Targeting regulatory T cells by E7777 enhances CD8 T-cell-mediated anti-tumor activity and extends survival benefit of anti-PD-1 in solid tumor models.

Introduction: Regulatory T cell (Treg)-targeting cancer immunotherapy aims to transiently deplete Treg cells in the tumor microenvironment, without affecting effector T cells (Teff), thus both enhancing anti-tumor activity and avoiding autoimmunity. This study evaluated whether adding E7777 (a new formulation of denileukin diftitox [DD]) improved the efficacy of anti-PD-1 antibody therapy. DD is a recombinant protein containing the hydrophobic and catalytic portions of diphtheria toxin fused to full-length human IL-2. E7777 has the same amino acid sequence and brief circulatory half-life as DD, but with greater purity and potency. Methods: Subcutaneous syngeneic murine solid tumor models (colon cancer CT-26 and liver cancer H22) were used to evaluate safety, efficacy, and overall survival with E7777 and anti-PD-1 antibodies, each administered as monotherapy or in concurrent or sequential combination. In Experiment 1, treatments were compared to assess anti-tumor activity at various time points, with tumors excised and dissociated and tumor leukocytes characterized. In Experiment 2, tumor growth, response, and overall survival were characterized for 100 days following a 3-week treatment. Results: E7777 administered in combination with anti-PD-1 led to significantly increased anti-tumor activity and durable, extended overall survival compared to either treatment alone. In both tumor models, the Treg cell infiltration induced by anti-PD-1 treatment was counterbalanced by co-treatment with E7777, suggesting potential synergistic activity. Combination therapy showed the most favorable results. Treatment with E7777 was safe and well-tolerated. Discussion: Combined E7777 and anti-PD-1 therapy was well tolerated and more effective than monotherapy with either drug.

Durvalumab as monotherapy and in combination therapy in patients with lymphoma or chronic lymphocytic leukemia: The FUSION NHL 001 trial.

BACKGROUND: Studies suggest that immune checkpoint inhibitors may represent a promising strategy for boosting immune responses and improving the antitumor activity of standard therapies in patients with relapsed/refractory hematologic malignancies. AIMS: Phase 1/2 FUSION NHL 001 was designed to determine the safety and efficacy of durvalumab, an anti-programmed death ligand 1 (PD-L1) antibody, combined with standard-of-care therapies for lymphoma or chronic lymphocytic leukemia (CLL). METHODS AND RESULTS: The primary endpoints were to determine the recommended phase 2 dose of the drugs used in combination with durvalumab (durvalumab was administered at the previously recommended dose of 1500 mg every 4 weeks) and to assess safety and tolerability. Patients were enrolled into one of four arms: durvalumab monotherapy (Arm D) or durvalumab in combination with lenalidomide ± rituximab (Arm A), ibrutinib (Arm B), or rituximab ± bendamustine (Arm C). A total of 106 patients with relapsed/refractory lymphoma were enrolled. All but two patients experienced at least one treatment-emergent adverse event (TEAE); those not experiencing a TEAE were in Arm C (diffuse large B-cell lymphoma [DLBCL]) and Arm D (DLBCL during the durvalumab monotherapy treatment period). No new safety signals were identified, and TEAEs were consistent with the respective safety profiles for each study treatment. Across the study, patients with follicular lymphoma (FL; n = 23) had an overall response rate (ORR) of 59%; ORR among DLBCL patients (n = 37) was 18%. Exploratory biomarker analysis showed that response to durvalumab monotherapy or combination therapy was associated with higher interferon-γ signature scores in patients with FL (p = .02). CONCLUSION: Durvalumab as monotherapy or in combination is tolerable but requires close monitoring. The high rate of TEAEs during this study may reflect on the difficulty in combining durvalumab with full doses of other agents. Durvalumab alone or in combination appeared to add limited benefit to therapy.

ROBUST: A Phase III Study of Lenalidomide Plus R-CHOP Versus Placebo Plus R-CHOP in Previously Untreated Patients With ABC-Type Diffuse Large B-Cell Lymphoma.

PURPOSE: Patients with the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) historically showed inferior survival with standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Phase II studies demonstrated that adding the immunomodulatory agent lenalidomide to R-CHOP improved outcomes in ABC-type DLBCL. The goal of the global, phase III ROBUST study was to compare lenalidomide plus R-CHOP (R2-CHOP) with placebo/R-CHOP in previously untreated, ABC-type DLBCL. METHODS: Histology and cell-of-origin type were prospectively analyzed by central pathology prior to random assignment and study treatment. Patients with ABC-DLBCL received lenalidomide oral 15 mg/d, days 1-14/21 plus standard R-CHOP21 versus placebo/R-CHOP21 for six cycles. The primary end point was progression-free survival (PFS) per independent central radiology review. RESULTS: A total of 570 patients with ABC-DLBCL (n = 285 per arm) were stratified by International Prognostic Index score, age, and bulky disease, and randomly assigned to R2-CHOP or placebo/R-CHOP. Baseline demographics were similar between arms. Most patients completed six cycles of treatment: 74% R2-CHOP and 84% placebo/R-CHOP. The most common grade 3/4 adverse events for R2-CHOP versus placebo/R-CHOP were neutropenia (60% v 48%), anemia (22% v 14%), thrombocytopenia (17% v 11%), and leukopenia (14% v 15%). The primary end point of PFS was not met, with a hazard ratio of 0.85 (95% CI, 0.63 to 1.14) and P = .29; median PFS has not been reached for either arm. PFS trends favoring R2-CHOP over placebo/R-CHOP were seen in patients with higher-risk disease. CONCLUSION: ROBUST is the first DLBCL phase III study to integrate biomarker-driven identification of eligible ABC patients. Although the ROBUST trial did not meet the primary end point of PFS in all patients, the safety profile of R2-CHOP was consistent with individual treatments with no new safety signals.

Biomarkers for Risk Stratification in Patients With Previously Untreated Follicular Lymphoma Receiving Anti-CD20-based Biological Therapy.

Follicular lymphoma (FL) is an indolent B-cell neoplasm of germinal center origin. Standard treatment regimens consist of anti-CD20 therapy with or without chemotherapy. While high response rates to initial therapy are common, patients ultimately relapse or have progressive disease. Clinical risk factors such as the Follicular Lymphoma International Prognostic Index (FLIPI) have been identified, but there is a need for prognostic and predictive biomarkers. We studied markers of lymphoma cells and tumor microenvironment by immunohistochemistry in tissue samples from patients enrolled in 1 of 4 phase 2 trials of anti-CD20-based biological therapy for previously untreated grades 1 to 2 or 3A FL. Results were correlated with progression-free survival (PFS) and PFS status at 24 months. The 4 trials included 238 patients (51.1% male, median age: 55 y) with stage III, IV, or bulky stage II disease. By FLIPI, 24.6% had low-risk, 56.8% had intermediate-risk, and 18.6% had high-risk disease. The outcome differed significantly for patients treated with lenalidomide and rituximab (CALGB 50803) compared with the other 3 trials (median: PFS not reached vs. 3.0 y, hazard ratio=3.47, 95% confidence interval: 2.11-5.72); therefore, data were stratified by clinical trial (CALGB 50803 vs. all others) and adjusted for FLIPI risk group. Among 154 patients with available tissue, interfollicular BCL6 positivity, interfollicular CD10 positivity, and elevated Ki67 proliferation index ≥30% within neoplastic follicles were each associated with inferior PFS and a high risk of the early event by PFS status at 24 months. We identify promising biomarkers for FL risk stratification that warrant further validation in phase 3 trials.

Population Pharmacokinetics of an Anti-PD-L1 Antibody, Durvalumab in Patients with Hematologic Malignancies.

BACKGROUND AND OBJECTIVES: Durvalumab, a human monoclonal antibody targeting programmed cell death ligand 1, has been approved for urothelial carcinoma and stage III non-small cell lung cancer by the US Food and Drug Administration and is being evaluated in various malignancies. The objective of this study was to develop a population-pharmacokinetic model of durvalumab in patients with various hematologic malignancies and to investigate the effects of demographic and disease factors on the pharmacokinetics in this population. METHODS: A total of 1812 concentrations from 267 patients with myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma were included in the analysis. RESULTS: The pharmacokinetics of durvalumab was adequately described by a two-compartment model with first-order elimination. A decrease in durvalumab clearance over time was mainly explained by incorporation of time-dependent changes in albumin (in all patients) and immunoglobulin G (in patients with multiple myeloma) into the model. For multiple myeloma, patients with immunoglobulin G ≥ 20 g/L showed a 30% lower area under the concentration-time curve at cycle 1 compared with patients with immunoglobulin G < 20 g/L. The impact of any baseline covariates on durvalumab pharmacokinetics did not appear to be clinically relevant. The pharmacokinetics of durvalumab in hematologic malignancies was generally consistent with previously reported pharmacokinetics in solid tumors. CONCLUSIONS: These results support the same dosing regimen (1500 mg every 4 weeks) for both solid tumors and hematologic malignancies from the perspective of adequate exposure. Additionally, total immunoglobulin G level could be a critical covariate for the pharmacokinetics of monoclonal antibodies in patients with multiple myeloma.

The prognostic significance of PFS24 in follicular lymphoma following firstline immunotherapy: A combined analysis of 3 CALGB trials.

Follicular lymphoma (FL) patients treated with firstline R-CHOP who experience progression of disease (POD) within 2 years have a shorter survival than those who do not have POD within 2 years. Whether this observation holds for patients treated initially with biologic immunotherapy alone is unknown. We performed a retrospective analysis of 174 patients pooled from three frontline rituximab (R)-based nonchemotherapy doublet trials: R-galiximab (Anti-CD80, CALGB 50402), R-epratuzumab (Anti-CD22, CALGB 50701), and R-lenalidomide (CALGB 50803) to determine outcomes of early progressors and risk factors for early POD, defined as progression within 24 months from study entry. Twenty-eight percent (48/174) of patients had early POD. After adjusting for the Follicular Lymphoma International Prognostic Index (FLIPI), patients with early POD from study entry had a worse OS compared with patients who did not progress within 2 years (HR = 4.33 (95% CI 1.50-12.5), P = 0.007). For early POD, the 2-year survival was 80% vs 99% for nonearly POD, and the 5-year survival was 74% vs 90%, respectively. These findings suggest that the adverse survival of patients with early POD may be independent of initial treatment modality.

Up-regulation of hexokinase II contributes to rituximab-chemotherapy resistance and is a clinically relevant target for therapeutic development.

In order to identify cellular pathways associated with therapy-resistant aggressive lymphoma, we generated rituximab-resistant cell lines (RRCL) and found that the acquirement of rituximab resistance was associated with a deregulation in glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. Hexokinase II (HKII), the predominant isoform overexpressed in cancer cells, has dual functions of promoting glycolysis as well as inhibiting mitochondrial-mediated apoptosis. We found that RRCL demonstrated higher HKII levels. Targeting HKII resulted in decreased mitochondrial membrane potential, ATP production, cell viability; and re-sensitization to chemotherapy agents. Analyzed gene expression profiling data from diffuse large B-cell lymphoma patients, high-HKII levels were associated with a shorter progression free survival (PFS) and/or overall survival (OS). Our data suggest that over-expression of HKII is associated with resistance to rituximab and chemotherapy agents in aggressive lymphoma and identifies this enzyme isoform as a potential therapeutic target.

A Phase II Trial of Rituximab Combined With Pegfilgrastim in Patients With Indolent B-cell Non-Hodgkin Lymphoma.

BACKGROUND: To explore the role of augmenting neutrophil function in B-cell lymphoma, we conducted a phase II study evaluating the safety and clinical efficacy of pegfilgrastim and rituximab in low-grade CD20+ B-cell non-Hodgkin lymphoma (B-NHL). PATIENTS AND METHODS: Twenty patients with indolent B-NHL were treated with rituximab (375 mg/m2) every other week for 4 doses, followed by every 2 months for 4 additional doses. Pegfilgrastim was administered subcutaneously 3 days before each dose of rituximab. Clinical activity and tolerability were assessed using standard criteria. Biologic monitoring included phenotype characteristics of the host neutrophils, changes in oxidative burst, and functional assays. RESULTS: The patient demographics included median age of 64 years, 70% were male, 70% had follicular lymphoma, and 90% had stage III-IV disease. The median number of previous therapies was 2 (range, 0-5); 90% had received previous anti-CD20 monoclonal antibody therapy. The addition of pegfilgrastim to rituximab did not increase rituximab-related toxicities. The overall response rate was 60% (12 of 20), with a complete response (CR) rate of 35% (7 of 20). The median progression-free survival (PFS) duration was 17.9 months (95% confidence interval, 9.9-27.6 months); the median overall survival was not reached. A shorter time-to-peak oxidative burst after the first dose of pegfilgrastim was associated with greater CR rates (P = .04) and longer PFS (P = .03). CONCLUSION: The pegfilgrastim-rituximab combination was well tolerated, with favorable outcomes compared with historical controls. A shorter time-to-peak oxidative burst was associated with higher CR rates and longer PFS. Our results support further evaluation of strategies that enhance the innate immune system to improve rituximab activity in B-NHL.

A Phase 2/3 Multicenter, Randomized, Open-Label Study to Compare the Efficacy and Safety of Lenalidomide Versus Investigator's Choice in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma.

Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator's choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL).Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype [germinal center B-cell (GCB) vs. non-GCB; determined by immunohistochemistry (IHC)] and then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis [GCB vs. activated B-cell (ABC)] using gene expression profiling (GEP) were exploratory endpoints.Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n = 54; GCB, n = 48) received ≥1 dose of lenalidomide or IC. Hematologic treatment-emergent adverse events with lenalidomide versus IC included neutropenia (42.6%; 36.4%), anemia (33.3%; 47.3%), thrombocytopenia (24.1%; 43.6%), and leukopenia (5.6%; 12.7%), respectively. Overall, lenalidomide-treated patients had an ORR of 27.5% versus 11.8% in IC (ORRs were similar regardless of IHC-defined DLBCL subtype). Median PFS was increased in patients receiving lenalidomide (13.6 weeks) versus IC (7.9 weeks; P = 0.041), with greater improvements in non-GCB patients (15.1 vs. 7.1 weeks, respectively; P = 0.021) compared with GCB (10.1 vs. 9.0 weeks, respectively; P = 0.550).Conclusions: The clinical benefit of lenalidomide monotherapy in DLBCL patients was more evident in the non-GCB subtype. Exploratory analyses suggest that this preferential benefit was more pronounced in the GEP-defined ABC population, demonstrating a need for additional studies of lenalidomide in DLBCL using GEP subtyping. Clin Cancer Res; 23(15); 4127-37. ©2017 AACR.

Mitotic catastrophe and cell cycle arrest are alternative cell death pathways executed by bortezomib in rituximab resistant B-cell lymphoma cells.

The ubiqutin-proteasome system (UPS) plays a role in rituximab-chemotherapy resistance and bortezomib (BTZ) possesses caspase-dependent (i.e. Bak stabilization) and a less characterized caspase-independent mechanism-of-action(s). Here, we define BTZ-induced caspase-independent cell death pathways. A panel of rituximab-sensitive (RSCL), rituximab-resistant cell lines (RRCL) and primary tumor cells derived from lymphoma patients (N = 13) were exposed to BTZ. Changes in cell viability, cell-cycle, senescence, and mitotic index were quantified. In resting conditions, RRCL exhibits a low-proliferation rate, accumulation of cells in S-phase and senescence. Exposure of RRCL to BTZ reduces cell senescence, induced G2-M phase cell-cycle arrest, and is associated with mitotic catastrophe. BTZ stabilized p21, CDC2, and cyclin B in RRCL and in primary tumor cells. Transient p21 knockdown alleviates BTZ-induced senescence inhibition, G2-M cell cycle blockade, and mitotic catastrophe. Our data suggest that BTZ can induce apoptosis or mitotic catastrophe and that p21 has a pivotal role in BTZ activity against RRCL.

18F-FDG PET for Measurement of Response and Prediction of Outcome to Relapsed or Refractory Mantle Cell Lymphoma Therapy with Bendamustine-Rituximab.

In a single-arm, phase 2 clinical trial, bendamustine-rituximab (BR) demonstrated an overall response rate of 82% among 45 patients with relapsed or refractory mantle cell lymphoma (MCL), with manageable tolerability. A prespecified 18F-FDG PET analysis was conducted to assess the predictive value of the metabolic response to BR compared with the response by International Working Group (IWG) criteria. METHODS: Adult patients with relapsed or refractory MCL underwent 18F-FDG PET at screening and after 6 cycles of BR therapy. Scans were reviewed by a central facility and scored using the 5-point Deauville scale, comparing uptake to the liver and mediastinum in up to 6 lesions, to determine metabolic response rates, indicated by negative posttreatment scans. Metabolic responses were compared with study outcomes assessed by IWG criteria. RESULTS: Complete 18F-FDG PET data were available for 32 of 45 patients. All patients had positive baseline scans, with baseline scores ranging from 4 to 5. Complete metabolic responses (CMR) were observed in 24 (75%) patients after 6 cycles of BR. Patients attaining a CMR had a 96% overall response rate by IWG criteria, with 62.5% achieving a complete response. Of the 8 patients not attaining a CMR, 6 responded to BR but none achieved a complete response. CMR was associated with a greater 1-y progression-free survival of 91.5%, compared with 12.5% without CMR; a longer median duration of response of 20.6 mo, compared with 7.8 mo; and improved overall survival at 1 y. 18F-FDG PET data from patients with refractory or advanced disease demonstrated CMR in more than half. CONCLUSION: Compared with positive end-of-treatment 18F-FDG PET, negative scans, indicating a CMR, were predictive of improved 1-y survival, duration of response, and overall survival for patients with relapsed or refractory MCL receiving BR.

Phase 1 trial of rituximab, lenalidomide, and ibrutinib in previously untreated follicular lymphoma: Alliance A051103.

Chemoimmunotherapy in follicular lymphoma is associated with significant toxicity. Targeted therapies are being investigated as potentially more efficacious and tolerable alternatives for this multiply-relapsing disease. Based on promising activity with rituximab and lenalidomide in previously untreated follicular lymphoma (overall response rate [ORR] 90%-96%) and ibrutinib in relapsed disease (ORR 30%-55%), the Alliance for Clinical Trials in Oncology conducted a phase 1 trial of rituximab, lenalidomide, and ibrutinib. Previously untreated patients with follicular lymphoma received rituximab 375 mg/m2 on days 1, 8, 15, and 22 of cycle 1 and day 1 of cycles 4, 6, 8, and 10; lenalidomide as per cohort dose on days 1 to 21 of 28 for 18 cycles; and ibrutinib as per cohort dose daily until progression. Dose escalation used a 3+3 design from a starting dose level (DL) of lenalidomide 15 mg and ibrutinib 420 mg (DL0) to DL2 (lenalidomide 20 mg, ibrutinib 560 mg). Twenty-two patients were enrolled; DL2 was determined to be the recommended phase II dose. Although no protocol-defined dose-limiting toxicities were reported, a high incidence of rash was observed (all grades 82%, grade 3 36%). Eleven patients (50%) required dose reduction, 7 because of rash. The ORR for the entire cohort was 95%, and the 12-month progression-free survival was 80% (95% confidence interval, 57%-92%). Five patients developed new malignancies; 3 had known risk factors before enrollment. Given the increased toxicity and required dose modifications, as well as the apparent lack of additional clinical benefit to the rituximab-lenalidomide doublet, further investigation of the regimen in this setting seems unwarranted. The study was registered with www.ClinicalTrials.gov as #NCT01829568.

High Body Mass Index in Elderly Patients With DLBCL Treated With Rituximab-Containing Therapy Compensates for Negative Impact of Male Sex.

BACKGROUND: The impact of patient body habitus and sex on outcomes in diffuse large B-cell lymphoma (DLBCL) remains controversial. We investigated the impact of body mass index (BMI), body surface area (BSA), age, and sex on clinical outcomes in patients with DLBCL treated in the rituximab era. PATIENTS AND METHODS: Patients with de novo DLBCL (n=1,386) diagnosed between June 2000 and December 2010 treated with rituximab-containing chemotherapy were identified from the NCCN Oncology Outcomes Database for Non-Hodgkin's Lymphoma. Progression-free survival (PFS) and overall survival (OS) at 3 years were analyzed based on sex, age, and baseline BMI/BSA. RESULTS: High BMI was associated with a lower risk of disease progression or death than low or normal BMI, whereas male sex was associated with poor clinical outcomes, especially among elderly patients (age >60 years). Compared with elderly women, elderly men experienced worse PFS (3-year hazard ratio [HR], 1.5) and OS (3-year HR, 1.6), but these differences diminished with increases in BMI and BSA. In multivariable analysis, normal BMI compared with high BMI was independently associated with poor outcomes (3-year PFS HR, 1.5; OS HR, 1.6) after adjusting for sex. Notably, only 13% of elderly men had BMI less than 25 kg/m2 and only 26% had BSA less than 2 m2 CONCLUSIONS: Analysis of unselected patients with DLBCL treated with rituximab-containing chemotherapy confirmed an age-dependent disadvantage to male sex in treatment outcomes, but this effect is abrogated by higher levels of BMI and BSA in most North American men.

Cardiotoxicity as indicated by LVEF and troponin T sensitivity following two anthracycline-based regimens in lymphoma: Results from a randomized prospective clinical trial.

Anthracycline-induced cardiotoxicity influences treatment selection and may negatively affect clinical outcomes in lymphoma patients. While epirubicin induced cardiotoxicity less often than the same dose of doxorubicin in breast cancer, higher doses of epirubicin are required in lymphoma regimens for equivalent efficacy. Whether a higher dosage of epirubicin also induces cardiotoxicity less often than doxorubicin in lymphoma remains unknown. We therefore administered 6-8 cycles of cyclophosphamide, vincristine and prednisone (CEpOP) +/- rituximab (R) with either epirubicin (CEpOP) or doxorubicin (CHOP) to patients (N=398) with untreated diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma grade 3 (FLG3). Left ventricular ejection fraction (LVEF) and high-sensitivity serum cardiac troponin T (HsTnT) were assessed at baseline and after 4 cycles of treatment. Epirubicin (70 mg/m2/dose) was equivalent to doxorubicin (50 mg/m2/dose) in terms of 3-year progression-free survival. The risk of decreased LVEF was similar between the two regimens. CEpOP+/-R induced HsTnT elevation less often than CHOP+/-R. We conclude that CEpOP+/-R is a more acceptable regimen with short-term efficacy similar to CHOP+/-R in lymphoma patients. Longer follow-up is needed to monitor the risk of cardiac dysfunction and determine whether differences in the induction of elevated HsTnT between epirubicin and doxorubicin justify changes in clinical practice.

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

PURPOSE: 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. METHODS: 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 48 h 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. RESULTS: 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. CONCLUSIONS: 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.

Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo.

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 µM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL.

Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL.

Obinutuzumab is a novel glycoengineered Type-II CD20 monoclonal antibody. CD20 is expressed in approximately 100% of children and adolescents with Burkitt lymphoma (BL) and 40% with precursor B-cell acute lymphoblastic leukaemia (pre-B-ALL). We evaluated the anti-tumour activity of obinutuzumab versus rituximab against rituximab-resistant (Raji 4RH) and -sensitive (Raji) BL and pre-B-ALL (U698-M) cells in vitro and in human BL or Pre-B-ALL xenografted mice. We demonstrated that obinutuzumab compared to rituximab significantly enhanced cell death against Raji 35·6 ± 3·1% vs. 25·1 ± 2·0%, (P = 0·001), Raji4RH 19·7 ± 2·2% vs. 7·9 ± 1·5% (P = 0·001) and U-698-M 47·3 ± 4·9% vs. 23·2 ± 0·5% (P = 0·001), respectively. Obinutuzumab versus rituximab also induced a significant increase in antibody-dependent cellular cytotoxicity (ADCC) with K562-IL15-41BBL expanded NK cells against Raji 73·8 ± 8·1% vs. 56·81 ± 4·6% (P = 0·001), Raji-4RH 40·0 ± 1·6% vs. 0·5 ± 1·1% (P = 0·001) and U-698-M 70·0 ± 1·6% vs. 45·5 ± 0·1% (P = 0·001), respectively. Overall survival in tumour xenografted mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to those receiving 30 mg/kg of rituximab in BL; Raji (P = 0·05), Raji4RH (P = 0·02) and U698-M (P = 0·03), respectively. These preclinical data suggest obinutuzumab is significantly superior to rituximab in inducing cell death, ADCC and against rituximab-sensitive/-resistant BL and pre-B-ALL xenografted mice. Taken together, these preclinical results provide evidence to suggest that future investigation of obinutuzumab is warranted in patients with relapsed/refractory CD20(+) BL and/or pre-B-ALL.

Circulating CD4(+)CXCR5(+) T Cells Exacerbate B Cell Antibody Production in Crohn's Disease Through IL-21 Secretion.

Interleukin-21 (IL-21) upregulation was observed in Crohn's disease (CD) patients and was shown to contribute to ongoing mucosal inflammation in CD patients through stabilizing Th1 cell differentiation and IFN-γ production. Given the role of IL-21 in mediating adaptive B cell antibody responses in healthy individuals, we examined the effect of IL-21 upregulation in B cell responses in patients with active CD, including ileum, ileocolonic and colon subtypes, defined by the primary site of CD involvement. We first observed an upregulation of blood plasma IL-21 concentration and IL-21 production from CD4(+) T cells in CD patients compared to healthy individuals. The IL-21-expressing T cells were more concentrated in the CD4(+)CXCR5(+) compartment, both in unstimulated medium and after stimulation with SEB. ICOS and PD-1 expressions were also concentrated in the CD4(+)CXCR5(+) subset in CD patients. Since peripheral blood CD4(+)CXCR5(+) T cell-mediated antibody secretion is IL-21-dependent, we examined the plasma antibody concentration in CD patients and healthy controls. We found that CD patients had significantly higher plasma Ig level than healthy patients, with no significant differences between different CD subtypes. Higher plasma IL-21 concentration and increased IL-21 production from CD4(+) T cells were directly correlated with higher plasma antibody levels. Moreover, we found that IL-21 and CD4(+)CXCR5(+) T cells can directly enhance B cell antibody response in CD patients. Depletion of secreted IL-21 by sIL-21R addition compromised the CD4(+)CXCR5(+) T cell-mediated increase in antibody production. Together, our results demonstrated a novel role of IL-21 in mediating B cell inflammation in CD development.

Phase II study of bendamustine combined with rituximab in relapsed/refractory mantle cell lymphoma: efficacy, tolerability, and safety findings.

In most cases of relapsed/refractory mantle cell lymphoma (MCL), patients respond to salvage therapy, though typically responses are partial and/or transient followed by disease progression, even with newer agents (e.g., ibrutinib). In this multicenter, open-label, single-arm, phase II study, patients with relapsed/refractory non-blastoid MCL received bendamustine 90 mg/m(2) (days 1 and 2) and rituximab 375 mg/m(2) (day 1) for 6 planned 28-day cycles. Functional imaging with 18-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) was conducted at baseline and after cycle 6. Forty-five patients were enrolled (median age, 70 years; 82 % stage IV disease; median number of prior chemotherapies, 2 [range, 1-4]), showing an overall response rate (ORR; primary efficacy measure) of 82 % (complete response [CR], 40 %; partial response, 42 %). In the 32 patients with complete 18F-FDG PET/CT data, 75 % achieved a complete metabolic response. Median duration of response was 1.6 years, 1-year progression-free survival was 67 %, and 3-year overall survival was 55 %. Main non-hematologic adverse events were nausea (69 %), fatigue (56 %), decreased appetite (42 %), constipation (38 %), diarrhea (36 %), vomiting (36 %), and decreased weight (31 %). Grade 3/4 neutropenia and lymphopenia occurred in 44 and 89 % of patients, respectively. ORR and CR rate compared favorably with single-agent ibrutinib (ORR, 67 %; CR, 23 %); bendamustine-rituximab is an effective therapy with manageable toxicity in relapsed/refractory MCL.

Randomized Trial of Lenalidomide Alone Versus Lenalidomide Plus Rituximab in Patients With Recurrent Follicular Lymphoma: CALGB 50401 (Alliance).

PURPOSE: Lenalidomide and rituximab (LR) are active agents in follicular lymphoma (FL). Combination regimens have not been previously assessed in randomized studies. PATIENTS AND METHODS: The Cancer and Leukemia Group B (Alliance) 50401 trial is a randomized phase II trial studying rituximab (375 mg/m(2) weekly for 4 weeks), lenalidomide (15 mg per day on days 1 to 21, followed by 7 days of rest, in cycle 1 and then 20 mg per day on days 1 to 21, followed by 7 days of rest, in cycles 2 to 12), or LR. The rituximab-alone arm was discontinued as a result of poor accrual. Eligibility included recurrent FL and prior rituximab with time to progression of ≥ 6 months from last dose. Aspirin or heparin was recommended for patients at high thrombosis risk. RESULTS: Ninety-one patients (lenalidomide, n = 45; LR, n = 46) received treatment; median age was 63 years (range, 34 to 89 years), and 58% were intermediate or high risk according to the Follicular Lymphoma International Prognostic Index. In the lenalidomide and LR arms, grade 3 to 4 adverse events occurred in 58% and 53% of patients, with 9% and 11% of patients experiencing grade 4 toxicity, respectively; grade 3 to 4 adverse events included neutropenia (16% v 20%, respectively), fatigue (9% v 13%, respectively), and thrombosis (16% [n = 7] v 4% [n = 2], respectively; P = .157). Thirty-six percent of lenalidomide patients and 63% of LR patients completed 12 cycles. Lenalidomide alone was associated with more treatment failures, with 22% of patients discontinuing treatment as a result of adverse events. Dose-intensity exceeded 80% in both arms. Overall response rate was 53% (20% complete response) and 76% (39% complete response) for lenalidomide alone and LR, respectively (P = .029). At the median follow-up of 2.5 years, median time to progression was 1.1 year for lenalidomide alone and 2 years for LR (P = .0023). CONCLUSION: LR is more active than lenalidomide alone in recurrent FL with similar toxicity, warranting further study in B-cell non-Hodgkin lymphoma as a platform for addition of novel agents.