ALBERT

Description: Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and is characterized by its genetic and clinical heterogeneity. Patients can develop DLBCL de novo or as a transformation from other lymphoid malignancies, most commonly follicular lymphoma. For patients with relapsed/refractory DLBCL (rrDLBCL), prognosis is extremely poor with 2-year overall survival of 20-40%. While numerous treatments are under investigation to improve patient outcomes, the success of these treatments has been limited as the genetic mechanisms underpinning treatment resistance are largely unknown. Identifying genomic alterations which contribute to relapse may improve salvage therapy for patients with rrDLBCL or allow patients to be stratified prior to frontline treatment. Methods To identify genomic alterations which contribute to R-CHOP resistance, we previously collected samples from patients enrolled in four clinical trials exploring candidate salvage therapies for patients with rrDLBCL as well as a retrospective rrDLBCL cohort, totalling 193 cases (133 de-novo DLBCL, 60 transformed). Plasma samples were collected from each patient upon relapse along with diagnostic tissue biopsies where available. A combination of exome sequencing and target-panel sequencing of lymphoma associated genes was performed on circulating tumour DNA and tissue biopsies (if available). Mutations implicated in R-CHOP resistance were identified through two complimentary strategies. First, the mutation frequency of recurrently mutated genes across de novo rrDLBCL samples was compared to a cohort of unrelated diagnostic DLBCL cases (n=1691) to identify genes enriched for mutations. Second, the genomic landscape and tumor clonal structure was compared prior to and following R-CHOP to identify mutations in each patient that underwent clonal expansion following therapy. Anti-CD20 antibody binding affinity of MS4A1 mutants was evaluated using flow cytometry on transfected CHO-S cells. Results We have identified five genes enriched for mutations in our rrDLBCL cohort relative to diagnostic DLBCL: KMT2D (Mutated in 49%, Q=0.0385), TP53 (47%, Q=1.07x10-9), FOXO1 (11%, Q=0.0727), NFKBIE (11%, Q=0.0385), and MS4A1 (8%, Q=0.0522). Consistent with its characterization as a poor prognostic marker, mutations in TP53 were typically present at diagnosis and remained stable following R-CHOP therapy for both de novo and transformed DLBCL (23/27 cases, 85%). Recurrent mutations affecting Arg248 of TP53 (6.8%, Q=0.0413) were also clonally stable and have previously been associated with poor overall survival across several cancer types. The histone methyltransferase KMT2D is dominated by nonsense and frameshift mutations which were stable or underwent clonal expansion following R-CHOP (17/19, 89%). Recurrent missense mutations in MS4A1 targeted the small loop and adjacent transmembrane domains of CD20, including several patients with a Tyr86 mutation. Transfected cells carrying Tyr86Cys or Leu66Arg mutations were not bound by rituximab or other anti-CD20 antibodies including obinituzumab and ofatumumab. Subclonal populations containing MS4A1 mutations underwent clonal expansion (6 cases) or were stable (1 case) following treatment, including one case with multiple MS4A1 mutations in distinct subclonal populations which both underwent clonal expansion. In another unique case, a series of ctDNA samples were available prior to and following R-CHOP and salvage therapy, where we again observed convergent evolution of two mutually exclusive clonal subpopulations containing MS4A1 mutations. The first subpopulation underwent clonal expansion following frontline therapy but was extinguished following salvage therapy, while the other subpopulation underwent clonal expansion following salvage therapy and harboured a transmembrane domain mutation. Conclusion Mutations in TP53 and truncating mutations in KMT2D are generally present prior to treatment and will be investigated as biomarkers of treatment failure. Additional mutations are not always present at diagnosis, but their emergence can be detected in ctDNA and relapsed tissue, specifically mutations in MS4A1. As mutations in MS4A1 attenuate rituximab binding and are recurrently associated with clonal expansion, they likely impart a selective advantage and lead to resistance against anti-CD20 antibodies. Disclosures Michaud: Epizyme: Employment. Daigle:Epizyme: Employment. Jain:Kite/Gilead: Consultancy. Kuruvilla:Roche: Honoraria; Astra Zeneca: Honoraria; Novartis: Honoraria; Merck: Honoraria; Karyopharm: Honoraria; Gilead: Honoraria; Celgene: Honoraria; BMS: Honoraria; Amgen: Honoraria; Seattle Genetics: Consultancy; Roche: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Gilead: Consultancy; Janssen: Research Funding; Roche: Research Funding; BMS: Consultancy; Abbvie: Consultancy; Seattle Genetics: Honoraria; Janssen: Honoraria. Assouline:Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau. Scott:NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding. Johnson:BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; 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.

Description: Introduction Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) are treated with standard frontline immunochemotherapy (R-CHOP). However, for cases where R-CHOP fails (relapsed-refractory DLBCL, rrDLBCL), prognosis is extremely poor, with 2-year overall survival of 20-40%. The successful development of new therapies may be hampered by our limited understanding of the genetic and molecular mechanisms underpinning treatment resistance. For example, recent data from our group has highlighted novel mutations that emerge following treatment with R-CHOP. The contribution of copy-number variations (CNVs) towards treatment resistance has not yet been thoroughly explored. A more complete characterization of these genetic alterations may lead to new prognostic biomarkers or treatment strategies. Methods We analyzed exome sequencing data from 59 rrDLBCL cases derived from either tissue biopsies or liquid biopsies collected after relapse, including both unpublished and previously published cases (Schmitz et al. (2018) NEJM 378:1396-1407 and Morin et al. (2016) Clin Can Res 22(9)). We separately performed low-pass whole-genome sequencing (lpWGS, 0.1-1x coverage) on 45 rrDLBCL liquid biopsies with ctDNA levels insufficient for exome-based analysis, for a total of 104 cases with copy-number information. We identified CNVs from exome and lpWGS data using Sequenza and ichorCNA, respectively. Next, we identified significant peaks of recurrent gains and losses using GISTIC2. Comparison of these peaks to CNVs in a previously published diagnostic DLBCL cohort (Schmitz et al. (2018) NEJM 378:1396-1407) enabled the identification of events that were significantly more prevalent in rrDLBCL. Results Overall, the landscape of CNVs in rrDLBCL is reminiscent of diagnostic DLBCL, with recurrent amplifications of chromosome 7 (43/104, 41.3%) and 18q (42/104, 40.4%) and recurrent deletions of 6q (25/104, 24.0%) and 17p13 (39/104, 37.5%). We identified nine regions enriched for recurrent amplifications or deletions among rrDLBCLs. These include deletions of 17p13.1 (20.4% in diagnostic biopsies vs 41.3% of rrDLBCLs, q=8.53x10-5) and recurrent amplifications of 8q24 (18.5% vs 42.3%, q=5.72x10-7) and 7p22 (27.2% vs 57.9%, q=6.29x10-8). Many of these peaks represent focal events that are exceedingly rare in diagnostic DLBCL and do not contain established lymphoma-associated genes, including amplifications affecting 700kb of 6p11.2 (2.03% vs 7.69%, q=0.0178) and 500kb of 19p13.3 (6.7% vs 31.7%, q=9.99x10-10). Notably, the 6p11.2 amplifications were associated with inferior progression-free survival following R-CHOP (p=0.02), with most tumors harboring this alteration relapsing within 12 months. We also identified a novel, recurrent deletion affecting a 20mb region of 5q (2.78% vs 10.6%, q=0.00604) which was significantly deleted in rrDLBCL. For tumors with additional samples collected prior to R-CHOP and following salvage therapy, deletions of 5q appeared to emerge following frontline therapy and persisted after subsequent treatments, suggesting they may contribute to treatment resistance. Discussion The 17p13.1 deletion enriched in rrDLBCL encompasses TP53, which is a common target of somatic point mutations in rrDLBCL and associated with inferior treatment outcomes. The amplification of 8q24 and 7p22 include MYC and GNA12/CARD11, respectively, although these large events encompass numerous additional genes which may be the target of such events. Curiously, the focal 6p11.2 amplification only overlaps a handful of genes including miR_598, which has been predicted to target CD27 and CD38 and whose expression is upregulated in B-cell cell lines (Lawrie et al. (2008) Leukemia 22:1440-2446). Further investigation and validation of these events and their corresponding targets will provide insight into the biology of rrDLBCL and may reveal novel therapeutic targets. Disclosures Michaud: Epizyme: Current Employment. Daigle:Epizyme: Current Employment. Jain:Kite/Gilead: Consultancy; Novartis: Consultancy. Kuruvilla:Merck: Consultancy, Honoraria; Bristol-Myers Squibb Company: Consultancy; Celgene Corporation: Honoraria; AstraZeneca Pharmaceuticals LP: Honoraria, Research Funding; AbbVie: Consultancy; Gilead: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria; Antengene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; TG Therapeutics: Honoraria. Crump:Servier: Consultancy; Roche: Consultancy; Kite/Gilead: Consultancy. Assouline:BeiGene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding; Pfizer: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding. Steidl:Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy; Bayer: Consultancy; Curis Inc: Consultancy. Johnson:AbbVie: Research Funding; Roche/Genentech, Merck: Honoraria; Roche/Genentech, Merck, Bristol-Myers Squibb, AbbVie: Consultancy. Scott:NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; Janssen: Consultancy, Research Funding; Roche/Genentech: Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Celgene: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Morin:Celgene: Consultancy.