Visco, Carlo

Abstract Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2–targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. Implications: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell–like compared with the germinal-center B-cell–like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL.

Introduction: The phase 3 international, randomized, double-blind ROBUST clinical trial (NCT02285062) compared progression-free survival between patients with previously untreated ABC-type DLBCL treated with placebo plus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (placebo-R-CHOP) versus lenalidomide + R-CHOP (R2-CHOP) for 6 21-day cycles. HRQoL, as measured by the Functional Assessment of Cancer Therapy-Lymphoma (FACT-Lym) and the EuroQoL 5-dimension 3-level (EQ-5D-3L) patient-reported questionnaires, was evaluated as a secondary endpoint for the 2 treatment arms. Methods: For each treatment group, FACT-Lym and EQ-5D-3L scores were measured at baseline (BL), mid-, end-of-treatment (EOT), and post-treatment, in 12-week intervals from Week 34 onward. Patients were evaluated if they had completed BL and ≥ 1 post-BL calculable FACT-Lym subscale measurements. Non-inferiority (NI) of R2-CHOP versus placebo-R-CHOP was measured for 4 key FACT-Lym subscales: Physical Well-Being (PWB), Additional Concerns (AC), Functional Well-Being (FWB), and Trial Outcome Index (TOI). NI was assessed at EOT (typically 3-4 weeks after completing Cycle 6) and at post-treatment follow-up at Week 34 by analysis of covariance (ANCOVA). The NI margins were prespecified as 1 less than the higher published minimal important difference (MID) for each subscale. Since a range of MIDs have been published, a more conservative value was also used as a sensitivity analysis. Time to improvement (TTI), using published responder definitions, was also assessed for the FACT-Lym subscales. EQ-5D-3L scores were summarized descriptively. Results: Demographic and baseline disease characteristics were balanced across the 2 treatment arms (N = 285 per arm). The percentage of patients who completed the FACT-Lym out of the expected population remained > 85% at all time points with > 50 patients still on the study in each treatment arm up to Week 142. Completion rates and reasons for missing assessments were balanced across the 2 treatment arms. FACT-Lym subscale scores were generally higher after EOT, similar for both treatment groups, and mean changes from BL were close to or above MID (where defined), except for the Social/Family Well-Being subscale. Mean differences in FACT-Lym TOI between treatment arms are shown in Table 1. As positive treatment differences favor R2-CHOP and -10 was the predefined NI margin, NI was demonstrated for the FACT-Lym TOI subscale at both EOT and Week 34 assessments. Superiority of R2-CHOP was demonstrated at EOT as the confidence interval (CI) lies completely above 0 (P < 0.04); however, this difference was not clinically meaningful. Furthermore, NI was also demonstrated when using the sensitivity analysis margin of -4.5. ANCOVA models were also fitted for the other key subscales of PWB, AC and FWB (Table 2). NI was demonstrated for all NI margins at both time points for the PWB and AC subscales. The FACT-Lym FWB subscale demonstrated NI for both margins at EOT and Week 34 for the primary NI margin of -2 based on the MID minus 1, but did not demonstrate NI when an NI margin of -1, based on an alternative published MID, was used. The TTI curves and associated summary statistics were similar for the 2 treatment arms, with a median TTI for the FACT-Lym TOI of 21 weeks (95% CI 15-22) for patients treated with R2-CHOP and 18 weeks (95% CI 15-22) for patients treated with placebo-R-CHOP. Mean EQ-5D-3L visual analogue scale (VAS) and Utility Index (UK weights) change from baseline scores were higher in both treatment arms and the mean changes were above, or close to, the published MID (7 for VAS, 0.1 for Utility Index) at EOT assessments. Prespecified sensitivity and subgroup analyses supported the conclusions from the primary HRQoL analyses. Conclusions: The predefined NI levels were met for the 4 key FACT-Lym subscales. Similar results were observed across the other HRQoL assessments, sensitivity, and subgroup analyses. Adding lenalidomide to the R-CHOP regimen did not adversely affect the HRQoL of patients with DLBCL as measured by FACT-Lym and EQ-5D-3L. These data serve as a benchmark for future clinical trials combining next-generation immunomodulatory drugs with standard R-CHOP to improve clinical efficacy while maintaining HRQoL in patients with DLBCL. HRQoL analyses should become an integral part of future clinical trials of novel combination regimens in DLBCL. Disclosures Chiappella: Roche: Speakers Bureau; Teva: Speakers Bureau; Servier: Other: advisory board, Speakers Bureau; Janssen: Other: advisory board, Speakers Bureau; Celgene: Other: advisory board, Speakers Bureau. Cocks:Adelphi Values: Employment; Amgen: Consultancy; BMS: Consultancy; Celgene Corporation: Consultancy; Endomag Ltd.: Consultancy. Greenwood:Adelphi Values: Employment. Williams:Adelphi Values: Employment. Scott:Celgene: Consultancy; Roche/Genentech: Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Janssen: Consultancy, Research Funding. Yamamoto:SymBio: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria; Sanofi: Honoraria; Astra-Zeneca: Consultancy, Research Funding; Sumitomo Dainippon: Honoraria; Chugai: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Gilead Sciences: Research Funding; Solasia Pharma: Research Funding; Bayer: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; ARIAD: Research Funding; Pfizer: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Kyowa Kirin: Honoraria; MSD: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Meiji Seika Pharma: Consultancy, Honoraria; HUYA/IQVIA Services Japan: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; Incyte: Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Jurczak:Bayer: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morphosys: Research Funding; Incyte: Research Funding; Celgene Corporation: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celtrion: Research Funding; Novo Nordisk: Research Funding; Servier: Research Funding; TG Therapeutics: Research Funding. Özcan:Sanofi: Other: Travel support; Abdi Ibrahim: Other: Travel support; BMS: Other: Travel support; Takeda: Honoraria, Other: Travel support, Research Funding; Archigen: Research Funding; Roche: Other: Travel support, Research Funding; Bayer: Research Funding; Janssen: Other: Travel support, Research Funding; Celgene Corporation: Research Funding, Travel support; AbbVie: Other: Travel support, Research Funding; MSD: Research Funding; Novartis: Research Funding; Jazz: Other: Travel support; Amgen: Honoraria, Other: Travel support. Belada:Roche: Consultancy; Gilead Sciences: Consultancy; Takeda: Consultancy. Margunato-Debay:Celgene Corporation: Employment, Equity Ownership. Czuczman:Celgene Corporation: Employment, Equity Ownership. Zhai:Celgene Corporation: Employment, Equity Ownership. Braverman:Celgene Corporation: Employment. Vitolo:Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; F. Hoffmann-La Roche: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding.

Abstract Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Abstract Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Key PointsPhenotypic and genotypic profiling of MDM2 in DLBCL. MDM2 as a negative regulator of p53 tumor suppressor function.