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1.  The mTORC1/mTORC2 inhibitor AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells 
Neuro-Oncology  2013;16(1):29-37.
The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs).
mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation.
Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments.
These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.
PMCID: PMC3870843  PMID: 24311635
AZD2014; glioblastoma; mTOR; orthotopic xenograft; Radiation; tumor stem cell
2.  The ATP-Competitive mTOR Inhibitor INK128 enhances in vitro and in vivo radiosensitivity of pancreatic carcinoma cells 
Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given mTOR's regulatory role in gene translation, in this study we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines.
Experimental Design
Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity on 3 pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation and tumor growth delay.
INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells, but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap-complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation.
These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair.
PMCID: PMC3947297  PMID: 24198241
Radiosensitization; mTOR; pancreatic carcinoma; INK128; xenograft
3.  Inhibition of PLK1 in glioblastoma multiforme induces mitotic catastrophe and enhances radiosensitization 
Glioblastoma multiforme (GBM) is the most common primary brain tumor in the USA with a median survival of approximately 14 months. Low survival rates are attributable to the aggressiveness of GBM and a lack of understanding of the molecular mechanisms underlying GBM. The disruption of signaling pathways regulated either directly or indirectly by protein kinases is frequently observed in cancer cells and thus the development of inhibitors of specific kinases has become a major focus of drug discovery in oncology. To identify protein kinases required for the survival of GBM we performed a siRNA-based RNAi screen focused on the human kinome in GBM. Inhibition of the polo-like kinase 1 (PLK1) induced a reduction in the viability in two different GBM cell lines. To assess the potential of inhibiting PLK1 as a treatment strategy for GBM we examined the effects of a small molecule inhibitor of PLK1, GSK461364A, on the growth of GBM cells. PLK1 inhibition arrested cells in the mitotic phase of the cell cycle and induced cell kill by mitotic catastrophe. GBM engrafts treated with GSK461364A showed statistically significant inhibition of tumor growth. Further, exposure of different GBM cells to RNAi or GSK461364A prior to radiation resulted in an increase in their radiosensitivity with dose enhancement factor ranging from 1.40 to 1.53 with no effect on normal cells. As a measure of DNA double strand breaks, γH2AX levels were significantly higher in the combined modality as compared to the individual treatments. This study suggests that PLK1 is an important therapeutic target for GBM and can enhance radiosensitivity in GBM.
PMCID: PMC3755049  PMID: 23790466
Glioblastoma multiforme; PLK1; GSK461364A; siRNA; radiation
4.  Visualizing Molecular Profiles of Glioblastoma with GBM-BioDP 
PLoS ONE  2014;9(7):e101239.
Validation of clinical biomarkers and response to therapy is a challenging topic in cancer research. An important source of information for virtual validation is the datasets generated from multi-center cancer research projects such as The Cancer Genome Atlas project (TCGA). These data enable investigation of genetic and epigenetic changes responsible for cancer onset and progression, response to cancer therapies, and discovery of the molecular profiles of various cancers. However, these analyses often require bulk download of data and substantial bioinformatics expertise, which can be intimidating for investigators. Here, we report on the development of a new resource available to scientists: a data base called Glioblastoma Bio Discovery Portal (GBM-BioDP). GBM-BioDP is a free web-accessible resource that hosts a subset of the glioblastoma TCGA data and enables an intuitive query and interactive display of the resultant data. This resource provides visualization tools for the exploration of gene, miRNA, and protein expression, differential expression within the subtypes of GBM, and potential associations with clinical outcome, which are useful for virtual biological validation. The tool may also enable generation of hypotheses on how therapies impact GBM molecular profiles, which can help in personalization of treatment for optimal outcome. The resource can be accessed freely at (a tutorial is included).
PMCID: PMC4091869  PMID: 25010047
5.  Bringing the heavy: carbon ion therapy in the radiobiological and clinical context 
Radiotherapy for the treatment of cancer is undergoing an evolution, shifting to the use of heavier ion species. For a plethora of malignancies, current radiotherapy using photons or protons yields marginal benefits in local control and survival. One hypothesis is that these malignancies have acquired, or are inherently radioresistant to low LET radiation. In the last decade, carbon ion radiotherapy facilities have slowly been constructed in Europe and Asia, demonstrating favorable results for many of the malignancies that do poorly with conventional radiotherapy. However, from a radiobiological perspective, much of how this modality works in overcoming radioresistance, and extending local control and survival are not yet fully understood. In this review, we will explain from a radiobiological perspective how carbon ion radiotherapy can overcome the classical and recently postulated contributors of radioresistance (α/β ratio, hypoxia, cell proliferation, the tumor microenvironment and metabolism, and cancer stem cells). Furthermore, we will make recommendations on the important factors to consider, such as anatomical location, in the future design and implementation of clinical trials. With the existing data available we believe that the expansion of carbon ion facilities into the United States is warranted.
PMCID: PMC4002206  PMID: 24679134
Radiotherapy; Radiobiology; Carbon ions; Hypoxia; α/β ratio; Tumor microenvironment; Tumor metabolism; Cancer stem cells
6.  External Beam Radiation Therapy in Treatment of Malignant Pheochromocytoma and Paraganglioma 
Frontiers in Oncology  2014;4:166.
Purpose: Pheochromocytomas (PCCs) are neuroendocrine tumors arising from the adrenal medulla or as paraganglioma (PGL) from extra-adrenal sites. While usually benign, a small fraction is malignant. Multi-modality therapy is used in treating malignant disease; however, little data exist on the role of external beam radiation therapy (EBRT). In this retrospective review, we assessed response to EBRT in malignant PCCs or PGLs.
Methods and Materials: Records of patients treated at the National Institutes of Health who received EBRT between 1990 and 2012 were studied. Patients were assessed for symptomatic control, biochemical response, local and distant control by response evaluation criteria in solid tumors v1.1 or stable disease on imaging reports, toxicity by radiation therapy oncology group (RTOG) criteria, and survival.
Results: There were 24 patients treated who received EBRT to lesions of the abdomen (n = 3), central nervous system (n = 4), and bone (n = 40). Lesions were treated with 3D conformal EBRT to a mean dose of 31.8 Gy in 3.3 Gy fractions, or fractionated stereotactic radiosurgery to 21.9 Gy in 13.6 Gy fractions. Patients experienced acute (n = 15) and late (n = 2) RTOG toxicities; no patient experienced acute toxicity ≥4 or late toxicity ≥2. Symptomatic control was achieved in 81.1% of lesions. Stable radiographic response was achieved in 86.7% of lesions with progression in 13%. Distant progression was observed overall in 75% of patients and average survival was 52.4 months.
Conclusion: Malignant PCC and PGL often do not respond well to current systemic therapies. In these cases, EBRT can be considered in patients with symptomatic, localized disease progression.
PMCID: PMC4073229  PMID: 25019060
radiation; pheochromocytoma; paraganglioma; malignant; neuroendocrine
7.  Radiation Therapy in Management of Sporadic and Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors 
Frontiers in Oncology  2014;4:324.
Introduction: Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas in which complete surgical resection is the mainstay of therapy. However, the recurrence rate is high and few options remain for refractory or metastatic MPNST. This study examines the outcomes of adjuvant radiation therapy in MPNST in patients with and without neurofibromatosis type 1 (NF1) and reviews the literature on use of radiation for MPNST.
Methods: A retrospective review of 33 MPNST patients between 1990 and 2012 evaluated at the NIH. All diagnoses were pathologically confirmed at the NCI. Clinical presentation, treatment, and survival were analyzed.
Results: Thirty-three patients were included 18 NF1-associated, 15 sporadic tumors. Tumor location included extremity (58%), trunk (36%), and head/neck (6%). Histologic grade showed 25 high-grade tumors compared to 7 low-grade tumors. Twenty patients were treated with radiation therapy (median total dose of 58.5 Gy with 1.8 Gy/fraction). A median survival of all patients was 46.5 months and 43.7% overall 5-year survival. Prognostic factors include extent of resection, tumor location, and histology grade. Radiation was not found to be a prognostic factor for overall survival.
Conclusion: This study is consistent with previous studies regarding the role of radiation in the management of MPNST. Prospective evaluation of adjuvant radiation will allow to more fully define the role of radiation in MPNST.
PMCID: PMC4233912  PMID: 25452937
malignant peripheral nerve sheath tumor; malignant schwannoma; neurofibrosarcoma; neurogenic sarcoma; radiation therapy
8.  Ionizing Radiation and Glioblastoma Exosomes: Implications in Tumor Biology and Cell Migration12 
Translational Oncology  2013;6(6):638-648.
Exosomes are nanometer-sized lipid vesicles released ubiquitously by cells, which have been shown to have a normal physiological role, as well as influence the tumor microenvironment and aid metastasis. Recent studies highlight the ability of exosomes to convey tumor-suppressive and oncogenic mRNAs, microRNAs, and proteins to a receiving cell, subsequently activating downstream signaling pathways and influencing cellular phenotype. Here, we show that radiation increases the abundance of exosomes released by glioblastoma cells and normal astrocytes. Exosomes derived from irradiated cells enhanced the migration of recipient cells, and their molecular profiling revealed an abundance of molecules related to signaling pathways important for cell migration. In particular, connective tissue growth factor (CTGF) mRNA and insulin-like growth factor binding protein 2 (IGFBP2) protein levels were elevated, and coculture of nonirradiated cells with exosomes isolated from irradiated cells increased CTGF protein expression in the recipient cells. Additionally, these exosomes enhanced the activation of neurotrophic tyrosine kinase receptor type 1 (TrkA), focal adhesion kinase, Paxillin, and proto-oncogene tyrosine-protein kinase Src (Src) in recipient cells, molecules involved in cell migration. Collectively, our data suggest that radiation influences exosome abundance, specifically alters their molecular composition, and on uptake, promotes a migratory phenotype.
PMCID: PMC3890698  PMID: 24466366
9.  Predictive value of tumor recurrence using urinary vascular endothelial factor levels in patients receiving radiation therapy for Glioblastoma Multiforme (GBM) 
Biomarker Research  2013;1:29.
Glioblastoma Multiforme (GBM) is the most common primary malignant tumor of the central nervous system. Standard of care includes maximal resection followed by chemoradiotherapy. Tumors need adequate perfusion and neovascularization to maintain oxygenation and for removal of wastes. Vascular endothelial growth factor (VEGF) is a well characterized pro-angiogenic factor. We hypothesized that the increases in urinary VEGF levels would occur early in the course of tumor recurrence or progression. We examine the feasibility of collecting and analyzing urinary VEGF levels in a prospective, multi-institutional trial (Radiation Therapy Oncology Group, RTOG, 0611) as well as the role of VEGF as a marker of tumor recurrence.
We evaluated VEGF levels in urine specimens collected post-operatively, at the conclusion of radiation therapy (RT) and one month following RT. Urinary VEGF levels were correlated with tumor progression at one year. VEGF levels were measured by enzyme-linked immunosorbant assay in urine specimens and normalized to urinary creatinine levels. Sample size was determined based on a 50% 1-year recurrence rate. With a sensitivity and specificity of 80%, the expected 95% confidence interval was (0.69, 0.91) with 100 patients. A failure was defined as documented disease progression, recurrence or death before one year.
202 patients were enrolled between February-2006 and October-2007. Four patients were ineligible as they did not receive RT. Of the remaining 198 patients, 128 had all three samples collected. In this group, 35 patients (27.3%) did not progress, 89 (69.5%) had progression and 4 (3.1%) died without evidence of progression. Median VEGF levels at baseline were 52.9 pg/mg Cr (range 0.2- 15,034.4); on the last day of RT, 56.6 (range 0–2,377.1); and at one month follow-up, 70.0 (range 0.1-1813.2). In patients without progression at 1-year, both baseline VEGF level and end of RT VEGF level were lower than those of patients who progressed: 40.3 (range 0.2-350.8) vs. 59.7 (range 1.3-15,034.4) and 41.8 (range 0–356.8) vs. 69.7 (range 0–2,377.1), respectively. This did not reach statistical significance. Comparison of the change in VEGF levels between the end of RT and one month following RT, demonstrated no significant difference in the proportions of progressors or non-progressors at 1-year for either the VEGF increased or VEGF decreased groups.
Urine can be collected and analyzed in a prospective, multi-institutional trial. In this study of patients with GBM a change in urinary VEGF levels between the last day of RT and the one month following RT did not predict for tumor progression by one year.
PMCID: PMC4177620  PMID: 24252135
VEGF (vascular endothelial factor); Glioblastoma Multiforme (GBM); Urinary biomarker; Radiation therapy; Tumor recurrence
10.  Chemoirradiation for Glioblastoma Multiforme: The National Cancer Institute Experience 
PLoS ONE  2013;8(8):e70745.
Standard treatment for glioblastoma (GBM) is surgery followed by radiation (RT) and temozolomide (TMZ). While there is variability in survival based on several established prognostic factors, the prognostic utility of other factors such as tumor size and location are not well established.
Experimental Design
The charts of ninety two patients with GBM treated with RT at the National Cancer Institute (NCI) between 1998 and 2012 were retrospectively reviewed. Most patients received RT with concurrent and adjuvant TMZ. Topographic locations were classified using preoperative imaging. Gross tumor volumes were contoured using treatment planning systems utilizing both pre-operative and post-operative MR imaging.
At a median follow-up of 18.7 months, the median overall survival (OS) and progression-free survival (PFS) for all patients was 17.9 and 7.6 months. Patients with the smallest tumors had a median OS of 52.3 months compared to 16.3 months among patients with the largest tumors, P = 0.006. The patients who received bevacizumab after recurrence had a median OS of 23.3 months, compared to 16.3 months in patients who did not receive it, P = 0.0284. The median PFS and OS in patients with periventricular tumors was 5.7 and 17.5 months, versus 8.9 and 23.3 months in patients with non-periventricular tumors, P = 0.005.
Survival in our cohort was comparable to the outcome of the defining EORTC-NCIC trial establishing the use of RT+TMZ. This study also identifies several potential prognostic factors that may be useful in stratifying patients.
PMCID: PMC3733728  PMID: 23940635
11.  A Phase I Study of the Combination of Sorafenib With Temozolomide and Radiation Therapy for the Treatment of Primary and Recurrent High-Grade Gliomas 
Despite recent advances in the management of high-grade and recurrent gliomas, survival remains poor. Antiangiogenic therapy has been shown to be efficacious in the treatment of high-grade gliomas both in preclinical models and in clinical trials. We sought to determine the safety and maximum tolerated dose of sorafenib when combined with both radiation and temozolomide in the primary setting or radiation alone in the recurrent setting.
Methods and Materials
This was a preclinical study and an open-label phase I dose escalation trial. Multiple glioma cell lines were analyzed for viability after treatment with radiation, temozolomide, or sorafenib or combinations of them. For patients with primary disease, sorafenib was given concurrently with temozolomide (75 mg/m2) and 60 Gy radiation, for 30 days after completion of radiation. For patients with recurrent disease, sorafenib was combined with a hypofractionated course of radiation (35 Gy in 10 fractions).
Cell viability was significantly reduced with the combination of radiation, temozolomide, and sorafenib or radiation and sorafenib. Eighteen patients (11 in the primary cohort, 7 in the recurrent cohort) were enrolled onto this trial approved by the institutional review board. All patients completed the planned course of radiation therapy. The most common toxicities were hematologic, fatigue, and rash. There were 18 grade 3 or higher toxicities and no grade 5 events. The median overall survival was 18 months for the entire population.
Sorafenib can be safely combined with radiation and temozolomide in patients with high-grade glioma and with radiation alone in patients with recurrent glioma. The recommended phase II dose of sorafenib is 200 mg twice daily when combined with temozolomide and radiation and 400 mg with radiation alone. To our knowledge, this is the first publication of concurrent sorafenib with radiation monotherapy or combined with radiation and temozolomide.
PMCID: PMC3635494  PMID: 22687197
12.  A Phase II Study of Pegylated Interferon Alfa-2b (PEG-Intron®) in Children with Diffuse Intrinsic Pontine Glioma (DIPG) 
Cancer  2011;118(14):3607-3613.
Interferon-alpha is a cytokine with demonstrated activity in patients with supratentorial gliomas, but the ideal dose and schedule of administration is unknown. Studies suggest low-dose continuous exposure is more efficacious than intermittent high doses. We performed a Phase II study of PEG-Intron® in children with DIPG, a population with dismal survival despite decades of clinical investigation. The primary objective was to compare 2-year survival to a historical cohort treated with radiation therapy alone.
Patients received weekly subcutaneous PEG-Intron® at a dose of 0.3 μg/kg beginning 2–10 weeks after completing radiation therapy until disease progression. Patients were evaluated clinically and radiographically at regular intervals. Serum and urine were assayed for biomarkers prior to each cycle. Quality of life (QOL) evaluations were administered at baseline and prior to every other cycle of therapy to parents of patients ages 6–18 years.
Thirty-two patients (median age 5.3 years, range 1.8–14.8) enrolled and received a median of 7 cycles of therapy (range 1–70+). PEG-Intron® was well tolerated and no decrease in QOL scores was noted in the subset of patients tested. The two-year survival rate was 14%, which was not significantly improved compared to our historical cohort. However, median time to progression (TTP) was 7.8 months, which favorably compares to recent trials reporting TTP of 5 months in a similar population.
Although low dose PEG-Intron® therapy did not significantly improve 2-year survival in children with DIPG compared to a historical control population, it may delay time to progression.
PMCID: PMC3290731  PMID: 22086404
brainstem; glioma; interferon; pontine; children
13.  Biopsy Needle Artifact Localization in MRI-guided Robotic Transrectal Prostate Intervention 
Recently a number of robotic intervention systems for magnetic resonance image (MRI) guided needle placement in the prostate have been reported. In MRI-guided needle interventions, after a needle is inserted, the needle position is often confirmed with a volumetric MRI scan. Commonly used titanium needles are not directly visible in an MR image, but they generate a susceptibility artifact in the immediate neighborhood of the needle. This paper reports the results of a quantitative study of the relationship between the true position of titanium biopsy needle and the corresponding needle artifact position in MR images, thereby providing a better understanding of the influence of needle artifact on targeting errors. The titanium needle tip artifact extended 9 mm beyond the actual needle tip location with tendency to bend towards the scanner’s B0 magnetic field direction, and axially displaced 0.38 mm and 0.32 mm (mean) in scanner’s frequency and phase encoding direction, respectively.
PMCID: PMC3675798  PMID: 22481805
Needle artifact; prostate intervention; robotic intervention; transrectal biopsy
14.  Competitive but Not Allosteric mTOR Kinase Inhibition Enhances Tumor Cell Radiosensitivity1 
Translational Oncology  2013;6(3):355-362.
The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according γH2AX foci. PP242 exposure did not influence the initial level of γH2AX foci after irradiation but did significantly delay the dispersal of radiation-induced γH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.
PMCID: PMC3660805  PMID: 23730416
15.  Caloric restriction augments radiation efficacy in breast cancer 
Cell Cycle  2013;12(12):1955-1963.
Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.
PMCID: PMC3735710  PMID: 23708519
caloric restriction; breast cancer; radiation; IGF; tumor regression; cytotoxic therapy
16.  Genotype and Tumor Locus Determine Expression Profile of Pseudohypoxic Pheochromocytomas and Paragangliomas12 
Neoplasia (New York, N.Y.)  2013;15(4):435-447.
Pheochromocytomas (PHEOs) and paragangliomas (PGLs) related to mutations in the mitochondrial succinate dehydrogenase (SDH) subunits A, B, C, and D, SDH complex assembly factor 2, and the von Hippel-Lindau (VHL) genes share a pseudohypoxic expression profile. However, genotype-specific differences in expression have been emerging. Development of effective new therapies for distinctive manifestations, e.g., a high rate of malignancy in SDHB- or predisposition to multifocal PGLs in SDHD patients, mandates improved stratification. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggest novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies.
PMCID: PMC3612915  PMID: 23555188
17.  Correlation of Plasma FL Expression with Bone Marrow Irradiation Dose 
PLoS ONE  2013;8(3):e58558.
Ablative bone marrow irradiation is an integral part of hematopoietic stem cell transplantation. These treatment regimens are based on classically held models of radiation dose and the bone marrow response. Flt-3 ligand (FL) has been suggested as a marker of hematopoiesis and bone marrow status but the kinetics of its response to bone marrow irradiation has yet to be fully characterized. In the current study, we examine plasma FL response to total body and partial body irradiation in mice and its relationship with irradiation dose, time of collection and pattern of bone marrow exposure.
C57BL6 mice received a single whole body or partial body irradiation dose of 1–8 Gy. Plasma was collected by mandibular or cardiac puncture at 24, 48 and 72 hr post-irradiation as well as 1–3 weeks post-irradiation. FL levels were determined via ELISA assay and used to generate two models: a linear regression model and a gated values model correlating plasma FL levels with radiation dose.
At all doses between 1–8 Gy, plasma FL levels were greater than control and the level of FL increased proportionally to the total body irradiation dose. Differences in FL levels were statistically significant at each dose and at all time points. Partial body irradiation of the trunk areas, encompassing the bulk of the hematopoietically active bone marrow, resulted in significantly increased FL levels over control but irradiation of only the head or extremities did not. FL levels were used to generate a dose prediction model for total body irradiation. In a blinded study, the model differentiated mice into dose received cohorts of 1, 4 or 8 Gy based on plasma FL levels at 24 or 72 hrs post-irradiation.
Our findings indicate that plasma FL levels might be used as a marker of hematopoietically active bone marrow and radiation exposure in mice.
PMCID: PMC3591371  PMID: 23505536
18.  Molecular Profiling Indicates Orthotopic Xenograft of Glioma Cell Lines Simulate a Subclass of Human Glioblastoma 
Cell line models have been widely used to investigate glioblastoma multiforme (GBM) pathobiology and in the development of targeted therapies. However, GBM tumors are molecularly heterogeneous and how cell lines can best model that diversity is unknown. In this report, we investigated gene expression profiles of three preclinical growth models of glioma cell lines, in vitro and in vivo as subcutaneous and intracerebral xenografts to examine which cell line model most resembles the clinical samples. Whole genome DNA microarrays were used to profile gene expression in a collection of 25 high grade glioblastomas, and comparisons were made to profiles of cell lines under three different growth models. Hierarchical clustering revealed three molecular subtypes of the glioblastoma patient samples. Supervised learning algorithm, trained on glioma subtypes predicted the intracranial cell line model with one glioma subtype (r = 0.68; 95% bootstrap CI −0.41, 0.46). Survival analysis of enriched gene sets (p < 0.05) revealed 19 biological categories (146 genes) belonging to neuronal, signal transduction, apoptosis, and glutamate mediated neurotransmitter activation signals that are associated with poor prognosis in this glioma subclass. We validated the expression profiles of these gene categories in an independent cohort of patients from “The Cancer Genome Atlas” project (r = 0.62, 95% bootstrap CI: −0.42, 0.43). We then used this data to select and inhibit a novel target (glutamate receptor) and showed that LY341595 a glutamate receptor specific antagonist could prolong survival in intracerebral tumor-implanted mice in combination with irradiation, providing an in vivo cell line system of preclinical studies.
PMCID: PMC3164941  PMID: 21595825
Supervised model; glioblastoma; orthotopic model; cell lines; xenograft
To identify, within the framework of a current Phase I trial, whether factors related to intraprostatic cancer lesions (IPLs) or individual patients predict the feasibility of high-dose intraprostatic irradiation.
Methods and Materials
Endorectal coil MRI scans of the prostate from 42 men were evaluated for dominant IPLs. The IPLs, prostate, and critical normal tissues were contoured. Intensity-modulated radiotherapy plans were generated with the goal of delivering 75.6 Gy in 1.8-Gy fractions to the prostate, with IPLs receiving a simultaneous integrated boost of 3.6 Gy per fraction to a total dose of 151.2 Gy, 200% of the prescribed dose and the highest dose cohort in our trial. Rectal and bladder dose constraints were consistent with those outlined in current Radiation Therapy Oncology Group protocols.
Dominant IPLs were identified in 24 patients (57.1%). Simultaneous integrated boosts (SIB) to 200% of the prescribed dose were achieved in 12 of the 24 patients without violating dose constraints. Both the distance between the IPL and rectum and the hip-to-hip patient width on planning CT scans were associated with the feasibility to plan an SIB (p = 0.002 and p = 0.0137, respectively).
On the basis of this small cohort, the distance between an intraprostatic lesion and the rectum most strongly predicted the ability to plan high-dose radiation to a dominant intraprostatic lesion. High-dose SIB planning seems possible for select intraprostatic lesions.
PMCID: PMC3580994  PMID: 20932672
IMRT; Prostate cancer; Dose escalation; Radiotherapy; Treatment planning
20.  The relationship of Vascular endothelial growth factor gene polymorphisms and clinical outcome in advanced gastric cancer patients treated with FOLFOX: VEGF polymorphism in gastric cancer 
BMC Cancer  2013;13:43.
The aim of this study is to evaluate the associations between vascular endothelial growth factor (VEGF) Single-nucleotide polymorphisms (SNPs) and clinical outcome in advanced gastric cancer patients treated with oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX).
Genomic DNA was isolated from whole blood, and six VEGF (−2578C/A, -2489C/T, -1498 T/C, -634 G/C, +936C/T, and +1612 G/A) gene polymorphisms were analyzed by PCR. Levels of serum VEGF were measured using enzyme-linked immunoassays.
Patients with G/G genotype for VEGF -634 G/C gene polymorphism showed a lower response rate (22.2%) than those with G/C or C/C genotype (32.3%, 51.1%; P = 0.034). Patients with the VEGF -634 G/C polymorphism G/C + C/C genotype had a longer progression free survival (PFS) of 4.9 months, compared with the PFS of 3.5 months for those with the G/G (P = 0.043, log-rank test). By multivariate analysis, this G/G genotype of VEGF -634 G/C polymorphism was identified as an independent prognostic factor (Hazard ratio 1.497, P = 0.017).
Our data suggest that G/G genotype of VEGF -634 G/C polymorphism is related to the higher serum levels of VEGF, and poor clinical outcome in advanced gastric cancer patients.
PMCID: PMC3573956  PMID: 23374220
VEGF; Polymorphism; Gastric cancer
21.  Astrocytes Enhance the Invasion Potential of Glioblastoma Stem-Like Cells 
PLoS ONE  2013;8(1):e54752.
Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133− counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133− cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133− cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.
PMCID: PMC3551925  PMID: 23349962
22.  Opposing Effects of Pigment Epithelium-Derived Factor on Breast Cancer Cell versus Neuronal Survival: Implication for Brain Metastasis and Metastasis-Induced Brain Damage 
Cancer Research  2012;72(1):144-153.
Brain metastases are a significant cause of cancer patient morbidity and mortality, yet preventative and therapeutic options remain an unmet need. The cytokine PEDF is downregulated in resected human brain metastases of breast cancer compared to primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a pro-survival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish that PEDF as both a metastatic suppressor and a neuroprotectant in the the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences.
PMCID: PMC3254209  PMID: 22215693
PEDF; breast cancer; brain metastasis; neurons; neuronal injury
23.  Preclinical models in radiation oncology 
As the incidence of cancer continues to rise, the use of radiotherapy has emerged as a leading treatment modality. Preclinical models in radiation oncology are essential tools for cancer research and therapeutics. Various model systems have been used to test radiation therapy, including in vitro cell culture assays as well as in vivo ectopic and orthotopic xenograft models. This review aims to describe such models, their advantages and disadvantages, particularly as they have been employed in the discovery of molecular targets for tumor radiosensitization. Ultimately, any model system must be judged by its utility in developing more effective cancer therapies, which is in turn dependent on its ability to simulate the biology of tumors as they exist in situ. Although every model has its limitations, each has played a significant role in preclinical testing. Continued advances in preclinical models will allow for the identification and application of targets for radiation in the clinic.
PMCID: PMC3549821  PMID: 23270380
Preclinical models; Radiation oncology; Radiosensitizer; Orthotopic xenograft model
24.  StRAP: An Integrated Resource for Profiling High-Throughput Cancer Genomic Data from Stress Response Studies 
PLoS ONE  2012;7(12):e51693.
The increasing availability and maturity of DNA microarray technology has led to an explosion of cancer profiling studies for identifying cancer biomarkers, and predicting treatment response. Uncovering complex relationships, however, remains the most challenging task as it requires compiling and efficiently querying data from various sources. Here, we describe the Stress Response Array Profiler (StRAP), an open-source, web-based resource for storage, profiling, visualization, and sharing of cancer genomic data. StRAP houses multi-cancer microarray data with major emphasis on radiotherapy studies, and takes a systems biology approach towards the integration, comparison, and cross-validation of multiple cancer profiling studies. The database is a comprehensive platform for comparative analysis of gene expression data. For effective use of arrays, we provide user-friendly and interactive visualization tools that can display the data and query results. StRAP is web-based, platform-independent, and freely accessible at
PMCID: PMC3524254  PMID: 23284744
25.  A phase Ib trial of the combination of the anti-angiogenic agent sunitinib and radiation therapy for patients with primary and metastatic central nervous system malignancies 
Cancer  2011;117(24):5548-5559.
This phase I trial evaluated sunitinib combined with radiation therapy (RT) for primary or metastatic central nervous system (CNS) malignancies.
Experimental Design
Eligible patients had CNS malignancies requiring a (minimum) two week course of RT. Sunitinib (37.5 mg) was administered daily for the duration of radiotherapy treatment with optional treatment extension of a month. Urine was collected at 3 time points for correlative biomarker studies. The primary endpoint was acute toxicity defined by the Common Toxicity Criteria version 3.
Fifteen patients were enrolled (12 CNS metastasis; 3 primary tumors). RT doses ranged from 14–70 Gy (1.8–3.5 Gy per fraction). Acute toxicities included hematologic, nausea, hyperglycemia, fatigue, hypocalcemia, and diarrhea. Six patients (40%) developed grade ≤2 toxicities. Grade 3 toxicities occurred in 7 (47%) patients including hematologic, fatigue, deep vein thrombosis, dysphasia, hyperglycemia and hyponatremia. No grade 3–5 hypertensive events or intracerebral hemorrhages occurred. Two Grade 5 adverse events attributed to disease progression occurred. Median follow up was 12.4 months. Two (13%) patients had partial response, 9 (60%) patients had stable disease and 2 (13%) patients had progressive disease. Six-month progression-free survival of brain metastasis patients was 58%. Grade 3 hematologic toxicity correlated with higher VEGF changes between baseline and completion of RT.
Continuous 37.5 mg sunitinib combined with RT in CNS malignancies yields acceptable toxicities and adverse events. Changes in urine VEGF were associated with hematologic toxicity and should be analyzed in a larger cohort. The feasibility, safety, and early response results warrant a Phase II trial.
PMCID: PMC3170674  PMID: 21647871
sunitinib; RT; central nervous system; anti-angiogenesis; brain metastases

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