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1.  Randomized Phase III Trial of ABVD Versus Stanford V With or Without Radiation Therapy in Locally Extensive and Advanced-Stage Hodgkin Lymphoma: An Intergroup Study Coordinated by the Eastern Cooperative Oncology Group (E2496) 
Journal of Clinical Oncology  2012;31(6):684-691.
Purpose
Although ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) has been established as the standard of care in patients with advanced Hodgkin lymphoma, newer regimens have been investigated, which have appeared superior in early phase II studies. Our aim was to determine if failure-free survival was superior in patients treated with the Stanford V regimen compared with ABVD.
Patients and Methods
The Eastern Cooperative Oncology Group, along with the Cancer and Leukemia Group B, the Southwest Oncology Group, and the Canadian NCIC Clinical Trials Group, conducted this randomized phase III trial in patients with advanced Hodgkin lymphoma. Stratification factors included extent of disease (localized v extensive) and International Prognostic Factors Project Score (0 to 2 v 3 to 7). The primary end point was failure-free survival (FFS), defined as the time from random assignment to progression, relapse, or death, whichever occurred first. Overall survival, a secondary end point, was measured from random assignment to death as a result of any cause. This design provided 87% power to detect a 33% reduction in FFS hazard rate, or a difference in 5-year FFS of 64% versus 74% at two-sided .05 significance level.
Results
There was no significant difference in the overall response rate between the two arms, with complete remission and clinical complete remission rates of 73% for ABVD and 69% for Stanford V. At a median follow-up of 6.4 years, there was no difference in FFS: 74% for ABVD and 71% for Stanford V at 5 years (P = .32).
Conclusion
ABVD remains the standard of care for patients with advanced Hodgkin lymphoma.
doi:10.1200/JCO.2012.43.4803
PMCID: PMC3574266  PMID: 23182987
2.  Gene Expression–Based Model Using Formalin-Fixed Paraffin-Embedded Biopsies Predicts Overall Survival in Advanced-Stage Classical Hodgkin Lymphoma 
Journal of Clinical Oncology  2012;31(6):692-700.
Purpose
Our aim was to reliably identify patients with advanced-stage classical Hodgkin lymphoma (cHL) at increased risk of death by developing a robust predictor of overall survival (OS) using gene expression measured in routinely available formalin-fixed paraffin-embedded tissue (FFPET).
Methods
Expression levels of 259 genes, including those previously reported to be associated with outcome in cHL, were determined by digital expression profiling of pretreatment FFPET biopsies from 290 patients enrolled onto the E2496 Intergroup trial comparing doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and Stanford V regimens in locally extensive and advanced-stage cHL. A model for OS separating patients into low- and high-risk groups was produced using penalized Cox regression. The model was tested in an independent cohort of 78 patients enriched for treatment failure but otherwise similar to patients in a population-based registry of patients treated with ABVD. Weighted analysis methods generated unbiased estimates of predictor performance in the population-based registry.
Results
A 23-gene outcome predictor was generated. The model identified a population at increased risk of death in the validation cohort. There was a 29% absolute difference in 5-year OS between the high- and low-risk groups (63% v 92%, respectively; log-rank P < .001; hazard ratio, 6.7; 95% CI, 2.6 to 17.4). The predictor was superior to the International Prognostic Score and CD68 immunohistochemistry in multivariate analyses.
Conclusion
A gene expression–based predictor, developed in and applicable to routinely available FFPET biopsies, identifies patients with advanced-stage cHL at increased risk of death when treated with standard-intensity up-front regimens.
doi:10.1200/JCO.2012.43.4589
PMCID: PMC3574267  PMID: 23182984
3.  Prospective Study of Sequential Reduction in Immunosuppression, Interferon Alpha-2B, and Chemotherapy for Posttransplantation Lymphoproliferative Disorder 
Transplantation  2008;86(2):215-222.
Background
Several interventions can cure posttransplant lymphoproliferative disease (PTLD); a sequential approach is usual, starting with reduction in immunosuppressives (RI). The efficacy of RI remains poorly defined, particularly in adults. We assessed an algorithm starting with a defined course of RI in all patients, escalating to interferon (IFN) alpha2b, and finally to chemotherapy, in a prospective multicenter phase II study of adult solid organ transplant recipients. The design predated rituximab.
Methods
Reduction in immunosuppressives: cyclosporine or tacrolimus reduction by 50% for 2 weeks; a further 50% reduction for 1 week if not in complete remission (CR). Intravenous acyclovir was given for the duration of all RI. Patients with less than CR, or any rejection, resumed immunosuppressives and proceeded to IFN 3 MIU/m2/day for up to 3 months; if less than CR, ProMACE-CytaBOM chemotherapy.
Results
Twenty patients were registered over 60 months; 16 patients with biopsy-proven PTLD were eligible (13 heart, 3 kidney recipients). Median age was 47 (24–75) years. Reduction in immunosuppressives resulted in only 1 of 16 partial responses (12.5%), no CR. Progressive disease occurred in 8 of 16 (50%) and 6 of 16 (38%) experienced rejection. Only 1 of 13 (7%) patients achieved durable CR with IFN. Seven eligible patients received ProMACE-CytaBOM chemotherapy, five of seven (67%) achieving CR, four of five durable beyond 2 years.
Conclusions
Reduction in immunosuppressives produced no CR, progressive disease and rejection were frequent; response to IFN was rare. A strong case can be made for adding rituximab to RI as initial therapy. Chemotherapy resulted in 57% durable CR, data that are relevant for the up to two thirds of PTLD patients who are refractory to rituximab.
doi:10.1097/TP.0b013e3181761659
PMCID: PMC4029101  PMID: 18645482
PTLD; Lymphoma; Immunosuppressive reduction; S9239
4.  The Efficacy and Tolerability of ABVD and Stanford V in Older Hodgkin Lymphoma Patients: A Comprehensive Analysis from the North American Intergroup Trial E2496 
British journal of haematology  2013;161(1):76-86.
SUMMARY
There is a lack of contemporary prospective data examining the ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) and Stanford V (SV; doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin, etoposide, prednisone) regimens in older Hodgkin lymphoma (HL) patients. Forty-four advanced-stage, older HL patients (aged ≥60 years) were treated on the randomized study, E2496. Toxicities were mostly similar between chemotherapy regimens, although 24% of older patients developed bleomycin lung toxicity (BLT), which occurred mainly with ABVD (91%). Further, the BLT-related mortality rate was 18%. The overall treatment-related mortality for older HL patients was 9% versus 0.3% for patients aged <60 years (p<0.001). Among older patients, there were no survival differences between ABVD and SV. According to age, outcomes were significantly inferior for older versus younger patients (5-year failure-free survival: 48% vs 74%, respectively, p=0.002; 5-year overall survival: 58% and 90%, respectively, p<0.0001), while time-to-progression (TTP) was not significantly different (5-year TTP: 68% versus 78%, respectively, p=0.37). Furthermore, considering progression and death without progression as competing risks, the risk of progression was not different between older and younger HL patients (5 years: 30% and 23%, respectively, p=0.30); however, the incidence of death without progression was significantly increased for older HL patients (22% versus 9%, respectively, p<0.0001). Thus, the marked HL age-dependent survival differences appeared attributable primarily to non-HL events.
doi:10.1111/bjh.12222
PMCID: PMC3906856  PMID: 23356491
Hodgkin lymphoma; elderly; treatment-related toxicity; bleomycin lung toxicity
5.  Combination of a selective activator of the glucocorticoid receptor Compound A with a proteasome inhibitor as a novel strategy for chemotherapy of hematologic malignancies 
Cell Cycle  2013;12(1):133-144.
Glucocorticoids are widely used for the treatment of hematological malignancies; however, their chronic use results in numerous metabolic side effects. Thus, the development of selective glucocorticoid receptor (GR) activators (SEGRA) with improved therapeutic index is important. GR regulates gene expression via (1) transactivation that requires GR homodimer binding to gene promoters and is linked to side effects and (2) transrepression-mediated via negative GR interaction with other transcription factors. Novel GR modulator Compound A (CpdA) prevents GR dimerization, retains glucocorticoid anti-inflammatory activity and has fewer side effects compared with glucocorticoids in vivo. Here we tested CpdA anticancer activity in human T- and B-lymphoma and multiple myeloma cells expressing GR and their counterparts with silenced GR. We found that CpdA in GR-dependent manner strongly inhibited growth and viability of human T-, B-lymphoma and multiple myeloma cells. Furthermore, primary leukemia cell cultures from T-ALL patients appeared to be equally sensitive to glucocorticoid dexamethasone and CpdA. It is known that GR expression is controlled by proteasome. We showed that pretreatment of lymphoma CEM and NCEB cells with proteasome-inhibitor Bortezomib resulted in GR accumulation and enhanced ligand properties of CpdA, shifting GR activity toward transrepression evaluated by inhibition of NFкB and AP-1 transcription factors. We also revealed remarkable GR-dependent cooperation between CpdA and Bortezomib in suppressing growth and survival of T- and B-lymphoma and multiple myeloma MM.1S cells. Overall, our data provide the rationale for novel GR-based therapy for hematological malignancies based on combination of SEGRA with proteasome inhibitors.
doi:10.4161/cc.23048
PMCID: PMC3570502  PMID: 23255118
glucocorticoid receptor; selective GR activator (SEGRA); lymphoma; leukemia; multiple myeloma; proteasome inhibitor
6.  Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma  
PLoS ONE  2013;8(11):e81333.
Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.
doi:10.1371/journal.pone.0081333
PMCID: PMC3842257  PMID: 24312289
7.  Statin-dependent activation of protein kinase C delta (PKCδ) in APL cells and induction of leukemic cell differentiation 
Leukemia & lymphoma  2012;53(9):1779-1784.
Statins are HMG-CoA reductase inhibitors, which block the conversion of HMG-CoA to mevalonate and have potent cholesterol lowering properties. Beyond their importance in generation of lipid lowering effects, the regulatory effects of statins on the mevalonate pathway have significant impact on multiple other cellular functions. There is now extensive evidence that statins have anti-inflammatory and anti-neoplastic properties, but the precise mechanisms by which such responses are generated are not well understood. In the present study we demonstrate that statins engage a member of the PKC family of proteins, PKCδ, in acute promyelocytic leukemia (APL) cells. Our studies show that atorvastatin and fluvastatin induce proteolytic activation of PKCδ in the APL NB4 cell line which expresses the t(15;17) translocation. Such engagement of PKCδ results in induction of its kinase domain and downstream regulation of pathways important for statin-dependent leukemia cell differentiation. Our studies show that the function of PKCδ is essential for statin-induced leukemic cell differentiation, as demonstrated by studies involving selective targeting of PKCδ using siRNAs. We also demonstrate that the potent enhancing effects of statins on ATRA-induced gene expression for CCL3 and CCL4 requires the function of PKCδ, suggesting a mechanism by which statins may promote ATRA-induced antileukemic responses. Altogether, our data establish a novel function for PKCδ as a mediator of statin-induced differentiation of APL cells and antileukemic effects.
doi:10.3109/10428194.2012.668287
PMCID: PMC3616499  PMID: 22356114
8.  Dasatinib therapy results in decreased B cell proliferation, splenomegaly, and tumor growth in a murine model of lymphoma expressing Myc and Epstein-Barr virus LMP2A 
Antiviral Research  2012;95(1):49-56.
Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8 weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.
doi:10.1016/j.antiviral.2012.05.003
PMCID: PMC3377840  PMID: 22609829
Burkitt lymphoma; dasatinib; Epstein-Barr virus (EBV); latent membrane protein 2A (LMP2A); Lyn; post-transplant lymphoproliferative diseases (PTLD)
9.  The genetic landscape of mutations in Burkitt lymphoma 
Nature genetics  2012;44(12):1321-1325.
Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.
doi:10.1038/ng.2468
PMCID: PMC3674561  PMID: 23143597
10.  Nano-Encapsulation of Arsenic Trioxide Enhances Efficacy against Murine Lymphoma Model while Minimizing Its Impact on Ovarian Reserve In Vitro and In Vivo 
PLoS ONE  2013;8(3):e58491.
Advances in cancer therapy have increased the rate of survival of young cancer patients; however, female lymphoma patients frequently face a temporary or permanent loss of fertility when treated with traditional cytotoxic agents. The potential loss of fertility is an important concern that can influence treatment decisions for many premenopausal cancer patients. The negative effect of chemotherapeutic agents and treatment protocols to patients’ fertility–referred to as fertotoxicity–are thus an increasingly important cancer survivorship issue. We have developed a novel nanoscale formulation of arsenic trioxide, a potent drug for treatment of hematological malignancies, and demonstrate that it has significantly better activity in a murine lymphoma model than the free drug. In parallel, we have developed a novel in vitro assay of ovarian follicle function that predicts in vivo ovarian toxicity of therapeutic agents. Our results reveal that the nanotherapeutic agent is not only more active against lymphoma, but is fertoprotective, i.e., it is much less deleterious to ovarian function than the parent drug. Thus, our in vitro assay allows rapid evaluation of both established and experimental anticancer drugs on ovarian reserve and can inform the selection of efficacious and fertility-sparing treatment regimens for reproductive-age women diagnosed with cancer.
doi:10.1371/journal.pone.0058491
PMCID: PMC3603968  PMID: 23526987
11.  Activity of Topotecan 21-Day Infusion in Patients with Previously Treated Large Cell Lymphoma: Long-term follow-up of an Eastern Cooperative Oncology Group Study (E5493) 
Leukemia & lymphoma  2012;53(6):1137-1142.
The purpose of this study was to determine the activity of topotecan given by 21-day continuous infusion in patients previously treated with one prior therapy for a diffuse large cell lymphoma or immunoblastic lymphoma. Patients with appropriate histology and measurable disease who had been treated with one prior chemotherapy regimen were eligible for study. Slides of tumor biopsies were submitted for central review of pathology. Patients were required to have ECOG performance status of 0, 1, or 2 and adequate bone marrow function. Patients were treated with continuous infusion topotecan, 0.4 mg/m2/day × 21 days. Therapy could be escalated to 0.5 and then 0.6 mg/m2/day in subsequent cycles if there was no dose limiting toxicity at the initial dose level. Patients were treated with 2 cycles after achieving complete response or until disease progression or unacceptable toxicity occurred. Thirty seven patients were enrolled, however, only 26 cases were eligible due to performance status of > 2 (2), more than one prior chemotherapy (1), and wrong histology on review (8). Due to the unexpectedly high ineligibility rate, two sets of analysis were done for all the 37 patients enrolled and for the 26 eligible patients, respectively. Of the 37 patients (15 males and 22 females), international prognostic index included 11% low risk, 30% low-intermediate risk; 46% high-intermediate risk, and 8% high risk. The median follow up was 77 months. A total of 136 cycles of therapy were given with a median of 3 cycles per patient. Grade 4 toxicities included: 14% grade 4 thrombocytopenia; 14% grade 4 granulocytopenia, 8% leukopenia, 3% each anemia, hemorrhage, infection, vomiting, thrombosis, liver toxicity, and neuromotor toxicity. The response analysis including all 37 patients showed 5 complete remissions (CR) and 4 partial remissions (PR) for a total response rate of 24% (90% two-stage confidence interval 13–39%). Median progression free survival (PFS) was 3.7 months, with one and two year PFS of 21% and 6% respectively (90% confidence interval 11–34% and 2–15%). Median overall survival (OS) was 10.5 months, with one and two year OS 41% and 27% respectively (90% confidence interval 27–53% and 16–39%). Analysis including only eligible patient showed similar response rates and survival outcomes. Single agent topotecan has moderate activity for previously treated high grade lymphoma equivalent to that of several newer agents, and should be considered for incorporation into multi-drug salvage chemotherapy programs.
doi:10.3109/10428194.2011.643406
PMCID: PMC3568670  PMID: 22111940
13.  Curcumin nanodisk-induced apoptosis in mantle cell lymphoma 
Leukemia & lymphoma  2011;52(8):1537-1543.
Mantle cell lymphoma (MCL) is a pre-germinal center neoplasm characterized by cyclin D1 overexpression resulting from t(11;14)(q13;q32). Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. In the present study, we investigated a novel drug delivery nanovehicle enriched with the bioactive polyphenol, curcumin (curcumin nanodisks; curcumin-ND). Cells treated with curcumin-ND showed a dose-dependent increase in apoptosis. This was accompanied by enhanced generation of reactive oxygen species (ROS). The antioxidant, N-acetylcysteine, inhibited curcumin-ND induced apoptosis, suggesting that ROS generation plays a role in curcumin action on MCL cells. Curcumin-ND decreased cyclin D1, pAkt, pIκBα, and Bcl2 protein. In addition, enhanced FoxO3a and p27 expression as well as caspase-9, -3, and poly(ADP-ribose) polymerase (PARP) cleavage were observed. Curcumin-ND treatment led to enhanced G1 arrest in two cultured cell models of MCL.
doi:10.3109/10428194.2011.584253
PMCID: PMC3321639  PMID: 21699455
Nanodisks; mantle cell lymphoma; curcumin; cell cycle; apoptosis
14.  Curcumin nanodisks: formulation and characterization 
Nanodisks (ND) are nanoscale, disk-shaped phospholipid bilayers whose edge is stabilized by apolipoproteins. In the present study, ND were formulated with the bioactive polyphenol, curcumin, at a 6:1 phospholipid:curcumin molar ratio. Atomic force microscopy revealed that curcumin-ND are particles with diameters <50 nm and thickness of a phospholipid bilayer. When formulated in ND, curcumin is water-soluble and gives rise to a characteristic absorbance spectrum with a peak centered at 420 nm. Fluorescence spectroscopy of curcumin-ND provided evidence of self-quenching. Incubation of curcumin-ND with empty-ND relieved the self-quenching, indicating redistribution of curcumin between curcumin loaded- and empty-ND. In HepG2 cells, curcumin-ND mediated enhanced cell growth inhibition compared to free curcumin. In a cell culture model of mantle cell lymphoma, curcumin-ND were a more potent inducer of apoptosis than free curcumin. The nanoscale size of the complexes, combined with their ability to solubilize curcumin, indicates ND may have in vivo therapeutic applications.
doi:10.1016/j.nano.2010.08.002
PMCID: PMC3030927  PMID: 20817125
Curcumin; Nanodisk; Phospholipid; Apolipoprotein; Delivery
15.  Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone Andrographolide, the active component of Andrographis paniculata 
Purpose
Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, anti-viral and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines.
Experimental Design
We studied the Burkitt p53 mutated Ramos cell line, the mantle-cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1 and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL.
Results
We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI) in the presence and absence of the antioxidant N-acetyl-L-cysteine (NAC), the glutathione-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by PARP cleavage and activation of caspases 3, 8 and 9. We measured BAX conformational change, and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEFBax−/−/Bak−/−), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and in patient samples.
Conclusions
Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted.
doi:10.1158/1078-0432.CCR-10-0883
PMCID: PMC2948634  PMID: 20798229
non-Hodgkin lymphoma; andrographolide; apoptosis; oxidative stress; ROS; caspase
16.  All trans retinoic acid nanodisks enhance retinoic acid receptor mediated apoptosis and cell cycle arrest in mantle cell lymphoma 
British journal of haematology  2010;150(2):158-169.
Summary
Mantle cell lymphoma (MCL) is characterized by translocation t(11;14)(q13;q32), aggressive clinical behaviour, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel biological pathways. All trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. The present study evaluated cell killing effects of ATRA-enriched nanoscale delivery particles, termed nanodisks (ND), on MCL cell lines. Results show that ATRA-ND induced cell death more effectively than naked ATRA (dimethyl sulphoxide) or empty ND. ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. The antioxidant, N-acetylcysteine, inhibited ATRA-ND induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 growth arrest, up-regulated p21and p27, and down regulated cyclin D1. At ATRA concentrations that induced apoptosis, expression levels of retinoic acid receptor-α (RARα) and retinoid X receptor-γ (RXRγ) were increased. Compared to naked ATRA, ATRA-ND significantly stimulated transcriptional activity of RARA in a model carcinoma cell line. Furthermore, the RAR antagonist, Ro 41-5253, inhibited ATRA-ND induced ROS generation and prevented ATRA-ND induced cell growth arrest and apoptosis. In summary, incorporation of ATRA into ND enhanced the biological activity of this retinoid in cell culture models of MCL.
doi:10.1111/j.1365-2141.2010.08209.x
PMCID: PMC2907750  PMID: 20507312
Nanodisks; Mantle Cell Lymphoma; ATRA; Reactive Oxygen Species; Apoptosis
17.  Hypoxia-Inducible Factor-1 α Expression Predicts Superior Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With R-CHOP 
Journal of Clinical Oncology  2010;28(6):1017-1024.
Purpose
Hypoxia-inducible factor (HIF) controls the expression of genes in response to hypoxia, as well as a wide range of other cellular processes. We previously showed constitutive stabilization of HIF-1α in the majority of patients with diffuse large B-cell lymphoma (DLBCL). To our knowledge, the prognostic significance of HIF in lymphoma has never been investigated.
Patients and Methods
We studied the immunohistochemical protein expression of HIF-1α on tissue microarrays from 153 patients with DLBCL treated in sequential cohorts with cyclophosphamide, doxorubicin, oncovin, and prednisone (CHOP) or rituximab-CHOP (R-CHOP) from 1999 to 2002. Results were correlated with patient outcome.
Results
Median follow-up for all patients was 80 months. Among all patients, HIF-1α was expressed in 62% of germinal center and 59% of non–germinal center patients. With HIF-1α analyzed as a dependent variable, there were no survival differences in CHOP-treated patients. In the R-CHOP group, however, HIF-1α protein expression correlated with significantly improved progression-free survival (PFS) and overall survival (OS). Five-year PFS for HIF-1α–positive patients was 71% v 43% for HIF-1α–negative patients (P = .0187), whereas 5-year OS was 75% and 54%, respectively (P = .025). In multivariate analysis with International Prognostic Index criteria, HIF-1α remained a significant predictor for PFS (P = .026) and OS (P = .043). Compared with other biomarkers, HIF-1α correlated only with BCL6 (P = .004). In terms of gene expression, we found several common gene associations of HIF-1α and the stromal-1 signature with genes predominantly involved in regulation of the extracellular matrix (eg, BGN, COL1A2, COL5A1, and PLOD2).
Conclusion
The expression of HIF-1α protein is an important independent favorable prognostic factor for survival in patients with DLBCL treated with R-CHOP.
doi:10.1200/JCO.2009.24.1893
PMCID: PMC2834428  PMID: 20048181
18.  Multicenter Analysis of 80 Solid Organ Transplantation Recipients With Post-Transplantation Lymphoproliferative Disease: Outcomes and Prognostic Factors in the Modern Era 
Journal of Clinical Oncology  2010;28(6):1038-1046.
Purpose
Adult post-transplantation lymphoproliferative disease (PTLD) has a reported 3-year overall survival (OS) of 35% to 40%. The impact of rituximab on the outcome of PTLD is not well defined.
Methods
We examined the clinical features and outcomes among a large cohort of solid organ transplantation (SOT) –related patients with PTLD who were recently treated at four Chicago institutions (from January 1998 to January 2008).
Results
Eighty patients with PTLD were identified who had a median SOT-to-PTLD time of 48 months (range, 1 to 216 months). All patients had reduction of immunosuppression as part of initial therapy, whereas 59 (74%) of 80 patients received concurrent first-line rituximab with or without chemotherapy. During 40-month median follow-up, 3-year progression-free survival (PFS) for all patients was 57%, and the 3-year overall survival (OS) rate was 62%. Patients who received rituximab-based therapy as part of initial treatment had 3-year PFS of 70% and OS 73% compared with 21% (P < .0001) and 33% (P = .0001), respectively, without rituximab. Notably, of all relapses, only 9% (4 of 34 patients) occurred beyond 12 months from PTLD diagnosis. On multivariate regression analysis, three factors were associated with progression and survival: CNS involvement (PFS, 4.70; P = .01; OS, 3.61; P = .04), bone marrow involvement (PFS, 2.95; P = .03; OS, 3.14; P = .03), and hypoalbuminemia (PFS, 2.96; P = .05; OS, 3.64; P = .04). Furthermore, a survival model by multivariate CART analysis that was based on number of adverse factors present (ie, 0, 1, ≥ 2) was formed: 3-year PFS rates were 84%, 66%, 7%, respectively, and 3-year OS rates were 93%, 68%, 11%, respectively (P < .0001).
Conclusion
This large, multicenter, retrospective analysis suggests significantly improved PFS and OS associated with early rituximab-based treatment in PTLD. In addition, clinical factors at diagnosis identified patients with markedly divergent outcomes.
doi:10.1200/JCO.2009.25.4961
PMCID: PMC2834429  PMID: 20085936
19.  The Novel Expanded Porphyrin, Motexafin Gadolinium, Combined with Ytrrium-90 Ibritumomab Tiuxetan for Relapsed/Refractory Non-Hodgkin Lymphoma: Pre-Clinical Findings and Results of a Phase I Trial 
Purpose
Therapeutic strategies to enhance the efficacy of radioimmunotherapy have not been explored. Motexafin gadolinium (MGd) is a novel anti-cancer agent that targets redox-dependent pathways and enhances sensitivity of tumor cells to ionizing radiation.
Experimental Design
We performed pre-clinical studies examining MGd combined with rituximab and/or radiation in lymphoma cells. We subsequently completed a phase I clinical trial combining escalating doses of MGd concurrently with standard yttrium-90 (90Y)-ibritumomab tiuxetan for patients with relapsed/refractory non-Hodgkin lymphoma.
Results
In HF1 lymphoma cells, MGd and rituximab resulted in synergistic cytotoxicity (combination index 0.757) through a mitochondrial-mediated caspase-dependent pathway, while cell death in Ramos and SUDHL4 cells was additive. MGd/rituximab combined with radiation (1–3Gy) resulted in additive apoptosis. Twenty-eight of 30 patients were evaluable on the phase I clinical trial. Median age was 65 years (47–87), and histologies were: marginal-zone (n=1), mantle-cell (n=3), diffuse large-cell (n=6), and follicular lymphoma (n=18). 86% of all patients were rituximab-refractory. Therapy was well-tolerated and no dose limiting toxicity was seen. Overall response rate (ORR) was 57% (complete remission (CR) 43%) with median time-to-treatment failure (TTF) of 10 months (1–48+) and median duration-of-response of 17 months. Of note, all responses were documented at 4 weeks. Furthermore, in rituximab-refractory follicular lymphoma (n=14), ORR was 86% (CR 64%) with median TTF of 14 months (2–48+).
Conclusions
This represents the first report of a novel agent to be combined safely concurrently with radioimmunotherapy. Further, tumor responses with 90Y-ibritumomab tiuxetan/MGd were prompt with a high rate of CRs, especially in rituximab-refractory follicular lymphoma.
doi:10.1158/1078-0432.CCR-09-0905
PMCID: PMC2763343  PMID: 19825958
lymphoma; radioimmunotherapy; rituximab-refractory; oxidative stress; MGd
20.  Glutathione Depletion Enhances Arsenic Trioxide-Induced Apoptosis in Lymphoma Cells Through Mitochondrial-Independent Mechanisms 
British journal of haematology  2010;150(3):365-369.
doi:10.1111/j.1365-2141.2010.08197.x
PMCID: PMC2910124  PMID: 20408844
non-Hodgkin lymphoma; arsenic trioxide; apoptosis; oxidative stress; ROS
21.  Hypoxia-Inducible Factor-Alpha Activation in Lymphoma and Relationship to the Thioredoxin Family 
British journal of haematology  2008;141(5):676-680.
Hypoxia-Inducible-Factors (HIFs) activate oncogenic pathways, while thioredoxins, including thioredoxin-1 (Trx1) and thioredoxin reductases-1 and -2 (TrxR1 and TrxR2), promote HIF-α stabilization. Elevated levels of thioredoxin or HIF have been associated with poor outcomes in solid tumors and each represent potential therapeutic targets. In lymphoma cell line immunoblotting studies, we found that Raji and SUDHL4 cells exhibited normoxic HIF-2α protein-stabilization. Furthermore, five lymphoma cell-lines showed increased TrxR1 expression, while only Namalwa, HF1, and SUDHL4 had Trx1 and TrxR2 activation. Utilizing tissue microarrays from diffuse large B-cell (DLBCL) and follicular lymphoma (FL) patient specimens (n=82), we found different frequency of HIF expression in FL versus DLBCL as well as differing HIF-1 versus HIF-2 expression within each histologic subgroup. Forty-four percent of DLBCL versus 11% of FL cases had moderate-to-high expression of both HIF-1α and HIF-2α (p=0.0017), while 56% of DLBCL and 32% of FL samples had at least low HIF-1α and HIF-2α expression (p=0.042). Survival analysis among newly-diagnosed DLBCL cases showed 44% 2-year event-free survival (EFS) and 67% overall survival (OS) with high HIF-1α and HIF-2α expression compared with 67% and 76%, respectively, (p=0.10 and p=0.64, respectively) without high expression. These data demonstrate that HIF and the thioredoxins are activated in lymphoma and that HIF expression may influence prognosis.
doi:10.1111/j.1365-2141.2008.07093.x
PMCID: PMC2894542  PMID: 18422776
22.  The Histone Deacetylase Inhibitor PCI-24781 Induces Caspase and ROS-Dependent Apoptosis Through NF-kB and is Synergistic with Bortezomib in Lymphoma Cells 
Purpose
We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase inhibitor (HDACi), PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) cell lines and primary lymphoproliferative (CLL/SLL) cells.
Experimental design
Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, while caspase-activation was determined by Western blot. NF-kB-related mRNAs were quantified by RT-PCR, NF-kB-related proteins by Western blotting, and NF-kB DNA binding-activity by electromobility shift assay. Finally, gene expression profiling (GEP) was analyzed.
Results
PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G0/G1 arrest, decreased S-phase, increased p21 protein, and increased ROS in HL and NHL cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all NHL lines (combination indices: 0.19-0.6) and was additive in HL and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation vs either agent alone. GEP showed that PCI-24781-alone significantly downregulated several antioxidant genes, proteasome components, and NF-kB pathway genes, effects which were enhanced further with bortezomib. RT-PCR confirmed downregulation of NF-kB1 (p105), c-Myc, and IkB-kinase subunits, while NF-kB DNA-binding activity was decreased.
Conclusion
We show that PCI-24781 results in increased ROS and NF-kB inhibition, leading to caspase-dependent apoptosis. We also demonstrate that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.
doi:10.1158/1078-0432.CCR-08-2365
PMCID: PMC2704489  PMID: 19417023
HDAC inhibition; reactive oxygen species; NFκB; apoptosis; lymphoma
23.  Yttrium-90 Ibritumomab Tiuxetan Doses Calculated to Deliver up to 15 Gy to Critical Organs May Be Safely Combined With High-Dose BEAM and Autologous Transplantation in Relapsed or Refractory B-Cell Non-Hodgkin's Lymphoma 
Journal of Clinical Oncology  2009;27(10):1653-1659.
Purpose
To determine the maximum-tolerated radiation-absorbed dose (RAD) to critical organs delivered by yttrium-90 (90Y) ibritumomab tiuxetan in combination with high-dose carmustine, etoposide, cytarabine, and melphalan (BEAM) chemotherapy with autologous transplantation.
Patients and Methods
Eligible patients had relapsed or refractory CD20+ non-Hodgkin's lymphoma (NHL). Individualized 90Y activities were based on dosimetry and were calculated to deliver cohort-defined RAD (1 to 17 Gy) to critical organs with three to six patients per cohort. The therapeutic dose of 90Y ibritumomab tiuxetan was followed by high-dose BEAM and autologous transplantation.
Results
Forty-four patients were treated. Thirty percent of patients had achieved less than a partial remission to their most recent therapy and would not have been eligible for autologous transplantation at most centers. The toxicity profile was similar to that associated with high-dose BEAM chemotherapy. Two dose-limiting toxicities occurred at the 17 Gy dose level, which made 15 Gy the recommended maximum-tolerated RAD. Although eight patients received at least twice the conventional dose of 0.4 mCi/kg, a weight-based strategy at 0.8 mCi/kg would have resulted in a wide range of RAD; nearly 25% of patient cases would have received 17 Gy or more, and many would have received less than 10 Gy. With a median follow-up of 33 months for all patients, the estimated 3-year progression-free and overall survivals were 43% and 60%, respectively.
Conclusion
Dose-escalated 90Y ibritumomab tiuxetan may be safely combined with high-dose BEAM with autologous transplantation and has the potential to be more effective than standard-dose radioimmunotherapy. Careful dosimetry is required to avoid toxicity and undertreatment.
doi:10.1200/JCO.2008.19.2245
PMCID: PMC2668971  PMID: 19255322
24.  Maintenance Rituximab After Cyclophosphamide, Vincristine, and Prednisone Prolongs Progression-Free Survival in Advanced Indolent Lymphoma: Results of the Randomized Phase III ECOG1496 Study 
Journal of Clinical Oncology  2009;27(10):1607-1614.
Purpose
To determine if maintenance rituximab (MR) after standard chemotherapy improves progression-free survival (PFS) in advanced-stage indolent lymphoma.
Patients and Methods
Patients with stage III-IV indolent lymphoma with responding or stable disease after cyclophosphamide, vincristine, and prednisone (CVP) chemotherapy were stratified by initial tumor burden, residual disease after CVP (minimal or gross), and histology, and randomly assigned to observation (OBS) or MR 375 mg/m2 once per week for 4 weeks every 6 months for 2 years. PFS was the primary end point.
Results
Three hundred eleven (282 with follicular lymphoma) evaluable patients who received CVP were randomly assigned to OBS (n = 158) or MR (n = 153). Best response improved in 22% MR versus 7% OBS patients (P = .00006). Toxicity was minimal in both study arms. Three-year PFS after random assignment was 68% MR versus 33% OBS (hazard ratio [HR] = 0.4; P = 4.4 × 10−10 [all patients]) and 64% MR v 33% OBS (HR = 0.4; P = 9.2 × 10−8 [patients with follicular lymphoma]). There was an advantage for MR regardless of Follicular Lymphoma International Prognostic Index score, tumor burden, residual disease, or histology. In multivariate analysis of MR patients, minimal disease after CVP was a favorable prognostic factor. OS at 3 years was 92% MR versus 86% OBS (HR = 0.6; log-rank one-sided P = .05) and, among patients with follicular lymphoma, OS was 91% MR versus 86% (HR = 0.6; log-rank one-sided P = .08). A trend favoring MR was observed among patients with high tumor burden (log-rank one-sided P = .03).
Conclusion
The E1496 study provides the first phase III data in untreated indolent lymphoma that MR after chemotherapy significantly prolongs PFS.
doi:10.1200/JCO.2008.17.1561
PMCID: PMC2668968  PMID: 19255334

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