Collaborative Trial In Relapsed Aggressive Lymphoma Research Articles

Comparison of overall survival of lisocabtagene maraleucel (liso‐cel) versus standard of care (SOC) adjusting for crossover in second‐line (2L) R/R large B‐cell lymphoma

Introduction: TRANSFORM (NCT03575351) is a multinational, phase 3 study that compared efficacy and safety of liso-cel versus SOC, including overall survival (OS) as a key secondary endpoint, as a 2L treatment for patients (pt) with relapsed/refractory (R/R) large B-cell lymphoma who were eligible for transplant. The study design permitted pts treated with 2L SOC to receive liso-cel as third line (3L) treatment if protocol-defined conditions were met and requested by investigators. The intention-to-treat (ITT) analysis of OS for liso-cel versus SOC in TRANSFORM addressed a specific research question that did not account for treatment switching. Additionally, as real-world use of chimeric antigen receptor (CAR) T cell therapies in 3L may be less common compared with TRANSFORM, the ITT analysis may provide a conservative estimate of the treatment effect for liso-cel versus SOC on OS. Here, we conducted a complementary analysis of OS to adjust for crossover and estimate the relative effect of liso-cel versus SOC in the absence of 3L CAR T cell therapy. Methods: An external control arm based on external data from the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) phase 3 study (NCT00137995) was created using the inverse probability of treatment weighting (IPTW) method. CORAL provided relatively mature survival data and reflects OS in the absence of CAR T cell therapies, which were not available at the time CORAL was conducted. After aligning inclusion/exclusion criteria of the 2 studies, 258 pts from CORAL were retained. To control for confounding, the 2L SOC cohort from CORAL was weighted to match the baseline distributions of prognostic factors and treatment effect modifiers of the liso-cel population in TRANSFORM. Relative efficacy was estimated by fitting a weighted Cox regression model to survival data for the external SOC arm from CORAL and the liso-cel arm from TRANSFORM. Results: After population adjustment via IPTW, the effective sample size (ESS) for SOC (CORAL) was 43.6% of the unadjusted sample size. The IPTW approach generally reduced imbalances in baseline characteristics between the 2 populations. The adjusted hazard ratio (HR; 95% CI) for liso-cel versus SOC was 0.50 (0.32–0.78), indicating that liso-cel was associated with a survival benefit in comparison with SOC in the absence of treatment switching (Table). Conclusions: These analyses demonstrated that liso-cel is associated with prolonged OS in comparison with SOC under a scenario of no 3L CAR T cell therapy, and the consideration of external data is a suitable alternative to existing statistical methods to adjust for treatment switching. The research was funded by: This study was funded by Bristol Myers Squibb. All authors contributed to and approved the abstract; writing and editorial assistance were provided by Emily Burke, PhD, of The Lockwood Group (Stamford, CT, USA), funded by Bristol Myers Squibb. Keyword: Cellular therapies Conflicts of interests pertinent to the abstract. F. Morschhauser Consultant or advisory role: Roche, Gilead, Abbvie Other remuneration: Membership on an entity's Board of Directors or advisory committees: Roche, Gilead, Novartis, BMS, Abbvie, Genmab, Miltenyi, Allogene therapeutics, AstraZeneca, Janssen H. Ghesquieres Consultant or advisory role: Gilead Sciences, Roche Honoraria: Gilead Sciences, Roche, Takeda Educational grants: Abbvie M. Kamdar Consultant or advisory role: AstraZeneca, Adaptive Biotechnologies, Abbvie, BeiGene, ADC Therapeutics, Syncopation Life Sciences, Bristol-Myers Squibb, Genetech/Roche Research funding: Novartis Other remuneration: Speakers' Bureau: Seattle Genetics F. F. Liu Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb C. Chandler Employment or leadership position: Conor Chandler is employed by Evidera, an independent research company that provides consulting services to life science companies; in his salaried position, he works with a variety of companies and is precluded from receiving payments or honoraria directly from these organizations for services rendered. Evidera received payment from Bristol Myers Squibb for the conduct of this study. A. Crotta Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb S. L. Klijn Employment or leadership position: Bristol Myers Squibb Consultant or advisory role: Bayer, Bristol Myers Squibb, Kite, Janssen - Payments made to OPEN Health Stock ownership: Bristol Myers Squibb A. Elsada Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb A. Previtali Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb I. Proskorovsky Employment or leadership position: Full-time employee of Evidera, which received funding from Bristol Myers Squibb to conduct this research. N. Schmitz Honoraria: Allogene Educational grants: Allogene, Miltenyi Other remuneration: Grants from Janssen, AstraZeneca, Abbvie J. S. Abramson Consultant or advisory role: Celgene, Novartis, Abbvie, Kite, Genetech, EMD Serono, MorphoSys, Alimera Sciences, Karyopharm Therapeutics, Bristol-Myers Squibb, C4 Therapeutics, BeiGene, AstraZeneca, Incyte, Bluebird Bio, Kymera, Epizyme, Genmab, MustangBio, Ono Pharmaceutical, Century Therapeutics, Lilly, Caribou Biosciences, Janssen, Takeda Honoraria: Regeneron, AstraZeneca, Janssen, Bristol-Myers Squibb Research funding: Seattle Genetics, AI Therapeutics, Bristol-Myers Squibb/Celgene

Indirect comparison of tisagenlecleucel and historical treatments for relapsed/refractory diffuse large B-cell lymphoma

AbstractNo head-to-head trials have compared the efficacy of tisagenlecleucel vs historical treatments for adults with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). This study indirectly compared the overall survival (OS) and overall response rate (ORR) associated with tisagenlecleucel, using data from the JULIET study (Study of Efficacy and Safety of CTL019 in Adult DLBCL Patients; #NCT02445248), vs historical treatments assessed in the CORAL (Collaborative Trial in Relapsed Aggressive Lymphoma) study follow-up population. To assess treatment effects in the treated (full analysis set [FAS]) and enrolled (intention-to-treat [ITT]) study populations, the JULIET FAS vs the CORAL follow-up FAS and JULIET ITT vs CORAL follow-up ITT populations were separately compared. Propensity score weighting using standardized mortality ratio weight (SMRW) and fine stratification weight (FSW) was used to compare OS and ORR, adjusting for baseline confounders. The results indicated that tisagenlecleucel was associated with a lower hazard of death among the FAS (adjusted hazard ratio [95% confidence interval], both FSW and SMRW, 0.44 [0.32, 0.59]) and ITT populations (FSW, 0.60 [0.44, 0.77]; SMRW, 0.57 [0.44, 0.73]; all, P < .001). Median OS was 12.48 months (JULIET) vs 4.34 to 4.40 months (CORAL) for the FAS, and 8.25 (JULIET) months vs 4.04 to 4.86 (CORAL) months for the ITT populations. Tisagenlecleucel was associated with a significantly higher ORR compared with historical treatments among the FAS (adjusted response rate difference [95% confidence interval], both FSW and SMRW, 36% [22%, 0.48%]; P < .001) and among the ITT populations after SMRW adjustment (11% [0%, 22%]; P = .043). This analysis supports that improved response and OS are achieved in patients with r/r DLBCL treated with tisagenlecleucel compared with those treated with alternative historical treatments.

The conditional survival analysis of relapsed DLBCL after autologous transplant: a subgroup analysis of LY.12 and CORAL

AbstractThe conditional survival of patients after frontline therapy for diffuse large B-cell lymphoma (DLBCL) approaches that of the general population once patients have survived disease free for 2 years. We sought to determine the conditional survival of patients among patients with relapsed de novo DLBCL successfully undergoing an autologous stem-cell transplant (ASCT) after first relapse. A total of 478 patients with de novo DLBCL, relapsed after 1 treatment from the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) and LY.12, were included. Patients were followed prospectively after ASCT for a median of 5.3 and 8.2 years, respectively. Individual patient data were analyzed for event-free survival (EFS) and overall survival. Standardized mortality ratios (SMRs) were estimated using French and Canadian life tables. The EFS estimates declined with each year of follow-up after ASCT and were 50.1% (95% confidence interval [CI]: 43.7% to 56.3%) and 43.4% (95% CI: 36.7% to 49.9%) at 5 years in CORAL and LY.12, respectively. The rate of death stabilized once patients achieved at least 4 years of EFS. Compared with the age- and sex-matched population, the SMR was significantly higher until 5 years after ASCT, when values were no longer statistically significant. Patients undergoing ASCT for relapsed DLBCL continue to have a higher rate of death at least until they have survived event free for 5 years. These observations can help to determine endpoints for future clinical trials in this population and for patient counseling. This trial was registered at www.clinicaltrials.gov as #NCT00078949.

Hexokinase II Expression As a Prognosis Marker in Diffuse Large B-Cell Lymphoma (DLBCL) Pre- and Post- the Incorporation of Rituximab to Standard CHOP Chemotherapy

Purpose: The results of the prospective multicenter phase III Collaborative trial in relapsed aggressive lymphoma (CORAL) study stressed the need to identify cellular pathways associated with poor clinical outcomes and to develop novel therapeutic strategies for DLBCL. We previously demonstrated that over-expression of HKII contribute to the acquirement of resistance to rituximab and chemotherapy agents in DLBCL B-cell lymphoma in pre-clinical models. Using gene expression profiling, we also demonstrated that higher mRNA levels of HKII correlated with a shorter progression free survival (PFS) and overall survival (OS) in previously untreated DLBCL in the pre- and post-rituximab era. In current study, we aimed to assess HKII expression by immunohistochemistry (IHC) and analyze the prognostic significance in 97 diffuse large B cell lymphoma patients pre- and post-rituximab era treated in a different Country/Institution. Methods: We retrospectively identified 97 newly diagnosed DLBCL treated at Fudan University Shanghai Cancer Center, Shanghai, China from March 2009 to December 2012. Clinical, demographic and pathological characteristics for each patient were collected. Patients were treated with standard doses of CHOP (N=41) or rituximab and CHOP (N=56). Pathological archived material was submitted for immunohistochemistry (IHC) for the determination of cell of origin (COO) (Han's algorithm) and HKII levels. Co-relation between HKII expression and COO or impact on overall response rate (ORR), PFS and OS were analyzed. Result: Demographic and clinical characteristic of gender, stage and IPI score distribution were similar between patients treated with CHOP or R+CHOP. As expected, the clinical outcome of DLBCL patients treated with R+CHOP was superior than those treated with CHOP. HKII expression was observed in 62 DLBCL patients (63.9%). There was a correlation between HKII expression and COO. Expression of HKII was observer in 84.6% of non-germinal center B-cell (non-GCB) DLBCL and 60% of GCB DLBCL (P=0.027). There was no correlation between HKII expression and ORR or PFS in DLBCL patients treated with CHOP or R-CHOP. In patients treated with CHOP chemotherapy, HKII expression was associated with an inferior OS, 100% (HKII negative) vs. 67% (HKII positive) (P=0.025). In rituximab era, additionally rituximab to CHOP results significantly improved OS in HKII positive patient compared to CHOP only patients (91% vs 67%, P=0.05), but R+CHOP did not change OS between HKII negative patients. Conclusion: As previously observed, HKII expression is more prevalent in non-GCB DLBCL patients. In pre-rituximab era, HKII expression was associated with a poor clinical outcome after CHOP chemotherapy. The negative effect of HKII expression appears to be overcome by the addition of rituximab to CHOP. (This work was supported by National Natural Science Foundation of China (81670177 and 81773203)). Disclosures No relevant conflicts of interest to declare.

How I manage patients with relapsed/refractory diffuse large B cell lymphoma.

SummaryDespite progress in the upfront treatment of diffuse large B cell lymphoma (DLBCL), patients still experience relapses. Salvage chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard second‐line treatment for relapsed and refractory (R/R) DLBCL. However, half of the patients will not be eligible for transplantation due to ineffective salvage treatment, and the other half will relapse after ASCT. In randomized studies, no salvage chemotherapy regimen is superior to another. The outcomes are affected by the secondary International Prognostic Index at relapse and various biological factors. The strategy is less clear in patients who require third‐line treatment. A multicohort retrospective non‐Hodgkin lymphoma research (SCHOLAR‐1) study conducted in 636 patients with refractory DLBCL showed an objective response rate of 26% (complete response 7%) to the next line of therapy with a median overall survival of 6·3 months. In the case of a response followed by transplantation, long‐term survival can be achieved in DLBCL patients. There is clearly a need for new drugs that improve salvage efficacy. Encouraging results have been reported with chimeric antigen receptor ‐T cell engineering, warranting further studies in a well‐defined control group of refractory patients. The Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) was used as a handy framework to build the discussion.

VEGF121, is predictor for survival in activated B-cell-like diffuse large B-cell lymphoma and is related to an immune response gene signature conserved in cancers.

Tumor microenvironment including endothelial and immune cells plays a crucial role in tumor progression and has been shown to dramatically influence cancer survival. In this study, we investigated the clinical relevance of the gene expression of key mediators of angiogenesis, VEGF isoforms 121, 165, and 189, and their receptors (VEGFR-1 and R-2) in a cohort of patients (n = 37) with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) from the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL). In patients with ABC-like DLBCL, but not in patients with GCB-like DLBCL, low VEGF121 expression was associated with a significantly better survival than in those with high VEGF121 level: 4-year overall survival at 100% vs 36% (p = .011), respectively. A specific gene signature including 57 genes was correlated to VEGF121 expression level and was analyzed using a discovery process in 1,842 GSE datasets of public microarray studies. This gene signature was significantly expressed in other cancer datasets and was associated with immune response. In conclusion, low VEGF121 expression level was significantly associated with a good prognosis in relapsed/refractory ABC-like DLBCL, and with a well-conserved gene-expression profiling signature related to immune response. These findings pave the way for rationalization of drugs targeting immune response in refractory/relapsed ABC-like DLBCL.

Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models.Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (Δψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs.Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole.Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. DisclosuresNo relevant conflicts of interest to declare.

Palbociclib (PD-0332991), a Potent Selective CDK4/6 Inhibitor, Overcomes Therapy-Resistant and Sensitizes Diffuse Large B-Cell Lymphoma Pre-Clinical Models to Chemo-Reagents

Background: Results from the Collaborative trial in relapsed aggressive lymphoma (CORAL) study highlighted the poor clinical outcome of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). We found that RRCL exhibited a unique cell cycle distribution (accumulation in S-phase) and an increase in cyclin D Kinase-4 (CDk4) mRNA/protein levels. In our current work, we evaluated Palbociclib, a novel orally bioavailable CDK4/6 inhibitor recently approved by the Food and Drug Administration for the treatment of breast cancer in rituximab-sensitive and -resistant lymphoma pre-clinical models.Methods: A panel of rituximab-sensitive cell lines (RSCL) or RRCL were exposed to escalating dosages of PD0332991 (0-80µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently RSCL or RRCL were exposed to PD0332991 or vehicle control and doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Changes in cell viability were determined by the Presto Blue assay. Coefficient of synergy was calculated using the CalcuSyn software.Results: Palbociclib induced dose- and time-dependent decrease in cell viability in both RSCL and RRCL. Of interest, palbociclib was more potent in RRCL than RSCL. The IC50 values were lower in RRCL Raji-4RH and RL-4RH (12.12µM and 13.43µM) than in RSCL Raji and RL cells (14.7µM and 19.99µM). In vitro exposure of lymphoma cells to palbociclib resulted in G1 cell cycle arrested and it was more evident in RRCL than in RSCL. Synergistic activity was observed by combining palbociclib with doxorubicin, dexamethasone and proteasome inhibitors (bortezomib and carfilzomib) in vitro. Our data suggests that, palbociclib is active against in rituximab-sensitive or resistant pre-clinical models. In addition, CDK4/6 inhibition resulted in synergistic effects with several chemotherapeutic agents. A better understanding in the molecular events triggered by palbociclib is necessary to better develop optimal combination strategies using this novel agent in future clinical trials. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund). DisclosuresCzuczman:Celgene: Employment; MorphoSys: Consultancy; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.

Vorinostat, a histone deacetylase (HDAC) inhibitor, promotes cell cycle arrest and re-sensitizes rituximab- and chemo-resistant lymphoma cells to chemotherapy agents.

Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab-chemotherapy-resistant preclinical models. A panel of rituximab-chemotherapy-sensitive (RSCL) and rituximab-chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were evaluated by Western blotting. Finally, cell lines were pre-exposed to vorinostat for 48h and subsequently exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine); changes in cell viability were determined by CellTiter-Glo(®) luminescence assay (Promega, Fitchburg, WI), and synergistic activity was evaluated using the CalcuSyn software. Vorinostat induced dose-dependent cell death in RRCL and in primary tumor cells. In addition, in vitro exposure of RRCL to vorinostat resulted in an increase in p21 and acetylation of histone H3 leading to G1 cell cycle arrest. Vorinostat exposure resulted in apoptosis in RSCL cell lines but not in RRCL. This finding suggests that in RRCL, vorinostat induces cell death by alternative pathways (i.e., irreversible cell cycle arrest). Of interest, vorinostat was found to reverse acquired chemotherapy resistance in RRCL. Our data suggest that vorinostat is active in RRCL with a known defective apoptotic machinery, it can active alternative cell death pathways. Given the multiple pathways affected by HDAC inhibition, vorinostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B cell lymphoma.

Up-Regulation of Hexokinase II (HK) Alters the Glucose Metabolism and Disrupts the Mitochondrial Potential in Aggressive B-Cell Lymphoma Contributing to Rituximab-Chemotherapy Resistance and Is a Clinically Relevant Target for Future Therapeutic Development

Abstract The results of the prospective multicenter phase III Collaborative trial in relapsed aggressive lymphoma (CORAL) study stressed the need to identify cellular pathways associated with poor clinical outcomes and to develop novel therapeutic strategies for relapsed/refractory aggressive lymphoma. To this end, we generated several pre-clinical models of rituximab resistance and previously reported that the acquirement of rituximab resistant was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. Hexokinase (HK) is the first step rate-limiting enzyme of the glycolytic pathway and mediates the phosphorylation of glucose to glucose-6-phosphate (G-6-P), promoting the glycolic pathway. Four isoforms of HK had been characterized. Hexokinase II (HKII), the predominant isoform overexpressed in cancer cells, has a catalytic and a biding domain. The catalytic domain of HKII promotes glycolysis while the binding domain protects tumor cell from mitochondrial outer membrane permeability (MOMP) and inhibits mitochondrial-mediated apoptosis. In our current work, we further study the biological and clinical significance of HKII expression in aggressive B-cell lymphoma. First, we analyzed differences in HKII gene and protein expression in a panel of rituximab-chemotherapy sensitive (RSCL) or resistant (RRCL) diffuse large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma (BL) cell lines. RRCL were found to have higher HKII mRNA and protein levels as determined by qRT-PCR and Western blotting. Subcellular fractionation of the mitochondria and cytoplasm revealed that most of the HKII was localized in the mitochondria in RRCL. Moreover, immunoprecipitation studies demonstrated that HKII was bound to the voltage dependent anion channel (VDAC). Of interest, by targeting HKII using either 1) a competitive substrate inhibitor (2DG), 2) a kinase inhibitor (lonidamine), 3) disruption its interaction with VDAC (HKII/VADC competitive binding peptide assay), or 4) complete silencing of HKII (using HKII siRNA interference or metformin) resulted in a decrease in the mitochondrial membrane potential (MMP), reduction in ATP production, decrease in cell viability and re-sensitization of RRCL to chemotherapy agents. To further assess the contribution of HKII to rituximab/chemotherapy resistance in a more clinically relevant setting, we analyzed gene expression profiling data from 401 patients with DLBCL treated with CHOP (N=181) or R+CHOP (N=220) as front-line therapy. High levels of HKII correlated with a poor prognosis in DLBCL patients. In patients receiving CHOP chemotherapy, high levels of HKII correlated with a shorter overall survival (OS) (P=0.0012). Similarly, high-HKII mRNA levels were associated with a shorter progression free survival (PFS) (P=0.039) and OS (P=0.043) in R-CHOP treated DLBCL. Our data suggest that over-expression of HKII contribute to the acquirement of resistance to rituximab and chemotherapy agents in DLBCL B-cell lymphoma. It appears that HKII binding to VDAC and to a lesser degree HKII catalytic domain, contribute to therapy resistance in lymphoma pre-clinical models. Clinically, higher levels of HKII correlate with a shorter PFS and OS in previously untreated DLBCL in the pre- and post-rituximab era. Targeting HKII appears to be a challenging but attractive strategy to potentiate the anti-tumor activity of current available monoclonal antibodies or chemotherapy agents in aggressive lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

MLN4924 An Investigational Inhibitor Of NEDD8 Activating Enzyme (NAE) Is Active In Pre-Clinical Models Of Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma

The results of the Collaborative trial in relapsed aggressive lymphoma (CORAL) study suggest that diffuse large B-cell lymphomas (DLBCL) that relapse or fail to respond to rituximab-chemotherapy in the front-line possess a more resistant disease and represent an emerging challenge for clinicians treating aggressive B-cell lymphomas. It also stresses the need to further study and define at the molecular level the mechanisms by which DLBCL are developing resistance to chemo-immunotherapy. We previously demonstrated that the ubiquitin-proteasome system (UPS) plays an important role in the development of rituximab-chemotherapy resistance. Targeting the UPS has become an important therapeutic strategy in relapsed/refractory DLBCL. MLN4924, a NAE inhibitor selectively blocks the UPS up-stream by preventing the activation of a subset of ubiquitin ligases known as cullin-ring ligases. We evaluated the activity of MLN4924 in a panel of rituximab-chemotherapy (RSCL) sensitive and resistant (RRCL) germinal center B-cell (GCB) and ABC-DLBCL cell lines. RSCL and RRCL were exposed to MLN4924 (0.5μM and 1.0μM) for 24-72 hrs. Changes in cell viability, cell cycle and expression of key regulatory proteins of the cell cycle, Bcl-2 family members, and the UPS were evaluated using the cell titer glo assay, flow cytometry and western blotting respectively. MLN4924 induced cell death in ABC-DLBCL cell lines (both RSCL and RRCL) and to a lesser degree in GCB-DLBCL cell lines. Anti-tumor activity plateau was seen after 48 hrs of drug exposure. In MLN4924 sensitive cells we consistently observed cell cycle arrest in G1 phase, down-regulation of Bcl-XL and PARP cleavage. We also observed down regulation of NEDD8 protein across all treated cell lines. Bcl-XL down-regulation appears to be regulated at the transcriptional level. MLN4924 exposure in vitro resulted in a decrease in Bcl-XL mRNA as determined by quantitative polymerase chain reaction (qPCR), perhaps due to the inhibition of NFkB activity as demonstrated in MLN4924-exposed cells by p65 co-localization studies using the imagestream technology. Our data suggests that MLN4924 is active in ABC-DLBCL by inducing cell cycle arrest in G1 phase and rendering lymphoma cells more susceptible to apoptosis. MLN4924 lowers the apoptotic threshold of lymphoma cells by negatively regulating Bcl-XL levels at the transcription level. Selective inhibition of the NFkB transcription factor most likely play a role in the down-regulation of Bcl-XL observed in all cell lines tested. Ongoing studies aimed to further define the molecular mechanisms of action of MLN4924 can potentially assist scientists and clinicians in the optimal design of clinical trials incorporating this agent in relapsed/refractory DLBCL patients. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

What Is the Role of Stem-Cell Transplantation for Follicular Non-Hodgkin Lymphoma in the Rituximab Era?

Defining the best treatment for follicular lymphoma (FL) is still a challenge, partly because the available treatment options are often associated with prolonged overall survival (OS) as a result of the long natural history of this disease. Many clinical studies have used progression-free survival (PFS) as their end point but suffer from insufficient follow-up or lack of statistical power to assess OS, which is the indisputably best end point. Until the introduction of rituximab, standard chemotherapy (varying from low-dose oral chlorambucil to intensive multidrug schedules) was effective in producing responses but did not result in a survival benefit. The introduction of autologous peripheral-blood stem-cell transplantation with its inherent low morbidity and mortality in comparison with autologous bone marrow transplantation resulted in three trials applying high-dose therapy as first-line treatment for FL. All these studies showed an improvement of antitumor efficacy, but none had a significant impact on OS. This is most likely the result of the inability to select patients who might benefit most from this procedure or, again, insufficient follow-up. Patients who experience relapse, especially early relapse, have shown that they have a lymphoma with a clinically less benign behavior. In this patient population, several phase II studies suggested an improved survival, which was confirmed by the only randomized study in relapsed FL that showed a significantly improved OS in patients who underwent transplantation as compared with standard chemotherapy. Several authors have described a relatively high incidence of myelodysplasia, attributed to the high-dose therapy. However, this may also be the result of extensive pretreatment, suggesting that high-dose therapy might be of greater benefit earlier in the treatment course, as opposed to after multiple relapses. After the introduction of rituximab, several groups chose to investigate whether OS was improved with the incorporation of rituximab in standard-dose, first-line treatment of FL. The general conclusion is that PFS was improved in all, but OS was prolonged in only some, of these trials. Likewise, incorporating rituximab into highdose chemotherapy as part of first-line therapy resulted in better PFS but an OS benefit was observed in only one such trial. In the article that accompanies this editorial, Pettengel et al describe the results of the randomized European Group for Blood and Marrow Transplantation (EBMT) LYM-1 study. Rituximab-naive patients with relapsed chemosensitive FL were randomly assigned to analyze the efficacy of in vivo purging and maintenance therapy with rituximab in combination with high-dose therapy and autologous stem-cell transplantation. In 7 years, 280 patients were included. The authors conclude that in vivo purging did not improve outcome; however, maintenance therapy did improve PFS, but not OS at the time of this report. This study was not designed to assess the value of high-dose therapy because all patients received this treatment, which the authors considered standard therapy in relapsed FL. Although an important observation, it is important to realize that the patients in the EBMT LYM-1 study all were rituximab naive, which limits conclusions on current applicability. In addition, from the French Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) study, we have learned that the results of high-dose therapy in patients with diffuse large B-cell lymphoma who experience relapse after first-line rituximab-containing therapy are significantly inferior, especially in the situation of an early relapse. Whether this is also the case in FL is an open question, but data from another French study in FL may be reassuring that this is not necessarily the case in FL. We can conclude that high-dose therapy changes the natural behavior of FL in relapsed disease and, probably, also in first-line therapy if PFS is a convincing end point. However, it is not clear that PFS is the best end point in FL. How can we improve the outlook for patients with FL? One approach is to wait for the current studies to mature and report OS data. Alternative options are new and more effective monoclonal antibodies or monoclonals conjugated to either radionuclides or toxins. Currently, there are no randomized data in support of these expensive options. Is allogeneic transplantation the panacea? Although complicated by a high treatment-related mortality (TRM), myeloablative transplants resulted in convincing evidence of cure in patients for whom other intensive therapies had failed. With the introduction of nonmyeloablative transplants, the decreased TRM resulted in better outcomes, even in patients who had previously undergone autograft. The high TRM after allografting in comparison with autografting limits its role in first-line salvage. It remains to be seen whether tandem transplantation combining the efficacy of highdose therapy with the immunological graft versus lymphoma effect will lead to the desired more favorable outcome. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 13 MAY 1 2013

Is there any role for transplantation in the rituximab era for diffuse large B-cell lymphoma?

Salvage chemotherapy followed by high-dose therapy and autologous stem cell transplantation is the standard of treatment for chemosensitive relapses in diffuse large B-cell lymphoma. The addition of rituximab to chemotherapy has improved the response rate and failure-free survival after first-line treatment and relapses. Fewer relapses are expected, although there is no consensus on the best salvage regimen. The intergroup Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) set the limits for this standard of treatment after first comparing 2 salvage regimens: rituximab, ifosfamide, etoposide, and carboplatin (R-ICE) and rituximab, dexamethasone, aracytine, and cisplatin (R-DHAP). There was no difference in response rates or survivals between these salvage regimens. Several factors affected survival: prior treatment with rituximab, early relapse (< 12 months), and a secondary International Prognostic Index score of 2-3. For patients with 2 factors, the response rate to salvage was only 46%, which identified easily a group with poor outcome. Moreover, patients with an ABC subtype or c-MYC translocation responded poorly to treatment. More than 70% of patients will not benefit from standard salvage therapy, and continued progress is needed. Studies evaluating immunotherapy after transplantation, including allotransplantation, new conditioning regimens with radioimmunotherapy and other combinations of chemotherapy based on diffuse large B-cell lymphoma subtype, are discussed herein. Early relapses and/or patients refractory to upfront rituximab-based chemotherapy have a poor response rate and prognosis. A better biological understanding of these patients and new approaches are warranted.

Is there any role for transplantation in the rituximab era for diffuse large B-cell lymphoma?

Abstract Salvage chemotherapy followed by high-dose therapy and autologous stem cell transplantation is the standard of treatment for chemosensitive relapses in diffuse large B-cell lymphoma. The addition of rituximab to chemotherapy has improved the response rate and failure-free survival after first-line treatment and relapses. Fewer relapses are expected, although there is no consensus on the best salvage regimen. The intergroup Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) set the limits for this standard of treatment after first comparing 2 salvage regimens: rituximab, ifosfamide, etoposide, and carboplatin (R-ICE) and rituximab, dexamethasone, aracytine, and cisplatin (R-DHAP). There was no difference in response rates or survivals between these salvage regimens. Several factors affected survival: prior treatment with rituximab, early relapse (&lt; 12 months), and a secondary International Prognostic Index score of 2-3. For patients with 2 factors, the response rate to salvage was only 46%, which identified easily a group with poor outcome. Moreover, patients with an ABC subtype or c-MYC translocation responded poorly to treatment. More than 70% of patients will not benefit from standard salvage therapy, and continued progress is needed. Studies evaluating immunotherapy after transplantation, including allotransplantation, new conditioning regimens with radioimmunotherapy and other combinations of chemotherapy based on diffuse large B-cell lymphoma subtype, are discussed herein. Early relapses and/or patients refractory to upfront rituximab-based chemotherapy have a poor response rate and prognosis. A better biological understanding of these patients and new approaches are warranted.

The Germinal Center/Activated B-Cell Subclassification Has a Prognostic Impact for Response to Salvage Therapy in Relapsed/Refractory Diffuse Large B-Cell Lymphoma: A Bio-CORAL Study

To evaluate the prognostic value of the cell of origin (COO) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBLC), prospectively treated by rituximab, dexamethasone, high-dose cytarabine, and cisplatin (R-DHAP) versus rituximab, ifosfamide, carboplatin, and etoposide and followed by intensive therapy plus autologous stem-cell transplantation on the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) trial. Among the 396 patients included on the trial, histologic material was available for a total of 249 patients at diagnosis (n = 189 patients) and/or at relapse (n = 147 patients), which included 87 matched pairs. The patient data were analyzed by immunochemistry for CD10, BCL6, MUM1, FOXP1, and BCL2 expression and by fluorescent in situ hybridization for BCL2, BCL6 and c-MYC breakpoints. The correlation with survival data was performed by using the log-rank test and the Cox model. Characteristics of immunophenotype and chromosomal abnormalities were statistically highly concordant in the matched biopsies. In univariate analysis, the presence of c-MYC gene rearrangement was the only parameter to be significantly correlated with a worse progression-free survival (PFS; P = .02) and a worse overall survival (P = .04). When treatment interaction was tested, the germinal center B (GCB) -like DLBCL that was based on the algorithm by Hans was significantly associated with a better PFS in the R-DHAP arm. In multivariate analysis, independent prognostic relevance was found for the GCB/non-GCB the Hans phenotype interaction treatment (P = .04), prior rituximab exposure (P = .0052), secondary age-adjusted International Prognostic Index (P = .039), and FoxP1 expression (P = .047). Confirmation was obtained by gene expression profiling in a subset of 39 patients. COO remains a major and independent factor in relapsed/refractory DLBCL, with a better response to R-DHAP in GCB-like DLBCL. This needs confirmation by a prospective study.