synergistic effect on U-87 cell death (65%) when pretreated with topotecan but not with temozolomide (10%). Furthermore, pretreatment of U-87 with topotecan resulted in enhanced U-87 cell eradication by human cytotoxic CD8+ T cells even at effector-to-target ratios of 1:1 within 24 hours. Studies are currently underway to validate these findings in a syngeneic murine model of established intracerebral tumor by up-regulating Fas on the intracerebral tumor followed by immunotherapy to determine if this approach may be applicable to human patients. The combination of Fas upregulation, potentially with chemotherapy, and clonal expansion of cytotoxic CD8+ T cells secondary to immunotherapy may increase immune clearance, thus maximizing the chemotherapeutic effect and representing a potential synergistic approach.


Jill E. Dusak,1,2 Prasanna Kumar,3 J. Mark Braughler,3 and Hideho Okada1,2; 1Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 2Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; and 3RheoGene Inc., Norristown, PA, USA

We have previously demonstrated that dendritic cell (DC)-based delivery of interferon (IFN)-α into central nervous system (CNS) tumors facilitates the tumor-homing and therapeutic efficacy of Type-1 cytotoxic T lymphocytes (CTLs) in an IFN-inducible protein (IP)-10–dependent manner. This approach also facilitates migration of antigen-presenting cells (APCs) from CNS tumor sites to the draining cervical lymph nodes, where these cells cross-prime tumor antigen (TA)-specific CTLs. In addition, interleukin (IL)-12 is a cytokine that has a key role in activating natural killer (NK) cells, promoting CTL maturation, and biasing CD4+ T cells towards Type-1 differentiation. We hypothesized that DC-production of both these cytokines within the tumor microenvironment would promote antitumor immunity and CTL induction. The rationale for clinical translation of this approach would be further strengthened if expression of inflammatory cytokines can be tightly regulated, thereby minimizing the risk of autoimmunity in the CNS. The novel RheoSwitch Therapeutic System (RTS) allows maximum control of gene expression in mammalian cells by the small- molecule activator drug, RG-115830 (inducer ligand) and its responsive gene promoter. We have created adenoviral vectors (Ad) encoding murine IFN-α and IL-12 and green fluorescence protein (GFP) downstream of the inducible promoter (Ad-RTS vectors). In vitro, we have confirmed that transgene expression by bone marrow (BM)-derived DCs transduced with the Ad-RTS vectors is highly specific and sensitive to RG-115830. In vivo, C57/BL6 mice bearing syngeneic i.c. GL261 gliomas received i.t. injection of PKH26– red-labeled DCs that had been transduced ex vivo with Ad-RTS-GFP. Subsequent intraperitoneal injection of RG-115830 resulted in a dose-dependent induction of GFP signal in PKH-26–red-labeled DCs based upon histologic evaluations of GL261 glioma tissues derived from treated mice. Glioma tissues from mice without the RG-115830 treatment for at least 3 days demonstrated the presence of injected DCs (PKH-26–red-positive) with no GFP expression, indicating that the RTS system allows effective and tight ligand-dependent induction of transgene in the CNS tumor environment. Additionally, C57/BL6 mice bearing syngeneic i.c. GL261 gliomas received i.t. injections of DCs transduced with Ad-RTS vectors encoding IFN-α and IL-12 followed by i.p. injections of RG-118530 on the subsequent day and every other day for a total of 4 injections. Glioma-bearing mice treated with Ad-RTS-transduced DCs and RG-118530 demonstrated significantly prolonged survival compared with mice treated with transduced DCs but without the ligand and with mice that had no treatment. These data suggest that Ad-RTS vector-based cytokine-gene delivery may represent a safe and efficient strategy for immunogene therapy for gliomas.


Giulia Fulci,1,2 Laura Breymann,1 Davide Gianni,1 Sarah S. Rhee,3 Daniel J. Brat,4 Anat Stemmer-Rachamimov,5 Jianhua Yu,6 David N. Louis,5 Ralph Weissleder,3 Michael A. Caligiuri,6 and E. Antonio Chiocca1,2,6; 1Molecular Neuro-Oncology Laboratories, Neurosurgery Service, 3Center for Molecular Imaging Research, 5Pathology Service, Massachusetts General Hospital-East Building, Charlestown, MA, USA; 2Dardinger Center for Neuro-Oncology and Neurosciences, Department of Neurological Surgery, James Cancer Hospital and Solove Research Institute, The Ohio State University Medical Center, Columbus, OH, USA; 4Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA; 6The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA

Advances in virology and tumor biology have enabled development of oncolytic viruses (OV), which replicate selectively in tumor cells. OV progeny propagate their oncolysis throughout the tumor and spare surrounding normal cells. Phase I clinical trials have shown that OV therapy is safe, but it has limited efficacy. A rapid host response to OV therapy has been observed, which includes intratumoral immune cells and acute-phase reaction to intravascular virus. Currently, the role of host immune responses in the efficacy or toxicity of OV therapy is thought to be beneficial because oncolysis stimulates adaptive immunity, setting up an anticancer vaccination effect. However, initial innate responses to OV may reduce its anti-cancer effects. For example, we have shown a herpes simplex virus type 1 (HSV1)-based OV therapy to be more efficient when cyclophosphamide (CPA) is present, and this heightened efficiency is credited to the immunosuppressive action of CPA. Here, we show that in the absence of CPA immunosuppressive action, OV replication is inhibited and viral particles are cleared from the tumor within 72 hours of delivery. We have explored the mechanisms behind this finding and show that, in a syngeneic rat glioma model, intratumoral OV administration is associated with a rapid increase of natural killer (NK) cells, microglia/macrophages (CD68+ and CD163+), and interferon gamma (IFNγ). Pretreatment with CPA enhances OV replication and oncolysis and reduces an OV-mediated increase in CD68+ and CD163+ cells and intratumoral IFNγ. Experiments performed on glioma cells grown in vitro demonstrate that IFNγ directly inhibits viral replication capacity and CPA inhibits NK cell-mediated release of IFNγ. Molecular imaging shows that CPA pretreatment inhibits OV-induced infiltration in tumor-associated phagocytic cells, which is associated with decreased clearance of intratumoral viral particles. In conclusion, our results reveal the reason why OV therapy for brain tumors has resulted in low efficiency thus far and have uncovered molecular and cellular mechanisms that inhibit intratumoral spread of OV. These data suggest a new therapeutic path to improve the efficacy of virotherapy of cancer and a novel role of innate immunity for the therapy of brain tumors. The relevance of this discovery to human patients was demonstrated by the fact that infiltration of CD68+ and CD163+ cells is also significant in OV-treated human gliomas.


Roberta P. Glick,1 Terry Lichtor,1 Amla Chopra,2 Lisa Feldman,1 Julian Hardman,1 Britt Borden,1 InSug O-Sulllivan,2 and Edward P. Cohen2; 1Department of Neurosurgery, John H. Stroger Hospital and Rush University Medical Center; 2Department of Microbiology and Immunology, University of Illinois at Chicago; Chicago, IL, USA

We have recently reported encouraging preliminary results of a novel immunogene therapy using a unique DNA-based vaccine to treat malignant brain tumors. However, brain tumors escape recognition by the classic immune response by secreting immunosuppressive factors or by stimulating immunosuppressive cells, thereby limiting the effectiveness of most immunotherapies. Recently, a unique population of regulatory T cells (Tregs) has been identified. These regulatory cells are CD4+, CD25+, and the FoxP3 transcription factor+. These Treg cells suppress T cell-mediated immune response and also regulate other arms of an effective immune response. In studies outside the CNS, these cells have been found to directly inhibit NK cell-mediated tumor rejection and NK-mediated cytolysis largely by a TGFβ-dependent mechanism and independent of IL-10, which is a known tumor suppressor. Furthermore, Treg inhibition has been associated with enhanced antitumor activity. Thus, Tregs are one mechanism of immuno-suppression that may be responsible for the limited effectiveness of tumor immunotherapy that can be targeted for enhanced immunogenicity. In this study, C3H/He mice were injected weekly X 2 near the fat pad with either a unique DNA-based vaccine or a control. After 3 days, the spleens and lymph nodes were removed and the cells were prepared for immunofluorescent staining and cytofluorometric measurements by FACS with the following markers: CD4, CD8, CD25, CD62L, B7–1, B7–2, CTLA-4, and FoxP3. The results demonstrate that the highly immunogenic vaccine significantly downregulated CD25 and Fox P3 in the spleen and lymph node. In an intracerebral tumor model, a breast carcinoma cell line (SB5b) was injected i.c. into C3H/He mice through a unique micro-cannula system, followed 2 days later by treatment with the DNA-based vaccine cells into the tumor bed through the cannula on weekly intervals. FACS analysis of the spleen cells taken from the animals after 2 weeks reveal a marked increase in CD4 cells and a moderate increase in CD8 cells in the treated animals. In addition, there was a mild downregulation of CD25 cells and the Fox P3 transcription factor in the spleen cells of the treated animals. Survival was prolonged in mice with an intracerebral tumor treated with an intracerebral cytokine-secreting allogeneic DNA vaccine. These data demonstrate the effectiveness of immunogene therapy for CNS tumors and suggest that one of the mechanisms of increased immunogenicity of the DNA-based vaccine may be via the inhibition of Tregs and inhibition of tumor-induced immunosuppression. This study suggests a new target for enhancing immunotherapy.


Kazunori Haga,1 German G. Gomez,2 Christopher R. Logg,1 Takahiro Kimura,1 Kei Hiraoka,1 Thomas C. Chen,3 Linda M. Liau,1 Carol A. Kruse,2 and Noriyuki Kasahara1; 1University of California Los Angeles, Los Angeles, CA, USA; 2La Jolla Institute for Molecular Medicine, San Diego, CA, USA; and 3University of Southern California, Los Angeles, CA, USA

Gene therapy strategies for glioblastoma multiforme (GBM) employing conventional replication-defective retrovirus vectors have resulted in therapeutically inadequate levels of transduction in clinical trials. RCR vectors would be more efficient, because each successfully transduced tumor cell would itself become a virus producer cell (VPC) sustaining further transduction events after initial administration while retaining the intrinsic inability of retroviruses to infect quiescent normal cells. We previously demonstrated that i.t. injection of RCR vectors achieved efficient tumor-restricted suicide gene transfer in intracranial glioma models without detectable spread to normal tissues, achieving significantly prolonged survival without systemic side effects. We have now improved the efficiency of this approach by engineering alloCTLs to become RCR-VPCs. AlloCTLs are sensitized to tumor host human leukocyte antigens, the expression which is largely absent on normal brain cells but highly expressed by glioma cells. AlloCTLs also traffic through tissue and can act directly as cytolytic effector cells. The clinical feasibility and safety of intratumoral alloCTLs for adoptive immunotherapy of glioma has been previously confirmed in a phase I study. We propose that alloCTL/VPCs will act as motile cellular delivery platforms that will not only penetrate the tumor mass but facilitate multifocal spread of the replicating vectors to infiltrating glioma cells. First, RCR vectors expressing GFP and pseudotyped with amphotropic murine leukemia virus (MLV) or Gibbon ape leukemia virus (GALV) envelope proteins were tested for their ability to transduce primary human alloCTLs and convert them into VPCs under various conditions, including chondroitin sulfate precipitation, transduction on fibronectin-coated plates, spinoculation, or co-culture with VPCs. AlloCTLs co-cultured with human glioma cells producing RCR vectors resulted in effective re-sensitization while resulting in efficient viral transduction of > 80% of the alloCTLs in a dose-dependent manner. When the transduced alloCTLs were placed into culture with naïve glioma cells to which the alloCTLs were sensitized, highly efficient secondary horizontal transduction of the RCR vector from the alloCTLs to the glioma cells was observed in a dose-dependent and a time-dependent manner. Next, alloCTL/VPCs were prepared for delivery of RCR vectors carrying the yeast cytosine deaminase (CD) suicide gene using the optimized conditions. The alloCTL/VPCs were exposed to glioma cells at a ratio of 1:10. After 1 week of co-incubation, PCR analysis confirmed that the CD suicide gene had spread from the alloCTLs to the glioma cell population, and the pro-drug 5–fluorocytosine could efficiently kill both the alloCTLs and transduced glioma cells. These results confirm that alloCTL/VPCs efficiently promote RCR vector spread in glioma cells and impart them with susceptibility to suicide gene therapy, demonstrating the feasibility of combining adoptive immunotherapy with viroreplicative gene therapy for gliomas.


Shuang-Yin Han, Stephen Skirboll, Jian Cui, Holgado-Madruga Marina, and Albert Wong; Department of Neurosurgery, Stanford University Medical Center, Stanford, CA, USA

The cancer stem cell hypothesis states that tumors are initiated and maintained exclusively by a small fraction of cells with stem cell-like properties. Because this hypothesis predicts that it is only necessary to eradicate cancer stem cells (CSCs) for therapeutic efficacy, novel treatment strategies have been formulated to target CSCs. Cancer stem cells have now been confirmed in several cancers, including glioblastoma (GBM). The critical stem cell marker for GBM is CD133. Because CD133 is also shared by neural/hematopoietic stem cells, it would be highly desirable to also employ a marker specific to tumors. EGFRvIII is a tumor-specific variant of the EGF receptor that is expressed at high levels in GBM and has not been found in the normal brain or in the hematopoietic system. CSCs are thought to accumulate all genetic alterations, and EGFRvIII is the result of a gene deletion; therefore, CSCs should express EGFRvIII. We first confirmed that GBMs can co-express CD133 and EGFRvIII. Primary tumor specimens that were proven EGFRvIII-positive by immunohistochemistry were dissociated and cultured for 2–3 days prior to sorting. Using double-labeling FACS with a monoclonal antibody against CD133 and against EGFRvIII, we calculated that the fraction of cells for 5 tumor samples that were (CD133+EGFRvIII+)/(total CD133+) ranged from 1.83% to 58.33% and the fraction that were (CD133+EGFRvIII+)/(total EGFRvIII+) ranged from 8.47% to 57.4%. These data demonstrate that CD133 and EGFRvIII can indeed be co-expressed on GBM cells; however, there was some variability. Since GBM samples rapidly lose EGFRvIII expression in culture, a dissociation protocol was developed that preserves cell surface markers enabling cell sorting within 2 hours. For 1 sample dissociated by this method, we found the fraction of (CD133+EGFRvIII+)/(total EGFRvIII+) was 87.91% and for (CD133+EGFRvIII+)/(total EGFRvIII+), it was 79.13%, indicating a high degree of concordance between these 2 markers. To generate a CSC-specific reagent, we have started to develop a bispecific antibody (BsAb). By virtue of combining 2 binding specificities, a BsAb enhances the selectivity and efficacy of targeting. We therefore designed a BsAb that will simultaneously target CD133 and EGFRvIII. First, single-chain antibody fragments for CD133 and EGFRvIII were cloned into the pET32a vector and expressed in E. Coli. The binding affinity and specificity of the scFv were confirmed by ELISA and were within an order of magnitude of the original monoclonal antibody. Then, the 2 single-chain antibody fragments were fused to a modified hinge region containing the CH2 and CH3 domain of human IgG1 with a knob-in-hole structure to promote dimerization and then cloned into a bicistronic vector (pBudCE4.1). The BsAb were expressed in mammalian cells (HEK293) and purified. Analyses using Coomassie stain, ELISA, and western blot showed good purity, affinity, and specificity. This antibody can now be tested for its ability to target GBM and promote tumor regression in animal models.


Edward W.B. Jeffes, Jian Gang Zhang, Neil Hoa, Animesh Petkar, Christina Delgado, Samuel Chong, Andre Obenaus, Ramon Sanchez, Sakineh Khalaghizadeh, Tetyana Khomenko, Brandon A. Knight, Reza Alipanah, Tuong-Vi Nguyen, Chirag Shah, Seema Vohra, Jing-Li Zhuang, Jessie Liu, H. Terry Wepsic and Martin R. Jadus; Veterans Affairs Medical Center, Long Beach, CA, USA; University of California, Irvine, Irvine, CA, USA; Non-Invasive Imaging Laboratory Radiobiology Program, Loma Linda University, Loma Linda, CA, USA

Combining a T9/9L glioma vaccine that expresses the membrane form of macrophage colony-stimulating factor (mM-CSF) with a systemic anti-angiogenic drug-based therapy theoretically targeted towards growth factor receptors within the tumor vasculature, successfully treated > 90% of the rats bearing 7-day-old intracranial T9/9L gliomas. The antiangiogenic drugs included DMBI (a platelet- derived growth factor receptor B and a fibroblast growth factor receptor 1 kinase inhibitor) and oxindole (a vascular endothelial growth factor receptor 2 kinase inhibitor). Twenty percent to 40% of the animals treated with the antiangiogenic drugs alone survived, whereas all nontreated controls and all tumor vaccine-treated rats died within 40 days. In vitro, these drugs inhibited endothelial cells from proliferating in response to the angiogenic factors produced by T9/9L glioma cells and prevented endothelial cell tubulogenesis. FITC-labeled tomato lectin staining demonstrated fewer and constricted blood vessels within the intracranial tumor after drug therapy. MRI scans demonstrated that the intracranial T9 glioma grew much slower in the presence of these antiangiogenic drugs. These drugs did not affect in vitro glioma cell growth or T cell mitogenesis. Histologic analysis revealed that tumor destruction occurred at the margins of the tumor where there was a heavy lymphocytic infiltrate. A real-time PCR assay showed more interleukin-2–specific MRNA was present within the gliomas in the vaccinated rats treated with the drugs. Animals that rejected the established T9/9L glioma by the combination therapy proved immune against an intracranial rechallenge by T9/9L glioma but showed no resistance to an unrelated breast cancer.


T.G. Johns,1 R.M. Perera,1 R.B. Luwor,1 W.K. Cavenee,2 A.M. Scott,1 and F.B. Furnari2; 1Ludwig Institute for Cancer Research, Austin Hospital, Heidelberg, Australia; 2Ludwig Institute for Cancer Research, San Diego Branch, University of California at San Diego, La Jolla, CA, USA

The EGFR is frequently overexpressed or mutated in glioma and appears to contribute to its progression. The most common mutation, the de2-7 EGFR (EGFRvIII), is characterized by a 267 amino acid deletion from the extracellular domain that renders this receptor constitutively active. Potential EGFR-specific therapeutics include monoclonal antibodies (mAbs) and small molecular weight tyrosine kinase inhibitors (TKIs). Mechanisms causing resistance and susceptibility to EGFR-targeted TKIs have been studied extensively in many tumor types, whereas factors affecting the efficacy mAbs directed to the EGFR remain relatively unknown, especially in gliomas. We therefore examined the efficacy of 2 EGFR- specific mAbs (mAb 806 and 528) that differ in their modes of action against a panel of U87MG-derived glioma xenografts expressing different variants of the EGFR. Using this approach allowed us to keep the genetic background constant — only changing the status of the EGFR. Both mAbs behaved in a similar fashion. The efficacy of mAb did correlate with wild-type (wt) EGFR number, but the most important determinant of antitumor activity was activation of the EGFR. In particular, U87MG xenografts expressing the constitutive de2-7 EGFR responded to mAb treatment, whereas xenografts exhibiting a dead kinase (DK) version of the de2-7 EGFR (generated by a single intracellular point mutation) were completely refractory to therapy. Further increasing expression levels of the de2-7 EGFR lead to a corresponding increase in mAb-mediated anti-tumor activity, suggesting that the more dependent a xenograft becomes on the EGFR system, the greater it responds to EGFR mAbs. Significantly, a de2-7 EGFR variant containing tyrosine to phenylalanine mutations at all the major autophosphorylation sites also responded to mAb therapy. This observation, combined with the lack of activity seen against the de2-7 EGFR DK xenografts, suggests that the kinase activity, as opposed to auto-phosphorylation, correlates with responsiveness to antibody therapy. Thus, these mAbs probably function by blocking the transphosphorylation of a target molecule associated with the de2-7 EGFR. Because all the U87MG-derived xenografts used to test the efficacy of mAb 806 and 528 co-express the de2-7 variants and the wt EGFR, a state that mimics the situation on glioma patients, we assessed their therapeutic efficacy against NR6 xeno-grafts expressing the de2-7 EGFR in isolation. Although mAb 806 showed robust antitumor activity against these xenografts, mAb 528 therapy was ineffective. This finding suggests that mAb 528 functions by preventing the de2-7 EGFR-mediated transphosphorylation of the wt EGFR, while mAb 806 must disrupt another interaction. The therapeutic consequences of these observations will be discussed in detail.


Seok-Gu Kang, Sin Soo Jeun, Yong Gil Hong, Sang Won Lee, Pil Woo Huh, and Chun Kun Park; Department of Neurosurgery, The Catholic University of Korea College of Medicine, Uijeongbu St. Mary’s Hospital, Uijeongbu, Korea

Marrow stromal cells (MSCs) have been shown to have the capacity for orthodox and unorthodox plasticity. In this study, the authors tried to differentiate MSCs into immune effector cells and also to assess in vitro cytotoxicity of MSCs from rat models. Rat MSCs (rMSCs) were isolated by standard methodology and were activated by interleukin (IL)-2, IL-15, colony-stimulating factor GM-CSF, and combinations of these. The phenotypes of these various activated cells were determined using flow cytometry. The secreted protein from effector cells was estimated using an enzyme-linked immunosorbent assay. Cytotoxicity of rMSCs and activated rMSCs against the target cells (9L rat glioma cell line) were estimated using a visual survival cell assay. The expression of immune response-related genes in activated cells was measured. After the cytokine activation of rMSCs, the populations of immune effector cells (CD8, CD161a) and immune reaction-related proteins (IL-4, γ-INF) were increased. There was a significant cytotoxicity of rMSCs activated with various cytokine combinations. Apoptosis may be one of the lysis mechanisms of target cells by activated rMSCs. The contributing genes could be γ-INF, FasL, and perforin. This study suggests that rMSC may differentiate into immune effector cells and have cytotoxic ability against malignant glioma cells; however, we must investigate orthotopic animal studies for the proper translation.


Terry Lichtor,1 Roberta P. Glick,1 Alessandra Spagnolo,2 Edward P. Cohen,3 and Douglas L. Feinstein2; 1Department of Neurosurgery, Rush University Medical Center; 2Departments of Anesthesiology and 3Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL, USA

In this study, we explored the benefits of treating C57Bl/6 mice with an established intracerebral glioma by combining immunotherapy with IL-2–secreting syngeneic/allogeneic fibroblasts administered into the tumor bed along with the chemotherapeutic agent pioglitazone, a thiazolidinedione (TZD). TZDs are agonists of the peroxisome proliferator-activated receptor gamma (PPARγ). They have been found to exert antiproliferative effects on several transformed cell lines. Previous studies by this laboratory have revealed the immunotherapeutic properties of the IL-2–secreting fibroblasts in treating intracerebral gliomas in mice. The sensitivity of Gl261 glioma cells and primary astrocytes to pioglitazone was determined in vitro by incubating the cells with increasing amounts of pioglitazone. Viability was assessed by measuring lactate dehydrogenase release, and the effects on metabolism were determined by measuring superoxide production and levels of superoxide dismutase. Gl261 cells were injected i.c. into C57Bl/6 mice, followed by treatment with pioglitazone either orally or intracerebrally into the tumor bed. The effect of the combined therapy was determined by injecting C57Bl/6 mice with an established intracerebral Gl261 glioma with IL-2-secreting allogeneic fibroblasts and pioglitazone directly into the tumor bed through a unique cannula system. Pioglitazone was found to induce cell death in Gl261 glioma cells grown in vitro and cause only modest damage to astrocytes. Pioglitazone also resulted in a significantly greater induction of cellular superoxide in glioma cells than in astrocytes, which can activate apoptotic pathways. Pioglitazone administered i.c. (P < 0.05), but not orally, was found to prolong survival in mice with an intracerebral glioma. Spleen cells from mice injected with IL-2 secreting cells showed a stronger response to glioma cells than controls, as measured by an ELISPOT interferon-γ assay; pioglitazone did not enhance this activity. Synergistic effects of combination therapy on prolonging survival were found in mice receiving both pioglitazone and IL-2 secreting fibroblasts (P < 0.005 vs. untreated animals). Pioglitazone induces metabolic and oxidative stresses that are tolerated by astrocytes but not by glioma cells, which could account for selective vulnerability and increased sensitivity to IL-2. These findings suggest the potential for the use of this FDA-approved drug in the treatment of brain tumors. The data indicate the beneficial effects of combination therapy using pioglitazone and immunotherapy in mice with intracerebral glioma.


Tiefu Liu, Jiaozhong Cai, Denise Gibo and Waldemar Debinski; Wake Forest University School of Medicine, Brain Tumor Center of Excellence, Winston-Salem, NC, USA

We have revealed that interleukin (IL)-13Rα2, a restricted receptor for (IL)-13, is overexpressed in most patients with glioblastoma multiforme (GBM). We previously generated a wild type IL-13–based Pseudomo-nas exotoxin A (PE)-containing recombinant cytotoxic protein (hIL13-PE38QQR), which was made for comparative purposes to an IL-4–based cytotoxin and which was not intended for brain tumor therapy. However, we subsequently found that hIL13-PE38QQR is useful in molecular targeted therapy of malignant gliomas, because malignant glioma cells appeared to be extremely susceptible to the cytotoxin and it demonstrated a significant antitumor effect in in vivo models of gliomas. Clinical trials using hIL13-PE38QQR in patients with GBM are currently ongoing. We have also documented that IL-13–directed cytotoxins target GBM cells through monomeric IL-13Rα2. They may, however, also induce toxicity by binding to its receptor that is shared with IL-4, a heterodimeric signaling complex of IL-13Rα1 and IL-4Rα, which is found in normal tissues, including the brain. We have documented that the binding of IL-13 to IL-13Rα1 and IL-13Rα2 chains is done primarily through its C-terminus, whereas the binding to the IL-4Rα chain is through its N-terminus. IL-13 is fused to PE through its C-terminal end but must be linked to diphtheria toxin (DT), another bacterial toxin suitable for making recombinant cytotoxins, through the N-terminus. Thus, in the present study, we used DT rather than PE for IL-13–based cytotoxins in order to leave the C-terminus IL-13Rα2–binding region (helix D) more accessible by the receptor. We also minimized the interaction of IL-13 with IL-13Rα1/IL-4Rα and maximized the interaction with IL-13Rα2 through amino acid substitutions. Thus, we generated a novel, variant IL-13 molecule, IL-13QM, by replacing Glu13, Arg66, and Ser66, the key amino acids for IL-4Rα binding, and Lys105, the key site for IL-13Rα2 affinity with Lys, Asp, Asp and Arg, respectively. The IL-13, IL-13QM, DT-IL-13, and DT-IL-13QM proteins were expressed in E Coli and purified using FPLC. TF1 lymphoblast cells express IL-13Rα1/IL-4Rα signaling receptor but not IL-13Rα2, and wild type IL-13 stimulated their proliferation potently at IC50 of 42 pM, whereas IL-13QM showed no proliferative effect. However, IL-13QM blocked the cytotoxicity of hIL-13–PE38QQR on IL-13Rα2–expressing human GBM cells very effectively. Furthermore, both DT-IL-13 and DT-IL-13QM demonstrated a profound cytotoxic effect on GBM cell lines with IC50s <5 pM. The killing of GBM cells by both DT-IL-13 and DT-IL-13QM was specific to IL-13Rα2, because it was neutralized by IL-13 and not by IL-4. Both cytotoxins have shown little, if any, cytotoxicity on IL-13Rα1/IL-4Rα-expressing, but IL-13Rα2–lacking, epidermoid carcinoma A431 cells or on RBE4 normal rat brain microvascular endothelial cells, even at high concentrations of the cytotoxins, whereas the IC50 of hIL-13–PE38QQR on A431 cells was 28 nM. Our results suggest for the first time that it is possible to generate a rationally designed quadruple mutant of IL-13, IL-13QM, which is functional alone or in fusion with DT. IL-13QM-directed N-terminally-fused DT cytotoxin may yield increased specificity, decreased toxicity, or both in patients with GBM.


Brian Patrick O’Neill, Ahmet Dogan, and Caterina Giannini; Mayo Clinic, Rochester, MN, USA

The central nervous system (CNS) is a frequent site for development of Epstein-Barr virus (EBV)-mediated B cell lymphoproliferative disorders in the context of systemic immunosuppression, particularly in patients who undergo solid organ transplantation. In this study we describe the clinical, imaging, and pathologic observations of a CNS disorder histologically similar to posttransplant lymphoproliferative disorders that occurred in 4 patients with autoimmune disease who were treated with mycophenolate mofetil (MM).

Age (years)57655888
AIDDermatomyositisRelapsing PolychondritisCNS vasculitisMyasthenia Gravis
Duration (years)6595
MM dose1,000 mg BID1,000 mg BID1,000 mg BID1,000 mg BID
Duration (months)8114637
SyndromeEncephalopathyHemisphericPosterior fossaHemispheric
Duration (months)1322
ImagingMultiple, irregular c/eMultiple, r/eMultiple, c/eSingle, r/e
PathologyPolymorphous (LD)Polymorphous (LD)Mono-morphous (LBCL)Mono-morphous (LBCL)

Key: MM: mycophenolate mofetil; mg: milligrams; BID: twice daily; c/e: contrast-enhancing; r/e: ring-enhancing; LD: lymphoproliferative disorder; LBCL: diffuse large B cell lymphoma; EBV: Epstein-Barr virus; DMX: dexamethasone; RTX: rituximab; CR: complete response; PROG: progression.

New immunosuppressive regimens such as MM have a more selective and more profound effect on pathways of lymphocyte regulation. Because of its favorable toxicity profile, MM is now being used as steroid-sparing immunomodulatory therapy in autoimmune disorders. Based on our experience presented herein, we recommend caution in patient selection and close surveillance of those patients for development of EBV-mediated B cell lympho-proliferative disorders.


Robert M. Prins,1,2 Chengyi J. Shu,2 Haumith Khan-Farooqi,1 Pilar DeLa Rocha,3 Dan D. Vo,3 Caius Radu,2,4 Owen Witte,2,4,5 Antoni Ribas,3,6,7 and Linda M. Liau1,7; 1Division of Neurosurgery, 2Department of Microbiology, Immunology and Molecular Genetics, 3Division of Surgical Oncology, 4Department of Medical & Molecular Pharmacology, 5Howard Hughes Medical Institute, 6Department of Hematology/Oncology, 7Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Immunotherapeutic, antigen-specific targeting of solid tumors, whether the tumors are located systemically or in the brain, must usually involve the ability of T cells to overcome peripheral tolerance to self tumor antigens before killing tumor cells. Historically, preclinical models of immunotherapeutic, antigen-specific targeting have used xenogeneic or viral antigen targets. Thus, most models do not recapitulate the constraints normally faced in patients to effectively induce effective antitumor immunity. We have recently discovered that both human and mouse central nervous system (CNS) gliomas express immunologically relevant concentrations of melanoma-associated antigens (MAA), which retain significant immunological tolerance. To create a preclinical system that recreates the clinical impediments to generating effective antitumor immune responses to self, tumor-associated antigens (TAA), we have targeted MAA on CNS gliomas by the adoptive transfer of clonal, gp100-specific CD8+ T cells and dendritic cell (DC) vaccination. In addition, we have also created a model whereby we can noninvasively visualize both the CNS tumor growth and the trafficking of gp100-specific CD8+ T cells using bioluminescent and micro-PET imaging. Mice are implanted with CNS tumors: B16 melanoma and GL26 glioma. After whole-body irradiation (500 cGy XRT) to induce transient lymphopenia, mice are adoptively transferred with lentiviral-transduced, gp100-specific CD8+ T cells and vaccinated with gp100 peptide-pulsed DC and high-dose IL-2. CNS tumor progression is noninvasively monitored via firefly bioluminescent imaging. Similarly, tumor-specific T cell trafficking is monitored with Renilla luciferase bioluminescent and micro-PET imaging. Our studies demonstrate the ability to generate significant antitumor immunity to both subcutaneous and CNS tumors as well as noninvasively image the trafficking of the tumor antigen-specific CD8+ T cells in the process. The adoptive transfer of labeled gp100-specific CD8+ T cells into lymphopenic, tumor-bearing mice results in robust expansion through lymphoid organs and the ability to induce clinically relevant survival in subcutaneous and CNS tumor-bearing mice. These studies demonstrate that peripheral tolerance can be overcome to treat CNS tumors. Future studies can now analyze in detail the fundamental mechanisms by which effective antitumor immunity can be achieved.


J. Rodrigues, G. Gonzalez, J. Kelly, V.W. Yong, P.A. Forsyth, and I.F. Parney; University of Calgary, Canada

Malignant glioma patients are immunosuppressed with deficits in lymphocyte signaling and cytokine production compared with healthy individuals; however, malignant gliomas are highly infiltrated by monocytes and macrophages. In other cancers, circulating CD14 immunosuppres-sive myelomonocytic lineage cells, termed myeloid suppressor cells (MSC), have been identified that are inversely correlated with patient survival. We hypothesize that glioma exposure causes normal monocytes to assume an MSC-like phenotype and that glioma patients have increased levels of circulating MSCs. CD14+ monocytes were purified from normal donor PBMC by magnetic beads and co-cultured with human glioma cell lines or normal human astrocytes (NHA). CD14 and CD11b expression was determined by flow cytometry before and after co-culture. Glioma (or NHA)-conditioned monocytes were purified by CD11b magnetic beads, and MSC (CD33+/HLA-DR/Lineage) frequency was determined by flow cytometry (Lineage = CD3/CD14/CD19/CD56). Phagocytic ability was assessed by incorporation of FITC-labeled E. coli cell wall particles. Apoptosis was measured in activated lymphocytes exposed to glioma-conditioned monocytes using Annexin V/7AAD staining. Monocyte and MSC frequency was determined in PBMC from glioma patients and healthy control subjects. Monocytes downregulated CD14 after exposure to glioma cell lines but not NHA. This downregulation was nearly complete for some cell lines (U251, A172) and partial for others (U87, T98). CD11b expression was preserved. Glioma-conditioned monocytes could be purified (90%) from co-cultures with CD11b beads. Increased levels of MSCs were seen in U251–conditioned monocytes compared with controls (13% vs. 3%). Glioma-conditioned monocytes had reduced phagocytic ability. They produced increased activated lymphocyte apoptosis (16%–27% vs. 7%–12% with controls). Patients with glioma tended to have decreased circulating monocyte levels compared with healthy controls (7% vs. 15% of PBMC), but despite this finding, patients with glioma had increased MSC levels (mean, 14%; range, 4%–24% of monocytes vs. mean, 1.7%; range, 0%–4%). Normal human monocytes co-cultured with glioma cells assume an immunosuppressive phenotype and surface marker profile similar to MSCs seen in other cancer types. MSCs are present in glioma patients and may contribute to the immunosuppressed phenotype. Further studies will determine the MSC cytokine expression profile. Additional patient data will be required to further elucidate the relationship of MSCs and immunosuppression in glioma patients.


Kotaro Sasaki,2 Xinmei Zhu,1,3 Mitsugu Fujita,1,3 Fumihiko Nishimura,1,3 Jill E. Dusak,1,3 Walter J. Storkus,2 and Hideho Okada1,3; Departments of 1Neurological Surgery, 2Dermatology and Immunology, University of Pittsburgh School of Medicine, 3Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Development of effective immunotherapeutic strategies for central nervous system (CNS) tumors requires a firm understanding of the factors that regulate the trafficking of tumor antigen-specific cytotoxic T lymphocytes (CTLs) into CNS tumors. Using C57BL/6 mice bearing i.c. ovalbumin-transfected melanoma (M05), we previously demonstrated the preferential CNS tumor homing and therapeutic efficacy of i.v. infused Tc1 cells compared with Tc2 cells. Further characterizing the expression of homing and chemokine receptors on Tc1 and Tc2 cells, we have found that Tc1 cells express significantly higher levels of very late antigen (VLA)-4 (heterodimer of CD49d and CD29) than Tc2 cells [mean fluorescence intensity (MFI); CD49d: 799 vs. 196, CD29: 1090 vs. 513 for Tc1 and Tc2, respectively]. Other activation markers, such as function-associated antigen (LFA)-1 and CD25, demonstrated similar expression levels on Tc1 and Tc2 cells, suggesting that the difference of VLA-4 expression is not merely the differential activation status between Tc1 and Tc2 cells. Although CD49d, which is also known as α4–integrin, can comprise heterodimers with both β1 (CD29) and β7 integrins, α4β7 complex was not expressed on Tc1 cells nor on Tc2 cells, suggesting that CD49d comprises the heterodimer with β1 (CD29) to form VLA-4. In accordance with these observations, Tc1 cells demonstrated a remarkable adhesion activity against plastic plate-coated with VCAM-1–Ig fusion protein (63.3% ± 7.1% of the total plated cells adhered), whereas Tc2 cells demonstrated only a background level of adhesion (7.25% ± 1.25%). Furthermore, treatment with anti-VLA-4 monoclonal antibodies (mAbs) significantly blocked Tc1 cell binding to VCAM-1–Ig, supporting the specificity of VCAM-1–VLA-4–mediated Tc1 cell adhesion. Finally, the significance of VLA-4 expression on Tc1 was determined in mice bearing i.c. M05 tumors. Mice bearing day 10 M05 received i.v. infusions of Tc1 cells pretreated ex vivo with anti-CD49d mAb or isotype IgG. Forty-eight hours after the infusion, an evaluation of tumor-infiltrating lymphocytes revealed that pretreatment with anti-VLA-4 mAbs dramatically diminished the CNS-tumor homing of OVA-specific Tc1 cells. Collectively, these data indicated the critical role of VLA-4 in the effective CNS-tumor homing of Tc1 cells and led us to employ the VLA-4 expression-status on glioma-antigen- specific T cells as a surrogate marker in our immunological monitoring plans in an ongoing vaccine trial. These data also prompt us to develop more effective vaccines and ex vivo T cell activation regimens that promote Type-1 phenotype, including VLA-4 expression on effector T cells.


Ariane Söling,1 Eva-Maria Plugge,1 Marc Schmitz,2 Bernd Weigle,2,3 Roland Jacob,1 Jörg Illert,1 Hans-Jürgen Holzhausen,4 and Nikolai G. Rainov1,5; 1Department of Neurosurgery and 4Institute of Pathology, Martin Luther University Halle-Wittenberg, Halle, Germany; 2Institute of Immunology, Technical University Dresden, Dresden, Germany; 3Eucodis GmbH, Vienna, Austria; and 5Department of Neurosurgery, Central Clinic Augsburg, Augsburg, Germany

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is frequently expressed in cancers, including brain tumors. Survivin may be associated with tumor progression and poor prognosis of patients with meningiomas and gliomas. We asked whether the tumor-associated antigen survivin is capable of eliciting a humoral immune response in patients with meningiomas and gliomas and whether it represents a diagnostic marker in these patients. This study used ELISA and immunoblot analyses to test serum samples from patients with gliomas and meningiomas for immuno-reactivity against purified recombinant survivin. Tumor samples from the patients were immunohistochemically stained for survivin. Survivin-specific antibodies were detected in 11.9% (5/42) of patients with meningioma and in 8.6% (3/35) of patients with malignant gliomas (WHO grades III and IV), but they were not detected in healthy controls. Tumor samples from patients with detectable antisurvivin antibodies demonstrated survivin expression in at least 20% of tumor cells. We conclude that patients with meningiomas and malignant gliomas can mount a high-titer IgG immune response against the universal tumor-associated antigen survivin. Antisur-vivin antibodies may represent attractive tools for diagnosing brain tumors and for monitoring the course of the disease.


Jian Gang Zhang,1 Junichi Eguchi,2 Carol A. Kruse,3 German G. Gomez,3 Habib Fakhrai,4 Stephanie Schroter,5 Wenxue Ma,5 Neil Hoa,1 Boris Minev,5 Christina Delgado,1 H. Terry Wepsic,1 Hideho Okada,2 and Martin R. Jadus1; 1Veterans Affairs Medical Center, Long Beach, CA, University of California, Irvine, Irvine, CA, USA; 2University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 3La Jolla Institute for Molecular Medicine, San Diego, CA, USA; 4NovaRx Corporation, San Diego, CA, USA; 5University of California, San Diego Cancer Center, La Jolla, CA, USA

Immunotherapy for brain tumors can involve active vaccination, passive cellular therapy approaches, or both. Use of glioma cell lines that are partially matched at class 1 human leukocyte antigen (HLA) loci and that display known tumor-associated antigens (TAA) or antigenic precursors (TAPP) could be used for extraction of TAA peptides to make dendritic cell (DC) vaccines. After confirming the neuroectodermal origin of 20 human glioma cell lines, we proceeded to characterize their class 1 HLA-A, HLA-B, HLA-C, and/or class II HLA-DR, DQ by molecular phenotyping, and the levels of HLA class 1 by flow cytometry. Twelve of the 20 cell lines were also characterized for protein and/or mRNA expression of 16 TAPP or TAA. All cell lines exhibited multiple expressions. Most glioma cells expressed B-Cyclin, EPHA2, GP100, GNT-V, IL13Rα2, HER2/NEU, hTERT, MAGE-1, MART-1, and survivin. Cytotoxic T lymphocytes (CTLs) were generated in vitro from patient-derived CD8+T cells specifically sensitized to HLA-A2+ restricted TAA by autologous dendritic cells. The CTLs lysed T2 cells loaded with those specific antigens or gliomas that were HLA-A2+ and were also positive for MART-1, GP100, EPHA2, HER2/NEU, and tyrosinase. We confirmed these findings with tumor infiltrating lymphocyte (TIL) cell lines that were restricted to the GP100 and the tyrosinase peptides, and we showed that they specifically lysed glioma cells that were positive for HLA-A2 and the peptide but not for those cells negative for HLA-A2 or lacking the specific epitope. These data provide proof-in-concept for the use of allogeneic, partially HLA patient-matched glioma cells for vaccine generation.


Xinmei Zhu,1,2 Fumihiko Nishimura,1,2 Junichi Eguchi,1,2 Jill E. Dusak,1,2 Talal El-Hefnawy,2 Ian F. Pollack,1,2 Andres Salazar,3 and Hideho Okada1,2; 1Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 2Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; and 3Oncovir, Inc., Washington, DC, USA

Toll-like receptors (TLRs) play a crucial role in host defense against invading microorganisms by recognizing pathogen-associated molecular patterns, such as double-strand RNA for TLR3. As TLR3 signaling promotes Type-1 adaptive immunity, and TLR3 is most abundantly expressed in the CNS among the TLRs, we hypothesized that stimulation of TLR3 signaling would enhance the effect of peripheral vaccinations directed against glioma-associated antigen (GAA)-derived, cytotoxic T lymphocyte (CTL)-epitopes by potentiating both induction and effector phases of immune responses. To this end, we employed the TLR3 ligand, polyinosinic-polycytidylic acid (poly-IC) stabilized with poly-lysine and carboxy-methylcellulose (poly-ICLC), which has been extensively characterized in patients with glioma as a single agent. C57BL6 mice bearing syngeneic i.c. GL261 gliomas received subcutaneous vaccinations with synthetic peptides designed in GL261-derived CTL epitopes (TRP-2[180–188], hGP100[25–33], and mEphA2 [671–679]) emulsified in Incomplete Freund Adjuvant (IFA) in combination with i.m. injections of poly-ICLC twice weekly. In some mechanistic evaluations, to improve the sensitivity of evaluations for antigen-specific CTLs, CD8+ T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT-1 mice were adoptively transferred into syngeneic mice bearing i.c. OVA-expressing M05 melanoma before receiving IFA-emulsified OVA-vaccines and i.m. poly-ICLC. Our data demonstrate that this combination strategy promotes the following: (1) systemic induction of GAA-specific CTLs and survival of mice without inducing auto-immunity; (2) persistence of antigen-specific CTLs in hosts by repetitive poly-ICLC injections following the peptide-vaccines; (3) i.c. tumor-infiltration of antigen-specific CTLs that also express interferon (IFN)-γ; and (4) the number of antigen-specific CTLs expressing very late antigen (VLA)-4, which we separately demonstrated as a critical homing receptor to CNS tumors. Furthermore, real-time RT-PCR analyses of i.c. glioma tissues in poly-ICLC/GAA-vaccine-treated mice demonstrated up-regulation of major histocompatibility complex (MHC) class I, chemokine CXCL10 and vascular-endothelial cell adhesion molecule (VCAM-1), which is the ligand for VLA-4, suggesting that i.m. delivered poly-ICLC modulates i.c. tumor microenvironment in a way that vaccine-induced GAA-reactive CTLs can efficiently traffic to the tumor-site and exert their antitumor effects. Taken together, poly-ICLC, which has been previously clinically evaluated, can be effectively combined with antigen-specific vaccine strategies, thereby providing a greater index of therapeutic efficacy.



Samson Amos and Isa M. Hussaini; Department of Pathology and Neuroscience, University of Virginia, Health System, Charlottesville, VA, USA

Glioblastoma multiforme (GBM) is the most malignant astrocytoma; is characterized by uncontrolled, aggressive cell proliferation and infiltrative growth within the brain; and is resistant to conventional therapy. The molecular and cellular mechanisms governing astrocytic tumor invasion remains poorly understood. We determined the role of low-density lipoprotein receptor-related protein (LRP) in glioblastoma invasive growth. In this study, we demonstrated that activation of protein kinase C (PKC) in astrocytic cell cultures downregulated the expression of LRP and increased the secretion of urokinase (uPA) into conditioned medium. Pretreatment of cell cultures with PKC inhibitors (BIM, Gö 6976) and PI3-kinase inhibitor (LY 294002) and gene silencing with PKCα siRNA abrogated PMA-induced downregulation of LRP, decreased the level of expression of uPA, and inhibited astrocytic tumor cell invasion. Confocal microscopy studies revealed the co-localization of PKC-α and LRP in glioblastoma cell lines. In a Boyden Chamber invasion assay, LRP-deficient glioblastoma cells were more invasive (40%) than LRP-expressing cells, whereas uPA-deficient GBM cells had decreased invasive capacity. Our data show that LRP expression inversely correlates with uPA secretion and GBM invasion. Taken together, our data strongly suggest the involvement of PKCα/PI3 kinase signaling pathways in the regulation of LRP-mediated astrocytoma invasion.


Marie E. Beckner, Naomi R. Agostino, Wendy Fellows-Mayle, Glenn T. Gobbel, Zhe Zhang, Billy W. Day, Ian F. Pollack; University of Pittsburgh, Pittsburgh, PA, USA

The invasiveness of astrocytomas is largely responsible for failed resections and possibly resistance to other therapies. The potential of astrocytoma cells for invasive migration is remarkably robust in regard to hostile tissue microenvironments. Their escape from tethered connections to the microvasculature implies tolerance of intermittent loss of mitochondrial energy production when the oxygen supply is interrupted and vigorous mechanisms to prevent intracellular acidosis. The purpose of this study was to develop methods for identifying and testing drugs to suppress gycolytic migration and invasion of astrocytoma cells. In vitro, inhibition of mitochondria established glycolytic conditions for invasion by human U87 and LN229 cells through filters coated with gelatin or Matrigel that were comparable to those of normoxic invasion. In several week-long assays, U87 cells, highlighted with a fluorescent dye, Dil, demonstrated widespread dispersion of single cells migrating through 1-mm rat brain slices in response to a gradient of hepatocyte growth factor (HGF) and serum with a mitochondrial inhibitor present. In addition to mitochondria, the vascular supply, systemic immune function, and bacterial contamination were eliminated as variables. In separate studies, a 1-D gel analysis with mass spectrometry of pseudopodia formed by U87 cells in Boyden chambers demonstrated increased amounts of ATP citrate lyase (ACL) compared with unmigrated cells. Because mitochondria release the first intermediate of the Kreb’s cycle, citric acid, to the cytosol when inactivated, we hypothesized that a mechanism to prevent accumulation of citric acid with its 3 carboxylic acid groups within pseudopodia would aid in the preservation of their functional integrity during hypoxic (glycolytic) migration. Thus, metabolism of citric acid via ACL was proposed as a target for suppression of tumor cell migration when mitochondria are inhibited or nonfunctional. Two inhibitors of ACL, radicicola and (-) hydroxycitric acid, demonstrated significant (P < 0.05) suppression of U87 cell migration in Boyden chambers, responding to HGF under glycolytic conditions. Glycolytic compared with normoxic cell migration was more susceptible to (-) hydroxycitric acid. Ongoing studies indicate that the effects of (-) hydroxycitric acid are even stronger in LN229 cells. Growth factor-stimulated anaplerotic pathways for citric acid, as well as lactic acid, leading to synthesis of lipids, glycogen, and peptides for proteins, are possible means to remove metabolic acids within tumor cell pseudopodia, with the accompanying energy debts paid by other regions of the cells or paid later when the oxygen supply is reestablished. ACL inhibitors, such as the anti-obesity drug (-) hydroxycitrate, are candidate drugs to be combined with other agents that oppose anaplerotic pathways, such as metformin, an inhibitor of gluconeogenesis, that has also been to shown inhibit the glycolytic migration of astrocytoma cells. These drug combinations can now be tested in vitro in the novel rat brain slice invasion model with minimized variables to develop strategies for in vivo use. The goals are suppressing tumor cell invasion, forcing tumor cells to remain connected to the vasculature for continuous exposure to traditional drugs, and complementing anti-angiogenesis treatments. Support for these studies was provided by the Nick Eric Wichman Foundation and the Bcez Foundation.


Peter D. Canoll, Christopher Beadle, and Steven S. Rosenfeld; Departments of Pathology and Neurology, Columbia University, New York, NY, USA

The invasiveness of gliomas remains a vexing problem that limits the efficacy of local therapies. We have previously shown that glioma invasion, migration, and attachment could be blocked by non-toxic inhibitors of myosin light chain kinase (MLCK), an enzyme required for the activation of non-muscle myosin II. We concluded that myosin II is an essential and targetable component in the motile and invasive apparatus of these tumors. To test this hypothesis, we examined the effects of blebbistatin on glioma migration and invasion in vitro and in a brain slice preparation. Unlike MLCK inhibitors, blebbistatin directly and specifically inhibited myosin II and reduced the tension generated by the cell cortex. As with the MLCK inhibitors, blebbistatin is non-toxic and effectively blocked the perivascular migration of GFP-labeled glioma cells in a rat brain slice preparation. Furthermore, we observed that dividing glioma cells failed to complete cytokinesis — the final stage of mitosis — and became multi-nucleated. Invasion in vitro, as assayed by Transwell chambers, was also inhibited by blebbistatin. More careful evaluation of the Transwell chambers revealed that in the presence of blebbistatin, leading cell processes, including invadopodia, were able to pass through the 3-micrometer pores of the chamber. However, the more bulky posterior of the cell, containing the nucleus, could not traverse these pores in the presence of the drug. This suggested to us that the role of myosin II in glioma invasion is restricted to a discrete function—to produce the posterior-generated force needed to squeeze the nucleus through the tight spaces that characterize the brain extracellular space. Consistent with this hypothesis, we found that the migration of glioma cells on a cover slip, with few spatial constraints on cell movement, was not affected by bleb-bistatin. We conclude from these results that myosin IIB is responsible for pushing glioma cells through the tight extracellular spaces that characterize brain white matter and that this function represents a specific adaptation of these tumors allow migration through the tightly packed extracellular space that characterizes normal brain tissue.


Charles Conrad, Yongjie Ji, Mark Emmett, Maja Puchades, Xiaoyang Sheng, Kenneth Aldape and Carol Nilsson; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, USA; Institute of Neuroscience and Physiology, Sahlgrenska Academy, Goteborg University, Mol Endal, Sweden

We identified, using proteomics approaches, a lectin-binding protein (galectin-1) that is subject to extremely large expression level changes after glioblastoma cell lines are transfected with wild-type p53. Galectins are a family of proteins that recognize extracellular glycoproteins and are known to confer a number of biologic effects, including cell motility, apoptosis, mRNA splicing, and cell cycle control. We also found that the expression of galectin-1 significantly influenced the invasive behavior of glioblastoma cell lines in matrigel invasion assays. All glioblastoma cell lines tested to date expressed a high level of galectin-1. Additionally, cell survival and clonogenic colony growth in soft agar was also significantly reduced by inhibition of galectin-1 through siRNA treatment. Finally, tumor samples from patients with high-grade malignant gliomas demonstrated that survival is inversely correlated with galectin-1 expression level (P < 0.0005 by the Wilcoxon signed-rank test). Taken together, these results, which will be presented in detail, demonstrate that galectin-1 is an important target for both glioma survival and invasiveness and represents a potentially excellent therapeutic target for drug development.


T. Demuth, L.B. Reavie, M. Nakada, S. Nakada, J.L. Rennert, C. Beaudry, D.B. Hoelzinger and M.E. Berens; Translation Genomics Research Institute, Phoenix, AZ, USA

The early and pervasive tendency of glioma cells to invade into peritumoral normal brain underlies patients’ poor prognosis. This malignant dissemination of glioma prevents complete surgical resection and places tumor cells behind an intact blood brain-barrier and outside the field of radiation, inevitably leading to tumor recurrence. Laser capture microdissection was used to collect phenotypically homogenous populations of invasive and stationary glioma cells using a high-throughput, 3-dimensional in vitro invasion assay. Whole human genome expression profiling was performed, followed by technical, clinical, and biologic validation. Mitogen-activated protein kinase kinase 3 (MKK3), a member of the MAP kinase family, was significantly upregulated in invasive glioma cells. QRT-PCR (technical) validation confirmed this microarray finding. Immunohistochemical analysis (IHC) of a tissue microarray (TMA) of invasive glioma cells clinically validated the in vitro observation and confirmed the activity of MKK3 (pMKK3) and the activation of its downstream target p38 in invasive glioma cells in situ. Immunoblotting on > 50 surgical specimens revealed activated MKK3 and p38 to be drivers of glioma progression; in a comprehensive glioma expression data set of 170 glial tumors, MKK3 was identified as a strong predictor of poor patient survival (Cox hazard ratio = 0.17, P < 0.05). Inhibition of MKK3 by siRNA and p38 by small molecule revealed decreased in vitro and ex vivo invasiveness in organotypic rat brain slice assay while sensitizing glioma cells to apoptosis induction through temozolomide, confirming the significance of this pathway in glioma invasion and survival. MKK3 and p38 were identified as important drivers of glioma invasion, and MKK3 was found to strongly predict patient survival. Small molecule inhibition of p38 resulted in decreased invasiveness and heightened glioma cells’ susceptibility to undergo apoptosis, rendering MKK3 and p38 novel targets for anti-invasive therapies in combination with cytotoxic agents.


A.G. Gilg, S. Tye, S. Ghatak, S. Misra, R. Visconti, J. Duncan, M. Kindy, S. Ramamoorthy, B. Toole and B.L. Maria; Charles P. Darby Children’s Research Institute, Medical University of South Carolina, Charleston, SC, USA

Hyaluronan, a large polysaccharide constitutively expressed in the brain extracellular matrix, is involved in the invasiveness and drug resistance of malignant cells through its interactions with receptor tyrosine kinases (RTKs). Hyaluronan oligomers (O-HA) that inhibit hyaluronan/CD44 interactions suppress the activities of several RTKs and the PI3K/Akt, RAF-1/ERK, and FAK pathways in malignant cells. The purpose of this study was to target hyaluronan/CD44 interactions in drug-resistant glioma progenitor cells. Side populations (SP) of C6 glioma progenitor cells were isolated by FACS analysis on the basis of their expression of ABCG2 (breast cancer resistance protein, BCRP). C6 glioma progenitor cells (C6SPs) were 1000 × more resistant to methotrexate, and targeted inhibition of methotrexate efflux by the BCRP inhibitor KO143 decreased drug resistance by 30%. Nestin-positive C6SPs were highly tumorigenic in vivo, and cells exhibited characteristic properties of invasive human glioma cells in the white matter, the subpial region, and around the hyaluronan-rich perineuronal nets in an established spinal cord glioma model. Inhibition of hyaluronan/CD44 interactions with hyaluronan oligomers in C6SPs decreased phosphorylation of EGFR, c-MET, and Akt and decreased BCRP production. Hyaluronan oligomers injected into the engrafted C6 tumor reduced tumor growth and invasiveness, and hyaluronan oligomers were nontoxic and nonimmunogenic in vivo. On the basis of these findings, we propose that hyaluronan oligomers decrease invasiveness and enhance drug sensitivity in glioma stem cell-like cells and that this mechanism is mediated by the suppression of EGFR and c-MET activity and Akt-mediated BCRP function.


Jakub A. Godlewski, M. Oskar Nowicki, E. Antonio Chiocca and Sean E. Lawler; Dardinger Laboratory for Neurosciences and Neuro-Oncology, Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA

The aim of this study was to identify gene expression changes during glioma invasion. We used a modified 3-dimensional spheroid invasion assay with dissociated glioblastoma cell flank tumors from athymic mice, allowing us to obtain milligram quantities of RNA. Samples were obtained during the course of cell migration into a collagen I matrix, followed by quantitative RT-PCR. We initially examined the candidate tumor suppressor genes associated with tumor progression, which have not been examined in gliomas. A group of genes transcriptionally downregulated several-fold during glioma cell migration includes antagonists of the Wnt signaling pathway. These genes (sFRP-1, 3, and 4 and DKK-1) encode for secreted proteins that are able to bind either directly to Wnt molecules or to their transmem-brane receptors (Frizzled or LRP5/6), serving as competitive antagonists of Wnt signaling. The other 2 genes significantly downregulated in migrating glioma cells were 14-3-3σ and its upstream transcriptional regulator, p53. Interestingly, migration correlates well with the level of p53 expression and with the degree of 14-3-3σ disappearance. We are currently assessing the importance of these molecules in glioma invasion and the mechanism by which their expression is regulated.


Lorin M. Henrich and Isa M. Hussaini; Department of Pathology, University of Virginia, Charlottesville, VA, USA

Glioblastoma multiforme (GBM) is the most common astrocytoma and is associated with a poor prognosis and limited therapeutic options. One hallmark of GBM is aggressive invasion of the surrounding brain tissue, resulting in rapid tissue destruction. Increased matrix metalloproteinase 9 (MMP9) expression has been associated with increased astrocytoma progression, and inhibition of MMP9 secretion results in decreased tumor invasiveness. However, the mechanisms involved in regulation of MMP9 expression and activity are poorly understood. We provide data suggesting a novel role of the alpha-1A adrenergic receptor (α1AADR) as a key regulator of MMP9 expression and activity. The expression of α1AADR and GAPDH in normal human astrocytes (NHAs), GBM cell lines, and GBM surgical specimens was evaluated by reverse-transcriptase PCR (RT-PCR). The expression of MMP9, MMP2, and GAPDH was evaluated in U1242 GBM cells by RT-PCR. For MMP9 activity, U1242 cells were treated with phenylephrine (PE) or pretreated with the α1ADR antagonist, prazosin, or the α1AADR antagonist, RS100329. The conditioned media was concentrated and analyzed by zymography. For the invasion assays, Boyden chamber inserts were coated with type IV collagen and seeded with U1242 cells prior to treatment with PE or pretreatment with RS100329. α1AADR-specific transcript expression was detected by RT-PCR in RNA extracted from NHAs and the GBM tumor cell lines U1242, U251, U87, and U373. α1AADR expression was also detected in RNA extracted from 4 GBM surgical specimens. In unstimulated U1242 cells, expression of MMP9 was undetectable by RT-PCR. Stimulation with PE resulted in induction of MMP9 expression by 3 hrs, with decreased expression by 24 hrs. The level of expression of MMP2 or GAPDH did not change with PE treatment, and pretreatment of the cells with prazosin or RS100329 blocked PE-induced expression of MMP9, suggesting that the effect is specific to α1AADR. Treatment with PE resulted in enhanced activity of secreted MMP9, which was blocked by pretreatment with RS100329. Furthermore, stimulation of U1242 cells with PE resulted in a 2-fold increase in cell invasion compared with the vehicle control, and the increase in invasion was inhibited by 69% after pretreatment with RS100329. These data show that α1AADR is expressed in normal astrocytes, glioblastoma cell lines, and patient tumor specimens. Specific activation of α1AADR induces upregulation of MMP9 expression and activity and enhances the invasive capacity of glioblastoma cells in vitro. Our novel findings suggest a role for α1AADR-mediated regulation of MMP9 activity and invasion in glioblastoma and provide a potential new target for therapeutic intervention.


D. Langhans, D. Zirkel, M. Westphal, K. Lamszus, and O. Heese; Department of Neurosurgery, University Medical Center, Hamburg-Eppendorf, Germany

Various in vivo studies have demonstrated that neural stem cells (NSCs) have a migration tendency towards intracranial gliomas, making these cells a potential carrier for the delivery of therapeutic genes to disseminated gliomas. Little is known about the direct effects of NSC on glioma biology. We analyzed glioma and NSC migration in response to conditioned media of the corresponding cell type in 3 different in vitro assays. Five glioma cell lines were exposed to conditioned media of the murine neural stem cell line C17.2, and spheroid proliferation (15 days) and migration (5 days) were assessed. With the identical experimental set-up, C17.2 neurospheres were exposed to conditioned media of the 5 glioma cell lines. In addition, in an organotypic brain slice assay, the effects of either conditioned media of glioma cells on NSC migration or the effects of NSC conditioned media on glioma migration were evaluated using a confocal laser microscope on day 2, 6, and 12. NSCs and glioma cells were identified inside the murine brain slice by pre-implantation staining with DiI and DiO. In 3 of 5 glioma cell lines, migration and invasion were augmented by NSC-conditioned media. No inhibitory effect of NSC-conditioned media on glioma migration was seen at all. On the other hand, the conditioned media of glioma cells augmented NSC migration heterogeneously, ranging from almost no stimulation in 2 glioma cell lines to strong stimulation in 1 glioma cell line. Co-culturing of NSCs and glioma cells inside the brain slice resulted in a directed migration of both cell types towards each other in 3 of 5 glioma cell lines. In 3 different in vitro assays, we demonstrated a stimulatory effect of NSC-conditioned media on glioma cell migration and invasion, making the postulated hypothesis of an intrinsic glioma-inhibitory effect of NSC questionable. On the other hand, migration of NSCs towards gliomas in our assay system seems to depend on individual phenotypic characteristics and the growth factor release patterns of the target glioma tissue.


Laura J. Lewis-Tuffin and Panos Z. Anastasiadis; Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA

p120 catenin is a member of the Armadillo-domain family of proteins, which play a variety of roles in modulating cell-cell adhesion and cell motility. p120 itself interacts with the cytoplasmic tail of classical cadherins, regulating cadherin clustering and intercellular junction stabilization. p120 also regulates the activity of the small GTPases RhoA, Rac, and Cdc42, which organize the cytoskeleton to regulate cell adhesion and cell motility. Under certain circumstances, p120 can also be found in the nucleus, where it interacts with the transcriptional repressor Kaiso to regulate gene expression. p120 is increasingly recognized as an important protein in the processes of epithelial tumor cell invasion and metastasis. Our previous data indicate that p120 exerts its pro-invasive actions, in part, through its interaction with mesenchymal cadherins. Little is known about the role of the cadherin-catenin complex in gliomagenesis or glioma invasiveness. Our initial studies focused on the characterization of the cadherin-catenin complexes in a large panel of human glioma cell lines that were grown on plastic or propagated as xenografts in nude mice. Almost all cell lines tested predominantly expressed the full-length p120 isoform 1. The level of p120 expression and the relative abundance of the shorter p120 isoform 3 varied among the different lines. With 1 exception, no glioma cell lines expressed E-cadherin. Almost all expressed N-cadherin, with many co-expressing cadherin 11 and some expressing cadherin 6 as well. Levels of p120 expression correlated well with the relative expression levels of mesenchymal cadherins, suggesting that p120 stabilizes these cadherins in glioma cells. To address the hypothesis that p120 and mesenchymal cadherins promote the motile phenotype of gliomas, we are now testing the migration and invasiveness of p120- or cadherin-depleted glioma cells in vitro in response to serum or EGF. We anticipate that an understanding of the relative significance of cadherin-catenin interactions in glioma cell invasiveness will lead to novel targets for glioma therapy.


Ta-Jen Liu,1 Tiffany LaFortune,1 Ningyi Tiao,1 Yihong Zhou,2 John de Groot,1 Heidi Lane,3 Greg Fuller,1 Zhimin Lu,1 and W.K. Alfred Yung1; 1Brain Tumor Center, Departments of Neuro-Oncology and NeuroPathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2Neurobiology and Developmental Science, Arkansas Cancer Research Center, University of Arkansas for Medical Science, Little Rock, AR, USA; 3Novartis Pharma AG, Basel, Switzerland

Neuropilin-1 (NRP-1) was initially identified as a receptor that controls axon guidance during neuronal development. It was subsequently revealed that NRP-1 acts as a co-receptor of VEGF and regulates tumor angiogenesis. An analysis of SAGE data from the Cancer Genome Anatomy Project (CGAP) demonstrated an inverse correlation between tumor grade and NRP-1 expression, suggesting that NRP-1 has a role in tumor progression. We investigated the functional role of NRP-1 in gliomagenesis. Upon exposure to hypoxic growth, expression of NRP-1 was stimulated in glioma cells, with a concomitant increase of the HIF-1α level, suggesting that NRP-1 is a hypoxic responsive gene. Accordingly, 3 putative hypoxia-inducible factor (HIF-1) binding sites were identified in the 5′ un-translated region of the NRP-1 gene by sequence analysis and were further confirmed by the in vivo occupation of HIF-1 protein via a chromatin immunoprecipitation assay (Chip). Binding of HIF-1α to all 3 sites increased significantly when cells were treated with CoCl2 or subjected to hypoxic growth. Forced NRP-1 expression stimulated glioma cell migration and invasion and enhanced anchorage-independent growth in vitro and tumor formation in vivo, supporting its role in promoting tumor progression. Conversely, inhibition of endogenous NRP-1 expression by siRNA attenuated glioma cell invasion. Furthermore, pharmacologic inhibition of mammalian target of rapamycin (mTOR) activity with RAD001 led to a reduction of NRP-1 expression and cell invasion, supporting the idea that HIF-1 regulates NRP-1 expression. Exogenous NRP-1 expression promoted both the in vitro anchorage-independent and in vivo intracranial growth of glioma cells. More importantly, staining of in vivo tumor sections with synaptophysin revealed a profound perivascular infiltration of NRP-1 expressing cells compared with parental cells. NRP-1 expression resulted in activation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK), suggesting that NRP-1 signaling activates genes whose activity favors glioma proliferation and invasion.


Yevgeniy Lukyanov, Tona Schnee, Mine Esencay, Elizabeth W. Newcomb, and David Zagzag; Microvascular and Molecular Neuro-Oncology Laboratory, Departments of Pathology and Neurosurgery, NYU School of Medicine, New York, NY, USA

HSPs are cellular chaperone proteins required for essential housekeeping functions such as protein folding, assembly, and transportation across different cell compartments. HSP90 is ubiquitously and abundantly expressed and involved in maintaining the correct conformation and stability of its client proteins. Numerous proteins involved in the control of physiologic and pathophysiologic processes require HSP90 for their biogenesis, regulation, and functionality. The expression of HSP90 is increased in cancer compared with normal tissues, suggesting it has a role in maintaining malignant transformation. Because some HSP90 client proteins include Akt, Her2/Neu, and Raf-1, important participants in pathways driving tumor progression, HSP90 would make an excellent target for cancer therapy. Geldanamycin and its analogue, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), inhibit HSP90 function, which in turn, inhibits several critical steps involved in cell invasion. Here, we investigated the effects of 17AAG on the migration of murine GL261 glioma cells using an in vitro migration assay. GL261 cells were plated on the chamber inserts in the absence or presence of 17AAG for 16 h, and the number of cells that migrated through was assessed. In the presence of 200nM 17AAG, the migration of GL261 cells was inhibited by approximately 50% (P < 0.01). One HSP90 client, matrix metal-loproteinase (MMP)-2, plays a critical role in the degradation of extracellular matrix proteins and promotes glioma cell invasion. GL261 cells were grown in the absence or presence of increasing concentration of 17AAG for 24 h, and the level of MMP-2 protein secreted into the conditioned medium was measured by gelatin zymography. The results showed that 17AAG treatment decreased the secretion of MMP-2 in a dose-dependent manner. Because extracellular signal-regulated kinases (ERKs) 1/2 are associated with cellular migration in a wide variety of cell types, we next determined the effect of 17AAG treatment on ERK phosphorylation. We performed a Western blot analysis and observed that 17AAG downregulated ERK phosphorylation as early as at 24 h. In summary, 17AAG significantly reduced glioma cell migration, and secretion of MMP2. 17AAG significantly reduced the expression of phospho-ERK. Collectively, our data show that 17-AAG may be of therapeutic value for the treatment of patients with high-grade gliomas.


Debra A. Mayes,1 Yuanjie Hu,1 Yue Teng,1 Xiaosong Wu,1 Kishori Panda,1 Longjian Liu,1 Eric Siegel,1 Fang Tan,2 W.K. Alfred Yung,2 and Yi-Hong Zhou1; 1 University of Arkansas for Medical Sciences Arkansas Cancer Center, Little Rock, AR, USA; 2 The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Glioblastoma (GBM) is the most invasive brain tumor. We previously reported that the transcription factor PAX6 suppresses the tumorigenicity of GBM cells. By an in vitro matrigel invasion assay on 2 GBM cell lines stably transfected with wild-type and/or 2 mutant forms of PAX6, we found the first evidence that PAX6 inhibits the invasiveness of GBM cells and that the DNA-binding domain is required for this effect. By real-time quantitative reverse transcription (QRT) PCR, gelatin zymography, and immunohistochemistry assays, the expression of the gene-encoding matrix metalloproteinase-2 (MMP2) in GBM cell lines grown in vitro or in intracranial (i.c.) xenografts in nude mice was shown to be repressed by either stable or adenovirus-mediated overexpression of PAX6. Luciferase promoter and electrophoretic mobility shift assays revealed that PAX6 bound directly to the MMP2 promoter and regulated the promoter activity. The knockdown of MMP2 in cells transfected with a dominant-negative mutant of PAX6 displayed a significant decrease in invasiveness, but it was not as low as that of PAX6 transfectant. The Spearman rank correlation test showed significant reverse correlations (P < 0.05) between PAX6 and MMP2 expression, as quantified by real-time QRT-PCR in human tissue specimens (41 GBMs, 43 anaplastic astrocytomas, and 7 adjacent normal tissues). Interestingly, the degree and significance of the reverse correlation was increased after excluding anaplastic astrocytomas, but it became insignificant after excluding GBMs. All statistical tests were 2-sided. Overall data revealed a mechanism for PAX6’s suppression function in GBM via suppressing cell invasiveness. MMP2 is one of the PAX6 target genes mediating its suppression of invasion.


Jessica McCready,1 Zendra E. Zehner2 and Helen L. Fillmore1,3; Harold F. Young Neurosurgical Center, Departments of 1Anatomy and Neurobiology, 2Biochemistry and 3Neurosurgery, Virginia Commonwealth University, Richmond, VA, USA

A functional single nucleotide polymorphism (SNP) at position -1607 in the matrix metalloproteinase-1 (MMP-1) promoter results in significantly higher MMP-1 transcriptional activity in glioma cell lines. This SNP consists of the presence (2G) or absence (1G) of a guanine nucleotide at position -1607. We recently reported that the distribution of the MMP-1 genotype differed significantly between the healthy population and the glioblastoma patient population, with the 2G/2G genotype being more prevalent in glioblastoma patients. In addition, MMP-1 mRNA and protein levels in a select group of tissues were significantly higher than those in normal brain control tissues. The additional guanine nucleotide creates a binding site for ETS transcription factors and, combined with an adjacent AP-1 binding site at position –1602, creates a Ras-responsive element (RRE), which is responsible for synergistic increases in transcription when stimulated by Ras. We determined the potential signaling mechanisms responsible for increases in MMP-1 transcription due to the presence of the RRE in response to hepatocyte growth factor/scatter factor (HGF/SF) treatment. Human glioma cells T98 (1G/1G) and U251 (2G/2G) expressed the cMet receptor for HGF/SF, as assessed by Western blot analysis. HGF/SF-treated T98 cells had a 2-fold increase in MMP-1 mRNA levels. In contrast, MMP-1 levels were increased by more than 10-fold in HGF/SF-treated U251 cells. Total ERK protein levels did not change in response to HGF/SF treatment; however, phosphoERK appeared in the nucleus within 10 minutes of stimulation. The addition of the MEK inhibitor U0126 prevented the activation of phosphoERK. In addition, HGF/SF led to significant increases in MMP-1 transcription via the MAP kinase ERK pathway. The levels of the AP-1 transcription factor proteins cJun and cFos were increased in response to HGF treatment, but the levels of Ets-1 and ETV-1 did not increase. The addition of U0126 inhibited the increase in AP-1 protein levels. Results from chromatin immuno-precipitation assays demonstrated that cFos and cJun bound to both the 1G and 2G promoters after HGF/SF treatment; however, the amount of cJun associated with the 2G promoter was significantly higher. HGF/SF also led to an increase in Ets-1 binding to the 2G MMP-1 promoter. ETV1 bound only to the 1G promoter, and this binding was not affected by HGF/SF. Treatment with the MEK inhibitor U1026 inhibited protein binding to both the 1G and 2G promoters The results from our study indicate that HGF/SF induces binding of cJun, cFos, and Ets-1 to the additional RRE in the MMP-1 2G distal promoter. The data presented herein reveals 1 possible mechanism for the difference in transcriptional activity between the 1G and 2G MMP-1 promoters in glioma cells. The MMP-1 SNP (-1607) may contribute to tumor function and glioma invasion, especially in response to growth factors such as HGF/SF.


Masaki Morishige, Shigeru Hashimoto, Tatsuya Abe, Hidenori Kobayashi and Hisataka Sabe; Department of Neurosurgery, School of Medicine, Oita University, Oita, Japan; Departments of Molecular Biology, Osaka Bioscience Institute, Osaka, Japan

Glioblastoma multiforme (GBM) is the most invasive form of glioma and is extremely refractory to therapy. Invasive phenotypes are considered to be a principal predictor of poor prognosis. Determining which molecules regulate invasion will thus contribute to improved GBM treatments. We have previously showed that Arf6 plays an important role in the invasive activities of human breast cancer and that AMAP1, an effector of GTP-Arf6, is involved in the invasive mechanism by binding Cortactin and Paxil-lin in breast cancer. We found that AMAP1 was highly expressed in primary human gliomas, and the AMAP1-mediated trimeric protein complex was also detected in GBM cells. Furthermore, blockage of this complex formation by cell-permeable proline peptide derived from the AMAP1 inhibited GBM invasion in vitro. Our results indicate that Arf6 and its regulator are the major components of cancer invasive activities and may be novel pharmaceutical targets for preventing GBM invasion.


Mitsutoshi Nakada,1 Kelsey L. Drake,1 Tim Demuth,1 Linsey B. Reavie,1 Satoko Nakada,1 Jean-Claude Zenklusen,2 Howard A. Fine,2 Tom Mikkelsen,3 and Michael E. Berens1; 1The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA; 2National Cancer Institute, Bethesda, MD, USA; 3Henry Ford Hospital, Detroit, MI, USA

To determine the molecular biologic drivers of the malignant phenotype of glioma, it is crucial to identify the major molecules and gene products that contribute to glioma invasion. Two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. The bidirectional receptor/ligand signaling system, EphB/ephrin-B, was associated with the invading cell phenotype, as determined by pathway enrichment analysis. Eph/ephrin, whose mutual activation causes dispersive effects on cell-cell contact, represents the largest family of tyrosine kinases in humans, and its signaling is involved in neurodevelopmental processes, including morphogenesis, cell migration, and vascular formation. The clinical relevance of EphB/ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of certain EphB/ephrin-B family members’ mRNA, including ephrin-B1 and –B2, were significantly higher in GBM samples (n = 82) than in normal brain tissues (n = 24). A Kaplan-Meier analysis demonstrated that ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short-term survival in malignant astrocytoma patients (n = 97, P = 0.016). On an immunohistochemical analysis, ephrin-B2 was localized primarily in GBM cells and not in normal brain tissue. A moderately invasive glioma cell line, U87, expressed higher levels of ephrin-B2 than did less invasive glioma cell lines (U251, T98G, G112, U118, and SF767). The invasion of U87 was accelerated by the addition of EphB2/Fc chimera, which activates ephrin-B. U87 cells transfected with ephrin-B2 siRNA decreased invasion in vitro, whereas ephrin-B1 siRNA did not affect invasion activity. The depletion of endogenous ephrin-B2 expression abrogated the increase of invasion by EphB2/Fc stimuli, indicating that increased invasion is dependent on ephrin-B2 activation. Concomitant with this data, increased Akt phosphorylation was observed in the presence of EphB2/Fc, where reduction of ephrin-B2 by siRNA negated the increased phosphorylation of Akt caused by the addition of EphB2/Fc, indicating that increased Akt phosphorylation is directly linked to ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a powerful predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion, making this signaling pathway a potential therapeutic target.


M. Oskar Nowicki, Jennifer L. Cutter, E. Antonio Chiocca and Sean Lawler; The Dardinger Laboratory for Neuro-Oncology and Neurosciences, Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA

Infiltration of normal brain tissue by invading tumor cells is a major factor in the recurrence and poor prognosis of malignant gliomas. Therapeutic strategies to prevent the invasion process or target invading cells are, therefore, highly sought after. Here, we report that the mood-stabilizing drug lithium potently inhibits glioma cell invasion and that this inhibition is mediated by glycogen synthase kinase-3 (GSK-3). Lithium chloride (LiCl) treatment blocked invasion in all the cell lines we have evaluated by spheroid invasion, transwell migration, and scratch assays. The inhibition was dose-dependent and reversible even after prolonged (96-hour) LiCl exposure. One of the best-characterized targets of lithium action is GSK-3. We found that sphere expansion was blocked by 2 specific GSK-3 inhibitors, verifying that GSK-3 inhibition plays a role in invasion. Microscopic studies of drug-treated cells revealed a change in morphologic characteristics, with the cells no longer sending out protrusions at the leading edge. These data suggest that targeting GSK-3 or GSK-3-related pathways may be relevant in the treatment of invasive brain tumors.


Baisakhi Raychaudhuri and Michael A. Vogelbaum; Brain Tumor Institute, Cleveland Clinic, Cleveland, OH, USA

We previously showed that the latent transcription factor NF-κB strongly mediated the invasive behavior of malignant glioma cell lines in vitro. Interruption of NF-κB activation by IκB-super repressor (IκB-SR) significantly compromised the migration and invasion of glioma cell lines, as measured by a matrigel-Boyden chamber assay. Interleukin-8 (IL-8) is a pleiotropic chemokine that is aberrantly expressed in many GBM cell lines and is a known target of NF-κB. We found that expression of this chemokine in gliomas is, in large part, attributable to an aberrant, persistent activation of the transcription factor NF-κB. Levels of IL-8 released into a culture medium paralleled the levels of aberrant NF-κB activation. Over-expression of IκB-SR, a potent inhibitor of NF-κB activity, significantly reduced IL-8 mRNA transcription and protein release into the cell culture medium. These findings led us to hypothesize that IL-8 is responsible for the regulation of NF-κB-dependent invasion by glioma cell lines. In support of this hypothesis, we have found that treating glioma cell lines with an IL-8-neutralizing antibody markedly decreased their invasiveness in the matrigel-Boyden chamber assay compared with cells treated with control IgG or those left untreated. Ongoing studies are focused on inhibitors of the IL-8 receptor. These data are the first to link the invasiveness of GBM cells to aberrant expression of IL-8.


L.B. Reavie,1 T. Demuth,1 D.B. Hoelzinger,1 J.L. Rennert,1 R. Bristol,1 S. Nakada,1 J.C. Zenklusen,2 H.A. Fine,2 T. Mikkelsen,3 and M.E. Berens1; 1The Translational Genomics Research Institute, Phoenix, AZ, USA; 2The National Cancer Institute, Bethesda, MD, USA; and 3Henry Ford Hospital, Detroit, MI, USA

The clinical management of glial tumors is confounded by the propensity of these malignant cells to disperse into the brain, frequently seeding distant sites of recurrence. This represents a central behavior in the malignant progression of these cells, but dispersion is poorly addressed by current therapies. Failure to manage these invading cells leaves patients vulnerable to recurrence. We hypothesized that the invasive behavior of glioma cells is driven by a distinct gene expression profile and that among the invasion-associated genes are novel therapeutic targets. The aim of this study was to broaden the scope of a pilot study that successfully identified genes previously unassociated with glioma invasion and to mature the biologic validation of candidate genes using 2-dimenstional and 3-dimensional endpoints of glioma dispersion. Whole human genome expression profiling of stationary and dispersive cells in glioma biopsy specimens (n = 19) isolated by laser capture microdissection (LCM) was performed. T-testing (P < 0.001) identified 3 novel genes not previously associated with glioma. In a clinically annotated data set of 171 glial tumors, these genes were identified as markers of glioma progression and predictors of patient survival. Our results demonstrate that biopsy-based LCM-collected glioma core and rim cells can be used as a discovery platform for identifying novel genes that may serve as prognostic markers for or therapeutic targets of malignant glial tumors. A key driver for progress in maturing candidates into therapeutic targets of invasive glioma cells is the ability to validate the biologic effects using mid- and high-throughput assays of glioma dispersion. This study is supported by grants NS42262 and CA085139.


B. Salhia,1 N. Tran,2 M. Nakada,2 A. Chan,3 M. Berens,1 J.T. Rutka,1 and M. Symons3; 1The Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, The University of Toronto, Toronto, Ontario, Canada; 2Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix, AZ, USA; and 3Center for Oncology and Cell Biology, The Feinstein Institute for Medical Research at North Shore-LIJ, Manhasset, NY, USA

The invasion of tumor cells into regions of normal brain tissue limits current therapies for malignant astrocytoma. We recently demonstrated a critical role of Rac1, a member of the Rho family of small GTPases, in glioma invasion. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). There are 26 known Rac-GEFs in the human genome. To identify Rac-GEFs that contribute to glioma invasion, we mined microar-ray data obtained from 111 human malignant astrocytoma specimens and 24 nonneoplastic brain specimens. Three Rac-GEFs, Ect2, Trio, and Vav3, displayed consistent association with high-grade tumors and poor survival, whereas the expression levels of Rac1 were similar across the panel examined. We performed quantitative PCR to validate the expression of these Rac-GEFs in independent clinical specimens. An immunohistochemical analysis of Rac-GEF expression in human brain specimens is in progress. Using both radial migration and ex vivo brain slice invasion assays, we also showed that the siRNA-mediated depletion of Ect2, Trio, or Vav3 in glioblastoma cell lines significantly inhibited their invasive properties. The depletion of any GEF caused no significant changes in glioblastoma cell proliferation. We hypothesize that the respective Rac-GEFs mediate the activation of Rac by specific receptors. In summary, our results suggest that Rac-GEFs may be a novel target in the treatment of astrocytoma.


Susobhan Sarkar,1 Robert K. Nuttall,2 Shuhong Liu,1 Dylan R. Edwards,2 and V. Wee Yong1, 3; Departments of 1Oncology and 3Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada; 2School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom

The ability of glioma cells to extensively invade the central nervous system is a major cause of the high morbidity of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to the extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished, in part, by matrix metalloproteinases (MMPs) within a 3-dimensional (3D) milieu of the brain parenchyma. Most studies have used a 2D monolayer culture system; however, we used a 3D matrix of collagen type 1 (CL) gel to address glioma-secreted proteases, ECM, and the invasiveness of glioma cells in vitro. Previously, we found that tenascin-C (TN-C), a commonly elevated ECM in high-grade gliomas, stimulated glioma cell invasion when incorporated into the 3D CL matrix. In the present study, we determined the role of MMPs in CL/TN-C-stimulated glioma invasion, the modulation by inflammatory cytokines known to be present in the tumor microenvironment, and the signaling cascade involved. The TN-C mediated invasion in the 3D CL matrix was blocked by metalloproteinase inhibitors BB-94, GM6001, and TIMP-1, but this did not involve the gelatinases (MMP-2 and -9) commonly implicated in 2D glioma growth. A thorough analysis of 21 MMPs and 6 ADAM members as determined by Taqman real-time PCR analyses showed that MMP-12 was increased in gliomas by TN-C in a 3D matrix. An elevated level of MMP-12 transcripts was also detected in high-grade GBM specimens compared with low- or mid-grade GBM or normal brain tissue. A Western blot analysis of the conditioned medium showed increased expression of the pro and active forms of MMP-12 in U251 or U178 glioma cell lines when grown in a 3D CL/TN-C matrix compared with the 3D CL control or 2D poly-ornithine (PO) coatings. Moreover, function-blocking antibodies to MMP-12 and small interfering RNA (siRNA) to MMP-12 attenuated the TN-stimulated glioma invasion, ascertaining a role for MMP-12 in regulating glioma invasiveness through interaction with TN-C. We tested the role of IL-1β, a microglia/monocyte derived cytokine, and found this to further stimulate the invasiveness of glioma cells embedded in the CL/TN-C 3D matrix. Glioma invasiveness was blocked by pharmacologic inhibitors with relative selectivity for protein kinase C (PKC), myosin light chain kinase, and src tyrosine kinase pathways. Calphostin C, a relatively selective inhibitor for PKC, was found to decrease TN-C mediated glioma invasion in a dose-dependent manner. Rottlerin, a PKC delta-specific inhibitor, showed a similar result. In addition, subcellular studies for PKC translocation as an indicator of PKC activation strongly implicated PKC alpha, delta, and epsilon isoforms in CL/TN-C-mediated glioma invasion. Overall, the results of this study suggest that in 3D growth, TN-C is a favorable substrate for glioma invasiveness and that its effect is mediated through MMP-12 and PKC and further modulated by inflammatory cytokines in the glioma microenvironment.


Charles B. Stevenson, Karen K. Deal, Stephanie M. Miller, Juan G. Valadez, Jason A. Winston, Reid C. Thompson, and Moneeb Ehtesham; Department of Neurological Surgery, Vanderbilt University Medical Center, Nashville, TN, USA

The prognosis of patients with malignant gliomas remains dismal. The clinical aggressiveness and treatment-refractory nature of these neoplasms is derived, in large part, from their highly proliferative and infiltrative nature. As such, the development of an effective therapeutic modality for these tumors will require a better understanding of the specific biologic cues that drive glioma growth and invasiveness. We determined whether the in vitro and in vivo association of the cell surface chemokine receptor, CXCR4, was associated with histologic grade and the development of an invasive phenotype in glioma. Using fully quantitative real-time PCR, we analyzed the expression levels of CXCR4 and its corresponding ligand CXCL12 in 90 unique patient-derived glioma tissue samples. The expression of CXCR4 and CXCL12 was further verified at the protein level using immunohistochemical analysis. We then determined the functional role of CXCR4 in glioma by assessing the contribution of this receptor to tumor cell invasiveness. Using an experimental rodent model of intracranial glioma, we isolated infiltrative glioma cells by means of laser capture microdissection and once again analyzed CXCR4 expression levels using quantitative real-time PCR. Subsequently, glioma cells were treated with CXCR4 neutralization antibody or small-interference RNA technology, and their infiltrative capabilities were characterized by an in vitro matrigel invasion assay. We demonstrated that CXCR4 expression correlated strongly with increasing tumor grade in World Health Organization grade II through IV gliomas (P = 0.0006) and found a significant association between CXCR4 and CXCL12 expression levels in a given grade of tumor. In addition, we found substantially elevated expression of CXCR4 in infiltrative glioma cells compared with noninvasive tumor cells. Importantly, abrogation of the CXCR4 function significantly impaired glioma cell invasiveness in matrigel-based tumor infiltration assays. Together, these findings demonstrate a strong association between expression of CXCR4 and histopathologic aggressiveness, as well as assumption of an invasive phenotype in glioma cells. In light of this, these data underscore the importance of CXCR4 as a potential therapeutic target for the treatment of malignant glioma.


Nhan L. Tran,1 Wendy S. McDonough,1 Benjamin A. Savitch,1 Shannon P. Fortin,1 Jeffrey A. Winkles,2 Marc Symons,3 Mitsutoshi Nakada,1 Heather E. Cunliffe,1 Galen Hostetter,1 Dominique B. Hoelzinger,1 Jessica L. Rennert,1 Jennifer S. Michaelson,4 Linda C. Burkly,4 Christopher A. Lipinski,5 Joseph C. Loftus,5 Luigi Mariani,6 and Michael E. Berens1; 1Cancer and Cell Biology Division, Translational Genomics Research Institute, Phoenix, AZ, USA; 2Departments of Surgery and Physiology and the Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA; 3Center for Oncology and Cell Biology, The Feinstein Institute for Medical Research at North Shore-LIJ, Manhasset, NY, USA; 4Biogen Idec Inc., Cambridge, MA, USA; 5Mayo Clinic Scottsdale, Scottsdale, AZ, USA; and 6University Hospital, Inselspital, Bern, Switzerland

Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion into normal brain. Understanding the genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. We found that the fibro-blast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, was expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 over-expression stimulated glioma cell migration and invasion, and depletion of the small GTP-binding protein, Rac1, by siRNA inhibited this cellular response. Activation of Fn14 signaling by its ligand, TNF-like weak inducer of apoptosis (TWEAK), stimulated migration and upregulated expression of Fn14; this TWEAK effect required Rac1 and nuclear factor κB (NF-κB) activity. The Fn14 promoter region contains NF-κB binding sites that mediate positive feedback, causing sustained overexpression of Fn14 and enduring glioma cell invasion. Fn14 gene expression levels increased with glioma grade; in GBM specimens, the levels of Fn14 expression were inversely correlated with patient survival. These results demonstrate that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Currently, we are testing the applicability of the inhibition of the Fn14 pathway by functional blocking monoclonal antibodies against Fn14 as a means to selectively target invasive glioma cells. An analysis of Fn14 protein expression on glioma xenograft tissue microarrays revealed 2 xenografts with high Fn14 expression. Orthotopic xenograft studies using these two human glioblastomas are in progress to assess the effects of biologic inhibitors of the Fn14 pathway on the induction of cell death by a cytotoxic agent, temozolomide (Temodar), and the effects on the extent of tumor invasion. A histologic assessment of tumor size, invasion pattern, and cell death will be used to determine the effectiveness of the biologic inhibitors to Fn14. These studies are currently underway to assess the suitability of Fn14 as a targeted therapy against invasive human glioma cells.


George K. Wang, Limei Hu, Gregory N. Fuller, and Wei Zhang; Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Previous studies have established IGFBP2 as one of the most frequently overexpressed genes in high-grade gliomas. Our in vitro studies also showed that IGFBP2 promotes cell mobility and cell invasion. Enhancement of glioma cell invasion is at least partially attributed to elevation of MMP2 expression by IGFBP2, but it is not clear how IGFBP2 augments cell mobility. Our previous microarray studies showed that IGFBP2 activates the expression of integrin α5. A structural analysis revealed that IGFBP2 has an Arg-Gly-Asp (RGD) domain, which is a known integrin-binding motif. Therefore, we hypothesized that IGFBP2 enhances cell motility via interaction and activation of integrin α 5. We confirmed our microarray results by demonstrating that the expression of integrin α 5 is upregulated at the protein level in IGFBP2-overexpressing SNB19 glioma cells. Co-immunoprecipitation confirmed that IGFBP2 does indeed interact with integrin α 5. To further confirm that IGFBP2 interacts directly with integrin _5 through the putative RGD domain on IGFBP2, we created an RGD → RGE mutant (D306E) IGFBP2. Co-immunoprecipitation then showed that D306E-IGFBP2 had no detectable binding with integrin α 5. We further observed that IGFBP2-overexpressing cells displayed extensive cell surface lamellipodia, whereas D306E-IGFBP2-overexpressing cells showed abundant cell surface focal adhesions. Consistent with this, a phenotype analysis showed that IGFBP2-overexpressing cells had elevated migration rates compared with the vector control; in contrast, D306E-IGFBP2-overexpressing cell migration rates were not elevated and were comparable to that of the vector control. Using siRNA to knock down the expression of integrin α 5, we further established the necessity of both IGFBP2 and integrin α 5 in this cell mobility pathway. We further demonstrated that this pathway required the cells to be sufficiently anchored to a surface and be in the presence of a specific extracellular matrix component, fibronectin, to be activated. We conclude that 1 pathway by which IGFBP2 activates glioma cell mobility is through its interaction with integrin α 5; this interaction is specifically mediated through an integrin-binding domain on IGFBP2, and the activation of this pathway requires the presence of a fibronectin.


Wesley J. Whitson, M. Oskar Nowicki, Nina Dmitrieva, Rachel Kantosky, E. Antonio Chiocca and Sean E. Lawler; Dardinger Laboratory for Neurosciences and Neuro-Oncology, Department of Neurological Surgery, The Ohio State University Medical Center, Columbus, OH, USA

The microtubule-binding protein doublecortin (DCX) regulates neuronal migration during development and has recently been identified as a potential pro-invasive gene in glioblastoma multiforme (GBM). In this study, we determined the expression of DCX and the related molecule doublecortin-domain containing protein 1 (DCDC1) in glioma cell lines and patient tumor specimens. We observed 5- to several hundred-fold upregulation of both genes in glioma cell lines compared with normal astrocytes and in 4 of 6 (DCX) and 6 of 6 GBM samples (DCDC1) compared with normal brain tissue. In these specimens, increased expression of up to 100-fold was observed for both genes. Upregulation was confirmed by immunostaining for DCX and Western blotting for DCDC1. In ongoing studies, we are assessing the functional importance of these genes in glioma biology, particularly with regard to cell migration/invasion.



Mary Lou Affronti, Jeannette M. Dowell, James E. Herndon II, Joan Cahill, Jeremy N. Rich, Jennifer A. Quinn, David A. Reardon, James J. Vredenburgh, Annick Desjardins, Sridharan Gururangan, and Henry S. Friedman; The Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA

We conducted a retrospective data analysis to determine the overall survival rate of 82 primary GBM patients who were diagnosed at Duke between 2000 and 2004 and who received radiation therapy and concurrent temozolomide, followed by adjuvant rotational multiagent chemotherapy (consisting of temozolomide, CCNU, and CPT-11) for 12 months. Our adjuvant approach used multiple chemotherapeutic agents with different tumoricidal mechanisms to prevent tumor resistance. Twenty-seven percent of the patients were women, and 73% were men. The mean age was 52 years (median age, 54 years; range, 21–76 years); 39% were [less-than-or-eq, slant] 50 years. Eighty-two percent of patients were white, 4% were African-American, and 14% were other races. Fifteen percent of patients had an ECOG performance status score of 0, 61% an ECOG score of 1, 20% an ECOG score of 2, and 4% an ECOG score of 3. Overall survival was 57% (95% CI, 47%–69%) at 1 year and 31% (95% CI, 21%–46%) at 2 years. Median survival was 63.4 weeks (95% CI, 49–80) with a median follow-up of 86 weeks (range, 14–163). Seventy-five percent of patients underwent surgical resection, and 25% underwent biopsy alone. A previous meta-analysis of 12 randomized trials comparing additional adjuvant chemotherapy to radiation alone demonstrated only a 5% increase (from 15% to 20%) in the 2-year survival rate (Stewart, Lancet, 2002). A multi-institutional phase III trial recently reported a significantly higher 2-year survival rate (26.5%) for patients receiving radiation with concomitant temozolomide and 6 cycles of adjuvant temozolomide than for patients receiving radiation therapy alone (10.4%) (Stupp et al., N Engl J Med, 2005). Although the entry criteria for our and Stupp et al.’s patient cohorts are similar, direct comparison of survival rates is problematic because of possible differences between the 2 cohorts in the case mix. Therefore, the survival experience of our cohort is examined within specific patient subgroups defined by important prognostic factors: ECOG performance status and age. Within these patient subgroups, the Kaplan-Meier estimator is used to generate estimates for 1-, 2- and 3-year survival rates with standard deviations. Additional analyses and updates of overall survival, progression-free survival and O6-alkyl-guanine-DNA alkyltransferase (AGT) status are presented. To date, we have determined that the utilization of a multi-drug regimen — in contrast to single-agent adjuvant therapy — at increased dose intensity (52 weeks) results in encouraging overall survival at 1 year (57%) and 2 years (31%). Despite the limitations of historical data analysis, these results will assist in the development the future adjuvant treatment approaches for patients with primary GBM and potential subsequent randomized trials.


Michael A. Badruddoja,1 Asha Das,2 Ray M. Chu,2 Eli Gabyan,3 Heather Trimm,2 Diane Trycieky,2 John Yu,2 Carol Hurwitz,3 Keith L. Black2; 1Center for Neurosciences and Department of Radiation Oncology, University Medical Center, University of Arizona, Tucson, AZ; 2Departments of Surgery and 3Hematology/Oncology, Cedars Sinai Medical Center-Maxine Dunitz Neurosurgical Institute, Los Angeles, CA, USA

Gefitinib (Iressa) is a small-molecule inhibitor that irreversibly inhibits tyrosine kinase activity of EGFR in micromolar concentrations. Rapamycin binds FKB-12, inhibits the activity of p-AKT, and inhibits the p70S kinase and 4E-binding protein (4E-BP1), which subsequently limits translation. Dysregulation of the EGFR and intracellular second messengers associated with this pathway are important in the pathogenesis associated with glioblastoma. Resistance to EGFR antagonists has been associated with loss of activity on an important regulatory phosphatase, PTEN. Gefitinib as a single agent has had only modest activity against malignant glioma. This study was designed to determine the efficacy and toxicity associated with the combination of gefitnib and rapamycin for patients with recurrent glioblastoma. Gefitinib was given at a dose of 500 mg daily for patients not on enzyme-inducing anti-convulsants (EIACs), while those who were on EIACs received doses titrated to a target dose of 1,500 mg daily. Rapamycin was initated at 2 mg daily and was titrated to a plasma level of 4–12 μg/ml. A treatment cycle was defined as 4 weeks. Patients were evaluated at 8–week intervals with laboratory data and brain imaging. Patients remained on therapy until tumor progression, unacceptable toxicity, or patient withdrawal occurred. A total of 21 patients were screened for enrollment, and 18 patients were evaluable. The mean age was 51.6 years. Thirteen patients were men. Six patients had biopsy only, 2 patients had subtotal resections, and 10 patients had gross total resections. The mean KPS was 77%. One patient experienced grade III rash. A second patient experienced grade III diarrhea, grade III renal failure, grade IV hypotension, grade IV dyspnea, grade IV metabolic acidosis, grade III coagulopathy. A third patient experienced grade III wound infection requiring debridement and antibiotics. A fourth patient experienced grade III elevated LFT, intratumoral hemorrhage, seizure, and hypertension. The rate of 6-month progression-free survival was 16.6%. The mean time to tumor progression was 3 months, and there were 2 minor responses. Gefitinib plus rapamycin for patients with recurrent glioblastoma is well-tolerated in a majority of patients treated. The combination produced 2 minor responses and conferred disease stability in a substantial percentage of patients.


Prasanta Banerji and Pratip Banerji; PBH Research Foundation, Kolkata, India

The recurrence of brain tumors after surgery, radiation therapy, and chemotherapy is a common problem, although many conventional and alternative therapies are available. The use of Ruta 6 and Calcarea Phosphorica 3X to treat brain tumors has gained popularity in India, and most of our patients are undergoing this treatment without any other form of conventional treatment. In other countries, however, this treatment is used mainly to prevent tumor recurrence. We analyzed the records of patients (76 from India, 144 from the United States, and 27 from other countries including the United Kingdom, Canada, Spain, Italy, Sweden, Australia, New Zealand, The Netherlands, Turkey, Israel, Malaysia, Pakistan, etc.) who underwent our treatment without undergoing any concurrent conventional therapy to prevent tumor recurrence only. The analysis showed that our treatment has a definite role in preventing the recurrence of brain tumors: 66.7% of the cases studied did not have recurrence. Prevention in oligodendroglioma was excellent but was comparatively less so in brainstem gliomas.


D.T. Blumenthal,1,2 C. Rankin,3 K. Stelzer,4 S. Schulman,5 A. Sloan,6 E. Rushing,7 L. Gavigan,8 and A. Spence9; 1Huntsman Cancer Institute and 5Health Sciences Center, University of Utah, Salt Lake City, UT, USA; 2Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; 3Cancer Research and Biostatistics, Seattle, WA, USA; 4Mid-Columbia Medical Center, The Dalles, OR, USA; 6Moffitt Cancer Center at the University of South Florida, Tampa, FL, USA; 7Armed Forces Institute of Pathology, Washington, DC, USA; 8Cancer Research and Biostatistics, Seattle, WA, USA; 9University of Washington Medical Center, Seattle, WA, USA

Despite multimodality treatment with surgery, radiation therapy, and chemotherapy, the prognosis for GBM is poor, with an average survival time of approximately 1 year. Previous SWOG studies have shown that the level of O6-alkylguanine-DNA alkyltransferase (AGT) in tumor tissue may be an important predictor for survival in patients treated with alkylating chemotherapy. AGT is a DNA repair enzyme that provides cancer cell resistance to O6-alkylating chemotherapy. High levels of this enzyme correlate with the resistance of glioma cell lines to alkylating chemotherapy. O6-benzylguanine (O6-BG) is a potent inactivator of AGT. We studied the clinical impact and toxicity of O6-BG in addition to BCNU and radiation in the treatment of newly diagnosed GBM. The study was activated in September 2001 and closed in November 2005. Eligible patients had histologically confirmed GBM or gliosarcoma. Patients were stratified by age (> 50 years or < 50 years), performance status (0–1 versus 2), and surgery of biopsy versus resection. The study was closed after an interim analysis did not show benefit of O6-BG to BCNU + RT. One hundred eighty-three patients were registered, 93 in the experimental O6-BG arm and 90 in the standard BCNU + RT arm. The median overall survival (OS) was 9 months for the standard group and 11 months for the experimental group. A 40% improvement at formal interim analysis was rejected for OS of BCNU+ RT versus O6-BG + BCNU + RT, P = .002, with a hazard ratio of 0.84, 99% confidence interval (.53–1.33). The median progression-free survival (PFS) was 4 months for both groups. A 40% improvement in PFS was ruled out at P = .001, with a hazard ratio of 0.84 and 99% confidence interval (.54–1.29). One hundred seventy patients were assessable for toxicity. Three treatment-related deaths occurred on the experimental arm: 1 patient from neutropenic sepsis, the second from febrile neutropenia, and the third from renal failure and adult respiratory distress syndrome (ARDS). Forty-five additional patients experienced primarily hematologic grade IV toxicities. Three treatment-related deaths occurred on standard therapy: 2 patients died from respiratory infection and one from ARDS. Seventeen additional patients suffered grade IV toxicities. The addition of O6-BG to the standard regimen of RT + BCNU did not improve overall survival or progression-free survival in patients with newly-diagnosed GBM in this phase III trial. Furthermore, increased toxicity was seen in the experimental group. Maturation of data and further analysis of subgroups, long-term survivors, and the effect of other prognostic factors are ongoing.


Felix Bokstein and Deborah T. Blumenthal; Neuro-Oncology Service, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

Response rates to second-line chemotherapy in recurrent high-grade glial tumors are very low, and new, effective treatments are needed. To evaluate response rates and tolerability of chemotherapy with bevacizumab and irinotecan in recurrent high-grade glial tumors, we treated 12 patients with recurrent gliomas with bevacizumab (5 mg/kg) and irinotecan (125 mg/m2) every 2 weeks. Ten patients had glioblastoma multiforme, 1 had grade III oligodendroglioma, and 1 had grade III oligoastrocytoma. All patients had been previously treated with least 1 chemotherapy regimen. Four patients began treatment immediately after surgery (gross total removal of recurrent tumor in 2 patients and subtotal removal in an additional 2 patients). The therapy was initiated in 8 patients after the previous treatment failed. Enzyme-inducing antiepileptic drugs were withdrawn, and patients were placed on nonenzyme-inducing antiepileptic medications before starting treatment. Response was evaluated (using Macdonald criteria) by gadolinium-enhanced MRI performed every 4 cycles of treatment. Seventy-five percent of patients (8/12) responded to treatment: 2 showed complete radiological response (CR), and 7 others showed a very impressive partial response (PR) with a more than 50% decrease in volume of enhancing tumor. One of the patients with CR completed the treatment after 16 cycles (8 months) and was stable for 1 month. One patient with a partial response after 4 cycles of treatment had further progression after an additional 8 cycles and eventually died of progressive disease. As of May 2006, all but 1 of the responding patients continued to receive treatment after 4 to 12 cycles of chemotherapy. Three patients developed rapid progression of disease after 2 to 4 cycles of treatment, all of them with radiological signs of leptomeningeal or ependymal involvement. The treatment was well tolerated: 2 patients developed transient diarrhea, 1 developed focal seizures, and 1 developed transient weakness and fever. The adverse effects were mild and did not go beyond grade II. These preliminary data show a promising high response rate of recurrent high-grade gliomas to chemotherapy with bevacizumab and irinotecan. This regimen is associated with minimal toxicity.


S.R. Burzynski, R.A. Weaver, B. Szymkowski, T.J. Janicki, M.I. Khan, and V. Dolgopolov; Burzynski Clinic, Houston, TX, USA

VHL disease is associated with neoplasms in multiple organs — among these are brain tumors. This disease is characterized by biallelic loss of the VHL gene. In less than 20% of cases, the VHL function is lost due to hypermethylation; therefore, hypomethylating agents have been proposed for VHL disease treatment. This report describes a rare case of VHL with multicentric brain tumors, including a diffuse intrinsic brainstem tumor successfully treated with ANP, which contained a hypomethylating agent. A 40-year-old white man developed ataxia, aphasia, and right facial nerve paralysis in January 2003, and MRI and PET scans revealed progressive lesions in the brainstem, cerebellum, and right internal capsule described as hemangioblastomas. A CT scan showed a left renal cyst. A brain biopsy was not recommended because of the associated risk. Molecular genetic studies (MGS) at the Children’s Hospital in Philadelphia (CHP) revealed the complete deletion of 1 allele of the VHL gene. On July 30, 2003, the patient was admitted for administration of intravenous ANP according to an FDA- and IRB-supervised protocol and subsequently attained maximum dosages of A10 10.72 and of AS2–1 0.33 g/kg/d. He discontinued i.v. ANP on January 25, 2004. From May 9, 2004, to July 13, 2004, he received oral A10 and AS2–1, both 0.15 g/kg/d and is off ANP at present. He was never given corticosteroids with ANP. His initial symptoms resolved after 4 months of ANP treatment. A brain PET scan after 5 months of ANP treatment and repeat MRIs after 11, 15, 21, and 27 months were within normal limits and the renal cyst was no longer seen on a CT after 15 months. Repeat MGS at CHP after 3 months of treatment did not show a detectable lesion or point mutation in the VHL gene. Treatment with ANP was well tolerated with only minor adverse experiences. The patient continues to be in complete response for approximately 2 years. In conclusion, this report describes a puzzling case of a complete response of VHL disease to ANP treatment. The patient initially had the complete deletion of one allele of the VHL gene and was suspected to have methylation of the second copy, which responded to the treatment with the hypomethylating agent, ANP. It is also possible that he had VHL mosaicism. More definite conclusions may be reached after the treatment of additional cases.


Robert Cavaliere; The Ohio State University, Columbus, OH, USA

Hemangioblastomas often present as multifocal tumors. Subarachnoid dissemination, however, is rare. We report a patient who initially presented with a sporadic solitary hemangioblastoma that subsequently disseminated throughout the neural axis. The patient is a 42–year-old man who initially presented in 1998 with gait instability, nausea, and headaches. Imaging scans revealed a solitary left cerebellar hemispheric mass that was completely resected; a pathologic assessment indicated hemangioblastoma. The patient developed regional recurrences in 2003 and April 2005 that were treated with resection and stereotactic radiosurgery, respectively. In August 2005, he presented with neck and shoulder pain. Imaging scans revealed multifocal disease throughout the posterior fossa and spine. A dominant and presumably symptomatic lesion was resected from his cervical spine. Despite surgery, he continued to decline, developing incontinence and paraparesis, at which time he was referred to Ohio State University. Spinal MRI showed diffuse, nodular spinal leptomeningeal enhancement. Hematocrit was 58%. The patient has been treated with 200 mg thalidomide daily. He has been on treatment for 6 months and has remained radiographically and clinically stable. His hematocrit declined to 39%. His course has been complicated by deep vein thrombosis and pulmonary embolism, for which he is now anticoagulated. He also has had moderate fatigue effectively managed with methylphenidate. Hemangioblastoma rarely disseminates through subarachnoid space. Thalidomide, an oral agent with antiangiogenic and immunomodulatory properties, may be an effective, well-tolerated treatment. Hematocrit may be useful as a disease marker. Although a causative link has not been established, a heightened awareness for thromboembolic events is necessary for patients receiving thalidomide. A review of the literature and representative imaging will be presented.


A. Chakravarti, B. Berkey, I. Robins, A. Guha, W. Curran, D. Brachman, C. Shultz, and M. Mehta; USA

The epidermal growth factor receptor (EGFR) pathway is commonly deregulated in glioblastomas (GBM), and its activity has been associated with treatment resistance in preclinical models. Accordingly, the Radiation Therapy Oncology Group (RTOG) recently conducted a phase I/II study of gefitinib, an EGFR tyrosine kinase inhibitor, in combination with radiotherapy for patients with newly diagnosed GBM. One hundred seventy-eight patients with GBM were entered on RTOG 0211 (phase I: 31 patients and phase II: 147 patients). The maximum tolerated dose (MTD) of gefitinib was determined to be 500 mg in non-EIACD patients, and the phase II component of RTOG 0211 was continued at this dose level during radiation and as maintenance for 18 months afterward or until disease progression. One hundred nineteen (out of 147) patients completed treatment per protocol or completed protocol with acceptable deviation or both. The median survival time for all patients in the study was 11.0 months. Progression-free survival was 5.1 months for all patients. When considering only patients who were treated per protocol, the median survival of RTOG 0211 patients was 11.5 months compared with 11.0 months for historical controls treated in previous RTOG studies (P = 0.14). Patients in RPA Class IV appeared to derive the greatest benefit from gefitinib when combined with radiotherapy compared with historical control groups, although not reaching statistical significance. Molecular and genetic profiling efforts are underway to identify patients who might derive the greatest benefit from gefitinib in the upfront setting, which will be reported at the time of the annual meeting. These include markers such as EGFRvIII and PTEN, which have been recently reported to be associated with response to anti-EGFR agents in the recurrent setting, and members of key signal transduction pathways regulated by EGFR. The observed survival advantage of newly diagnosed GBM patients treated with gefitinib in combination with radiotherapy compared with historical control groups treated on previous RTOG studies does not reach statistical significance. This work was supported by grant number RTOG U10 CA21661, CCOP U10 CA37422, Stat U10 CA32115 from the National Cancer Institute.


Marc Chamberlain,1 Sajeel Chowdhary,1 Deborah Blumenthal,2 Michael J. Glantz3; 1H. Lee Moffitt Cancer Center, Tampa, FL, USA; 2University of Utah, Salt Lake City, UT, USA; 3University of Massachusetts, Worcester, MA, USA

A prospective phase II study of CPT-11 was conducted in adult patients (pts) with recurrent temozolomide-refractory anaplastic astrocytoma (AA) with a primary objective of evaluating 6-month progression-free survival (PFS). Thirty patients (20 men, 10 women), ages 29–60 years (median, 43 years), with radiographically recurrent AA were treated. All patients had been previously treated with surgery, involved-field radiotherapy, and adjuvant chemotherapy (temozolomide [TMZ] in 20, BCNU in 10). Thirteen patients were treated at first recurrence with an alternative chemotherapy (TMZ in 10, all previously treated with BCNU, and one each with CCNU, sorafenib, or TMZ). All patients were treated at either first or second recurrence with CPT-11 administered intravenously once every 3 weeks (350 mg/m2 for patients on nonenzyme-inducing anticonvulsants, 600 mg/m2 for patients on enzyme-inducing anticonvulsants). A single dose of CPT-11 was operationally defined as a cycle. Neurological and radiographic evaluations were performed every 8 weeks. All patients were evaluable for toxicity, 29 for response. A total of 218 cycles of CPT-11 (median 4 cycles [range, 2–12] was administered). Toxicities included diarrhea in 19 patients (3 grade III), leukopenia in 17 (2 grade III), fatigue in 11 (2 grade III), anemia in 6 (1 grade III), delayed nausea/vomiting in 5 (2 grade III), neutropenia in 5 (2 grade III), and renal failure in one (1 grade V). Two patients (6.7%) required PRBC transfusions, 6 (20%) demonstrated a radiographic complete (1) or partial (5) response, 15 (50%) demonstrated stable disease, and 8 (27%) had progressive disease following 3 cycles of CPT-11. The time to tumor progression ranged from 0.5 to 12 months (median = 5, 95% CI, 3.8 ± 6.2 months). Survival ranged from 0.5 to 24 ± months (median 7, CI, 4.8 ± 9.2 months); 6-month and 12-month PFS rates were 43% and 3%, respectively. CPT-11 demonstrated modest efficacy with acceptable toxicity in this cohort of adult patients with recurrent anaplastic astrocytoma, all of whom had failed previous TMZ chemotherapy.


Charles Conrad, Christina Meyers, Timothy Madden, Waldemar Priebe, Patricia Gaupp, Karen Rohan, Christiane Baud; The University of Texas M.D. Anderson Cancer Center, Houston, TX; Reata Pharmaceuticals, Dallas, TX, USA

RTA 744 is a novel anthracycline that has been shown in preclinical studies to cross the blood-brain barrier and improve survival in an orthotopic murine model of glioblastoma multiforme (GBM). The first human trial of RTA 744 was initiated at M.D. Anderson Cancer Center in patients with primary high-grade gliomas. Patients receive RTA 744 as a 2-hour intravenous infusion on each of the first 3 days of a 21-day cycle. Dose escalation is proceeding according to an accelerated titration design with single patient cohorts and 100% dose escalation steps until first evidence of drug-related grade II or greater toxicities. The MTD is defined as the dose at which [less-than-or-eq, slant] 1/6 patients experiences a DLT with at least 2/6 experiencing a DLT at the next higher dose level. The MTD is being determined first in patients who do not take enzyme-inducing anti-convulsants. A second cohort of patients who do take these medications is planned. Pharmacokinetic samples are being taken multiple times on days 1–5 of Cycle 1. Tumor activity is being assessed according to the MacDonald criteria. As of May 2006, RTA 744 has been administered to a total of 7 patients (pts) at dose levels of 1.2 (1 pt), 2.4 (3 pts), 4.8 (2 pts), and 9.6 mg/m2/day (1 pt) (corresponding to 3.6, 7.2, 14.2, and 28.4 mg/m2/cycle). No grade 2 or greater drug-related toxicities have been observed at doses of 4.8 mg/m2 and under; results at 9.6 mg/m2 are pending. The pharmacokinetic profile indicates dose proportionality with some accumulation by day 3. Plasma half-life of RTA 744 is approximately 30 hours. Three of the first 4 patients received at least 4 cycles, and one of these patients remains enrolled in the study. Evidence of clinical activity was also seen in the first 4 patients, including 2 minor responses (both GBM pts at 2.4 mg/m2) and 1 stable disease (AO pt at 1.2 mg/m2). The most recent patient received a dose 4 times the level at which tumor regression was first documented. The results of MRI scans from the 2 most recently enrolled patients are pending. RTA 744 is well tolerated at doses of 4.8 mg/m2/day, has good pharmacokinetics, and shows early evidence of activity. Dose escalation should continue to define the MTD. Full results of this trial should be available by the fall of 2006. Based on the activity observed to date, phase II studies of this novel agent in primary brain tumors appear to be warranted.


P. Costello,1 W. McDonald,2 D. MacDonald,3 R. Hammond,4 and J. Megyesi5; 1Pathology Department, University of Western Ontario, London, Canada; 2Onco-Screen Inc., London, Canada; 3Neurology and Neuro-Oncology Departments, 4Division of Neuro-Pathology, Department of Pathology, 5Division of Neurosurgery, Department of Surgery, London Health Sciences Centre, London, Ontario, Canada

Improved treatment for brain tumors is needed. Most models assessing chemotherapies fail to incorporate heterogeneity of patient responses. Tumor progression is dependent on the ability of the tumor to invade and grow into surrounding local tissue and metastasize to distant sites in the body. In this study, a surgical sample of each patient’s primary brain tumor was obtained at the time of surgical resection and assessed while exposed to a panel of clinically relevant chemotherapies using an ex vivo model of invasion and growth. This invasion and growth of the representative tissue fragments is hypothesized to be reflective and predictive of a patient’s clinical response to therapy. Tissue specimens were placed into a nutrient-rich collagen matrix and monitored microscopically to measure the actual distance tumors invaded in the presence of chemotherapeutic agents for 5 days after surgical removal. Each therapy was applied as an overlay directly to the tissue samples. Four samples were tested for each treatment and control conditions. All samples were preserved for examination of markers related to tumor growth, invasion, and viability. Twenty-nine brain tumor patient’s therapy sensitivity profiles have been assessed thus far. Each tumor displayed a unique and significant (P < .05) invasion and response profile. Meningioma and ependymoma tumor samples from 6 patients did not migrate significantly into the matrix as was expected for benign and noninvasive tumors. Nine patient’s malignant tumors were not significantly sensitive to any therapy tested. Eight malignant tumors responded to docetaxel, 4 responded to procarbazine, 7 responded to vincristine and only 2 to temozolomide. Results will continue to be compared with patient response, time to recurrence, and survival for up to 2 years. Individual response to chemotherapy is highly variable both clinically and in our ex vivo assessment. Prescreening each patient’s responsiveness to chemotherapies using this unique method could lead to a more individualized and, therefore, a more effective approach to the treatment of brain tumurs.


Annick Desjardins, James Vredenburgh, Jennifer Quinn, Jeremy Rich, Sith Sathornsumetee, Sridharan Gururangan, Allan Friedman, David Reardon and Henry Friedman; Duke University Medical Center, Durham, NC, USA

The prognosis for malignant gliomas remains poor. Once these tumors have recurred, the prognosis is even poorer. Knowing that malignant gliomas have high concentrations of VEGF receptors, and the higher the VEGF receptor concentration, the worse the prognosis, we decided to evaluate the efficacy of bevacizumab in patients with malignant brain tumors. Bevacizumab is a humanized IgG1 monoclonal antibody to VEGF, which is synergistic with chemotherapy for most malignancies. We performed a phase II study combining bevacizumab with irinotecan for patients with recurrent malignant gliomas and observed an unprecedented response rate of 63%. Building on these results, we treated a number of our patients who were not eligible for a study with bevacizumab and different chemotherapeutic agents. All patients received bevacizumab at 10 mg/kg i.v. every 14 days. The chemotherapy combined with bevacizumab was chosen depending on prior therapeutic history. As of May 10, 2006, 26 patients with recurrent malignant brain tumors had been treated with a combination of irinotecan and bevacizumab, 8 patients with a combination of VP-16 and bevacizumab, 4 patients with daily low-dose temozolomide and bevacizumab, 2 patients with daily low-dose cyclophosphamide and bevacizumab, and 2 patients with temozolomide and bevacizumab. Of the 26 patients treated with irinotecan and bevacizumab, 2 had an intratumoral bleed (8%) compared with 2 of the 8 patients on the combination of VP-16 and bevacizumab (25%). Two patients on the VP-16 and 1 patient on daily low-dose temozolomide had deep venous thrombosis. Two patients had grade III leukopenia and hypertension. However, the main side effect reported by 48% (20) of the patients, with no relation to the chemotherapeutic agent used, was grade II fatigue. Fifty-four percent of the patients treated with bevacizumab and irinotecan (14/26) had a PR. Of the 42 patients treated off protocol, 20 remain on treatment, for a median of 4 (range, 1–7) cycles. Studies are necessary to assess the feasibility, safety, and efficacy of bevacizumab and various chemotherapeutic agents, but the preliminary data suggest that bevacizumab can be safely combined with a number of agents with encouraging efficacy.


Annick Desjardins, James Vredenburgh, Jennifer Quinn, Jeremy Rich, Sith Sathornsumetee, Allan Friedman, David Reardon, and Henry Friedman. Duke University Medical Center, Durham, NC, USA

Treating patients with newly diagnosed malignant glioma who have tumors not amenable to surgical resection (secondary to the location and the extent of the tumor) presents a challenge. Given the side effects of radiation to extensive areas of the brain, reducing the tumor volume before radiation therapy is an area of interest. Building on the results of our phase II study combining bevacizumab with irinotecan for patients with recurrent malignant gliomas who demonstrated a response rate of 63%, we decided to treat a number of our patients who had voluminous, unresectable disease with bevacizumab and temozolomide or irinotecan as an upfront regimen. Bevacizumab is a humanized IgG1 monoclonal antibody to VEGF, which is synergistic with chemotherapy for most malignancies. Temozolomide is an oral methylating agent known to be effective for treating primary malignant brain tumors. Irinotecan is a topoisomerase I inhibitor. All patients received bevacizumab at 10 mg/kg i.v. every 14 days. Patients with MGMT levels < 20% received temozolomide 200 mg/m2 for 5 days followed by 23 days off for a maximum of 4 cycles. Patients with MGMT levels > 20% received irinotecan i.v. every 14 days (125 mg/mg2 for nonenzyme-inducing anticonvulsants and 340 mg/m2 for enzyme-inducing anticonvulsants) for a maximum of 3 cycles. Six patients have been treated thus far with the combination of temozolomide and bevacizumab. One patient demonstrated dramatic regression of a butterfly glioblastoma multiforme after 4 cycles of treatment. Three others had to discontinue treatment: 2 patients secondary to pneumonia and 1 patient for rupture of the esophagus. Two patients have not yet completed their fourth cycle. Two patients have been treated thus far with the combination of irinotecan and bevacizumab and are tolerating it well after having completed 2 cycles. No incidence of hemorrhage or arterial thrombosis has been observed thus far. The combination of chemotherapy with bevacizumab is feasible in patients with newly diagnosed high-grade malignant glioma who are not amenable to resection. However, the toxicity profile is significant and should be carefully considered. Studies to determine the feasibility and efficacy of those combinations should be performed, including an evaluation of the influence of MGMT to determine the treatment plan.


A. Desjardins, J.A. Quinn, J.N. Rich, J.J. Vredenburgh, S. Sathornsumetee, S. Gururangan, A.H. Friedman, W. Berg, M. J. Egorin, A. Salvado, H.S. Friedman, and D.A. Reardon; University of Pittsburgh Cancer Institute, Pittsburgh, PA; Novartis Pharmaceutical Corporation, East Hanover, NJ; Duke University Medical Center, Durham, NC, USA

This study attempts to extend the antiglioma activity of imatinib mesylate (IM) plus hydroxyurea (H), by adding RAD001 (R), an orally bioavailable inhibitor of mTOR, which is a critical intracellular mediator of signal transduction and metabolism. We employ a 3 + 3 dose escalation design to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of IM + H + R administered daily in adult patients with recurrent malignant glioma. Eligibility criteria include [less-than-or-eq, slant] 3 previous recurrences, a KPS > 60, and adequate organ function. The patients are stratified based on concurrent enzyme-inducing anticonvulsant use (EIAC), and both strata are independently escalated. The initial dose level for each stratum is as follows: IM, 400 mg/day; H, 500 mg bid; R, 2.5 mg/day. Each treatment cycle is 28 days. Response is evaluated every other cycle. Pharmacokinetic studies are performed on days 1 and 28 of cycle 1. Twenty-eight patients with recurrent GBM have enrolled. All patients progressed after at least XRT and temozolomide treatment. The median age is 51 years (range, 30–75 years), 50% are men, and 46% are on EIACs. One DLT (thrombocytopenia) occurred among 5 patients on dose level 1 (non-EIAC stratum). No other DLTs have occurred. The dose escalation schema has been amended to include alternate day R dosing. The pharmacokinetic results of IM were consistent with those previously reported for patients on IM and H. IM clearance on day 1 was 492 ± 247 ml/min in the EIAC stratum and 231 ± 100 ml/min in the non-EIAC stratum. On day 28, IM clearance was decreased in both strata (243 ± 93 ml/min in the EIAC stratum and 116 ± 47 ml/min in the non-EIAC stratum). Pharmacokinetic results for H and R are pending. Fifteen patients continue on the study, including 5 who have received 6 or more cycles of therapy. One partial response has been observed and accrual is ongoing. Further accrual is warranted. An update of outcome, toxicity, and pharmacokinetic analyses will be presented.


E.M. Dunbar, T. Siddiqui, T.A. Yachnis, T. Eskin, J. Bennett, and A.M. Shahlaee; University of Florida, Gainesville, FL, USA

Erdheim-Chester Disease (ECD) is a rare and debilitating non-Langerhans histiocytic disorder characterized by diffuse bony, visceral, endocrine, and neurologic manifestations. Rarely, it presents with intraparenchymal CNS lesions, and even more rarely, with dominant CNS symptoms. Although treatment approaches have included the use of vinca-alkaloids, anthracyclines, steroids, resection, or radiotherapy, no treatment standard exists or has shown acceptable efficacy. Even less understood are treatment options for intraparenchymal CNS-ECD. We present a case report highlighting additional diagnostic and therapeutic strategies that warrant further discussion and confirmation by the neuro-oncology community: a 62-year-old white woman presented after 4 months of left trigeminal neuralgia, right-sided paresis, and fatigue. An MRI scan revealed multiple enhancing lesions, predominantly at the gray-white matter junctions, largely within the left cerebrum, left cerebellum, left caudate, optic chiasm, pons, hypothalamus, and pituitary. A FLAIR sequence demonstrated significant edema. Spectroscopy revealed elevated choline and decreased NAA peak. A multi-institutional review of a brain biopsy confirmed ECD with characteristic histiocytic markers (CD68 positive, S-100 equivocal, and CD1a-negative). Hematologic and endocrine lab results were normal. A bone scan was equivocal. A body CT showed only bilateral sclerotic change of the hips; however, a PET scan revealed strongly positive FDG-uptake at the intracranial lesions. After an initial 4-month course of steroids, the patient decompensated, prompting a trial of weekly INF-alpha, which resulted in 3 months of stable disease. Unfortunately, debilitating neurologic and radiologic progression necessitated a trial of another agent. Considering a case report of 2-CDA improving systemic ECD disease, and given the knowledge that 2-CDA, a purine antagonist, crosses the blood-brain barrier, we favored 2-CDA over other options, such as cytoxan, methotrexate, or radiotherapy. After 2 cycles of 2-CDA at 0.14 mg/kg/day, days 1–5, q 4 weeks, the patient demonstrated an objective response on re-imaging (MRI), including dramatic improvement of the CNS lesions. To our knowledge, this case represents the first time that ECD has been characterized by strong PET positivity and highlights the potential importance of PET scans for both the diagnosis and monitoring of ECD, including cases with CNS involvement. Likewise, to our knowledge, this is the first time that 2-CDA has been shown to improve intraparenchymal CNS disease and offers consideration of 2-CDA as a second line treatment after failure of IFN or potential combination therapy. Lastly, this case report emphasizes the importance of multi-institutional collaboration to confirm the diagnosis of ECD and a multimodality approach to optimizing treatment.


Rebecca Fisher,1 Michael Glantz,1 Deborah Blumenthal,2 Julia Batten,2 Alixis Van Horn,1 Lisa Chalmers,3 and Marc Chamberlain3; 1University of Massachusetts, Worcester, MA; 2University of Utah, Salt Lake City, UT; 3H. Lee Moffitt Cancer Center, Tampa, FL, USA

PCNSL is an uncommon (but an increasingly occurring) primary brain tumor with an initial responsiveness to both chemotherapy and radiation that belies its almost inevitable recurrence and lethality. The long-term side effects of whole brain irradiation, the apparent efficacy of MTX-containing regimens, and the prominent role rituximab has assumed in lymphoma therapy have stimulated investigations of chemotherapy-alone treatment strategies. Many questions remain, including the optimum combination of agents and the importance of intra-CSF chemotherapy. Forty-eight consecutive patients with newly diagnosed, histologically or cytologically confirmed PCNSL (age [gt-or-equal, slanted] 18 years, KPS [gt-or-equal, slanted] 50) were treated with intravenous MTX (8 g/m2) on days 1, 14, 28, 42, and then monthly for 1 year. Rituximab (375 mg/m2) was administered with MTX for the first 6 months. At one of the 3 participating institutions, all (19/48) evaluable patients also received 10 doses of intraventricular DepoCyt (50 mg) at the time of MTX treatment. The patients were followed up clinically (every 4 weeks) and radiographically (every 8 weeks). Patients ranged in age from 18–93 years (median, 56 years) with 21 patients [gt-or-equal, slanted] 60 years, and the overall KPS was 50–100 (median, 80). Tumors were multifocal in 41% (20/49) of patients. Lymphomatous meningitis was documented prior to treatment in 42% (19/45) of all patients but in 78% (14/18) of patients from whom large ventricular CSF samples were obtained. Diagnosis was established by biopsy in 38 patients, resection in 8 patients, and other means (cytology, vitrectomy) in 3 patients. In 267 treatment cycles (range, 2–12; median 4), toxicity [gt-or-equal, slanted] grade III occurred in 11 cycles (4.2%) and included anemia (1), neutropenia (3), thrombocytopenia (1), nephrotoxicity (2), and mucositis (1). Of the 48 evaluable patients, 31 (65%) had a complete initial response to treatment, 12 had a partial response, 1 stable disease, and 4 progressive disease. After a median of 19 months of follow-up, median survival has not been reached. One- and 2-year survival rates for all 48 patients are 83% and 64%, respectively, and did not differ between patients who received or did not receive intrathecal chemotherapy. The median time to progression was 12 months (range, 2–66 months), Time to progression was longer in patients treated at diagnosis with intrathecal DepoCyt (13 months; range, 2–57+ months) than in patients not treated intrathecally (7 months; range, 2–66+ months). In a multivariate analysis, positive CSF cytology and multifocal tumor predicted worse outcomes but did not reach statistical significance. This regimen is effective and well tolerated in patients with PCNSL. CSF involvement at diagnosis appears more common than previously demonstrated but documentation may require multiple large volume ventricular CSF sampling. A positive CSF cytology and multifocal tumor may represent poor prognositic signs. Intra-CSF chemotherapy at diagnosis appears to delay time to recurrence, but a survival benefit has not been demonstrated.


A. Fitzsimmons, T. Batchelor, A. O’Neill, and J. Henson; Massachusetts General Hospital, Boston, MA, USA

Temozolomide (TMZ), an oral alkylating agent and first-line therapy for glioblastoma (GBM), is being increasingly used, often over protracted periods of time, in patients with low-grade and anaplastic gliomas These tumors are often associated with prolonged survival. Treatment-related myelodysplastic syndrome and acute myelogenous leukemia (t-MDS/AML) are life-threatening complications of alkylating agents that have been rarely reported in patients receiving TMZ for primary brain tumors, but there is little understanding of the degree of risk or of the role of other therapies. We report 3 patients with primary brain tumors who developed t-MDS/AML following treatment with TMZ. Patient 1 was a 66-year-old woman who was diagnosed with early-stage breast cancer and treated with adjuvant radiotherapy (RT) 2 years before developing an anaplastic oligodendroglioma. She received RT and 1 cycle of adjuvant procarbazine, lomustine, and vincristine (PCV) complicated by myelosuppression. She developed progressive disease 12 months later and was treated with 22 cycles of TMZ (150 mg/m2) over 23 months before developing persistent thrombocytopenia. She received 3 additional cycles at 4-month intervals before developing t-MDS, which was confirmed by bone marrow biopsy. She progressed to AML 8 months later and died 5 months later. Patient 2 was a 54-year-old woman who had bilateral breast cancer 10 years before presenting with GBM. The breast cancer had been treated with 2 cycles of CMF, 4 cycles of doxorubicin and cyclophosphamide, and RT to each breast. The GBM was treated with RT followed by 20 cycles of TMZ over 27 months (5 cycles at 200 mg/m2, 15 at 150 mg/m2). Coincident with the last cycle of TMZ, the patient developed transfusion-dependent anemia, which was likely representing t-MDS. Two months later, a bone marrow biopsy showed t-AML, and she died 7 days later. Patient 3 was a 56-year-old woman who was treated with PCV for a progressive grade II oligodendroglioma with high MIB-1 and a 1p/19q deletion. PCV treatment was stopped after 3 cycles because of myelosuppression. She was then treated with 12 cycles of TMZ (1 cycle at 200 mg/m2, 11 cycles at 150 mg/m2). Fourteen months after completion of TMZ, a bone marrow biopsy showed M6 AML (erythroleukemia). The median age at diagnosis of AML for these patients was 59 years (range, 57–66 years). All 3 patients were women. The mean number of TMZ cycles was 19 (SD, 6.6). The mean time from initiation of TMZ to diagnosis of AML was 39 months (SD, 19). Mean time elapsed from cessation of TMZ to the diagnosis of AML was 10 months (range, 2–14 months). All 3 patients had received prior treatment with cytotoxic chemotherapy and 2 had a history of breast cancer treated with chemotherapy (1 patient) and RT (both patients). Protracted administration of TMZ may be associated with t-MDS/AML in brain tumor patients. Older age and previous treatment with alkylating agents and RT may also increase the risk.


P.A. Forsyth,1,3 G. Roldan,2 D. George,4 C. Wallace,5 D.G. Morris,2 G. Cairncross,1,3 M. Vallee Matthews,2 J. Markert,8 Y. Gillespie,8 M. Coffey,7 B. Thompson,7 and M. Hamilton8; 1Departments of Oncology and Clinical Neurosciences, University of Calgary and Tom Baker Cancer Centre, Calgary, AB; 2Department of Oncology, Tom Baker Cancer Centre; 3Clark Smith Integrated Brain Tumor Research Centre; 4Departments of Pathology, 5Diagnostic Imaging, Foothills Medical Centre, Calgary; 6Department of Neurosurgery and Clinical Neurosciences, Foothills Medical Centre and University of Calgary; 7Oncolytics Biotech, Calgary, AB, Canada; 8Division of Neurosurgery, University of Alabama at Birmingham, AL, USA

Reovirus is an oncolytic virus that replicates preferentially in transformed cells with activated Ras signaling pathways. Reovirus has shown promising activity in in vivo models of MG and in a phase I trial in patients with cutaneous metastases from systemic cancer. We performed this dose-escalation trial of IT reovirus administration to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) in patients with recurrent MG. The secondary endpoints were response, survival, and time to progression (TTP). The patients were aged [gt-or-equal, slanted] 18 years, had a KPS [gt-or-equal, slanted] 60, received prior radiotherapy with or without chemotherapy, had a histologically proven recurrence of MG, and had [less-than-or-eq, slant] 3 instances of recurrence. Reovirus was administered IT stereotactically at 1 of 3 dose levels (1×107, 1×108, or 1×109 TCID 50) in a volume of 0.9 ml. Twelve patients were treated at 3 dose levels. Seven patients were men, the median KPS was 80, and the median age was 53.5 years. Ten patients had glioblastoma multiforme, 1 had anaplastic astrocytoma, and another had anaplastic oligoastrocytoma. Recurrences were the first, second or third in 6, 5, and 1 patients, respectively. During IT viral administration, all patients were treated with prophylactic anticonvulsants, and 6 (50%) were receiving corticosteroids. The first, second, and third cohorts contained 3, 6, and 3 patients, respectively. No grade III-IV adverse events occurred definitely or probably related to the administration of reovirus. A transient increase in GGT was possibly related to reovirus administration, and 1 patient experienced grade III motor weakness possibly related to postinjection edema. Viral shedding and systemic immune responses were examined but results are pending. There were no complete or partial responses; 1 patient had stable disease, 10 progressive disease, and one was not evaluable. The median survival was 20 weeks (range, 6–171 weeks), 6 patients survived > 6 months and 3 are alive 5, 6, and 40 months from the time of the reovirus injection. The mediann TTP was 4.3 weeks (range, 3.4–39 weeks). An MTD was not reached. The intratumoral administration of reovirus was well tolerated in patients with recurrent MG. Phase II studies in MG are planned.


David Fortin, Cathy Gendron, and Marie Boudrias; CHUS — Sherbrooke University, Canada

The incidence of metastasis is estimated to be in the range of 200,000 new cases per year in the USA and is steadily increasing. When diagnosed with multiple lesions, or a non-operable lesion, patients are treated with whole brain radiation as a palliative measure. The literature reports a median survival in the range of 2–7 months. This report details our experience with blood-brain barrier disruption (BBBD)-enhanced chemotherapy for the treatment of brain metastasis. This prospective study enrolled 28 patients with brain metastasis distributed in 5 groups based on tumor histology. Patients were treated with intra-arterial chemotherapy, with or without BBBD, depending on the mass effect. A methotrexate-based regimen was offered to the systemic lymphoma patients, whereas a carboplatin-based regimen was offered to all other groups. Each cycle was repeated on a monthly basis. Forty-two patients were enrolled. Ten patients were excluded from the analysis because they had disease progression after a single cycle or were lost to follow-up, which left 32 patients for analysis (23 women, 9 men). The median age was 55.9 years. Sixty percent of patients had been exposed to radiation therapy and 72% to chemotherapy prior to enrollment. These 32 patients were submitted to a total of 167 procedures. The median survival (MS) was 10.4 months for lung carcinoma (N = 15), 10.5 months for adenocarcinoma (N = 10), and 8.8 months for the oat cell group (N = 5). For ovarian carcinoma (N = 5), the MS was 21 months, whereas the MS for breast carcinoma patients was 8.8 months (N = 3). For the systemic lymphoma group (N = 7), an MS of 16.3 months was reached. This treatment seems promising in the treatment of the following metastatic cancers to the brain: lung carcinoma (adenocarcinoma and small cell), ovarian carcinoma, and systemic lymphomas.


M.R. Gilbert, J. Gonzalez, K. Hunter, K. Hess, E. Chang, V. Puduvalli, M. Groves, H. Colman, C. Conrad, V. Levin, S. Woo, A. Mahajan, S. Hsu, J. de Groot, and W.K.A. Yung; The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Concurrent TMZ and external beam radiation (XRT) followed by 6 cycles of adjuvant TMZ (5 of a 28 day cycle) prolongs survival in patients with newly diagnosed GBM. Additional studies suggest that dose-dense scheduling of TMZ (7 days on, 7 days off) may enhance efficacy. The addition of cytostatic agents to TMZ may also improve efficacy. Therefore, we developed a clinical trial to test a dose-dense schedule of TMZ as a single agent or in varied combinations with Thal, CRA, and Cel. This phase I study tested triplet and quadruplet combinations in preparation for an 8-arm, phase II randomized factorial design protocol that will evaluate TMZ alone, and doublet, triplet, and quadruplet regimens. Eligibility criteria included newly diagnosed GBM, age > 10 years, KPS > 60, and adequate renal, hepatic, and bone marrow function. All patients received conventional XRT using conformal planning. Sixty Gy was delivered in 2 Gy fractions over 6 weeks with TMZ (75 mg/m2 daily × 42 days). An MRI scan was done 1 month after chemoradiation. Patients with stable disease or response were treated with TMZ alone (150 mg/m2/day, 7-day on/7-day off schedule) or TMZ with doublet combinations of Thal, CRA, or Cel, or all 3 other agents. Toxicities, measured after week 4 of treatment, were scored using the NCI Common Toxicity Criteria, volume 3. Fifty-four patients were accrued into the 5 treatment arms. The median patient age was 52 years (range, 18–76 years), and the median KPS was 90. Adjuvant treatment was not given to 12 patients (22%): 10 patients had disease progression, 1 patient had complications, and 1 patient withdrew. The full dose was well tolerated for TMZ alone, for TMZ + Cel + CRA, and for TMZ + Thal + Cel. The TMZ + CRA + Thal group had 1 episode of grade III fatigue and grade III rash. With all 4 agents, 1 episode of grade IV neutropenia was noted. Venous thrombosis occurred in 7 patients, 4 of whom were on Thal. Grade III-IV lymphopenia developed in 63% of patients without any related infections. From the time of study entry, the 6–month progression-free survival rate was 63%, and the median progression-free survival time was 8.2 months (95% CI, 5.3–11.1 months). These results demonstrate that multiple cytostatic agents can be safely combined with dose-dense TMZ and suggest that these treatment strategies may enhance efficacy. This study established the dosing regimens for an 8-arm, phase II factorial design randomized trial, now underway using TMZ and the 3 cytostatic agents to test the effect of varied combinations as adjuvant therapy for newly diagnosed GBM.


Jon Glass,1 Brian A. Berkey,2 Christopher Schultz,3 Daniel J. Brat,4 Nancy L. Bartlett,5 Paul Brown,6 Elizabeth Gore,3 Paul Sperduto,7 and Minesh Mehta8; 1Fox Chase Cancer Center, Philadelphia, PA, USA; 2Radiation Therapy Oncology Group, Philadelphia, PA, USA; 3Medical College of Wisconsin, Milwaukee, WI, USA; 4Emory University, Atlanta, GA, USA; 5Washington University, St. Louis, MO, USA; 6Mayo Clinic, Rochester, MN, USA; 7CCOP Metro-Minnesota, Minneapolis, MN, USA; 8University of Wisconsin, Madison, WI, USA

The RTOG 9310 trial showed that a pre-radiation chemotherapy regimen consisting of intravenous and intrathecal methotrexate (MTX), pro-carbazine, and vincristine for primary central nervous system lymphoma (PCNSL) improved progression-free and overall survival times. Responses have been seen with temozolomide (TMZ) and rituximab (RTX) in recurrent PCNSL. A phase I study was performed to determine the maximum tolerated dose of TMZ in combination with MTX. A phase II study is ongoing. RTX 375 mg/m2 was administered 3 days before the first cycle of i.v. MTX, 3.5 g/m2 with leucovorin rescue given on weeks 1, 3, 5, 7, and 9 for a total of 5 cycles. TMZ was given daily for 5 days on weeks 4 and 8. The initial dose was 100 mg/m2 (Arm 1) with planned escalation to 150 mg/m2 (Arm 2) and then 200 mg/m2 (Arm 3). Hyperfractionated whole brain radiation therapy (WBRT; 1.2 Gy b.i.d. fractions) was delivered 5 days/week during weeks 11, 12, and 13 for a total of 36 Gy. TMZ 200 mg/m2 per day for 5 days was administered on weeks 14, 18, 22, 26, 30, 34, 38, 42, 46, and 50 for a total of 10 cycles. Six patients were treated on Arm 1 and 7 patients were treated on Arm 2. One patient in Arm 2 was deemed ineligible due to carmustine (Gliadel) wafer placement. At 100 mg/m2 of TMZ, there was 1 dose-limiting toxicity (DLT) (grade IV renal toxicity). At 150 mg/m2 of TMZ, there were 3 DLTs (grade III-IV hepatic toxicity in 2 patients, grade III nephrotoxicity in 1 patient). The maximum tolerated dose of TMZ in PCNSL patients treated with regimen is 100 mg/m2. This dose is being used in the phase II portion of this trial


Jon Glass; Fox Chase Cancer Center, Philadelphia, PA, USA

The purpose of this study is to evaluate the safety and efficacy of carboplatin dosing using the Calvert formula for recurrent glioblastoma multiforme (GBM). Carboplatin has been used as monotherapy and in combination therapy for recurrent GBM. Previously performed clinical trials have used body surface area rather than the Calvert formula for the dosing calculation. The area under the curve (AUC) dosing via the Calvert formula provides a more consistent exposure to carboplatin and allows for less variability in toxicity, particularly thrombocytopenia. No previous mono-therapy trial has used this dosing regimen. Patients eligible for this trial had a diagnosis of glioblastoma multiforme that recurred after radiation therapy and had undergone at least 1 previous chemotherapeutic regimen. All patients had measurable disease and an ECOG performance score of 2 or higher. Patients received carboplatin at a dose of AUC of 6 every 4 weeks. The glomerular filtration rate was estimated using the traditional Cockcroft-Gault formula [(140 − age(yrs)) × lesser of (IBW(kg) or actual weight(kg))/(72 × serum creatinine(mg/dl))]. MRI scans were performed every 8 weeks. Tumor responses were determined using routine MacDonald criteria. Thirty-four patients were treated; 21 were men and 13 were women with a median age of 60 years (range, 31–86 years). A median of 3 cycles were administered (range, 1–9). Disease stability was seen in 20 patients (59%) and progressive disease was seen in 14 patients (41%). No objective responses (CR/PR) were noted. The median survival time was 35 weeks; 5 patients are alive. A total of 126 cycles were administered. Grade III-IV leukopenia was seen in 6 cycles. Grade III-IV thrombocytopenia was seen in 5 cycles. Grade III anemia was seen in 2 patients. There were no nonhematologic toxicities and 1 case of febrile neutropenia. Carboplatin dosing using the Calvert formula for calculation of area under the curve for the treatment of recurrent glioblastoma multiforme is well tolerated and results in similar progression-free and overall survival rates when compared with studies using body surface area for dosage calculation. Toxicity was purely hematologic and compared favorably with trials utilizing body surface area for dosing. Although carboplatin has mild activity in the treatment of GBM, the use of this dosing scheme in combination therapy trials may allow for improved toxicity.


S.A. Grimm, J.S. Pulido, A. Omuro, J.Y. Blay, A.J.M. Ferreri, N. Doolittle, T. Batchelor, T. Siegal, K. Jahnke, T.N. Shenkier, A.J. Hall, F. Graus, U. Herrlinger, D. Schiff, J. Raizer, J. Rubenstein, N. Laperiere, and L.E. Abrey; Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Hospital Pitie-Salpetriere, Paris, France; International Extranodal Lymphoma Study Group; Oregon Health & Sciences University, Portland, OR, USA; Massachusetts General Hospital, Boston, MA, USA; Hadassah Hebrew University Hospital, Tel Aviv, Israel; Mayo Clinic, Rochester, MN, USA; British Columbia Cancer Agency, Vancouver, Canada; Royal Melbourne Hospital, Melbourne, Australia; Barcelona, Spain; University of Tuebingen, Tuebingen; University of Virginia, Charlottesville, VA, USA; Northwestern, Chicago, IL, USA; UCSF, San Francisco, CA, USA; Princess Margaret Hospital, Toronto, Canada

Approximately 15% to 25% of PCNSL patients have ocular involvement at the time of diagnosis. Because these patients represent a small fraction of patients with a rare disease, the natural history and optimal treatment of PCNSL with ocular dissemination are unknown. A retrospective study of 225 patients with ocular lymphoma was assembled from 15 centers in 8 countries. All patients had parenchymal brain lymphoma in addition to ocular involvement; none had systemic lymphoma. The median age at diagnosis was 58 years (range, 16–82 years). Fifty-seven percent of the patients were women. The median ECOG performance status was 2. The median latency period from symptom onset to diagnosis was 3 months (range, 0–48 months). A Slit lamp exam was positive in 145 patients, negative in 21, and not performed in 59. One hundred twelve patients underwent an ocular surgical procedure at diagnosis: 93 had a positive vitrectomy, choroidal or retinal biopsy; vitrectomy was negative in 19 patients. CSF cytology was positive in 35 patients (23%), negative in 120, and not performed in 70. Initial treatment information was available for 164 patients. Ninety-two patients received focal ocular therapy in conjunction with treatment for cerebral disease. Nineteen patients received intraocular chemotherapy, 72 ocular radiation, and 1 received both modalities. Seventy-two patients received PCNSL therapy that did not include focal ocular therapy. Progression free survival (PFS) and overall survival (OS) were 18.7 months and 33.1 months, respectively. Treatment had no impact on OS, but patients who were treated with a combination of focal ocular therapy with specific PCNSL therapy had significantly longer PFS (19 months vs. 14.2 months, P = 0.008). Recurrent or progressive PCNSL was the cause of death in 71% of patients. In this series, treatment type did not affect OS; however, patients who received more aggressive ocular treatment had an improved PFS. Therefore, the optimal initial therapy should include intraocular chemotherapy or ocular radiotherapy. Prospective clinical trials are needed to optimize the management of this disease.


S.A. Grimm, J.S. Pulido, K. Jahnke, D. Schiff, A.J. Hall, T.N. Shenkier, T. Siegal, N.D. Doolittle, T. Batchelor, U. Herrlinger, E.A. Neuwelt, N. Laperriere, J.Y. Blay, A.J.M. Ferreri, A. Omuro, and L.E. Abrey; Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Mayo Clinic, Rochester, MN, USA; University of Virginia, Charlottesville, VA, USA; British Columbia Cancer Agency, Vancouver, Canada; Hadassah Hebrew University Hospital, Tel Aviv, Israel; Oregon Health & Sciences University, Portland, OR, USA; Massachusetts General Hospital, Boston, MA, USA; University of Tuebingen, Tuebingen; Princess Margaret Hospital, Toronto, Canada; International Extranodal Lymphoma Study Group; Hospital Pitie-Salpetriere, Paris, France

PIOL is a hemopoietic tumor that arises in the retina, vitreous, or optic nerve head and carries a high risk of ocular and CNS relapse. The natural history and optimal management are unknown. A retrospective study of 81 patients with PIOL was assembled from 15 centers in 7 countries. Only patients with isolated ocular lymphoma were included; none had brain, spinal cord, or systemic lymphoma at diagnosis. The median age at diagnosis was 65 years (range, 24–85 years). Fifty-eight percent of the patients were women. The median ECOG performance status was 0, and only 3 patients had a score > 1. The median latency period from symptom onset to diagnosis was 6 months (range, 0–36 months). A Slit lamp exam was positive in 51 patients, negative in 6, and not reported in 24. Vitrectomy was positive in 72 patients and negative in 2. Six patients had a positive choroidal or retinal biopsy, and 1 had no ocular surgery. CSF cytology was positive in 10 patients (17%), negative in 48, and unknown in 23. Twenty-one patients received local therapy at diagnosis: 6 intraocular methotrexate (400 μg), 14 ocular radiation (median 3600 cGy), and 1 received both modalities. Fifty-two patients received more extensive therapy, including systemic chemotherapy alone in 20, and a combination of chemotherapy and radiotherapy in 32. Five patients received no treatment, and details are unknown in 3. Forty-seven patients (58%) relapsed after initial therapy. The relapse sites included the brain (47%), eyes (30%), brain and eyes (15%), and systemic (8%). Patients treated with ocular therapy alone did not have an increased risk of failing in the brain (P = 0.6). Progression free survival (PFS) and overall survival (OS) were 29.6 months and 57 months, respectively, and were unaffected by the choice of therapy. CNS disease was the cause of death in 19 patients (58%). In this series, treatment type did not affect sites of relapse, PFS, or OS in patients with PIOL. To minimize toxicity, the best initial therapy should be limited to intraocular chemotherapy or focal radiotherapy. Prospective clinical trials are needed to improve our understanding and treatment of this disease.


Ricky Kalra, Michael Zahner, Lisa K. Nicoletti, Keith M. Rich, Robert L. Grubb, Jr., Joshua L. Dowling, Neill M. Wright, Gregory J. Zipfel, Ralph G. Dacey, Jr., and Michael R. Chicoine; Departments of Neurosurgery, Washington University School of Medicine, and the Alvin J. Siteman Cancer Center, Saint Louis, MO, USA

The implantation of carmustine wafers (Gliadel) at the time of initial tumor resection and resection for tumor recurrence has been shown in randomized placebo-controlled clinical trials to prolong survival for patients with malignant gliomas. We retrospectively reviewed the records of all patients treated with Gliadel at our institution to determine its utility in a neuro-oncology practice beyond the constraints of a clinical trial. All patients underwent tumor resection and implantation of Gliadel for malignant gliomas at Barnes-Jewish Hospital from 1997 to 2006. Survival was assessed for all patients and further analyzed according to tumor histology. The 48 patients (31 men, 17 women) included in the study had a mean age of 54 years (range, 27–85 years) and a mean KPS of 82. Pathologic diagnoses included glioblastoma multiforme (GBM) in 36 patients, anaplastic astrocytoma (AA) in 3, anaplastic oligodendroglioma (AO) in 6, and gliosarcoma (GS) in 3. Gliadel was implanted at the time of initial tumor resection in 23 patients, at the time of recurrence in 24 patients, and in 1 patient both initially and at recurrence. The mean overall survival to date for the group (22 censored) was greater than 15 months (range, 0–77 months). The mean overall survival was 14 months for GBM (range, 0–49 months), 34 months for AA (range, 2–77 months), 19 months for AO (range, 3–54 months), and 6 months for GS (range, 3–13 months). Five patients (14%) with GBM survived longer than 2 years after surgery; 3 of the 5 patients survived over 3 years, and 1 of the 5 patients survived over 4 years. The mean survival for both recurrent and primary GBM patients was 14 months. Additionally, patients younger than 45 years with GBM had a more prolonged survival than patients older than 45 years (P = 0.05). Side effects potentially attributable to the implantation of Gliadel included 4 wound infections (8.3%), 3 new onsets of seizures (6.3%), and 3 cases of worsening cerebral edema (6.3%). In clinical practice, the implantation of Gliadel for patients with malignant gliomas provides patient survival rates and complication rates similar to those achieved in randomized clinical trials. Gliadel remains a treatment option for patients with malignant gliomas. Further analysis may reveal subgroups of patients for whom this treatment strategy is optimal.


John J. P. Kelly, Peter Forsyth, Michael Eliasziw, Mark G. Hamilton, and Ian F. Parney; The University of Calgary, Calgary, AB, Canada

Nonrandomized studies suggest that surgical resection of newly diagnosed malignant gliomas prolongs survival. Unfortunately, results from these studies are confounded by selection bias. A large randomized, controlled trial of biopsy versus resection is needed to properly address this issue. We wished to assess how many potentially eligible patients with malignant glioma present annually to our institution. This assessment is an important preliminary step toward establishing the feasibility of such a trial. A protocol for a randomized, controlled trial assessing biopsy versus resection for newly diagnosed malignant gliomas was developed. The projected number of patients required for this study is 312. We estimated 40 patients would be eligible for enrollment and randomization preoperatively. A retrospective chart review of all adult patients with glioma undergoing surgery at the University of Calgary between February 1, 2003, and January 31, 2006, was performed. The number of potentially eligible trial patients was determined. Over the 3-year study period, we have identified 227 patients with a new pathologic diagnosis of glioma. To date, we have reviewed all 125 newly diagnosed gliomas (WHO grade II-IV) over the 18-month period between July 2004 and February 2006. We identified 102 newly diagnosed enhancing gliomas. Of these patients, 59 met eligibility criteria for randomization. Forty-three patients were ineligible. Twelve of these patients were excluded based on clinical evidence for raised intracranial pressure, 12 had bilateral tumors not amenable to resection, 11 demonstrated multifocal lesions on imaging, 6 had previously undergone biopsy of a low-grade glioma, 5 did not meet a minimum KPS score of 50, 3 had a prior systemic malignancy, 3 had infratentorial lesions, 4 had lesions not amenable to greater than 75% resection, and 1 patient was medically unfit for craniotomy. Thirty patients were excluded based on 1 exclusion criterion alone, 11 based on 2 criteria, and 2 based on 3 of the exclusion criteria. Three of the eligible patients would have been excluded after their surgical procedure based on a final pathologic diagnosis inconsistent with malignant glioma. Six additional patients in the study period had nonenhancing mass lesions consistent with low-grade glioma but final pathologic diagnosis confirmed malignant glioma. Based on this series of patients, approximately 39 patients per year meet eligibility criteria for randomization and 5% of these would be excluded post-procedure for a diagnosis other than malignant glioma. We predict that total enrollment for a trial comparing biopsy versus resection of malignant glioma will need to be 344 (312 + 32 additional patients to account for 5% diagnosis other than malignant glioma and 5% loss to follow-up). Assuming 50% enrollment of potentially eligible trial candidates, we predict annual enrollment of 19 or 20 patients at our institution. A multicenter trial with 10 similar institutions could reach accrual in less than 2 years. We plan to proceed with a pilot trial at 2 institutions (the University of Calgary and the University of Alberta).


S. Kesari,1 D. Schiff,2 L. Doherty,1 D.C. Gigas,1 T.T. Batchelor,3 A. Muzikansky,3 A. O’Neill,3 J. Drappatz,1 A. Chen-Plotkin,1 N. Ramakrishna,1 S. Weiss,1 B. Levy,1 J. Bradshaw,1 P.M. Black,1 J. Folkman,4 M. Kieran,1,4 and P.Y. Wen1; 1Dana-Farber/Brigham and Women’s Cancer Center, Boston, MA, USA; 2University of Virginia, Charlottesville, VA, USA; 3Massachusetts General Hospital, MA, USA; 4Children’s Hospital, Boston, MA, USA

Preclinical evidence suggests that continuous low-dose daily (metronomic) chemotherapy may inhibit tumor endothelial cell proliferation (angiogenesis) and prevent tumor growth. We conducted a phase II study of continuous low-dose etoposide (VP-16), alternating with cyclophosphamide (CP), in combination with thalidomide (T) and celecoxib (C) in adult patients with recurrent malignant gliomas. Patients received VP-16 [35 mg/m2 (maximum 100 mg/day) daily for 21 days] alternating with CP [2 mg/kg (maximum 100 mg/day) for 21 days]. Thalidomide was started at 200 mg daily and increased by 100 mg weekly (maximum 1200 mg/day). Celecoxib was started at 200 mg twice daily (maximum 400 mg twice daily). MRI scans were performed every 6 weeks. Patients were treated until tumor progression or unacceptable toxicity. Serum and urine samples were collected for measurement of angiogenic peptides. Forty-eight patients were enrolled (15 women, 33 men). Twenty-eight patients had glioblastomas (GBM), and 20 had anaplastic gliomas (AG). The median age was 53 years, and the median KPS was 70. Thirty-two percent of patients had undergone 1 previous chemotherapy treatment, 34% had 2, and 34% had 3 or more previous chemotherapies. The therapy was reasonably well tolerated in this heavily pretreated population. No treatment-related deaths occurred. Fatigue was common but usually mild. Two percent of patients had a partial response (PR), 9% had a minor response (MR), 59% had stable disease (SD), and 30% progressed (PD) at their first scan. For GBM patients, the median progression-free survival (PFS) time was 11 weeks, the 6-month PFS rate was 9%, and the median overall survival (OS) time was 21 weeks. For AG patients, the median PFS time was 14 weeks, the 6-month PFS rate was 26%, and the median OS time was 41.5 weeks. When comparing responders (SD + MR + PR) with non-responders (PD), there was a significant difference in OS (P = 0.049) with median point estimates for responders and non-responders of 33 and 20 weeks, respectively; PFS was also significantly different (P < 0.0001) with 6-month PFS estimates of 21% vs. 0% in the responder and the non-responder groups, respectively. Some of the patients had adequate serial serum (N = 25) or urine (N = 31) specimens for angiogenic peptide analysis. In this limited set, serum and urine angiogenic peptides did not correlate with response or survival (P > 0.05). This 4-drug, oral antiangiogenic chemotherapy regimen was well tolerated. Angiogenic peptide levels did not correlate with survival or response. Although there were some responders, the regimen did not significantly improve OS in this heavily pretreated group of patients who were generally not eligible for conventional protocols. Further studies using antiangiogenic chemotherapy combined with more potent antiangiogenic agents in patients with less advanced disease may be warranted.


R.V. La Rocca, J. Hodes, W.G. Villanueva, T.W. Vitaz, D.J. Morassutti, M.J. Doyle, S. Glisson, A. Cervera, L. Stribinskiene, P. New, and N.S. Litofsky; Kentuckiana Cancer Institute, PLLC, Louisville, KY, USA; Hodes Neurosurgery, Louisville, KY, USA; Louisville Neurosurgery, Louisville, KY, USA; University of Louisville, Department of Neurological Surgery, Louisville, KY, USA; Center for Neurosurgical Care, Louisville, KY, USA; Neurosurgical Group of Greater Louisville and Southern Indiana, Louisville, KY, USA; Baylor College of Medicine, Houston, TX, USA; University of Missouri, Columbia School of Medicine, Division of Neurological Surgery, Columbia, MO, USA

Previous studies have demonstrated the effectiveness of both BCNU wafers (Gliadel Wafer [polifeprosan 20 with carmustine implant], MGI PHARMA) and TMZ (Temodar [temozolomide], Schering Corporation) as adjuvant therapies to resection and radiation therapy (RT) in patients with initial high-grade malignant glioma (MG). This phase II multicenter trial assessed the safety and efficacy of BCNU wafers in combination with TMZ. Eligible patients were aged 18–72 years with surgically operable, initial high-grade MG. Enrollment began in July 2003 and is ongoing. After resection and BCNU wafer insertion, patients received concomitant treatment with daily radiotherapy 2 Gy (total [less-than-or-eq, slant] 60 Gy) + TMZ 75 mg/m2 ([less-than-or-eq, slant] 45 days) followed by monthly TMZ (200 mg/m2 daily × 5 every 28 days, [less-than-or-eq, slant] 18 cycles). The endpoints include survival and progression-free survival (PFS), which were estimated with the Kaplan-Meier method. As of March 2006, 33 patients have been enrolled at 3 centers. Glioblastoma multiforme was diagnosed in 32 patients. The median age is 56 years (range, 29–74 years), and the median KPS is 100 (range, 70–100). After a median follow-up of 8.1 months (range, 1.0–22.9 months), 22 patients have had a recurrence and 15 patients have died. The cause of death in 12 patients was disease progression. Twelve patients remain on active treatment, and 1 patient withdrew consent. The median survival is 18.5 months and the median PFS is 6.4 months. The 1-year survival and PFS rates are 56.8% and 20.7%, respectively. Adverse events include deep vein thrombosis (5 patients), pulmonary embolism (4 patients), sterile brain abscess (1 patient), and bacterial pneumonia (1 patient). No cases of acute respiratory distress syndrome or Pneumocystis carinii pneumonia have been experienced. These data suggest that combination therapy with BCNU wafers followed by RT/TMZ is an effective therapy regimen in patients with initial high-grade resectable MG. Randomized trials will be needed to ultimately assess efficacy compared with RT/TMZ alone. Adverse events were similar to those observed in patients undergoing treatment for high-grade glioma.


A.B. Lassman,1 T.F. Cloughesy,2 L.M. DeAngelis,1 E. Tara,1 T. Sardharwala,1 and L.E. Abrey1; 1Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 2University of California Los Angeles, Los Angeles, CA, USA

The management of newly diagnosed anaplastic oligodendroglioma (AO) and anaplastic mixed glioma (AMG) varies significantly. Recent results demonstrate that chemotherapy (CT) with procarbazine, lomustine, and vincristine (PCV) administered before or after radiotherapy (RT) improves progression-free survival but not overall survival (OS). However, many patients receive CT alone as the initial therapy, and it is unclear whether deferring RT affects progression or survival. We retrospectively reviewed records of patients treated for AO or AMG from 1986 to 2006) at Memorial Sloan-Kettering Cancer Center or the University of California, Los Angeles. Patients were grouped by their initial treatment strategy: observation, RT alone, CT alone, CT then RT, RT then CT, or RT with concurrent CT. The CT regimen (PCV, temozolomide, other) was also recorded. Patients without histologic confirmation of AO or AMG were excluded as were patients who previously received RT or CT for a low-grade glioma. Two hundred forty patients (132 men, 108 women) with treatment-naïve AO (N = 139) or AMG (N = 101) had a median age of 41 years (range, 19–81 years). The median OS for all patients was 10.8 years with a median follow-up of 3.0 years for surviving patients (N = 167). The median time to progression (TTP) for all patients was 2.9 years. Combining RT with CT (in either order or concurrently) prolonged TTP relative to treatment with CT alone (median 5.3 vs. 2.3 years, P < 0.05) but without an OS benefit (median 9.6 years vs. not-reached, P = 0.63). Combining RT with CT as initial therapy for AO and AMG may improve TTP without prolonging OS. As many patients survive long enough to experience potential cognitive impairment from brain RT, our results suggest that deferring RT until disease progression may be reasonable in patients responding favorably to CT. We are collecting additional data from Oligodendroglioma Study Group institutions to assess the effect of different CT regimens (e.g., PCV vs. temozolomide), 1p/19q loss of heterozygosity, and histologic subtype on both TTP and OS.


F. S. Lieberman,1 B. Berkey,2 C. Tsien,3 W. Curran,4 M. Werner-Wasik,4 R. Smith,1 L. Grossheim,5 E. Hug,6 and M. Mehta5; 1University of Pittsburgh, Pittsburgh, PA, USA; 2Radiation Therapy Oncology Group, Philadelphia, PA, USA; 3University of Michigan, Ann Arbor, MI, USA; 4Thomas Jefferson University, Philadelphia, PA, USA; 5Medical College of Wisconsin, Milwaukee, WI, USA; 6University of Dartmouth, Hanover, NH, USA

Irinotecan and temozolomide compare favorably with regimens tested in recurrent GBM. The RTOG 04-20 study intensifies the Stupp adjuvant regimen by using irinotecan and temozolomide in place of temozolomide alone. Adult patients with newly diagnosed, histologically confirmed, supratentorial GBM were eligible. Subjects began temozolomide 75mg/m2 daily the night before initiation of EBRT and continued until the final day of RT. Pneumocystis prophylaxis was started before RT and for 2 weeks following RT. Within 6 weeks after EBRT, subjects with stable or improved MRI were scheduled to receive temozolomide 150 mg/m2 on days 1–5, and irinotecan 200 mg/m2 on days 1 and 15 of 28 day cycles × 12. Clinical assessments and post contrast MRI are required before EBRT, after RT, and after every 2 treatment cycles. Only nonenzyme-inducing anticonvulsants were allowed. The accrual of 170 patients was completed in September 2005, and 140 patients are currently evaluable. The median age is 57 years, and 80% of patients were RPA class III or IV. Before cycle 1 of adjuvant therapy, 32 subjects withdrew (20 progressed, 4 toxicity). Of the first 25 subjects receiving irinotecan, 10 experienced grade III-IV hematologic toxicities in the initial 3 cycles. The protocol was modified by dose-reducing irinotecan to 100 mg/m2 in cycle 1, escalating to 150 and 200 mg/m2 in subsequent cycles only if no dose-limiting hematologic toxicity occured. One treatment-related death has occurred. Grade III-IV hematologic toxicities have occurred during the first 3 adjuvant cycles in 11 subjects entered onto the modified regimen. Diarrhea and constitutional symptoms are the most common nonhematologic toxicities. Although irinotecan and temozolomide were well tolerated as treatment for recurrent GBM, grade III and IV myelosuppression was seen even after irinotecan dose reduction in the initial 3 cycles of adjuvant treatment given after EBRT with concomitant low-dose temozolomide. The preliminary data for time to progression and overall survival will be available for presentation.


Mark K. Lyons, Alyx B. Porter, and E. Peter Bosch; Mayo Clinic Arizona, Scottsdale, AZ, USA

Intraspinal tumors rarely cause raised intracranial pressure. In such cases, an elevated cerebrospinal fluid (CSF) protein is frequently found. This case is unique because of the initial presentation of papilledema, an absence of CSF protein elevation, the late appearance of myelopathy, and the eventual development of metastatic high-grade astrocytoma to the brain. A 19–year-old right-handed man presented with transient visual obscurations. An examination revealed bilateral papilledema. An MRI scan of the brain was normal, with CSF opening pressure of 43 cm H2O, WBC 1, protein 48 mg/dl, and glucose 41 mg/dl. All viral tests, connective tissue markers, and heavy metal screens were negative. The patient was diagnosed with pseudo-tumor cerebri and underwent LP shunting. The patient subsequently developed lower extremity weakness, was diagnosed with Devic’s syndrome, and was referred to our institution. Examination revealed optic disc pallor, afferent pupillary defect, asymmetric spastic paraparesis, hyperreflexia, bilateral Babinski signs, LE vibratory loss, and a T5 sensory level. An MRI scan demonstrated a non-enhancing swollen spinal cord from C7–T4, and the patient underwent spinal cord biopsy and ligation of the LP shunt. A pathologic assessment confirmed grade fibrillary astrocytoma. The patient received 48.6 Gy in 27 fractionated sessions and temozolomide. Persistent headaches responded to VP shunting. Several months later, he developed new onset symptoms and a follow-up MRI of the brain demonstrated an intraaxial lesion that was resected and confirmed as high grade astrocytoma by pathologic assessment. The initial presentation of a cervicothoracic malignant spinal astrocytoma with intracranial hypertension and visual loss without elevated CSF protein or myelopathy symptoms is rare. The subsequent development of a metastatic cranial high grade astrocytoma further illustrates the poor prognosis of these tumors.


Mark K. Lyons; Mayo Clinic Arizona, Scottsdale, AZ, USA

Pilocytic astrocytomas are relatively uncommon tumors found predominately in children and they are considered as essentially benign gliomas in the pediatric population. Reports of these tumors are extremely rare in the elderly. Their histopathologic features do not correlate with an aggressive clinical course or poor prognosis. Intracranial hemorrhage in pilocytic astrocytoma has been reported primarily in the young. Lones and Verity reported a 69-year-old woman with a fatal hemorrhage of a pilocytic astrocytoma in the thalamus. Supratentorial pilocytic astrocytomas appear most commonly as solitary lesions in the temporal lobes. Imaging characteristics of pilocytic astrocytomas are generally well demarcated with variable degrees of enhancement. Pathologic studies have demonstrated that some of these tumors infiltrate the surrounding parenchyma. The presence of nuclear pleomorphism, mitotic activity, and endothelial proliferation occur in pilocytic astrocytomas but do not carry the same poor prognostic significance. The MIB-1 and p53-labeling indexes are generally negative. Lieu reported a review of brain tumors with intracranial hemorrhage and found that the highest rate of hemorrhage for the primary brain tumor group occurred in pilocytic astrocytomas. Gottfreid reported the case of an intracranial hemorrhage secondary to a pilomyxoid astrocytoma, which has been identified as a more aggressive form of pilocytic astrocytoma with a less favorable clinical outcome in pediatric patients. Pilocytic astrocytoma in an elderly adult is extremely rare. Temporal lobe pilocytic astrocytomas may arise from nodular heterotopia and may act as a precursor for the formation of a pilocytic astrocytoma. Consequently, some of these tumors may be essentially dormant for decades. Pilocytic astrocytomas are almost always benign in their clinical course. Although most commonly found in children, pilocytic astrocytomas can occur in adults. The potential microscopic infiltration of pilocytic astrocytomas into the adjacent parenchyma may influence the likelihood of spontaneous hemorrhage.


James M. Markert, Michael D. Medlock, Samuel D. Rabkin, Yancey Gillespie, Matthias Karrasch, Eunice Braz, Axel Mescheder, and Robert L. Martuza; Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, AL; Department of Neurosurgery, Massachusetts General Hospital, Boston, MA; USA; MediGene AG, Munich, Germany

G207 is a modified oncolytic herpes simplex virus (oHSV) type 1 that has been genetically engineered to replicate in and kill cancer cells while sparing normal cells. The unique construction of G207 allows it to be inoculated directly into malignant gliomas. The results of this study have been published previously, but we now provide updated results, including updated outcome data. Sixteen patients with glioblastoma multiforme (GBM), 4 patients with anaplastic astrocytoma (AA), and 1 patient with an anaplastic mixed glioma were stereotactically inoculated with G207. Three patients were entered into each of 7 dosing cohorts. Dosing started at 1 × 106 pfu (plaque forming units) of G207 and was escalated up to 3 × 109 pfu. An assessment of potential acute toxicities was permitted by having a 10-day waiting period before inoculation of the next patient within each dose cohort as well as a 28-day waiting period between each dose cohort. Vital signs, neurological examinations, general physical examinations, MRI, HSV antibody status, and tests for shedding of herpes simplex virus were performed on a regular basis. We have previously reported that 1 month after G207 injection, all patients were alive and their mean KPS had diminished only by 0.4% and the Mini Mental Status Examination only by 3.5%. Two patients had a partial response (at least a 50% decrease in contrast-enhancing tumor volume) at any time after G207 injection, and 16 patients showed stable disease. One patient could not be evaluated by MRI because of the use of a pacemaker. One GBM patient died of a stroke 9 months after treatment. An autopsy did not demonstrate evidence of residual tumor or virus-related toxicity. New to this report, 1 AA patient survived for approximately 63 months after treatment. The final surviving GBM patient developed a recurrence requiring reoperation 7 years after treatment and died 6 months later. No long-term evidence of toxicity was identified, either by MRI or by clinical examination. This study showed that doses up to 3 × 109 pfu of G207 can be inoculated safely into malignant gliomas without development of encephalitis or occurrence of toxicity related to this mutant herpes virus. Patients did develop complications frequently associated with malignant glioma, including death, but none of these complications could unequivocally be ascribed to G207; no dose-limiting toxicity was observed. No evidence of long-term toxicity was seen in a patient surviving greater than 7 years post-treatment. Follow-up studies of G207 treatment for malignant glioma are underway.


Marlon S. Mathews,1 Mark E. Linskey,1 Franklin D. Westhout,1 Anton N. Hasso,2 and John P. Fruehauf3; Departments of 1Neurosurgery, 2Radiology, and 3Medicine, University of California-Irvine, Orange, CA, USA

Approved as a first-line therapy for metastatic colon cancer in 2004, bevacizumab is being used off-label to treat a number of cancers, including metastatic breast and lung cancers. Bevacizumab is a monoclonal antibody that binds to and inactivates the vascular endothelial growth factor (VEGF), which stimulates neoangiogenesis. Case report: A 54-year-old woman with a history of metastatic infiltrating ductal breast carcinoma treated with bilateral mastectomies, chemotherapy, and radiation, was found to have left occipital and right parietal intra-axial contrast-enhancing masses on surveillance MRI. She underwent left occipital and right parietal craniotomies for microsurgical resection of these lesions. After the procedures, she was placed on bevacizumab for control of the systemic disease. Six months after being placed on bevacizumab, a right occipital nonenhancing lesion was detected on FLAIR sequence MRI. Based on the nonenhancing character of the new lesion and the absence of surrounding edema, nonneoplastic processes such as granuloma, inflammation, and previous hemorrhage were considered in the differential diagnosis, although a metastatic lesion could not be ruled out considering the patient was taking an antiangiogenic agent. After discontinuing bevacizumab therapy, the patient underwent microsurgical resection of the lesion. Histopathologic examination of the right occipital lesion was consistent with metastatic breast cancer. Six weeks after discontinuing bevacizumab therapy and 3 weeks after microsurgical resection, a new contrast-enhancing mass was noted in the right temporal lobe using T1W MRI. Studies using dynamic contrast-enhancing MRI (DCE-MRI) have shown decreased uptake of contrast in bevacizumab treated tumors. As the off-label use of bevacizumab has increased, patients have been receiving this drug and developing new cerebral metastases. It is important for clinicians to recognize that this drug may alter usual patterns of blood-brain barrier breakdown and tumor vasculature seen in cerebral metastases. This alteration may in the future also be seen with new drugs such as small molecule inhibitors of neoangiogenesis.


L. Mechtler,1 J. Alksne,2 E. Wong,3 E. Arenson,4 L. Recht,5 N. Avgeropolous,6 D. Eisenstat,7 A. Hormigo,8 J. Perry,9 J. Raizer,10 W. Shapiro,11 L. Taylor,12 M. Shulman,13 and L. Carr14; 1DENT Neurologic Institute, Buffalo, NY, USA; 2Moores UCSD Cancer Center, La Jolla, CA, USA; 3Beth Israel Deaconess Medical Center, Boston, MA, USA; 4Colorado Neurological Institute, Englewood, CO, USA; 5Stanford University, Stanford, CA, USA; 6Hospital Cancer Institute, Orlando, FL, USA; 7CancerCare Manitoba, Winnipeg, Canada; 8Memorial Sloan Kettering Cancer Center, New York, NY, USA; 9Sunnybrook and Women’s College Health Sciences Centre, Toronto, Canada; 10Northwestern University, Chicago, IL, USA; 11Barrow Neurological Institute, Phoenix, AZ, USA; 12Virginia Mason Medical Center, Seattle, WA, USA; 13BioMedical Resources, San Francisco, CA, USA; 14Neurobiological Technologies, Inc., Emeryville, CA, USA

The aim of this study is to evaluate the long-term safety, tolerability, and steroid-sparing potential of Xerecept (corticorelin acetate injection), a synthetic peptide with an amino acid sequence identical to that of human corticotropin-releasing factor (hCRF), in patients with primary or secondary brain tumors (BT) and peritumoral brain edema. Following participation in one of 2 randomized, double-blind phase III trials comparing Xerecept with placebo or Xerecept with dexamethasone, 20 patients completed, and 2 patients failed to complete, at least 4 weeks of treatment with subcutaneous Xerecept 1.0 mg bid in an ongoing open-label study. We reduced dexamethasone maximally as tolerated; the protocol did not require maximum reduction during the first 4 weeks. We assessed patients at abaseline and bafter 4 weeks of treatment (or at early study drug discontinuation [SDD]), and c4 weeks after early SDD: body weightb,c; vital signsb,c; EKGb,c; physical examb,c; neurologic measuresb,c; Fact-Br QOLb; concomitant medicationsa,b,c; AEs a,b,c, including steroid-related side effects a,b,c; dexamethasone dosea,b,c; and brain MRIb. The first 20 patients who completed at least 4 weeks of treatment with Xerecept 1.0 mg bid included 14 men and 6 women; mean age 53.5 years (range, 33–67 years); white 19, African-American 1; glioblastoma multiforme 11, metastatic BT 3, meningioma 2, astrocytoma 1, anaplastic oligoastrocytoma 1, other 2. For these 20 patients, we will present an interim report of AEs and changes in mean dexamethasone dose and steroid-related side effects from baseline to week 4 and duration of treatment to date. For the 2 patients who did not complete at least 4 weeks of treatment, we will present the reasons for early SDD; AEs; changes from baseline to early SDD in mean dexamethasone dose, steroid-related side effects, and neurologic measures; and brain MRI results following early SDD.


Tom Mikkelsen, Tom Doyle, Nina Paleologos, Lonni Schultz, and David Croteau; Henry Ford Hospital, Detroit, MI, USA

We are conducting a clinical trial to evaluate the safety and efficacy of single-agent temozolomide chemotherapy for patients with newly diagnosed anaplastic oligodendrogliomas (AOs) or anaplastic oligoastrocytomas (AOAs). Treatment assignment is based on the identification of chromosome combined chromosome 1p/19q LOH. Those patients with this favorable prognostic finding are allowed to continue with chemotherapy alone for 1 year, whereas those patients not showing LOH are treated with concurrent chemo-radiation and adjuvant chemotherapy. Our primary intentions are (1) to demonstrate the safety of single-agent chemotherapy in this population, (2) to show the noninferiority of this regime when compared with published accounts of therapy in this disease, and (3) to describe the incidence of adverse events associated with radiation/chemotherapy in non-LOH patients. The goal is to demonstrate that single-agent chemotherapy is a rational treatment choice with regard to safety and efficacy and should be considered as a treatment arm in large randomized trials. To date, 41 patients have been accrued, 27 with 1p/19q LOH, 10 without LOH, and 4 pending results. With a median time of follow-up of 24.3 months (range, 0–57.9 months), current safety and efficacy data will be presented.


Nimish A. Mohile, Andrew B. Lassman, David N. Louis, Tarun Kewalramani, Peter Forsyth, Douglas Stewart, Nina Paleologos, Jeffrey J. Raizer, Lisa M. DeAngelis, J. Gregory Cairncross, and Lauren E. Abrey; Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Massachusetts General Hospital, Boston, MA, USA; University of Calgary, Calgary, AB, Canada; Northwestern University, Evanston, IL, USA; Northwestern University, Chicago, IL, USA; and London Regional Cancer Center, London, ON, Canada

Anaplastic oligodendrogliomas (AO) and anaplastic mixed gliomas (AMG) are incurable tumors with demonstrated chemosensitivity. A previous phase II trial demonstrated the efficacy of intensive PCV (I-PCV) followed by myeloablative single-agent thiotepa with ASCR. We report the preliminary results of a new trial using an intensified myeloablative regimen: busulfan and thiotepa. Twenty patients (16 AO, 4 AMG) from 4 institutions with a median age of 46 years (range, 19–60 years) and a KPS of 90 (range, 70–100) were treated with 4 cycles of I-PCV. Patients with a complete response (CR) to I-PCV or who continued to be free of enhancing disease after surgery and I-PCV (CCR) were eligible for transplant. Prospective testing of 1p/19q LOH was performed. Fourteen patients (70%) had a CR or CCR to I-PCV and underwent transplant. Ten of these patients are alive with no evidence of progression at a median follow-up of 19 months (range, 8–32 months). Two patients relapsed after transplant: 1 died of sepsis > 100 days from ASCR and 1 died from complications of veno-occlusive disease (VOD). Two patients who underwent transplantation with no evidence of disease also developed VOD but with no permanent sequelae. Six patients were ineligible for transplant: 4 did not respond to I-PCV, 1 developed an intratumoral hemorrhage before I-PCV and 1 patient with a CR did not undergo transplantation; 3 died from tumor progression. The results of 1p/19q testing are available for 17 patients: 11 had co-deletions of 1p/19q, 3 had deletions of 1p, and 3 had both 1p and 19q intact. Seven 1p/19q-deleted patients and all patients with other genetic status had a CR or CCR to I-PCV. To date, there is no apparent difference in outcome based on molecular genetic status. Half of all patients who enrolled achieved a durable response and were able to defer radiotherapy. However, 3 patients developed VOD likely related to the addition of busulfan to the conditioning regimen. Although patients numbers in this study are small, the 1p/19q LOH results suggest that patients with intact chromosomes may also derive benefit from this regimen.


Pamela New; The Methodist Hospital Neurological Institute, Houston, TX, USA

Primary central nervous system lymphoma (PCNSL) in immunocom-promised patients carries a dismal prognosis for survival. Treatment has traditionally involved radiotherapy and reduction in immunosuppressive agents. The incidence in the human immunodeficiency virus (HIV)(+) population seems to be decreasing in the era of antiretroviral therapy, whereas the incidence in patients who undergo organ transplants is stable despite an increasing number of transplant recipients. Treatment has traditionally involved radiation therapy because of concern over the tolerance of chemotherapeutic agents. Patients are often in the later stages of the disease when the diagnosis is made. This abstract describes the treatment of 2 immunosuppressed patients with high-dose methotrexate, the response to date, and the side effects experienced. The first patient is a 52-year-old woman who had undergone 2 renal transplants and was diagnosed with PCNSL involving the cervical spinal cord. She was treated with high-dose methotrexate (MTX) at 8 g/m2, occasionally adjusted for creatinine clearance, for 6 induction cycles, followed by 11 maintenance cycles. The second patient is a 34-year-old man who presented with a 3-week history of confusion and was found to have a large ring-enhancing mass. The results of the biopsy confirmed the diagnosis. This patient is still in the induction phase of MTX treatment. The first patient completed over a year of chemotherapy with high-dose MTX and experienced grade II nausea and vomiting with no myelosuppression. Renal function has remained normal as has neurological function. The patient has remained on immunosuppressive agents throughout this period. She is presently at 21 months postdiagnosis without recurrence. The second patient has tolerated induction therapy without experiencing nausea, vomiting, or myelosuppression thus far. Grade II fatigue has been the most significant toxicity. He also remains on retroviral therapy. At the time of the HIV/PCNSL diagnosis, his presenting symptom and finding was cognitive decline, which has remained stable thus far. There are no reports of chemotherapeutic trials in patients with immunosuppression and a diagnosis of PCNSL. Although the incidence may be declining, PCNSL still occurs in the HIV(+) and organ transplant populations. Treatment with radiotherapy has resulted in an increase in median survival from 13 weeks to 3 months in some reports. As the addition of MTX chemotherapy has proven effective in the nonimmunocompromised PCNSL population, a trial is warranted in patients who are immunosuppressed. The risk of further cognitive impairment secondary to radiotherapy is also a reason to explore the option of chemotherapy for these individuals, as they survive longer. A formal neuropsychiatric testing as part of a clinical trial of high-dose MTX in patients with HIV is being designed.


Teri D. Nguyen, Andrew B. Lassman, Eric Lis, Neal Rosen, David R. Shaffer, Howard I. Scher, Lisa M. DeAngelis, and Lauren E. Abrey; Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Overexpression of EGFR and PTEN loss in the majority of GBMs leads to increased AKT signaling and cellular proliferation. mTOR is a downstream target of AKT, which is blocked by RAD-001. The addition of an mTOR inhibitor to EGFR blockade by gefitinib may augment downregulation of AKT. Nineteen patients with GBM were enrolled in a phase I/II protocol of gefitinib and RAD-001, open to patients with either hormone-refractory prostate cancer or recurrent GBM. Patients on enzyme-inducing anti-epileptic drugs (EIAEDs) were excluded, patients who had previous treatment with an EGFR inhibitor were allowed, and there were no limitations on the number of previous relapses. All patients received gefitinib 250 mg daily. Two patients enrolled in a dose-escalation arm and received RAD-001 30 mg or 50 mg weekly; 17 patients received the maximum tolerated dose of RAD-001, 70 mg weekly. Baseline and follow-up MRI scans were reviewed by 3 independent reviewers using modified RECIST criteria. The primary endpoints were radiographic response and progression-free survival (PFS). Eleven men and 8 women with a median age of 53 years (range, 22–72 years) and a median KPS of 80 (range, 70–100) enrolled in the study. Seventeen patients (89%) enrolled for their second or greater recurrence. The most frequently occurring grade I and II toxicities were thrombocytopenia, transaminitis, rash, anemia, hypercholesterolemia, and diarrhea. The most frequent grade III and IV toxicities were elevated ALT and lymphopenia. Six patients (32%) had a radiographic response (2 partial, 4 minor), and 3 (16%) achieved stable disease. Among the responders, 1 patient was treated at third recurrence, 2 at fourth recurrence, and 1 patient had progressed through previous gefitinib therapy. The median PFS was 2.6 months, with 1 patient progression-free at 6 months. The median overall survival was 6.5 months. The combination of RAD-001 and gefitinib demonstrated activity in 47% of patients with recurrent GBM (32% partial or minor response, 16% stable disease). Most subjects were heavily pretreated and were expected to have resistant disease. The observed radiographic responses were not well captured by conventional response criteria and require an alternative method of assessment. In order to improve the durability of response, alternate dosing or treatment earlier in the course of disease should be considered in future studies of this promising combination.


D. Peereboom,1 C. Brewer,1 D. Schiff,2 P. Fisher,3 M. Chamberlain,4 S. Panullo,5 H. Newton,6 R. Prayson1, G. Stevens,1 M. Vogelbaum,1 S. Toms,1 P. Elson,1 and G. Barnett1; 1Cleveland Clinic, Cleveland, OH; 2University of Virginia, Charlottesville, VA; 3Stanford University, Palo Alto, CA; 4Moffitt Cancer Center, Tampa, FL; 5Columbia Medical College, New York, NY; 6Ohio State University, Columbus, OH, USA

The standard initial therapy for patients with pure and mixed anaplastic oligodendrogliomas (AO/MAO) has included chemotherapy and radiation therapy. These gliomas have particular sensitivity to chemotherapy, which varies according to the molecular genetics of the tumor. Due to the chemo-responsiveness of these tumors, this trial has used a dose-intense regimen of temozolomide and has reserved RT for patients with disease progression. This study sought to determine the progression-free survival, response rate, and quality of life (QOL) in patients (pts) with newly diagnosed AO/MAO treated with temozolomide every other week and to determine outcomes according to tumor cytogenetic status. Eligible pts had newly diagnosed AO/MAO with no prior chemotherapy or RT. All pathology had central review and tumor assay for 1p deletion using FISH. The analysis was stratified by 1p status. Temozolomide was given 150 mg/m2 days 1–7 and 15–21, every 28 days. Therapy was given for up to 8 cycles. Responses and QOL (FACT-BR and EORTC brain module) were measured every 8 weeks. Sixty pts have enrolled from 7 centers. One patient withdrew before starting treatment and has thus been excluded from all analyses. Four pts are still receiving therapy and are not yet evaluable. Patient characteristics are as follows: median age, 42 years (range, 18–83 years); KPS, 100 in 18 pts, 90 in 29 pts, 70–80 in 12 pts; histology, AO in 38 pts, MAO in 21 pts, and 1p deletion in 33 pts (26/38 with AO and 7/21 with MAO). Patients have received 0–8 cycles (median, 8) of temozolomide (38 pts completed 8 cycles, 9 withdrew consent for toxicity or patient choice prior to completion, 8 withdrew for progressive disease before completion, 4 continue with stable disease on therapy). Three pts have required dose reduction for toxicity (20% reduction for thrombocytopenia). Of 22 pts with measurable disease and response data available, 2 (AO with 1p loss) (9%) achieved complete remission, 9 (41%) had stable disease, and 11 (50%) had progression. Thirty-seven pts remain free from progression with a median progression-free survival of 27 months (range, 0–39 months; 95% confidence interval 10.8–37.2 months). Patients with intact 1p tended to have shorter progression-free survival than pts with 1p loss (median 10.7 vs. 32.6 months, P = .06). Fifty pts remain alive. Median survival has not been reached, and it is too early to assess the impact of 1p status on this endpoint. Seven pts experienced grade III-IV neutropenia, 5 grade III thrombocytopenia, and 13 with grade III lymphopenia. Grade I and II toxicities have included fatigue, nausea, vomiting, and constipation. QOL data will be presented upon study completion. This trial supports the concept of using chemotherapy alone as initial therapy for patients with chemotherapy-responsive gliomas. The durability of responses is not yet known. Overall survival data is immature. Toxicity in this trial has been manageable. Most patients with newly diagnosed AO/MAO receiving temozolomide can delay the need for radiation therapy for a median of over 2 years. Temozolomide offers a reasonable alternative to initial RT for patients with newly diagnosed AO/MAO.


D.M. Peereboom, C.J. Brewer, J.H. Suh, G.H. Stevens, G.H. Barnett, S.A. Toms, P. Elson, M.A. Vogelbaum, and R.J. Weil; Cleveland Clinic Brain Tumor Institute, Cleveland, OH, USA

Approximately 40%–50% of glioblastomas (GBM) overexpress EGFR, which activates tyrosine kinase to promote GBM cell infiltration and proliferation in vitro. Radioresistance of some GBMs has been correlated with EGFR overexpression. Erlotinib is a specific and potent EGFR tyrosine kinase inhibitor (TKI) with activity against refractory GBM. Furthermore, the occurrence of erlotinib-induced rash of [gt-or-equal, slanted] grade (G) II has been correlated with improved survival in non-small cell lung cancer. A phase II trial of erlotinib with concurrent RT-temozolomide (RT-TMZ) was therefore initiated with pharmacodynamic dosing of erlotinib. Eligible patients (pts) had newly diagnosed GBM; age [gt-or-equal, slanted] 18 years; KPS [gt-or-equal, slanted] 60; no cytochrome P-450 enzyme-inducing anticonvulsants; no prior treatment with temozolomide (TMZ), TKI, or cranial RT. Pts received RT 60 Gy in 30 fractions with concurrent TMZ (75 mg/m2/day for 42 days). Four weeks after RT-TMZ, pts received TMZ 150–200 mg/m2/day for 5 days, every 28 days for 12 cycles. Pts received erlotinib daily from day 1 of RT until disease progression. Erlotinib dosing started at 50mg/day for each patient with escalation every 2 weeks by 50 mg/day until the occurrence of grade II rash or maximum dose of 150 mg/day. We used the FISH assay for EGFR amplification of all tumors. Twenty-eight patients were enrolled in this study. One patient never received treatment and was therefore excluded from all analyses. The median age was 52 years (range, 18–74 years); KPS = 90 (range, 70–100). Seven pts had gross total resection, 8 had subtotal resection, and 12 had biopsy only. Nine pts were EGFR amplified, 17 pts were EGFR not amplified, and 1 pt had no assay. The maximum dose of erlotinib reached before GII rash: 50 mg/d (1 pt), 100 mg/d (5 pt), and 150 mg/day (21 pts). Twenty-one patients have progressed (78%) and 8 (30%) have died. The median progression-free survival was 3.6 months. Six patients are alive and progression-free at 4.4+, 8.3+, 11.5+, 14.7+, 15.8+, and 21.8+ months, respectively. Four deaths occurred on study, three of which were definitely related to treatment. One patient died of pneumocystis carinii pneumonia despite prophylaxis with pentamidine. Two patients died of refractory bone marrow aplasia and one died of non-neutropenic sepsis. The trial was therefore terminated after the accrual of 27 of 30 planned patients. Other treatment-related GIII-IV toxicities were (GIII/GIV): thrombocytopenia (4/4), anemia (3/1), ANC (4/0), lymphopenia (15/0), fatigue (2/0), and febrile neutropenia (3/0). Additional events unrelated to therapy have included wound infection, spontaneous pneumothorax, and rectus sheath hematoma. The combination of erlotinib with RT-TMZ using dose escalation to a pharmacodynamic endpoint is feasible in patients with newly diagnosed GBM. Unfortunately, the activity was modest and toxicity was substantial. Whether erlotinib contributed to the adverse events seen in this trial is unclear. The cytostatic activity of erlotinib possibly antagonizes the cytotoxic activity of RT and temozolomide when administered concurrently. Future trials that combine cytotoxic and cytostatic therapy should perhaps employ these mechanisms of action in a sequential rather than in a concurrent fashion with cytotoxic therapy first.


Marta Penas-Prado and Vinay K. Puduvalli; Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Adult medulloblastoma (MB) is uncommon. Combined treatment with surgery, craniospinal radiation (CSRT), and chemotherapy has improved its outcome. Because of the paucity of prospective data, the treatment approach is usually derived from results of retrospective studies and trials in children. However, the optimal treatment in adult MB remains to be determined. To study the clinical characteristics, imaging and laboratory findings, and treatment and outcome of a series of adult patients ([gt-or-equal, slanted] 18 years) with MB, a retrospective review was conducted using data from a subgroup of adult patients with MB treated at MDACC between 1995 and 2004 who were identified in the neuro-oncology patient longitudinal database. Data collected included the modalities of treatment, such as surgery, radiation therapy, and chemotherapy at various stages of the disease (neoadjuvant, chemoradiation, adjuvant, and at recurrence). Of the 34 adult patients with MB who were identified, 25 were newly diagnosed (19 average-risk and 6 high-risk), and 9 were seen at recurrence. The median age at diagnosis was 29 years (range, 18–38 years). Extent of surgery was GTR in 19 patients (56%), STR in 12 (35%), and biopsy in 1 (3%). Thirty-three patients received CSRT. Twelve (35%) received chemotherapy at diagnosis and 14 received chemotherapy at recurrence. The median TTP was 60 months. The PFS at 3 and 5 years was 69% and 47%, respectively. The median OS was 84 months, and OS at 5 years was 71%. Among these 34 patients, we identified a subset of 18 who were seen at diagnosis and who were then followed up at MDACC alone. The median age of this subgroup was 30 years. GTR was performed in 10 patients, STR in 7, and biopsy in 1. Thirteen were average-risk, and 5 were high-risk (M0 = 2; M3 = 3). All patients but one received CSRT (30–36 Gy) followed by a posterior fossa boost (54–56 Gy). Seven patients (39%) received chemotherapy at diagnosis (4 neoadjuvant, 1 chemoradiation and adjuvant, and 2 adjuvant). Of the various chemotherapy regimens, cyclophosphamide, etoposide, and platinum derivatives were the most frequently used agents. In this group of 18 patients, a total of 6 (33%) developed recurrence and received salvage chemotherapy. The most frequent combination was cyclophosphamide, etoposide and cisplatin. PFS was 81% at 3 years and 72% at 5 years. The median survival was not reached after a median follow-up of 55 months. OS was 94% at 3 years and 78% at 5 years. Although no definite conclusions could be drawn regarding efficacy of the therapies because of the small number of patients, combined treatment at diagnosis appeared to be effective in high-risk patients with metastatic disease (the 3 patients were alive and free of recurrence at 48, 79, and 107 months). Most average-risk patients did not receive any chemotherapy at diagnosis (10 out of 13). Among these patients, 4 recurred and 3 died at 41, 47, and 84 months. The PFS and OS in our series are similar to those reported in children. The better outcome in the subset followed up closely at MDACC may be due to referral bias but points to the fact that there continues to be a high variability in the management of the disease and a lack of a standard treatment approach in adult patients. In this retrospective series, only a small number of patients received chemotherapy at diagnosis, and thus no comparison to different modalities was possible. Valid information on the efficacy and timing of chemotherapy in the adult patient with MB is very difficult to obtain from retrospective series. In addition, whether chemotherapy has a role in the initial treatment of average-risk patients remains to be determined. Large, multi-institutional prospective trials in adult patients with MB are warranted to provide more definitive treatment guidelines.


S. Phuphanich, J. Supko, K.A. Carson, S.A. Grossman, L.B. Nabors, T. Mikkelsen, G. Lesser, M. Rosenfeld, S. Desideri, and J. Olson; for the New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium, Baltimore, MD, USA

Bortezomib is a novel anticancer agent with a small molecule that selectively inhibits the proteasome by binding tightly to the enzyme’s active sites. The objective is to determine the maximum tolerated dose (MTD) and evaluate the safety, toxicity, and biologic activity of bortezomib for the treatment of recurrent malignant glioma (MG). Eligible patients (pts) had supratentorial progressive MG and had undergone previous radiation therapy and [less-than-or-eq, slant] 1 regimen of chemotherapy. Dose escalation was conducted separately for pts taking enzyme inducing anti-seizure drugs (EIASD+) and for those who were not (EIASD-). The starting dose in both groups was 0.9 mg/m2, cycle length was 6 weeks, and intrapatient dose escalation was not allowed. After 2 dose levels, the 6-week cycle length was deemed too toxic and was reduced to 3 weeks with a bortezomib i.v. push twice a week on weeks 1 and 2, and dose escalation started at 0.9 mg/m2. The 20S proteasome activity was determined in whole blood lysates collected at screening (1–7 days before treatment), shortly before and at 1, 4, and 24 hours after the first bortezomib dose. Sixty-three evaluable pts (45 men, 18 women) have been enrolled. The median age was 51 years; median KPS was 90%; 78% had GBM, and all but 2 had received 1 prior chemotherapy regimen. Five dose levels of bortezomib were tested in the EIASD- group: 0.9 mg/m2 (N = 3 for 6 weeks; N = 3 for 3 weeks), 1.25 mg/m2 (N = 2 for 6 weeks; N = 3 for 3 weeks), 1.5 mg/m2 (N = 3), 1.7 mg/m2 (N = 3), and 1.9 mg/m2 (N = 6). Two pts had DLTs at 1.90 mg/m2: a grade III thrombocytopenia and grade III fatigue and sensory neuropathy. Eight dose levels have been tested in the EIASD+ group: 0.9 mg/m2 (N = 3 for 6 weeks; N = 3 for 3 weeks), 1.25 mg/m2 (N = 3 for 6 weeks; N = 3 for 3 weeks), 1.5 mg/m2 (N = 3), 1.7 mg/m2 (N = 3), 1.9 mg/m2 (N = 3), 2.1 mg/m2 (N = 3), 2.3 mg/m2 (N = 3), and 2.5 mg/m2 (N = 3). One pt at 2.5 mg/m2 developed severe thrombocytopenia (DLT), hence this dose level was expanded for 3 additional pts. Ten additional pts (EIASD-) were treated at MTD of 1.7 mg/m2 and only one developed DLT for severe headache and sensory neuropathy. The most common adverse events were thrombocytopenia and sensory peripheral neuropathy. The extent of proteasome inhibition in whole blood increased in a dose-dependent manner in both treatment groups. Mean proteasome inhibition in EIASD+ pts 1 hour after receiving 2.1 mg/m2 of bortezomib (77 ± 12%) was similar to EIASD- pts treated with a dose of 1.7 mg/m2 (79 ± 6%). Fifty-one pts died of tumor progression, 3 died of other causes (cardiac arrest, aspiration, sepsis), and 9 pts are alive. The median survival time was 6.1 months (95% CI, 3.9, 7.4 months). In conclusion, 3-week intravenous dosing of bortezomib schedule is feasible in pts with recurrent MG. The MTD of bortezomib in EIASD- pts is 1.7 mg/m2. The EIASD+ group MTD has not been determined, but it is at least 2.3 mg/m2, which is higher than a standard dose of 1.3 mg/m2 for the treatment of multiple myeloma. The maximum inhibition of 20S proteasome activity in whole blood after administering bortezomib appears to be diminished in EIASD+ pts.


Surasak Phuphanich, Zhoabin Zhang, Daniel Brat, Ellen McKenzie, and Jeffrey Olson; Emory University, Atlanta, GA, USA

Biodegradable carmustine (BCNU)-containing polymer therapy increases the median survival time of malignant glioma recurrence from 23 weeks to 31 weeks. Sixteen percent of recurrent malignant gliomas have a partial response to IFNα alone. Neither treatment is curative. This investigation’s objective was to assess the toxicity of biodegradable BCNU-containing polymer followed by systemic IFNα after recurrent malignant glioma resection. Secondary assessment of tumor genetic expression profiles and methylguanine methyltransferase (MGMT) expression in relationship to observed response was done. Patients (KPS [gt-or-equal, slanted] 50) with recurrent malignant gliomas requiring surgery whose frozen section documented high grade gliomas with necrosis were eligible for biodegradable BCNU-containing polymer implantation followed 1 week later by an 8-week cycle of IFNα therapy. The doses (number of subjects) studied were 3 Mu/m2 (3), 6 Mu/m2 (3) and 9 Mu/m2 (4), administered subcutaneously 3 times a week. Tumor samples were acquired for gene expression analysis and qualitative MGMT immunohistochemical analysis. Two dose-limiting toxicities in the form of grade III fatigue/malaise occurred at 9 Mu/m2. The maximum tolerated dose was 6 Mu/m2. Two partial responses lasting over 2 years in each case were observed in the 3 Mu/m2 dosage. Tumor analysis with a Genome U133 Plus 2.0 array with 54,000 probe sets in 2 responders and 4 nonresponders provided observably different genetic expression profiles. Two hundred two genes had 4-fold or greater differences in expression between the responders and nonresponders including expected markers such as the Akt gene. Two of the 10 cases were MGMT negative (< 10% positive nuclei) and one responded. One responder was intensely MGMT positive. This data justifies a phase II study using IFNα at 6 Mu/m2 after biodegradable BCNU-containing polymer implantation in patients who are surgical candidates. Alternative dosing with 3 Mu/m2 could be used, as responses were observed at that level. The correlative genetic and enzyme expression data provides provocative but not statistically significant information. These analyses are feasible and demonstrate enough variation in this small sample of cases to suggest predictive significance may be reached in a phase II study.


Alyx Porter-Umphrey,1 Caterina Giannini,2 Timothy Kaufmann,3 Claudia Lucchinetti, John L. D. Atkinson,4 and Brian Patrick O’Neill1; 1Departments of Neurology, 2Pathology, 3Radiology, and 4Neurosurgery, Mayo Clinic Rochester (MCR), Rochester, MN, USA

Current practice suggests refraining from CS administration in suspected cases of PCNSL unless there is significant mass effect, based on the belief that CS induces apoptosis of neoplastic cells and renders the subsequent biopsy nondiagnostic. This study, with Mayo Foundation IRB approval, sought to determine if CS administration at the time of biopsy influenced PCNSL pathology. The study used a retrospective review of clinical, imaging, pathology, and outcomes of immunocompetent PCNSL patients from 2000 to 2005 with pathologically confirmed PCNSL at MCR and excluded patients who did not meeting criteria or who lacked research consent. One hundred eight PCNSL patients treated from January 1, 2000, to December, 31, 2005, were identified. Fifty-seven patients did not meet criteria, leaving 51 patients, 49 having B-cell lymphoma. Thirty-one patients received CS before diagnosis, and 24 of those patients continued CS at the time of biopsy. Forty-six (of 51) patients had presenting and preoperative neuroimaging; 23 received CS. Seventeen (of 23) had no significant change on neuroimaging pre- and post-initiation of CS. There were no cases of CS-induced disappearance of contrast enhancement or re-emergence of enhancement after CS withdrawal. In this pilot study, we found that administration of CS in patients with PCNSL does not appear to affect biopsy results nor does it prolong the diagnosis and initiation of treatment. The use of CS should be defined by clinical circumstance rather than concern of obscuring PCNSL diagnosis.


Nader Pouratian, Jaime Gasco, Mark Shaffrey, David Schiff; Departments of Neurological Surgery and Neurology, University of Virginia, Charlottesville, VA, USA

Protracted low-dose temozolomide (75 mg/m2 on days 1–21 of 28 days) offers advantages over standard temozolomide schedules (200 mg/m2 on days 1–5 of 28 days), including greater cumulative drug exposure and depletion of O6-alkylguanine DNA alkyltransferase levels, potentially overcoming intrinsic chemoresistance. We review our experience using protracted low-dose temozolomide in patients with low-grade gliomas (LGG) to quantify its toxicity and chemotherapeutic efficacy. We retrospectively reviewed 25 patients with pathologically proven LGG (age range, 21–76 years, median, 48 years) who were treated with protracted low-dose temozolomide (10 patients treated for progressive disease, 15 as part of the initial treatment). Diagnoses included oligodendroglioma (15), oligoastrocytoma (6), astrocytoma (3), and unspecified LGG (1). None were treated with radiation. Toxicities were graded according to the NCI Common Toxicity Criteria. Tumor response was graded based on changes in tumor size on MRI, steroid requirements, and clinical exam, using established response criteria. Two hundred forty-three cycles of protracted low-dose temozolomide were administered to 25 patients. Three patients (12%) were changed to standard temozolomide dosing because of chemotherapeutic side effects, including intractable nausea (2) and multiple cytopenias (1). Toxicities generally occurred between the first and sixth cycle. The most frequent chemotherapeutic side effects were fatigue (76%), lymphopenia (72% total [24% grade II, 48% grade III]), constipation (56%), and nausea (52%). Other grade III-IV toxicities included secondary malignancy (MALT lymphoma [cycle 12]), pruritis, hyponatremia, neutropenia, leukopenia, and cognitive decline (N = 1 for each). Low-grade toxicities, in order of decreasing frequency, included leukopenia, transaministis, vomiting, neutropenia, pruritis, hyponatremia, rash, hyperkalemia, depression, arthralgia, rash, weight loss, thrombocytopenia, and visual phenomena. The overall tumor response (including partial response [PR], minimal response [MR], and stable disease [SD]) was 88%, (32% PR, 24% MR, and 32% SD). The mean Kaplan-Meier progression-free survival (PFS) estimate was 19.9 months (95% CI, 16.4–23.4 months). Six-month and 12-month PFS rates were 92% and 76%, respectively. Response rates and PFS were independent of pathologic subtype, deletion status, and the indication for chemotherapy. Protracted low-dose temozolomide is well tolerated in the majority of patients without significant adverse consequences attributable to chemotherapeutic toxicities. Based on this small sample, protracted low-dose temozolomide may result in improved tumor response rates and PFS than standard dosing.


V.K. Puduvalli, P. Giglio, M.D. Groves, K.R. Hess, M.R. Gilbert, S. Mahankali, E. Jackson, V.A. Levin, C.A. Conrad, S. Hsu, H. Colman, M. Ritterhouse, S. Ichtech, and W.K.A. Yung; Departments of Neuro-Oncology, Biomathematics and Imaging Physics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

To determine the efficacy and toxicity of the combination of irinotecan and thalidomide in adults with recurrent glioblastoma multiforme (GBM) not on enzyme-inducing anticonvulsants (EIAED), we studied patients with recurrent GBM with no more than 2 prior relapses after surgery and first-line radiation therapy. The primary endpoint was progression-free survival at 6 months (PFS-6); secondary endpoints included response rate, overall survival, and toxicity. The study was powered to detect an improvement in PFS-6 from 0.15% to 0.35% compared with historical controls. Patients were treated in 6-week cycles with irinotecan at 125 mg/m2 weekly times 4 followed by 2 weeks off treatment and thalidomide starting at 100 mg daily and increased as tolerated to a maximum dose of 400 mg daily. Patients were evaluated with an MRI scan, including dynamic contrast enhanced images, before and after each cycle of therapy. Thirty-two evaluable patients were enrolled in the study. Eight patients were alive and progression free at 6 months. The PFS-6 was 25% (95% CI, 14%, 46%) and the median progression-free survival was 13 weeks (95% CI, 10, 24). The best responses were CR in 1 patient, PR in 1, and SD in 19. The overall survival (OS) at 6 months was 62% (95% CI, 48%, 81%) and was 34% at 1 year (95% CI, 21%, 56%). The median overall survival was 36 weeks (95% CI, 24, 56). Adverse events (grade III or IV) included diarrhea and abdominal cramps, lymphopenia, neutropenia, and fatigue. Four patients died while on treatment; of these, 2 deaths were considered possibly attributable to treatment-related toxicity. The combination of irinotecan and thalidomide shows promising activity in patients with recurrent GBM not on EIAEDs. The results of our study suggest that combining cytotoxic and antiangiogenic agents is an effective strategy for the treatment of recurrent GBM; they also provide a basis for exploring combination therapies similar to the one in this study utilizing newer targeted antiangiogenic agents.


J.A. Quinn, A. Desjardins, J.N. Rich, J.J. Vredenburgh, D.A. Reardon, S. Gururangan, S. Sathornsumetee, A. Walker, K.N. Lavin, R. Birch, A.H. Friedman, H.S. Friedman; Duke University Medical Center, Durham, NC; Keryx Biopharmaceuticals, Inc., Memphis, TN, USA

We conducted a phase I clinical trial in patients with progressive glioblastoma multiforme (GBM). This trial was designed to define the maximum- tolerated dose (MTD) of temozolomide (Temodar) administered for 5 consecutive days in combination with O6-benzylguanine (O6-BG). Two different dosing regimens of temozolomide were explored. On schedule 1, patients received 200 mg/m2/day on day 1 and 25 mg/m2/day (dose escalations by 25 mg/m2/day) on days 2–5. On schedule 2, patients received the same dose on all 5 days. The first dose level for schedule 2 was dependent on the MTD found in schedule 1. O6-BG was administered both as a bolus infusion (120 mg/m2), over 1 hour on day 1 and repeated every 48 hours on days 3 and 5, and as a continuous infusion (30 mg/m2/day) on days 1–5. Temozolmide was administered orally within 60 minutes of the end of the first 1-hour infusion of O6-BG and then every 24 hours during the continuous infusion of O6-BG. Treatment cycles were 28 days long. On schedule 1, the MTD was established at 200 mg/m2/day on day 1 and at 50 mg/m2/day on days 2–5 with dose-limiting toxicities (DLTs) limited to myelosuppression. On this schedule, 16 of 17 patients were evaluable for toxicity. The first DLT was experienced at the second temozolomide dose level (50/mg/m2/day) with 1 episode of grade IV neutropenia in 6 evaluable patients. However, at the third temozolomide dose level (75 mg/m2/day), 3 of 5 evaluable patients experienced one of the following DLTs: grade IV neutropenia, grade IV thrombocytopenia, or grade IV leukopenia. Likewise, the other 2 of 5 evaluable patients at this dose level both experienced grade III neutropenia that was treated with filgrastim (Neupogen) before establishing the nadir. On schedule 2, the MTD has yet to be established with a current temozolomide dose of 75 mg/m2/day. Once the MTD has been established on both schedules, this study will provide the foundation for a phase II trial of temozolomide in combination with O6-BG in temozolomide-resistant malignant glioma.


H. Ian Robins,1 Anne O’Neill,2 Ronald Sapiente,3 Mark Olsen,4 Mark Gilbert,5 Brian Berkey,6 and Minesh Mehta MD1; 1Univ. WI Comprehensive Cancer Center, Madison, WI, USA; 2Dana Farber Cancer Inst., Boston, MA, USA; 3Carle Clinic Assoc., Effingham, IL, USA; 4 St Francis Hospital: OK CCOP; 5M.D. Anderson Cancer Center, Houston TX, USA; 6RTOG, Philadelphia, PA, USA

Clinical experience and preclinical studies support the concept that low molecular weight heparin could provide prophylaxis for thromboembolic disease in patients with GBM as well as serve as an angiogenesis inhibitor, providing both antineoplastic effects and radiosensitization. In this phase II trial, dalteparin (5000 IU subQ daily) was given with and after conventional XRT (59.4 Gy/33 fx) in patients with newly diagnosed GBM. No systemic therapy was permitted until disease progression. At the time of progression, patients could continue dalteparin in addition to salvage regimens. Survival was compared with the RTOG database, and thromboembolic events (TEE) were prospectively documented. Forty-five patients were accrued between August 2002 and September 2004; 3 were ineligible and not included in the efficacy results. Pretreatment characteristics included the follwing: median age, 61 years; ECOG Performance Status, 0 = 38%, 1 = 57%, 2 = 5%; partial resection, 43%; total resection, 45%. The grade III toxicities experienced pre-progression included fatigue and neurological complications. No patients experienced grade III-IV bleeding or thrombocytopenia. No TEEs occurred while patients were receiving dalteparin. The median time on dalteparin was 6.3 months, the median time to progression was 3.9 months (95% CI, 3–6 months), and the median survival time (MST) was 11.9 months (95% CI, 10–14 months). MST was compared with the historical GBM database of the Radiation Therapy Oncology Group (RTOG) using the RTOG Recursive Partitioning Analysis (RPA). After controlling for RPA class, the observed MST did not exhibit a substantial advance over previous studies with various XRT/drug regimens including carmustine. As dalteparin does not have significant overlapping toxicities with most other drugs, its testing in a combined modality approach with other medications may be justified in future clinical trials. Historically, the incidence of TEE in GBM patients is approximately 30%. The lower-than-expected incidence seen in the context of this trial suggests its potential utility for prophylaxis. This study was supported by PHS grants CA23318, CA66636, CA21115, CA21076, CA13650 from NCI, NIH, and DHHS and the Kathleen Reader Memorial Research Fund.


Sith Sathornsumetee, Jeremy N. Rich, James J. Vredenburgh, Annick Desjardins, Jennifer A. Quinn, Sri Gururangan, Allan H. Friedman, Merrill J. Egorin, August Salvado, Henry S. Friedman, and David A. Reardon; Duke University Medical Center, Durham, NC; University of Pittsburgh Medical Center, Pittsburgh, PA; Novartis Pharmaceutical Corporation, East Hanover, NJ, USA

Imatinib mesylate (Gleevec), a kinase inhibitor of the PDGF receptor, c-kit, and bcr/abl, has demonstrated promising anti-glioma activity in combination with chemotherapy, hydroxyurea. Imatinib mesylate has been shown to decrease tumor interstitial pressure and may thus increase chemotherapy delivery to tumors. The combination of imatinib mesylate with temozolomide, a standard chemotherapeutic agent for malignant glioma, seems warranted. This phase I trial is designed to determine the maximum tolerated dose (MTD) and the dose-limiting toxicity (DLT) of imatinib mesylate when combined with standard-dosed temozolomide. Eligibility criteria include histologically confirmed malignant glioma; age at least 18 years; KPS of at least 60%; less than grade II intratumoral hemorrhage; adequate hepatic, renal, and bone marrow function; and a lack of prior failure or significant toxicity after treatment with either imatinib mesylate or temozolomide. Temozolomide is dosed at 200 mg/m2 on days 4–8 of each 28-day cycle. Imatinib mesylate is administered on days 1–8 of each cycle and the dose is escalated in successive cohorts of 3–6 patients via a standard “3 + 3” dose escalation design. Patients are stratified based on concurrent use of enzyme-inducing anticonvulsants (EIAC; phenytoin, carbamazepine, oxcarbazepine and phenobarbital) and both strata are independently escalated. To date, 56 patients have been enrolled, including 46 with GBM, 9 with anaplastic gliomas and 1 with pleomorphic xanthoastrocytoma with anaplastic features. Median age is 53.6 years (range, 28–72 years); 66% are men and 48% are on EIAC. The MTD has yet to be defined for either stratum. One patient in the EIAC stratum developed a DLT of possible interstitial nephritis. One patient in the non-EIAC stratum developed a DLT of elevated liver transaminases. Three patients discontinued therapy due to toxicities, with 1 asymptomatic intracerebral hemorrhage and 2 severe hematologic toxicities. Pharmacokinetic sampling has been performed in approximately half of the patients. Four partial responses were observed as best response. Three patients completed the study (12 cycles) with stable disease. Ten patients remain on study and 3 have undergone more than 10 cycles of therapy with stable disease. Thirty-two patients (57%) have developed progressive disease and have discontinued therapy. The combination of imatinib mesylate and temozolomide is safe and well tolerated. Further patient accrual and dose escalation are ongoing.


Meryl A. Severson, III,1 Jamie Weydert,1 Zita Sibenaller,1 Mohammad Vasef,2 Timothy Ryken,1; 1Departments of Neurosurgery and Pathology, Carver College of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA; 3Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

Primary intracranial Hodgkin’s lymphoma is an extremely rare disease with only 22 reported cases in the literature. Treatment has generally consisted of surgical resection followed by radiotherapy. Adjuvant chemotherapy has not been routinely prescribed in published reports. We report the only case, to our knowledge, in which a patient with primary intracerebral Hodgkin’s lymphoma (PIHL) has been treated with carmustine (BCNU) wafers as well as extirpation and radiotherapy. Additionally, this is the first report to examine the genetic profile of a primary cerebral Hodgkin’s lymphoma lesion. Our patient is a 76-year-old right-handed man who presented in September 2001 with word-finding difficulty and diplopia. An MRI scan revealed a 3 × 4 cm left parieto-occipital ring-enhancing mass with surrounding vasogenic edema. Tumor extirpation with the assistance of image guidance was performed and intraoperative frozen section was consistent with a high-grade glial neoplasm. The resection cavity was then lined with 6 carmustine-containing polymeric wafers for chemotherapeutic effect. The patient’s postoperative course was unremarkable and he was discharged 3 days after surgery. The intraoperative pathologic diagnosis was amended to malignant lymphoma consistent with Hodgkin’s lymphoma after immunohistochemical analysis. Pre- and postoperative metastatic work-ups did not reveal any evidence of systemic disease and the patient was additionally treated with whole-brain radiotherapy (WBRT). Because of the extremely rare nature of PIHL, the transcription profile of this patient’s tumor was investigated using microarray techniques. He has been closely observed and 57 months have passed since his initial diagnosis. He continues to do well, lives independently, and is free of tumor recurrence. This case represents the longest reported follow-up, the only reported case in the literature of PIHL treated with carmustine wafers, and the first time this extremely rare tumor has been genetically profiled. As our patient has done well and remains disease-free nearly 5 years later, we advocate consideration of carmustine wafer implantation in cases of PIHL, as well as in instances of intracerebral metastatic Hodgkin’s disease.


G.D. Shah,1,2 J.S. Silver,3 I. Gavrilovic,1 L.E. Abrey,1 and A.B. Lassman1; Departments of 1Neurology and 2Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 3Department of Medicine, Saint Francis Hospital and Medical Center, Hartford, CT, USA

Imatinib inhibits platelet-derived growth factor receptor (PDGFR) as well as other targets. Preliminary studies have shown that imatinib mono-therapy is generally ineffective for treating recurrent GBM. In an attempt to improve efficacy, 2 recent trials combined imatinib with hydroxyurea, a ribonucleoside diphosphate reductase inhibitor that disrupts DNA synthesis. Objective responses were observed in up to 20% of patients, and the 6-month progression-free survival rate was approximately 30% with grade III-IV hematologic toxicity in < 15% of patients. We retrospectively reviewed our results treating 14 patients (7 men, 7 women; median age, 47 years; range, 20–69 years; median prior chemotherapy treatments, 2.5) with imatinib (400 mg oral daily) and hydroxyurea (500 mg oral twice daily) for recurrent malignant glioma (9 GBM, 2 anaplastic astrocytoma, 2 anaplastic oligodendroglioma, 1 gliosarcoma). Eleven patients were evaluable for best radiographic response by MRI after at least 1 month of treatment and were evaluable for toxicity. Partial response (PR) was observed in 2 patients (18%), stable disease (SD) in 3 (27%), and progressive disease (PD) in 6 (55%). The median progression-free survival was 9 weeks (range, 2–25 weeks) and the overall survival was 18 weeks (range, 2–43 weeks). One patient with a PR developed therapy-limiting hematologic toxicity on day 19 of treatment, progressing to grade 4 on day 64, and persisting until death on day 127 despite discontinuing both drugs. The other patient with a PR and 2 of 3 patients with SD also developed therapy-limiting hematologic toxicity. None of the patients with PD suffered therapy-limiting hematologic toxicity. Combining imatinib with hydroxyurea is effective in a subset of patients with malignant glioma. However, myelosuppression can persist for weeks to months after discontinuing the regimen, precluding further chemotherapy. Our data also suggest a possible relationship between hematologic toxicity and disease control, implying that glioma and marrow stem cells may share a common sensitivity to this chemotherapy regimen. If additional patients experience therapy-limiting myelosuppression in the setting of response or prolonged disease stabilization, consideration should be given to the collection of peripheral stem cells before treatment. Autologous stem cell rescue has been used to maintain treatment with imatinib for leukemia patients with marrow aplasia and may also allow continuation of imatinib and hydroxyurea treatment for responding glioma patients.


W.R. Shapiro,1 L.S. Ashby,1 S. Phan2; 1Barrow Neurological Institute, Phoenix, AZ, USA; 2Pharmacyclics, Sunnyvale, CA, USA

MGd is a novel antineoplastic agent that targets tumors, inhibits thio-redoxin reductase, and generates reactive oxygen species (ROS) by redox cycling. Preclinical models show that MGd enhances the cytotoxic activity of several chemotherapy drugs including temozolomide. This phase I trial evaluated the safety and tolerability of MGd in combination with temozolomide in patients with recurrent malignant gliomas. Patients with malignant gliomas and adequate bone marrow, hepatic, and renal function were eligible. Cohorts of 3 to 6 patients were treated with increasing doses of MGd, starting at 2.5 mg/kg i.v. followed by temozolomide at 150 mg/m2 (first cycle if temozolomide-naive) or 200 mg/m2 (subsequent cycles if no myelosuppresion or prior temozolomide dose) > 60 minutes later. Treatments were repeated q4 weeks. Twenty patients were treated with MGd in 4 cohorts (dose levels 1.0, 3.0, 5.0, 7.5 mg/kg) and temozolomide. Eleven patients were men and 9 were women (median age, 57 years; range 23–69 years). Diagnoses included 9 patients with glioblastoma multiforme (GBM), 3 with anaplastic astrocytoma (AA), 7 with anaplastic oligodendroglioma (AO), and one with another diagnosis. Eleven patients had received prior systemic therapy including 6 who previously received temozolomide. All patients had previously received radiation therapy. No dose-limiting toxicities occurred. The MGd-related toxicities that were reported in > 10% of patients include digital skin discoloration and blisters, nausea, diarrhea, vomiting, fatigue, and pruritis. Adverse events [gt-or-equal, slanted] grade III were arthralgia, extremity pain, and nail-bed tenderness in 1 patient each. Two patients discontinued treatment for drug-related adverse events (hypertension and extremity pain). Patients received a median of 4 cycles (range, 1–10). The maximum therapeutic response was stable disease: GBM 5, AA 3, AO 5. MGd in combination with temozolomide was well tolerated and did not increase expected temozolomide toxicity.


E.G. Shaw, B. Berkey, S.W. Coons, D. Brachman, J.C. Buckner, K.J. Stelzer, G.R. Barger, P.D. Brown, M.R. Gilbert, and M. Mehta; Wake Forest University School of Medicine, Winston-Salem, NC; Radiation Therapy Oncology Group, Philadelphia, PA; Arizona Oncology Services Foundation, Phoenix, AZ; Mayo Clinic, Rochester, MN; Swedish Hospital, Celilo Cancer Center, The Dalles, WA; Wayne State University School of Medicine, Detroit, MI; M.D. Anderson Cancer Center, Houston, TX; University of Wisconsin School of Medicine, Madison, WI, USA

In 1998, the Radiation Therapy Oncology Group (RTOG) initiated Protocol 9802 for adults with supratentorial low-grade glioma (LGG). Patients were divided into 2 groups based on risk. Low risk patients were defined as those aged < 40 years who underwent a gross total resection (GTR), as determined by the neurosurgeon, of a WHO grade II astrocytoma (A), oligodendroglioma (O), or an oligoastrocytoma (OA). These patients were observed postoperatively with serial MRI scans and clinical follow-up. Initial results, reported at the WFNO in 2005, are updated data herein. Eligibility criteria included a histologically proven WHO grade II A, O, or OA based on a central pathology review, age [gt-or-equal, slanted] 18 years and < 40 years, KPS [gt-or-equal, slanted] 60, Neurologic Function Score (NFS) [less-than-or-eq, slant] 3, supratentorial tumor location, GTR, available pre- and postoperative MRI scans, and a signed consent form. MRI scans were obtained every 6 months. The prognostic factors analyzed for their effect on overall survival (OS), progression-free survival (PFS), and tumor recurrence included age (< 30 years vs. [gt-or-equal, slanted] 30 years), sex (men vs. women), KPS ([less-than-or-eq, slant] 90 vs. 100), NFS (0 vs. 1–3), histology (A = astrocytoma or astrocytoma-dominant OA vs. O = oligodendroglioma or oligodendroglioma-dominant OA), contrast enhancement on preoperative MRI scan (present vs. absent), preoperative tumor diameter (< 4 cm vs. [gt-or-equal, slanted] 4 cm), and baseline mini-mental status exam (MMSE) score (< 30 vs. 30). Between 1998 and 2002, 111 eligible and analyzable patients were entered on the study. The OS and PFS at 3 years for all patients was 97% and 69%, respectively. The only 2 prognostic factors predicting for significantly poorer PFS in univariate and multivariate analyses were histology (univariate P = 0.0064, multivariate P = 0.03, hazard ratio = 2.05) and preoperative tumor diameter (univariate P = 0.0001, multivariate P = 0.0004, hazard ratio = 3.04). The crude incidence of tumor recurrence was 54% for A vs. 32% for O and 63% for tumors [gt-or-equal, slanted] 4 cm vs. 27% for tumors < 4 cm. The 3-year PFS was 90% for O tumors < 4 cm vs. 41% for A tumors [gt-or-equal, slanted] 4cm. These data suggest that patients age < 40 years with a supratentorial WHO grade II astrocytoma [gt-or-equal, slanted] 4 cm in diameter who undergo a neurosurgeon-defined GTR have a nearly 60% risk of tumor progression 3 years after resection, warranting close postoperative follow-up and consideration for adjuvant treatment. Data on the postoperative MRI-defined extent of surgical resection will be presented at the meeting.


R. Soffietti,1 R. Rudà,1 E. Laguzzi,1 F. Giunta,2 A. Pace,3 C. Carapella,3 M. Salvati,4 M. Scerrati,5 A. Silvani,6 L. Fariselli,6 and R. Merli7; 1Neuro-Oncology, Torino; 2Neurosurgery, Brescia; 3Neurology and Neurosurgery, Roma Regina Elena Cancer Institute; 4Neurosurgery Roma University; 5Neurosurgery Ancona; 6Neurological Institute, Milano; 7Neurosurgery, Bergamo, Italy

This study sought to assess the efficacy and toxicity of temozolomide in patients with gliomatosis cerebri, a diffusely growing neuroepithelial tumor whose optimal treatment is unclear. Since 1999, 41 patients with histologically confirmed (biopsy or partial resection) gliomatosis cerebri were treated with temozolomide either upfront or at the time of progression after previous radiotherapy/chemotherapy. Tissue specimens were diagnosed as glioblastoma in 3 cases, malignant glioma in 6, anaplastic astrocytoma in 7, gemistocytic astrocytoma in 2, astrocytoma in 12, anaplastic oligoastrocytoma in 1, oligoastrocytoma in 1, oligodendroglioma in 4, and glial proliferation typical of gliomatosis cerebri in 5. Patient characteristics were as follows: median age, 49 years, median KPS at diagnosis, 80. Presenting symptoms were as follows: seizures (20 patients), intracranial hypertension (8), motor deficits (7), mental status changes (2), drowsiness and diplopia (3), dizziness and vomiting (1). Nineteen pretreatment MRI scans demonstrated some contrast enhancement. Twenty-two patients were treated upfront, wheras 19 received either radiation therapy or nitrosourea-based chemotherapy before temozolomide. All patients were treated with temozolomide 200 mg/m2 per day for 5 days every 4 weeks until progression or unacceptable toxicity. Response was evaluated according to Macdonald criteria on MRI T1-weighted gadolinium and FLAIR images. The median number of cycles was 7 (range, 1–20). Two patients (5%) showed a CR of the contrast enhancing area, 2 patients (5%) a PR of the FLAIR hyperintense area, 5 (12%) a minor response, 16 (39%) an SD and 16 (39%) a PD. The overall response rate (CR + PR + minor response) was 22%. The median time to tumor progression (TTP) was 9 months (range, 1–27 months), and the median survival time was 13 months (range, 3–123 months). The Progression-free survival rate at 6 months was 66% and at 12 months was 43%. Oligodendroglial tumors showed a 43% response rate and a TTP of 11 months. A clinical benefit was observed in 12 patients (29%), consisting mainly of a reduction of seizures. Responses prevailed in patients treated at progression compared with those treated upfront (14% versus 32%). Four patients showed grade III-IV hematologic toxicity (WBC and PLT). Temozolomide seems to be moderately effective and safe in treating gliomatosis cerebri. To improve the efficacy of temozolomide in an upfront setting, a phase II study with a dose-dense schedule has been launched.


Joohee Sul, Lisa M. DeAngelis, Joachim Yahalom, Lauren E. Abrey; Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Half of PCNSL patients are older than age 60 at diagnosis, and advanced age is a poor prognostic factor. Older patients are poorly represented in clinical trials and their prognosis and optimal therapy are unknown. We performed a retrospective analysis of 148 patients [gt-or-equal, slanted] age 65, treated at our institution from 1985 to 2005; patients were identified from an institutional database of 382 PCNSL patients. Median overall survival (OS) and progression-free survival (PFS) were calculated using the Kaplan-Meier method. The chi-square analysis was used to compare discrete variables. Sixty-eight patients (46%) were men, and 80 (54%) were women. The median age at diagnosis was 72 years. Forty-five patients (30%) had a history of previously cured cancer (18 lymphoma, 27 solid tumors). The median KPS at diagnosis was 70, and the median creatinine clearance was 73. Eighteen patients (12%) had positive CSF cytology, and 25 (17%) had ocular involvement. Ninety-four percent of initial chemotherapy regimens included high-dose methotrexate. Intrathecal chemotherapy was given to 43 patients as part of the initial treatment. Six patients did not receive treatment. Consolidation chemotherapy, usually high-dose cytarabine, was given to 52 patients. At last follow-up, 47 patients were alive and 101 were dead.

TreatmentMedian OS (months)Median PF (months)Neurotoxicity
All patients (N = 148)24.514.724
Chemotherapy Only (N = 96)24.010.83
Chemotherapy 1 RT (N = 30)22.421.117
RT Only (N = 16)24.517.94

PCNSL was the most common cause of death (64.4%). However, 14% of deaths were attributed to treatment-related toxicity. Older patients treated for PCNSL had a median overall survival time of approximately 2 years, regardless of initial treatment. This is substantially longer than the median OS of 7.6 months reported for patients [gt-or-equal, slanted] age 60 receiving whole brain RT alone. However, patients treated with chemotherapy alone had a high risk of relapse, whereas those who received RT as a component of initial therapy had a significantly higher risk of treatment-related neurologic toxicity (P < 0.0000001).


W. Taa1,l C. Zonnenberg,2 B.A. Zonnenberg,3 J.E. Bromberg,1 T.J. Postma,2 J.M. Geitenbeek,4 W. Boogerd,5 J.M. Kros,1 M.C.M. Kouwenhoven,1 and M.J. van den Bent1; 1Erasmus MC, Rotterdam; 2VUMC, Amsterdam; 3UMCU, Utrecht; 4UMCN, Nijmegen; 5Netherlands Cancer Institute, Amsterdam, The Netherlands

In several studies, it has been shown that temozolomide (TMZ) is effective in treating patients with a recurrent high-grade glioma. A few studies have examined the effect of TMZ on recurrent low-grade astrocytoma after surgery and radiotherapy, but none included a homogeneous group of patients. For this retrospective, multicenter study, we have evaluated a cohort of patients treated with TMZ in 5 hospitals in The Netherlands between 1995 and 2005 for progression after radiotherapy of an originally low-grade astrocytoma. None of the evaluated patients underwent previous chemotherapy and all had disease progression after surgery and radiation therapy. The patients were treated with TMZ 200 mg/m2/day for 5 days on a 28–day cycle for a maximum of 12 cycles or until tumor progression. Toxicity was scored using the NCI Common Toxicity Criteria, Version 2. Response was assessed by bi-monthly MRI and clinical assessment. Responses were assessed using the MacDonald criteria, using change in the product of two perpendicular diameters through the tumor, as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The progression-free survival and overall survival were stipulated with the Kaplan-Meier method. Fifty-five patients were treated with first-line TMZ for a recurrent low-grade astrocytoma. The median number of TMZ cycles was 8 (range, 2–18). Nine patients had transient grade III/IV hematologic toxicities, but no one had to stop the cycles because of toxicity. After 6 and 12 months, respectively, 67% and 30% of the patients were still progression free; the median overall survival time was 16 months. Of the 49 patients that were evaluable for response, 12 had a CR, 15 PR, 17 SD, and 5 PD. The results of this retrospective study indicate that TMZ has single-agent activity with mild toxicity in patients with a progressive low-grade astrocytoma after radiotherapy.


Mizuhiko Terasaki, Shintaro Fukushima, Kiyohiko Sakata, Minoru Shigemori; Department of Neurosurgery, Kurume University School of Medicine, Fukuoka, Japan

The optimum treatment of recurrent malignant gliomas remains undetermined. Interest in the use of temozolomide has increased, but only a limited number of patients respond to this therapy. Eleven patients with a mean age of 42 years (range, 15–74) who had recurrent or treatment-induced malignant gliomas were treated with combined temozolomide and oral VP-16 chemotherapy. Diagnoses included treatment-induced PNET (1), recurrent brainstem glioma (3) (1 malignant transformation and 2 recurrence), recurrent anaplastic astrocytoma (3), and recurrent glioblastoma (4). Ten (91%) patients showed an objective response to treatment. The progression-free survival rate was 4 months. The histologic subtype of the tumor, its location, and its maximum response to chemotherapy did not have an effect on the duration of disease control. Our data suggested that treatment with temozolomide and oral VP-16 is effective in controlling recurrent or treatment-induced malignant gliomas.


Ivo W. Tremont-Lukats,1 and Zoran Rumboldt2; 1Culicchia Neurological Clinic, Marrero, LA, USA; 2Medical University of South Carolina, Charleston, SC, USA

A 19-year-old man with primary diffuse meningeal gliomatosis began treatment with adjuvant temozolomide (TMZ, 150 mg/m2 on days 1–5 every 28 days). One month before, he had finished craniospinal irradiation with concurrent TMZ (75 mg/m2 orally). On day 3 of treatment, he developed headaches, confusion, and seizures. On admission, the patient had a blood pressure of 141/105 mm Hg. He was confused and had a mini-mental state exam (MMSE) score of 20. Funduscopy, visual fields by confrontation, and visual acuity were normal. The patient had a symmetrical, intentional hand tremor. No laboratory abnormalities or evidence of infection were present. An MRI scan of the brain on admission was compared with a baseline MRI taken two days before the onset of cycle 1 with TMZ, showing bilateral subcortical and cortical lesions in parieto-occipital and posterior frontal lobes with increased apparent diffusion coefficients. We stopped treatment with TMZ and started with levetiracetam 250 mg twice daily. Three days after admission, the patient was clinically better and was discharged. We followed up 1, 3, and 8 weeks after discharge. His mental status improved but never returned to baseline. We restarted TMZ for cycle 2 at 100 mg/m2. A follow-up MRI 6 weeks after admission showed complete disappearance of the hyperintense lesions. The patient continued treatment with TMZ but had disease progression and died 7 months after admission. Posterior reversible encephalopathy syndrome (PRES) is the acute and variable presentation of headaches, delirium, seizures, and visual deficits associated with bilateral cortical and subcortical vasogenic edema predominantly in the posterior areas of the brain. The most common causes of PRES are hypertensive encephalopathy, eclampsia, and immunosuppressive drugs in transplant patients. PRES has been described in adult and pediatric cancer patients treated with CHOP (cyclophosphamide, adriamycin, vincristine, prednisone), l-asparaginase, fludarabine, ARA-C, gemcitabine, and cisplatin, but we did not find published reports of PRES associated with TMZ in MEDLINE or in TOXNET, the toxicology database of the National Library of Medicine. Complete resolution of symptoms is the rule after stopping the causative drug, but there are exceptions. We conclude that temozolomide was directly associated with PRES in this patient because of the close temporal relation between the onset of treatment with temozolomide and symptoms without any other modification to the patient’s drug list; the radiographic changes by MRI taken before chemotherapy and during admission days later; the resolution of the syndrome after withholding TMZ; and the growing association between cytotoxic drugs and PRES. Whether this case was an idiosyncratic reaction or a consequence of increased permeability of the blood-brain barrier after craniospinal radiation with concurrent TMZ is unclear. However, our case report illustrates that TMZ alone can cause severe but potentially reversible neurotoxicity.


A. Ty, S.J. See, M.C. Wong; National Cancer Center and the National Neuroscience Institute (Singapore General Hospital Campus), Singapore

The role of temozolomide (TMZ) in brain tumor therapy has been established. However, the optimal dosing schedule is undefined. Preclinical data suggest that a protracted dosing schedule improves efficacy by exhausting AGT, thus overwhelming cellular repair systems. A recent clinical experience with a dose of 75 mg/m2 for 21/28 days reported more lymphopenia but less thrombocytopenia and reported more infections and infection-related deaths compared with conventional doses of 150–200 mg/m2 for the 5/28-day schedule. This report details our experience. From January 2003 to March 2006, 11 patients (8 men; median age, 50 years) with histologically proven malignant gliomas (5 glioblastoma multiforme [4 chemotherapy-naïve, 1 recurrent disease], 3 anaplastic astrocytoma [1 chemotherapy-naïve, 2 recurrent disease], 1 recurrent anaplastic oligodendroglioma, 1 recurrent PXA with anaplastic features and 1 recurrent oligodendroglioma) received TMZ using a low-dose protracted schedule (63 mg/m2/day for 20/28 days). All patients were [gt-or-equal, slanted] 18 years of age, had a Karnofsky Performance Score [gt-or-equal, slanted] 60, and had adequate hematologic, renal, and hepatic function. Patients received a median of 6 cycles (range, 5–24) of TMZ. The median dose was 63 mg/m2 for 20 days (1260 mg/28 day cycle) and a total of 107 cycles were administered. Three patients received concurrent radiation therapy and TMZ before adjuvant TMZ. Four patients (1 GBM, 2 anaplastic astrocytoma, 1 oligodendroglioma) were heavily pretreated with [gt-or-equal, slanted] 6 cycles of PCV before second-line TMZ chemotherapy. Adverse events (AE) were graded according to NCI-Common Toxicity Criteria, Version 3. No grade III-IV AEs or deaths related to treatment were observed. Grade I-II AEs complicated 13 cycles (12%). The most common grade I-II AE was lymphopenia (4%) followed by fatigue (3%), anemia (2%), and constipation (2%). In our group of patients, protracted low-dose TMZ (63 mg/m2 for 20/28 days) was safe, with no grade III-IV AEs or infection-related deaths. More data are needed for clinicians to assess the frequency and risk of serious AEs in protracted TMZ regimens under evaluation.


M.A. Vogelbaum,1 J.H. Sampson,2 S. Kunwar,3 S.M. Chang,3 F.F. Lang,4 M. Shaffrey,5 A.L. Asher,6 D. Croteau,7 K. Parker,7 J.L. Dul,7 J.W. Sherman,7 and R.K. Puri8; 1Cleveland Clinic Foundation, Cleveland, OH, USA; 2Duke University Medical Center, Durham, NC, USA; 3University of California, San Francisco, CA, USA; 4The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 5University of Virginia, Charlottesville, VA, USA; 6Carolina Neurosurgery and Spine Associates, Charlotte, NC, USA; 7NeoPharm, Inc., Lake Forest, IL, USA; 8CBER, U.S. FDA, Bethesda, MD, USA

Cintredekin besudotox (CB), a recombinant protein consisting of IL13 and truncated Pseudomonas exotoxin, binds selectively to IL13Rα2 receptors overexpressed by malignant glioma (MG). This study assessed the safety of CB administered by convection-enhanced delivery (CED) followed by standard external beam radiotherapy (EBRT) with or without temozolomide (TMZ) in patients with newly diagnosed MG. After a gross total resection of the tumor, 2 to 4 intraparenchymal catheters were sterotactically placed and CB (0.25 μg/mL or 0.5 μg/mL) was infused for 96 hours. Ten to 14 days later, EBRT (5940–6100 cGy, 5 days/week for 6–7 weeks) was given with or without TMZ (75 mg/m2/day, 7 days/week during EBRT). Safety was assessed over an 11-week observation period after catheter placement. Twenty-two patients (12 men, 10 women; median age, 55 years; 21 GBM, 1 AOA) were enrolled. No patients experienced dose-limiting toxicities (DLTs) in the first 2 cohorts (0.25 μg/mL CB + EBRT [N = 3] and 0.25 μg/mL CB + EBRT + TMZ [N = 3]). One patient experienced a DLT (seizure) in the third cohort (0.5 μg/mL CB + EBRT [N = 6]). Four patients in the final cohort (0.5 μg/mL CB + EBRT + TMZ [N = 10]) completed treatment, and 2 patients are currently receiving treatment with no DLTs reported. Four patients were not considered evaluable for dose decision and were replaced. CB-related adverse events that occurred in more than 1 patient were cognitive disorder, asthenia, and sensory disturbance. No Grade III-IV hematologic toxicities were observed. The overall survival extends up to 86 weeks to date. The convection-enhanced delivery of CB followed by EBRT ± TMZ appears to be well tolerated in adults with newly diagnosed MG.


James Vredenburgh, Annick Desjardins, James E. Herndon II, David Reardon, Jennifer Quinn, Sith Sathornsumetee, Sridharan Gururangan, Allan Friedman, Darell Bigner, and Henry Friedman; Duke University Medical Center, Durham, NC, USA

The prognosis for recurrent malignant gliomas is poor, with a median survival time less than 10 months. Malignant gliomas have high concentrations of VEGF receptors, and the higher the VEGF receptor concentration, the worse the prognosis. Bevacizumab is a humanized IgG1 monoclonal antibody to VEGF, which is synergistic with chemotherapy for most malignancies. Irinotecan is a topoisomerase 1 inhibitor and has modest activity against recurrent malignant gliomas. We report an FDA-approved phase II trial of bevacizumab and irinotecan for the treatment of recurrent malignant gliomas. Sixty-eight patients were enrolled: 32 with grade IV tumors (glioblastoma multiforme) and 36 with grade III tumors (anaplastic astrocytomas or oligodendrogliomas). All patients had progressive disease and underwent previous radiation therapy and chemotherapy. The first 32 patients were treated every other week with bevacizumab 10 mg/kg and irinotecan 125 mg/m2 (for patients not taking enzyme-inducing anti-epileptic drugs [non-EIAED]) or 340 mg/m2 for patients taking enzyme-inducing anti-epileptic drugs (EIAED). The last 36 patients were treated with irinotecan 125 mg/m2 (non-EIAED) or 350 mg/m2 (EIAED) on days 1, 8, 22, and 29 and bevacizumab 15 mg/kg on days 1 and 22. The regimen was well tolerated with no CNS hemorrhages or systemic hemorrhages greater than grade I. Eight patients were taken off the study because of thrombotic complications: 4 pulmonary emboli, 2 deep venous thromboses, 1 thrombotic thrombocytopenic purpura, 1 thrombotic stroke. Two of these patients died. Two patients discontinued treatment secondary to grade II protein-uria, and 3 discontinued treatment because they required non-neurosurgical surgery. The response rate was 63% in the first 32 patients (19 PRs and 1 CR). The median progression-free survival (PFS) was 24 weeks, and the 6-month PFS was 39%. The median overall survival has not been reached and exceeds 6 months. The follow-up for the second cohort is shorter, the efficacy is similar, but there was more toxicity. Nine patients in the second cohort were removed from the study in cycles 1 or 2 secondary to toxicity. Nineteen patients died from disease progression. The combination of bevacizumab and irinotecan is safe and one of the most active regimens against malignant gliomas.


P.Y. Wen,1 S.M. Chang,1 J. Kuhn,1 K. Lamborn,1 H.I. Robins,1 T. Cloughesy,1 M.R. Gilbert,1 W.K.A. Yung,1 M. Mehta,1 L.M. DeAngelis,1 L.E. Abrey,1 S. Kesari,1 J. Drappatz,1 A.B. Lassman,1 J. Dancey,2 M.D. Prados1; 1North American Brain Tumor Consortium; 2Cancer Therapy Evaluation Program, NCI, Bethesda, MD, USA

Glioblastomas frequently have amplification and mutation of epidermal growth factor receptors (EGFR) and inactivation of the tumor suppressor gene PTEN, leading to increased signaling through the MAP kinase and Akt/PI3kinase/mTOR pathways. Studies using single-agent EGFR and mTOR inhibitors have shown only modest activity. Combinations of EGFR inhibitors with mTOR inhibitors may possibly lead to greater antitumor activity. The North American Brain Tumor Consortium (NABTC) is conducting a phase I/II study of the EGFR inhibitor erlotinib in combination with the mTOR inhibitor temsirolimus (CCI-779) in patients with recurrent malignant gliomas. The eligibility criteria in the phase I component were histologically proven glioblastomas (GBM) and anaplastic gliomas (AG), radiologic evidence of progression, age [gt-or-equal, slanted] 18 years, life expectancy [gt-or-equal, slanted] 8 weeks, KPS [gt-or-equal, slanted] 60, adequate bone marrow reserve, and organ function. There was no limit on the number of prior therapies. Because both erlotinib and temsirolimus are metabolized by cytochrome P450 enzymes, patients could not be receiving enzyme-inducing antiepileptic drugs. The dose of erlotinib was fixed at 150 mg daily. Patients initially received temsirolimus 50 mg intravenously once weekly and the dose was adjusted based on toxicities. Dose-limiting toxicities (DLT), determined during the first 4 weeks of therapy, were defined as any grade IV hematologic toxicity, except for grade III thrombocytopenia, and any grade III or unacceptable grade II nonhematologic toxicities. Escalation was performed in standard groups of 3. The maximum tolerated dose (MTD) was defined as the dose at which DLTs occurred in no more than 1 out of 6 patients. To date, 15 eligible patients have been enrolled (10 GBM, 5 AG). Eight patients were men, and 7 were women (median age, 55 years; range, 33–74 years). The median KPS was 90 (range, 70–100); the median number of prior chemotherapy regimens was 1 (range, 0–3). Two of the 3 patients receiving 50 mg of temsirolimus developed DLTs (intolerable grade II rash and mucositis, grade III liver function abnormalities, and grade III rash and dehydration). Three of the 6 patients receiving 25 mg of temsirolimus weekly experienced DLTs (1 patient with grade III rash; 1 patient with grade III rash, diarrhea, and dehydration,; and 1 patient with grade III mucositis and infection). Two of 6 patients receiving 15 mg of temsirolimus weekly experienced grade III rash. The combination of erlotinib and temsirolimus was associated with a higher-than-expected incidence of rash and mucositis. The final MTD, pharmacokinetics, and response data will be presented.


P.Y. Wen,1 W.K.A. Yung,1 K. Lamborn,1 T. Cloughesy,1 L.M. DeAngelis,1 H.A. Fine,1 S.M. Chang,1 H.I. Robins,1 K. Fink,1 L.E. Abrey,1 A.B. Lassman,1 M. Mehta,1 S. Kesari,1 L. Kim,1 C. Stiles,2 M. Egorin,3 R. Kaplan,4 A. Murgo,4 and M.D. Prados1; 1 North American Brain Tumor Consortium (NABTC); 2Dana Farber/Brigham and Women’s Cancer Center, Boston, MA; 3 University of Pittsburgh, Pittsburgh, PA; 4Cancer Therapy Evaluation Program, NCI, Bethesda, MD, USA.

Platelet-derived growth factor (PDGF) and its receptors (PDGFR) are frequently expressed together in meningiomas, raising the possibility that an autocrine/paracrine loop contributes to the pathogenesis of these tumors. Imatinib mesylate is an inhibitor of PDGFR and may have therapeutic potential in meningiomas. The NABTC conducted a phase II study of imatinib in patients with recurrent meningiomas (NABTC 01-08). Patients were stratified into two cohorts: (1) grade I meningiomas and (2) atypical and malignant meningiomas. The primary endpoint was 6-month progression-free survival (PFS). Because imatinib is metabolized by the cytochrome P450 system (3A4), patients could not be receiving enzyme-inducing anti-epileptic drugs. All patients had a histologic diagnosis of meningiomas and radiographic evidence of recurrence or progression. There was no limit in the number of previous therapies. Patients were initially treated with 600 mg of imatinib for the first 4-week cycle. If this initial treatment was well tolerated, the dose was increased to 800 mg/d for subsequent cycles. Twenty-three patients were enrolled in the study (13 meningiomas, 5 atypical meningiomas, 5 malignant meningiomas), of which 22 were eligible. Thirteen patients were women and 10 were men with a median age of 58 years (range, 26–75 years). The median KPS was 80 (range, 60–100). All patients previously underwent multiple surgeries and 20 previously underwent radiotherapy. Seven patients had 1 prior chemotherapy regimen, and 3 had 1 prior hormonal therapy regimen. Two patients discontinued treatment as a result of toxicities, but otherwise, the drug was generally well tolerated. No patient had grade IV toxicities. One patient had a small aymptomatic intratumoral hemorrhage. One patient each had grade III anemia, elevated SGPT, dizziness, dehydration, neutropenia and leukopenia, and 2 patients had grade III hypophosphatemia. Nineteen patients were evaluable for response. Ten patients had disease progression at the first scan, 9 were stable, and none had a partial response. Overall median progression-free survival (PFS) was 2 months (range, 0.7 to 18 months); 6-month PFS was 29.4%. For grade I meningiomas, the median PFS was 3 months (range, 1.1 to 17.8 months); 6-month PFS was 45%. For the atypical and malignant meningiomas, the median PFS was 2 months (range, 0.7 to 3.7 months); 6-month PFS was 0%. Single-agent imatinib was reasonably well tolerated but had minimal activity in meningiomas. Treatment of meningiomas with imatinib in combination with hydroxyurea or with other targeted molecular agents directed at EGFR and/or VEGF and VEGFR may be more effective.


Franklin D. Westhout, Laura S. Paré, and Mark E. Linskey; Department of Neurological Surgery, University of California Irvine School of Medicine, Orange, CA, USA

The peripheral causes of foot drop are well recognized. Central nervous system causes are rare and an important, albeit underappreciated, differential etiology. Patient 1: A 46-year-old man with a history of lumbar spine fracture and L4-5 instrumentation-fusion presented with progressive weakness and numbness of the left foot, followed within 3 months by similar symptoms in the right foot. Lumbar spine imaging failed to reveal compressive nerve root pathology. An electromyogram revealed complete left and incomplete right peroneal neuropathy. An MRI scan of the upper spine revealed significant spinal stenosis at C4–C7 and T11–T12. Patient 2: A 66-year-old man presented with a progressive right foot drop over 2 months. Spine imaging results were normal. An MRI scan of the brain revealed a left parasagittal meningioma. The first patient underwent spinal decompression at all 3 levels. His gait improved with marked resolution of his right foot drop and significant improvement of his left foot drop. The second patient underwent craniotomy for microsurgical tumor resection. His foot drop was moderately improved during hospitalization and continues to improve as an outpatient. Foot drop does not always originate from spinal nerve root compression or peripheral neuropathy. Central causes, although rare, need to be considered. Central causative lesions usually occur at locations where pyramidal tract connections are condensed and specific, and the function is soma-topographically organized. Common locations include the inter-hemispheric motor cortex homunculus (mass lesion, ACA stroke), internal capsule (lacune), cerebral peduncle (lacune), and the spinal cord pyramidal tract (myelopathy). These cases confirm that good results can be achieved if correctable central causes of foot drop are recognized.


Jun-ping Zhang and Zhong-ping Chen; Cancer Center, Sun Yat-sen University, Guangzhou, China

Chemotherapy sensitivity and resistance assays (CSRAs) offer the potential of selecting cancer treatments based on the responsiveness of individual tumors as measured using in vitro assays. CSRAs provide the possibility of tailoring treatment to individual patients by using effective agents while eliminating unnecessary ones. The cytotoxic effects of nitrosourea and temozolomide (TMZ) are principally mediated by alkylation of the O6-position of guanine in DNA. Malignant gliomas resistant to these agents are frequently associated with high levels of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Patients who underwent surgery for malignant gliomas in the last 5 years at The Cancer Center, Sun Yat-sen University were enrolled in this study. The tumor tissues obtained during surgery were immediately sent to the neuro-oncology laboratory for CSRA testing (using the MTT assay). The histologically confirmed glioma samples were evaluated for MGMT protein expression by immunohistochemistry. After radiation therapy, the patients received chemotherapy. The chemotherapy regimen was determined based on the results of the CSRA and the MGMT protein expression. The sensitive drugs were selected, and the resistant drugs were eliminated from the treatment plan. Nitrosourea and TMZ were not included in the chemotherapeutic regimens for patients with MGMT-positive tumors, but treatment for MGMT-negative patients was not limited in this way. Forty-two patients who received at least 4 cycles of chemotherapy were evaluated. Seven patients received 2 chemotherapy regimens consecutively, and thus, 49 cases overall were evaluable. Complete response (CR) was achieved in 6 patients (12%), partial response (PR) in 10 (20%), and stable disease (SD) in 20 (41%). Thirteen patients (27%) had progressive disease (PD). Objective response rate (CR+PR) was 33%, and response plus stable disease rate (CR+PR+SD) was 73%. Hematologic toxicity was the main side effect observed in this series. One patient experienced neutropenic fever; another patient with grade IV thrombocytopenia received platelet transfusion. Nonhematologic toxicities were mainly nausea/vomiting, fatigue, and alopecia. Grade III and IV toxicities included anemia (1%), leukopenia (28%), thrombocytopenia (8%), fatigue (3%), nausea/vomiting (3%), and alopecia (11%). Chemotherapy for patients with malignant glioma based on in vitro CSRAs and MGMT expression of the tumor could improve the overall response rate.


J. Zhu; Tufts University, New England Medical Center, Boston, MA, USA

The combination of MTX-based chemotherapy with whole brain radiation has improved the median survival of patients with PCNSL to 40 months. Neoadjuvant chemotherapy using HD-MTX has a comparable result. Old age is an independent negative prognostic factor of survival and tolerance to chemotherapy. We studied the response and adverse effects of HD-MTX in patients who were 70 years or older at the time of PCNSL diagnosis. We identified 32 eligible patients diagnosed with PCNSL involving the brain parenchyma, the eyes, or both, who were treated at Massachusetts General Hospital between 1992 and 2004. Twenty-seven patients (median age, 73 years; range, 70–82 years) received HD-MTX (range 3.5 gm/m2–8 gm/m2) chemotherapy with dose adjustment based on kidney function as the initial treatment. The response to HD-MTX and toxicity involving hematologic, renal, and gastrointestinal systems were analyzed by reviewing medical records and neuroimaging. The performance status before and after chemotherapy and prognostic factors including results of spinal fluid cytology and protein and serum lactate dehydrogenase were also investigated. The patients received a total of 343 cycles of HD-MTX-based chemotherapy as the initial treatment. A median of 6 cycles per patient was given. Excluding 1 patient who developed myocardial infarction after 1 cycle of MTX, the overall response rate was 96.1%, with 16 complete responses (61.5%), 9 partial responses (34.6%), and 2 progressive disease (7.7%). The median overall survival time was 438 days (mean, 829 days). The median progression free survival time was 205 days (mean, 412 days). About 56% of all patients suffered side effects. XX patients developed multiple adverse reactions. The most commonly observed toxicities were gastrointestinal disturbances (46%), hematological complications (46%) with 1 grade III neutropenia, 1 grade IV thrombocytopenia, renal insufficiency (40%), and other complications, including mucositis, pneumonitis, and conjunctivitis (30%). We conclude that HD-MTX chemotherapy is efficacious for treating PCNSL in patients 70 years or older, and although adverse effects are common, most of them are transient and reversible.


Zion Zibly,1 Dvora Nass,2 Jacob Barham,3 Roberto Spiegelmann,1 Moshe Hadani,1 and Zvi R. Cohen1; 1Department of Neurosugery, 2Institute of Pathology, and 3The Oncology Center, Sheba Medical Center, Israel

Central nervous system involvement in AIDS patients is a well known phenomenon and is present in approximately 40 to 60% of patients at some stage of the disease. The causes of the neuropathy are various, ranging from infectious, inflammatory, neoplasm and other. Usually when a brain lesion is detected on imaging study an empiric therapy is given. When no clinical and radiological improvement is shown, a brain biopsy is suggested. This study analyzes the data on 10 patients with AIDS who underwent stereotactic brain biopsy at the Sheba Medical Center between 1997 and 2006. The diagnostic yield of brain biopsy in these patients and the alteration in the course of the disease is evaluated. A retrospective analysis of patient’s demographics, neurologic status, radiologic imaging and pathologic results was performed, and the clinical outcomes were recorded. Nine patients were men; the mean age at onset of neurological manifestation was 42 years. The clinical manifestations of the disease were mainly cognitive changes and motor deficits. The mean CD4 count before biopsy was 48. The lesions that were targeted were located in the basal ganglia in 6 patients, the parietal lobe in 2, the temporal lobe in 1, and the frontal lobe in 1. Pathologic examination revealed parasitic infections in 4 patients (aspergillosis in 1, toxoplasmosis in 3), progressive multifocal leukoencephalopathy in 3, AIDS encephalopathy in 1, and lymphoma in 2 (1 had toxoplasmosis within the same specimen). One patient had a nondiagnostic biopsy. Four biopsies in 3 patients revealed pathologic specimens; however, a diagnosis was not established. Two patients underwent a repeated biopsy that was eventually diagnostic. The modality of treatment was modified in 8 patients and led to clinical and radiologic improvement in 4 patients. Two have since died from GI complications.



Justin M. Bachmann, Karen K. Deal, J. Gerardo Valadez, Reid C. Thompson, and Moneeb Ehtesham; Vanderbilt University Department of Neurological Surgery, Nashville, TN, USA

Bmi1 is a polycomb-group transcriptional repressor that is necessary for the self-renewal of hematopoietic and neural stem cells. Bmi1 is an inhibitor of tumor markers such as p16 and p14 and has been shown to be highly expressed in medulloblastomas. Preliminary microarray analysis suggests that Bmi1 may serve as a stem-cell marker and prognostic indicator in various tumors, including prostate and breast cancer. We hypothesize that Bmi1 may also be a useful tumor marker in malignant glioma, correlating with histological grade. cDNA libraries were obtained from 44 tumors (6 grade I, 10 grade II, 13 grade III, and 15 grade IV) and 4 normal cortex samples resected from epilepsy patients. A cDNA mixture containing 18S, a validated housekeeping gene, and a Bmi1 plasmid were generated for use as standard curves with quantitative PCR. The tumor cDNA was subjected to real-time singleplex quantitative PCR with 18S and Bmi1 primers and SYBR green master mix using a Bio-Rad MyIQ Icycler. Bmi1 concentrations were determined relative to that of 18S. When normalized to non-neoplastic brain tissue samples, the average Bmi1 expression in grade I tumors was 16.3 times that of normal brain. Grade II tumors had a 6.3-fold increase in Bmi1 expression, and both grade III and grade IV samples had a 10.2-fold increase in Bmi1. Previous reports have demonstrated that medulloblastomas highly express Bmi1, but Bmi1 had not been characterized in gliomas. We demonstrate that Bmi1 expression rises as the histological grade of tumors increases from II to III. This increase in expression could reflect an increase in the malignant stem-cell population among these tumors. It is plausible that as the stem-cell component of a glioma increases, the tumor becomes more resistant to chemotherapy, with a shorter interval to progression.


Daniela A. Bota,1 Kathy L. Newell,2 Marsha Danley,2 Marilyn Davis,2 Richard Dubinsky,1 and Sarah A. Taylor3; 1Department of Neurology, 2Department of Pathology, and 3Division of Clinical Oncology, University of Kansas Medical Center, KS, USA

Glioblastoma multiforme (GBM) is an aggressive cancer. The median reported survival for patients with this disease is between 1 and 2 years. However, there is high heterogeneity in patients’ response to treatment, disease-free survival, and overall survival (OS), which cannot be accurately predicted at the time of diagnosis. Moreover, most GBM studies have included patients that received different treatments, making it hard to extrapolate the data to the standard treatment. Our objective was to study the OS-predictive value of various molecular markers representing different pathways of oncogenesis in a group of patients with primary GBM that received the current standard of treatment. We identified 19 GBM patients who were treated in the KU Oncology Center between January 1, 2000, and July 30, 2004, with surgical resection followed by conformal radiation and temozolomide per Stupp. All patients but one continued on temozolomide until progression. The antibodies used targeted different pathways, such as proliferation (MIB-1), tumor progression and resistance to apoptotic signals (p53), cell migration and invasion (MMP2), control of cell cycle and transcriptional regulation (MAPK), proliferation (EGFR), angiogenesis (VEGF), and resistance to temozolomide (O-6–methylguanine DNA transferase). Both semiquantitative and quantitative (ACIS II, Clarient, San Juan Capistrano, CA, and ISI) methods were used to evaluate the immunostained sections, and these results were correlated to length of survival. The data were analyzed with PC-SAS software (SAS Institute, Cary NC) using stepwise logistic regression analysis for survival (defining trends for P < 0.1). The patients included in our study had a median age of 53 years (range, 29–71 years), a median performance score of 80% (range, 50–100%), and a mean survival of 601 days (range, 159–1,189 days). The only markers that showed trends toward correlating with increased survival were high p53 levels (P = 0.07) and low MAPK (P = 0.08), although low MMP2 was close to the threshold (P = 0.14). The other molecular markers, including EGFR, VEGF and MGMT, did not correlate with patient survival. Of the four patients who lived more than 30 months, all had negative EGFR staining, and three had negative p53 and very low or absent MGMT. This pilot study suggests that the most important pathways for tumor activity in patients treated with temozolomide are those involved in apoptosis (p53), cell cycle control (MAPK), and possibly local migration (MMP2). The ability to predict patient survival and response to treatment at the time of the initial diagnosis would be an invaluable tool both for planning future therapies and for patients’ treatment and quality-of-life decisions. We will further test whether a panel of 10 molecular markers involved in different aspects of tumorigenesis can be used to achieve greater accuracy in predicting patients’ long-term prognosis.


Zhong-ping Chen and Wei-Ying Yu; Cancer Center, Sun Yat-sen University, Guangzhou, China

It has been noted that low-grade gliomas (LGG; WHO grades I or II) may progress rapidly or remain stable for many years. In this study, we retrospectively investigated alterations of several cell cycle-associated proteins in LGG and compared clinical outcomes of the patients to identify possible protein markers of malignant transformation. This study included 36 patients initially diagnosed with grade II astrocytoma, allocated into three groups: (1) those with malignant transformation (10 cases), in whom the tumor was recurrent, the patient received a second surgery, and the tumor was histologically confirmed to be grade III or IV; (2) those without malignant transformation (10 cases), in whom the tumor was recurrent but histological diagnosis at the second surgery was still grade II; and (3) those who were progression free (13 cases), in whom there was no evidence of recurrence in a 5-year follow-up period. All of the archival tissues (paired samples of recurrent cases) were analyzed for expression of Ki-67, p53, p27, p21, Cox-2, iNOS, and VEGF through immunohistochemical staining. The expression of Ki-67 was significantly higher in the malignant transformation group than in the progression-free group (P < 0.05), whereas there was no difference between the group without malignant transformation and the malignant transformation or progression-free groups (P > 0.05). p53 expression was also higher in the malignant transformation group than in the progression-free group and the group without malignant transformation (chi-square test, P < 0.05), whereas p53 expression was comparable between the progression-free group and the group without malignant transformation (P > 0.05). Expression of p27, p21, Cox-2, iNOS, and VEGF was detected in all of the cases, but there was no significant difference among each group and thus, it seems, no predictive value. p53 and Ki-67 could be predictive molecular markers of malignant transformation in LGG.


Gilbert J. Cote,1 Hannah C. Cheung,1 Lynda J. Corley,2 Gregory N. Fuller,2 and Ian E. McCutcheon3; Departments of 1Endocrine Neoplasia and Hormonal Disorders, 2Pathology, and 3Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Polypyrimidine tract-binding protein (PTB) is a multifunctional RNA-binding protein with known roles in alternative splicing, 3′-end formation, polyadenylation, and mRNA stability. PTB is expressed in developing mammalian astrocytes, absent in mature adult astrocytes, and aberrantly elevated in gliomas. It is unclear whether PTB is a coincidental marker of tumor progression or a significant mediator of tumorigenesis. In developing Drosophila, absence of the PTB homologue hephaestus results in increased Notch activity. Since Notch is a well-known inducer of glial cell fate, we determined whether overexpression of PTB in glial cell tumors provides a selective growth advantage by inhibiting activated Notch (Notch1IC)-mediated differentiation. To do this, we performed immunohistochemical analysis of expression of PTB, Notch1IC, Hes1 (a downstream Notch target), and GFAP on an extensive human tissue microarray that included 246 gliomas, 10 gliosarcomas, and 10 normal brains. Statistically significant PTB overexpression was seen in all glioma grades, with the highest increase in grade IV tumors. Notch1IC was also abnormally expressed in gliomas except in a subset of grade IV tumors, in which it was absent. This decrease in Notch1IC was not associated with increased PTB expression. We also examined the effect of PTB ablation on Notch1 activation in glioblastoma cell lines. Using siRNA oligonucleotides, we depleted PTB from SNB19 and U251MG glioblastoma cells. By immunocytochemistry, we observed only a slight or no increase in activated Notch1 upon PTB ablation. We conclude that PTB and Notch1 are independent and functionally unlinked markers of glioma progression and that PTB is not a sole inhibitor of Notch pathway signaling.


Wagner G. Dos Santos, Helen L. Fillmore, and William C. Broaddus; Department of Neurosurgery, Harold F. Young Neurosurgical Center, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA, USA

Resistance to ionizing radiation continues to be one of the major factors contributing to the poor prognosis of patients with malignant gliomas. However, the mechanism underlying this remarkable resistance is still unsolved. Here, we approached this issue by exposing glioma cells to single or fractionated doses of radiation (5 × 6 Gy and 2 × 60 Gy) and characterizing the morphological and molecular changes in the surviving cell population. Surviving U87MG and VC95G cells, which both have wild-type p53, showed sustained growth arrest and a striking change in morphology characterized by increased size (10- to 15-fold compared to untreated control), flattening, increased projections, and cytoplasmic vacuolization typically associated with senescence and autophagy. Viable surviving cells could be maintained in culture for at least 1 year and were able to re-enter the cell cycle, but at a low rate compared to the untreated parental cells, as indicated by positive staining for the Ki67 proliferation marker. At the molecular level, the viable cells showed reduced levels of Rb protein but increased levels of survivin and p21Waf1/Cip1/Sdi1. In contrast, T98G cells, which have mutant p53, did not show the same alterations in morphology or in the expression of these proteins and did not survive as long as U87MG and VC95G cells. The results suggest that although radiation-induced senescence and autophagy can lead to cell death, they can also provide a mechanism for survival of glioma cells, depending on their genetic background. The ability of some cells to remain viable after radiation treatment may have implications for tumor recurrence commonly observed in malignant gliomas.


Wagner G. Dos Santos, Josephine Fernando, Timothy VanMeter, Helen L. Fillmore, and William C. Broaddus; Department of Neurosurgery, Harold F. Young Neurosurgical Center, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA, USA

Malignant gliomas are heterogeneous and invasive tumors with very poor prognosis. Although much is understood about the molecular and genetic changes occurring during the progression of these tumors, little is known about the mechanisms by which the brain environment influences tumor progression. Lysyl oxidases (LOX) are enzymes involved in the cross-linkage of collagen and elastin and have been shown to have oncogenic functions. Based on microarray results showing enhanced expression of LOX-L2, a lysyl oxidase family member, in malignant glioma tumor tissues compared to normal brain, we sought to further investigate the expression of this enzyme in gliomas both in vitro and in vivo. Our results revealed higher levels of LOX-L2 expression in a panel of brain tumor tissues, including anaplastic astrocytomas, oligodendrogliomas, ependymomas, pilocytic astrocytomas, and glioblastoma multiforme, compared to normal brain. Interestingly, LOX-L2 was undetectable in glioma cell lines isolated from the tumor tissue and maintained in vitro. The increased levels of LOX-L2 expression in brain tumors are comparable to those observed in puncture-injured brains, suggesting a role for lysyl oxidases in the remodeling of extracellular matrix. We present for the first time evidence of increased lysyl oxidase expression in brain tumors and suggest a putative role for lysyl oxidases in brain tumor pathology and progression. The detection of lysyl oxidases in tumor tissue, but not in isolated tumor cells, is an example of how the brain microenvironment may be important in providing molecules that influence tumor development.


Michael L. Edgeworth,1,3 Sarah A. Schwartz,2,3 Sara L. Frappier,2,3 Deming Mi,3,4 Reid C. Thompson,5 and Richard M. Caprioli2,3; Departments of 1Neurology, 2Biochemistry, 3Mass Spectrometry Research Center, 4Biostatistics, and 5Neurosurgery, Vanderbilt University School of Medicine, Nashville, TN, USA

The prediction of tumor response to chemotherapy is currently based on histology and a limited number of genetic markers. As new therapies become available, new methods to predict response to chemotherapy are needed to personalize treatment decisions for individual patients. We hypothesize that proteomic analysis using direct-tissue MALDI-MS can predict survival in glioma patients treated with adjuvant chemotherapy. Informed consent was obtained from patients undergoing tumor resection in an IRB-approved protocol. Samples were collected and snap-frozen in liquid nitrogen at the time of surgery and stored at −80°C until the time of analysis. The frozen tissue samples were cut into 12-μm-thick sections, thaw-mounted onto gold-plated MALDI target plates, and spotted with a small organic matrix compound. Histologic diagnoses were made by a neuropathologist from serial H&E-stained sections according to the 2000 WHO classification. Matrix droplets were analyzed on a MALDI time-of-flight Voyager DE-STR mass spectrometer. Optical section images were taken to align MS analysis regions with cellular morphology determined by histology. Spectra were baseline corrected, normalized, and aligned using common peaks in ProTS Data. An average spectrum was obtained for each patient, and peak lists were then acquired and binned together. The area under the curve was then computed for each bin per patient over the average spectrum. A multivariate Cox proportional hazards model was used to evaluate the association between the 151 candidate features and patient survival, adjusting for clinical prognostic indicators. Fifty-one tissue samples were analyzed from individual patients who received postoperative chemotherapy. Histologic diagnoses included astrocytoma, oligodendroglioma, and mixed oligoastrocytoma, with 16 grade III and 35 grade IV tumors. One patient had a grade IV gliosarcoma. From the proteomic profiles, three features were identified at m/z values of 4350, 4390, and 5766. Using a multivariate Cox proportional hazards model, the peak intensities at each m/z value were positively associated with patient survival after being adjusted for age, tumor grade, histology, extent of surgery, and radiation therapy (P < 0.05). Hazard ratios for each feature were 0.14, 0.30, and 0.16, respectively. Postoperative radiation therapy and lower tumor grade also remained significantly associated with longer patient survival (P < 0.01). Proteomic profiles can predict response to chemotherapy in patients with high-grade glioma independent of tumor histology, tumor grade, age, extent of resection, and treatment with radiation therapy.


G.N. Fuller,1 H. Takei,1 K.D. Aldape,1 J.C. Cangelosi,2 and G.A. Campbell2; 1The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, and 2The University of Texas Medical Branch, Galveston, TX, USA

Previous studies have suggested that elevated indices of cell proliferation, such as mitotic index, PCNA labeling index, and Ki-67 antigen labeling index, may predict early recurrence of subtotally resected choroid plexus papilloma. The most aggressive form of choroid plexus tumor, choroid plexus carcinoma, is associated with increased mitotic activity. Choroid plexus tumors with intermediate levels of cell proliferation have been termed atypical choroid plexus papillomas, but the prognostic significance of this feature remains unresolved, possibly in part because of the relative difficulty of reliably quantitating mitotic figures on hematoxylin- and eosin-stained slides. The recent introduction of mitosis-specific anti-phosphohistone H3 (pHH3) antibodies permits rapid and unambiguous identification and quantitation of mitotic figures. This study was designed to assess the feasibility and reliability of using anti-pHH3 to determine the mitotic index in a series of choroid plexus tumors, including papillomas, atypical papillomas, and carcinomas. Thirteen tumors were evaluated for mitotic index, and the results were correlated with the Ki-67 (MIB-1) labeling index. Patients’ ages ranged from 5 days to 39 years; six patients were male and seven female. Anatomic locations included the lateral ventricle (6 cases), third ventricle (1 case), fourth ventricle (5 cases), and cerebellopontine angle (1 case). Ki-67 labeling indices ranged from 0.1% to 11.5%, and pHH3 mitotic indices ranged from 0 to 126 per 10 high-power (× 400) fields. The Ki-67 and pHH3 indices correlated with each other and with WHO tumor grade. Moreover, mitotic index determination by anti-pHH3 immunocytochemistry was both more rapid and more reproducible between independent evaluators than either traditional mitotic figure counting on hematoxylineosin slides or Ki-67 labeling index quantitation on MIB-1–immunostained tissue sections. The results of this study demonstrate that anti-pHH3 immunostaining for mitotic figures permits rapid, reliable determination of cell proliferation in choroid plexus neoplasms. Further studies are warranted to determine potential clinical utility.


Bronner P.A. Goncalves, Mário H.G. Faria, Manoel O. Moraes-Filho, and Silvia H.B. Rabenhorst; Department of Pathology and Forensic Medicine, Federal University of Ceará, Fortaleza-Ceará-Brazil

Despite recent advances in glioma chemotherapy, survival for the majority of patients with high-grade tumors remains unchanged. The failure of adjuvant therapy is attributed in part to genetic alterations acquired during development and/or progression of these tumors, characterizing the primary chemoresistance phenomenon. Differential expression of enzymes that play a central role in DNA biosynthesis (O6-methylguanosine-DNA-methyl-transferase [MGMT], thymidylate synthase [TdS], and topoisomerase IIα [TopoIIα]) and that catalyze cell detoxification (glutathione-S-transferase π [GSTπ]) have been shown to determine resistance to antineoplastic drugs. The aim of the present study was to evaluate the expression of described chemoresistance-related proteins in human astrocytic tumors, correlate the findings with histopathological grade (WHO), and disclose possible therapeutic implications. An immunohistochemical study of MGMT, TdS, GSTπ, and TopoIIα using the avidin-biotin-peroxidase method was performed in 55 astrocytomas (13 grade I, 14 grade II, 7 grade III, and 21 grade IV) and 5 samples of non-tumor brain tissue (control group). Positive indices (PIs) for MGMT (100%), GSTπ (92%), and TdS (79%) were high and similar in all graduations. TopoIIα PIs tended to increase according to malignant progression (22% for grade I, 60% for grade II, 71% for grade III, and 94% for grade IV). Labeling indices (LIs) for MGMT (68.07, 72.50, 63.14, and 73.10 for grades I-IV, respectively), GSTπ (59.80, 61.46, 55.00, and 46.71), and TdS (24.16, 21.66, 18.33, and 31.66) were similar in all histopathological grades, except for the lowest GSTπ and higher TdS expressions in grade IV tumors. TopoIIα LIs (4.33, 31.60, 18.28, and 29.22) demonstrated no tendency relating to astrocytoma gradation, despite elevated scores observed in diffuse tumors (grades II-IV). These enzymes were not detected in non-tumor astrocytes, except for MGMT. The results indicate that MGMT is constitutively and highly expressed in astrocytic cells, which in part supports the frequent failure of alkylating-based therapy in astrocytomas. On the other hand, GSTπ and TdS expression appears to result from the neoplastic process (demonstrating high levels since low-grade tumors), which is in accordance with the habitual resistance of astrocytic tumors to cytotoxic drugs detoxified by glutathione (e.g., alkylating agents, antibiotics) and to folate-pathway inhibitors (e.g., 5–fluorouracil, methotrexate), respectively. In contrast, high levels of TopoIIα in diffuse astrocytomas may constitute a favorable situation for chemotherapeutic drugs that stabilize the cleavable complex formed between TopoII and DNA (e.g., anthracyclines, ellipticins, epipodophyllotoxins). The conflicting results in grade IV tumors reinforce their heterogeneity. Finally, we suggest that this protocol may be of predictive value in selecting appropriate drugs for chemotherapy and avoiding drugs expected to be ineffective because of enhanced expression of specific resistance factors.


Bronner P.A. Gonçalves, Germano P.V. Lima, Mário H.G. Faria, and Silvia H.B. Rabenhorst; Department of Pathology and Forensic Medicine, Federal University of Ceará, Fortaleza-Ceará-Brazil

Molecular studies of astrocytic tumors have delineated specific genetic alterations that are distinguished in the neoplastic context. p53 mutations and EGFR overexpression are hallmarks of astrocytoma tumorigenesis, defining mutually exclusive pathways (especially for glioblastomas). However, there is a subset of tumors that cannot be properly explained by these alterations. In the last decade, the c-MYC oncogene has been identified as a centerpiece of the tumorigenic process of various human cancers. Recent evidence reinforces the direct and indirect participation of the c-MYC protein in regulating the cell cycle, differentiation, apoptosis, genomic instability, and angiogenesis, although the pathways are not completely understood. c-MYC expression has been described in glial neoplasms and correlated with tumor progression, but its impact on astrocytoma tumorigenesis remains unclear. The aim of the present study was to evaluate the contribution of c-MYC expression in astrocytic tumors compared to p53 mutation and EGFR overexpression. Immunohistochemical analysis of the p53/p21WAF1/CIP1, EGFR, and c-MYC proteins using the avidin-biotin-peroxidase method was performed in 55 astrocytomas (13 grade I, 14 grade II, 7 grade III, and 21 grade IV) and 5 samples of non-tumor brain tissue (control group). p53 positive indices (PIs; 46% for grade I, 50% for grade II, 57% for grade III, and 67% for grade IV) and labeling indices (LIs; 7.69, 9.64, 15.28, and 22.95, respectively) showed a tendency to increase according to malignant progression, while p21 PIs (38%, 28%, 14%, and 57%) and LIs (10.38, 12.14, 5.71, and 24.79) demonstrated the opposite inclination, except for high scores in grade IV tumors. The p53/p21 profile revealed a tendency to higher incidence of p53 mutations in agreement with gradation (30%, 43%, 57%, and 38%), despite the low value for grade IV. EGFR PIs (46%, 21%, 0%, and 61%) and LIs (27.30, 3.92, 0.00, and 19.28) displayed the impact of EGFR overexpression in pilocytic astrocytomas and glioblastomas. Nuclear c-MYC (expected as functional) PIs (15%, 43%, 57%, and 86%) and LIs (5.46, 10.42, 10.00, and 30.04) tended to increase according to histopathological grade. After establishing the main molecular alteration in each tumor through quanti-qualitative comparisons among these markers, we found that c-MYC expression is able to explain astrocytoma tumorigenesis in the majority of cases when p53 and EGFR alterations are absent or not significant. These proteins were not detected in non-tumor astrocytes. These results confirm p53 mutation as the main genetic alteration in astro-cytic tumors, appearing as an initial, strong potential predictor of tumor progression. EGFR overexpression is reinforced as an important disruption responsible for a subset of glioblastomas. Moreover, the intriguing EGFR expression in pilocytic astrocytomas reveals a new principle to better understanding these tumors. Finally, c-MYC expression emerges as a candidate to explain astrocytoma tumorigenesis as an alternative to p53 mutation and EGFR overexpression, representing a new molecular pathway.


Andreas F. Hottinger,1 Bin Gu,2 Martin Fleisher,2 Sasan Karimi,3 Lauren Abrey,1 Eric Holland,4 Lisa M. DeAngelis,1 and Adília Hormigo1; 1Department of Neurology, 2Clinical Laboratories, 3Department of Neuroradiology, and 4Department of Cancer Biology and Genetics and Neurosurgical Service of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

In PCNSL, disease status is currently determined by brain MRI. However, MRI can be inconclusive, and non-invasive tests to evaluate disease status could be useful. Serum markers can support diagnosis, monitor treatment, and predict prognosis in some patients with cancer. Matrix metalloproteinases have been associated with angiogenesis, tumor infiltration, and metastasis formation in systemic lymphoma. This study was designed to determine whether circulating levels of MMP-9 could be related to disease status in PCNSL. Circulating levels of MMP-9 were determined by ELISA and correlated with disease status assessed by MRI in a prospective longitudinal study. The disease status was classified as no evidence of disease (NED) versus active disease (AD). Eighteen PCNSL patients have been enrolled since August 2002, and 93 blood samples have been obtained (median, 6 per patient, range, 1–10). In the NED group (N = 12 patients; 59 samples), the mean MMP-9 level was 205.5 ± 19.1 ng/ml (median, 162.3 ng/ml; range, 35.9–649.1 ng/ml). In the AD group (N = 16 patients; 34 samples), MMP-9 levels were significantly higher, with a mean of 638.2 ± 140.7 ng/ml (median, 287.5 ng/ml; range, 24.7–3340.6 ng/ml; P = 0.0001). Paired samples were obtained from 10 patients, before and after therapy. Patients in the AD group had a mean of 1223.9 ± 336.5 ng/ml (median, 770.1 ng/ml; range, 284.1–3340.6 ng/ml) before treatment; when complete response was reached, levels fell to a mean of 143.5 ± 38.4 ng/ml (median, 86.7 ng/ml; range, 28.1–356.5 ng/ml; P = 0.005). During follow-up, 4 of the 18 patients developed a total of 5 recurrences; mean MMP-9 levels increased from 154.22 ± 34.9 ng/ml (median, 162.4 ng/ml; range, 64.5–260.3 ng/ml) to 324.4 ± 98.5 ng/ml (median, 251.5 ng/ml; range, 159.5–709.7 ng/ml; P = 0.14) at relapse. PCNSL patients with active disease have circulating MMP-9 levels that are significantly higher than those in patients with no evidence of disease. A significant drop in MMP-9 compared to baseline was seen after successful treatment. MMP-9 levels increased with disease recurrence but did not reach statistical significance in this small population. MMP-9 has the potential to complement neuroimaging to confirm remission or progression.


F.M. Iwamoto,1 L. Nicolardi,1 A. Demopoulos,1 V. Barbashina,2 M. Ladanyi,2 L.M. DeAngelis,1 and A. Hormigo1; Departments of 1Neurology and 2Pathology, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, USA

Loss of 1p and 19q chromosomes occurs in 40–60% of oligodendrogliomas and is associated with a better prognosis. However, most case series have a small number of patients and limited follow-up. We retrospectively evaluated patients with glioma who had 1p/19q molecular studies performed at MSKCC from January 2000 to December 2004. DNA was extracted from tumor tissue and germline material and evaluated by PCR using at least three markers for each chromosome. We obtained both clinical and molecular pathology information for 208 patients with WHO grade II (N = 127) or grade III (N = 81) gliomas. There were 113 men and 95 women, with a median age at tumor diagnosis of 39.5 years (range, 9 to 83 years). Median follow-up was 67 months (range, 2 to 301 months) for grade II patients and 39 months (range, 2 to 180 months) for grade III patients. Thirty-eight patients had astrocytoma (AII), 58 had oligodendroglioma (OII), 31 had oligoastrocytoma (OAII), 21 had anaplastic astrocytoma (AIII), 37 had anaplastic oligodendroglioma (OIII), and 23 had anaplastic oligoastrocytoma (OAIII). 1p chromosome analysis was performed in all patients and detected deletions in 105 patients (76% of OII, 42% of OAII, 21% of AII, 89% of OIII, 17% of AOIII, and 14% of AIII). 19q chromosome studies were performed in 118 patients and detected deletions in 46 patients (56% of OII [14/25], 44% of OAII [12/27], 26% of AII [5/19], 77% of OIII [13/17], 11% of OAIII [2/19], and 0% of AIII [0/11]).

Median PFS (months)
Grade IIGrade III
Chromosome 1p deleted79.8P = 0.00253.4P = 0.04
Chromosome 1p intact45.417.9
Chromosome 19q deleted79.4P = 0.0356.5P = 0.01
Chromosome 19q intact37.618
Oligodendroglioma74.1P = 0.253.4P = 0.1
Median OS (months)
Grade IIGrade III
Chromosome 1p deletedNRP = 0.004NRP = 0.009
Chromosome 1p intact22851.6
Chromosome 19q deletedNRP = NSNRP = 0.004
Chromosome 19q intact22841.8
Oligodendroglioma236P = 0.3112.6P = 0.1

Our data suggest that patients who have a deletion of 1p or 19q have a longer progression-free survival. However, there was no statistical difference in overall or progression-free survival of patients with oligodendrogliomas compared to those with astrocytomas and oligoastrocytomas.


K.A. Jaeckle, P.A. Decker, K.V. Ballman, P.J. Flynn, C. Giannini, B.W. Scheithauer, R.B. Jenkins, and J.C. Buckner; Mayo Clinic, Jacksonville, FL; Mayo Clinic, Rochester, MN, USA; Metro MN, St. Louis Park, MN, USA

Although pathologists estimate that gliomas undergo anaplastic transformation in over 25% of cases, there are few comprehensive analyses that have examined the frequency of transformation within individual patients in tumor tissues resected at initial diagnosis and time of recurrence. We performed a central review of paired glioma tissues from initial diagnosis and recurrence (> 90 days) in 208 patients enrolled in prospective phase II and III NCCTG clinical trials. Groups were compared using Kaplan-Meier, log-rank, ANOVA, and chi-square tests. High-grade (WHO grades 3–4) recurrence after a prior initial diagnosis of a low-grade (WHO grade 2) tumor occurred in 14 (70%) of 20 patients with astrocytomas, 28 (68%) of 41 patients with oligoastrocytomas (OA), and 18 (43%) of 42 patients with oligodendrogliomas (P = 0.031). The median overall survival (measured from initial diagnosis) differed between those presenting with GBM at initial diagnosis (1.7 yrs; 95% CI: 1.5–2.2 yrs), those presenting with low-grade tumor and then GBM at recurrence (3.7 years; 95% CI: 2.8–4.2 yrs), and those with non-GBM both initially and at recurrence (5.5 yrs; 95% CI: 4.9–7.0 yrs; P<0.001). The mean time to recurrence (yrs ± SD) also differed between these three groups (1.1 ± 1.1, 2.9 ± 1.8, and 4.0 ± 2.9 yrs, respectively; P < 0.001). Overall survival from time of initial diagnosis also differed significantly when patients presenting with GBM initially were compared to those who developed GBM at recurrence (P = 0.041). Overall survival from time of recurrence differed significantly between patients recurring with high-grade gliomas as a function of histologic subtype [astrocytoma grade 3–4, 0.7 yrs (95% CI: 1.8–5.1 yrs); OA grade 3–4, 1.0 yrs (95% CI: 0.8–1.3 yrs); oligodendroglioma grade 3, 2.1 yrs (95% CI: 0.9–3.0 yrs); P = 0.02]. The median overall survival from time of recurrence was 0.7 yrs (95% CI: 0.5–1.1 yrs) for patients with GBM at initial diagnosis, 0.6 yrs (95% CI: 0.5–1.0 yrs) for patients with GBM at time of recurrence, and 1.6 yrs (95% CI: 1.1–2.1 yrs) for those with non-GBM initially and at recurrence (P < 0.001). Overall survival from time of recurrence differed significantly when patients presenting with GBM at initial diagnosis were compared with those who developed GBM at recurrence (P=0.017). Progression from initial low-grade glioma to high-grade glioma at recurrence was observed in over two thirds of patients with low-grade astrocytoma and nearly one third of patients with low-grade oligodendroglioma and was more frequent in patients with astrocytoma or mixed glioma than in those with oligodendroglioma. These groups had significant differences in overall survival, whether measured from the time of initial diagnosis or the time of recurrence. The overall survival of patients presenting initially with GBM also differed significantly from patients who developed GBM at recurrence, whether measured from the time of initial diagnosis or the time of recurrence. These data have important implications for the design of clinical trials involving recurrent glioma and for the interpretation of survival data following treatment of newly diagnosed glioma patients.


D.A. Jellinek,1 D. Levy,1 E. Maltby,1 N. Atkey,1 S. Hibberd,1 D. Crimmins,1 K. Stoeber,2 G.H. Williams,2 and S.B. Wharton3; 1Sheffield Teaching Hospitals, Sheffield, UK; 2University College London, UK; 3University of Sheffield, UK

It is uncertain why oligodendrogliomas with deletions from chromosomes 1p and 19q (Del+) result in better survival and response to chemotherapy than those without (Del−). We have investigated whether Del+ and Del– oligodendrogliomas differ in their apoptotic and kinetic indices. FISH was used to determine the 1p, 19q status of 54 oligodendrogliomas (25 WHO grade II and 29 WHO grade III). Quantification was performed for (1) apoptosis, using an index of apoptotic bodies; (2) licensed but nonproliferating cells, using an index of cells expressing the Mcm2 licensing protein minus the Ki67 labeling index (Mcm2-Ki67); and (3) the geminin-to-Ki67 ratio, as an index of G1-phase cells. Protein expression was determined by immunohistochemistry, and labeling indices were determined as the percentage of immunolabeled cells in at least 1000 cells counted. Del+ oligodendrogliomas showed a higher level of apoptosis but did not differ from Del− tumors in Mcm2-Ki67 or geminin/Ki67 labeling indices. WHO grade III tumors showed a higher proportion of licensed, nonproliferating cells than did grade II tumors. An increased susceptibility to apoptosis is a candidate mechanism to account for the better survival and chemoresponsiveness of oligodendrogliomas with 1p, 19q deletions.


Randy Jensen, Jeannette Flynn, and David Gillepsie; Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA

Hypoxic regions within glioblastoma multiforme (GBM) tumors may account for the resistance to radiation and chemotherapy and ultimate poor prognosis of these tumors. Hypoxia plays a role in the regulation of gene expression for a number of proteins that may mediate the malignant progression of GBM. We hypothesize that measures of hypoxia and vascularity, using both biochemical markers and imaging, might predict patient survival and response to treatment modalities. We also examined the effect of inhibition of hypoxia-inducible factor-1α (HIF-1α) on tumor growth, proliferation, apoptosis, and angiogenesis in a malignant glioma mouse model. We examined 175 human gliomas for expression of hypoxia-regulated proteins, including HIF-1α and its downstream-regulated proteins, by immunohistochemistry. We examined a subset of these patients for overall survival, markers of apoptosis, cellular proliferation, and microvascular density. This information was correlated with preoperative imaging, including measures of necrotic areas, peritumoral edema, perfusion imaging, and MR spectroscopy. A number of malignant glioma cell lines, as well as a U251 cell line with a luciferase expression vector under the control of a hypoxia response element, were transfected with siRNA constructs directed against the HIF-1α gene. These cells were assayed for in vitro and in vivo growth studies and luciferase activity. Tumors were harvested and MIB-1 labeling index, apoptotic index and microvascular density measurements were performed. VEGF, CA-IX, GLUT-1, and HIF-1 expression correlated positively with increasing tumor grade (P = 0.007) and negatively with survival. Proliferative index predicted tumor grade, but microvascular density score did not correlate with grade or survival. We have preliminary results to suggest that perfusion and MRS can predict expression of these same molecules; relationship with overall survival will be determined. Inhibition of HIF-1 by siRNA resulted in significant growth inhibition and decreased luciferase activity compared with negative controls in the mouse model. Cellular proliferation and microvascular density were also reduced significantly, while apoptotic index was increased in these tumors. Hypoxia-related proteins are elevated in malignant gliomas. HIF-1α expression affects glioma tumor proliferation, apoptosis, angiogenesis, and growth. Measures of tumor hypoxia, vascularity, and proliferation could be used to predict survival and response to current therapeutic measures. In addition, our mouse experiments suggest the potential for treatment of malignant gliomas with RNAi directed against HIF-1α.


L.B. Kilburn,1 M.F. Okcu,1 Y. Cao,2 A. Renfro,2 L.E. Wang,2 P. Adatto,2 M. Gilbert,2 K. Aldape,2 Q. Wei,2 and M. Bondy2; 1Baylor College of Medicine, Houston, TX, USA; and 2The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

With the established prognostic factors, it is impossible to adequately predict which patients with anaplastic glioma (WHO grade III) will benefit from treatment. Glutathione S-transferases (GSTs) are polymorphic phase II metabolic enzymes that are responsible for glutathione conjugation of multiple alkylating agents and scavenging of free oxygen radicals created by radiation therapy. We hypothesized that patients who have drug-metabolizing genotypes that encode for no or low-activity enzymes will have longer overall survival than patients with genetically determined higher detoxification activity. The objective of this study was to clarify the patients’ survival potential by investigating the role of polymorphisms in GST-family enzymes in predicting the survival of 220 patients with primary malignant brain tumors diagnosed with WHO grade III gliomas. The 220 patients in this study were a subset of patients with primary malignant gliomas who participated in a study of malignant gliomas between 1994 and 2000 and had available DNA samples. Genetic variants for the GSTM1, GSTT1, and GSTP1 enzymes were determined using PCR and restriction fragment-length polymorphisms. Medical records were reviewed for patient and treatment characteristics, including extent of surgery, type and duration of chemotherapy, and dose and duration of radiation therapy. Comparisons for overall survival were analyzed using Kaplan-Meier and Cox proportional hazards analyses, adjusted in the latter for age, sex, histology, extent of surgery, chemotherapy, and radiation therapy. In univariate analyses, patients who were older, who only had a biopsy for surgical treatment, and who had three or more surgeries had a significantly shorter survival. In multivariable analyses of the anaplastic astrocytoma group (N = 122), compared to patients with the GSTP1 *B/*B genotype, patients with all other GSTP1 genotypes had 7.8 times higher risk of death (95% CI, 1.6–38.3). Among patients with anaplastic oligodendroglioma and anaplastic mixed oligoastrocytic tumors (N = 98), those with the GSTT1 null genotype had 3.2 times higher risk of death than did those with the GSTT1 not-null genotype (95% CI, 1.5–6.7). In support of our hypothesis, patients with anaplastic astrocytoma and the GSTP1 *B/*B genotype had improved survival, likely because of decreased metabolism of chemotherapeutic agents and oxygen free radicals. In contrast, patients with anaplastic oligodendroglial tumors and the GSTT1 null genotype had shorter survival. We propose that this finding is a result of modifications in therapy secondary to increased toxicity. Analyses of toxicity data and 1p/19q status in the anaplastic oligodendroglial subgroup are ongoing.


G. Kumar,1 C.A. Kang,2 and C. Giannini3; Departments of 1Internal Medicine, 2Neurology, and 3Pathology, Mayo Clinic, Rochester, MN, USA

A 74-year-old farmer presented with a 6–month history of worsening headaches. He had an episode of diplopia that resolved after 6 weeks, which was thought to be caused by mononeuropathy of the third nerve. He then developed worsening disequilibrium, gait unsteadiness, and writing difficulties. On examination, he was found to have an ataxic gait with a tendency to fall to the right, right-sided dysmetria, and dysdiadochokinesis. MRI initially showed an abnormal signal within the right inferior and superior cerebellar hemisphere, with areas of contrast enhancement concerning for subacute infarct or infiltrating malignancy. The patient had a repeat MRI scan 1 month later that revealed a right cerebellar mass with a heterogeneously enhancing lesion on T2-weighted imaging. A right suboccipital craniotomy and biopsy were performed, and histopathology showed noncaseating granulomas suggestive of sarcoidosis. The patient was then treated with steroids; in view of complicating factors such as diabetes, hypertension, and hyperlipidemia, he was switched to infliximab. On last follow-up, the patient reported improvement in symptoms. Sarcoidosis is a rare multisystem inflammatory disease of unclear etiology that affects the CNS in about 5–15% of patients. It usually manifests as a granulomatous inflammation of the basal meninges resulting in cranial nerve palsies. It can mimic a tumor, on rare occasion, as in this patient. In addition, in the absence of significant systemic disease, it is unusual for neurosarcoidosis to be isolated in the CNS. There have been no controlled studies addressing the treatment of neurosarcoidosis, although there are many case reports that document excellent responses to corticosteroids. Medical therapy alone may be inadequate in clearing a mass of inflammatory tissue. Thus, surgery plays an important role both in obtaining the diagnosis and in treatment. Infliximab has shown great promise as a steroid-sparing agent in neurosarcoidosis. We present this case of neurosarcoidosis presenting as a cerebellar mass as a reminder that not all cerebellar mass lesions are tumors and that clinicians should be alert to potential differential diagnoses. There have been other patients in whom extensive surgery has been performed for non-malignant disease, and we report this case to increase awareness of this “great mimicker.”


I. Lavon,1,2 B. Zelikovitsh,1,2 Daniel Zrihan,1,2 Y. Fellig,3 D. Soffer,3 and T. Siegall1,2; 1Gaffin Center for Neuro-Oncology and Departments of 2Neurology and 3Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel

Allelic losses on chromosomes 1p/19q are well recognized in OT, while loss of 10q is more frequent in high-grade astrocytomas. MGMT promoter methylation (PM) is associated with improved survival in GBM treated with alkylating agents. In OT, the status of MGMT-PM and its association with other genetic alterations is not well characterized, and even less is known about the temporal evolution of such changes detected at tumor progression (TP). The objective of this study was to evaluate the status of chromosomes 1p/19q/10q and MGMT-PM in the early phase of OT and again at TP. Forty-six tumors from 23 patients were obtained at an early phase of the disease course and again at TP. Initial diagnosis included 17 OT and 6 oligoastrocytic tumors. Chromosome 1p36, 19q13, and 10q22-26 status was evaluated from paired tumor-blood DNA samples using PCR-based microsatellite analysis. MGMT promoter status was determined by methylation-specific PCR analysis. At initial evaluation, 61% of the tumors were low grade (WHO grade II), compared to only 17% at TP (P = 0.0000). Of 17 tumors that were initially described as OT, 13 (76.5%) remained in this lineage regardless of their grade, while 23.5% changed to primarily astrocytic tumors. Of the 6 mixed tumors, 4 (67%) transformed to astrocytic tumors (P = 0.02). The cell type of all 10 tumors initially characterized as OT remained unchanged if they contained 1p/19q deletions, while only 1 of 6 mixed tumors with 1p/19q deletion remained phenotypically unchanged (P = 0.008). Of the 15 tumors with early 1p loss, 80% remained OT at TP, compared with the 8 tumors without 1p loss, in which 75% changed to the astrocytic phenotype (P = 0.01). Allelic loss of 10q was uncommon in both early tumors (18%) and at TP (13%). The proportion of MGMT-PM increased from 19% at the early phase to 71% at TP (P = 0.000). MGMT was uniformly unmethylated in early tumors with an intact 1p, whereas 31% of tumors with 1p deletion contained MGMT-PM (P = 0.04). The proportional gain in MGMT-PM at TP was limited to 31% for tumors with 1p deletion, unlike tumors with an intact 1p that had an 87.5% gain of MGMT-PM at TP (P = 0.006). Our findings indicate that OT with 1p/19q deletion tend to retain their cell phenotype and genetic profile at TP, unlike tumors with no deletions. MGMT-PM is more pronounced at TP, particularly in tumors with an intact 1p. It is suggested that the chemosensitivity of OT is not related to MGMT-PM and that there should be other contributing factors that are yet to be discovered.


Victor A. Levin, Jacob L. Jochec, Lisa M. Shantz, Kenneth R. Hess, and Kenneth D. Aldape; Departments of Neuro-Oncology and M.D. Anderson Clinical Oncology Program, Biostatistics & Applied Mathematics, and Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA, USA

This study was intended to assess the relationship between progression-free survival (PFS) in patients whose malignant gliomas were treated with combination α-difluoromethylornithine (DFMO) and PCV (procarbazine, CCNU, and vincristine) chemotherapy and the level of ornithine decarboxylase (ODC) in the original tumor biopsy prior to chemotherapy. Formalin-fixed tumor pathology slides were requested from pathologists for all evaluable patients with anaplastic gliomas (AG) treated on CCOP protocol DM92–035. Tissue blocks or slides were obtained from 27% of eligible patients. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647) and a previously validated technique. PFS values were calculated by the CCOP Data Management Office as of March 30, 2006. Ab-ODC-Alexa 647 intensity was measured in three ×40 magnification fields for each tumor in triplicate. Values were compared to those from standardized transgenic heart samples of known ODC activity (nmol/30 min/μg protein) to calculate tumor ODC activity values. Tumor ODC levels were variably heterogeneous among tumors and histologic types. Using multivariate analyses and a Cox proportional hazards model, we found that PFS was inversely related to age (P < .0001) and median tumor ODC activity (P < .001). Furthermore, median PFS for patients with AG and glioblastoma (N = 41) was 10 years if median tumor ODC activity was ≤ 33 nmol/30 min/μg protein and only 39 weeks if ODC activity was ≤ 33 nmol/30 min/mg protein. Of the AG tumors, 20% had ODC levels > 33 nmol/30 min/μg protein. For patients receiving only PCV chemotherapy (N = 17), PFS was 3.9 years if ODC activity < 33 and 37 weeks for ODC activity > 33 nmol/30 min/μg protein. In conclusion, measurement of Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate for ODC biochemical activity and, as such, is inversely predictive of response to DFMO-based chemotherapy. Partial funding for this study was provided by the Commonwealth Foundation for Cancer Research and NIH grant U10 CA 45809 (CCOP).


Marlon S. Mathews,1 Christopher Duma,1,2 and Franklin D. Westhout1; 1Department of Neurosurgery, University of California Medical Center, Irvine, Orange, CA, USA; 2Hoag Memorial Hospital Presbyterian, Newport Beach, CA, USA

Central neurocytomas (CN) are rare and mostly benign tumors found primarily in the lateral ventricles of the brain. CNs are composed of uniform round cells exhibiting neuronal differentiation and are found almost exclusively in adults between 15 and 60 years of age. Over a 2-month period, an 81-year-old male presented with decreasing appetite, confusion, gait instability, and incoordination. Other than a slightly unsteady gait, his physical and neurological exams were within normal limits. A CT scan demonstrated an irregularly marginated low-density focus at the atrium of the right lateral ventricle. MRI showed a heterogeneously enhancing mass at the same location. The patient underwent a stereotaxic biopsy of the lesion. Histopathologic examination revealed a central neurocytoma. Following the biopsy, the patient underwent gamma-knife radiosurgery of the lesion. The histologic appearance of the tumor suggested an oligodendroglioma or a CN. Because of the patient’s age, lymphoma and metastatic tumors were also considered. Strong diffuse cytoplasmic staining for synaptophysin and a paucity of GFAP staining were consistent with the diagnosis of CN. The diagnosis of CN was further strengthened by detection of neuron-specific enolase and neuronal nuclear antigen in the tumor cells. CNs are rare tumors afflicting the CNS of young adults and represent less than 0.5% of all primary CNS tumors. Up to 70% of patients present between the ages of 20 and 40 years. The occurrence of a symptomatic CN in an 81-year-old patient is unique and probably represents the oldest reported case of a CN. The optimal treatment for CN is unclear, and management ranges from surgery (subtotal or total resection), standard external-beam radiation, radio-surgery, and chemotherapy. This article highlights the fact that age should not be used as single criterion to rule out CNs in elderly patients.


Marlon Mathews,1 Christopher Duma,1,3 Anton Hasso,2 David Klein,3 and Denise Vanhorn3; Departments of 1Neurosurgery and 2Radiology, University of California Medical Center, Irvine, Orange, CA, USA; 3Hoag Memorial Hospital Presbyterian, Newport Beach, CA, USA

Hematopoiesis outside the bone marrow is known to occur in patients with severe anemia, leukemia, polycythemia, or myelofibrosis and in patients suffering from chronic poisoning by marrow-toxic substances. The liver, spleen, and lymph nodes are the most common sites. A 66-year-old, right-handed male complained of 4 days of terrible right-sided, sharp headaches for which he saw his primary care provider. Outside of being obese, his physical and neurological examinations were within normal limits. Routine laboratory examination showed a WBC count of 30,800 cells/μl, with a differential WBC count of 76% lymphocytes and 24% neutrophils. All other routine investigations were within normal limits. Noncontrast head CT scan and MRI scans showed a large right fronto-temporal, extra-axial, dural-based mass with associated recent intramural hemorrhage and evidence of midline shift and uncal herniation. The mass showed heterogeneous uptake of gadolinium contrast agent with frontal and temporal dural tailing. The mass was resected using a right-sided extended craniotomy with anterior and middle fossa approach. The patient’s postoperative course was uneventful. Hematoxylin- and eosin-stained biopsy specimen showed whorls of tumor cells, diagnostic of a meningioma. Interspersed within the tumor bulk were nucleated red blood cells, representing areas of extramedullary erythropoiesis within the meningioma. No other areas of extramedullary erythropoiesis were found. The patient underwent flow cytometric evaluation, which confirmed the clinical suspicion of an underlying chronic lymphocytic leukemia. Occurrence of hematopoiesis within a meningioma is extremely rare. In 1983, Gregorios et al. reported a case of extramedullary hematopoiesis occurring within a malignant meningioma. Although meningiomas are known to stimulate bone formation, osteogenic foci were not found in the resected tissue in either our patient or in the patient in the aforementioned report. Various theories may explain the occurrence of extramedullary hematopoiesis within a meningioma in our patient, such as in situ growth and development of hematopoietic tissue from multipotent mesenchymal cells, direct extension of hematopoietic activity from the neighboring marrow cavity, displacement of stem cells from bone marrow that settle and develop in tissues where capillaries and blood vessels proliferate (meningioma), or congenital heterotopia of totipotent connective tissue cells, which under certain circumstances may transform into hematopoietic tissue.


K.L. McDonald,1 J.F. Parkinson,1,3 H.T. Wheeler,2 C.A. Cook,1 R.J. Cook,3 M.T. Biggs,3 N.S. Little,3 and B.G. Robinson1; 1Cancer Genetics Unit, Kolling Institute of Medical Research, University of Sydney, Australia; 2Northern Cancer Institute, Sydney, Australia; 3Department of Neurosurgery, Royal North Shore and North Shore Private Hospitals, Sydney, Australia

The methylation status of the promoter region of the MGMT gene was recently demonstrated to be important in assessing the likelihood of a favorable response to temozolomide in patients with glioblastoma (GBM). Methylation-specific PCR (MSP PCR), a commonly used method, uses primers that amplify a small region of the promoter and bind selectively depending on the methylation status of four CGs within this region. This targeting of such a small region in the promoter is prone to false positives and negatives. Full sequencing of the CpG-rich region of the MGMT promoter provides a more accurate representation of the methylation status of the MGMT promoter. In this study, we surveyed GBM tumors and determined the percentage methylation using sequence analysis. We looked for hotspots of methylation and assessed the accuracy of MSP PCR. In addition, our aim was to determine the degree of methylation needed to perturb mRNA expression of the gene and downstream protein expression. Tumor samples collected as part of the Australasian Brain Tumour Bank were accessed. DNA was extracted, treated with bisulfite, and assessed using MSP PCR. To investigate a larger region of the MGMT promoter containing 25 CpG islands, PCR of a 316 base-pair region of the MGMT promoter was performed and the products sequenced. The extent of methylation on sequencing was assessed by comparison to universally methylated and unmethylated control DNA. RNA has also been extracted using a QIAzol-based protocol. Tumor RNA was then subjected to analysis using quantitative real-time PCR. Among the 32 tumor samples analyzed, large variation observed (4%–92% methylation). Unexpectedly, the overwhelming majority of the tumors studied were unmethylated. Upon examination of the GBMs that did show some degree of methylation, no hotspots of methylation were identified. Sequencing and MSP PCR did not concur in 20% of the cases. Upon closer examination of the sequence, the four CG regions targeted by the MSP PCR primers were not methylated, but CGs were methylated elsewhere. This highlights the need to sequence large sections of the promoter when examining methylation status. Results at the mRNA and protein expression levels will shed light on this.


A. Misra,1 I. Smirnov,1 S. VandenBerg,1 M. Ware,1 C. Hong,1 Y. Hirose,2 E. Mirvish,1 L. Kapp,1 S. Kharbanda,3 J. Nigro,1 E. Pan,4 L. Prestegarden,1 R. Yeh,1 J. Costello,1 A. Yates,5 D. Pearl,5 B. Scheithauer,6 P. Burger,7 C. Giannini,6 H. Phillips,3 K. Aldape,8 and B.G. Feuerstein1; 1Univ. of California, San Francisco, CA, USA; 2Keio University School of Medicine, Tokyo, Japan; 3Genentech Inc., South San Francisco, CA, USA; 4Florida Hospital Cancer Institute, Orlando, FL, USA; 5Ohio State University, OH, USA; 6Mayo Clinic, Rochester, MN, USA; 7Johns Hopkins Medical Center, Baltimore, MD, USA; 8The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Pathologic grade predicts outcome in astrocytic tumors. WHO grading is based on cellular atypia, mitotic activity, endothelial proliferation, and necrosis. Grade 2 tumors (low grade, median survival > 5 yr) usually have cellular atypia, while grade 3 tumors (anaplastic, median survival ~3.5 yr) usually have both atypia and mitotic activity. However, some patients with grade 2 and 3 tumors have poor survival, and others have good survival. There is no known marker to detect the atypical survivors. We used DNA microarrays to study 21 consensus grade 2 and 41 consensus grade 3 astrocytomas. Consensus was defined as diagnostic agreement among at least four of six neuropathologists. We used Pearson’s correlation analyses to identify highly correlated chromosomal aberrations, cluster analysis to identify genetic groups, significance analysis of microarrays to identify genetic differences between grades, and the Cox proportional hazards model to identify genetic markers associated with survival. Our preliminary analyses suggest that major interchromosomal correlations for copy-number loss and gain occur in grade 2 tumors between (1) chromosome (chr) 4 and chrs 7, 9, 14, and 17; (2) chr 7 and chrs 9, 14, and 17; and (3) chrs 1 and X; and in grade 3 tumors between (1) chrs 1 and 6; (2) chrs 4 and 7/8; (3) chrs 6 and 16/17; (4) chrs 7 and 10; and (5) chrs 9 and 15. Our findings also suggest that losses on chr 10 encompass the PTEN gene and that the chr 13 loss includes a ~5-Mb region at 13q22.2. Kaplan-Meier analysis suggests that PTEN loss segregates poor and better survivors more efficiently (log-rank = 0.0094 and Gehan-Wilcoxon P = 0.00942) than does tumor grade (P = 0.0166). We plan to collect another set of patients to validate this information. This work was supported by NIH grants NS42927 and CA85799, the National Brain Tumor Foundation, and the Barrow Neurologic Institute.


J.F. Parkinson, K.L. McDonald, H. Wheeler, R.J. Cook, M.T. Biggs, N.S. Little, and B.G. Robinson; Cancer Genetics Unit, Kolling Institute of Medical Research, University of Sydney, Australia; Northern Cancer Institute, Sydney, Australia; Department of Neurosurgery, Royal North Shore and North Shore Private Hospitals, Sydney, Australia

The methylation status of the promoter region of the MGMT gene has recently been demonstrated to be important in assessing the likelihood of response to temozolomide in patients with glioblastoma (GBM). However, very little is known about changes in the methylation status of the MGMT promoter over time and in response to treatment. In addition, variability within individual tumors has not been explored. GBM samples from five patients were analyzed. There were two serial samples from four of the patients and samples from three consecutive surgeries from one of the patients. Tumor samples were snap-frozen in liquid nitrogen and stored at −80°C until use. DNA was extracted, treated with bisulfite, and then assessed using methylation-specific PCR assay. To investigate a larger region of the MGMT promoter containing 25 CpG islands, PCR of a 316 base-pair region of the MGMT promoter was performed and the products sequenced. The extent of methylation on sequencing was assessed by comparison to universally methylated and unmethylated control DNA. Further, multiple samples were taken from different regions of a large tumor in a different patient and subjected to the above analysis. Clinical information from each patient, particularly interval treatment, was gathered and considered. There was no change in methylation between samples in one of the patients. However, in the remaining four patients, there was variation in the extent of methylation between samples. Investigation of multiple sites within the same tumor showed that three of the four sites examined were totally unmethylated, while the fourth site showed 16% methylation. The methylation status of the MGMT promoter may be important when considering treatment for patients with GBM, but caution must be used when interpreting results. The potential reasons for alterations in methylation status will be discussed. Regional variation within an individual tumor is also a factor for consideration. Correlation between the above findings, mRNA expression, and MGMT protein expression and activity are also important. It is unclear from these results whether methylation status depends on sampling site or whether variability occurs over time or with progression. These results do, however, suggest that treatment decisions based on methylation status need to be questioned and clinically validated.


Christopher E. Pelloski,1 Li Zhang,2 E. Lin,2 Krishna Baht,3 J. Matthew McDonald,3 W.K. Alfred Yung,4 Howard Colman,4 Shiao Y. Woo,1 Amy B. Heimberger,5 Dima Suki,5 Michael Prados,6 Susan Chang,6 Fredrick Barker III,7 Jan C. Buckner,8 Karla V. Ballman,9 Alfred F. Furth,10 C. David James,11 and Kenneth D. Aldape4; Departments of 1Radiation Oncology, 2Biostatistics and Applied Mathematics, 3Pathology, 4Neuro-Oncology, and 5Neurosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 6Department of Neurosurgery, University of California San Francisco School of Medicine, San Francisco, CA, USA; 7Neurosurgical Service, Massachusetts General Hospital, Boston, MA, USA; Divisions of 8Medical Oncology, 9Biostatistics, 10Cancer Center Statistics, and 11Experimental Pathology, Mayo Clinic, Rochester, MN, USA

The EGFRvIII variant is the most common EGFR mutation in glioblastoma (GBM) and is present in one quarter to one third of all cases. It is currently a therapeutic target in these tumors, but the clinical characteristics of patients with EGFRvIII expression and its relationship to other key markers in GBM are not clear. EGFRvIII expression was assessed by immunohistochemistry (IHC) in 649 cases (268 in initial set; 381 in validation set) of newly diagnosed GBM from three different institutions. These data were compared with expression of phospho-intermediates of the downstream Akt and Ras pathways (Akt, mTOR, p70S6K, and MAP kinase) and YKL-40, as well as known clinical risk factors. EGFRvIII expression was also determined in a set of tumors for which gene expression profiling data (Affymetrix) were available. The presence of phosphorylated intermediates of the AKT and Ras pathways was predictive of poorer outcome in EGFR-vIII-negative cases. Clinical covariates (determined using the Radiation Therapy Oncology Group recursive partitioning analysis) were also predictive. These prognostic associations were not observed in EGFRvIII-positive cases. Survival analyses using pairwise combinations of markers identified EGFRvIII and YKL-40 as a pair of markers with prognostic significance. In particular, EGFRvIII-negative/YKL-40–negative cases had a markedly improved outcome compared to other combinations of the two markers (85 vs. 47–56 weeks; P < 0.001). A characterization and regression tree analysis revealed new possible risk-stratification schemes based on clinical and molecular features of GBM. Lastly, a set of genes was identified that was associated with EGFRvIII expression and confirmed using real-time RT PCR and IHC. The presence of EGFRvIII in GBM defines a unique subtype of this disease in which traditional clinical risk factors and previously established molecular factors are not predictive of outcome. EGFRvIII is an independent molecular marker of survival, along with YKL-40. The EGFRvIII status of patients should be considered in future clinical trials and therapy development.


B. Pollo, E. Maderna, C. Calatozzolo, M. Gelati, C. Marras, A. Silvani, D. Croci, A. Boiardi, and A. Salmaggi; Istituto Nazionale Neurologico C. Besta, Milano, Italy

Various prognostic factors have been reported in low-grade gliomas, including histotype, tumor size and location, age, neurological signs, entity of surgical resection, and genetic pattern. The study of clinical and biological prognostic factors in gliomas with an oligodendroglial component could improve the management of these patients. Microvessel density (MVD) has been correlated to clinical outcome in astrocytomas, but its impact in oligodendrogliomas and mixed tumors is not clear. The proangiogenic chemokine stromal cell-derived factor (SDF-1/CXCL12) has a role in angiogenesis and chemotaxis of endothelial cells through the activation of its receptor CXCR4, and in low-grade gliomas is related to a shorter time to tumor progression (TTP). The intermediate filament Nestin is expressed in proliferating vessels in gliomas. Platelet-derived growth factor B (PDGF-B) and its receptor PDGFR-β are involved in tumorigenesis, angiogenesis, and malignant progression in gliomas. The aim of our study was to retrospectively investigate 40 patients with low-grade gliomas (11 oligodendrogliomas and 29 oligoastrocytomas) for a pattern of factors related to angiogenesis and glioma growth—such as MVD and immunohistochemical expression of CXCL12, CXCR4, PDGF-B, PDGFR-β, and Nestin—and their relationship to clinical course. The impact of clinical patient-related variables (including age, presurgery enhancement in CT/MRI, KPS, and extent of resection) and tumor-related features (MVD and the expression of CXCL12, CXCR4, PDGF-B, PDGFR-β, and Nestin) on TTP and survival time was evaluated with statistical analysis by Kaplan-Meier survival curves, and differences in these parameters between subgroups of patients were analyzed by log-rank and Fisher tests. In our study, oligodendroglioma histotype was associated with a trend to more prolonged TTP than were mixed tumors (P = 0.12). In immunohistochemical analysis, CXCL12 showed the highest number of positive samples (66%) in vessels, and, in most cases, endothelial and neoplastic cell immunoreactivity was associated. Positivity for CXCL12 on tumor/endothelial cells was the only factor associated with a significantly shorter TTP (P = 0.011). CXCL12 was more frequently expressed by tumor cells in oligoastrocytomas than in oligodendrogliomas and was predictive of a shorter survival time (P = 0.014). Moreover, CXCL12 is not only related to angiogenesis but also exerts an antiapoptotic effect, so CXCL12 may contribute to tumor progression and endothelial escape from apoptotic mechanisms. In conclusion, further research is required to assess the role of this chemokine in malignant progression of oligodendroglial tumors. CXCL12 expression on these tumors might add prognostic information to available clinical and biomolecular indexes, contributing to optimal management in patients with variable life expectancy.


Jingxin Qiu,1 Mingli Yang,2 W. Stratford May,2 Chen Liu,1 William A. Friedman,3 and Anthony Yachnis1; 1Department of Pathology, 2UF Shands Cancer Center, and 3Department of Neurosurgery, University of Florida College of Medicine, Gainesville, FL, USA

JAZ is a nuclear/nucleolar zinc finger protein that has been suggested to play a role in tumor cell death. Although its normal cellular function is not completely understood, JAZ does have a unique high binding affinity to dsRNA. Previous studies showed that JAZ is highly expressed in the mouse brain, but the expression pattern and cell type-specific distribution of JAZ in the normal and neoplastic human CNS is unknown. As a first step in exploring a potential role of JAZ in glioma tumorigenesis, we studied the expression pattern of JAZ in the human CNS. Immunohistochemical (IHC) studies were performed using a tissue microarray containing 90 tissue cores representing a spectrum of infiltrating gliomas and non-neoplastic brain tissue. IHC findings were confirmed and extended using immunofluorescence, subcellular fractionation, and western blotting. In contrast to the nuclear localization observed in a number of non-CNS mammalian cell lines, we found that JAZ was predominantly expressed in the cytoplasm, particularly the mitochondria, of mature human CNS neurons. While normal glial cells in general do not express JAZ, it was abundantly expressed in malignant gliomas, with a predominant cytoplasmic pattern of immunoreactivity. Grade II, III, and IV astrocytic tumors had stronger JAZ reactivity than did anaplastic oligodendrogliomas (AOs). However, recurrent AOs (S/P radiation or chemotherapy) tended to show higher JAZ expression than did primary AOs. Cytoplasmic (mitochondrial) expression of JAZ in the human CNS, as well as upregulation and differential expression in gliomas, are novel findings of this study. Nucleocytoplasmic shuttling of JAZ was recently reported and was suggested to play a role in the processing of microRNAs, a group of tiny noncoding RNA molecules that are believed to be involved in oncogenesis. Our finding of unusual predominant cytoplasmic JAZ expression suggests the involvement of this unique zinc finger protein in glioma tumorigenesis.


Fausto J. Rodriguez,1 Bernd W. Scheithauer,1 and John D. Port2; 1Department of Laboratory Medicine and Pathology, and 2Division of Neuroradiology, Mayo Clinic, Rochester, MN, USA

Malignant glioneuronal tumors of the brain are rare and poorly characterized. Here we report the clinicopathologic features of three examples with unusual morphologies featuring both glial and neuronal differentiation. Clinical features were abstracted from retrospective chart review. Pre-operative imaging studies included MRI of the brain (two cases) and CT without contrast (one case). H&E slides were reviewed in all cases, and immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue using antibodies against GFAP, S100, synaptophysin, neu-N, chromogranin, neurofilament protein, EMA, p53, and Ki-67. Transmission electron microscopy was performed on formalin-fixed (N = 1) and paraffin-embedded (N = 2) tissues. Ultrastructural analysis using the immunogold technique for GFAP was also performed. Two patients were male and one was female; their ages were 84, 66, and 34 years, respectively. Radiologic studies demonstrated hyperdensity on CT (N = 3), multicentricity (N = 2), and a cortical-based solid component with a cystic extension into the white matter (N = 2). One lesion was preoperatively considered a hematoma. At surgery, the tumors were superficial and relatively circumscribed. Histologically, they were composed of large epithelioid cells (N = 3), spindle cells (N = 1), and poorly differentiated smaller cells with high nuclear/cytoplasmic ratios (N = 1). Coagulative nonpalisading necrosis and brisk mitotic activity were present in all cases. Endothelial proliferation was absent. The tumors were immunopositive for GFAP (N = 3), S100 (N = 3), synaptophysin (N = 3), chromogranin (N = 3), neu-N (N = 2), and neurofilament protein (N = 2). EMA stains were negative. Electron microscopy demonstrated convincing neurosecretory granules in one case, some in filament-containing cells immunogold labeled for GFAP. Clinical follow-up was available in two patients, both of whom died 3–5 weeks postoperatively. True malignant neoplasms with glial and neuronal differentiation do occur in the CNS of adults and may pursue a highly aggressive course. Their diagnostic features might not be readily apparent on routine histologic sections but are evident at the immunohistochemical and ultrastructural level. The use of minimal diagnostic criteria, such as immunoreactivity for a single antigen (e.g., neurofilament protein) may not be sufficient and should be discouraged.


T. Shechori,1 B. Hu,1 S.S. Silver,1,2 A. Marchevsky,2 X. Fan,2 and W.H. Yong1,2; 1Department of Pathology, UCLA Medical Center, Los Angeles, CA, USA; 2Department of Pathology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA

Analysis of pathology reports stored in pathology information systems is an important element in identifying and assessing prognostic tumor markers. However, the study of pathology reports from hospital pathology information systems is often limited because of technical constraints and privacy considerations, including those of institutional review boards and issues related to HIPAA. A National Cancer Institute-sponsored consortium called the Shared Pathology Informatics Network (SPIN) has attempted to develop software tools to improve access to pathology data and tissue specimens. To test and validate such software tools, we propose that it is useful to create a virtual pathology report database free of the privacy concerns of a real database. As a model, we have constructed a software program that can generate virtual oncologic neuropathology reports using the current WHO classification of CNS tumors and the College of American Pathologists brain and spinal cord cancer protocol. This program has generated a database of 5,000 virtual neuropathology reports and has the potential to build many more. Oncology informaticians can use the resulting virtual database to test the efficacy of software, including scrubbers (for removing patient identifiers), autocoders, and data mining tools, prior to applying them to real patient databases. Fictional or virtual patients, complete with birthdates, medical record numbers, and gender designations, are created. There are corresponding neuropathology reports with virtual physicians, electronic signatures, and all the diagnostic and descriptive elements of the standard cancer pathology report. We have designed a virtual database that includes all the CNS tumor types in the WHO classification, with common tumors represented in proportions reflecting their incidence as reported by the Central Brain Tumor Registry of the United States 2005–2006 report. Rare tumor entities are included in quantifiable numbers as consult cases. Parameters including median age, age range, common locations, and male: female ratio for each tumor type are programmed where known. Bimodal age distributions for some tumors are incorporated. In a subset, clinical history, tumor images, and adjunctive immunohistochemical or molecular studies are linked to the relevant tumor case. The underlying data elements are stored in a Microsoft Access database. Visual Basic for Applications programs compile the elements into individual coherent pathology reports in Microsoft Word format. The creation of a virtual database with known parameters that can be well characterized permits testing of software tools in a relatively controlled environment compared to a real database. In summary, a virtual neuropathology database is a novel experimental substrate potentially useful for the evaluation of anonymization and oncology data mining software.


Zita A. Sibenaller, Carey L. Allen, Craig J. Kilburg, and Timothy C. Ryken; University of Iowa Carver College of Medicine, Iowa City, IA, USA

Tumorigenesis is determined not only by the DNA sequence of specific genes but also by the epigenetic code that regulates their expression. One way that expression of genes is epigenetically regulated is through DNA methylation, particularly in the promoter region. It has been reported that the MGMT promoter is methylated in approximately 40% of grade III and IV glioma tissue, which prevents transcription of the gene. The loss of the MGMT protein results in the cell’s inability to repair alkylation adducts in the DNA, culminating in cell death. Also reported is a survival benefit for patients treated with an alkylating agent and radiotherapy in which the MGMT gene is inactivated. There is an ongoing debate as to the better representation of the epigenetic code: tissue samples made up of different cell types, or cell lines established from the tumor tissue containing one cell type, grown in vitro, that might have undergone a change in the epigenetic tags. Using microarray technology, we examined MGMT expression in 20 primary cell lines established from patients diagnosed with glioblastoma multiforme. Although MGMT expression varied between each sample, our data identified a patient group with an average 10–fold decrease in MGMT gene expression. Each patient received BCNU wafers and an alkylating agent at the time of their initial surgery and the standard course of follow-up therapy, but their prognosis was poor. While the sample size was small, the data suggest that the effect of MGMT methylation may be separate from that of alkylating agents.


M.M. Souweidane,1 I.J. Dunkel,2 M.A. Edgar,3 M. Manaqibwala,4 Paul J. Christos,5 L. Becker,6 and J.T. Rutka7; 1,5The Weill Medical College of Cornell University, New York, NY, USA; 1,2,3Memorial Sloan Kettering Cancer Center, New York, NY, USA; 4Robert Wood Johnson Medical School, New Brunswick, NJ, USA; 6,7Hospital for Sick Children, Toronto, Canada

Cerebrospinal fluid (CSF) and arachnoid sampling during tumor resection may provide prognostic information. CSF and arachnoid tissue were sampled at the time of primary posterior fossa tumor resection. The results were tabulated by tumor type. Patients were assigned to group A (tumors with high metastatic potential) or group B (those without high metastatic potential). The results of intraoperative staging for group A patients were compared with conventional staging methods (MRI and lumbar puncture) and analyzed with respect to disease outcome. From a cohort of 73 patients, 67 patients had CSF, 69 had arachnoid, and 63 had both sampled. Tumor types varied, with pilocytic astrocytoma (34.3%), medulloblastoma (30.1%), and ependymoma (9.6%) representing the majority. There were 29 (40.3%) patients in group A and 43 (59.7%) in group B. Median follow-up was 21.8 months (range: 0.2–122.9 months). Arachnoid infiltration was identified in 11 (15.9%) and CSF was positive in 17 (25.4%). Arachnoid infiltration and CSF cytology were found in 25.0% and 38.1% for medulloblastoma, 8.3% and 4.4% for pilocytic astrocytoma, and 0.0% and 40.0% for ependymoma, respectively. For group A, arachnoid infiltration was found in 29.6% of patients and CSF was positive in 46.4%, compared with 4.9% (P = 0.005) and 7.9% (P = 0.0003) of patients in Group B. Arachnoid infiltration and CSF sampling were congruous with M stage in 69.0% (P = 0.04) and 82.8% (P = 0.001) of patients, respectively. Patients that had positive arachnoid had a 54.6% incidence of disease progression or recurrence, compared to 23.6% among patients with negative arachnoid (P = 0.04). Similarly, progression or recurrence occurred in 68.8% of patients with positive CSF sampling, compared to 18.0% when CSF was negative (P = 0.0001). When either positive arachnoid sampling or CSF cytology was integrated into a prognostic grade, the predictive value of disease progression was 66.7% or 87.5%, respectively, compared with 66.7% when conventional staging was used. Arachnoid infiltration or CSF dissemination found at the time of tumor resection in patients with posterior fossa brain tumors is frequent. Intraoperative staging correlates well with conventional staging methods and enhances the predictive value of conventional risk categories. The integration of arachnoid or CSF sampling into prognostic grading should be studied in larger prospective studies.


Roy Torcuator, Ian Lee, Lisa Scarpace, and Tom Mikkelsen; Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA

Oligodendrogliomas with the genotypic profile of allelic loss of 1p and 19q (loss of heterozygosity, LOH) have been associated with chemosensitivity resulting in improved prognosis. Published studies have demonstrated the benefit of temozolomide, an oral alkylating agent, in the treatment of progressive low-grade and anaplastic oligodendrogliomas. Furthermore, the DNA repair enzyme MGMT has been shown to induce resistance to alkylating agents. It is unclear whether there is a correlation between LOH status and MGMT methylation status. Patients with low-grade and anaplastic oligodendrogliomas were identified using the Hermelin Brain Tumor Database. Of these patients, 29 had sufficient tissue for analysis of MGMT gene promoter methylation using methylation-specific PCR. 1p/19q status (LOH) was determined by 0-paired tumor-blood PCR-based analysis. Of the 29 patients, 16 had sufficient data to be analyzed for time to progression and survival time. Of the 16 patients with LOH, 7 had methylated MGMT. In the group with maintained 1p/19q, 2 of 11 had methylated MGMT. In patients with sufficient follow-up time for analysis, four of the eight patients with LOH had MGMT methylation. Of the two patients who progressed, a shorter time to progression was noted in the patient with methylated MGMT (491 vs. 600, respectively; median follow-up, 40 months). In the group with sufficient time for analysis, two of the eight patients with intact 1p/19q showed methylation. Half of the patients with intact 1p/19q progressed regardless of methylation status. However, of the patients who progressed, a shorter time to progression was noted in the unmethylated group (789 vs. 498.3, respectively). A greater proportion of patients with LOH had stable disease (six of eight) compared to patients with intact 1p/19q (four of eight). In both subgroups, methylation status did not affect the proportion of patients who progressed. From this preliminary data, MGMT methylation status does not correlate with LOH with regard to progression-free survival. Further analysis will use increased follow-up time and additional patients.


J.G. Valadez,1 M. Ehtesham,2,3,4 A. Sarangi,1 S. Chanthaphaychith,2 V. Grover,1 M.W. Becher,5 R.C. Thompson,2,4 and M.C. Cooper1; Departments of 1Neurology, 2Neurosurgery, 3Cancer Biology, and 5Pathology and 4The Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA

The Hedgehog (Hh) signaling pathway regulates progenitor cell fate in embryogenesis and tumorigenesis of multiple organ systems. Prompted by the requirement for Sonic hedgehog (Shh) signaling in the regulation of neural progenitor cells, we investigated the activity of this pathway in adult gliomas. Here we provide evidence that the Hh pathway is operational in grade II (GII) and III (GIII) gliomas but not in grade IV (GIV) gliomas. We found that mRNA expression of the Hh receptor Patched (PTCH) was elevated only within GII and GIII gliomas. PTCH protein was detected within a subset of GII and GIII glioma tumor cells, many of which coexpressed the proliferation marker Ki67 and the stem cell marker Bmi-1. Hh pathway responsiveness was measured only in primary cell lines derived from GII and GIII gliomas and with culture conditions that favored the maintenance of progenitor cells, not under conditions that favored progenitor cell differentiation. In light of the recent identification of tumor-initiating progenitor cells from adult GIV gliomas, these findings may indicate a role for Shh signaling in their regulation within clinically distinct intermediate-grade gliomas.


M.C. van Kouwenhoven, Th. Gorlia, J.M. Kros, J-Y. Delattre, A.A. Brandes, M.J.B. Taphoorn, A. Allgeier, D. Lacombe, and M.J. van den Bent; Department of Neurology/Neuro-Oncology, Daniel den Hoed Cancer Center/Erasmus University Hospital; on behalf of the EORTC Brain Tumor Group

Between 1995 and 2002, the EORTC Brain Tumor Group conducted a randomized trial 368 patients to investigate the effect of adjuvant PCV chemotherapy on the outcome of anaplastic oligodendroglioma and mixed oligoastrocytoma. In the present study, we investigated the influence of clinical and molecular factors on the outcome of patients. Clinical, treatment, neuroradiological, histological, and molecular factors (1p and/or 19q loss) were available for most patients entered into the trial. Cox proportional hazards models (Cox PH) with stepwise selection at 1% significance were fitted to screen factors. The probability of inclusion in a multivariate model was estimated by bootstrap for each factor. The variables of the final Cox PH were entered into a recursive partitioning analysis (RPA). In the Cox PH on 278 patients with all available data, including 1p/19q assessment, age (< 45, 45–54, and > 54 years), extent of surgery (biopsy vs. resection), WHO performance status (0, 1, or 2), combined 1p/19q loss, and endothelial proliferation and necrosis (both as determined at central review) were found to be prognostic independent factors. The absence of tumor enhancement, good MMSE, and previous resection for a low-grade tumor were not related to a favorable outcome. In RPA using these factors, the first node was made by 1p/19q status; for patients without 1p/19q loss, the second node was the presence of necrosis. These results show that pretreatment tumor characteristics have a large impact on the prognosis of these patients. Tumors with 1p/19q loss constitute a different entity. The prominent role of necrosis in non-1p/19q deleted tumors suggests that some of these tumors behave like glioblastomas. A better molecular characterization of non-1p/19q deleted tumors is needed. A nomogram developed on this model that allows assessment of prognosis in individual patients will be presented.


Franklin D. Westhout,1 Marlon Mathews,1 Laura Paré,1 William B. Armstrong,2 and Mark E. Linskey1; Departments of 1Neurological Surgery and 2Head and Neck Surgery, School of Medicine, University of California Irvine, Orange, CA, USA

Schwannomatosis has become a new recognized classification of neurofibromatosis. Although the genetic loci are on chromosome 22, schwannomatosis lacks the classical bilateral vestibular schwannomas seen in NF2. Cervical plexus tumors are rare, and schwannomas in schwannomatosis tend to be large and cystic. We present the surgical treatment of three patients at our institution for probable schwannomatosis: one brother, his sister, and a middle-aged gentleman. The brother presented with progressively enlarging tumors, including large bilateral neck masses. All patients had spinal intradural, extramedullary nerve sheath tumors and some degree of functional impairment. Surgery in all patients was multistaged and consisted of exploration and excision of forearm tumors, laminectomies with tumor dissection and resection, and right-neck exploration. The schwannomas had cystic and solid features with enhancing and nonenhancing features on MR imaging. The right-neck tumor in case 1 originated from an upper cervical plexus sensory twig. Postoperatively, this patient had marked respiratory improvement and increased range of motion in the neck. Pathologic analysis of specimens from all cases and stage procedures demonstrated schwannoma. Cases 1 and 2 met the diagnostic criteria for schwannomatosis (MacCollin et al.). Schwannomatosis-affected patients have a better prognosis due to lower incidence of intracranial tumors compared to NF2. Schwannomas of the cervical plexus are rare and can mimic brachial plexus tumors. The predominant sensory nerve involvement on peripheral nerves and the degree of interdigitation of rootlets contributes to the low morbidity of surgical treatment, which should be conserved for symptomatic cases or impending functional impairment.


Joseph C. Wiley, Zia Karim, Ajay Pandita, and Abhijit Guha; The Hospital for Sick Children, Toronto, ON, Canada

Neurofibromatosis-1 (NF1) is the most common tumor-predisposing syndrome in humans, with an incidence of approximately 1:3500. Neurofibromin, the protein encoded by Nf1, acts as a p21–Ras-GAP by directly interacting with and inactivating p21–Ras through its GAP-related domain (GRD). However, evidence suggests that non-Ras-GAP functions of neurofibromin, mediated through interactions with domains outside of the GRD, are of importance. These domains include the CSRD, TBD, Sec14, and SYNBD1 and 2 domains, with only a few interacting proteins identified to date. GST-fusion proteins containing neurofibromin domains were used along with gel-based mass-spectrometric (MS) proteomic analysis to identify novel neurofibromin-interacting proteins. To date, four of six major domains have been cloned into an N-terminal tagging GST expression vector (PGEX4T series). GST-fusion proteins were purified on a GSH-agarose column and incubated with protein obtained from the derivative cell types of the prevalent NF1–associated tumors, Schwann cells and astrocytes. Proteins that associated with the fusion construct were eluted and separated via 1D SDS-PAGE. Gels were Coomassie stained, and unique bands were excised, tryptically digested, and subjected to MS for identification. We optimized the expression of our fusion domains, confirmed the identity of the expressed domains by MS, and initiated experiments with the human MPNST cell line ST8814. The GST-tagged tubulin-binding domain (TBD) identified alpha-tubulin and two other previously unreported interacting proteins, the mitochondrial protein LRPPRC and the microtubule-associated motor protein Dynein, as potential interactors. Current investigations are centered on confirming these neurofibromin interactions in vivo via colocalization and reverse-bait immunoprecipitation experiments. Ongoing studies to identify additional proteins that interact with TBD and the other neurofibromin domains will lead to a better understanding of how loss of neurofibromin expression leads to the plethora of clinical findings, including a variety of tumors, in NF1 patients.



Dolly G. Aguilera, Peter Zage, Johannes Wolff, and Vidya Gopalakrishan; Department of Pediatrics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Medulloblastomas are malignant pediatric tumors of the posterior fossa and often have a poor prognosis. Because various studies have shown that human medulloblastomas have extensive epigenetic modification of gene expression that may be linked to the pathogenesis of the tumor, we have evaluated the use of small molecule inhibitors of enzymes that modulate chromatin structure and gene expression. Histone deacetylases (HDACs) are a family of enzymes that deacetylate chromosomal histones and repress gene expression. Here, we have evaluated the effect of the histone deacetylase inhibitor (HDACi) MS-275 on the human medulloblastoma cell lines Daoy and D-283 and examined the mechanism of action of this drug. Our experiments using MTT assays carried out with various concentrations and times of exposure to the drug have revealed a significant decrease of Daoy and D283 cell viability. Flow cytometric analysis revealed that MS-275 causes an initial accumulation of cells in the G1 phase of the cell cycle leading to eventual apoptotic cell death. These effects also occur in a dose and exposure-dependent manner. We further characterized the mechanism of apoptosis by carrying out caspase assays. These experiments reveal an elevation of Caspase-3 activity, suggesting that it may have an important role in executing MS-275 mediated apoptosis. Because Caspase-3 is a terminal mediator of apoptosis, we are in the process of determining upstream modulators of Caspase-3 that may play a role in MS-275–induced cell death in medulloblastoma cells. The effect of MS-275 on other cell cycle proteins and chromatin structure is also being evaluated. We conclude that the HDACi, MS-275, has a significant antitumor effect on medulloblastoma cells and has the potential to be a novel therapeutic agent for these tumors.


D. Aguilera,1 N. Khang,1 J. Wolff,1 S. Majumder,2 and V. Gopalakrishnan1; 1Division of Pediatrics and 2Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Although medulloblastomas are the most common malignant tumors in children, the molecular basis of these tumors is largely unknown. We have identified the RE1 Silencing Transcription Factor (REST), a previously identified regulator of neuronal development, as a critical factor in medulloblastoma tumorigenesis. Using mouse models, we have shown that REST cooperates with the oncogene Myc to promote tumor formation in the murine cerebellum but not in the forebrain. Mechanistically, REST contributes to tumor formation by blocking neuronal differentiation and providing proliferation advantage to cells overexpressing the protein. We have also determined that a number of human medulloblastomas express elevated levels of the REST protein. Importantly, countering REST function using a dominant positive mutant of REST called REST-VP16 abrogates the tumorigenic potential of these cells. Thus, REST appears to have potential as a novel therapeutic target for medulloblastomas. Because REST-mediated repression occurs through epigenetic modification of target gene expression by histone deacetylation and DNA methylation, we have tested the ability of the histone deacetylase (HDAC) inhibitor, MS-275, and the DNA methylation (DNMT) inhibitor, 5-Azacytidine, to modulate REST activity in both human medulloblastoma cell lines and in immortalized Myc-expressing mouse cells that conditionally overexpress REST (NSC-M-R). Our experiments reveal that although MS-275 decreases cell viability in a concentration-dependent and time-dependent manner, 5-Azacytidine had relatively little effect on cell viability under the conditions of our study. Immunofluorescence assays with human medulloblastoma cells and NSC-M-R mouse cells revealed the induction of neuronal differentiation markers, such as type III beta tubulin, a REST target gene, and neurofilament, a nontarget gene upon treatment with MS-275. The de-repression of REST target gene expression suggests that REST activity is inhibited by MS-275 in these tumor cells. However, these cells also continued to express the neural stem cell marker nestin, which suggests an aberrant activation of the neuronal differentiation program. Treatment with MS-275 also promoted cell cycle arrest followed by caspase-mediated cell death. In conclusion, our results indicate that the HDAC activity of REST most likely is more important for medulloblastoma tumorigenesis and that HDAC inhibitors may have promise as therapeutic agents for medulloblastomas that express REST in an aberrant manner.


Joann Ater, Emi Holmes, Tianni Zhou, Claire Mazewski, William Roberts, Timothy Booth, Alice Ettinger, David Freyer, Regina Jakacki, Richard Kadota, Kenneth Lazarus, Jennifer Pearce, Ian Pollack, Michael Prados, Richard Sposto, Gilbert Vezina, Jeffery Wisoff, Alan Yates, Roger Packer; for the Children’s Oncology Group (COG), Arcadia, CA, USA

The treatment of centrally located, unresectable and progressive low-grade gliomas in young children has always been difficult because of the high risks of surgery and serious late effects of radiation. The primary aim of A9952 was to compare event-free survival in these children as a result of treatment with either Regimen A, consisting of carboplatin and vincristine (CV), or Regimen B, consisting of a combination of thioguanine, procarbazine, CCNU, and vincristine (TPCV). The secondary aims were to evaluate quality of survival with respect to intellect, growth, vision, neurological impairments, and endocrine function. Eligible patients were randomized at the time of study entry to 1 of 2 treatment regimens stratified by tumor location and histology. Children with tumors involving the optic chiasm and posterior optic pathway were eligible without biopsy. Patients with tumors of the optic nerve without chiasmal involvement were not eligible. All patients were required to have a residual tumor to be eligible. Patients with neurofibromatosis type 1 (NF) were nonrandomly assigned to Regimen A (CV) and were required to have documented radiographic progression. From April 1997 to January 2005, 422 eligible patients were registered: 146 on CV, 146 on TPCV, and 130 NF patients nonrandomly assigned to CV. The number of patients per strata assignment at study entry were 253 hypothalamic/chiasmal (123 non-NF, 130 with NF) and 169 in other sites (95 pilocytic astrocytoma, 28 fibrillary grade II astrocytoma, 46 other histologies). For non-NF patients, the 5-year event-free survival (EFS) is 36% +/− 4.3% and overall survival (OS) is 87% +/− 2.7%, with a median time to progression of 46 months. Follow-up is currently not sufficiently mature to report or draw conclusions about the relative efficacy of the 2 chemotherapy regimens. For NF patients, the 5-year EFS is 66% +/− 5.7% and OS 98% +/− 1.4%. The EFS was significantly better for the NF patients when compared with all non-NF patients (P [less-than-or-eq, slant] 0.0001) and with the non-NF patients in the hypothalamic/chiasmal strata (P [less-than-or-eq, slant] 0.0001). Both regimens were successful in controlling the tumor for a median time of more than 3 years, thus allowing a delay of radiotherapy or other treatment. At this point, there was no significant difference in efficacy between regimens, but further follow-up is needed. The significantly improved EFS in the children with NF also requires further investigation. An MRI review that will further define tumor location, characteristics, and response was initiated but is not yet complete. The data on quality of survival were collected during the study but require further analysis. A long-term late-effects study that includes a quality of life measurement may be needed in the future to determine if treatment of initial progression with chemotherapy was of benefit in these children who often receive multiple successive treatments throughout their lifetimes.


Ute Bartels,1 Uri Tabori,1 Jing Ma,2 Michael Carter,3 Eric Bouffet,1 and Cynthia Hawkins2 ; Divisions of 1Hematology/Oncology, 2Pathology and 3Neurosurgery, The Hospital for Sick Children, Toronto, Canada

Ependymomas are the third most common brain tumor in the pediatric population. The main challenges in ependymoma treatment are the lack of markers predicting tumor behavior and the limited treatment options, especially in cases of recurrent disease. Radiation is the mainstay of treatment, and adjuvant chemotherapy has thus far not improved survival rates. This study evaluated microvessel density (MD) and angiogenic features as potential independent markers of outcome and to establish a basis for antiangiogenic therapy. Patients with a pathologic diagnosis of intracranial ependymoma who underwent surgery at the Hospital for Sick Children in Toronto between 1985 and 2004 were retrospectively identified through the pathology and oncology databases. For each patient, all pathological blocks and corresponding slides were obtained and reviewed for diagnostic accuracy and tissue adequacy. Ependymoma tissue microarrays were constructed and immunostained with factor VIII and specimens were evaluated for MD. Kaplan-Meier analysis was used to look for an effect on progression-free survival (PFS). Cox regression was used for multivariate analysis. Seventy-five patients were identified with sufficient clinical data and adequate pathological material. The mean age at presentation was 5.8 years +/− 0.5 years. Forty patients (53%) had at least 1 recurrence, including 29 who had at least a second sample available for MD comparison between the time of diagnosis and the time of recurrence. The 5-year overall survival rate was 57% +/− 7% (10-year OS rate, 38% +/− 9%) and the 5-year PFS rate was 34% +/− 6% (10-year PFS rate, 30% +/− 7%). MD showed a median of 16 vessels/1.2 mm2 with a wide range of variation (SD, 11). No differences in MD were observed between classic (N = 29) versus anaplastic (N = 46) ependymomas (median, 17 vessels/1.2 mm2 for both) and between tissue acquired at the time of initial diagnosis versus at recurrence (median, 16 vessels/1.2 mm2 for both). We did not find any correlation between age and MD. There was a trend toward longer PFS in those patients with low MD (< 16 vessels/1.2 mm2) at the time of initial diagnosis (P = 0.073), which was significant in those patients over the age of 3 years (P = 0.011). MD in recurrent tumors was not informative. Low MD is predictive of longer PFS in children older than 3 years at the time of their initial ependymoma diagnosis, independent of tumor grade and extent of surgical resection. Evaluation of angiogenic features in these tumors may identify a subgroup amenable to antiangiogenic therapy.


S.R. Burzynski, T.J. Janicki, R.A. Weaver, B.J. Szymkowski, M.I. Khan, V. Dolgopolov; Burzynski Clinic, Houston, TX, USA

Antineoplastons (ANP) are synthetic analogues of naturally occurring phenylacetylglutamine, phenylacetylisoglutamine, and phenylacetate. Previous reports have described a significant percentage of objective responses and increased overall survival rates in patients with newly diagnosed and recurrent diffuse intrinsic brainstem gliomas and multicentric gliomas. This study describes the treatment of a group of patients involved in phase II trials of ANPs who had both brainstem gliomas and multicentric tumors (MBSG). These trials were monitored by the FDA and the Institutional Review Board. Nineteen evaluable patients diagnosed with MBSG were treated with ANP. The median age was 9.2 years (range, 3.9–40.8 years) and 90% were less than 18 years old. Diffuse intrinsic brain stem glioma was diagnosed in 95% of patients and cervicomedullary tumor in 5%. A biopsy was performed in 37% of patients, and pathology results confirmed 4 with low-grade and 3 with high-grade gliomas. Tumor recurrence after previous standard treatment was documented in 60% of patients. ANP was given intravenously daily through a subclavian venous catheter and a double-channel infusion pump. The median duration of i.v. ANP was 4½ months, and the median of average dosages of A10 was 9.2 g/kg/day and AS2-1 was 0.32 g/kg/day. Responses were assessed using gadolinium-enhanced MRI scans and confirmed using PET scans in some cases. A complete response (CR) was determined in 11%, partial response (PR) in 5%, stable disease (SD) in 37%, and progressive disease (PD) in 47%. The overall survival rate (OS) at 1 year was 53%, at 2 years was 32%, and at 5 years was 16%. The progression-free survival rate (PFS) was 26% at 1 year and 16% at 2 years. The maximum survival time is 9+ years. The patients did not experience any serious toxicities (grades III–IV), and there were no chronic toxicities. The results of the study showed favorable responses and survival data in a small group of patients diagnosed with difficult-to-treat brain tumors.


Navjot Chaudhary and Paula Klurfan; McMaster University, Hamilton, Ontario, Canada

Medulloblastoma is the most common primary central nervous system tumor that arises in childhood. There is strong evidence that medulloblastoma is very difficult to control, despite the use of radiation and chemotherapy. Children who undergo surgical resection have a higher overall rate of survival. The evolution of stereotactic radiosurgery (SRS) may offer hope for patients with medulloblastoma. The objective of this study was to determine the efficacy of SRS compared with surgery for patients with medulloblastoma. We searched 3 electronic databases: the Cochrane Central Register of Controlled Trials (CENTRAL, 2004 issue 2), MEDLINE (1966 to present), and EMBASE (1974 to present). Comparative studies that assessed the efficacy of SRS in children with medulloblastoma were included. Outcome measures included progression-free survival (PFS), overall survival, and tumor recurrence. Despite extensive searching, no randomized controlled trials (RCT) were found. The electronic search identified 27 studies. A total of 15 studies were selected for further evaluation. Seven articles met eligibility criteria and were included in the final review (N = 144). The weighted mean for the PFS median was 10.7 months (95% confidence interval [CI], 9.4–12 months), and the weighted mean for overall PFS and survival at a minimum of a 2-year follow-up was 16.8% (95% CI, −47.7%–81.2%) and 39.0% (95% CI, 6.3%71.8%), respectively. The rate of tumor recurrence was 29.8% (95% CI, −0.1–59.8). SRS appears to reduce the proportion of first failures occurring locally and is associated with better outcome when implemented as a part of initial treatment. Some patients with unresectable relapsed disease benefit from SRS; however, SRS treatment for multiple lesions does not appear to be curative. Local recurrence can be controlled by SRS with chemotherapy but the survival of patients with metastases may not be prolonged effectively by SRS in conjunction with aggressive chemotherapy. A limitation in the literature is the lack of RCTs. Cohort or single-arm studies only provide partial information and have the risk of significant bias. An appropriate RCT should be designed to determine which technique is superior for patients with medulloblastoma. The treatment of medulloblastoma with SRS, either alone or in combination, merits further investigation before clear recommendations can be made.


Samuel H. Cheshier,1 Laurie E. Ailles,2 Michael Lim,1 Paul Laddis,1 Victor Tse,1 Irving L. Weissman,2,3 and Stephen Huhn1; Departments of 1Neurosurgery, 2Pathology, and 3the Institute of Medicine, Stanford University School of Medicine, Stanford, CA, USA

The recent identification of neuroepithelial stem cells (NSC) in human fetal tissue has led to the opportunity to determine the relationship of these cells to brain tumor cells. To better understand the relationship between pediatric tumors and normal neural progenitors, we prospectively determined the frequency of tumor cells expressing NSC-associated cell surface proteins in a series of pediatric brain tumor specimens. Brain tumors isolated from pediatric patients were disassociated, and isolated cells were then stained with monoclonal antibodies specific for cell surface molecules. The markers that we analyzed included CD133, CD24, and CD45. The cells were analyzed using Becton Dickinson FACSDiva flow cytometry software (Becton Dickinson Biosciences, NJ). Fifteen pediatric tumors were analyzed, including 4 medulloblastomas, 2 ependymomas, 2 pilocytic astrocytomas, and 1 each of the following tumors: low-grade glioma (WHO II), anaplastic astrocytoma, atypical teratoid rhabdoid tumor (ATRT), astroblastoma, malignant teratoma, ganglioglioma, and nongerm-cell germinoma. CD133 was expressed in all 9 high-grade (WHO III or IV) tumors with an average of 14.7% (SD, 18.5%). We found no detectable expression of CD133 in 5 low-grade tumors (WHO I or II). One ependymoma (WHO II) had 41% CD133 expression. In 6 cases, the staining results enabled us to determine the frequency of cells expressing the specific NSC phenotype (CD133+/CD24−/CD34−/CD45−). An astroblastoma, an ATRT, and a malignant teratoma contained 24%, 1.4%, and 0.22% CD133+/CD24−/CD34−/CD45− cells, respectively. Two pilocytic astrocytomas and 1 non-germ-cell germinoma failed to express the NSC phenotype. In this series, high-grade pediatric tumors expressed higher levels of CD133 than low-grade tumors. Furthermore, some tumors contained cells expressing the full pattern of cell surface proteins established for normal fetal NSC. These results suggest a potential relationship between normal progenitor cells and neuro-oncogenesis and highlight the possible significance of CD133 expression and tumor anaplasia.


Chandra M. Das, Peter Zage, Johannes Wolff, and Vidya Gopalakrishnan; Deparment of Pediatrics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Medulloblastoma is a malignant pediatric brain tumor that occurs mostly in the cerebellum. Conventional treatment of medulloblastoma, including surgery, craniospinal radiation therapy, and chemotherapy, can diminish the size of the primary tumor, but recurrence and metastasis are common. Thus, new or combinatorial therapeutic approaches to the treatment and management of this disease are necessary. In this study, we propose using valproic acid (VPA), an established antiepileptic drug, as an adjuvant therapy to etoposide (VP16) for the treatment of medulloblastomas. Valproic acid has recently been identified as a histone deacetylase inhibitor (HDACI) and has been shown to alter the expression of cell cycle genes by affecting the levels of acetylated histone H3 and H4 at these genes. Etoposide is a commonly used chemotherapeutic drug that functions as a topoisomerase II inhibitor. Therefore, we hypothesize that VPA could potentiate the cytotoxicity of etoposide by increasing chromatin decompaction, leading to an increased frequency of DNA breaks, to an upregulation of genes mediating cell cycle arrest and apoptosis, or both. We examined the cytotoxic effects of VPA and VP16 alone and in combination on 2 human medulloblastoma lines (DAOY and D283) using MTT assays. Valproic acid and VP16 both decrease cell viability in a time-dependent and a dose-dependent manner. We next examined the ability of VPA and VP16 at doses at which neither drug alone had a significant effect on cell viability to synergize and increase cell killing. A dramatic increase in cell killing was observed when medulloblastoma cells were co-exposed to VPA and VP16. This enhancement of cytotoxicity by VPA was observed at all tested doses of VP16. A flow cytometric analysis of these cells revealed a predominantly G1 accumulation of cells upon exposure to VPA and a G2 arrest in cells exposed to VP16. An enhancement in the appearance of cells with a sub-G1 DNA content was observed upon treatment with VPA and VP16 together compared with cells treated with each agent alone. Our in vitro studies have shown that a combination of VPA and VP16 has significantly elevated cytotoxicity at doses and incubation periods compared with either drug alone. The mechanism of apoptosis and the effect of VPA on pathways detecting DNA damage are also currently under evaluation. We conclude that VPA can be used as a chemosensitizer in medulloblastoma therapy.


E.M. Dunbar, B.T. Hoang, W.A. Friedman; University of Florida, Gainesville, FL, USA

Ependymomas of the fourth ventricle are rare tumors with an infratentorial location that poses therapeutic challenges. The existing literature reveals conflicting data on the significance of various patient and treatment factors; therefore, a retrospective review was performed at the University of Florida to examine the factors affecting survival of patients with ependymomas of the fourth ventricle. We conducted an IRB-approved retrospective review of the medical records of 48 patients with ependymomas of the fourth ventricle who underwent neurosurgical resection from 1960 to 2005. Forty-four patients were selected for analysis after 3 were removed because of unknown histology or extent of surgery. The median follow-up time was 44.3 months, ranging from 0.2 months to 986 months. The median age was 13 years, ranging from 1 year to 68 years. Standard neurosurgical definitions of total, near total, and subtotal extent of resection were used and confirmed by the neurosurgeon in 20.8% of cases (N = 10), by MRI scan in 48% (N = 23), and unknown in 31.2% (N = 15). The WHO criteria were used for the pathologic diagnosis of ependymoma and anaplastic ependymoma. Kaplan-Meier analysis was used to determine survival of all patients and then divided by histology. The overall actuarial survival rate was 1 year (yr) (93%), 2 yr (82%), 5 yr (58%), 10 yr (37%), 20 yr (21%) and 30 yr (21%). The significance of histology was studied by comparing the actuarial survival rate of patients with ependymomas at 1 yr (93%), 2 yr (89%), 5 yr (73%), 10 yr (58%), 20 yr (23%), and 30 yr (23%), with that of patients with anaplastic ependymomas at 1 yr (93%), 2 yr (68%), 5 yr (32%), 10 yr (11%), and no survivors at 20 yr and 30 yr. A multivariate analysis was done to determine whether the factors of histology, extent of resection, histology, patient age at resection, and use of adjuvant radiotherapy affected survival. The extent of surgical resection was significant [Hazard ratio (HR) = 0.51, P = 0.035]. The actuarial survival rate after total resection was 1 yr (89%), 2 yr (83%), 5 yr (76%), 10 yr (51%) versus any less-than-total resection (near total + subtotal) at 1 yr (96%), 2 yr (89%), 5 yr (49%), and 10 yr (29%). Histology was also significant (HR = 3.30, P = 0.015). The multivariate analysis did not reveal significant differences with respect to either patient age at the time of surgery (HR = 0.99, P = 0.61) or adjuvant radiotherapy (HR = 0.74, P = 0.59). In conclusion, these results suggest that the extent of resection and histology are important survival factors and that radiation therapy is of questionable value. The authors acknowledge the inherent limitations of this study’s retrospective nature and long duration, including evolving imaging capabilities (CT versus MRI), different surgeons, different radiation techniques, and selection biases.


Jason Fangusaro, Ignacio Gonzales, Gordon McComb, and Jonathan Finlay; Children’s Hospital Los Angeles, Los Angeles, CA, USA

CNS papillary glioneuronal tumors are unique extraventricular neurocytic tumors that feature pseudopapillary structures in the presence of both glial and neuronal elements. Approximately 20 cases have been described in the literature. These patients are typically treated with surgery alone. To our knowledge, there have been no reports of tumor recurrence. We describe 2 pediatric cases of papillary glioneuronal tumors that progressed after initial surgical resection. The first patient, a 14-year-old female, underwent partial resection of a left frontal lobe lesion revealing a papillary glioneuronal tumor. Postoperatively, the patient was observed with surveillance imaging. Four years later, she re-presented with new onset headaches, and an MRI scan revealed tumor progression. She received one cycle of oral temozolomide, and then she was lost to follow-up. She returned approximately 1 year later with progressive disease. She received local irradiation and achieved a complete radiographic response. The second patient, an 8-year-old female, underwent a complete surgical resection of a left paraventricular mass also diagnosed as a papillary glioneuronal tumor. She presented emergently 3 months after diagnosis with emesis and weakness. A CT scan revealed a hematoma that was surgically removed. Biopsies were taken from within the resection cavity and were positive for recurrent tumor. Six weeks after surgery, the patient is being observed closely with surveillance imaging. These 2 cases show that papillary glioneuronal tumors can behave more aggressively than described within the literature. We hypothesize that the high MIB index may be more predictive of the more aggressive lesions. These tumors should be observed very closely with surveillance imaging postoperatively. Our experience with the first patient also demonstrates that in progressive disease, radiation therapy may be a useful alternative treatment strategy.


W.A. Hall,1 N.D. Doolittle,2 L. Muldoon,2 D. Fortin,4 E.A. Neuwelt2, 3; 1Department of Neurosurgery, University of Minnesota Medical School, Minneapolis, MN; 2Departments of Neurology and 3Neurosurgery, Oregon Health & Science University, Portland, OR; 4Department of Neurosurgery and Neuro-Oncology, Sherbrooke University, Sherbrooke, Quebec, Canada

The prognosis for patients with diffuse pontine gliomas (DPG) remains poor. New treatments are needed for this disease. From 1984 to 1998, 8 patients (4 men, 4 women), median age 11 years, with DPG were treated with monthly osmotic blood-brain barrier disruption (BBBD) chemotherapy using intra-arterial carboplatin or methotrexate and intravenous cytoxan and etoposide. Patients presented for a median duration of 6 weeks with increased intracranial pressure, long tract signs, diplopia, ataxia, and nausea/vomiting. DPG was demonstrated on MRI scan in 7 patients and on CT scan in 1 patient. Two patients underwent tumor biopsy: one had an astrocytoma and the other had an anaplastic astrocytoma. The median number of chemotherapy cycles that were administered by BBBD was 10 (mean, 10). One patient who started on carboplatin was converted to methotrexate, and 5 started on the methotrexate protocol were converted to carboplatin. MRI demonstrated partial responses in 2 patients, stable disease in 5 patients, and disease progression in 1 patient. The median time to tumor progression was 15 months (range, < 1 month–40 months). The median survival from the time of diagnosis was 27 months (range, 7months–80 months). The median survival time from the first BBBD or intra-arterial treatment was 16.5 months (range, 5 months–69 months). One patient was lost to follow-up, date of death unknown. Although the sample size is small, the time to disease progression and survival times are longer than those previously reported in other DPG series. In addition, the ability to demonstrate stable disease or partial responses in DPG on MRI argues for the therapeutic benefit of BBBD chemotherapy.


K. Karakoula,1 B. Suarez-Merino,1 S. Ward,1 K. Phipps,2 W. Harkness,2 R. Hayward,2 D. Thompson,2 D.G.T. Thomas,3 and T.J. Warr1; 1Neuro-Oncology Group, Department of Molecular Neuroscience and 3Division of Neurosurgery, Institute of Neurology, London, UK; 2Department of Neurosurgery, Great Ormond Street Hospital, London, UK

Ependymomas are glial cell-derived tumors characterized by varying degrees of chromosomal abnormalities and variability in clinical behavior. Ependymomas are associated with a poor overall survival rate of only 50% at 5 years in children, with local relapse being the major source of therapeutic failure. At present, the molecular genetic alterations underlying the pathogenesis of pediatric ependymomas are poorly understood. A gain of chromosome 1q and a deletion of chromosome 22 have been reported as common chromosomal aberrations by cytogenetic and comparative genomic hybridization analyses. In our previous study of pediatric ependymomas using microarray expression analysis, we observed overexpression of 10 genes ([gt-or-equal, slanted]3–fold, P < 0.05) located at 1q21–q32 and underexpression of 17 genes ([gt-or-equal, slanted]2–fold, P < 0.002) mapping to 22q12.3–13.3. We have investigated amplification of these genes in a series of 57 pediatric ependymomas using real-time quantitative PCR analysis (Q-PCR) with TaqMan probes. Q-PCR analysis was performed using the ABI PRISM 7000 Sequence Detector System and reactions were carried out in triplicate following the delta-delta Ct method to determine gene copy number changes in tumor samples relative to normal blood controls. The results showed that 58.5% (24/41) of the samples had additional copies of at least 1 gene on chromosome 1q, of which the calcium binding protein calgizzarin, the adaptor protein SCH1, and the human JTB and TPR genes had the highest incidences of gain. Furthermore, a gain of JTB was observed more frequently in recurrent ependymomas compared with primary tumors. The overexpression of all 4 of these genes has been previously demonstrated in other human cancers. On chromosome 22, 79% (45/57) of the cases had a loss of 1 or more genes. Four genes, RAC2, C22orf2, MKL1, and EP300, were deleted in 47%, 41%, 37%, and 32%, respectively, of samples and 5 samples (9%) had homozygous deletions of at least 1 gene. This study provides further evidence that genes on chromosome 1q and 22 are critically involved in the development of pediatric ependymoma.


B.K. Kleinschmidt-DeMasters, Mark A. Lovell, Andrew M. Donson, Steven O. Addo-Yabo, Kevin O. Lillehei, and Nicholas K. Foreman; University of Colorado Health Science Center and The Children’s Hospital, Denver, CO, USA

Many sarcomas are too rare to accumulate case series. One such sarcoma is intracranial malignant ectomesenchymoma (EM), a pediatric tumor thought to arise from neural crest cells with the potential to give rise to mesodermal and neuroectodermal tissues. The dominant elements in ectomesenchymoma (also called gangliorhabdomyosarcoma) are mature ganglion cells and rhabdomyosarcoma (RMS). We sought to investigate a case of EM by molecular, cytogenetic, and gene array analyses and compare results with conventional RMS and with malignant peripheral nerve sheath tumor (MPNST), another sarcoma type with potential for divergent differentiation, especially skeletal muscle elements in individuals with neurofibromatosis type I. Real-time quantitative PCR testing for the classic gene rearrangements seen in RMS [t(2;13)-PAX3/FKHR] and Ewing’s sarcoma ([t(11;22) & t(21;22)–EWS/FLI-1 & EWS/ERG) was used for characterization of the EM, with gene array analyses on all tumor types, using an unbiased comparison of the entire gene-expression profile. A standard cytogenetic analysis was also undertaken. Gene rearrangement studies were negative in EM. Surprisingly, gene array analyses showed tight clustering of the EM with the MPNST but showed divergence from the RMS as well as divergence from gene-expression profiles of other types of pediatric brain tumors (N = 52). Ectomesenchymoma and MPNST were hyperdiploid, with complex karyotypes, but without diagnostic translocations. Ectomesenchymoma karyotype was 84–87,XXX,-X,-1,der(2)t(1;2) (q12;q14.1),-4,-5,-5,der(5)t(5;?;5)(p15;?;q13) x2,-9,-9,del(11)(q22)x2,-17,-19,-21,der(21)t(17;21)(q21;q22),-22,-22,+r,+mar1,+mar2,+mar3[cp10]. MPNST karyotype was 53–71,XXX,-1,add(1)(p36.3),der(1)t(1;4)(p36.3;q 13),+2,add(4)(q21)x2,add(6)(q16), add(7)(q36),+8,der(9)add(9)(p12),add(9)(q34),+10,add(11)(p11.2),-12,+add(13)(q34), +14, add(15)(p11.2), dup(15)(q21q23),+dup(15),-16,add(16)(p13.3)x2,-17,-18,add(18)(p11.2),+add(18),-20,-20,add(20)(p11.2), -22,+r,+1-3mar[cp18]/123–130,idemx2[cp2]. Despite the presence of malignant skeletal muscle differentiation in the EM, gene array testing showed no overlap with RMS but rather with MPNST. This finding suggests a common stem cell origin or embryonic gene recapitulation for these tumors and provides novel insight into their underlying biological characteristics.


Joseph L. Lasky III,1 Theodore Moore,1 and Linda Liau2; Departments of 1Pediatric Hematology/Oncology and 2Neurosurgery, UCLA, Los Angeles, CA, USA

High-grade malignant gliomas are relatively rare in children, but as in adults, they carry a dismal prognosis even after standard therapy. A further understanding of the molecular biology and genetics of these tumors will hopefully result in more successful therapies. Here, we report a case of a 15-year-old male with a progressive history of lower extremity weakness. A laminectomy and a biopsy were performed, revealing the histologic diagnosis of a primary spinal glioblastoma arising at T11 to L1 levels. The tumor was GFAP-, Vimentin-, EGFR-, and nuclear P53-positive. Because of nonspecific CD99 staining in some areas, a FISH assay was done using a EWSR1 breakapart rearrangement probe (VYSIS; Des Plaines, IL) on a paraffin-embedded section revealing 3 intact 22q12 region signals and 1 split EWSR1 signal, suggesting a loss of the 5′ EWSR1 flanking region in 72.5% (290/400) of cells. Just prior to therapy, a repeat MRI scan of the spine revealed leptomeningeal spread to T3/T4. Treatment was initiated with radiation (whole spine to 36 Gy and tumor boost to 56 Gy) and concurrent temozolomide therapy (90 mg/m2/day). Although we planned to start at a higher dose of temozolomide, the patient then presented with seizures and hydrocephalus, and an MRI revealed extensive intracranial ventricular and subependymal disease. Whole brain radiation therapy (to 43 Gy) was then initiated with concurrent temozolomide. After radiation therapy was completed, daily temozolomide therapy was continued (90 mg/m2/day). Unfortunately, the disease progressed both intracranially and in the spine. Treatment was initiated with rapamycin and erlotinib; however, the disease continued to progress and the patient died about 8 months after diagnosis. Cytogenetic abnormalities in glioblastoma are many and varied. Abnormalities of chromosome 22q in glioblastoma are well described and predominantly involve losses. Candidate genes in this region that may play some role in gliomagenesis include, for example, NF2 or TIMP-3. The EWS gene product is able to bind RNA and has been shown to be involved with splicing and transcription complexes. Fusion proteins with EWS are found in a variety of solid tumors, most notably the Ewing’s family of sarcomas (EWS-FLI1). Here we describe the first reported case of a pediatric glioblastoma with abnormalities at this locus. We also review the role for chromosome 22 abnormalities and other cytogenetic features in glioblastoma pathogenesis and discuss the possible biological implications of EWS and other chromosome 22 abnormalities in this disease.


N. Potter,1 R. Poh,1 S. Ward,1 K. Phipps,2 R. Hayward,2 W. Harkness,2 D. Thompson,2 D.G.T. Thomas,3 J. Rees,1 J. Darling,4 and T. Warr1; 1Neuro-Oncology Group, Department of Molecular Neuroscience, 3Division of Neurosurgery, Institute of Neurology, London, UK; 2Department of Neurosurgery, Great Ormond Street Hospital, London, UK; 4Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton, Wolverhampton, UK

Brain tumors are the most frequently found solid tumor in children, 40% of which are astrocytomas. These are graded according to the WHO classification into the more common low-grade (I and II) and high-grade (III and IV) tumors. Little is known about the genetic basis underlying the development of pediatric astrocytomas. In this study, we have studied the correlation between abnormal gene expression in pediatric astrocytoma with genomic copy number changes. We used the Affymetrix HGU133A array to identify differentially expressed genes in a group of pediatric astrocytoma short-term cell cultures comprising 9 grade I, 11 grade II and 12 grade IV tumors. Data analysis was carried out using Genespring version 6.0 software. In addition, we used the Spectral Chip 2600 to generate array-comparative genomic hybridization (aCGH) profiles of each short-term cell culture. Chromosome regions of gain and loss were then compared with differential gene expression using Formatter software. Hierarchical clustering of the short-term cultures according to expression profile similarity showed that the tumors clustered into 3 clear groups that were independent of grade. Two groups were predominantly low-grade tumors, comprised of a mixture of grade I and II tumors with 3 grade IV tumors, and the third group contained predominantly high-grade tumors with 2 low-grade tumors. Genes involved in the phosphatidylinositol signaling system, the cell cycle pathway, and the regulation of the actin cytoskeleton, were significantly differentially expressed between the 3 groups. Differential disruption of these cell pathways may be associated with subtypes of pediatric astrocytoma. Most tumors in the third group (including the low-grade tumors) showed copy number changes that can be correlated with changes in gene expression. In specific tumors, the downregulation of TSB1 (thrombospondin-1) correlated with loss at 15q15. This gene has previously been found to be downregulated in astrocytoma and is involved in cell adhesion. This finding suggests that gene expression in a subset of pediatric astrocytomas is influenced by gene dosage.


Qin Shu,1 Kwong-Kwok Wong,2 Adekunle Adesina,3 Bobbie Antalffy,3 Jack Su,2 Lazlo Perlaky,2 Susan Blaney,2 Ching C. Lau,2 and Xiao-Nan Li1; 1Laboratory of Molecular Neuro-Oncology, 2Texas Children’s Cancer Center, 3Department of Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA

Animal models play crucial roles in both biological and preclinical studies of human cancers. For medulloblastoma, which is the most common malignant brain tumor that occurs in children, there is only limited availability of tumor models that reliably recapitulate the biology of this highly malignant neoplasm. To create mouse models that would faithfully mimic histopathological, immunophenotypical, and genetic characteristics of human medulloblastomas, we injected a fresh surgical specimen from an anaplastic medulloblastoma into the cerebrum or cerebellum of SCID mice. The developed xenografts have since been serially passaged in mouse brains for 8 generations while maintaining a constant tumorigenicity rate (100%) and reproducible animal survival times. Detailed characterization of the xenograft tumors revealed that they shared the same histological and immunohistochemical features as the original tumor. Using 10K SNP array, we further demonstrated that xenograft tumors (passage 1 and 5) retained the genetic profiles resembling those found in the primary tumor. To ensure a stable and timely supply of animal models, we optimized the protocol for long-term storage of xenograft tumor cells in liquid nitrogen, and we confirmed that these xenograft cells not only preserved the tumorigenicity (100%) but also produced tumors that kept the histological features of the original tumor. To reproduce the leptomeningeal spread that is often responsible for tumor recurrence in patients, we injected tumor cells through the cerebrum into subarachnoidal space and showed that the created seeding nodules were capable of invading neighboring normal brain tissues to develop into large tumors. In summary, we have developed a novel orthotopic xenograft medulloblastoma model that accurately replicates the histopathological, genetic, and behavioral features of anaplastic medulloblastomas.


Courtney Smith,1,2 Alison Vollmer,2 Bhargavi Rajan,2 Brian Rood,2 Tobey MacDonald,2 and Stanislav Vukmanovic2; 1Institute for Biomedical Sciences, George Washington University, Washington DC, USA; 2Children’s Research Institute, Children’s National Medical Center, Washington, DC, USA

Although the survival rate for medulloblastoma patients over the past decade has improved, it has come at the expense of quality of life. This indicates the need for an alternative method of treatment other than the conventional chemotherapy and radiation therapy. The use of immune-based treatments is a realistic alternative, but it is impeded by the lack in knowledge of whether and what kind of immune response to medulloblastoma may exist and be boosted. The goal of our research is to detect the signs and characteristics, if any, of the immune response to medulloblastoma. Because MHC class I expression is required for T cell recognition of a foreign or malignant cell and the loss of MHC class I expression is required for NK cell recognition of the same malignant cell, the level of expression of this surface molecule is indicative of the type of immune response that can occur. We therefore analyzed the expression of MHC class I on both medulloblastoma patients and cell lines. MHC class I expression in DAOY and D283 cell lines was detected using real-time PCR analysis and flow cytometry. We analyzed 18 patients for MHC class I expression using either immunohistochemistry or real-time PCR analysis. Both the patient group and the D283 medulloblastoma cell line show consistent negative expression of MHC class I. We then measured the degree of immune response to the tumor in the patient samples using immunohistochemistry for the common leukocyte antigen, CD45. Results showed varying degrees of leukocyte infiltration, warranting further analysis into the type of immune cells that were recruited. We are presently using immunohistochemistry to analyze a tissue microarray containing over 100 patients to increase our sample size. Ultimately, this insight into the immune response will allow for an informed decision about what type of immune-based therapy may be most beneficial for patients with medulloblastoma.


D. Stearns,1,2 A. Chaudhry,1 and C.G. Eberhart1; 1Department of Neuropathology, Johns Hopkins Medical Center, Baltimore, MD, USA; 2Department of Pediatrics, Drexel University School of Medicine/St. Christopher’s Hospital for Children, Philadelphia, PA, USA

Overexpression of the c-myc (MYC) oncogene has been associated with poor prognosis in medulloblastoma. Recently, we have shown that MYC can induce anaplasia in the established medulloblastoma cell lines DAOY and UW228. Using DAOY cell lines engineered to stably overexpress MYC in tumor xenografts, we have analyzed the changes in gene expression induced by MYC using the Affymetrix U133A array platform and the Genespring GX expression data analysis software. Nearly 1,500 probesets were differentially expressed in high MYC tumors more than twice as much as compared with low MYC tumors (524 up, 969 down). Using the NetAffx Gene Ontology Mining Tool, we analyzed these data sets and found that a remarkable number of extracellular matrix genes (ECM) were downregulated in the high MYC tumors (116/408, 28.4% total probesets in the GO 5578 classification). This included a large number of collagens (types I, III, IV, V, VI, IX, XI, and XIV) and collagen-associated genes. We compared this list of downregulated ECM genes to published data sets defining genes associated with survival, treatment failure, and the desmoplastic phenotype (Pomeroy et al., Nature, 2001). Desmoplasia in medulloblastoma can be detected as dense reticulin staining of collagen and other ECM components in internodular regions, and it has been associated with improved prognosis in some studies. Because MYC is associated with poor clinical outcomes in medulloblastoma, we hypothesized that the same genes upregulated in desmoplastic tumors might be downregulated by MYC. Of the 70 classifiers elevated in desmoplastic tumors, 13 were downregulated by MYC in DAOY, 7 of which were ECM genes. Consistent with this was a marked decrease in reticulin staining in the flank xenograft tumors derived from DAOY-myc lines. We examined expression of the COL1A2 gene using quantitative real-time PCR analysis and found a 45-fold reduction of expression in vivo and a 6-fold reduction in vitro. Furthermore, COL1A2 mRNA expression levels were extremely low in 2 other medulloblastoma cell lines known to have high levels of MYC (D425, D283) and were reduced 3-fold in vitro in UW228 cell lines stably transfected with MYC, indicating that this finding was not restricted to the DAOY line. COL1A2 mRNA levels were also high in primary desmoplastic nodular medulloblastomas. The origin of the impressive ECM production in desmoplastic medulloblastoma (tumor vs stroma) has not been characterized previously. Because the techniques we used detect only human transcripts, and high levels were detected in vitro as well as in vivo, our data would suggest a tumor cell origin versus a reactive phenomenon for at least a portion of the ECM material. In summary, our data support the concept that the ECM in medulloblastoma can originate from primary malignant cells and that MYC can modulate its production in some cell lines.


Hernan Vasquez,1 Wei Zheng,2 Vidya Gopalakrishnan,1 and Johannes Wolff1; 1Department of Pediatrics, The University of Texas M.D. Anderson Cancer Center, Houston TX, USA; 2Purdue University, School of Health Sciences, Neurotoxicology Division, IN, USA

Choroid plexus tumors arise from the epithelial cells that produce cereberospinal fluid in the brain and occur more frequently in children under the age of 1 year. The current treatments include radiation and chemotherapy; however, radiation causes long-term problems in the developing brain. Therefore, we have looked at using novel combinations of chemotherapeutic agents as treatments. Previous reports have shown that valproic acid (VPA), a commonly used anti-epileptic drug and histone deacetylase inhibitor, improves the efficacy of radiation therapy. In this study, we examined whether VPA has a similar effect on methotrexate (MTX). To evaluate the effect of VPA and MTX on transformed choroid plexus cells in culture, we carried out MTT assays with various concentrations of MTX and 1.5 mM of VPA. Methotrexate combined with VPA showed an unexpected cell survival curve. Valproic acid was able to protect cells from dying during a portion of the MTX concentration curve. Cell survival was 29% at 0.2 μM MTX alone and with VPA increased to 76%. This observation was more noticeable within 24 to 72 hours of recovery time after treatment. To test whether this VPA effect can be duplicated on other drug treatments, we selected a number of additional chemotherapeutic agents. When any of the following drugs, such as azacytidine, temozolomide, doxorubicin, or etopo-side were used, the toxic effect on choroid plexus increased when the drug was combined with VPA. In conclusion, VPA protects against methrotexate. If antiepileptic treatment is necessary under methotrexate treatment, an antiepileptic drug different from VPA should be chosen.


Andrew W. Walter and Jeff Campbell; Departments of Pediatric Hematology/Oncology and Neurosurgery, Jefferson Medical College and A. I. duPont Hospital for Children, Wilmington, DE, USA

Two children with soft-tissue masses of the scalp presented for treatment. Both were well, had no neurological signs or symptoms, and were thought to have scalp lesions. Both patients had locally destructive fibroblastic tumors with infiltration. Case 1: A 2-year-old male presented with an 8-month history of a firm mass in the left frontal region. On exam, he had a 2 × 2 cm firm, bony mass of the scalp. The surrounding bone was scalloped and everted at the edges of the mass. Plain films revealed a lytic lesion of the skull. Brain scans revealed erosion and fragmentation of the left frontal bone. There was a moderately large epidural soft tissue collection within the calvarium with compression of the adjacent brain parenchyma. An en bloc resection was performed. The bone margins were free of tumor but the tumor penetrated through the skull and invaded the dura. The pathologic diagnosis was cranial fasciitis. One year postoperatively, the patient underwent cranioplasty with a titanium malleable 1.3 mesh. No other treatments were given and the patient has remained free of disease since surgery. Case 2: A 6-year-old male presented with a 2-month history of a firm, bony, painless mass of the right temporal region. He was otherwise well. His exam was otherwise unremarkable. At surgery, the soft tissue component was resected without difficulty. The bone was eroded but unlike the previous case, the dura was not invaded. The child had a gross total resection and has done well since surgery. The pathologic diagnosis was cranial fasciitis. No further therapy has been given and there has been no evidence of disease since surgery. Cranial fasciitis is an invasive fibroblastic tumor of the scalp of young children. It belongs to a larger group of myofibroblastic diseases of childhood, which are slow growing, fibrous lesions. It is most commonly seen in children < 3 years of age and occurs predominantly in males. It usually erodes the underlying bone but typically does not breach the inner table of the skull. In some cases, including case 1 above, the dura can be involved thereby complicating resection. Recurrence rates are < 10% even if negative margins are not achieved.


Andrew W. Walter and Jeff Campbell; Jefferson Medical College, Departments of Pediatric Hematology/Oncology and Pediatric Neurosurgery, A. I. duPont Hospital for Children, Wilmington, DE, USA

A 4-year-old girl presented with sudden onset of nausea and vomiting with seizures. The results of her neurologic exam after resolution of the seizures were normal. An MRI of the brain revealed a subtle, nonenhancing, infiltrative lesion at the tip of the right temporal lobe, bright on FLAIR with decreased diffusion. Magnetic resonance spectroscopy showed increased lactate. At surgery, the tumor was noted to be rubbery but suckable using the Cavitron Ultrasonic Surgical Aspirator (Cooper Medical, Stamford, CT). A gross total resection was performed. The pathologic diagnosis was monomorphous angiocentric glioma. The MIB-1 index was 7%. An MRI scan of the spine and a spinal tap for cytology were both negative for tumor. The patient has remained free of disease since surgery. Monomorphous angiocentric glioma is a newly described neoplasm with features of both ependymoma and astrocytoma. Patients typically present in childhood with seizures. The lesions are distinctly infiltrative but their behavior is low grade with no further therapy typically required after complete resection. Only 8 cases have been described in the literature (Wang et al., J Neuropath Exp Neurol, 2005), and this entity has not yet been recognized in the WHO classification. Although these are low-grade lesions, 1 patient from the cited series (the only patient who did not present in childhood) died from recurrent disease.


Howard L. Weiner,1,4 Maria Pompeiano,1 Avinash Mohan,1 Richard Bakst,1 Leandro Piedimonte, Daniel Stephen,3 James S. Babb, David Zagzag,2 Daniel H. Turnbull,5 and Alexandra L. Joyner3; Skirball Institute of Biomolecular Medicine, Departments of 1Neurosurgery, 2Pathology, 3Cell Biology, 4Pediatrics, and 5Radiology, New York University School of Medicine, New York, NY, USA

Activation of the Sonic hedgehog (Shh) pathway in the developing cerebellum contributes to medulloblastoma formation. We previously showed that these tumors can be induced efficiently by infecting the embryonic mouse cerebellum with an Shh-expressing retrovirus. Here, we test whether the proliferating cells of the early postnatal cerebellum remain responsive to Shh induction of medulloblastomas. In contrast to the 76% of wild-type mice injected as embryos with Shh retrovirus that developed tumors, only 17% of those infected postnatally developed medulloblastomas, with many mice developing ectopic islands of internal granule cell layer-like cells. We also tested whether reduction of negative regulators of the Shh pathway, Ptc and Gli3, can increase tumorigenicity postnatally. In contrast to a lack of tumor formation in controls, 62% of Ptc+/ mice and 67% of Gli3+/ mice developed medulloblastomas. Our data indicate that mis-expression of Shh alone in the postnatal brain is not sufficient to induce tumors efficiently. However, by also removing 1 copy of Ptc or Gli3, medulloblastomas form at a significantly higher rate. This study represents the first evidence that Gli3 could normally play a role in the repression of medulloblastoma formation.


Jingying Xu, Ignacio Gonzalez-Gomez, Elizabeth Y. Melendez, Rex A. Moats, Anat Erdreich-Epstein, and C. Patrick Reynolds; Developmental Therapeutics Program, USC-UCLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children’s Hospital Los Angeles and Departments of Pediatrics and Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

The paucity of cell culture models for childhood brain tumors has prompted us to establish such cell lines for use in biological and preclinical developmental therapeutic studies. Brain tumors from pediatric patients were obtained at surgery or post-mortem and minced and cultured in lscoves Dulbecco’s Modified Eagle’s Medium (20% fetal bovine serum, insulin, selenium, and transferrin). Here we describe 3 new cell lines: CHLA-200 (anaplasic astrocytoma, at post mortem), CHLA-259 (medulloblastoma, at diagnosis) and CHLA-266 (atypical teratoid rhabdoid tumor with supratentorial metastasis, at diagnosis). All 3 cell lines had unique DNA fingerprints according to the ABI Profiler. All cell lines were adherent on tissue culture plastic but differed in morphology. The doubling times were 76 hours for CHLA-259, 45 hours for CHLA-266, and 55 hours for CHLA-200 compared with 29 hours for the commonly used medulloblastoma cell line DAOY. In contrast to fibroblasts, these 4 cell lines expressed telomerase reverse transcriptase (hTERT) and telomerase RNA template (hTR; quantitative RT-PCR, TaqMan, Applied Biosystems, Foster City, CA). CHLA-266, CHLA-259, and CHLA-200 strongly expressed the early neuronal marker PGP9.5 but did not express tyrosine hydroxylase. DAOY, CHLA-259, and CHLA-266 expressed MYCN mRNA, whereas CHLA-266 and CHLA-200 expressed cmyc. The cell lines were injected into the brains of nude mice, but only DAOY and CHLA-266 have proven tumorigenic to date. All 3 new lines showed striking multidrug resistance when compared with DAOY cells using a fluorescein diacetate and digital image microscopy cytotoxicity assay (DIMSCAN, Los Angeles, CA). The table shows concentrations of drugs that are lethal for 90% of cells (LC90). CHLA-271 (anaplastic ependenoma, at diagnosis) and CHLA-280 (same patient as CHLA-271, after chemotherapy) are new cell lines (characterization in progress). These new brain tumor cell lines will provide useful models for preclinical testing of novel drugs for possible activity against multidrug-resistant pediatric brain tumors. This study was supported by the Devin Hock Memorial Fund of the Michael Hoefflin Foundation and the Pediatric Brain Tumor Foundation.

LC90 (μg/ml)EtoposideTopotecanMelphalanVincristine
CHLA-266> 10> 1000> 209.0
CHLA-2001.7> 1000> 20> 1000
LC90 (μg/ml)CyclophosphamideCisplatinFenretinide
CHLA-2592.01.2> 12


Zeng-Jie Yang,1 Tracy-Ann Read,1 Tammy Ellis,2 Robert Machold,3 Gord Fishell,3 David H. Rowitch,4 Brandon J. Wainwright,2 and Robert J. Wechsler-Reya1; 1 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA; 2 Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia; 3 Developmental Genetics Program, Skirball Institute, NYU School of Medicine, New York, NY, USA; 4 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

Medulloblastoma is the most common malignant brain tumor in children. Although it is believed to originate from precursors in the cerebellum, the identity of these precursors remains unknown. Some studies have suggested that these tumors arise from lineage-restricted granule neuron precursors (GNPs), but more recent studies indicate that medulloblastomas express neural stem cell markers and can differentiate into both neurons and glia, raising the possibility that they may originate from multipotent neural stem cells (NSCs). The fact that stem cells from many tissues are sensitive to oncogenic transformation makes NSCs important candidates for the cell of origin of medulloblastoma as well. Here we examine the cell of origin for medulloblastomas resulting from mutations in the Sonic hedgehog-Patched signaling pathway. Using conditional knockout mice, we demonstrate that deletion of the patched gene in GNPs results in prolonged proliferation and expansion of the external germinal layer (EGL) where granule cells develop. Although this hyperplasia persists for a few weeks, the majority of patched-deficient GNPs eventually stop dividing and differentiate into neurons, implying that loss of the patched gene is not sufficient for transformation. However, in each animal, a cohort of cells continues to proliferate, and by 3 months of age, all conditional patched knockout mice develop medulloblastoma. Deletion of the patched gene in postnatal GNPs (using an inducible Cre recombinase) also results in medulloblastoma, indicating that even relatively mature progenitors are susceptible to transformation. Finally, tumor cells from conditional patched knockout mice can give rise to tumors after transplantation into SCID hosts, indicating that they are capable of long-term self-renewal in vivo. These studies indicate that the capacity for self-renewal and transformation is not restricted to stem cells but can also be induced in lineage-restricted progenitors. Identifying the cell of origin for medulloblastoma may provide important insight into the mechanisms of tumorigenesis and may allow us to develop more effective methods for eradicating this disease.



Samuel H. Cheshier,1 Laurie Ailles,2 Victor Tse,1 Stephen Skirboll,1 Stephen Huhn,1 and Irving Weissman2,3; Departments of 1Neurosurgery, 2Pathology, and 3The Institute of Medicine, Stanford University School of Medicine, Stanford, CA, USA

The study of primary human brain tumors in vivo has proven difficult, due to a lack of animal models allowing for reliable growth of freshly isolated tumor cells. Most systems currently employed require the injection of cells into the brain or flank of an immunocompromised adult mouse or rat. Tumor formation generally requires large numbers of input cells (on the order of millions) and the implantation of highly malignant clones of tumor cells obtained only after long-term culture or both. These difficulties may be due to a lack of developmental niches for tumors in adult mice, as well as a lack of complete immunosuppression. To overcome these obstacles, we injected freshly isolated glioblastoma multiforme (GBM) and medulloblastoma (MB) cells from patients into 1- to 3-day old RAG 2/common cytokine receptor gamma chain double knockout (RAG-γ dko) mice. RAG-γ mice have been definitively shown to completely lack T, B, or NK cells. The pups were injected with 200,000 fresh tumor cells or 2,000 FACS sorted cells into the right hemisphere and vermis for GBM and MB cells, respectively. The mice were analyzed at 3 months post injection and demonstrated tumors, which could be seen grossly with MRI imaging and staining with human specific antibody SC121. The resultant tumors were identical in histology to human tumors in vivo with GBM displaying morphologic features such as subependymal and subpial mounds and diffuse white matter infiltration, whereas MB growth followed CSF pathways. We plan to use this ability to reliably grow tumors with low cell input from freshly isolated GBM to help isolate tumor stem cells.


Samuel H. Cheshier,1 Laurie Ailles,2 Dominique M.O. Higgins,1 Michael Lim,1 M. Yashar S. Kalani,4 Simon Bababeygy,1 and Irving L. Weissman1,2,3; Departments of 1Neurosurgery, 2Pathology, 3The Institute of Medicine, and 4Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA

Cancer stem cell (CSC) isolation from glioblastoma multiforme (GBM) requires proteolytic-enzymatic digestion of tumors. Despite the use of careful techniques, most samples contain significant amounts of contaminating debris, and an analysis with sensitive instrumentation like Fluorescence Activated Cell Sorting (FACS) is fraught with difficulties such as frequent clogs and poor post-sort purity. Our analysis has revealed that most viable cells within tumor samples express CD90, a marker commonly used to isolate hematopoietic stem cells. We hypothesized that CD90 enrichment of tumor cells could enrich for cellular elements without segregating out the CSCs. Single-cell suspensions were made from 3 patient-derived GBM samples by enzymatic digestion. Cells were then stained with PE-conjugated anti-CD90 antibody, followed by anti-PE MACS magnetic microbeads. The cells were then enriched through magnetic separation columns and subjected to a limit-dilution tumor-sphere forming assay in 96 well plates. The cells were visualized with light microscopy and the degree of enrichment was assessed by using FACS. FACS demonstrated that more than 80% of tumors had CD90 cell expression. However, before enrichment, the samples were full of contaminating debris and cell fragments. After CD90 magnetic bead enrichment, the CD90-positive fraction contained significantly less debris and more single cells. In the limit-dilution experiments, tumor-sphere formation occurred only in the CD90-positive fraction. In one tumor, CD90-negative cells developed into fibroblast-like adherent cells but did not form any spheres, even in wells containing 25,000 cells. Enrichment of tumor samples using anti-CD90 magnetic column may be an effective method to purify the cellular elements from samples containing significant amounts of debris and cell fragments. This method may make cell counting more reliable as well as increase the accuracy of FACS sorting and analysis. It may also be utilized to mark human-derived tumor cells in xenotransplant models of tumor growth in rodents as human-specific monoclonal antibodies to CD90 exist. We plan to continue this analysis in a larger number of tumors to determine the general applicability of this technique.


G.N. Fuller,1 H. Wang,1 H. Wang,2 L.J. Corley,1 W. Zang,1 R.A. LeCouteur,3 A.W. Bollen,4 P.J. Dickinson,5 and R.J. Higgins3; Departments of 1Pathology and 2GI Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; Departments of 3Pathology, Microbiology and Immunology, and 5Surgery and Radiological Sciences, School of Veterinary Medicine, University of California Davis, CA, USA; 4Department of Pathology, School of Medicine, University of California San Francisco, San Francisco, CA, USA

We classified and graded 87 spontaneous canine diffuse gliomas using the current WHO 2000 criteria for human brain tumors as follows: grade II astrocytoma, N = 16; grade III astrocytoma, N = 11; grade IV astrocytoma, N = 16; grade II oligodendroglioma, N = 3; grade III oligodendroglioma, N = 15. A tissue microarray was constructed using duplicate cores from all tumors and immunocytochemistry was used to evaluate glioma expression of IGFBP2, a molecule of recently demonstrated importance in human diffuse gliomas.

Tumor TypeNumber of casesIGFBP2IGFBP2 +IGFBP2 ++IGFBP2 +++

A, astrocytoma (WHO grade II); AA, anaplastic astrocytoma (WHO grade III); GM, glioblastoma (WHO grade IV); O, oligodendroglioma (WHO grade II); AO, anaplastic oligodendroglioma (WHO grade III)

Overexpression of IGFBP2 correlated directly with tumor grade for both astrocytic and oligodendroglial tumors. These findings support the potential of the canine glioma model for directed pathway targeting studies.


M.A. Hunt,1 L.L. Muldoon,1 D. Palmieri,1 P.S. Steeg,1 and E.A. Neuwelt1,2; 1Women’s Cancers Section, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA; 2Oregon Health & Sciences University, Portland, OR, USA; and 3Portland VA Medical Center, Portland, OR, USA

Brain metastasis develops in 20% to 40% of patients with breast cancer. HER-2 positive status has been implicated in poorer prognosis from breast cancer and has also been associated with an increasing incidence of brain metastasis. The human MDA- MB-231BR brain seeking breast carcinoma cell line was used in this experiment. Two subclones of this cell line were previously created by transfection to express either low or high levels of HER-2 expression. Eight nude rats were sedated and underwent stereotactic implantation of either the native 231BR cell line (N = 2), the high HER-2– expressing subclone (N = 4), or the low HER-2–expressing cells (N = 2). Approximately 3.1 × 105 cells were implanted into the basal ganglia. The rats were followed for survival and were given routine examinations. When the rats became moribund, or demonstrated a significant neurologic deficit or severe weight loss, they were sacrificed and their brains were collected for pathologic examination. Tumor volumes were assessed after immuno-histochemical staining with CAM5.2. The overall survival rate was assessed for statistical significance using the Kruskal-Wallis test. The mean survival times for the native cell line, the low HER-2–expressing cell line, and the high HER-2–expressing cell line were 44 ± 0 days, 36 ± 0 days, and 19 ± 1.7 days, respectively. The differences between all groups were statistically significant (P = 0.046). Pathologic examination revealed large, well-circumscribed tumors in the parental 231BR cell line. The high HER-2 tumors were smaller but had more infiltrative margins. Our preliminary data suggest that HER-2 status correlates with decreased survival time in a rat model of brain metastasis from breast cancer. Additional clones will be examined. This model correlates with known clinical behavior of breast cancer and will allow evaluation of novel treatments for brain metastasis from breast cancer including combinations of trastuzumab with chemotherapy, and trastuzumab chemoimmunoconjugates.


D. Kamnasaran,1 C. Hawkins,1 and A. Guha1,2; 1Arthur & Sonia Labatts Brain Tumor Centre, Hospital for Sick Children Research Institute, Toronto; 2Division of Neurosurgery, Western Hospital, University of Toronto, Canada

Astrocytomas comprise over 60% of all primary human adult central nervous system neoplasms, and glioblastoma multiforme (GBM) is the most common and the most malignant. Our group previously created a Genetically Engineered Murine (GEM) model of astrocytoma by expressing oncogenic V12Ha-Ras under the control of the GFAP promoter. One strain, RasB8, is born normally and goes on to germline transmission, ultimately dying from the development of low- (80%) and high-grade (20%) astrocytomas by 3 to 4 months of age, which are pathologically similar to human secondary GBMs. We utilized this GEM as a “gene-discovery” reagent to screen for genetic modifiers that accelerate transformation of susceptible astrocytes. Retroviral gene trapping of non-transformed newborn (P0) V12Ha-Ras astrocytes trapped the transcription factor GATA6, resulting in transformation. GATA6 is a valid tumor suppressor gene (TSG) in murine and human astrocytomas based on (1) GATA6 expression was present in normal murine (P0) and in the transgenic RasB8 (P0) astrocytes, but it was absent in high-grade astrocytomas that developed by 3 months in the RasB8 GEM plus in RasB8–3mth established primary astrocyte cultures; (2) GATA6 expression was present in normal human astrocytes (immortalized with hTERT) but absent in several established human GBM lines and GBM explant xenografts; (3) GATA6 expression was absent in > 85% of high-grade and 11% of low-grade astrocytomas, and it was also absent in high-grade astrocytomas of a patient with secondary GBMs but not in low-grade astrocytomas of the same patient, consistent with a role in tumor progression rather than initiation; (4) among GBM operative specimens had loss of function mutations associated with loss of heterozygosity were identified; (5) stable reduction of GATA6 expression by siRNA in immortalized human astrocytes transfected with oncogenic V12Ha-Ras resulted in the formation of malignant intracranial astrocytomas in Nod-Scid mice; and (6) doxycycline induced over-expression of GATA6 in established human GBM glioma cell lines that lack endogenous GATA6 protein expression inhibited in-vitro and in-vivo transformed phenotype. Using GEMs and gene-trapping we have identified a novel TSG in human astrocytomas. GATA6 is a member of the mammalian GATA family of transcription factors and thus far is reported as a TSG in ovarian and adrenocortical tumors. Our data is the first to demonstrate GATA6 as being a TSG in the malignant progression of human astrocytomas, and a modifier with oncogenic p21–V12Ha-Ras. These results also demonstrate how GEMs, based on known human disease associated genetic alterations, can be utilized as a readily available “gene-discovery” reagent to identify novel genetic alterations in human diseases. Current study involves the creation of a conditional Gata6 GEM which will be bred to other glioma GEMs, and the search for transcriptional targets of GATA6 in astrocytes.


D. Kamnasaran,1 C. Hawkins,1 and A. Guha1,2; 1Arthur & Sonia Labatts Brain Tumor Centre, Hospital for Sick Children Research Institute, Toronto; 2Division of Neurosurgery, and Western Hospital, University of Toronto, Canada

The in vitro pluripotency of ES cells is determined by the ability to differentiate into derivatives of the 3 germ layers. We designate “synthetic astrocytes” as being differentiated from ES cells under artificial conditions. These cellular reagents are powerful alternative sources in the modeling of astrocytomas and have numerous advantages when compared with conventional methods. An optimal protocol was undertaken to utilize murine ES cells harboring wildtype (wt) and p53 (+/−) genetic backgrounds to produce “synthetic astrocytes” in the development of an alternative assay for the in vitro and in vivo transformation studies of candidate genes for astrocytomas. Astrocytic differentiation of the ES cells, with morphologies of Type-1 astrocytes, were GFAP-(astrocyte marker) positive in over 90% of cells. These astrocytes also expressed numerous markers (such as glutamate synthetase, vimentin, GABA-A,B) involved in astrocytic function. cDNA microarray analyses on Affymetrix arrays identified the transcriptosomes of synthetic astrocytes with wt or p53 (+/−) genetic backgrounds as being very similar (Pearson’s correlation coefficient (r) = 0.89, P < 0.001), with differentially expressed genes (P < 0.001) most likely representing p53 response genes. Transcriptosome fingerprinting was undertaken to compare the synthetic astrocytes with astrocytes isolated from different parts of the murine CNS and at different developmental stages. Both wt and p53 (+/−) synthetic astrocytes were most similar (r = 0.8, P < 0.001) to cortical astrocytes from newborn pups and the adult mice. Genetic modification of these astrocytes with p21–V12HaRas, plus other candidates for astrocytomas (MDM2, AKT and EGFR), demonstrated p53 null provided a proliferation and anchorage independent growth advantage over the wt, inclusive with additional genetic modifications with other astrocytoma candidates (MDM2, AKT and EGFR). Both synthetic astrocytes with genetic modifications of astroctyoma candidates developed high-grade intracranial tumors within a 6 month period, unlike the wildtype and p53 (+/−) synthetic astrocytes alone. We have developed an effective alternative assay for the analysis of the in vitro and in vivo transformation potentials of candidate genes for astrocytomas, using synthetic astrocytes. Our findings overwhelmingly demonstrate that these synthetic astrocytes are very similar to in vivo astrocytes, and when genetically modified, they can recapitulate the phenotype from in-vivo mouse models. Current efforts are focused on the use of random mutagenesis screens by gene trapping to identify novel genes that can modify the transformation potential of “synthetic astrocytes,” with subsequent verification for alterations in human glioma specimens.


Leon Kapp, Rodney Collins, Anjan Misra, Ezra Mirvish, Tarik Tihan,and Burt G. Feuerstein; Brain Tumor Research Center, and Departments of Neurological Surgery, Laboratory Medicine, and Neuropathology, University of California, San Francisco, CA, USA

We obtained a cell line derived from a GBM biopsy, serially passaged in mice, and designated, from David James at the Mayo Clinic. We grew the cells under standard tissue culture conditions (MEM supplemented with 10% FBS and non-essential amino acids) and after 6 months in culture, the line went through crisis and adapted to growth in culture. Its growth characteristics and behavior were similar to other standard GBM-derived cell lines such as U251, although cells have a longer generation time. Several weekly changes of medium over the confluent cells without passaging allowed foci to develop. Approximately 900 foci developed per T75 flask, suggesting that the cells generating these foci comprise about .05% to 0.1 % of the cell population. With repeated feeding, many of the foci developed into compact dense spheres. These spheres exhibited a very regular shape, and their size never exceeded 500–1000 cells, regardless of the time in culture. Additional studies suggested that: spheres removed from flasks and placed into new flasks have a 100% plating efficiency; cells quickly move out from the base of the spheres onto the new flask’s surface; spheres are tightly attached to the surrounding cell monolayer; and spheres maintain their shape even as cells grow out from their point of attachment to the flask surface. FGF and EGF had no effect on sphere growth and behavior. In efforts to grow isolated pure sphere populations, we found that parent cells plated into flasks coated with 1% agar do not attach, and spheres develop in suspension. We observed few single cells or groups of cells in the medium after 1–2 weeks under these conditions; thus, these conditions appear to favor the growth of spheres over the growth of parental single cells in culture. We are currently characterizing the growth and behavior of these spheres and evaluating them for various markers of neuroepithelial cells. This study is supported by NS42927, CA85799, CA97874, and the Barrow Neurologic Institute.


L.A. Lampson and C.A. Tripp; Department of Neurosurgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

The ventricle is an important site for brain tumors in several different contexts; some contexts are especially important in children. Choroid plexus carcinoma (CPC) is a rare but devastating tumor in children, for which new therapies are needed. Among more common pediatric tumors, new approaches are needed to attack residual tumor in the ventricle after conventional therapy. In adults and children, seeding of the cerebrospinal fluid (CSF) can be a route of tumor spread. Although these different tumors have different origins, the site of growth, rather than the origin, may be the more important feature for many studies. We describe a rat model that will be useful for testing new therapies against tumor in the ventricle, either in the choroid plexus or in the CSF. To aid detection of the smallest tumor foci, subclones of a rat cell line (MATB III) had been transduced with enzymes, b-galactosidase (b-gal) (MATB/lacZ) or placental alkaline phosphatase (PLAP) (MATB/ap), that are constitutively expressed and readily detected in tissue sections by simple histochemical stains. To deliver tumor while avoiding a needle wound to the brain, tumor cells were injected into the left common carotid artery. We have found that, in our rat model, this method delivers tumor efficiently to the ventricle on the injection side. To reveal tumor, sections through the brain were stained histochemically for b-gal (X-gal substrate) or PLAP, as appropriate. In different rats, graded doses of tumor cells were injected, and rats were sacrificed at different times. The results of this study show that (1) in tissue sections after histochemical stain, foci of MATB/lacZ tumor are bright blue and foci of MATB/ap are red. As an alternative, all tumor cells can be revealed by staining with monoclonal antibody for keratin (ABC method, DAB substrate); (2) tumor first appears in the choroid plexus of the lateral ventricle on the injection side; (3) the tumor foci grow larger with time. Growing tumor is seen within the choroid plexus and is also free within the ventricle. Expanding masses of tumor, and also free tumor cells in the CSF, are seen; (4) tumor masses are also seen in the third ventricle; (5) tumor masses are not seen in the lateral ventricle contralateral to the injection. Thus, for some studies, the contralateral ventricle will be a useful internal control; (6) as the tumor grows, increasing hydrocephalus is seen, as in human patients; and (7) these results confirm and extend earlier work in our laboratory, including work by Y. Kondo, R. Kapoor, and J. Durham. Although tumor in the ventricle can have different origins, the site, rather than the origin, may be the more important feature for some kinds of studies. The rat model described is useful for testing new therapies against tumor in the ventricle, whether in the choroid plexus or in the CSF. Positive features include the fact that it is a syngeneic, immunocompetent model, and tumor is introduced without a wound to the brain or skull. The amount of tumor can be controlled by varying the cell dose and time. The lateral ventricle opposite to the injection serves as an internal positive control. These features make the model appropriate for our own long-term goal of learning how best to exploit blood-borne responding cells (lymphocytes, monocytes) to attack tumor at different sites. The model will be useful for many other kinds of studies as well.


Kim A. Lopez,1 Allen E. Waziri,1 Marcela C. Assanah,1 Peter D. Canoll,2 and Jeffrey N. Bruce1; Gabriele Bartoli Brain Tumor Laboratory, Departments of 1Neurological Surgery and 2Pathology, Columbia University Medical Center, New York, NY, USA

Previous studies have shown the efficacy of convection-enhanced delivery (CED) of chemotherapeutic agents on tumors generated by transplanted glioma cell lines. However, these tumors do not resemble human gliomas histologically or physiologically nor have they proven to be reliable predictors of clinical outcome. We have developed a new model that uses a PDGF-expressing retrovirus to infect glial progenitors in the white matter of the adult rodent brain. Highly invasive tumors with marked vascular proliferation and pseudo-pallisading necrosis form rapidly and consistently, making this an ideal model to test new therapies. In this study, we treated the PDGF gliomas using CED of topotecan. We injected a PDGF-IRES-GFP retrovirus into the rostral subcortical white matter of adult rats. At 7 dpi, the rats were treated with continuous CED of either topotecan (N = 11) or PBS (N = 12) for 7 days (Alzet 2 mL osmotic mini-pump, 10 μL/hr). The rats were either sacrificed at 14 dpi (end of treatment), or allowed to survive until they showed signs of tumor burden. To document presence of tumor before treatment, a separate cohort of 6 rats were sacrificed at 7dpi. All of the rats sacrificed at 7 dpi had histopathologically evident tumors. By 17 dpi, 100% of the rats that received PBS died of large, infiltrative tumors. Immunohistochemistry showed the untreated tumors were composed of a mixture of infected (GFP+) and uninfected (GFP−) cells, as previously described (Assanah et al., 2006). Animals given topotecan and sacrificed on the last day of treatment (14 dpi) showed a robust response with massive tumor necrosis on histologic analysis. However, immunohistochemistry revealed rare GFP+ cells in both the ipsilateral and contralateral hemispheres. The topotecan-treated animals showed a significant survival advantage vs. controls (median = 58 days vs. 16 days; P = 0.0018; log-rank test), but tumors eventually recurred in all rats. The recurrent tumors histologically resembled the PBS-treated tumors but had infiltrated to a much greater extent into the contralateral hemisphere. Immunohistochemistry for GFP revealed marked regional heterogeneity within the recurrent tumors, with some areas containing a high percentage of GFP+ cells and other areas containing very few GFP+ cells. Although CED of topotecan was initially effective against our PDGF glioma model, a small subset of tumor cells survived treatment and eventually gave rise to recurrent tumors. The regional heterogeneity of GFP expression pattern within the recurrent tumors points to an oligo-clonal selection and re-expansion of these surviving cells. This is reminiscent of how human glioblastomas respond to therapy, making this an interesting model with which to study the mechanisms by which glioma cells escape therapy and give rise to recurrent tumors.


N.O. Schmidt, H. Günther, H. Meissner, M. Westphal, and K. Lamszus; Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum HH-Eppendorf, Hamburg, Germany

Previous reports have demonstrated that a variety of human tumor entities contain a small subpopulation of cells that display a stem-cell-like phenotype and seem to be responsible for driving tumor formation. Here, we describe the establishment and characterization of stable tumor stem cell lines isolated from human glioblastomas. Human glioblastoma sphere cultures were established from fresh surgical specimens and propagated using serum-free culture conditions with a variety of growth factors known to support the survival of stem cells. Long term cultures of tumor-derived neurospheres were extensively characterized using immunohistochemistry, real-time quantitative PCR analysis, FACS analysis and differentiation- and self-renewal assays. Tumorigenicity and in vivo growth characteristics were analyzed after intracerebral implantation in nude mice. We were able to establish 8 stable long-term cultures of tumor-stem cells derived from human glioblastomas. Almost 95% of these cells displayed simultaneous expression of the astroglial marker GFAP and various stem cell markers, such as nestin, CD133, sox2, oct4 and bmi1 in the absence of mature neuronal and oligodendroglial markers, confirming their relatively uncommitted stage. However, under differentiating culture conditions, neuronal and oligodendroglial differentiation was induced. In contrast to conventional intracerebral glioblastoma xenograft models, the intracerebral injection of tumor-stem cells resulted in highly invasive gliomas closely reflecting the growth pattern of tumors seen in patients. The time for tumor formation was cell line-dependent and reproducibly ranged between 3 and 8 months after cell injection. Human glioblastoma-derived tumor stem cells are a subpopulation of tumor cells with distinct biological features much more reflecting the overall heterogenic behavior of glioblastomas. These cells can be used to develop specifically tailored therapies and to test anti-invasive strategies in a highly invasive preclinical murine model.


C.A. Tripp, M.L. Hurley, and L.A. Lampson; Department of Neurosurgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

Brain metastases are as important clinically as primary tumors, but basic research has not focused on them. Our goal has been to develop a rat model that allows visualization and analysis of the earliest, smallest micro-metastases — too small to image or access by current standard methods. To aid detection of micrometastases, we transduced rat tumor cell lines with enzymatic markers that are constitutively expressed and readily detected in tissue sections. To favor delivery to the brain, we injected the cells into the left common carotid artery. In our first studies, we found that tumors grew well in the ventricles, but metastases were not seen consistently in the brain proper. Here we report conditions that result in reproducible, visualizable blood-borne metastases in the brain proper. In a previous study by Dr. Y. Kondo, MATB III, a highly metastatic variant of the well-characterized rat mammary carcinoma cell line 13762, had been made to constitutively express placental alkaline phosphatase (MATB/ap). In a simplification of our first procedure, 4 million MATB/ap cells were injected into the left common carotid artery without ligating side vessels. At different times (usually 1 week later), rats were euthanized by perfusion with fixative (2% paraformaldehyde), and their brains were cryoprotected. Sections through each brain were stained for tumor using a simple histochemical stain for the enzymatic marker. As a control for the injection method, 2 rats received 10u diameter polystyrene microspheres (beads) instead of tumor; the brains were fresh frozen immediately; and for analysis, sections were stained with H&E. The polystyrene beads defined the pattern that could be expected from cell-sized objects injected by our methods when cell division, cell death, and cell-cell interactions were not factors. The beads were widely distributed on the injection side. They appeared in all compartments (brain proper, meninges, choroid plexus) and anatomical regions, in general proportion to the area. The beads appeared singly, in pairs, or in small groups or clusters. Sometimes beads could be seen lined up within a vessel. After histochemical staining, tumor foci appeared red and could be detected at the lowest power (1× objective). A monoclonal antibody to keratin could also be used to stain tumors (ABC method, DAB substrate). At day 7, tumor foci were seen in the brain proper. They were sporadically distributed and visible in all anatomical regions; however, they were most common in the cortex of the hindbrain. In 2 rats analyzed later (days 9 or 10), fewer tumor foci were seen in the brain proper, but they were larger. This syngeneic, immunocompetent rat model consistently shows blood-borne metastases in the brain proper. The distribution of beads and tumor are consistent with the blood flow in the rat. The sporadic distribution of tumor in the brain proper parallels what is seen in human patients, in whom the location of blood-borne metastases cannot be predicted beforehand. The methods here differ in several ways from those we used previously. We do not yet know which variables are most important in determining whether tumors grow mainly in the ventricle (as previously reported) or reproducibly metastasize to the brain proper (as reported here). These studies are in progress.


Shuzhen Wang,1 Sonya Popoff,1 Martha Roemer,1 Joy Gumin,2 Helen Jiang,1 Fred Lang,2 Juan Fueyo,1 Candelaria Gomez-Manzano,1 Kenneth Aldape,3 Irene Newsham,1 Oliver Bogler,1,2 and Howard Colman1; Departments of 1Neuro-Oncology, 2Neurosurgery, and 3Neuropathology, Brain Tumor Center, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The cancer stem cell hypothesis proposes that a small subset of cells with properties reminiscent of normal stem cells (e.g., unlimited self-renewal, multi-lineage differentiation) may initiate and maintain cellularly heterogeneous tumors like glioblastoma multiforme (GBM). We aimed to determine if human GBM-derived tumor stem cells provide a more biologically relevant model than existing adherent cell lines for studies of tumor phenotype and treatment resistance. Utilizing serum-free culture conditions to expand tumor stem cells as “neurospheres” in the presence of FGF and EGF, we derived 6 GBM stem cell lines from either initial or recurrent tumors. LOH analysis for loci on chromosomes 3, 10, 11, 12, 13, 17, 19, and 22 demonstrated that, with rare exceptions, the allelotype of the GBM stem cell lines maintained in vitro for periods from 3 to 18 months matched the parent tumor at all loci. In particular, allelotyping for the loci on chromosome 10 in the region of the PTEN gene showed a 100% match in the 5 lines tested. An analysis of MGMT promoter methylation in the GBM stem cell lines and the parent tumors also demonstrated a 100% concordance (2 methylated, 4 unmethylated). The genotypic stability relative to the parent tumor of long-term GBM stem cell cultures contrasts with previous studies demonstrating a lack of genotypic similarity between the parent tumors and adherent cultures of primary GBM cells grown in serum containing media. The percentage of CD133 expressing cells (by FACS analysis) and expression of PTEN (by western blot) were also determined for each GBM stem cell line. The sensitivity of GBM stem cells to temozolomide treatment in vitro (40 to 1000μM) was determined both by counting of neurospheres and by MTS assay. Two lines were sensitive to temozolomide (LD50 < 40μM), 3 lines were resistant (LD50 > 200μM) and one line showed intermediate sensitivity. While no variables were significantly associated with temozolomide response in vitro in this data set, likely related to the small number of samples, the percentage of CD133 positive cells in each GBM stem cell line showed the strongest correlation to treatment resistance (r = 0.67, P = 0.22). Together, these data indicate that GBM-derived tumor stem cells represent a more biologically faithful phenocopy of the human tumor compared with existing glioma cell lines and primary adherent cultures, in terms of genetic and epigenetic stability. Isolation and expansion of additional GBM stem cell lines and further analyses are in progress to determine the extent to which these cells can be utilized as a patient-specific model for identification of molecular features associated with response to particular therapies.


Ming Zhang,1 Chandra Das,2 Hernan Vasquez,2 Dolly Aguilera,2 Vidya Gopalakrishnan,2 Peter Zage,2 and Johannes Wolff2; Departments of 1Biostatistics and Applied Mathematics and 2Pediatrics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

The kinetics of tumor cell repopulation after treatment may depend on the previous treatment and may offer a new endpoint for clinical trials. The mathematical description for a dying cell population is y(t) = a*exp(b*t), and it is y(t) = c*exp(d*t) for a growing population where all the parameters a, b, c, d are positive. Since both populations are involved in tumor repopulation after treatment, y(t) = a*exp(b*t) + c*exp(d*t) was tested in this project. Two different cell lines were tested with 2 different drugs. Human malignant glioma cells (U87 MG) cultured in DMEM plus 10% FCS were treated with etoposide at varying concentrations and allowed to regrow after treatment. Human medulloblastoma cells (DAOY) cultured in DMEM plus 10% fetal bovine serum, penicillin, and streptomycin were treated with MS275 at varying concentrations and allowed to regrow after treatment. Repeated MTT assays were performed to read the cell numbers. The data from these experiments validated the two-term exponential model described above. Cell number versus time data were fitted mathematically to the model. Parameters b and d were determined first and then used to obtain parameters a and c. The value of c decreased as the drug dose increased and their relationship was fitted to a sigmoid formula. The concentration independence of b and d indicated that the rates of cell number (in logarithm) change during cell proliferation and cell death were independent of drug treatment. This finding suggests constant times for cell cycle and apoptosis. The concentration dependence of c indicated that the recovery time, until tumor cells start regrowing after treatment, was dependent on the drug. This finding suggests repair mechanisms take more time after heavier damage. The two-term exponential model predicts tumor repopulation after treatment in the in vitro system. We will test other preclinical systems and clinical data to determine the relationship between tumor repopulation and drug dose in vivo.



T.S. Armstrong,1,2 T. Mendoza,2 I. Gring,2 C. Coco,2 M.Z. Cohen,1,2 L. Eriksen,1,2 M.A. Hsu,3 M.R. Gilbert,2 and C. Cleeland2; 1The University of Texas School of Nursing at Houston, Houston, TX, USA; 2The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Pfizer Pharmaceuticals, USA

The occurrence of symptoms has been shown to predict treatment course and survival in a number of patients with solid tumors. Patients with primary brain tumors (PBT) have unique neurologic symptoms. Currently, there are no instruments that can measure both neurologic and cancer-related symptoms. This study included 201 patients diagnosed with PBT. Data collection tools included a patient-completed demographic data sheet, an investigator-completed clinician checklist, and the core M.D. Anderson Symptom Inventory to which 18 neurologic symptoms were added (MDASI-BT). This study evaluated the reliability and validity of the MDASI-BT in patients with PBT. The mean symptom severity of items and cluster analysis were used to reduce the total number of items in the MDASI-BT to 22. Regression analysis showed that more than half (56%) of the variability in symptom severity was explained by the nine remaining brain tumor items. Factor analysis was then performed to determine the underlying constructs being evaluated by the remaining items. The 22–item MDASI-BT measures 6 underlying constructs: affective, cognitive, and focal neurologic deficit; constitutional and generalized symptoms; and a gastrointestinal-related factor. The internal consistency (reliability) of the sets of items comprising the six factors and the interference scale was .87, .82, .72, .81, .69, .67 and .91, respectively. Test-retest reliability was good in a subset of 19 patients that completed the instrument at two points in time. The MDASI-BT was sensitive to disease severity, according to KPS based on mean symptom severity (1.7 versus 3.8; P < .001) and mean symptom interference (2.2 versus 6.1; P < .001). The 22–item MDASI-BT demonstrated validity and reliability in patients with PBT. This instrument can be used to describe symptom occurrence throughout the disease trajectory and to evaluate interventions designed for symptom management.


Terri S. Armstrong,1,2 Tito Mendoza,1 Marlene Z. Cohen,1,2 Marilyn Morrissey,1 Mark R. Gilbert,1 and Charles Cleeland1; 1The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 2The University of Texas School of Nursing at Houston, Houston, TX, USA

The occurrence of multiple symptoms has been shown to impact outcomes, such as performance status and disease progression, of patients with solid tumors. The occurrence of multiple symptoms in patients with primary brain tumors (PBT) has not been routinely measured. The MDASI-BT was recently validated as a tool to measure symptoms in patients with PBT. The aim of this study was to report the association between symptom severity and interference with performance status and disease progression in patients with PBT, as measured by the MDASI-BT. Included in this study were 201 adult patients with PBT who presented to the clinic or inpatient unit at M.D. Anderson Cancer Center. Patients completed the MDASI-BT prior to learning their MRI results. Physician-documented performance status and radiology reports of MRI performed at the time of the visit were compared with symptom occurrence. Functional status was measured by KPS. Patients were categorized as having a ‘good’ (90 or 100) or ‘poor’ (80 or below) performance status. For disease status, we categorized patients as having newly diagnosed disease, stable disease, or recurrence, as documented by MRI. Of the 201 patients, 143 (71%) had good performance status and 58 (29%) had poor. As predicted, there was a significant difference in mean symptom severity (1.3 versus 2.79; P < .001) and mean symptom interference (1.75 versus 4.9; P < .001) between patients with good and poor performance status. Patients with recurrent tumors (N = 113) had significantly worse mean brain-tumor symptom severity scores, overall symptom severity scores, and interference scores than did those with newly diagnosed (N = 30) or stable (N = 58) disease. The newly diagnosed and recurrent tumor groups did not differ in their mean severity scores on the core items. The differences seen at recurrence further support the sensitivity of this instrument to changes in patients’ disease status. The cluster analysis showed a distinct co-occurrence of symptoms. These symptom clusters, which were associated with recurrence, will be reported at the time of presentation. The 22-item MDASI-BT is highly predictive of performance status and disease progression in patients with PBT. Among the core MDASI-symptom, brain-module, and interference items, the brain-module items showed the strongest association.


T.S. Armstrong, K. Hess, C. Coco, A.X. Ames, R. Manning, and M.R. Gilbert; Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Although myelosuppression is a dose-limiting toxicity of most cytotoxic chemotherapies, a reported benefit of TMZ is that myelosuppression is relatively uncommon. Most studies of TMZ report a 5–8% overall incidence of grade 3–4 myelosuppression, with slightly higher incidence in older women. In our practice, we recognized a trend of a higher incidence of myelosuppression in women. We therefore analyzed the hematologic toxicity in 685 consecutive patients after their first course of treatment with standard dose schedule of TMZ. We identified 1,600 patients with primary brain tumors in the M.D. Anderson Cancer Center brain tumor database as having a primary malignant glioma that was treated with TMZ. Complete data were available on 685 patients and included histopathologic diagnosis; TMZ treatment dates; treatment dose; height; weight; ideal body weight; body surface area; smoking status; concomitant medications; prior chemotherapies received; and laboratory studies (pretreatment and within 5 weeks after TMZ), including creatinine, white blood cell count, absolute neutrophil count, and platelet count. Women were more likely to experience grade 3 or 4 myelosuppression then were men (P = 0.015). A marked difference in the incidence of clinically significant neutropenia (grade 3 or 4) was noted between male (6.25%) and female patients (12.36%). Logistic regression analysis showed that risk of leukopenia was higher in women who had received one or fewer prior chemotherapies (P = 0.0009), weighed less than 50 kg, or were on enzyme-inducing anticonvulsants, but there was no association with age. Conversely, the leukopenia risk in men increased with age, treatment with two or more prior chemotherapy regimens, and/or use of medications for gastroesophageal reflux disease. Women were at higher risk of myelosuppression than were men. Factors associated with increased risk were different between the sexes. These intriguing findings are being further evaluated to establish a classification system for risk of myelosuppression that will then be validated in a prospective trial.


Beverly Barkon; School of Education, Carlow University, Pittsburgh, PA, USA

This presentation will describe the learning profiles of several children who have survived pediatric brain tumors and the effects that the disease and its treatment have had on their ability to perform in school. The major focus will be on interpreting educational evaluations to support children with cancer as they return to the classroom in order to assist physicians in making appropriate educational recommendations to their patients. The development of effective treatments for childhood cancers has resulted in an “epidemic of survival” for these children, with survival rates approaching 75%–80%. Currently, there are approximately 10,000 adult survivors of childhood cancer, and this number is increasing by 500 each year. Brain tumors comprise a high percentage of pediatric cancers; approximately 2,000 of the 11,000 children diagnosed annually will have brain tumors. Because brain tumors directly involve the nervous system, the effects of these tumors and their treatment on school performance and learning are profound. As the potential for survival has increased, there has been an increased interest in quality-of-life issues. For children, success in school is a major determinant of quality of life. Systematic study of the increasingly large population of pediatric cancer survivors is creating a research base reflecting the typical learning issues that face these children and young adults. The common problems shared by survivors include fluctuating abilities, fatigue, short-term memory deficits, and reading comprehension difficulties. These common problems are overlaid on the child’s own inherent pattern of strengths and needs. Detailed, thorough, and carefully done educational evaluations done by people who have an understanding of individual learning profiles, the typical profile of children with cancer, and the classroom environment are critical to the successful reintegration of survivors. The needs of survivors are not short-term. This presentation will illustrate the value of comprehensive educational evaluations for pediatric cancer survivors. Case studies that highlight the commonality and variety of educational challenges will be presented. This presentation is designed to provide physicians with guidance in making education-related recommendations to patients.


Susan Bell, John McGregor, Robert Vanecko, Sharon McDowell; Ohio State University Medical Center, Columbus, OH, USA

Intracranial neoplasms can be a significant source of disability that often prevents patients from returning home after surgery. The objective of this study was to measure functional outcomes of patients with brain tumors following acute inpatient rehabilitation and to identify any differences between tumor type, functional outcome, and discharge disposition. A retrospective review was performed of patients admitted to an inpatient rehabilitation center following craniotomy between 2004 and 2006. Outcome measures were admission and discharge functional independence measure (FIM), FIM subsets (activities of daily living [ADL], mobility, and cognition), FIM change, length of stay, and discharge disposition. The 41 patients were divided into 3 groups based on tumor type: metastatic (MET; N = 8; mean age = 64 years; age range = 37–80 years), high-grade glioma (HGG; N = 15; mean age = 58 years; age range = 34–78 years), and low-grade glioma (LGG; N = 18; mean age = 52 years; age range = 18–87 years). Mean length of stay for acute care and rehabilitation was similar for all three groups (MET: acute 8 days, rehab 11 days; HGG: acute 8 days, rehab 11 days; LGG: acute 9 days, rehab 13 days). The majority of patients in all groups were discharged to home (MET: 100%; HGG: 67%; LGG: 94%). There was a statistically significant improvement in mean total FIM from admission to discharge for all tumor groups (admission scores = 75, 68, and 71; discharge scores = 103, 84, and 99; P < 0.01, 0.05, and 0.01 for MET, HGG, and LGG, respectively). There was significant improvement (P < 0.01) in all the FIM subcategories (ADL, mobility, and cognition) for the MET and LGG groups. Patients in the HGG group had significantly improved ADL (P < 0.05) and mobility (P < 0.01) scores. Patients in all tumor groups experienced an overall increase in mean FIM at the time of discharge, with no significant difference noted between tumor types. Overall, 86% (N = 35) of the patients were discharged to home. Rehabilitation services may offer a unique opportunity to influence functional outcome in these individuals and influence caregiver burden once discharged to home


Jaishri Blakeley,1 Tien Peng,2 Xiaobu Ye,2 and Stuart Grossman2; Departments of 1Neurology and 2Oncology, Johns Hopkins Hospital, Baltimore, MD, USA

Steroids are critical in the management of high-grade gliomas (HGG) because of their ability to control tumor-related edema. However, steroids are associated with multiple toxicities. The most common and debilitating of these in patients with HGG is steroid myopathy (SM), which manifests as progressive proximal muscle weakness leading to loss of ambulation and, in severe cases, respiratory compromise. In this report, we describe the pattern of steroid use in patients with newly diagnosed HGG and the development of SM. We reviewed the charts of all adults with HGG diagnosed and treated exclusively at Johns Hopkins Hospital for the first 8 months after diagnosis between June 2004 and May 2005. Patients who died in less than 8 months were excluded. Starting steroid dose, subsequent documented doses, initiation date, and dates of change were recorded. Patients with the clinical diagnosis of “steroid myopathy” documented in their medical records were defined as SM positive. Twenty-eight patients met all inclusion criteria. The mean age of patients was 57.2 ± 8.7 years; 19 (67.8%) of the patients were males. All patients (100%) received steroids. Dexamethasone was used exclusively. Ninety-three percent of patients received >16 mg/day, and 36% were on >16 mg/day for at least a month. The average daily dose was 15.3 ± 7.1 mg. The mean cumulative dose was 2.8 ± 1.8 g. The median peak dose was 40 mg/day (range, 16–120 mg/day). The median number of days on steroids from diagnosis was 224 (range, 5–224); 67.8% of patients were on continuous steroids for 8 months. Eleven (39%; 95% CI, 22–59%) of the 28 patients were defined as having SM. Seven (63.6%) of the 11 SM patients required wheelchairs, compared to 2 (11.8%) without SM (P = 0.004). SM patients were on steroids significantly longer (214 ± 32 days) than were patients without SM (155 ± 81 days; P = 0.012). No strong evidence supported differences between the groups with respect to total dose, average daily dose, peak dose, or days on high-dose steroids. Patients with HGG are routinely exposed to high doses of steroids for long periods of time. Only 32% of patients were weaned off steroids in the first 8 months of treatment. SM was noted in 39% of our patients, and most required wheelchairs. These results are likely to significantly underestimate the true prevalence of SM in this population, given the amount of steroids administered and the retrospective nature of this study. Our data confirm prior reports of an association between duration of steroid use and SM. These findings highlight the high exposure risk of patients with HGG and have inspired clinical protocols to test treatment interventions for SM.


M.J. Bonner, K.K. Hardy, and V.W. Willard; Tug McGraw Quality of Life/Supportive Care Research Center of the Preston Robert Tisch Brain Tumor Center at Duke University Medical Center, Durham, NC, USA

Fatigue may be the most unrecognized symptom in the pediatric cancer population, particularly for survivors who are followed less closely. Indeed, it is not part of routine assessments in follow-up visits for survivors. Fortunately, recent interest in fatigue in children has led to better operationalization of fatigue as experienced by pediatric cancer patients and the development of new measures to assess fatigue in this population. For example, focus groups have yielded important qualitative findings and have increased recognition of pediatric fatigue as a critical area for further investigation. Investigations must now focus on identifying underlying causes of fatigue using quantitative methods of study. To this end, the current study aims to describe the pervasiveness and severity of fatigue in adolescent survivors of pediatric cancer and its relation to social functioning. Adolescent survivors were selected for two reasons: (1) adolescents are better able to report on the subjective experience of fatigue than are younger children; and (2) excessive fatigue experienced during adolescence may interfere with the attainment of critical developmental milestones, such as autonomy, identity development, and achievement of long-term educational and occupational goals. Such interference may occur in the context of fatigued adolescents’ reduced ability to participate in age-appropriate social, educational, employment, and recreational activities. Study objectives will be assessed using a sample of 100 adolescents who are survivors of pediatric cancer (including brain tumors and other cancers), are 13–18 years old, and have been off treatment for [gt-or-equal, slanted] 1 year. Participants will be mailed a study description, consent form, packet of questionnaires, and postage-paid envelope in which to return the completed questionnaires. Questionnaires included the PedsQL, PedsQL Multidimensional Fatigue Scale, Physical Activity Questionnaire for Children, and Social Skills Rating System. Relevant medical data will be collected from the charts of those who return the questionnaires. The objective of the study will be assessed by running descriptive statistics of the outcome measures (fatigue, physical activity, quality of life, and social functioning) to assess pervasiveness of fatigue in adolescent cancer survivors. t- and χ2–tests will then be used to compare sample to normative means for each outcome to assess prevalence and severity. Finally, demographic (age, age at diagnosis, time off treatment, gender), disease (diagnosis) and treatment (radiation, chemotherapy) related factors will be assessed as predictors of the experience of fatigue. Before needed interventions can be developed to improve quality of life in cancer survivors, it is critical to understand the factors that contribute to fatigue in this growing population.


Bart D. Brigidi, Renee H. Raynor, Melanie Bonner, Holly Schweitzer, Karen Carter, Bebe Guill, and Henry S. Friedman; The Tug McGraw Center for Quality of Life/Supportive Care Research in The Preston Robert Tisch Brain Tumor Center at Duke University Medical Center, Durham, NC, USA

Chronic, intrusive thoughts are known determinants of clinical levels of anxiety and depression in cancer patients. As such, we aimed to develop a self-report measure to screen for adult brain tumor patients at risk for developing anxiety and/or depression by determining the factor structure underlying cognitions related to their experience of diagnosis, treatment, and prognosis. An initial pool of items was generated by surveying 10 medical oncology providers (neuro-oncologists, medical psychologists, research nurses, midlevel clinical providers, clinical social workers, and family support personnel) about topics that would be of worry or concern for patients with brain tumors. This process resulted in a pool of 50 items, which were subsequently rated by five different medical oncology experts according to the relevance of each item to brain tumor patients (three-point Likert-type scale rated as 0, “not relevant”; 1, “possibly relevant”; or 2, “highly relevant.” Thirty-five items were identified as being at least “possibly relevant” by at least four of the five raters. A beta-test version of the DCCI-BT was administered to 363 consecutive male and female adult primary brain tumor patients (18 to 77 years old) seeking treatment at a large tertiary medical center specializing in CNS tumors. After removal of cases with missing data and/or multivariate outliers, data from 327 patients were available for analysis. Exploratory factor analytic procedures (EFA), including unweighted least-squares and principal component analysis, were run with both oblique and orthogonal rotations and used to extract robust factors from the 35 items. From the EFA procedures, we identified eight factors that were similar across analyses; however, a three-factor solution appeared to account for most of the variance explained in each analysis. In confirmatory factor analysis (CFA), a three-factor solution was requested using principal factors extraction with varimax rotation, given that most of the correlations in the factor correlation matrices were around or above .32. With a cut point of .20 for inclusion of a variable in interpretation of a factor, 2 of the 35 variables did not load on any factor. Results from the CFA were interpreted as factor 1 (integrity of self; e.g., “I am afraid that I will decline physically and become incapable of taking care of myself”), factor 2 (responsibility; e.g., “I worry that I may be responsible for my illness in some way”), and factor 3 (hope; e.g., “I trust myself and my family to manage the future, no matter what happens”). These factors accounted for 12%, 9%, and 5% of the variance, respectively. Cronbach alphas ranged from .65 to .86, which are considered adequate for research purposes. Test-retest (mean interval of 7 days) data and comparison data between DCCI-BT subscale scores and scores on established measures of psychopathology (e.g., Beck Depression Inventory and Beck Anxiety Inventory) for construct validity purposes are currently being collected and will be presented. Preliminary analyses suggest that brain tumor patients’ intrusive thoughts related to diagnosis, treatment, and prognosis appear underlain by separate factors of loss of integrity of the self and self-blame/guilt for acquiring illness. Additionally, it appears that brain tumor patients vary on a dimension that is associated with positive thoughts related to hope, increased meaning in life, and realization of being well-cared for by a higher being and/or significant others (e.g., family members and medical staff). These data represent an initial step to using a reliable and valid instrument to screen for brain tumor patients who may be at risk for developing anxiety and/or depression, and, therefore, who would be good candidates for early psychological intervention.


Bart D. Brigidi, Renee H. Raynor, and Henry S. Friedman; The Tug McGraw Center for Quality of Life/Supportive Care Research in The Preston Robert Tisch Brain Tumor Center at Duke University Medical Center, Durham, NC, USA

Neurocognitive assessment of patients with CNS tumors is often confounded by the joint effects of tumor and treatment. Existing studies show that right-hemisphere location of the tumor is associated with increased severity of cognitive deficits, but there is no difference in severity of neurocognitive deficits between patients with glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA). In this study, we sought to better understand the effects of tumor location and diagnosis on major areas of cognition in a sample of patients with high-grade CNS tumors. Retrospective chart review was conducted for all adult primary brain tumor patients with diagnoses of either AA or GBM who sought treatment at a large tertiary medical center and who completed neuropsychological evaluation. Patients completed standardized measures of verbal attention (Verbal Series Attention Test), verbal memory (Hopkins Verbal Learning Test-Revised), executive functioning (Trail Making Test Part B), verbal fluency (Controlled Oral Word Association Test), visuospatial skills (Benton Facial Recognition Test), and psychomotor skills (Trail Making Test Part A). Additionally, to account for the influence of mood on cognition, patients were included in the analysis only if they completed a screening measure of clinical depression (Beck Depression Inventory-II). After removal of cases with missing data and multivariate outliers, 277 cases (103 AA and 174 GBM) were available for analysis. Sixty-two percent of subjects were male, 97% were Caucasian, and 87% were right-handed. Subjects had a mean of 15 years of education (SD = 2.6 years) and a mean age of 45.80 years (SD = 11.26 years). The study is a 2 × 2 between-subjects multivariate analysis of covariance using 10 neurocognitive dependent variables and two independent variables [tumor hemisphere (right, left) and diagnosis (AA, GBM)]. Depression total score, time since diagnosis, and previous radiotherapy (yes, no) were entered as covariates. Neurocognitive raw test scores were converted to z-scores or standard scores, which were used as a proxy for differences in age and education. Data are being compiled to include radiotherapy as a covariate for all cases. However, in preliminary analyses with all other variables entered into the multivariate model, with the use of Pillai’s criterion, the combined neurocognitive variables were significantly affected by hemisphere [F (10, 262) = 6.12; P < .0001] but not by diagnosis or interaction of hemisphere X diagnosis. Left-hemisphere location was associated with increased severity of deficits in verbal attention speed of processing (P = .05), immediate verbal recall (P = .0001), delayed verbal recall (P = .0001), delayed verbal recognition (P = .0001), and verbal fluency (P = .0001). Preliminary results do not support previous findings that right-hemisphere tumor location is associated with increased severity of neurocognitive deficits. This result may be related to language deficits, or reduced left-hemisphere verbal abilities, that may have negatively impacted performance in other cognitive domains. Results are consistent with previous reports and support the notion that high-grade primary brain tumors may be underlain by neurotoxic processes that result in similar neurocognitive presentations. These results must be interpreted cautiously until the covariate of previous radiotherapy has been added into the model.


Paul D. Brown,1 Ashley Jensen,1 Sara J. Felten,1 Karla V. Ballman,1 Paul L. Schaefer,2 Kurt A. Jaeckle,1 and Jan C. Buckner1; 1Mayo Clinic and Mayo Foundation, Rochester, MN, USA; 2Toledo Community Hospital Oncology Program CCOP, Toledo, OH, USA

There is growing recognition that the primary cause of cognitive deficits in adult patients with primary brain tumors is the tumor itself and, more significantly, tumor progression. To assess the cognitive performance of patients with high-grade gliomas, we analyzed cognitive performance data collected in a consecutive series of prospective clinical trials. We studied 1,244 patients with high-grade brain tumors entered in eight consecutive North Central Cancer Treatment Group trials employing radiation and nitrosourea-based chemotherapy. The Folstein mini-mental status exam (MMSE) scores and ECOG performance scores recorded at baseline, 6, 12, 18, and 24 months were analyzed to assess cognitive and physical function over time. Patients who did not demonstrate radiographic tumor progression within 60 days of the assessment time were considered nonprogressors at that evaluation. A loss of more than three points in the MMSE was considered clinically significant deterioration. The proportion of patients without tumor progression who experienced clinically significant cognitive deterioration from baseline was stable at 6, 12, 18, and 24 months following study entry (18%, 16%, 14%, and 13%, respectively). Patients who demonstrated a significant decrease in their MMSE scores were significantly older than those who did not at 6 months (P < 0.01). ECOG performance scores were negatively correlated with MMSE scores throughout the study; this was statistically significant at 6, 12, and 18 months (P < 0.01). In patients without radiographic evidence of progression, clinically significant deterioration in MMSE scores was a strong predictor of a more rapid time to tumor progression and death. At 6 months, time to death was 225 days for patients with cognitive decline and 343 days for patients with stable or improved MMSE scores (P < 0.01); at 18 months, time to death was 435 days for patients with cognitive decline and 874 days for patients with stable or improved MMSE scores (P = 0.01). The proportion of high-grade glioma patients with cognitive deterioration over time is stable and most consistent with the constant pressure of tumor progression over time. Although poorer performance status and older age may contribute to cognitive decline, the predominant cause of cognitive decline seems to be subclinical tumor progression that precedes radiographic changes.


Robert Cavaliere,1 Edward R. Laws Jr.,2 Fred Anderson,3 Elana Farace,4 and the GO project investigators; 1Ohio State University, Columbus, OH, USA; 2University of Virginia, Charlottesville, VA, USA; 3University of Massachusetts, Worcester, MA, USA; 4Pennsylvania State University, Hershey, PA, USA

Radiation therapy has historically been the standard of care for patients with high-grade gliomas (HGG). Advanced age, however, is a poor prognostic factor among patients with these tumors, and survival is limited regardless of treatment. Furthermore, the putative toxicity and side effects of treatment, to which older patients may be more sensitive, may outweigh the small survival advantage conferred by radiation therapy. We hypothesized that although patients receiving radiation may live longer, there is questionable benefit to their quality of life (QOL). The GO data, a prospectively collected dataset of patients with HGG, were analyzed. Older patients were defined as those aged 65 years or older at time of diagnosis. Only newly diagnosed patients were included (the GO dataset also included patients who had had a recurrence). Patients were stratified according to whether or not they had had radiation by 3–month post-op. Survival analyses were conducted using Cox proportional hazards modeling, with established predictors of baseline KPS and tumor grade adjusted as covariates. QOL outcomes, including the SF-36, Glioma Outcomes Questionnaire, and DSM-IV definition of depression, were assessed at the 3–month post-op follow-up. Seventy-six patients aged 65 years or older with newly diagnosed HGG were identified, of whom 74% received radiation within 3 months of surgery. A survival analysis of length of survival with and without radiation was highly significant, revealing that over all ages, radiation was beneficial for extending length of life (P = .002), although the absolute difference between treatment groups was surprisingly small (356 versus 374 days of survival). There were, however, no differences in length of survival between older patients who did and did not receive radiation (P = .51). There were also no significant differences on any measured QOL variable. A comparison of younger to older people, all of whom had received radiation, revealed only that the older group reported significantly more difficulty with verbal expression than did the younger patients (F = 5.25; P = .02) in a one-way ANOVA. There does not appear to be a QOL benefit for radiation therapy for patients over age 65 in these data. Not surprisingly, the older patients report more neurocognitive compromise than do the younger patients. There also does not appear to be a survival benefit of irradiation in patients over age 65. These results call into question the “palliative” benefit of radiation therapy for older people with HGG. Limitations of the study are that this is an observational study, and missing data may bias results. Additional prospective studies incorporating neurocognitive and QOL endpoints are warranted to explore the issue further.


Edward F. Chang, G. Evren Keles, Matthew Potts, Kathleen R. Lamborn, Susan M. Chang, Nicholas M. Barbaro, and Mitchel S. Berger; Department of Neurological Surgery, University of California, San Francisco, CA, USA

Seizures play an important role in clinical presentation and quality of life after surgical resection of low-grade gliomas (LGGs). Our aim was to identify factors that (1) predispose patients with LGGs to develop seizures, (2) influence preoperative seizure control, and (3) predict postoperative seizure control. A retrospective chart review of all adult patients that underwent initial surgery for hemispheric LGGs at UCSF between 1997 and 2003 was performed. Patient demographics, details of the clinical presentation of seizures, radiographic findings, histopathology, and surgical and adjuvant treatments were recorded. Seizure status at 6 and 12 months was evaluated using the Engel scoring system for seizure control. Of the 332 patients included for analysis, 82% (N = 269) presented with one or more seizures (generalized: 33%; complex partial: 16%; simple partial: 22%; combination: 39%). Cortical location and oligodendroglioma and oligoastrocytoma subtypes were statistically more likely to be associated with seizures than were deeper midline locations and astrocytoma, respectively (multivariate logistic regression analysis [MVA] P = 0.017 and 0.001, respectively). Of the seizure patients, 49% (N = 132) had uncontrolled seizures before surgery. Uncontrolled seizures were more likely to be simple partial, to involve the temporal lobe, and to have longer duration from seizure onset (MVA P = 0.003, 0.04, and 0.01, respectively). For the cohort of patients that presented with seizures, 67% were seizure free, 17% had rare seizures, 7% showed meaningful improvement, and 8% showed no improvement or worsened at 6–12 months after surgery. Poor seizure control was more common in patients with longer seizure history (MVA P = 0.001) and in those that presented with simple partial seizures (MVA P = 0.004). Of the treatment-related variables, patients who underwent gross total resection were far more likely to have seizure control than subtotal resection or biopsy alone (OR = 16, 95% CI = 2.2–124; P < 0.006). Seizure recurrence was associated with tumor progression using both time-dependent Cox proportional hazard and Landmark models (P = 0.001). KPS was correlated with seizure control (P = 0.01). The majority of patients with LGG present with seizures, about half of which are uncontrolled before surgery. Postoperatively, over 90% are seizure free, have rare seizures, or have had meaningful improvement. A short history of seizures and gross total resection appear to confer a favorable prognosis for seizure control and overall quality of life.


Arnaldo N. Da Silva and David Schiff; University of Virginia, Neurology Department, Division of Neuro-Oncology, Charlottesville, VA, USA

Patients with brain tumors are susceptible to bone disease related to hemiplegia-associated osteopenia and the use of antiepileptic drugs, glucocorticoids, anticoagulants, chemotherapy, and radiation therapy. The authors review the pathophysiology of all these factors and their impact on bone integrity. Steps that can be taken to minimize or eliminate bone morbidity, including measurement of bone mineral density at treatment onset, adequate calcium intake, vitamin D and K supplementation, adequate sunlight exposure, weight-bearing exercises, fall prevention, avoidance of antiepileptic drugs linked to osteopenia, and judicious use and choice of glucocorticoids and anticoagulants are suggested. Medical treatment of osteoporosis related to cancer treatment in brain tumor patients using bisphosphonates, teriparatide, and calcitonin is discussed, as well as kyphoplasty for symptomatic vertebral compression fractures.


Arnaldo N. Da Silva and David Schiff; University of Virginia, Neurology Department, Division of Neuro-Oncology, Charlottesville, VA, USA

Glucocorticoids are the main drug used to control vasogenic edema in brain tumor patients. Rapid glucocorticoid taper may not only decompensate intracranial pressure but may also unmask secondary adrenal insufficiency, some symptoms of which can be mistakenly attributable to increased intracranial pressure or side effects of chemotherapy and radiation therapy. Five neuro-oncology patients with clinical and laboratory evidence of secondary adrenal insufficiency were identified in the University of Virginia Neuro-Oncology Database from February 2002 to January 2006. The most frequent symptom found among our patients was fatigue. High-dose cosyntropin test was used in all cases. All patients showed rapid improvement of their symptoms following glucocorticoid replacement. Although rare (1% in our experience), a healthy index of suspicion for this complication and use of pharmacologic testing will help avert misattribution of secondary adrenal insufficiency symptoms and deleterious consequences.


Mercedes D. Dickinson1 and C. Meyers2; 1University of Houston, Houston, TX, USA; 2The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Treatment for primary brain tumors has evolved over the last 20 years to include advances in surgery, radiation treatment, bioimmunotherapy, and chemotherapy. These therapies have been designed to attack the tumor itself, with lesser degrees of concern regarding how they affect surrounding brain parenchyma. However, there has been a shift to examine the cognitive effects of these treatments as well. This study explored the cognitive effects of the drug marimastat in the treatment of individuals with primary brain tumors. Marimastat is a matrix metalloproteinase inhibitor that has been implicated in slowing tumor angiogenesis and metastasis by destroying the extracellular matrix of tumor cells required for invasion, intravasation, extravasation, and migration. Given its proposed mechanism of action on tissue surrounding tumor cells, there exists the possibility of neurotoxic side effects when targeting brain tumors. The participants were 29 patients with primary brain tumors, all of whom received conventional first-line therapy (including surgical resection and one course of radiotherapy) prior to participation. In study 1, participants with glioblastoma multiforme-type brain tumors were randomly assigned in a double-blind fashion to receive marima-stat or placebo immediately following radiotherapy. In study 2, participants with other types of brain tumors (glioblastoma multiforme, astrocytoma, etc.) were given marimastat while receiving adjuvant chemotherapy. All participants were assessed with a neuropsychological battery prior to initiating marimastat and at 3-month follow-up. Analyses indicated that individuals receiving marimastat evidenced no declines in cognitive functioning above and beyond those attributable to standard treatment effects and disease progression. This was true when marimastat was directly compared to placebo and when given concurrently with chemotherapeutic agents. However, participants receiving marimastat in study 1 evidenced greater decline in upper extremity strength compared to placebo. This investigation revealed that there were no measurable neurotoxic effects of marimastat when used in combination with chemotherapeutic agents in the treatment of patients with primary brain tumors. However, participants receiving marimastat showed decreased upper extremity strength at follow-up. This finding may be related to the drug’s side-effect profile, which includes musculoskeletal complaints.


Elana Farace and Zarui Melikyan; Department of Neurosurgery, Pennsylvania State University, Hershey, PA, USA

We have previously shown that depression is a common problem in postoperative patients with primary malignant glioma (Litofsky, 2004). However, a careful analysis of patients who have received the presumptive diagnosis of a brain tumor but have not received any surgical, radiation, or chemotherapeutic treatment (the “reaction to diagnosis” period) has never been reported. Clinical depression may develop in reaction to anticipated losses and/or physiological changes from tumor or treatment, thus making depression a likely state variable. However, the personality or trait variables (positive and negative affect) provide a much richer understanding of the components of emotional outlook beyond clinical definition and have been shown to be independent constructs in a mixed cancer group and a palliative care setting. The purpose of this study was to examine whether depression or affect had more impact on patients’ activities of daily living (ADLs). Participants included 33 adult brain tumor patients (8 male, 25 female) with ages at diagnosis ranging from 20 to 84 years (mean = 56 years; SD = 15.4 years). Ninety-four percent of patients were Caucasian, 6% were African American, 85% were right-handed, and 18 were married (6 divorced, 4 widowed, and 4 single). The patients had a mean of 13 years of education (range = 8–22 years), and the group mean IQ estimate was in the average range (107; SD = 8.9). Tumor types included craniopharyngiomas, carcinomas, adenomas, meningiomas, primitive neuroectodermal tumors, central neurocytomas, glioblastomas, schwannomas, and all forms of glioma (grades I–IV) in a wide range of locations (left, right, callosal, skull base, etc.). Patients were sequentially referred for initial surgical resection of a solid primary brain tumor. All patients were given the Beck Depression Inventory–II (BDI) to assess DSM-IV-related depressive symptom severity; the Positive and Negative Affect Scale (PANAS), a measure of mood that gives information on both positive and negative mood states; and the Barthel ADL assessment. Spearman correlations were performed. A significant negative correlation was found between the Barthel and BDI (r = −.527), indicating that ADLs decrease as depression increases. However, there were much smaller relationships between the Barthel and the positive (r = .15) and negative (r = −.20) affect scales of the PANAS. This study provides preliminary evidence that affect and depression are likely independent constructs, which has previously been shown in mixed cancer group and palliative care settings. Given the literature on the deleterious effects of negative affect on outcomes in many forms of cancer, routine assessments of brain tumor patients may need to include measures of affect as well as depression.


Elana Farace,1 Jonas M. Sheehan,1 and Mark E. Shaffrey2; 1Department of Neurosurgery, Pennsylvania State University, Hershey, PA, USA; 2Department of Neurosurgery, University of Virginia, Charlottesville, VA, USA

Surgery is often the first treatment of choice for a brain tumor, particularly in patients with meningiomas and other accessible tumors. There is significant potential for surgical resection to increase or induce neuropsychological impairment in patients with brain tumors. On the other hand, surgery may relieve neurocognitive impairment, particularly that caused by mass effect or hydrocephalus. This study included adult patients (age 18 years or older) who were sequentially referred for initial surgical resection of a solid primary brain tumor and who could read and write English. Seven surgeons across two sites (UVA and PSU) contributed 27 patients (11 men and 16 women) who were seen for preoperative baseline and 6-week postoperative neuropsychological evaluations. Patients were given a standard neuropsychological assessment battery measuring a broad range of domains (attention, memory, executive function, visual and verbal memory, psycho-motor speed, and depression). Scores were adjusted for patient demographics using test norms as available. Median age at diagnosis was 49 years (range = 14–77 years), and mean education was 13 years (range = 9–20 years). All patients were Caucasian, 89% were right-handed, and 20 were married (4 single and 3 divorced). The group mean IQ estimate was in the average range (104; SD = 7.5). Tumor types included craniopharyngiomas, meningiomas, primary neuroectodermal tumors, central neurocytomas, and all forms of glioma (grades I-IV) in a wide range of locations (left, right, callosal, skull base, etc.). Only three patients had hydrocephalus. Eighty-four percent of patients showed improvement of at least 1 SD in at least one domain at 6 weeks post-surgery. Thirty-six percent of patients worsened by at least 1 SD in at least one neurocognitive domain (percentages overlap because some patients improved in one domain and worsened in another). The group mean psychomotor speed (pegboard and digit symbol) significantly improved. There were no other significant mean differences found and, most notably, no area in which mean scores declined. Specific predictors of decline will be discussed. Surgery was found to result, in certain conditions and in certain domains, in stable or improved neurocognitive function. While the neurocognitive improvement shown in these data could easily have been a result of practice from patients taking similar tests twice (although for memory tests, alternate forms were used), as some patients do clearly decline despite the practice, these two responses to surgery can be delineated. Although surgery has the potential to induce deficits through focal damage to surrounding tissue, increased risk of hemorrhage, etc., it may also improve function, perhaps through resolution of mass effect, relief of intracranial pressure, or even a benefit of debulking. A better understanding of the neurocognitive effects of surgical resection is needed.


David E. Gerber, Amandeep Salhotra, Jodi B. Segal, and Michael B. Streiff; Johns Hopkins University School of Medicine, Baltimore, MD, USA

Individuals with brain tumors have an increased risk of venous thromboembolism (VTE). Within this population, patients with meningiomas have been reported to have the highest incidence, exceeding 70% in one study (Sawaya et al., J Neurooncol, 1992). However, patients in that study did not receive pharmacologic VTE prophylaxis, and the utility of radio-labeled fibrinogen scans — the modality used to screen for DVT — has been questioned. We therefore conducted a retrospective review of postoperative meningioma patients to define VTE incidence and risk factors. Medical records were reviewed from all patients undergoing craniotomy for meningioma at Johns Hopkins Hospital between January 2004 and December 2005. The association between clinical characteristics and VTE was assessed using parametric and nonparametric statistical tests and survival analysis. A total of 224 individuals met criteria for this review. The mean age was 52 years (SD, 14 years), and 167 patients (75%) were women. Median follow-up time was 230 days (interquartile range [IQR], 89–428 days). Virtually all patients received mechanical (TED stockings and intermittent pneumatic compression devices placed preoperatively) and pharmacologic (unfraction-ated heparin, 5,000 units s.c. b.i.d. started 24 h postoperatively) VTE pro-phylaxis. VTE was diagnosed in 11 patients (4.9%; 95% CI, 2.5–8.6%) at a median of 16 days (IQR, 7–33 days) postoperatively. Six patients had deep venous thromboses, three patients had pulmonary emboli, and two patients had both. The development of VTE was associated with older age (mean, 68 years versus 52 years; P = 0.0001), male gender (P = 0.007), and nonam-bulatory status postoperatively (P < 0.0001). VTE was not associated with duration of surgery, tumor size, tumor location, tumor histologic grade, ABO blood type, or body mass index. Significant postoperative bleeding (defined as causing a neurologic change and/or necessitating re-operation) occurred in six patients (2.7%) overall. VTE occurs rarely in postoperative meningioma patients who receive combined-modality VTE prophylaxis. Pharmacologic prophylaxis with subcutaneous low-dose unfractionated heparin started 24 h postoperatively is safe in this patient population. VTE risk factors in these patients include advanced age, male gender, and non-ambulatory status postoperatively.


K.K. Hardy, M.J. Bonner, V.W. Willard, and K.C. Hutchinson; The Tug McGraw Center for Quality of Life/Supportive Care Research in The Preston Robert Tisch Brain Tumor Center at Duke University Medical Center, Durham, NC, USA

Given the consistent finding that family functioning is a significant predictor of psychological functioning in children with chronic illness, evaluation of parent/caregiver variables is important when conducting research with children diagnosed with brain tumors. Moreover, because parents are often used as proxies to describe the adjustment of their children, their own functioning and adjustment need to be considered. To date, several studies have investigated the relative levels of distress for parents of patients on versus off treatment, but prior studies have not examined parent adjustment as a function of tumor type. Rather, tumor types are typically combined and treated as a single variable. However, the identification of specific tumor types that put parents at greater risk for psychosocial distress has implications for clinical care and service delivery. The objective of this study was to assess differences in parent and family adjustment to pediatric brain tumors as function of tumor type. To assess distress associated with tumor type, data were extracted from a database of patients who received comprehensive evaluations as part of their routine care at DUMC. Data from the Brief Symptom Inventory and Family Environment Scale were used. Subjects included 87 caregivers of children diagnosed with a brain tumor. Of these, 77 (88.5%) identified themselves as mothers. The majority (N = 50; 57.2%) had at least some college education. Diagnosis type included medulloblastoma (N = 36; 41.4%), ependymoma (N = 15; 17.2%), pilocytic astrocytoma (N = 9; 10.3%), brainstem glioma (N = 16; 18.4%), and glioblastoma multiforme (N = 11; 12.6%). Children ranged in age from 1 to 19 years (M = 8.1 years; SD = 4.74 years). The majority (N = 64; 73.5%) were on treatment at the time of the evaluation. Consistent with prior studies, the current sample of parents did not exhibit high levels of depression or anxiety when compared to normative data. Broadly speaking, however, parents of children diagnosed with malignant tumors demonstrated higher levels of psychological distress than children with benign tumors. Moreover, for all tumor groups, time since diagnosis was negatively associated with psychological distress and positively associated with family cohesiveness. Finally, children’s age at evaluation was negatively correlated with family support and positively correlated with family conflict. Knowledge of a differential risk for parents of patients with various types of pediatric brain tumors will better inform intervention development and delivery and the distribution of resources, particularly for small programs with limited access to psychosocial support providers.


K.K. Hardy, M.J. Bonner, and V.W. Willard; Tug McGraw Quality of Life/Supportive Care Research Center of the Preston Robert Tisch Brain Tumor Center at Duke University Medical Center, Durham, NC, USA

Neurocognitive deficits in survivors of pediatric brain tumors have been well documented over the past two decades. A history of radiation treatment and young age at diagnosis are frequently cited as predictors of intellectual decline in survivors. However, because of highly variable diagnostic and treatment factors, assessing the relative contribution of additional disease and treatment-related risk factors, such as hydrocephalus, in a heterogeneous population of pediatric brain tumor patients has been difficult. Moreover, although there is substantial support for intellectual declines in the extant literature, academic functioning has been less well described, despite the impact of poor academic skills on survivors’ quality of life. The goal of the study was to assess the relationship between hydrocephalus treated with shunt and intellectual, academic, and memory functioning in survivors of medulloblastoma treated with radiation. Forty survivors (17 females, 23 males) of pediatric medulloblastoma who were at least 1 year post-treatment (mean, 2.4 years; SD, 1.97 years) were administered a neurocognitive battery as a part of routine follow-up care. All participants had received cranial radiation therapy as part of their care. Average age at diagnosis was 7.5 years (SD, 3.28 years; range, 1–14 years). Participants ranged in age at evaluation from 6.4 to 16.5 years (M, 11.3 years; SD, 3.06 years). Twelve participants (30%) had a history of hydrocephalus treated with ventriculoperitoneal (VP) shunt placement; participants with and without a history of hydrocephalous did not differ with respect to age at diagnosis, age at evaluation, race, and time since treatment. Compared to those without a history of hydrocephalus, participants with VP shunts demonstrated significantly lower intellectual and academic functioning, as measured by the Wechsler Intelligence Scale for Children–Third Edition and the Woodcock-Johnson Tests of Achievement–Revised. Of note, effect sizes were larger for academic than for intellectual differences between groups. However, short-term verbal and visual memory skills, as assessed by the Wide-Range Assessment of Memory and Learning, were not significantly different. In this small sample of medulloblastoma survivors, the presence of a VP shunt was associated with the presence of more severe intellectual and academic deficits. Indeed, patients treated for hydrocephalus performed over 1 SD below those without such history on many academic tasks. These results highlight the need to explore factors in addition to radiation and chemotherapy as risk factors for neurocognitive declines in survivors.


Y. Khakoo,1 T. Gershon,1 G. Solomon,2,3 and S. Merchant3; 1Departments of Pediatrics and Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Departments of 2Neurology and 3Pediatrics and Joan and Sanford I. Weill Medical College, Cornell University, New York, NY, USA

Pediatric oncology patients often have neurologic symptoms. In 1999, Antunes et al. reported that delirium and seizures were the second and third most common reasons after pain for pediatric neurologic consultation at Memorial Sloan-Kettering Cancer Center (MSKCC). In 2005, Khan et al. reviewed pediatric brain tumor patients with seizures and found that an EEG with slow waves predicted more difficult-to-control seizures. To ascertain the prevalence of EEG abnormalities in a population of pediatric cancer patients, we retrospectively reviewed all EEGs obtained from January 1, 2003, to April 1, 2006, at MSKCC. Seventy-eight EEGs were obtained on 53 patients. Ages ranged from 6 months to 30 years; 31 patients are alive. Twenty-four (45%) of the 53 patients had primary CNS tumors, while 26 (50%) had non-CNS primary malignancies. Three patients did not have a definitive malignancy. Of the 26 patients with non-CNS malignancies, 11 had leukemia/lymphoma, 5 had neuroblastoma, 4 had sarcomas, and 4 underwent bone marrow transplantation (2 for Fanconi anemia, 1 for beta-thalassemia, and 1 for osteopetrosis). Of the 26 patients with non-CNS malignancies, 7 had known CNS metastasis at the time of EEG. The majority of EEGs in the non-CNS tumor patients were ordered to evaluate alteration of mental status. In patients without CNS metastasis, the most likely etiology of the mental status change was toxic/metabolic. EEG proved helpful in diagnosing non-convulsive status epilepticus in some pediatric patients with cancer.


M. Klein,1 I. Bosma,2 L. Douw,1 J.C. Baayen,3 J.J. Heimans,2 T.J. Postma,2 B.W. van Dijk,4 J.C. Reijneveld,2 and C.J. Stam5; Departments of 1Medical Psychology, 2Neurology, 3Neurosurgery, 4MEG Center, and 5Clinical Neurophysiology, VU University Medical Center, Amsterdam, The Netherlands

Patients with low-grade glioma (LGG) suffer from neurocognitive defi-cits that cannot be explained by tumor localization alone. Previous research has shown that focal lesions in brain cancer patients give rise to disruption of the brain’s functional networks. The present study explores the relationship between network disruption and neurocognitive functioning in LGG patients. Magnetic fields were recorded during the resting state using a 151–channel whole-head MEG system in 17 LGG patients and 17 healthy controls matched for age, gender, and education. Synchronization likelihood (SL; a measure of synchronous activity between brain regions) scores were calculated between all channels and averaged to a number of local and global network synchronization scores. Mann-Whitney U tests were conducted to assess any differences in SL between patients and controls. A neurocognitive testing battery measuring (1) information processing speed, (2) psychomotor function, (3) attentional functioning, (4) verbal memory, (5) working memory, and (6) executive functioning was administered after MEG recording. Student’s t tests for independent samples were conducted between LGG patients and healthy controls to test for differences in neu-rocognitive performance. Pearson correlations were calculated between patient SLs and the six cognitive domains. Relative to healthy controls, LGG patients had significantly impaired information processing speed (P = 0.01), psychomotor function (P = 0.04), and working memory capacity (P < 0.01). LGG patients showed an increased local and long-distance SL in delta and theta (0,5–8 Hz) and beta and lower gamma (13–45 Hz) frequency bands. In lower (8–10 Hz) and upper (10–13 Hz) alpha bands, a decreased long-distance SL was found. Patients’ synchronous brain network activity in delta and theta bands showed negative correlations with neurocognitive functioning but not memory. Lower alpha SLs correlated negatively with attention and executive functions. Upper alpha SLs correlated with information processing speed and working memory. Strong negative correlations were found between upper gamma (55–80 Hz) SLs and neurocognitive functioning. Our findings indicate that (1) LGG alters functional connectivity in the brain; (2) these alterations are both focal and intrahemispheric; (3) effects differ per frequency band; and (4) brain network synchronicity and neurocognitive function are related in LGG patients.


K.R. Knight,1 D.F. Kraemer,3 C. Winter,1 and E.A. Neuwelt1,2; 1Oregon Health & Sciences University, Portland, OR, USA; 2Portland VA Medical Center, Portland, OR, USA; and 3Oregon State University, Portland, OR, USA

The purpose of this study is to describe progressive changes in hearing and cochlear function in children and adolescents treated with platinum-based chemotherapy and to begin preliminary evaluation of the feasibility of extended high frequency audiometry and distortion-product otoacoustic emissions (DPOAEs) for ototoxicity monitoring in children. Baseline and serial measurements of conventional pure-tone audiometry (.5–8 kHz) and evoked DPOAEs were conducted for 32 subjects aged 8 months to 20 years who were treated with cisplatin and/or carboplatin chemotherapy. Seventeen children also had baseline and serial measurement of extended high-frequency (EHF) audiometry (9–16 kHz). Audiologic data were analyzed to determine the incidence of ototoxicity using the ASHA criteria and the relationships between the different measures of ototoxicity. Of the 32 children, 20 (62.5%) had bilateral ototoxicity in the conventional frequency range during chemotherapy treatment, and 26 (81.3%) had bilateral decreases in DPOAE amplitudes and dynamic range. Of the 17 children with EHF audiometry results, 16 (94.1%) had bilateral ototoxicity in the EHF range. Pilot data suggest that EHF thresholds and DPOAEs show ototoxic changes before changes in hearing are detected by conventional audiometry. EHF audiometry and DPOAEs have the potential to reveal the earliest changes in auditory function during platinum chemotherapy.


Jin-shei Lai,1,2 David Cella,1,2 and Stewart Goldman2,3; 1Center on Outcomes, Research and Education, Evanston, IL, USA; 2Northwestern University School of Medicine, Chicago, IL, USA; 3Children’s Memorial Hospital, Chicago, IL, USA

Fatigue is a major concern for cancer patients at any age or disease stage. A scale that demonstrates stable measurement properties across the lifespan is important as it can facilitate the monitoring of fatigue longitudinally in a psychometrically sound manner. The purpose of this study was to validate such a scale — the pediatric Functional Assessment of Chronic Illness Therapy-Fatigue (pedsFACIT-F) — a component of the 51-item pediatric Fatigue Item-Bank, which was developed using literature review, patient/parent feedback, clinician evaluation, and a consensus meeting. PedsFACIT-F consists of 11 items (7 shared with the 13-item adult version of the FACIT-F) selected to maximize content validity and address developmental issues. One hundred thirty-three cancer patients (ages 8–17) who were on treatment or 4 months post-treatment were recruited. Of those patients, 57.5% were boys, 40.6% had leukemia, 22.6% had brain tumors, 98.3% received chemotherapy, 22.5% received radiation, 40% received surgery, and 74.8% had Karnofsky scores of 90 or 100. The mean hemoglobin was 11.5 (range, 6.7–16.4; 55.4% were anemic and 44.6% non-anemic). Alpha coefficients, item-total correlations, and exploratory factor analysis (EFA) were used to examine dimensionality. Rasch analysis was used to examine the scalability of the items (mean-square fit statistics [MnSq] between 0.6 and 1.4). ANOVA was used to examine differences between age groups (children vs. adolescents), anemic vs. non-anemic patients, genders, and disease types (leukemia, brain tumors, and others). Spearman correlations were used to compare PedsFACIT-F scores to pedsQL-fatigue subdomain scores. Good internal consistency was found (alpha, 0.87; item-total correlations, 0.47–0.70). EFA suggested one factor among the items (factor loading > 0.4 on the first factor with explained variance of 81.2%; one factor before the breakpoint in the screen-plot). All items had MnSq between 0.78–1.22 in the Rasch analysis. The anemic group reported significantly (P = .001) more severe fatigue than did the non-anemic group, but no differences were found among cancer types, between genders, or across age groups. Concurrent validity was supported (rho for general fatigue, sleep, and cognition was .84, .68, and .55, respectively). The pedsFACIT-F demonstrates acceptable psychometric properties using both classical and modern test theory criteria. Our next step is to test its measurement properties using Item Response Theory to evaluate the stability of the item parameter across the lifespan. Scores between pedsFACIT-F and adultFACIT-F can then be equated to enable its use in monitoring the quality of long-term care.


Jin-shei Lai,1,2 Jennifer Beaumont,2 David Cella,1,2 and Stewart Goldman2,3; 1Center on Outcomes, Research and Education, Evanston Northwestern Healthcare, Evanston, IL, USA; 2Northwestern School of Medicine, Chicago, IL, USA; 3Children’s Memorial Hospital, Chicago, IL, USA

Using parent proxies to report children’s health-related quality of life (HRQL) is common, as proxy ratings are thought to be more valid and sensitive to detecting change over time than children’s self-report. However, HRQL is a subjective perception emphasizing an individual’s phenomenological experiences. Thus, proxies may not truly reflect children’s HRQL. This study attempts to understand the relationship between proxy- and self-reported HRQL. Ninety-six cancer patients, aged 8–17 years, and their parents completed the pediatric Functional Assessment of Cancer Therapy (pedsFACT). The pedsFACT measures physical function (PF; eight items), positive illness experience (PI; four items), emotional distress (ED; six items), and social/family function (SF; eight items). Significant Cochran-Mantel-Haenszel (CMH) statistic (P < .05), Kappa statistic (>.3), and non-significant paired t-tests (NS, P > .05) were used to examine the relationship between patient and parent responses to each item. Proxy responses were considered comparable to self-report responses when CMH and Kappa were significant and the t-test was not significant. In PF, all items had significant CMH, seven of eight had Kappa > 0.3, and five had NS t-test. In PI, all had significant CMH, two had Kappa = 0.3, and two had NS t-test. In ED, three items had significant CMH, two had Kappa > 0.3, and four had NS t-test. In SF, three had significant CMH, one had Kappa > 0.3, and four had NS t-test. In general, parents responded more severely than did patients on all but one PI item. Responses to four PF items, one PI item, one ED item, and one SF item met all three criteria and were considered replicable between parents and patients. While patient and patient responses were significantly associated for most items, inconsistent results were found across analyses. As a result, the use of proxies to measure children’s HRQL using the complete pedsFACT is not recommended. While differences in responses reflect the differences in the perspectives of patients and their parents, proxy responses on a subset of seven items were found to be sufficiently representative of self-report to warrant their use. These items could serve as a foundation for a tool that could be used to measure HRQL in children. Despite the small sample, these results are promising. Clinical implications of this study will be discussed.


Jing Li,1 Soren Bentzen,1 Jialiang Li,1 Markus Renschler,2 and Minesh P. Mehta1; 1University of Wisconsin, WI, USA; 2Pharmacyclics, Sunnyvale, CA, USA

It is commonly speculated that deterioration of neurocognitive function (NCF) in patients with brain metastases (BM) who receive whole-brain radiation therapy (WBRT) will negatively impact quality of life (QOL). However, few retrospective or prospective studies have explored the relationship between NCF and QOL in this population. Two hundred and eight patients from the WBRT-alone (30 Gy) arm of a phase III trial (PCI-P120–9801) evaluating motexafin gadolinium in BM patients were analyzed. NCF was assessed by eight tests of memory, executive function, and fine motor coordination, and QOL was evaluated by the Barthel Index of activities of daily living (ADL) and the brain-specific Functional Assessment of Cancer Therapy–Brain subscale (FACT-Br). Correlations between these two variables at different time points were analyzed using Spearman’s rank correlation. The predictive effect of NCF from previous visits on QOL was studied using a linear mixed effects model. For lead-time analysis, NCF or QOL deterioration was defined statistically by population deterioration rate of ~33%. We defined a lead time as NCF deterioration before the date of QOL decline, while a lag time occurred when NCF deteriorated after QOL decline. The mean lead or lag time was estimated as the area under the Kaplan-Meier time-to-progression curve. A net lead time was calculated from the mean lead time and mean lag time. A two-sided P value <0.05 was considered statistically significant. At baseline, each NCF test showed weak to moderate, but statistically significant, correlation with the Bar-thel Index (r = 0.25–0.38; N = 175–204). At 4 months, these correlations became stronger (r = 0.29–0.51; N = 63–75). At 6 months, the correlations remained strong, although statistical significance was decreased as a result of the lower number of patients at risk (N = 41–46). Similar results were obtained with FACT-Br. Additionally, we found that scores from NCF tests from previous visits could be used to predict Barthel index and FACT-Br, with a predictive factor of 0.02–0.64 (P < 0.05 for all NCF tests). When testing the hypothesis that NCF deteriorated before FACT-Br decline, we found that all eight NCF tests deteriorated prior to Barthel Index, with a net lead time ranging from 61 to 153 days, and six of eight NCF tests deteriorated before FACT-Br, with a net lead time of 9–82 days. NCF and QOL were correlated in BM patients who received WBRT. NCF test scores from previous visits can be used to predict QOL measurements. NCF deterioration proceeded QOL decline by 9–153 days. To our knowledge, this is the first report to demonstrate such a sequential association between NCF and QOL in BM patients. These results demonstrate that any efforts to delay NCF decline will help to preserve QOL and therefore improve overall care for BM patients.


Flory L. Nkoy,1 Karen J. Valentine,2 and Ali K. Choucair3; 1University of Utah, Salt Lake City, UT, USA; Intermountain Healthcare, 2Cancer Services and 3Neuro-Oncology Service, Salt Lake City, UT, USA

PBTs often require aggressive treatments that are associated with various long-term side effects and functional impairment with little gain in survival. Symptom monitoring is therefore a critical part of patient care. In this study, we evaluated changes in QOL scores following routine assessment of QOL in patients with PBT. We also determined which global and subset QOL scores were associated with changes in clinical evaluations. With IRB approval, newly diagnosed patients with PBT referred to the Intermountain Healthcare Neuro-Oncology Service were prospectively enrolled from January 2003 to December 2004. Validated QOL measures (EORTC QLQ-C30, EORTC QLQ-BCM20, Spitzer QOL Index, Schwartz Fatigue Scale, and HADS) and standard clinical measures (KPS, MMSE, Neurological Function Status, neurological exam, and brain imaging) were collected from enrolled patients in 3-month intervals. No proxies were allowed. Repeated-measures analysis of variance was performed to determine whether patients experienced changes in QOL factors over time. Linear mixed models that adjusted for follow-up time and age were used to determine which clinical measures were associated with changes in QOL scores. We enrolled 54 patients during the study period. Of those, 49 (91%) completed the baseline questionnaires without assistance. Four patients did not complete any baseline questionnaires and were excluded from further analysis. The baseline characteristics of the remaining 50 patients are as follows: mean age was 42 ± 15 years (range, 18–71 years), 24 patients (48%) were female, mean follow-up time was 10 ± 1 months, 5 patients (10%) died, and 10 patients (20%) had recurrent tumors. The mean time to death or recurrence was 17 ± 2 months. QOL measures, global health status (P = 0.0028), role functioning (P [less-than-or-eq, slant] 0.0001), emotional functioning (P = 0.0116), and social functioning (P < 0.0001) significantly improved over time in these patients. The presence of pain (P = 0.0425) and insomnia (P = 0.0008) decreased over time. However, fatigue (P = 0.0193) and future uncertainty (P = 0.0001) fluctuated over time, and constipation problems (P = 0.0437) increased over time. QOL scores that were associated with the change in MMSE were diarrhea (P = 0.001), cognitive deficit (P = 0.006), visual disturbance (P = 0.026), and future uncertainty (P = 0.034). Cognitive functioning (P = 0.010), appetite loss (P = 0.031), financial difficulties (P = 0.046), motor dysfunction (P = 0.046), communication deficit (P = 0.002), headaches (P < 0.001), depression (P = 0.006), and Spitzer score (P = 0.001) were associated with neurological functioning. None of the global or subset QOL scores were associated with KPS. Correlation of imaging to QOL scores is currently under evaluation and will be reported in the future. Routine collection of QOL data improves the care of patients with PBT. Changes in MMSE and neurological functioning were associated with QOL changes, but KPS was not sensitive to the change in QOL scores.


Edward Pan and Susan Mitchell; Florida Hospital Neuroscience Institute, Orlando, FL, USA

Venous thromboembolism (VTE) is a common complication in patients with high-grade gliomas. The exact mechanism of development of VTE in brain tumor patients is unclear, but predisposing factors include hemiparetic limbs, chemotherapy, older age, and corticosteroid therapy. Brain tumor patients with acute VTE often receive inferior vena cava (IVC) filters rather than anticoagulation; however, complication rates are high after IVC filter placement, and recurrent deep-vein thromboses frequently occur after IVC filter placement without anticoagulation. Thus, there is no established standard of care for prophylaxis and treatment of VTE in brain tumor patients. Published reports have indicated a low incidence of intracranial hemorrhage (ICH) in brain tumor patients receiving anticoagulation. The optimum strategies for prophylaxis and treatment of acute VTE in brain tumor patients need to be delineated. The objectives of this study are (1) to determine the incidence of VTE and ICH in glioblastoma multiforme (GBM) patients, (2) to determine the mean time of VTE and ICH events from GBM diagnosis, and (3) to determine risk factors for predisposition of VTE and ICH in GBM patients. Retrospective analysis was performed on medical records of adult patients who were discharged from Florida Hospital Orlando or Florida Hospital Celebration with a primary or secondary diagnosis of GBM from 2001 to 2005. Incidences of VTE and ICH events and their correlating risk factors will be determined and analyzed for statistical significance. Analysis and reporting will include a compilation of statistics in addition to narrative reporting of the data. Descriptive statistics for continuous variables will consist of the mean, median, SD, and minimum and maximum values. For categorical variables, the number and percent of each category will be displayed. All data analyses will consist of total population tabulations and tabulations by categories, with the intent of making statistical inferences between the categories as well as comparison to the whole. Final results will be available prior to the annual meeting of The Society for Neuro-Oncology in November 2006.


Sumul N. Raval, Amy Rule, and Shirley S. Hwang; Leon Hess Cancer Center, Monmouth Medical Center, Long Branch, NJ, USA

Primary GBM exhibits overexpression or amplification of the epidermal growth factor gene. The combination of targeted therapy and chemotherapy may provide synergistic effects with better response rates. The effectiveness of bevacizumab and irinotecan in patients with relapsed GBM was first reported in 2005 (Stark-Vance et al., Neuro-Oncology, 2005). Bevacizumab is designed to inhibit vascular endothelial growth factor, a protein that plays an important role in tumor angiogenesis and maintenance of existing tumor vessels. In this report, we assess the effects of the combination of bevacizumab and irinotecan on overall responses, toxicity, cognitive function, and functional status in patients with recurrent GBM. Eight consecutive GBM patients who failed at least one prior chemotherapy regimen and had measurable disease on contrast-enhanced MRI from August 2005 to May 2006 were included in this analysis. Each patient received 5 mg/kg bevacizumab i.v. and 125 mg/m2 irinotecan i.v. infusion every 2 weeks until disease progression or development of unacceptable toxicity. The response was determined by MRI every two cycles. Cognitive function was assessed by Blessed Orientation-Memory-Concentration Test (BOMC); functional status was assessed by KPS, Barthel Index (BI), and Instrumental Activities of Daily Living (IADL) prior to each cycle of treatment. Descriptive statistics analysis was used. All patients failed temozolomide and radiation therapy, one patient had prior BCNU treatment, and four patients had prior irinotecan treatment. The median age was 51 years (range, 44–77 years). Pretreatment median scores were 75 for KPS (range, 40–90), 8 for BOMC (range, 1–18), 88.5 for BI (range, 46–100), and 7 for IADL (range, 0–14). Two patients exhibited mild and three patients exhibited moderate cognitive impairment by BOMC measure. The median number of cycles received was 6 (range, 1–16); six patients were evaluable for responses by MRI with 100% response rate (one complete response and five partial responses). One patient has died. The median length of follow-up was 3+ months (1+ to 9+ months). No grade 3 or 4 toxicities were reported. The most frequently reported grade 1 and 2 toxicities included fatigue in eight patients (100%), neutropenia in three (37.5%), anemia in two (50%), thrombocytopenia in two (25%), nausea in two (25%), diarrhea in two (25%), and lack of appetite in two (25%). All patients were evaluable for cognitive and functional response. Improvement in cognitive BOMC score was seen in four patients (50%), with median improvement by 1 point (range, 0–8 points); only two patients had mild cognitive impairment. Improvement in functional status was seen in seven patients (87.5%), with median improvement in KPS by 15 points (0–50 points), BI by 8 points (range, 0–54 points), and IADL by 4 points (range, 0–12 points). The combination of bevacizumab and irinotecan is well tolerated. The overall response rate was 100%, and improvements in cognitive functional and functional status were demonstrated. Further accrual and longer follow-up will determine the impact of this active combination.


M.J.B. Taphoorn, M.J. van den Bent, M. Mauer, C. Coens, J.-Y. Delattre, A.A. Brandes, C.C.D van der Rijt, H.J.J.A Bernsen, M. Frenay, C.C Tijssen, D. Lacombe, A. Allgeier, and A. Bottomley; Medical Center Haaglanden, The Hague, Netherlands

Little is known about the symptoms and health-related quality of life (HRQOL) of patients treated for newly diagnosed anaplastic oligodendro-gliomas. We investigated the impact of adjuvant procarbazine, CCNU, and vincristine (PCV) chemotherapy following radiotherapy (RT) compared to RT alone on both short- and long-term HRQOL. Adult patients with ana-plastic oligodendrogliomas, ECOG performance status 0–2, and adequate hematological, renal, and hepatic functions were randomly assigned to receive RT alone or RT plus PCV chemotherapy. HRQOL was assessed with the EORTC QLQ-C30 and Brain Cancer Module. Seven prespecified HRQOL endpoints were selected: global HRQOL, fatigue, nausea/vomiting, physical and social functioning, communication deficit, and seizures. We hypothesized that adjuvant chemotherapy would impair HRQOL during chemotherapy treatment compared to the no-treatment arm after RT, but HRQOL would be similar between groups once off treatments. Assessments were performed at randomization, at the end of RT, every 3 months for the first year following RT, and then every 6 months until progression. Of the 368 patients randomized, 58% were male, and the median age was 49 years. Compliance with HRQOL questionnaires was 78% at baseline and reasonable (55–71%) for each assessment up to 2.5 years post RT. As expected, initial analysis indicated that both groups showed substantial (but comparable) impairment at baseline for most scales. The RT plus PCV chemotherapy arm showed a significant and clinically meaningful increase in fatigue (P = 0.0016) and a decrease in physical functioning (P = 0.0063) during chemotherapy compared to the RT-alone arm. RT plus PCV chemotherapy also led to an increase in nausea/vomiting during and shortly after chemotherapy, but this was not clinically meaningful. The long-term results (after 1 year) showed no difference between arms. In combination with the clinical results that have already been reported, this study shows that RT plus adjuvant PCV chemotherapy increases progression-free survival compared to RT alone but leads to a short-term negative impact in two (fatigue and physical functioning) of the seven selected HRQOL endpoints. However, longer-term results show both groups to have comparable HRQOL.


P. Vanacker and G. Buyse; Dept. of Neurosurgery, AZ Damiaan, Ostend, Belgium

The objective of this study was to describe the effectiveness and health-related quality of life (HRQOL) of a patient with a recurrent tectal plate glioma (WHO grade III) during treatment with the alkylating agent temo-zolomide. The study was designed to analyze the role of temozolomide in the current management for brainstem tumors. The rehabilitation program consisted primarily of therapy with eight cycles of temozolomide (150 mg/m²/day for 5 days per 28–day cycle). Expecting a modest effect on absolute survival, it is essential to look not only at the effect on tumor reduction but also at the gain in HRQOL. The primary endpoints (effectiveness [MacDonald Criteria] and HRQOL) were evaluated at baseline and after the third, sixth, seventh, and eighth cycles. The secondary endpoints (side effects [NCI Common Toxicity Criteria version 2.0] and iatrogenic neurotoxicity [Medical Research Council Neurological Scale]) were also evaluated after each cycle. HRQOL was assessed using the two validated European Organization for Research and Treatment of Cancer (EORTC) questionnaires — core-30 (QLQ-C30) and the brain cancer module (EORTC BN-20) — covering multiple aspects of physical, psychological, and social functioning. On the primary endpoints, there was a progression in free survival of more than 9 months. MRI FLAIR images showed a stabilization of the tumor after the third, sixth, and eighth cycles. The global HRQOL outcomes assessment showed a significant improvement of 36.6% after three cycles of temozolomide and further progress during the therapy of 36.6% after six cycles, 44.6% after seven cycles, and 53.4% after eight cycles. On the secondary endpoint, there was an enhancement in the MRC Neurological Scale from score 4 to score 1 during the therapy. The treatment was very well tolerated by the patient. Side effects were asthenia (NCI grade 2 during the first six cycles and NCI grade 3 during cycles 7 and 8), raised aminotransferase levels (NCI grade 2 during cycle 7 and grade 1 during cycle 8), and continuous headaches (NCI grade 2). The effectiveness of the treatment in stabilizing the tumor, the improved HRQOL, and the acceptable safety profile make temozolomide a possible treatment for recurrent tectal plate glioma (WHO grade III). However, more case studies need to be analyzed systematically to determine the role of temozolomide in the rehabilitation program for recurrent high-grade tectal plate glioma. Such studies remain necessary as long as the newest molecular and genetic treatment modalities are still on a research level.


David Victorson,1 Cheryl Royce,2 David Draper,2 Irene Haggarty,2 Nancy Garren,2 Lyndon Kim,2 Jennifer L. Beaumont,1 David Cella,1 Astra M. Liepa,3 Donald E. Thornton,3 and Howard A. Fine2; 1Center on Outcomes, Research and Education, Evanston Northwestern Healthcare, Evanston, IL, USA; 2Neuro-Oncology Branch, National Cancer Institute, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; 3Eli Lilly and Co., Indianapolis, IN, USA

Enzastaurin (ENZ) is a targeted agent that inhibits protein kinase C β and the phosphatidylinositol 3-kinase/AKT pathways. In an ongoing phase II trial, daily oral ENZ has demonstrated a response rate of 23% in patients with recurrent high-grade gliomas (Fine et al., ASCO, 2005). To further evaluate the activity of ENZ in this population, health-related quality of life (HRQL) was also assessed in the trial. Patient eligibility included failed prior radiotherapy and KPS [gt-or-equal, slanted] 60. The Functional Assessment of Cancer Therapy-Brain (FACT-Br) was administered prior to start of therapy, at 3 weeks, at 6 weeks, and then every 6 weeks thereafter. For each of the FACT-Br scores, individual patients were classified as improved (IMPR), unchanged (UNCHG), or worsened by comparing change scores from baseline to the minimally important difference (MID). MIDs were estimated from this patient sample. Upper bounds of MID ranges were used to classify patients. For those patients classified as IMPR or UNCHG, duration of benefit was estimated based on 6-week cycles. Baseline FACT-Br data were available from 104 of 120 patients. Seventy-two percent of patients had a diagnosis of glioblastoma multiforme, 68% had KPS [gt-or-equal, slanted] 90 at baseline, and 84% had received at least two prior chemotherapy regimens.

Select FACT-Br parametersPatient classification3 weeks (%)N = 766 weeks (%)N = 5412 weeks (%)N = 21
Brain tumor subscale (BrS)IMPR/UNCHG4/599/5414/43
MID = 7 points on 92–point scaleWorsened373743
Trial Outcome Index (TOI; physical wellbeing + functional wellbeing + BrS)IMPR/UNCHG8/524/5310/52
MID = 11 points on 148–point scaleWorsened404338
Total FACT-BrIMPR/UNCHG15/434/555/57
MID = 11 points on 200–point scaleWorsened434238

The percentage of patients who experienced benefit for at least two cycles was 13% for BrS, 11% for TOI, and 10% for FACT-Br. Based on individual patient changes in FACT-Br scores, ENZ stabilized or improved HRQL parameters in the majority of patients at each assessment period. However, results reflect those patients who remained on therapy, and patient numbers declined over time because of disease progression.



D.T. Blumenthal,1 B. Berkey,2 D.Nelson,3 W. Curran,4 S. Leibel,5 L. Souhami,6 J. Michalski,7 B. Corn,1 A. Chakravarti,8 L. Rogers,9 and M. Mehta10; 1Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel; 2RTOG, Philadelphia, PA, USA; 3Mayo Clinic, Rochester, MN, USA; 4Thomas Jefferson University Hospital, Philadelphia, PA, USA; 5Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 6McGill University, Montreal, Canada; 7Washington University, St. Louis, MO, USA; 8Massachusetts General Hospital, Boston, MA, USA; 9GammaWest Radiation Therapy, Salt Lake City, UT, USA; 10University of Wisconsin, Madison, WI, USA

Minimal data exist in the medical literature on the effect of a delay in initiating radiation therapy for glioblastoma multiforme (GBM) on survival. Although prolonged delays of numerous weeks are believed to be detrimental, the effect of “short” delays is unclear. Some centers start radiation therapy within 1–2 weeks, as these tumors often proliferate rapidly; however, some delay the initiation of radiation therapy up to 4 or more weeks for several reasons. Only 1 retrospective single-institution report addresses the relevance of radiation therapy initiation timing in GBM. Most studies in other tumor types suggest that early initiation of radiation therapy improves prognosis. We performed a retrospective analysis of more than 2900 patients from 17 RTOG studies conducted between 1974 and –2002. All patients had undergone radiation therapy for newly diagnosed GBMs. Using a graded graph, we evaluated the time between surgery and initiation of radiation therapy as a multivariate factor for GBM prognosis. Overall survival comparisons were evaluated for 4 different time intervals from surgery to the start of radiation therapy ([less-than-or-eq, slant] 2 weeks, > 2–3 weeks, > 3–4 weeks, and > 4 weeks). These groups were further compared on the basis of their RPA classification. Other known prognostic factors (age, performance status [PS], and extent of resection) were evaluated using a multivariate model. RPA class, PS, age, and extent of resection were found to be significantly associated with overall survival. A comparison of the group with the shortest interval between surgery and radiation therapy ([less-than-or-eq, slant] 2 weeks) and the group with longest interval (> 4 weeks) revealed that the group with the longest interval had a statistically significant survival advantage (median, 9.2 versus 12.4 months; hazard ratio, 0.84; P = 0.0032). The respective 2-year survival rates were 10% and 16%. Contrary to our initial hypothesis that delaying radiation therapy may be aligned with diminished rates of survival, we found no such correlation within the relatively narrow timing parameters of this analysis. It is conceivable that clinical judgment prompted physicians to expedite treatment for patients who were more ill or who remained hospitalized beyond the expected recovery interval and that this biased the survival outcome. It also remains plausible that delays in radiation therapy beyond the scope of this study (i.e., exceeding 6 to 8 weeks) may yet adversely influence outcome.


P.D. Brown,1 T.B. Daniels,1 K. Ballman,1 S. Felton,1 J.C. Buckner,1 R.M. Arusell,1 W.J. Curran,2 R. Abrams,2 J.D. Earle,3 and E.G. Shaw2; 1NCCTG, Rochester, MN, USA; 2RTOG, Philadelphia, PA, USA; 3ECOG, Boston, MA, USA

A prognostic index for survival was constructed and validated from patient data from 2 EORTC radiation trials of low-grade glioma. We independently validated this prognostic index with a separate prospectively collected data set (Intergroup 86-72-51). Two hundred three patients were treated between 1986 and 1994 in an NCCTG-led trial that randomized patients with supratentorial low-grade glioma to 50.4 Gy or 64.8 Gy of radiation. Risk factors from the EORTC prognostic index were analyzed for prognostic value: histologic characteristics (oligo/mixed vs. astro), tumor dimension (< vs. [gt-or-equal, slanted] 6 cm), neurologic deficit (absent vs. present), age (