Shipp, Margaret Ann

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease characterized by multiple low-frequency alterations including somatic mutations, copy number alterations (CNAs) and chromosomal rearrangements. We sought to identify previously unrecognized low-frequency genetic events, integrate recurrent alterations into comprehensive signatures and associate these signatures with clinical parameters. For these reasons, our multi-institutional international group assembled a cohort of 304 primary DLBCLs from newly diagnosed patients, 87% of whom were uniformly treated with state-of-the-art therapy (rituximab-containing CHOP regimen) and had long term followup. Tumors were subjected to whole exome sequencing with an extended bait set that included custom probes designed to capture recurrent chromosomal rearrangements. In this cohort, 47% of samples had available transcriptional profiling and assignment to associated disease subtypes. Analytical pipelines developed at the Broad Institute were used to detect mutations (MuTect), CNAs (Recapseq+Allelic Capseq) and chromosomal rearrangements (dRanger+Breakpointer) and assess clonality (Absolute). To analyze formalin-fixed paraffin-embedded tumors without paired normals we developed a method which utilized 8334 unrelated normal samples to stringently filter recurrent germline events and artifacts. Significant mutational drivers were identified using the MutSig2CV algorithm and recurrent CNAs were assessed with GISTIC2.0. In addition, we utilized a recently developed algorithm, CLUMPS2, to prioritize somatic mutations which cluster in 3-dimensional protein structure. With this approach, we identified > 90 recurrently mutated genes, 34 focal amplifications and 41 focal deletions, 20 arm-level events and > 200 chromosomal rearrangements in the DLBCL series. Of note, 33% of the mutational drivers were also perturbed by chromosomal rearrangements or CNAs, underscoring the importance of a comprehensive genetic analysis. In the large DLBCL series, we identified several previously unrecognized but potentially targetable alterations including mutations in NOTCH2 (8%) and TET2 (5%). The majority of identified chromosomal rearrangements involved translocations of potent regulatory regions to intact gene coding sequences. The most frequently rearrangements involved Ig regulatory elements which were translocated to BCL2, MYC, BCL6 and several additional genes with known roles in germinal center B-cell biology. After identifying recurrent somatic mutations, CNAs and chromosomal rearrangements, we performed hierarchical clustering and identified subsets of DLBCLs with comprehensive signatures comprised of specific alterations. A large subset of tumors shared recurrent alterations previously associated with follicular lymphoma including mutations of chromatin modifiers such as CREBBP, MLL2, and EZH2 in association with alterations of TNFRSF14 and GNA13 and translocations of BCL2. This cluster was enriched in GCB-type DLBCLs and contained a subset with select genetic alterations associated with an unfavorable outcome. An additional cohort of tumors was characterized by alterations perturbing B-cell differentiation including recurrent BCL6 translocations or alterations of PRDM1. A subset of these DLBCLs had alterations of NOTCH2 and additional pathway components or mutations of MYD88 in association with TNFAIP3, CD70 and EBF1, a master regulator of B-cell differentiation. An additional group of DLBCLs exhibited frequent MYD88 mutations in association with alterations of CD79B, PIM1, TBL1XR1 and ETV6 and BCL2 copy gain; these tumors were highly enriched for ABC-type DLBCLs. This coordinate signature and additional alterations of p53 pathway components were associated with outcome. We explored bases for the identified genetic alterations in DLBCL by performing an in silico mutational signature analysis. The most frequent mutational signatures were those of spontaneous deamination (aging) and AID with rare cases of microsatellite instability. We also assessed the clonality of identified genetic features to define cancer cell fraction and establish the timing of specific genetic events. The comprehensive genetic signatures of clinically annotated DLBCLs provide new insights regarding approaches to targeted therapy. Disclosures Link: Kite Pharma: Research Funding; Genentech: Consultancy, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Pfreundschuh:Boehringer Ingelheim, Celegene, Roche, Spectrum: Other: Advisory board; Roche: Honoraria; Amgen, Roche, Spectrum: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

PURPOSE Nivolumab, an anti–programmed death-1 monoclonal antibody, has demonstrated frequent and durable responses in relapsed/refractory classic Hodgkin lymphoma (cHL). We report results from Cohort D of the CheckMate 205 trial, which assessed nivolumab monotherapy followed by nivolumab plus doxorubicin, vinblastine, and dacarbazine (N-AVD) for newly diagnosed cHL. METHODS Patients 18 years of age or older with untreated, advanced-stage (defined as III to IV and IIB with unfavorable risk factors) cHL were eligible for Cohort D of this multicenter, noncomparative, phase II trial. Patients received nivolumab monotherapy for four doses, followed by 12 doses of N-AVD; all doses were every 2 weeks, and nivolumab was administered at 240 mg intravenously. The primary end point was safety. Efficacy end points included objective response rate and modified progression-free survival, defined as time to disease progression/relapse, death, or next therapy. Chromosome 9p24.1 alterations and programmed death-ligand 1 expression were assessed in Hodgkin Reed-Sternberg cells in evaluable patients. RESULTS A total of 51 patients were enrolled and treated. At diagnosis, 49% of patients had an International Prognostic Score of 3 or greater. Overall, 59% experienced a grade 3 to 4 treatment-related adverse event. Treatment-related febrile neutropenia was reported in 10% of patients. Endocrine immune-mediated adverse events were all grade 1 to 2 and did not require high-dose corticosteroids; all nonendocrine immune-mediated adverse events resolved (most commonly, rash; 5.9%). At the end of therapy, the objective response rate (95% CI) per independent radiology review committee was 84% (71% to 93%), with 67% (52% to 79%), achieving complete remission (five patients [10%] were nonevaluable and counted as nonresponders). With a minimum follow-up of 9.4 months, 9-month modified progression-free survival was 92%. Patients with higher-level Hodgkin Reed-Sternberg programmed death-ligand 1 expression had more favorable responses to N-AVD ( P = .041). CONCLUSION Nivolumab followed by N-AVD was associated with promising efficacy and safety profiles for newly diagnosed, advanced-stage cHL.

Purpose Treatment options are limited for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Tumor cells can exploit the programmed death-1 checkpoint pathway to evade immune surveillance. In the current study, we evaluated the efficacy and safety of programmed death-1 blockade by nivolumab in patients with relapsed/refractory DLBCL. Methods In this phase II, open-label study, patients with relapsed/refractory DLBCL who were ineligible for autologous hematopoietic cell transplantation (auto-HCT) or who had experienced failure with auto-HCT received nivolumab 3 mg/kg every 2 weeks. We assessed the efficacy and safety of nivolumab as well as genetic alterations of 9p24.1. Results Among 121 treated patients, patients in the auto-HCT–failed cohort (n = 87) received a median of four nivolumab doses and a median of three doses were administered to those in the auto-HCT–ineligible cohort (n = 34). At a median follow-up of 9 months in the auto-HCT–failed cohort and 6 months in the auto-HCT–ineligible cohort, independently assessed objective response rates were 10% and 3%, and median durations of response were 11 and 8 months, respectively. Median progression-free survival and overall survival were 1.9 and 12.2 months in the auto-HCT–failed cohort and 1.4 and 5.8 months in the auto-HCT–ineligible cohort respectively. All three patients with complete remission—3% of the auto-HCT–failed cohort—had durable response (11 or more, 14 or more, and 17 months). Treatment-related grade 3 and 4 adverse events were reported in 24% of patients. The most common were neutropenia (4%), thrombocytopenia (3%), and increased lipase (3%). Of all evaluable samples for 9p24.1 analysis, 16% exhibited low-level copy gain and 3% had amplification. Conclusion Nivolumab monotherapy is associated with a favorable safety profile but a low overall response rate among patients with DLBCL who are ineligible for auto-HCT or who experienced failure with auto-HCT. Genetic alterations of 9p24.1 are infrequent in DLBCL.

Purpose Cancer cells can exploit the programmed death-1 (PD-1) immune checkpoint pathway to avoid immune surveillance by modulating T-lymphocyte activity. In part, this may occur through overexpression of PD-1 and PD-1 pathway ligands (PD-L1 and PD-L2) in the tumor microenvironment. PD-1 blockade has produced significant antitumor activity in solid tumors, and similar evidence has emerged in hematologic malignancies. Methods In this phase I, open-label, dose-escalation, cohort-expansion study, patients with relapsed or refractory B-cell lymphoma, T-cell lymphoma, and multiple myeloma received the anti–PD-1 monoclonal antibody nivolumab at doses of 1 or 3 mg/kg every 2 weeks. This study aimed to evaluate the safety and efficacy of nivolumab and to assess PD-L1/PD-L2 locus integrity and protein expression. Results Eighty-one patients were treated (follicular lymphoma, n = 10; diffuse large B-cell lymphoma, n = 11; other B-cell lymphomas, n = 10; mycosis fungoides, n = 13; peripheral T-cell lymphoma, n = 5; other T-cell lymphomas, n = 5; multiple myeloma, n = 27). Patients had received a median of three (range, one to 12) prior systemic treatments. Drug-related adverse events occurred in 51 (63%) patients, and most were grade 1 or 2. Objective response rates were 40%, 36%, 15%, and 40% among patients with follicular lymphoma, diffuse large B-cell lymphoma, mycosis fungoides, and peripheral T-cell lymphoma, respectively. Median time of follow-up observation was 66.6 weeks (range, 1.6 to 132.0+ weeks). Durations of response in individual patients ranged from 6.0 to 81.6+ weeks. Conclusion Nivolumab was well tolerated and exhibited antitumor activity in extensively pretreated patients with relapsed or refractory B- and T-cell lymphomas. Additional studies of nivolumab in these diseases are ongoing.

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88 , CARD11 , EZH2 , and CREBBP . We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B , MLL2 , BTG1 , GNA13 , ACTB , P2RY8 , PCLO , and TNFRSF14 . Further, we show that BCL2 mutations commonly occur in patients with BCL2 / IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS , BRAF , and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.