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Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit–like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.

Introduction: Genome- and transcriptome-wide analyses continue to enhance our understanding of the molecular pathogenesis of cancer. In lymphomas, this has enabled the identification of hundreds of recurrently mutated genes, highlighting genetic heterogeneity and relationships both within and among clinical entities. While the growing availability of lymphoma genomic data sets can be leveraged to integrate genomic analyses into diagnostic testing and clinical trials, the ability to rapidly process genomic data sets in a reproducible manner serves as a barrier to this goal. To this end, we developed a suite of tools Lymphoid Cancer Research modules (LCR-modules) to facilitate the discovery of novel drivers and molecular features in lymphoma cancers and perform quantitative comparisons between disease entities. We demonstrate here how this toolkit enabled a meta-analysis of lymphoma genomic data involving genome-wide profiles of 3330 patients. Methods: We assembled a collection of whole genome, whole exome, and RNA sequencing data from a combination of controlled-access repositories and ongoing projects at BC Cancer. The scope of genomic analysis of mature B-cell lymphomas (GAMBL) project includes cell lines and patient tumors from all common mature B cell neoplasms, comprising a total of 4612 samples from 3330 patients. To facilitate the project, we developed a suite of open-source and custom bioinformatics tools (https://github.com/LCR-BCCRC/lcr-modules) that leverages the Snakemake workflow management system and includes lymphoma-centric modules for the discovery and annotation of common mutation types, analysis of B-cell receptor repertoires and discovery of novel aSHM targets and relevant non-coding mutations, and RNA-seq analysis with batch correction and normalization. Individual modules are configured to create an automated, scalable, and reproducible workflow that runs each step as dictated by the availability of new data. The cohort-level integrative analysis and comparisons across entities are handled by our custom R package GAMBLR, which facilitates open-ended data analysis and custom visualizations. Results: Simple somatic mutations (SSM) were detected using a workflow that utilizes four algorithms to identify high-confidence variants with validated default thresholds for filtering of germline variants and common FFPE-associated artifacts, allowing for processing of samples without matched normal tissue. This automated and reproducible workflow facilitated the discovery of novel genes significantly mutated across lymphomas and broadened our understanding of the scope of aberrant somatic hypermutation (aSHM) and other non-coding mutations. Specifically, HNRNPU, STAT3, TFAP4, RRAGC were found to be mutated at relatively low frequencies, and their presence is a distinct feature of certain lymphomas or novel genetic subgroups within lymphoma types (Figure 1A). The aSHM analysis and discovery of novel hypermutated regions is handled by a custom tool Rainstorm. As a result, we were able to detect sites preferentially hypermutated in a single entity, such as the transcription start site of BACH2, mutated at lower rates than the other common target sites but significantly more in BL compared to other entities (Figure 1B). Combining aSHM at target sites discovered using our toolkit with other genetic features allowed us to explore and establish novel genetic subgroups within Burkitt lymphoma and follicular lymphoma. SV analysis can be conducted using Manta, GRIDSS, and JaBbA modules with downstream processing in GAMBLR. In B-cell lymphomas, the most common SVs identified using the automated workflow were targeting MYC, BCL2, and CCND1. Unsurprisingly, the most common translocation partner among B-cell lymphomas was the immunoglobulin heavy chain, but the novel BCL6-FOXP1, CD274-BACH2, BCL6-RHOH translocations in DLBCLs and MYC-BCL6 translocations in BLs were identified, among others (Figure 1C). Conclusions: We present here the modularized workflow for scalable and automated analysis of genomic and transcriptomic data and demonstrate that it can be successfully deployed across thousands of tumour samples for the discovery of known and novel lymphoma biology. This represents an important advancement in reproducibility that will facilitate clinical translation of genomic discoveries. Figure 1 Figure 1. Disclosures Grande: Sage Bionetworks: Current Employment. Coyle: Allakos, Inc.: Consultancy. Steidl: AbbVie: Consultancy; Trillium Therapeutics: Research Funding; Epizyme: Research Funding; Seattle Genetics: Consultancy; Curis Inc.: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding. Scott: Abbvie: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; Celgene: Consultancy; AstraZeneca: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Research Funding; Rich/Genentech: Research Funding; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. . Morin: Epizyme: Patents & Royalties; Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees.

Introduction Animal models of human cancers are an important tool for the development and preclinical evaluation of new treatments. Canine B-cell lymphoma (cBCL) is an appealing alternative to murine preclinical models due to its frequent, spontaneous incidence and its clinical and histological similarity to human B-cell non-Hodgkin lymphoma (NHL). The potential utility of cBCL as a veterinary model of human B-cell lymphomas would be bolstered by a more complete understanding of the genetic features found in cBCL. Methods To study the genetics of cBCL, we obtained fresh frozen and matched plasma/serum from 86 patients from the Canine Comparative Oncology Genomic Consortium(CCOGC) with 65 confirmed as B-cell lymphomas by immunophenotyping. Tumor DNA was prepared into libraries using the QIAseq FX DNA Library Kit (Qiagen). Plasma and serum DNA was prepared into libraries using the NebNext Ultra II DNA Library Prep Kit. Targeted hybridization enrichment was performed on the libraries using our custom baits and sequencing reads were aligned to canFam3.1 using Geneious and each mutation was visually confirmed. Variants were annotated with Variant Effect Predictor and human-dog pairwise alignments were extracted from Ensembl to identify the orthologous human amino acid for all canine variants. Results Our analysis confirmed the previously reported high frequency of mutations in TRAF3 and FBXW7. We also observed mutations in POT1, TP53, and SETD2 at similar frequencies to those reported in previous studies. DDX3X was mutated in 20% of cases, which is substantially higher than previously reported. MYC mutations were also more frequent (13%) than has been previously described in cBCL. In human lymphomas, MYC is commonly deregulated by translocation to a potent enhancer and these events are often associated with point mutations in MYC that are induced by aberrant somatic hypermutation (aSHM). Interestingly, we identified a more focal pattern of MYC mutations in cBCL that implies they do not result from aSHM and are likely functional. This finding implicates the conserved MYC phosphodegron sequence, a motif commonly mutated among additional aSHM-associated mutations, as the target of bona fide driver mutations in both human and cBCLs. Mutations in FBXW7 primarily affected the substrate recognition domain responsible for MYC degradation. The observation that MYC and FBXW7 mutations did not co-occur in any canine patient is consistent with the notion that FBXW7 mutations operate as an alternative path to MYC stabilization which is not frequently observed in human NHL. DDX3X was one of the most frequently mutated genes in our cohort (20%). DDX3X mutations are common in human Burkitt lymphoma and, though less abundant in hDLBCL, tend to be observed in samples with MYC translocations. In Burkitt lymphoma, these mutations display a sex-specific pattern, wherein females show mainly missense mutations, while males are affected by loss-of-function mutations. Interestingly, all DDX3X mutations in cBCL are missense variants and are presumed to be dominant acting. This lack of sex difference in DDX3X mutations is an important distinction between human and canine B-cell lymphomas that warrants further exploration. Conclusions Our study has revealed key differences in the mutational profiles of canine and human B-cell lymphomas and provides an impetus for enhanced genomic characterization of canine lymphomas as a model for human NHL, particularly in clinical trial settings. Disclosures Grande: Sage Bionetworks: Current Employment. Alcaide: GA Diagnostics AB: Current Employment. Morin: Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Epizyme: Patents & Royalties. Coyle: Allakos, Inc.: Consultancy.

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

Non-Hodgkin lymphomas (NHLs) are the most common cancer to affect pet dogs. In contrast to the many genes whose mutation contributes to lymphomagenesis in humans, relatively little is known about the acquired genetic alterations that lead to canine B-cell lymphomas (cBCLs). We performed a survey of 84 canine NHL tumors to identify genes affected by somatic point mutations. We found mutations affecting TRAF3, which encodes a negative regulator of nuclear factor (NF)-κB, to be a common feature of cBCLs, with mutations observed in 44% of tumors including a combination of somatic and rare germ-line variants. Overall, 30% of the tumors contained ≥1 somatic TRAF3 mutation. The majority of mutations are predicted to cause loss of TRAF3 protein including those impacting reading frame and splicing. To determine whether TRAF3 loss might be relevant to human NHL, we also analyzed 148 human diffuse large B-cell lymphoma (DLBCL) tumors and identified loss of TRAF3 as a common event, affecting ∼9% of DLBCLs, and reduced expression of TRAF3 among deleted cases. This study implicates mutations affecting NF-κB activity as a novel genetic commonality between human and canine NHLs and supports the potential utility of cBCLs with mutated TRAF3 as a model of the more aggressive activated B-cell subgroup of DLBCL.

Introduction: Leg ulcers (LUs) are a cutaneous complication of sickle cell anemia (SCA), whose etiology is considered multifactorial. In the search for new candidates for modulators of SCA clinical events, recent evidence suggests the significant role of mechanisms related to post-transcriptional regulation, especially microRNAs (miRNAs). Thus, the analysis of miRNAs miR-21 and miR-130a differential expression in patients with SCA becomes an interesting approach, since both act in the regulation of several biological mechanisms related to the pathophysiology of LU, especially the tissue repair process. In addition, these miRNAs have already been related to the regulation of serum leptin levels, a strong angiogenic pleiotropic hormone that acts in the healing process of skin lesions. Therefore, the aim of the study was to investigate the influence of miR-21 and miR-130a and serum leptin levels on the development of LUs in SCA patients. Methods: After analyzing medical records, 60 SCA patients were selected. Patients who presented some of the main clinical manifestations that may have etiology due to the underlying disease (for example: osteonecrosis, stroke, priapism and acute chest syndrome) were not included. Patients with a history of LU were considered cases, and those who did not develop this complication (n=20), were considered control (median age: 26 years, range: 19-61, 50% males). The control group was called "HbSS-Control" and the case group was divided into two subgroups: Active leg ulcer group, composed of 19 patients with active LU at the time of blood collection (median age: 35 years, range: 24-56, 68% males), and healed leg ulcer group, composed of 21 patients with healed LU at the time of blood collection (median age: 34 years, range: 22-52, 43% males). In addition, it was analyzed a group of 10 donors with normal hemoglobin profile (median age: 25 years, range: 20-30, 50% males), identified as "HbAA-Control". Expression levels of miRNAs extracted from peripheral blood, using mirVanaTM PARIS Kit (Invitrogen™) were evaluated by RT-qPCR technique utilizing TaqMan® probes. Serum leptin levels of the patients were evaluated employing the ELISA method (Human Leptin ELISA Kit, Millipore®). Mann-Whitney and Kruskal-Wallis tests were applied to compare continuous variables. Results: Up-regulation of both miRNAs was observed in the active leg ulcer group in contrast to the healed leg ulcer (miR-21: P Disclosures No relevant conflicts of interest to declare.

Objectives Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. We sought to extend our understanding of the molecular etiology of this malignancy using an integrative genomic analysis of diagnostic biopsies. Methods We performed exome sequencing on 51 frozen MCL tumors and analyzed these alongside previously published exome cohorts. We sequenced tumour genomes and matched constitutional DNA from 34 frozen MCLs, along with matched constitutional DNA, to more broadly identify the pattern of non-coding mutations. Based on mutations identified in this discovery cohort, we re-sequenced 18 recurrently-mutated genes in 212 archival MCLs, each having clinical follow-up data. We also performed RNA-seq on 110 of these cases and analyzed these data for alternative splicing and differential expression, including the differential splicing of HNRNPH1 in the context of recurrent intronic mutations. We investigated the functional and phenotypic effect of mutations and deregulated HNRNPH1 protein through ectopic expression of full-length HNRNPH1 and a mini-gene containing the exons and introns affected by mutations. Using custom droplet digital PCR (ddPCR) assays, we validated alternative splicing patterns in HNRNPH1 itself and other targets identified through re-analysis of available CLIP-seq data. Results In addition to confirming the prognostic association of TP53 and NOTCH1 mutations in MCL, we identified two additional genes associated with outcome: EWSR1 with poor outcome (HR = 5.6) and MEF2B with good outcome (HR = 0.2). By comparing mutation patterns to diffuse large B-cell lymphoma (DLBCL), we identified an MCL-specific missense hot spot in MEF2B, non-specific truncating mutations in EWSR1, and truncating mutations affecting the DAZAP1 C-terminus in both MCL and DLBCL. The DAZAP1 mutations are predicted to alter protein sub-cellular localization and disrupt protein-protein interactions. We also identified the focal recurrence of non-coding mutations surrounding a single exon of the HNRNPH1 gene that were largely restricted to MCL. These mutations affected a region bound by HNRNPH1 protein and disrupted the preferred binding motif of this protein. Intronic mutations were significantly associated with alternative splicing of the HNRNPH1 mRNA and appear to disrupt a negative regulatory loop that normally limits the level of HNRNPH1. Using cell-based assays, we have evaluated the role of HNRNPH1 in cell survival and proliferation. Our interrogation of alternative splicing events in downstream targets implicate HNRNPH1 as a master splicing regulator which may broadly perturb the transcriptome and proteome to favor lymphomagenesis in MCL. Conclusions We discovered three novel MCL-related genes with roles in RNA trafficking or splicing, namely EWSR1, DAZAP1, and HNRNPH1. Mutations in these RNA-binding proteins were identified in 49 of 291 (17%) samples analyzed. Our results improve the current understanding of the MCL mutational landscape, highlight the similarities and differences between MCL and DLBCL, and strongly implicate a role for aberrant regulation of RNA metabolism in MCL pathobiology. We elucidated a functional role for recurrent non-coding HNRNPH1 mutations specific to MCL and identified multiple downstream targets. We continue to explore putative trans targets of HNRNPH1, a novel oncoprotein in MCL. Disclosures Steidl: Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Bayer: Consultancy; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding. Connors:Bristol-Myers Squibb: Consultancy; Seattle Genetics: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Johnson:Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding. Scott:Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding.

BACKGROUND: Rd continuous is now a standard treatment option in patients (pts) with transplant-ineligible NDMM based on the results of the large phase 3 registrational FIRST trial showing significant PFS and OS benefits of lenalidomide (LEN) + low-dose dexamethasone until disease progression (Rd continuous) vs melphalan + prednisone + thalidomide (MPT). However, pts who relapse on frontline LEN represent a growing population for whom there is limited clinical evidence on subsequent treatment. Factors associated with relapse within 12 mos in Rd- and MPT-treated pts (LDH ≥ 200 U/L, ISS stage III, high-risk cytogenetics, ECOG PS ≥ 2, and baseline platelet count ≤ 150 × 109/L) indicate that the individual risk of progression may involve a combination of disease biology, genetics, and pt-specific factors (Facon, EHA 2019). Physicians may consider these early relapse factors when making treatment decisions. To characterize pts relapsing while receiving frontline LEN, this analysis evaluated Rd-treated pts from the FIRST trial who progressed while receiving LEN based on their time of relapse, type of relapse (CRAB [symptomatic] vs non-CRAB [nonsymptomatic]), and second-line treatment. METHODS: Pts randomized to Rd continuous (n = 535) or Rd for 18 cycles (n = 541) arms and who progressed while receiving LEN or within 60 days of treatment end (per IMWG criteria) were pooled and grouped according to their time of relapse after randomization (< 12 mos = early< 12; 12-18 mos = early12-18, and > 18 mos = late relapse). The data cutoff was January 2016. RESULTS: Of the 389 pts who relapsed, 203 had early< 12 relapse, 69 had early12-18 relapse, and 117 had late relapse. Pts who relapsed within the first 12 mos or between 12 and 18 mos had higher rates of elevated LDH, ISS Stage III disease, and ECOG PS 2 compared with those who relapsed beyond 18 mos (Table). Early< 12 and early12-18 relapses were also associated with poor PFS and OS outcomes. Median PFS in pts with early< 12, early12-18, and late relapse was 6.5, 15.9, and 26.4 mos, and median OS was 26.8, 41.6, and 78.0 mos, respectively (Figure). Pts with late relapse had more dose reductions prior to PD vs those with early relapse and were more likely to experience nonsymptomatic progression. Across the 3 groups, in pts experiencing a nonsymptomatic progression who started second-line treatment, 88% started second-line treatment prior to experiencing CRAB symptoms. The median time from PD to second-line treatment was shorter in those with CRAB relapse (1.4-2.5 mos) vs non-CRAB relapse (2.5-5.9 mos). Second-line treatment was reported in 170 (83.7%), 62 (89.9%), and 99 (84.6%) pts with early< 12, early12-18, and late relapse, respectively. The majority of these pts received bortezomib-based regimens (65.6% overall), most commonly bortezomib + dexamethasone, bortezomib + melphalan + prednisone, or bortezomib + alkylator (cyclophosphamide or bendamustine). Second PFS was similar regardless of time of relapse. CONCLUSIONS: This analysis focused on an emerging and clinically relevant population of transplant-ineligible pts relapsing on frontline LEN. Pts with late relapse on LEN more commonly had nonsymptomatic progression vs pts relapsing early. This raises the question on the possible impact of the immune stimulatory effects of LEN in pts able to remain on therapy and achieve a longer OS and warrants further investigation of the biological impact of IMiD agent-based therapies on long-term disease control. The increase in dose reductions in the late- vs early-relapse groups is also an important consideration for continuous treatment. Consistent with the prior analysis of the FIRST study exploring Rd and MPT-treated pts with early< 12 relapse (Facon, EHA), pts relapsing on LEN within 12 mos and between 12-18 mos both appeared to have functionally high-risk disease and represent a population with an unmet need that should be considered for inclusion in clinical trials investigating new therapeutic strategies. Due to the timing of the FIRST trial, limited novel-novel agent combination treatments were available at relapse, and the majority of pts received bortezomib-based second-line regimens. These results highlight the need for effective therapies for pts with early relapse. Further characterization of first- and second-line outcomes according to type of relapse are ongoing and will be reported at the meeting. Disclosures Dimopoulos: Sanofi Oncology: Research Funding. Hulin:celgene: Consultancy, Honoraria; Janssen, AbbVie, Celgene, Amgen: Honoraria. Leleu:Sanofi: Honoraria; Takeda: Honoraria; Oncopeptide: Honoraria; Karyopharm: Honoraria; Amgen: Honoraria; Carsgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Celgene: Honoraria; BMS: Honoraria; Merck: Honoraria. Delforge:Amgen, Celgene, Janssen , Takeda: Honoraria. Weisel:Adaptive Biotech: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Juno: Consultancy; Janssen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; GSK: Honoraria; Sanofi: Consultancy, Honoraria, Research Funding. Srinivasan:Celgene: Employment, Equity Ownership. Costa:Celgene: Employment, Equity Ownership. Robinson:Celgene: Employment, Equity Ownership. Facon:Celgene: 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; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees.

Introduction and background The treatment of patients with Multiple Myeloma (MM) with relapse or progressive disease after bortezomib, lenalidomide and high-dose therapy represents an important challenge. In the EMN02 collaborative trial newly diagnosed patients with symptomatic MM were randomized to receive VCD induction followed by HDM/ASCT or VMP, followed by a second randomization for VRD consolidation or no consolidation, followed by lenalidomide maintenance until progression (Cavo et al, ASH2017, abstract #397; Sonneveld et al, EHA2018, abstract #108). The present Phase 2 trial was designed for patients with refractory disease or first progression after inclusion in EMN02 in order to evaluate a salvage treatment with next generation proteasome inhibition and IMId, i.e., Carfilzomib, Pomalidomide and Dexamethasone. The primary endpoints were response and progression-free survival (PFS). This trial is registered at www.trialregister.nl as NTR5349 and EudraCT 2013-003265-34. Methods Patients who were included received four 28-days re-induction cycles of KPd, i.e. Carfilzomib (20/36mg/m2, days 1,2,8,9,15,16) with Pomalidomide (4 mg days 1-21) and Dexamethasone (20mg days 1,2,8,9,15,16). In patients who had not previously received HDM/ASCT, HDM(200 mg/m2) was administered followed by autologous stem cell transplantation with stem cells harvested during after induction therapy in the EMN02 trial. Consolidation consisted of 4 additional cycles of KPd, identical to the induction cycles. Patients with stable disease or better received Pomalidomide 4mg w/o Dexamethasone in 28 days cycles until progression. Results At the time of this first planned interim analysis 82 patients were registered and this analysis was performed in the first 60 patients. 48% were randomized prior HDM/ASCT and 42% VMP, and 10% were not randomized. Prior best responses in the EMN02 trial were 35% CR/sCR , 75% ≥VGPR, 97% ≥PR. The median follow-up from inclusion in EMN02 was 43 months (range 21 - 62 months). In 44 patients cytogenetic risk were known, 15 (34%) of them had high-risk FISH (del17p, t(14;16) or t(4;14)). 57 fifty-seven (95%) of patients had progressed during lenalidomide maintenance, 3 patient's data are not yet available. In the present trial 38 (63%) of patients achieved normal completion of treatment according to of the protocol. Twenty patients received their first HDM plus ASCT. Median time on therapy was 14 months. Full dose re-induction treatment according to protocol could be administered in 68% (for Carfilzomib) and 64% (for Pomalidomide) of patients respectively, while for consolidation this was 62% for both Carfilzomib and Best response on protocol was 31% CR/sCR, 65% ≥VGPR, 87% ≥PR, respectively, with no difference according to response on initial treatments. Median time to response (≥PR) was 2 months. At a median follow-up of 16.3 months (range 3 - 32 months) median PFS was 18 months with better outcome in standard risk cytogenetics (HR=0.27 (0.09, 0.83) 95% CIs vs NR) and in patients with prior VMP treatment (HR=0.49 (0.21, 1.16) 95% CIs vs NR). 48 (80%) of patients are alive and in follow-up. KPd-emerging non-hematologic grade 3 and 4 adverse events included cardiovascular (5%), respiratory (5%), infections (20%) and neuropathy (3%). There were 3 fatal SAEs not related to progression (1 patient cardiac failure, 2 patients pneumonia). KPd-emerging hematological toxicity grade 3 and 4 occurred in 30% of patients. Discussion This Phase 2 clinical trial demonstrates that KPd is a feasible, effective and safe triple drug regimen in RRMM patients who have been previously treated and/or are refractory to bortezomib and refractory to lenalidomide. A 87% overall response rate including 31% CR/sCR is clinically relevant in this population. Since median OS has not been reached, longer follow-up is needed. Acknowledgments This trial was conducted as an investigator sponsored trial in EMN and supported by independent grants and drug supply from Amgen and Celgene. Disclosures Sonneveld: BMS: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corp.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: 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. Cavo:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Corradini:Roche: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Novartis: Honoraria, Other: Advisory Board & Lecturer; Sandoz: Other: Advisory Board; Amgen: Honoraria, Other: Advisory Board & Lecturer; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Celgene: Honoraria, Other: Advisory Board & Lecturer. Patriarca:Janssen: Other: Advisory role; Celgene: Other: Advisory Role; Travel, accommodations, expenses; Jazz: Other: Travel, accommodations, expenses; MSD Italy: Other: Advisory Role; Medac: Other: Travel, accommodations, expenses. Minnema:Celgene: Consultancy, Research Funding; Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Servier: Consultancy. Costa:celgene: Employment. Iskander:amgen: Employment. Boccadoro:Mundipharma: Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding.

Recognizing biological heterogeneity in diffuse large B-cell lymphoma (DLBCL), significant effort has been made to define distinct molecular subgroups of prognostic importance which harbor potentially targetable biology. Reflecting this, in the recent revision of the WHO classification, DLBCL was divided into cell-of-origin molecular subtypes and a new entity was defined - high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH). ~8% of tumors with DLBCL morphology are HGBL-DH/TH and all HGBL-DH/TH with BCL2 translocations (HGBL-DH/TH-BCL2) are of the GCB molecular subtype. To explore specific biology operating in HGBL-DH/TH-BCL2, we analyzed RNAseq data from 157 de novo GCB DLBCL tumors (25 being HGBL-DH/TH-BCL2) with the aim of defining gene expression signatures that distinguish such cases from other GCB-DLBCLs. We identified 104 genes that were most significantly differentially expressed between HGBL-DH/TH-BCL2 and other GCB-DLBCLs, defined as having a 95% confidence interval of the Importance Score that did not cross 0. A model constructed from the expression of these genes clustered 42 tumors into one group ("double-hit signature" positive - DHITsig pos), and 115 tumors into the DHITsig neg group. 22 tumors were HGBL-DH/TH-BCL2 within the DHITsig pos group compared with only 3 tumors in the DHITsig neg group. We next assessed the clinical impact of the DHITsig within a uniformly R-CHOP treated cohort of de novo GCB-DLBCL drawn from a population-based registry, which included the discovery cases. The DHITsig pos group had significantly inferior outcomes for time to progression (TTP) and overall survival (OS) (P < 0.001 and P = 0.01, respectively) similar to ABC-DLBCL (Figs A, B). Notably, the non-HGBL-DH/TH-BCL2 cases sharing the DHITsig showed the same poor prognosis as the HGBL-DH/TH-BCL2 cases. A multivariate Cox model of TTP revealed that DHITsig remained prognostic, independent of IPI and MYC/BCL2 dual protein expression (HR = 3.1 [1.5 - 6.4], P = 0.002). We then applied this gene expression model to GCB-DLBCL in an independent dataset (n = 262 GCB-DLBCLs; Reddy et al,Cell 2017). Validating the prognostic significance, the DHITsig pos group had significantly inferior OS compared with other GCB-DLBCLs (P < 0.001) similar to ABC-DLBCL (Fig C). We then sought to determine whether differentially expressed genes, according to DHITsig, could inform on the biology of the DHITsig pos group. Gene set enrichment analysis (GSEA) strongly suggested a germinal centre dark-zone (DZ) cell-of-origin for the DHITsig pos tumors with significant enrichment of DZ and light-zone (LZ) gene signatures (Victora et al, Blood 2012) in DHITsig pos and neg tumors, respectively (FDR = 0.002 and < 0.001). Furthermore, the DHITsig pos group had up-regulation of pathways related to mitochondrial metabolism and RNA synthesis (both FDR < 0.001). We separately identified mutations associated with DHITsig pos cases within GCB-DLBCL. In addition to the expected enrichment of MYC and BCL2 mutations, chromatin modifiers EZH2 and CREBBP, as well as RFX7 and DDX3X (mutated in Burkitt lymphoma), were more frequently mutated in DHITsig pos tumors. In contrast, mutations of TNFAIP3, MYD88 and IRF4, more typical of ABC-DLBCLs, were more prevalent in DHITsig neg tumors. To enable application to FFPE biopsies, the DLBCL90 NanoString assay was developed by translating the DHIT gene expression signature into a 30-gene module that was then added to the Lymph2Cx assay. The DLBCL90 assay was applied to 171 DLBCL tumors (including 156 from the discovery cohort), yielding 26% DHITsig pos, 64% DHITsig neg, and 10% unclassified, with a frank misclassification rate of 3% against the RNAseq comparator. The prognostic significance of the groups was maintained (Fig D). Importantly, the DHITsig neg group had a disease specific survival of 91% at 5 years. To validate the association between the DHITsig and HGBL-DH/TH-BCL2 tumors, the DLBCL90 assay was applied to 113 transformed follicular lymphoma tumors. Within the DHITsig pos group, 19/34 tumors were HGBL-DH/TH-BCL2 compared with 0/58 in the DHITsig neg group. In conclusion, we have identified a clinically and biologically distinct subgroup of GCB-DLBCL tumors that are defined by the HGBL-DH/TH-BCL2 gene signature. The translation to an assay applicable to FFPE allows exploration of its utility to guide patient management within the context of clinical trials. Figure. Figure. Disclosures Sehn: Merck: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Steidl:Nanostring: Patents & Royalties: patent holding; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy. Connors:Cephalon: Research Funding; Amgen: Research Funding; F Hoffmann-La Roche: Research Funding; Roche Canada: Research Funding; Bristol Myers-Squibb: Research Funding; Janssen: Research Funding; Bayer Healthcare: Research Funding; Takeda: Research Funding; NanoString Technologies: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Seattle Genetics: Honoraria, Research Funding; Merck: Research Funding; Genentech: Research Funding; Lilly: Research Funding. Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Roche: Research Funding; Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding.

Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.