Abstract: Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. Emerging data suggests that cHLs use multiple genetic mechanisms to evade immune recognition. We previously found that HRS cells exhibit near-universal somatic copy number alterations (SCNAs) involving chromosome 9p24.1/PD-1-L1/PD-L2 and rare chromosomal rearrangements of PD-L1 or PD-L2. The 9p24.1 amplicon also includes JAK2, which increases JAK2 copy numbers, augments JAK2/STAT signaling and further induces PD-1 ligand expression. However, HRS cells also have inactivating mutations of B2M and decreased or absent MHC class I expression. In cHL, clinical responses to PD-1 blockade are unrelated to HRS cell expression of MHC class I but closely associated with HRS cell expression of MHC class II, highlighting the potential role of CD4+ T-cell effectors (J Clin Oncol 2018;36:942-50). To define genetic bases of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole exome sequencing (WES) of HRS cells. We first used a previously described multi-color flow cytometric sorting protocol (Methods 2012; 57:368-75) to obtain highly purified CD30+ HRS cells and normal B cells from the excisional biopsies of 25 newly diagnosed cHLs. The isolated HRS cells and paired normal B cells were then subjected to WES using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs). We used established analytical pipelines to identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV), SCNAs (GISTIC2.0) and SVs (4 algorithms). With improved methodology and purity (median of 80%) of the isolated HRS cells, we defined 15 significantly mutated CCGs, 21 recurrent SCNAs, including 6 CN gains (4 focal and 2 arm level) and 15 CN losses (14 focal and 1 arm level), and low frequency SVs. We identified 2 cHLs as hypermutators with MSI signatures due to splice site mutations in MSH2 or missense mutations in POLE. Excluding the 2 hypermutators, the analyzed cHLs had a median mutational density of 6.4 mutations/Mb, that falls within the top quartile of reported cancer mutational frequencies (Nature 2013 499:214). We also identified a previously unappreciated high incidence of ARID1A mutations (24%) in cHL. This is noteworthy because ARID1A deficiency increases mutational load and augments the efficacy of PD-1 blockade in murine models (Nature Med 2018;24:556). Together, the observed MSI signatures, relatively high mutational burden and newly identified ARID1A mutations in cHL represent additional potential genetic bases for the efficacy of PD-1 blockade. Notably, these cHLs also exhibited recurrent 9p24.1 copy gain (80%) and multiple genetic bases of enhanced JAK/STAT signaling including JAK2 copy gain (80%), STAT6 mutations (32%) involving known hotspots (D419 and N421) in the DNA-binding domain and frequent inactivating SOCS1 mutations (68%). We also identified multiple genetic bases for immune evasion, including B2M inactivating mutations (36%), HLA-B mutations (16%) and 6p21.32/HLA-B copy loss (28%), copy loss of the larger 6p21.32 region and inactivating CIITA SVs (8%). Additional signaling pathways were perturbed by multiple genetic mechanisms in these cHLs. For example, NF-κB pathway alterations included: TNFAIP3 mutations (24%) and 6q23.2/TNFAIP3 copy loss (56%), 12% biallelic; NFKBIE mutations (24%) and 6q21.32/NFKBIE copy loss (12%); and NFKBIA mutations (16%). The gene encoding the nuclear export protein, XPO1, was perturbed by E571K mutations (24%) and frequent 2p15/XPO1 copy gain (72%). Additionally, GNA13, an activator of RHOA and modifier of PI3K signaling, was mutated in 24% of cases. Of interest, cHL recurrent alterations including B2M, TNFAIP3, STAT6, and GNA13 mutations and 6q23.2 and 9p24.1 SCNAs were also identified in 〉 20% of examined primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases. In summary, comprehensive genomic analyses of purified HRS cells reveal new genetic bases of immune evasion, potential mechanisms of response and resistance to PD-1 blockade and additional targetable alterations. KW, BC, CS, AD and DW contributed equally. JF, GG and MS contributed equally. Disclosures Rodig: Affimed: Research Funding; KITE: Research Funding; Merck: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria.

Abstract: Large-scale cancer sequencing efforts worldwide have yielded numerous novel cancer drivers; however, how these genetic alterations functionally lead to cancer remains largely unknown. An indispensible approach for establishing the causal features of disease is through in vivo animal models. In chronic lymphocytic leukemia (CLL), only limited mouse models are currently available and most do not reflect the genetics of human CLL. Studies of whole-exome sequencing (WES) using CLL samples have consistently pointed to the common co-occurrence of mutations in the RNA splicing factor gene SF3B1 and mutations in the DNA damage response gene ATM or deletion of chromosome 11q (del(11q), whose minimally deleted region encompasses ATM). We therefore asked whether this combination of traits would be productive of CLL in mice. To this end, we modeled the effects of these combined alterations by crossing mice with conditional knockout of Atm and mice with a conditional knock-in allele of SF3B1 mutation (Sf3b1-K700E). We achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion by breeding these mice with CD19-Cre homozygous transgenic mice. We found that in vivo co-expression of these two mutations in B cells, but not of either single lesion alone, led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen (at low penetrance) in aged (18 to 24-month old) but not young mice. These malignant cells could be propagated by in vivo passaging, with detectable disease within 4 weeks following transfer, thus making this mouse line amenable to further drug discovery and biologic investigations. To better understand how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if RNA level changes are present in the double mutant mice. We performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, or singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease (n=2-6 samples, per group). Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that the splice variants in micewith mutated Sf3b1 alone (without CLL) were highly enriched at 3' splice sites (27 of 77 splice variants, t-test q 〈 0.05, absolute ΔPSI 〉 10%). On the other hand, mice with Atm single deletion displayed an RNA splicing pattern with enrichment of alternative first and last exons (11 and 12 of 52, chi-squared test, p=4.5 x 10-4). B cells with the combined Sf3b1 and Atm mutations displayed a combination of splicing patterns that comprised of both alternative 3' splice variants, as well as alternative first and last exons. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously demonstrated to be associated with CLL. We further investigated the differential gene expression between B cells from double mutant mice with and without CLL-like disease. We identified 1,875 CLL-specific genes (DESeq2, q 〈 0.01). Gene set enrichment analysis (GSEA) of these genes indicated their involvement in cellular processes such as IL2-STAT5 signaling and the interferon gamma response, both pathways implicated in human CLL. In parallel, we asked if there are DNA level changes in the doubly mutant mice. We examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. Preliminarily, we have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to cells with only single mutation. In summary, we have generated a genetically-engineered murine model that faithfully recapitulates human CLL genetics. This is the first demonstration that expression of putative CLL driver events identified from unbiased genome-wide sequencing indeed initiates CLL-like disease. Genome-wide DNA and RNA analysis using this model has revealed that altered RNA splicing, dysregulation of gene expression, and genomic instability all contribute to CLL leukemogenesis. We anticipate that further dissection of this murine model will shed light on mechanistic understanding of cooperation between Atm deletion and SF3B1 mutation in CLL. Disclosures No relevant conflicts of interest to declare.

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.

Abstract: A deep dive into genomic aberrations in 37 new diagnoses of primary mediastinal B-cell lymphoma (PMBL) reveals a surprisingly high number of driver mutations as well as genetic and epigenetic explanations for immune evasion by the tumor.

Abstract: Analyses of recurrent mutations, copy number alterations, and structural variants reveal complementary immune evasion mechanisms in cHL. The mutational burden in EBV– cHLs is among the highest reported, potentially contributing to the efficacy of PD-1 blockade.

Abstract: Primary mediastinal large B-cell lymphomas (PMBL) typically occur in young women who present with localized, large mediastinal masses. These tumors share certain clinical, pathomorphological and transcriptional features with classical Hodgkin lymphoma (cHL). To date, PMBL genetic analyses focused on limited sets of genes and recurrent somatic copy number alterations (SCNAs). Previously, we identified frequent 9p24.1/PD-L1/PD-L2 copy gains and increased expression of the PD-1 ligands as a genetically-defined immune escape mechanism in PMBL. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBL led to recent FDA approval and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. For these reasons, we obtained diagnostic biopsy specimens from 37 patients with PMBL (median age 34; female 70%) and performed whole exome sequencing (WES) with an expanded bait set to capture structural variants (SVs). Somatic alterations (mutations, SCNAs and SVs) were determined using established analytical pipelines including our algorithm for evaluating tumors without paired normal samples. Genes more frequently mutated than by chance, Candidate Cancer Genes (CCGs), were identified with MutSig2CV and recurrent SCNAs were defined with GISTIC2.0. SVs were characterized with a recently described 4-algorithm pipeline (Nature Medicine, 2018;24(5):679-690). First, we identified 15 CCGs (q-value 〈 0.1) including genes with known roles in PMBL, such as IL4R and TNFAIP3 and mutational drivers in additional B-cell lymphomas (B2M, GNA13, STAT6, IKZF3, XPO1, TP53, PAX5) and other cancers (TP53, ZNF217 and XPO1). Overlaying the predicted protein changes onto available 3D protein structures highlighted the likely biological functions of specific alterations, such as mutational clustering in the STAT6 DNA-binding domain. We next analyzed the PMBL mutational signatures and identified 3 cases as hypermutators with MSI signatures, including 2 with MLH1 frameshift mutations and 1 with a nonsense PMS2 mutation. Despite the young age of the PMBL patient cohort, the majority of remaining mutations were caused by spontaneous deamination at CpGs, a genetic signature usually associated with aging. The next most prevalent mutational signatures were APOBEC and, infrequently, AID. We observed a higher median mutational density in PMBL (7.56 mutations/MB), compared to diffuse large B-cell lymphoma (DLBCL) and most solid cancers, providing a potential basis for increased neoantigen production and responsiveness to PD-1 blockade. Next, we identified 18 recurrent SCNAs, including 10 copy gains (2 focal and 8 arm level) and 8 copy losses (7 focal and 1 arm level). Copy gains of 9p24.1/PD-L1/PD-L2 were detected in 70% of cases. SVs were defined at base-pair resolution and included infrequent (2/37) tandem duplications of both PD-1 ligands and inactivating CTIIA SVs (deletions and inversions) in 10% (4/37) of cases. Although PMBL had a higher mutational density than DLBCL, the PMBL alterations involved a smaller number of median genetic drivers (9 [PMBL] vs 17 [DLBCL] , respectively). Combined analyses of recurrent CCGs, SCNAs and SVs revealed that certain candidate driver genes were perturbed by multiple mechanisms. Examples include: TNFAIP3 (59% overall, 41% mutations, 24% copy loss, 6% biallelic); and B2M (51% overall, 30% mutations, 27% copy loss, 6% biallelic). Concurrent analyses of the 3 types of genetic alterations also revealed multiple bases of perturbing specific signaling pathways. In this PMBL series, 73% (27/33) of tumors exhibited one or more alterations of JAK/STAT pathway components: IL4R mutations (32%), JAK2 (9p24.1 focal copy gain [70%]) and STAT6 mutations (43%). Additionally, 59% of PMBLs had alterations of antigen presentation pathway components including B2M copy loss or mutations, copy loss of 6q21.33 (which includes the HLA class I/II loci) and SVs of CTIIA. These findings provide a genetic framework for analyzing the precise mechanism of action of PD-1 blockade in PMBL. Taken together, these findings underscore the importance of a comprehensive genomic analysis in PMBL and define additional candidate treatment targets and pathogenetic mechanisms in this disease. ____ BC, CS and AD contributed equally. GG and MAS contributed equally. Disclosures Rodig: Merck: Research Funding; KITE: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; AstraZeneca: Honoraria; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

Abstract: Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (PMBL) are aggressive tumors with distinct cells of origin and pathomorphological features. However, these lymphomas share certain transcriptional signatures and aberrant signaling pathways. CHLs and PMBLs both exhibit constitutive activation of NF-κB and JAK/STAT signaling and genetic bases of PD-1 mediated immune evasion including frequent 9p24.1/PD-L1/PD-L2 copy gains. In both lymphomas, PD-1 blockade is a FDA-approved therapy for relapsed/refractory disease. To characterize genetic bases of response to PD-1 blockade and identify complementary treatment targets in cHL and PMBL, we defined the comprehensive genetic signatures of both diseases. First, we obtained flow cytometry-sorted Hodgkin Reed Sternberg (HRS) cells from 23 biopsies of newly diagnosed cHLs and intact tumor biopsy specimens from 37 newly diagnosed PMBLs. The isolated HRS cells and paired normal DNAs and PMBL biopsy specimens were subjected to whole exome sequencing using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs), including translocations. We used newly developed and established analytical pipelines to analyze tumor samples without paired normals (PMBLs) and identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV, CLUMPS), SCNAs (GISTIC2.0) and SVs(4 algorithms) in both cHL and PMBL. In cHL, we identified 15 CCGs, 13 recurrent SCNAs, SVs in ETV6 and CIITA, complementary alterations of JAK/STAT, NF-κB and PI3K signaling pathway components and a median number of 11 genetic drivers per tumor. Previously unappreciated aspects of the cHL genetic signature included the increased incidence of driver mutational events in cHLs with ARID1A alterations (p=0.012). Analyses of co-occurring genetic events in EBV+ and EBV- cHLs confirmed that EBV- cHLs were significantly more likely to exhibit alterations of specific NF-κB signaling intermediaries (such as TNFAIP3 mutation and/or focal copy loss, p=0.006) and perturbations of MHC class I antigen presentation pathway components (inactivating B2M mutations, HLA-B mutations or focal copy loss of 6p21.32/HLA-B, p=0.008). The latter findings provide genetic bases for the reported differences in cell surface expression of MHC class I in EBV+ and EBV- cHLs. In PMBL, we defined 15 CCGs and more selective perturbations of specific epigenetic modifiers (ZNF217 and EZH2), transcription factors (PAX5 and IRF2BP2) and TP53, in comparison with cHL. The majority of these alterations were clonal supporting their role as early drivers. We identified 18 SCNAs and additional SVs in CIITA and PD-1 ligands, recurrent alterations of JAK/STAT and NF-κB signaling pathway components and a median of 9 genetic drivers per PMBL. Antigen presentation pathways in PMBL were perturbed by multiple recurrent alterations, including B2M mutations, focal copy losses of B2M and the MHCI/II loci, SVs of CTIIA and EZH2 mutations. There was a significant correlation between genetic perturbations of MHC class I pathway components and absence of MHC class I expression in PMBL, as previously described in cHL. Recurrent cHL alterations including B2M, TNFAIP3, STAT6, GNA13 and XPO1 CCGs and 2p/2p15/2p16.1, 6p21.32, 6q23.2 and 9p/9p24.1 SCNAs were also identified in & gt;20% of PMBLs, highlighting shared pathogenetic mechanisms in these diseases. These tumors of predominantly young adults (median age: cHL 26 yrs; PMBL 34 yrs) both had a high rate of spontaneous deamination of CpGs, a clock-like mutational signature that is typically associated with aging. CHLs and PMBLs both exhibited previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high mutational burdens, in comparison with other lymphoid and solid tumors. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that in carcinogen-induced cancers (melanoma and NSCLC). Additionally, both cHLs and PMBLs had an increased incidence of microsatellite instability and APOBEC mutational signatures, features associated with a more favorable response to PD-1 blockade. Taken together, these data define genetic similarities and differences in cHL and PMBL and establish a framework to comprehensively assess molecular bases of response to PD-1 blockade and develop rational combination therapies in these diseases. Disclosures Armand: Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Research Funding; Sigma Tau: Research Funding; Adaptive: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Affimed: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Pfizer: Consultancy; ADC Therapeutics: Consultancy; Infinity: Consultancy; Genentech: Research Funding; Tensha: Research Funding. Rodig:Merck: Research Funding; Affirmed: Research Funding; Kite, a Gilead Company: Research Funding; Bristol Myers Squib: Consultancy, Honoraria, Other: Travel Expenses, Speakers Bureau. Fromm:Merck, Inc.: Research Funding. Getz:Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Shipp:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Merck & Co.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.