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The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

Two articles in this week’s issue focus on the use of ipilimumab and decitabine for patients with myelodysplasia (MDS) and acute myeloid leukemia (AML) before and after hematopoietic stem cell transplantation (HSCT) for high-risk disease. In the first article, Garcia et al report on the results of a phase 1 trial of the combination in 54 patients, demonstrating overall response rate of 52% in patients who are HSCT-naïve and 20% in patients post-HSCT; responses are usually short-lived. In the second article, Penter and colleagues characterize gene expression responses to therapy and conclude that decitabine acts directly to clear leukemic cells while ipilimumab acts on infiltrating lymphocytes in marrow and extramedullary sites. Responses are determined by leukemic cell burden and by the frequency and phenotype of infiltrating lymphocytes. Increasing bone marrow regulatory T cells is identified as a potential contributor to checkpoint inhibitor escape.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

Background: CTLA-4 blockade with ipilimumab (IPI) has modest activity in hematologic malignancies relapsed post allogeneic hematopoietic cell transplantation (allo-HCT) (Davids M, NEJM). Adding decitabine (DAC) to IPI may increase efficacy of this approach in myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), and we further asked the extent to which alloreactivity contributed to this activity. Here, we present safety (Table 1) and clinical activity (Figure 1) from a phase I, multicenter, investigator-initiated study (CTEP 10026) of DAC plus IPI in pts with R/R MDS/AML with (Arm A) and without (Arm B) prior HCT. Methods: Pts ≥ 18 y with R/R MDS (≥ 5% blasts), R/R AML or untreated secondary AML were eligible. Prior HMA was permitted but trial was later amended to exclude those with disease progression on HMA therapy within 12 weeks of study entry. Pts post allo-HCT (Arm A) were required to be > 3 mo from donor cell infusion, have no prior acute GVHD Gr III or higher, be > 2 weeks off of immunosuppression and have donor T-cell chimerism ≥ 20%. Treatment was given in 28-day cycles with a lead-in single agent DAC (cycle 0) followed by combination DAC + IPI for up to a year. DAC infusion was given at 20 mg/m2 days 1-5. IPI infusion was given at 3 mg/kg (DL0), 5 mg/kg (DL1) or 10 mg/kg (DL2) on day 1 of cycles 1-4, 5, 7, 9 and 11. DLT was defined as ≥ Gr 3 non-hematologic adverse event (AE), acute GVHD, and steroid-refractory immune-related AE within 8 weeks of first IPI dose. Results: At data cut-off, 32 of 39 enrolled pts received at least 1 dose of DAC+IPI; 4 of 7 did not move on to C1D1 due to AML-related complications. Median age was 72 y (range 29-85); 25 had AML and 7 had MDS. Median number of prior regimens was 2 (range, 0-5). Twenty of 32 pts had received prior HMA therapy. A median of 3 IPI doses were given (range, 1-8). NGS performed on screening marrows using an 88-gene panel revealed mutations most commonly in ASXL1 (n=9), RUNX1 and STAG2 (n=6 each), and DNMT3A, NRAS and TP53 (n=5 each). Arm A (Post allo-HCT): Sixteen pts received DAC+IPI across the 3 DLs (7 at DL0, 3 at DL1, 6 at DL2; dose-expansion is open at DL2). The most common treatment emergent Gr 3/4 AEs were febrile neutropenia (n=3) and AST elevation, lymphocytopenia, neutropenia, thrombocytopenia, leukopenia (n=2 each). Seven of 16 pts experienced immune-related AE, including 1 with late-onset acute GVHD Gr III (colon/liver), 4 with chronic GVHD (mild, n=2; moderate, n=1; severe n=1), and 2 with Gr 2/3 ipi-induced colitis. All immune-related AEs were reversible with steroids, except for one case of steroid refractory Gr III acute GVHD complicated by fatal septic shock (DLT at IPI 10 mg/kg). In total, 4 of 16 evaluable pts achieved response, including 3 with CR and 1 with marrow CR; 2 of 4 responders had immune-related AE. For these 16 pts, the median OS is 12.8 mo (95% CI: 7.6-NA). Arm B (transplant-naïve): Sixteen pts received DAC+IPI across the 3 DLs (4 at DL0, 3 at DL1, 6 at DL2; 3 are in dose-expansion at DL2). The most common treatment emergent Gr 3/4 AEs were lymphocytopenia, thrombocytopenia and leukopenia (n=5 each); neutropenia (n=4); and anemia, fatigue, lung infection, and febrile neutropenia (n=3 each). In 6 of 16 pts that had ≥ Gr 2 immune-related AEs, all were reversible with steroids (including Gr 3 colitis (n=1), Gr 3 dermatitis (n=2), Gr 2 pneumonitis (n=1), Gr 3 hypophysitis/Gr 2 arthritis (n=1)), except for a case of Gr 2 dermatitis (resolved)/Gr 4 immune thrombocytopenia (refractory to steroids, transfusions and IVIG) (n=1; DLT at IPI 10 mg/kg). No study treatment-related deaths were observed. Objective responses were detected in 8 of 16 evaluable pts with 3 CR, 2 CRi, and 3 marrow CR. Only 2 of 8 responders had immune-related AEs. For these 16 pts, the median OS is 18.3 mo (95% CI: 11.7-NA). Overall, 2 pts discontinued study treatment due to AE. Dose expansion continues at DL2 (MTD) in Arm A and Arm B to further establish safety and tolerability. Correlative science including immunophenotyping is ongoing and will be presented. Conclusion: In this ongoing phase 1 study in pts with R/R or secondary MDS/AML, DAC plus IPI had expected AE profile and exhibited encouraging clinical activity. The rate of immune-related toxicity was frequent but similar to prior observations with IPI 10 mg/kg for melanoma. Further, high clinical activity was observed amongst pts who did not receive allo-HCT, suggesting that an alloreactive environment may not be required to benefit from CTLA-4 blockade. Disclosures Garcia: Pfizer: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eli Lily: Research Funding. Brunner:Astra Zeneca: Research Funding; Janssen: Research Funding; Xcenda: Consultancy; GSK: Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Jazz Pharma: Consultancy; Forty Seven, Inc: Consultancy; Celgene/BMS: Consultancy, Research Funding; Biogen: Consultancy; Acceleron Pharma Inc.: Consultancy. Neuberg:Celgene: Research Funding; Madrigal Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Steensma:Takeda: Consultancy; BMS/Celgene: Consultancy; Onconova: Consultancy; Arena: Current equity holder in publicly-traded company; CRISPR: Current equity holder in publicly-traded company; Aprea Therapeutics: Research Funding; Arrowhead Pharmaceuticals: Current equity holder in publicly-traded company; Astex Pharmaceuticals, Onconova Therapeutics, Pfizer, Stemline Therapeutics, Summer Road: Consultancy; H3 Biosciences: Research Funding. Rodig:Bristol Myers Squibb: Research Funding; Merck: Research Funding; Affimed: Research Funding; KITE/Gilead: Research Funding; Immunitas: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Lindsley:Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding; Bluebird Bio: Consultancy; MedImmune: Research Funding. Davids:Research to Practice: Honoraria; Eli Lilly: Consultancy; Adaptive Biotechnologies: Consultancy; Novartis: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Merck: Consultancy; Gilead Sciences: Consultancy; Surface Oncology: Research Funding; MEI Pharma: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Ascentage Pharma: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Sunesis: Consultancy; Zentalis: Consultancy; Syros Pharmaceuticals: Consultancy; Janssen: Consultancy; BeiGene: Consultancy; Celgene: Consultancy; Genentech: Consultancy, Research Funding; AbbVie: Consultancy; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Stone:Pfizer: Consultancy; Syros: Consultancy; Stemline: Consultancy; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gemoab: Consultancy; Syndax: Consultancy, Research Funding; Macrogenics: Consultancy; Trovagene: Consultancy; Biolinerx: Consultancy; Argenix: Other; Jazz: Consultancy; Aztra-Zeneca: Consultancy; Daiichi-Sankyo: Consultancy; Astellas: Consultancy; Syntrix: Other: DSMB; Novartis: Consultancy, Research Funding; Takeda: Other: DSMB; Arog: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Janssen: Consultancy; Celgene: Consultancy, Other. DeAngelo:Takeda: Consultancy; Blueprint Medicines Corporation: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy; Abbvie: Research Funding; Glycomimetics: Research Funding; Shire: Consultancy; Autolos: Consultancy; Amgen: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Forty-Seven: Consultancy; Jazz: Consultancy. Soiffer:Rheos Therapeutics: Consultancy; Celgene: Other; Juno: Other; Alexion: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Gilead: Consultancy; Be The Match/National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Decitabine plus Ipilimumab to be given in the context of a clinical trial

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation. CD70 and CD27 are homotrimer type II and homodimer type I transmembrane proteins and members of the TNF and TNF receptor superfamilies, respectively. CD70 is transiently expressed on certain normal B-cell and dendritic cell populations upon activation and constitutively expressed on multiple B-cell tumors. CD70 activation of CD27 + T cells promotes antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Patients with germline deficiencies of either CD70 or CD27 have an increased incidence of EBV-associated lymphoid malignancies, underscoring the importance of this co-stimulatory pathway. In our series, CD70 alterations were most common in Cluster 1 DLBCLs, which also exhibited recurrent BCL6 chromosomal translocations. The co-occurrence of CD70 and BCL6 genetic alterations was noteworthy because of the established role of CD8 + T-cell dependent immune surveillance in murine models of Bcl6-driven DLBCL [Nature Medicine 2014; 20:283-290]. To assess the consequences of Cd70 deficiency and perturbed CD70/CD27 co-stimulation on Bcl6-driven lymphomagenesis, we crossed the previously described Bcl6tg/+ and Cd70-/- mice to generate Cd70-/-; Bcl6tg/+ animals. In the aging cohorts, Cd70-/-; Bcl6tg/+ mice were more likely than Bcl6tg/+ animals (or the Cd70-/- or wild-type [WT] groups) to be euthanized for symptoms before the study endpoint (18 months [mo]) (5 of 18 Cd70-/-; Bcl6tg/+ euthanized for symptoms prior to the first of 9 Bcl6tg/+). Additionally, significantly greater total numbers and percentages of Cd70-/-; Bcl6tg/+ mice, in comparison to Bcl6tg/+ or Cd70-/- animals, were euthanized for symptoms (64% Cd70-/-; Bcl6tg/+ vs. 29% Bcl6tg/+, p=0.005 and 64% Cd70-/-; Bcl6tg/+ vs. 11% Cd70-/-, p=0.002). Almost all euthanized Cd70-/; Bcl6tg/+ and Bcl6tg/+ micehad massively enlarged spleens infiltrated with histopathologically confirmed DLBCLs characterized by clonal Ig gene rearrangements. Our findings indicate that genetic perturbation of Cd70 accelerates the onset and significantly increases the incidence of Bcl6-driven DLBCL. To characterize potential differences in the anti-tumor immune responses in Cd70-/-; Bcl6tg/+ and Bcl6tg/+ mice (and Cd70-/- and WT controls), we also harvested spleens from 6 animals in each of the aging cohorts at predetermined intervals (2, 6, 14 and 18 mo). We analyzed the composition of splenic-cell suspensions by flow cytometry and evaluated the intact splenic architecture and morphology with expert hematopathologists (VS, KW and SR). At 14 and 18 mo, spleens from WT and Cd70-/- animals were largely normal in appearance and size. In contrast, spleens from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ mice exhibited disordered architecture and abnormal pre-malignant lymphoid proliferation with expanded white pulp including morphologically and immunophenotypically aberrant B cells of small to large size and increased infiltrating T-cells. Additionally, significantly higher fractions of splenic CD8 + T cells from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals expressed the CD69 activation marker and exhibited terminal differentiation, consistent with an ongoing anti-tumor immune response. Interestingly, Bcl6tg/+ animals had significantly higher percentages of splenic TCRβ + T cells at the earlier time point (14 mo) with delayed-onset splenomegaly (at 18 mo), which is in marked contrast to the Cd70-/-; Bcl6tg/+ mice that had significantly lower percentages of splenic TCRβ + T cells at the earlier time point (14 mo) and early-onset splenomegaly (14 mo). These findings suggest that the initial T-cell mediated immune response was more effective in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ animals. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective anti-tumor immune response in Bcl6tg/+-driven DLBCL. Disclosures Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rodig: Affimed: Research Funding; Bristol-Myers-Squibb: Research Funding; Merck: Research Funding; Immunitas: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Consultancy; AstraZeneca: Consultancy, Research Funding; Merck: Research Funding; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding.

Nivolumab, an anti–programmed death-1 (PD-1) monoclonal antibody, showed promising activity in relapsed or refractory (R/R) follicular lymphoma (FL) in a phase 1 study. We conducted a phase 2 trial to further evaluate its efficacy and safety in patients with R/R FL and to explore biomarkers of response. Patients with R/R FL and at least 2 prior lines of therapy, each containing a CD20 antibody or an alkylating agent, were treated with nivolumab 3 mg/kg every 2 weeks. The primary end point was objective response rate (ORR) assessed by an independent radiologic review committee. Biomarker analyses included gene expression profiling and multiplex immunofluorescence studies of pretreatment tumor samples. A total of 92 patients were treated. After a minimum follow-up of 12 months, ORR was 4% (4 of 92 patients). Median progression-free survival (PFS) was 2.2 months (95% confidence interval [CI], 1.9-3.6 months). Median duration of response was 11 months (95% CI, 8-14 months). Exploratory analyses suggested that responders had significantly higher proportion of CD3+ T cells in the tumor microenvironment than nonresponders, but no significant differences in PD-1 or programmed death-ligand 1 expression were observed. High expression of a set of tumor-associated macrophage genes was associated with reduced PFS (hazard ratio, 3.28; 95% CI, 1.76-6.11; P = .001). The safety profile was consistent with previous reports of nivolumab. In conclusion, nivolumab monotherapy was associated with very limited activity in patients with R/R FL. Better understanding of the immune biology of this disease may facilitate the development of effective checkpoint-based strategies. This trial was registered at www.clinicaltrials.gov as #NCT02038946. Key Points: • Nivolumab monotherapy was associated with very limited activity in patients with R/R FL. • Understanding the tumor microenvironment may be necessary to develop effective checkpoint-based strategies in FL.

Classic Hodgkin lymphoma (cHL) is a tumor composed of rare, atypical, germinal center-derived B cells (Hodgkin Reed-Sternberg [HRS] cells) embedded within a robust but ineffective inflammatory milieu. The cHL tumor microenvironment (TME) is compartmentalized into "niches" rich in programmed cell death-1 ligand (PD-L1)-positive HRS cells and tumor-associated macrophages (TAMs), which associate with PD-1-positive T cells to suppress antitumor immunity via PD-L1/PD-1 signaling. Despite the exquisite sensitivity of cHL to PD-1 checkpoint blockade, most patients eventually relapse and need therapeutic alternatives. Using multiplex immunofluorescence microscopy with digital image analysis, we found that cHL is highly enriched for non-T-regulatory, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)-positive T cells (compared with reactive lymphoid tissues) that outnumber PD-1-positive and lymphocyte-activating gene-3 (LAG-3)-positive T cells. In addition, T cells touching HRS cells are more frequently positive for CTLA-4 than for PD-1 or LAG-3. We further found that HRS cells, and a subset of TAMs, are positive for the CTLA-4 ligand CD86 and that the fractions of T cells and TAMs that are CTLA-4-positive and CD86-positive, respectively, are greater within a 75 μm HRS cell niche relative to areas outside this region (CTLA-4, 38% vs 18% [P = .0001]; CD86, 38% vs 24% [P = .0007]). Importantly, CTLA-4-positive cells are present, and focally contact HRS cells, in recurrent cHL tumors following a variety of therapies, including PD-1 blockade. These results implicate CTLA-4:CD86 interactions as a component of the immunologically privileged niche surrounding HRS cells and raise the possibility that patients with cHL refractory to PD-1 blockade may benefit from CTLA-4 blockade.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) are extranodal large B-cell lymphomas (LBCLs) with inferior responses to current empiric treatment regimens. To identify targetable genetic features of PCNSL and PTL, we characterized their recurrent somatic mutations, chromosomal rearrangements, copy number alterations (CNAs), and associated driver genes, and compared these comprehensive genetic signatures to those of diffuse LBCL and primary mediastinal large B-cell lymphoma (PMBL). These studies identify unique combinations of genetic alterations in discrete LBCL subtypes and subtype-selective bases for targeted therapy. PCNSLs and PTLs frequently exhibit genomic instability, and near-uniform, often biallelic, CDKN2A loss with rare TP53 mutations. PCNSLs and PTLs also use multiple genetic mechanisms to target key genes and pathways and exhibit near-uniform oncogenic Toll-like receptor signaling as a result of MYD88 mutation and/or NFKBIZ amplification, frequent concurrent B-cell receptor pathway activation, and deregulation of BCL6. Of great interest, PCNSLs and PTLs also have frequent 9p24.1/PD-L1/PD-L2 CNAs and additional translocations of these loci, structural bases of immune evasion that are shared with PMBL.

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 >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.

Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the transformation of CLL to an aggressive lymphoma, or Richter's transformation (RT), remains a clinical challenge, as it responds poorly to standard therapies and shortens survival. Recent studies demonstrate that RT, but not underlying CLL, responds to PD-1 checkpoint blockade (CPB) with an overall response rate of 43-65%. Given the central role of T cells in anti-tumor immunity, we hypothesized that differences in T cell populations underlie response and resistance to CPB in RT. We focused on a discovery cohort of 6 patients with RT (4 responders, 2 non-responders) and 2 patients with relapsed/refractory CLL enrolled on a study in which patients were initiated with anti-PD1 therapy (nivolumab 3 mg/kg every 2 weeks), with subsequent concurrent ibrutinib (420 mg daily)(NCT 02420912). We examined a total of 15 serial study marrow specimens collected at treatment initiation and 3 month response evaluation, as well as 2 healthy marrow donors. To systematically discover the T cell populations and states associated with CPB response in RT, we performed single-cell RNA-sequencing (scRNA-seq, 10x Genomics) of non-lymphoma (CD5-CD19-) cells isolated by flow cytometry from marrow samples. A total of 60,727 T and NK cells were captured with average detection of 1001 genes/cell. Using the novel joint clustering approach Conos, 11 transcriptionally distinct clusters of lymphocytes were identified. We first contrasted baseline RT/CLL with normal marrow and observed differences across T cell populations, which we confirmed through the examination of publicly available marrow scRNA-seq data from 28 healthy donors. Compared to normal marrow, RT/CLL marrow was enriched for cytotoxic populations, including both CD8 effector/effector memory (E/EM) (p=0.001, t-test) and cytotoxic CD4 (p=0.001) T cells as well as for cells expressing multiple exhaustion markers, including PDCD1, LAG3 and TIGIT (p=0.001). In contrast, normal marrow contained increased T cells with a naïve-like phenotype (p=0.06). When we focused on the pre-treatment samples from RT patients, RT responders had a larger CD8 E/EM population (p=0.04) and fewer T regulatory cells (p=0.006, t-test) than RT non-responders. Using DESeq2 to compare clusters from all samples, we evaluated if there were differences in gene expression between RT responders and non-responders. CD8 E/EM T cells of RT non-responders showed increased expression of TOX, a recently uncovered master regulator of cell exhaustion (padj =0.00016), while this cell subtype in RT responders upregulated a contrasting program of activating transcription factors as well as the co-stimulatory gene CD226 (padj =0.04). As for CD4 T cells, RT responders revealed an enriched cytotoxic gene program compared to RT non-responders (padjPRF1 5.9 x 10-10, GZMH 6.0 x 10-6, NKG7 6.4 x 10-19). To investigate whether response to CPB therapy for RT was associated with changes in the T cell receptor (TCR) repertoire, and to obtain protein-level validation of transcriptional signatures, we performed single-cell TCR sequencing with paired gene and protein expression (10x Genomics) on pre- and post-therapy samples from a RT responder and a non-responder. Indeed, we confirmed our gene expression findings, including validation of cytotoxic CD4 T cells and the enrichment of CD226 protein in E/EM CD8 T cells in the RT responder. TCR clonal expansion was observed in the RT responder at baseline with persistence of enriched clonotypes following CPB, suggesting the presence of tumor-reactive T cell clones. In contrast, the RT non-responder displayed higher TCR diversity with enriched clonotypes showing increased exhaustion post-CPB (p In conclusion, we identified marrow T cell populations enriched in RT patients and described distinct T cell transcriptional programs that delineate RT responders from non-responders. We have thus discovered candidate gene biomarkers that may identify patients likely to respond to CPB therapies and uncovered a CD8 E/EM T cell population that is likely to underlie response to PD-1 CPB. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Neuberg:Celgene: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Pharmacyclics: Research Funding. Rodig:Bristol Myers Squib: Consultancy, Honoraria, Other: Travel Expenses, Speakers Bureau; Kite, a Gilead Company: Research Funding; Affirmed: Research Funding; Merck: Research Funding. Wierda:KITE pharma: Research Funding; Gilead Sciences: Research Funding; AbbVie: Research Funding; Acerta Pharma Inc: Research Funding; Genentech: Research Funding; GSK/Novartis: Research Funding; Pharmacyclics LLC: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Cyclcel: Research Funding; Loxo Oncology Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Juno Therapeutics: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma resistant to conventional chemotherapy. Although defined by the characteristic t(11;14) translocation, MCL has not been recapitulated in transgenic mouse models of cyclin D1 overexpression alone. Indeed, several genetic aberrations have been identified in MCL that may contribute to its pathogenesis and chemoresistance. Of particular interest is the frequent biallelic deletion of the proapoptotic BCL-2 family protein BIM. BIM exerts its pro-death function via its α-helical BH3 death domain that has the dual capacity to inhibit antiapoptotic proteins such as BCL-2 and MCL-1 and directly trigger proapoptotic proteins such as the mitochondrial executioner protein BAX. To evaluate a functional role for Bim deletion in the pathogenesis of MCL, we generated cyclin D1-transgenic mice harboring Bim-deficient B cells. In response to immunization, Eμ(CycD1)CD19(CRE)Bim(fl/fl) mice manifested selective expansion of their splenic mantle zone compartment. Three distinct immune stimulation regimens induced lymphomas with histopathologic and molecular features of human MCL in a subset of mice. Thus, deletion of Bim in B cells, in the context of cyclin D1 overexpression, disrupts a critical control point in lymphoid maturation and predisposes to the development of MCL. This genetic proof of concept for MCL pathogenesis suggests an opportunity to reactivate the death pathway by pharmacologic mimicry of proapoptotic BIM.

The present study aimed to determine the safety and activity of the histone deacetylase inhibitor panobinostat in patients with relapsed/refractory Waldenström macroglobulinemia (WM). Eligibility criteria included patients with relapsed/refractory WM with any number of prior therapies. Patients received panobinostat at 30 mg 3 times a week; 12 of 36 (33%) patients were enrolled at 25 mg dose. A total of 36 patients received therapy. The median age was 62 years (range, 47-80) and the median number of prior therapies was 3 (range, 1-8). All of the patients had received prior rituximab. Minimal response (MR) or better was achieved in 47% of patients (90% confidence interval [CI], 33-62), with 22% partial remissions and 25% MR. In addition, 18 (50%) patients achieved stable disease and none showed progression while on therapy. The median time to first response was 1.8 months (range, 1.7-3.2). The median progression-free survival was 6.6 months(90% CI, 5.5-14.8). Grade 3 and 4 toxicities included thrombocytopenia (67%), neutropenia (36%), anemia (28%), leukopenia (22%), and fatigue (11%). We conclude that panobinostat is an active therapeutic agent in patients with relapsed/ refractory WM. This study (www.clinicaltrials.gov identifier: NCT00936611) establishes a role for histone deacetylase inhibitors as an active class of therapeutic agents in WM.

VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain–containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative regulator of tumor growth by inhibiting angiogenesis.

We investigated the therapeutic potential of JQ1, an inhibitor of the BET class of human bromodomain proteins, in B-cell acute lymphoblastic leukemia (B-ALL). We show that JQ1 potently reduces the viability of B-ALL cell lines with high-risk cytogenetics. Among the most sensitive were lines with rearrangements of CRLF2, which is overexpressed in ∼ 10% of B-ALL. CRLF2 heterodimerizes with the IL7 receptor (IL7R) and signals through JAK2, JAK1, and STAT5 to drive proliferation and suppress apoptosis. As previously observed, JQ1 induced the down-regulation of MYC transcription, the loss of BRD4 at the MYC promoter, and the reduced expression of c-Myc target genes. Strikingly, JQ1 also down-regulated IL7R transcription, depleted BRD4 from the IL7R promoter, and reduced JAK2 and STAT5 phosphorylation. Genome-wide expression profiling demonstrated a restricted effect of JQ1 on transcription, with MYC and IL7R being among the most down-regulated genes. Indeed, IL7R was the only cytokine receptor in CRLF2-rearranged B-ALL cells significantly down-regulated by JQ1 treatment. In mice xenografted with primary human CRLF2-rearranged B-ALL, JQ1 suppressed c-Myc expression and STAT5 phosphorylation and significantly prolonged survival. Thus, bromodomain inhibition is a promising therapeutic strategy for B-ALL as well as other conditions dependent on IL7R signaling.

MSI2 is highly expressed in human myeloid leukemia (AML) cell lines, and high expression of MSI2 mRNA is associated with decreased survival in AML, suggesting its use as a new prognostic marker. To test this, we measured MSI2 protein level by immunohistochemistry in 120 AML patients. Most cases (70%) showed some nuclear or cytoplasmic positivity, but the percentage of positive cells was low in most cases. Despite this, MSI2 protein expression was negatively associated with outcome, particularly for patients with good cytogenetic subgroup. For practical diagnostic purposes, the strongest significance of association was seen in cases with > 1% of cells showing strong MSI2 staining, these having a very poor outcome (P < .0001). Multivariate analysis with cytogenetic category, age, white cell count, and French-American-British subtype demonstrated that nuclear MSI2 levels were independently predictive of outcome (P = .0497). These results confirm the association of MSI2 expression with outcome in AML at the protein level and demonstrate the utility of MSI2 protein as a clinical prognostic biomarker. In addition, although positive at some level in most cases, its prognostic power derived from few positive cells, supporting its role in control of normal hematopoietic stem cell function and highlighting its role in disease progression.

Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBVdriven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid Bcell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydratebinding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4 Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)–mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8 T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors. Fil: Ouyang, Ying. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Juszczynski, Przemyslaw. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Rodig, Scott J.. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Green, Michael R.. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: O´ Donnell, Evan. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Currie, Treeve. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Armant, Myriam. Immune Disease Institute/Harvard Medical School. Center for Human Cell Therapy; Estados Unidos Fil: Takeyama, Kunihiko. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Monti, Stefano. Broad Institute; Estados Unidos Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Ritz, Jerome. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos Fil: Kutok, Jeffery L.. Brigham and Women’s Hospital. Department of Pathology; Estados Unidos Fil: Shipp, Margaret A.. Dana-Farber Cancer Institute. Department of Medical Oncology; Estados Unidos

Background: Classical Hodgkin lymphoma (cHL) displays near-universal genetic deregulation of PD-1 ligand expression, and relapsed/refractory (R/R) cHL is uniquely sensitive to PD-1 blockade. However, such treatment for patients (pts) who relapse after or are ineligible for autologous stem cell transplantation (ASCT) appears to be rarely, if ever, curative. Deploying PD-1 blockade early after ASCT could leverage the remodeling immune landscape and minimal residual disease state to increase the cure rate of ASCT. We therefore conducted a phase 2 multi-center single-arm study of the anti-PD-1 monoclonal antibody pembrolizumab (pembro) in pts with chemosensitive R/R cHL after ASCT. Another arm of this study enrolled pts with R/R DLBCL and will be presented separately. Methods: Adult pts with R/R cHL who had received 2-3 lines of prior therapy and ASCT and who were chemosensitive prior to ASCT were enrolled on this study. In addition to meeting standard eligibility criteria for pembro treatment, pts had to have recovered from ASCT toxicities and begin study treatment within 60 days of stem cell infusion (goal within 21 days). They received pembro 200mg IV every 3 weeks for 8 cycles. PET-CT scans were obtained at post-ASCT baseline, after 3 and 7 cycles, then at 12 and 18 months post-ASCT. The primary endpoint was the progression-free survival rate (PFS) at 18 months after ASCT, assessed using International Harmonization Project 2007 criteria. Results: 31 pts were enrolled and 1 withdrew consent before starting treatment. Among the 30 eligible patients, median age was 33 (20-69). 26 pts (87%) were high-risk by virtue of primary refractory disease (57%), relapse within 12 months (17%), extranodal disease at relapse (27%) or absence of metabolic CR at ASCT (10%). At study baseline post-ASCT, 97% were in CR. 24 pts (80%) completed 8 cycles of pembro per protocol. 6 pts (20%) stopped pembro early for pt choice (n=2, including 1 pt with gr2 pneumonitis) or toxicity (n=4, including 2 pts with gr3 hepatitis, 1 with g3 pneumonitis, 1 with g2 diplopia). 9 pts (30%) experienced a total of 28 gr3 or higher adverse events (AEs). The most common gr4 AE was neutropenia (10%). 3 patients (10%) experienced 7 gr3-4 AEs at least probably related to pembro (gr3 diarrhea and gr3 eosinophilic colitis in 1 pt, gr3 leukopenia and gr4 neutropenia in 1 pt, gr3 leukopenia and gr3 ALT and AST elevation in 1 pt). 11 pts (37%) experienced at least one immune-related AE of gr2 or higher severity: pneumonitis (n=2 gr2, n=1 gr3), thyroid dysfunction (n=1 gr2), transaminitis (n=2 gr3), colitis/diarrhea (n=1 gr2, n=2 gr3), rash (n=2 gr2), pulmonary hemorrhage (n=1 gr3), arthritis (n=1 gr2), and increased creatinine (n=1 gr2). There was no treatment-related death. Among the 30 eligible pts, 2 were lost to follow-up after their 12m assessments (both in CR). 27 pts (90%) were evaluable for the primary endpoint (the last pt is still in follow-up and results will be updated for the meeting). 6 patients (20%) relapsed at a median of 8 months (3-18) from ASCT, and all other evaluable patients were in CR at the 18m timepoint. The KM estimate of 18m PFS for high-risk patients was 78% (95%CI 54-91). The 18m overall survival was 100%. Tumor biopsies from a pt who progressed on study demonstrated an increase in the %age of PD1+ T cells at progression, as well as an increase in the %age of PD-L1+ macrophages and PD-L1+ Reed-Sternberg cells (Figure). Other correlative studies including immune reconstitution and MRD analyses are ongoing. Conclusions: Pembrolizumab administered after ASCT in patients with R/R cHL has a safety profile that appears similar to its use in the R/R setting, although possibly with a higher rate of neutropenia. The 18-month progression-free rate in this high-risk cohort compares favorably with previous published studies, and supports the hypothesis that PD-1 blockade in this setting may increase the efficacy of ASCT. This should be tested in a randomized trial. Figure. Figure. Disclosures Armand: Pfizer: Consultancy; Affimed: Consultancy, Research Funding; Otsuka: Research Funding; Merck: Consultancy, Research Funding; Infinity: Consultancy; Adaptive: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Chen:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Consultancy; Magenta Therapeutics: Consultancy; REGiMMUNE: Consultancy. LaCasce:Humanigen: Consultancy, Honoraria; Research to Practice: Speakers Bureau; Bristol-Myers Squibb: Other: Data safety and monitoring board; Seattle Genetics: Consultancy, Honoraria. Jacobson:Pfizer: Consultancy; Humanigen: Consultancy; Precision Bioscience: Consultancy; Novartis: Consultancy; Kite: Consultancy; Bayer: Consultancy. Jacobsen:Merck: Consultancy; Seattle Genetics: Consultancy. Rodig:Bristol-Meyers-Squibb: Research Funding; KITE Pharma: Research Funding; Affimed Inc.: Research Funding; Merck & Co., Inc.: Research Funding. Shipp:AstraZeneca: Honoraria; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Merck: Research Funding. Herrera:BMS: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; KiTE Pharma: Consultancy, Research Funding; Seattle Genetics: Research Funding; Immune Design: Research Funding; Astra Zeneca: Research Funding.

Introduction: Patients (pts) with relapsed/refractory PMBCL (rrPMBCL) are typically treated like those with diffuse large B cell lymphoma (DLBCL), often with limited effective treatment options and poor outcomes. Unlike DLBCL and similar to classical Hodgkin lymphoma, PMBCL has frequent genetic abnormalities leading to over-expression of the programmed death (PD)-1 ligands, PD-L1 and PD-L2. This suggests that rrPMBCL should be sensitive to PD-1 blockade. In the phase 1b KEYNOTE (KN)-013 study (NCT01953692), pembrolizumab was associated with frequent and durable responses (Zinzani, Blood 2017) in pts with rrPMBCL. The international phase 2 KN170 (NCT02576990) study was conducted to extend these findings and evaluate correlative biomarkers of response. Here, we present updated results of all pts in KN013 (n=21) and the first full analysis of pts in KN170 (n=53). Methods: KN013 enrolled pts with rrPMBCL who had failed, were ineligible for, or refused autologous stem cell transplant (ASCT). KN170 enrolled pts with rrPMBCL who had relapsed after or were ineligible for ASCT with ≥2 lines of prior therapy. In KN013, the initial 10 pts received pembrolizumab 10 mg/kg Q2W; the remaining 11 patients and all patients on KN170 received pembrolizumab 200 mg Q3W for up to 2 years. Archival or fresh tumor tissue obtained before pembrolizumab initiation was used for correlative studies. Tumor response was assessed with PET/CT scans by IWG 2007 criteria. The primary endpoint of KN170 was objective response rate (ORR) by blinded independent central review (BICR). Secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety/tolerability. Exploratory endpoints included response by Lugano 2014 criteria and biomarker analyses. Data cutoff dates for this analysis were Apr 4, 2018 for KN013 and April 13, 2018 for KN170. Results: KN013 enrolled 21 pts with a median of 3 prior lines of therapy, of whom 13 (62%) were ASCT-ineligible. KN170 enrolled 53 pts also with a median of 3 prior lines, of whom 39 (74%) were ASCT-ineligible due to chemorefractoriness. In KN013, ORR was 48% (10/21; 95% CI, 26-70), with a CR rate of 33% (7/21). In KN170, ORR was 45% (24/53; 95%CI, 32-60), with a CR rate of 13% (7/53; 11/53 [21% by Lugano criteria]). In KN013, after a median follow-up duration of 29.1 mo (range, 0.6-49.6), median DOR was not reached (range, 1.9+ to 39.8+ mo) (Panel A). 2 patients in KN013 in CR at 2 years remained in CR after a further 12 and 18 mo of follow-up off therapy. After a median follow-up of 12.5 mo for KN170 (range, 0.1-25.6), median DOR was not reached (range, 1.1+ to 22.0+ mo) (Panel A). At data cutoff, no patient who achieved a CR on KN170 had relapsed. In KN013, median PFS was 10.4 mo (95%CI, 3.4 to not reached) with 12-mo PFS rate of 47%; median OS was 31.4 mo with 12-mo OS rate of 65% (Panel B). In KN170, median PFS was 5.5 mo (95%CI, 2.8-12.1) with 12-mo PFS rate of 38%; median OS was not reached (95% CI, 7.3 to not reached) with 12-mo OS rate of 58% (Panel B). In KN013, no new safety signals were observed compared with prior analyses. In KN170, 30 (57%) pts had a treatment-related AE (TRAE). Common (≥5%) TRAEs included neutropenia (19%), hypothyroidism and asthenia (8% each), and pyrexia (6%). 12 (23%) pts had a grade 3-4 TRAE, including 5 (9%) with grade 3 and 2 (4%) with grade 4 neutropenia. Six (11%) pts had an immune-mediated AE including 1 (2%) with grade 4 pneumonitis. There were no treatment-related deaths. Conclusion: Together with the longer follow-up results of KN013, KN170, the largest prospective clinical trial in rrPMBCL, establishes the robust antitumor activity of pembrolizumab in this disease, with exceptionally durable responses and survival in responding patients. These results provided the basis for the FDA accelerated approval of pembrolizumab in patients with rrPMBCL. Disclosures Armand: Otsuka: Research Funding; Adaptive: Research Funding; Merck: Consultancy, Research Funding; Affimed: Consultancy, Research Funding; Pfizer: Consultancy; Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Rodig:Merck & Co., Inc.: Research Funding; Affimed Inc.: Research Funding; KITE Pharma: Research Funding; Bristol-Meyers-Squibb: Research Funding. Özcan:Jazz: Other: Travel support; Bayer: Research Funding; BMS: Honoraria; MSD: Other: travel support, Research Funding; Janssen: Other: Travel Support, Research Funding; Jazz: Other; Celgene: Other: Travel support, Research Funding; Roche: Honoraria, Research Funding; Archigen: Research Funding; Novartis: Research Funding; MSD: Research Funding; Abbvie: Other: Travel payment; Takeda: Honoraria, Other: Travel payment, Research Funding. Fogliatto:Novartis: Consultancy; Roche: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding. Walewski:Roche, Celegene, Takeda, Janssen-Cilag, and Servier: Membership on an entity's Board of Directors or advisory committees; Roche, Celgene, Takeda, Janssen-Cilag, and Servier: Honoraria; Roche, GSK/Novartis, Takeda, and Janssen-Cilag: Research Funding. Gulbas:Pfizer: Other: Travel expenses; Roche and Janssen: Honoraria; Gilead: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees. Ribrag:Incyte Corporation: Consultancy; MSD: Honoraria; NanoString Technologies: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Other: travel; Gilead: Consultancy, Honoraria; Roche: Honoraria, Other: travel; Infinity: Consultancy, Honoraria; epizyme: Consultancy, Honoraria; Amgen: Research Funding; pharmamar: Other: travel; Servier: Consultancy, Honoraria; argenX: Research Funding. Christian:Immunomedics: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Acerta: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding. Perini:Janssen and Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen and Takeda: Speakers Bureau; Janssen and Takeda: Other: Travel expenses. Salles:Merck: Honoraria; Novartis: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; AbbVie: Honoraria; Acerta: Honoraria; Takeda: Honoraria; Pfizer: Honoraria; Morphosys: Honoraria; Servier: Honoraria; Amgen: Honoraria; Epizyme: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Svoboda:Pharmacyclics: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Kyowa: Consultancy; Regeneron: Research Funding; TG Therapeutics: Research Funding; KITE: Consultancy; Merck: Research Funding; Seattle Genetics: Consultancy, Research Funding. Chatterjee:Merck & Co., Inc.: Employment. Orlowski:Merck & Co., Inc.: Employment. Balakumaran:Amgen: Equity Ownership; Merck & Co., Inc.: Employment, Equity Ownership. Shipp:Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Merck: Research Funding. Zinzani:PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Background: The leading cause of death after high-dose chemotherapy and autologous stem cell transplantation (ASCT) for relapsed / refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains disease relapse. Except for specific subsets, DLBCL appears to have relatively low susceptibility to single-agent PD-1 blockade. However, administering PD-1 blockade early after ASCT could leverage the remodeling immune landscape and minimal residual disease state to increase the therapeutic effect of PD-1 blockade and decrease the relapse rate after ASCT. A prior study with pidilizumab showed encouraging results in this setting (Armand et al. JCO 2013;31:4199-206), but the specificity of that antibody is still under investigation. We therefore conducted a phase 2 multi-center single-arm study of the anti-PD-1 monoclonal antibody pembrolizumab (pembro) in patients with chemosensitive DLBCL after ASCT. Another arm of this study enrolled pts with R/R classical Hodgkin lymphoma and will be presented separately. Methods: Patients ≥ 18y with R/R DLBCL who had received no more than 3 lines of prior therapy and had undergone ASCT with chemosensitive disease were enrolled on this study. In addition to meeting standard eligibility criteria for pembro treatment, pts had to have recovered from ASCT toxicities and had to begin study treatment within 60 days of stem cell infusion, with a goal of starting treatment within 21 days of hospital discharge. All patients received pembro 200mg IV every 3 weeks for 8 cycles. PET-CT scans were obtained at post-ASCT baseline, after 3 and 7 cycles, then at 12 and 18 months post-ASCT. The primary endpoint was the progression-free survival rate (PFS) at 18 months after ASCT, assessed using the International Harmonization Project 2007 criteria, with the approach considered promising if 22 patients were alive and in remission at 18 months. Results: 31 pts were enrolled and 2 withdrew consent before starting treatment. Among the 29 eligible pts, median age was 57 (22-76). Prior to ASCT, 18 (62%) were in CR and 11 (38%) were in PR. At study baseline (post-ASCT), 25 (86%) were in CR. 18 pts (62%) completed all 8 cycles of pembro per protocol. 11 pts (38%) stopped pembro early for pt choice (n=1, after febrile neutropenia), toxicity (n=6, including 3 pts with gr3 pneumonitis, 1 with g3 hepatitis, 1 with gr4 aplastic anemia) or progressive disease (n=4). 23 pts (79%) experienced a total of 57 gr3 or higher adverse events (AEs). The most common gr4 AE was neutropenia (n=6, 21%). Concerning related AEs, 9 pts (31%) experienced 14 gr3-4 AEs at least probably related to pembro including neutropenia (4 gr3, 1 gr 4) and pneumonitis (2 gr3). 10 pts (34%) experienced at least one immune-related AE of gr2 or higher severity including pneumonitis (n=1 gr2, n=2 gr3), transaminitis (n=1 gr2, n=1 gr3) and rash (n=1 gr2, n=1 gr3). There were no treatment-related deaths. Among the 29 eligible pts, 1 patient withdrew consent after cycle 1 and 1 pt was lost to follow-up after the 12m assessment (in CR). 27 pts (93%) were evaluable for the primary endpoint. 10 patients (34%, 95% CI: 18-54%) experienced disease relapse at a median of 5 months (3-18) after ASCT, and all other evaluable patients (n=17, 59%, 95% CI: 39-76%) were in CR at the 18m timepoint. 2 pts have died, both of whom had relapse at 3m after ASCT. Correlative studies including immune reconstitution and MRD analyses are ongoing. Conclusions: Pembrolizumab administered after ASCT in patients with R/R DLBCL is feasible with toxicity similar to its use in the R/R setting for other hematological malignancies. The high rate of neutropenia on this study, which is not a common AE of pembro in other settings, may be related to the burden of prior therapy or possibly to an accentuated toxicity of pembro in this specific patient population. The 18-month progression-free rate did not meet the protocol-specified primary objective, and therefore does not support a larger confirmatory study. Future studies in this setting should likely focus on specific subsets of DLBCL, e.g. primary mediastinal BCL, EBV+ DLBCL, T cell histiocyte rich LCL, which may be especially sensitive to PD1 blockade. Disclosures Chen: REGiMMUNE: Consultancy; Magenta Therapeutics: Consultancy; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Consultancy. Armand:Pfizer: Consultancy; Adaptive: Research Funding; Merck: Consultancy, Research Funding; Infinity: Consultancy; Affimed: Consultancy, Research Funding; Otsuka: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Herrera:Seattle Genetics: Research Funding; KiTE Pharma: Consultancy, Research Funding; Gilead Sciences: Research Funding; Merck, Inc.: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Research Funding; Genentech: Consultancy, Research Funding; Immune Design: Research Funding. LaCasce:Humanigen: Consultancy, Honoraria; Bristol-Myers Squibb: Other: Data safety and monitoring board; Research to Practice: Speakers Bureau; Seattle Genetics: Consultancy, Honoraria. Jacobson:Pfizer: Consultancy; Precision Bioscience: Consultancy; Kite: Consultancy; Novartis: Consultancy; Bayer: Consultancy; Humanigen: Consultancy. Jacobsen:Merck: Consultancy; Seattle Genetics: Consultancy. Rodig:Merck & Co., Inc.: Research Funding; Affimed Inc.: Research Funding; KITE Pharma: Research Funding; Bristol-Meyers-Squibb: Research Funding. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

BACKGROUND: Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that shows efficacy in patients with refractory diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma and transformed follicular lymphoma after failure of conventional therapy. However, the exact mechanism of anti-tumor immunity is poorly understood, in part due to the dearth of data on the events in the tumor micro-environment (TME) that occur upon exposure to CAR-T cells. We sought to quantify and characterize both CAR-T cells and non-CAR T cells within the TME of DLBCL using tissue biopsy samples collected in the ZUMA-1 multicenter trial of CAR-T cell therapy for patients with refractory DLBCL. METHODS: Tumor samples obtained from patients 5-30 days (median 10 days) after CAR-T infusion ("CAR-treated", n=14) and randomly-selected untreated ("untreated ", n=15) archival DLBCL tissue samples were analyzed by multiplex immunofluorescence using formalin-fixed, paraffin embedded tissue sections, with successive labeling by the primary antibodies KIP-1 and/or KIP-3 (recognizing separate CD19 CAR epitopes), PAX5, PD-1, CD4, and CD8, followed by secondary amplification and tyramide-conjugated fluorophores. For each case, at least 3 representative 20x fields of view were selected and imaged using a multispectral imaging platform. Two specific image analysis algorithms were designed to accurately identify CD4 and CD8 T cells and PAX5+ DLBCL cells simultaneously, then to threshold PD-1 and KIP-1/-3 by relative fluorescent units (RFU) in each phenotype. RESULTS: We identified CAR T-cells within the fixed biopsy samples of CAR-treated DLBCLs by immunostaining with CAR T-cell specific antibody KIP-1; at the timepoints analyzed, CAR T-cells comprised only a small minority of total T- cells ( CONCLUSIONS: CD4+ and CD8+ CAR-T cells can be detected in CAR-treated DLBCL patient tissue biopsies by multiplex immunofluorescence. At the time points analyzed to date, CAR-T cells comprise only a small percentage of all T-cells ( *PH.C and M.L share equal contribution. Disclosures Armand: Otsuka: Research Funding; Affimed: Consultancy, Research Funding; Pfizer: Consultancy; Infinity: Consultancy; Adaptive: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Roberts:KITE: Employment. Rossi:KITE: Employment. Bot:KITE: Employment. Go:KITE: Employment. Rodig:Merck: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; KITE: Research Funding.

Introduction: Natural Killer cells (NK cells) play an important role in tumor immune-surveillance. NK-mediated cell killing can occur through distinct mechanisms: (1) by activating the receptor NKG2D and natural cytotoxicity receptors (NCR), and (2) through the potent activating receptor CD16 (FcγRIII) which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Therapeutic strategies to harness the ability of NK cells to induce an ADCC response are emerging. AFM13 is a CD30/CD16A targeting high affinity bispecific tetravalent antibody that engages and activates NK cells. This study evaluates the ability of AFM13 to engage innate immunity through specific activation and recruitment of NK cells to tumors expressing CD30 and the impact of these effects on clinical outcome. In addition, it examines the immunologic changes in the tumor and peripheral blood as a function of the dose and method of administration of AFM13 over time. Methods: Subjects with relapsed or refractory CD30 expressing lymphoma with cutaneous involvement were recruited into 3 treatment cohorts: (1) 1.5 mg/kg IV weekly, (2) 7 mg/kg IV weekly and (3) 7 mg/kg continuous intravenous infusion (CIVI) over 5 days weekly. Each cohort consisted of 3 patients. Subjects received 8 weeks of therapy each cycle. Response assessment was performed on week 11 of each cycle by mSWAT, photography, PET imaging and peripheral blood flowcytometry. A second cycle was administered if there was no progression of disease. Subjects underwent mandatory tumor biopsies and peripheral blood immunologic studies during the first cycle of therapy as follows: pretreatment, day 5 post first dose, week 4 and week 8 of therapy. An optional biopsy was also collected at end of study. Tumor biopsies were analyzed and evaluated by a pathologist and IHC image analyzer to characterize immune cell subpopulations. Peripheral blood samples were analyzed by flowcytometry. Results: Nine subjects were accrued. Their age ranged from 37-79 years, 3 of 9 where white and 6 of 9 were men. The median number of prior therapies was 4 (1-11), 2 patients had progressed on Brentuximab vedotin, and 5 patients received total skin electron beam radiotherapy. The disease etiologies were as follows: 4 patients with transformed mycosis fungoides, 2 patients with systemic anaplastic large cell lymphoma-ALK negative, 2 patients with non- transformed mycosis fungoides and 1 patient with cutaneous anaplastic large cell lymphoma. Among the 8 subjects who were evaluable for response 4 of 8 (50%) achieved an objective response rate (ORR). In cohort 1 we observed 1 complete response (CR), 1 partial response (PR) and 1 stable disease (SD). In cohort 2, we observed 3 SD. In cohort 3, we observed 2 PRs and one subject awaiting disease assessment. Infusion related reaction (IRR) were observed in 4 of 9 subjects and managed conservatively. Cellulitis was noted in 3 subjects of which 2 developed bacteremia requiring intravenous antibiotics, and was not considered related to AFM13 treatment. There were no other treatment-emergent adverse events. In the peripheral blood, we observed a decrease in circulating NK cells during therapy (Weeks 1- 8) with post therapy recovery (by week 11) by following cells that were CD56+ CD3- , CD56+ CD16+ and NKp46+ by Flow Cytometry. We observed an increase over time in the expression of the activation marker CD69 on NK cells from responders compared to non-responders. Similarly, tumor biopsies in responders showed increased infiltration of CD56+ NK cells as opposed to non-responders. Flow quantitation of circulating CD4+ CD25+ T cells (Tregs) shows a decrease in responders vs. non-responders. Conclusions: AFM13 demonstrated a high ORR of 50 % among a population of heavily pretreated patients with a CD30 positive lymphoproliferative T-cell malignancy. AFM 13 exhibited activity post Brentuximab vedotin failure. In addition, biological changes in NK cells may predict response. This data are the first to demonstrate the therapeutic advantages of this bispecific, and the first to correlate changes in NK cells as a function of dose and schedule. AFM13 is able to engage the innate immune system through a specific activation and recruitment of circulating NK cells to tumors expressing CD30. This immunological tumor response may correlate with clinical response. We have begun to expand the patient cohorts, and pursue additional comprehensive correlative studies in this population. Disclosures Rodig: KITE: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; Merck: Research Funding. O'Connor:Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

Introduction: T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive large B cell lymphoma that typically presents with disseminated disease. In contrast to diffuse large B-cell lymphoma, not otherwise specified (DLBCL), TCRLBCL is characterized histologically by rare malignant B-cells within a robust but ineffective inflammatory background composed of numerous T cells and macrophages. TCRLBCL shows a "tolerogenic" immune signature by gene expression profiling, as well as frequent upregulation of PD-L1 (Van Loo et al. PMID: 19797726; Chen et al. PMID: 23674495). Although these features suggest that active immune evasion is central to TCRLBCL pathogenesis, its mechanistic basis is poorly understood. Accordingly, we performed an integrated analysis of tumor genetics and cell-cell interactions within the tumor microenvironment to comprehensively study PD-1:PD-L1 interactions in a multi-institutional cohort of TCRLBCL. Methods: 34 cases of TCRLBCL were identified from the pathology archives of four academic medical centers. Control cohorts containing 21 cases of DLBCL and 106 cases of classic Hodgkin Lymphoma (CHL) were used as comparators. An established fluorescence in situ hybridization (FISH) assay was used to identify copy number changes and structural rearrangements of CD274 (PD-L1) and PDCD1LG2 (PD-L2) on chromosome 9p24.1, which represents the primary genetic mechanism of PD-L1/L2 expression in CHL (Roemer et al. PMID: 27069084). Tumor-specific expression of PD-L1 and PD-L2 protein was assessed by immunohistochemistry (IHC) and scoring by two pathologists using a modified H-score (percentage of positive tumor cells [0-100%] multiplied by the mean staining intensity [0-3+]). The topology of PD-L1/PD-1 expression and cell-cell interactions in the tumor microenvironment was determined by multispectral immunofluorescence (IF) microscopy and spatial image analysis, as previously performed for CHL (Carey et al. PMID: 28893733). Results: By FISH, copy gain or amplification of PD-L1 and PD-L2 was identified in 22/34 (64.7%) cases of TCRLBCL (Figure 1A) and was associated with a 4.9-fold increase in tumor PD-L1 expression relative to cases with disomy or polysomy (mean PD-L1 H-score 72 vs 14.7, p = 0.02). A rearrangement of PD-L2 was identified in one case and associated with diffuse expression of PD-L2. These findings contrasted with those observed in the DLBCL cohort, which showed a low overall frequency of 9p24.1 copy gain/amplification (5/21 cases, 23.8%) and only minimal tumor PD-L1 expression (mean PD-L1 H-score 15.6), and were intermediate to those observed in CHL, which shows near universal copy gain/amplification of 9p24.1 (98/106 cases, 92%) and extensive tumor PD-L1 expression (mean PD-L1 H-score 143.7; Figure 1B). By multispectral IF, TCRLBCL showed prominent infiltration by PD-L1+ tumor-associated macrophages (TAM) (Figure 1C), which were 5.5-fold increased relative to DLBCL and 6.6-fold increased relative to CHL (p < 0.001). TCRLBCL also showed marked infiltration by PD-1+ T cells, which were 12.3-fold increased relative to DLBCL and 3.4-fold increased relative to CHL (p < 0.001). By spatial analysis, PD-L1+ TAMs in TCRLBCL were located in closer proximity to tumor cells than PD-L1- TAMs (p < 0.001, Figure 1D-E) and also showed frequent direct interactions with PD-1+ T cells. These findings contrasted with those in DLBCL, where no local enrichment of PD-L1+ TAMs or PD-1+ T cells was identified, and were similar but more prominent than those observed in CHL. Conclusion: TCRLBCL is characterized by recurrent gains of PD-L1 and PD-L2 on chromosome 9p24.1 in association with tumor-specific expression of PD-1 ligands, as well as prominent infiltration by PD-L1+ TAMs and PD-1+ T cells. PD-L1+ TAMs in TCRLBCL are enriched around individual tumors cells and also show frequent direct interactions with PD-1+ T cells, consistent with the establishment of an immunoevasive-niche. These findings contrast with those observed in DLBCL and are most similar to those identified in CHL. Relative to CHL, however, TCRLBCL shows less frequent gains of 9p24.1 and tumor cell expression of PD-L1, and a greater degree of infiltration by PD-L1+ TAMs and PD-1+ T cells. These findings suggest that the PD-1:PD-L1 pathway is central to immune evasion in TCRLBCL and highlight the need to test the clinical efficacy of PD-1 blockade in this patient population. Disclosures Griffin: Moderna Therapeutics: Consultancy. Freeman:Novartis: Patents & Royalties; AstraZeneca: Patents & Royalties; Dako: Patents & Royalties; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Patents & Royalties; Merck: Patents & Royalties; EMD-Serono: Patents & Royalties; Roche: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Patents & Royalties; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Origimed: Membership on an entity's Board of Directors or advisory committees. Hodi:Merck: Consultancy. Shipp:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Bayer: Research Funding; AstraZeneca: Honoraria. Rodig:KITE: Research Funding; Affimed: Research Funding; Merck: Research Funding; Bristol Myers Squibb: Research Funding.

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 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.

Introduction: CD19 CAR T cells lead to durable responses in 40% of r/r aggressive B cell NHL patients. We performed a multicenter retrospective study of axicabtagene ciloleucel given in a real world setting where eligibility/management considerations may diverge from clinical trials. We evaluate efficacy and safety, and patient/disease factors associated with response and toxicity. Methods: Patient and treatment characteristics were summarized descriptively. Response and toxicity were reported with 95% exact binomial CIs. CyTOF was performed on frozen PBMCs using 38 metal-tagged mAbs. Multiplex IF was performed on FFPE tissue with standard, primary Abs sequentially, paired with a unique fluorochrome per published protocols. Standard IHC was done on FFPE whole tissue. Results: In total, 76 patients, median age 64, were included (Table 1). The majority had an ECOG PS ≤ 2. Twenty-one percent of patients had double/triple hit cytogenetics. Nearly half had an IPI ≥3 at treatment. Nearly 1/3 had a prior autologous transplant, 25% had prior ibrutinib, and 11% had prior lenalidomide. Twelve percent had bulky disease and 36% received bridging therapy following pheresis. Seventy-three patients were evaluable for response (Table 2). At 4m median f/u, best ORR and CRR was 64% and 41% among those treated. Six patients had 6m f/u, all PRs at 1m: 3 converted to CR and 3 had PD. Eleven patients (13%) were pheresed but not treated due to PD (6), infection (2), or non-conforming cells (3). By ITT analysis, the ORR and CRR were 57% and 36%. OS at 4m among those treated was 84%; PFS will be calculated with longer f/u. In univariate analysis, PS, tumor bulk, IPI, baseline CRP and prior ibrutinib were significantly associated with lack of response (Table 3). There was no association between response and double/triple hit cytogenetics, grade 3+ CRS or NT, or the use of tocilizumab/steroids. Among treated patients, 96% experienced CRS; in 17% this was ≥ grade 3. Two patients died from CRS (3%). Median time to onset was 1d; median duration was 6d (0-14d). NT was seen in 76% of patients; in 38% this was ≥ grade 3. One case of NT was fatal. Median time to onset was 5d; median duration was 8d (0-34d). Tocilizumab and steroids were given to 67% and 78% of patients. ICU care occurred in 30% of patients. Eleven treated patients have died: 6 from PD and 5 from toxicity. In univariate analysis, peak ferritin was associated with grade 3+ CRS and NT; peak ALC was associated with grade 3+ CRS, and peak CRP and prior autologous transplant were associated with grade 3+ NT (Table 4). Three of 4 patients who had a biopsy with CD19 staining after relapse were positive (Fig 1). All 3 patients with PDL1+ tumors were refractory to CAR T cell therapy. Multiplex IF and IHC were performed on 2 primary refractory patients at progression (Fig 1). One was CD19-/PDL1+; multiplex IF showed an abundance of CAR+ T-cells (Fig 1A,B). The second was CD19+/PDL1- and multiplex IF showed no CAR+ T-cells (Fig 1C,D). CyTOF analysis of PBMCs at serial timepoints was performed on 6 patients (4 CRs, 1 PD, 1 PD after CR)(Fig 2, Table 5). Peak CAR T cell levels were seen at day 7 in all patients with increased expression of PD1, 41BB, and Ki67, as well as CC3 indicating apoptosis, followed by a reduction in CAR T cells by day 14. Immune subsets that associate with response will be evaluated and reported. Results from additional patients and longer f/u will be presented. Conclusion: Retrospective analysis of a multicenter cohort treated in the real world with axi-cel reveals important distinctions from ZUMA-1. The ORR and CR rate are lower than the 82% and 54% reported on ZUMA-1. This may reflect inclusion of sicker patients with a poorer PS, and/or with different histologies (ie transformation from non-FL). Outcomes were significantly worse in high risk lymphomas, reflected by IPI, PS, tumor bulk, and baseline CRP. Rates of CRS and NT were similar to ZUMA-1, but toxicity was not associated with tumor bulk or response. It was associated with higher peak inflammatory markers and ALC, which may reflect peak CAR T cell levels, as shown previously. Progression biopsies highlight 3 potential resistance mechanisms: loss of target antigen, an inhibitory tumor/TME, and lack of CAR T cell tumor infiltration. Immunomodulatory molecules on CAR T cells that may affect their activity and survival are upregulated early. This suggests that unique combination approaches are necessary for specific patients/tumors. Disclosures Jacobson: Precision Bioscience: Consultancy; Kite: Consultancy; Pfizer: Consultancy; Bayer: Consultancy; Humanigen: Consultancy; Novartis: Consultancy. Rodig:Merck: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; KITE: Research Funding. Maus:novartis: Consultancy; agentus: Consultancy, Research Funding; crispr therapeutics: Consultancy, Research Funding; kite therapeutics: Consultancy, Research Funding; windmil therapeutics: Consultancy; adaptimmune: Consultancy. Chen:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; REGiMMUNE: Consultancy; Takeda Pharmaceuticals: Consultancy. Abramson:Verastem: Consultancy; Amgen: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Humanigen: Consultancy; Bayer: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Juno Therapeutics: Consultancy. Kline:iTeos: Research Funding; Merck: Honoraria, Research Funding. Cohen:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioInvent: Consultancy; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; BioInvent: Consultancy. Jaglowski:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Consultancy. Smith:Merck Sharp and Dohme Corp.: Consultancy, Research Funding; Acerta Pharma BV: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Incyte Corporation: Research Funding; Portola Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding. Maloney:Janssen Scientific Affairs: Honoraria; Seattle Genetics: Honoraria; Juno Therapeutics: Research Funding; Roche/Genentech: Honoraria; GlaxoSmithKline: Research Funding. Gopal:Spectrum: Research Funding; Seattle Genetics: Consultancy, Research Funding; Merck: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding; Gilead: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding; Brim: Consultancy; Teva: Research Funding; Aptevo: Consultancy; Incyte: Consultancy; Asana: Consultancy. Acharya:Teva: Honoraria; Juno Therapeutics: Research Funding.

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.

Classical Hodgkin lymphoma (cHL) and mediastinal large B-cell lymphoma (MLBCL) are lymphoid malignancies with certain shared clinical, histologic, and molecular features. Primary cHLs and MLBCLs include variable numbers of malignant cells within an inflammatory infiltrate, suggesting that these tumors escape immune surveillance. Herein, we integrate high-resolution copy number data with transcriptional profiles and identify the immunoregulatory genes, PD-L1 and PD-L2, as key targets at the 9p24.1 amplification peak in HL and MLBCL cell lines. We extend these findings to laser-capture microdissected primary Hodgkin Reed-Sternberg cells and primary MLBCLs and find that programmed cell death-1 (PD-1) ligand/9p24.1 amplification is restricted to nodular sclerosing HL, the cHL subtype most closely related to MLBCL. Using quantitative immunohistochemical methods, we document the association between 9p24.1 copy number and PD-1 ligand expression in primary tumors. In cHL and MLBCL, the extended 9p24.1 amplification region also included the Janus kinase 2 (JAK2) locus. Of note, JAK2 amplification increased protein expression and activity, specifically induced PD-1 ligand transcription and enhanced sensitivity to JAK2 inhibition. Therefore, 9p24.1 amplification is a disease-specific structural alteration that increases both the gene dosage of PD-1 ligands and their induction by JAK2, defining the PD-1 pathway and JAK2 as complementary rational therapeutic targets.

The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8+TCRαβ+CD44+CD122+Ly-6C+ T cellsthat closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8+ human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4+ CTCL-like phenotype when cells are transplanted in an MHC-I–deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.

Classical Hodgkin lymphomas (cHLs) include infrequent malignant Hodgkin Reed-Sternberg (HRS) cells within an extensive but ineffective inflammatory/immune cell infiltrate. HRS cells exhibit frequent copy number alterations (CNAs) of 9p24.1/CD274 (PD-L1)/PDCD1LG2(PD-L2), ranging from low-level polysomy to relative copy gain and high-level amplification, all associated with increased expression of programmed death receptor-1 (PD-1) ligands on the tumor cell. PD-1 ligands engage the PD-1 receptor on T cells, inhibiting T cell activation and anti-tumor immune responses. cHL patients (pts) with the highest-level 9p24.1 alterations, PD-L1/PD-L2 amplification, have inferior progression-free survival (PFS) after standard primary chemotherapy (Roemer et al. J Clin Oncol 2016). Given the demonstrated responsiveness of cHL to PD-1 blockade, we examined the prevalence and type of 9p24.1 genetic alterations, PD-L1 expression, and association of these alterations with clinical outcome in pts receiving nivolumab (nivo; anti-PD-1) for relapsed/refractory (R/R) cHL. CheckMate205 is a multicenter, multicohort, phase 2 trial of nivo in R/R cHL. This analysis focused on 2 cohorts: pts with recurrent cHL following autologous stem cell transplantation (ASCT) and subsequent brentuximab vedotin (BV) (cohort B), and pts with R/R cHL following ASCT and BV given pre- or post-ASCT (cohort C). Pts received nivo 3 mg/kg every 2 weeks. Best overall response (BOR) and PFS were assessed by an independent radiological review committee (IRRC). In pts with available tumor biopsies, 9p24.1 genetic alterations were evaluated via fluorescence in situ hybridization (FISH) assay; probes encompassed CD274 (PD-L1, red) or PDCD1LG2 (PD-L2, green)and included a centromeric control (aqua).Dual immunohistochemical staining of PD-L1/PAX5 was performed to delineate PD-L1 expression in PAX5dim+ HRS cells and PAX5- cells in the tumor microenvironment. A modified PD-L1 H-score (range 0-300) was calculated by multiplying the percentage of PAX5+ (malignant) or PAX5- (non-malignant) cells with positive staining (0-100%) and the average intensity of positive staining (1-3+; ≥50 RS cells counted). All p-values are nominal. 96 pts had evaluable baseline tumor biopsy specimens; all 96 had detectable 9p24.1 alterations: polysomy in 10/96 (10%), copy gain in 56/96 (58%), amplification in 28/96 (29%), and presumptive rearrangement (split-apart FISH signal) in 2/96 (2%) (Figure). There was a significant association between PD-L1 protein expression (H-score) and the level of 9p24.1 alterations in HRS cells (p=0.002) (Figure). We next evaluated the association between BOR and defined 9p24.1 alterations and PD-L1 H-scores. The level of 9p24.1 CNAs was significantly associated with BOR (p=0.01); no pts with progressive disease (PD) had genomic amplification and no pts with complete response (CR) had polysomy. Similarly, there was significant association between PD-L1 H-score in HRS cells and BOR (p=0.02); all pts with PD had PD-L1 H-scores in quartiles (Q) 1/2, whereas most pts with CR had PD-L1 H-scores in Q4. We also assessed the association of PFS with 9p24.1 status and PD-L1 H-scores. The level of 9p24.1 alterations was associated with PFS; pts with 9p24.1 amplification had the most favorable PFS with nivo. HRS cell PD-L1 H-score (by quartiles) was also significantly associated with PFS (p=0.048), as pts with PD-L1 H-scores in Q1/Q2 had significantly shorter PFS than those with scores in Q3/Q4 (p=0.01). In contrast, there was no association between PD-L1 expression on non-malignant cells in the tumor microenvironment and PFS (p=0.43). In conclusion, all evaluable pts in this study had genetic alterations of 9p24.1/PD-L1/PD-L2 and copy number-dependent increased expression of PD-L1 in HRS cells. Although high-level alterations of 9p24.1 and increased PD-L1 expression were previously linked with inferior response to standard primary chemotherapy, we now associate these parameters with more favorable outcomes to targeted PD-1 blockade. These analyses also highlight the importance of quantifying and delineating PD-L1 expression in HRS cells and non-malignant cells in the tumor microenvironment in cHL biopsy evaluation. While further research is needed to guide possible use in clinical practice, these data advance understanding of 9p24.1 alterations and PD-L1 expression as prognostic biomarkers in cHL. Disclosures Engert: Takeda, BMS: Consultancy, Honoraria, Research Funding. Zinzani:Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; MorphoSys: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Celegene: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Timmerman:Bristol-Myers Squibb, Kite Pharma, Valor Biopharmaceuticals, Janssen: Research Funding; Seattle Genetics, Genmab, Celgene: Consultancy, Honoraria. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Research Funding; Sequenta Inc: Research Funding; Roche: Research Funding; Merck: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy. Kuruvilla:BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Honoraria; Celgene: Consultancy, Honoraria; Merck: Honoraria; Roche Canada: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Lundbeck: Honoraria. Cohen:Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium/Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Collins:Takeda: Consultancy, Honoraria, Speakers Bureau. Trneny:Roche, Celgene, Takeda, Janssen, Gilead, Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche, Celgene: Research Funding. Farsaci:Bristol-Myers Squibb: Employment. Kato:Bristol-Myers Squibb: Employment. Sumbul:Bristol-Myers Squibb: Employment. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Shipp:Bayer: Research Funding; Merck, Gilead, Takeda: Other: Scientific Advisory Board; Cell Signaling: Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding.

Introduction:Programmed death-ligand 1 (PD-L1) contributes to tumor escape from immune surveillance by binding to programmed death-1 (PD-1), a negative regulator of T-cell responses. PD-L1 is expressed by both tumor cells and immune cells in the tumor microenvironment. In contrast, the role of PD-L2 in tumor immunity is unclear. To better understand the contribution of PD-L1/L2 to immune escape in marrow-based hematologic malignancies, we used multi-color flow cytometry and immunohistochemistry (IHC) to characterize cell-specific PD-L1/PD-L2 expression in patients with multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Results:PD-L1 was detectable (> 2% positive cells) in 100% of patients, with distinct disease-specific patterns. PD-L1 was expressed most widely in MM, predominantly by malignant plasma cells (median, 95% of plasma cells; n = 5) and lymphocytes (median, 12% of marrow lymphocytes; n = 5). In contrast, in MDS and AML, PD-L1 was more commonly expressed by non-tumor hematopoietic cells: MDS median, 36% of CD34− myeloid precursors and 47% of lymphocytes (n = 13) vs 12% of CD34+ myeloid blasts; AML median, 19% of CD34− myeloid precursors and 26% of lymphocytes (n = 7) vs 16% of CD34+ myeloid blasts were PD-L1+. A higher proportion of CD8+ T cells expressed PD-L1 compared with CD4+ T cells in all 3 malignancies: the CD8:CD4 ratio for PD-L1+ T cells was 3.02 in MM (n = 11), 1.92 in MDS (n = 10), and 1.29 in AML (n = 7). PD-L2 expression was largely absent in AML and MDS (< 2% of CD34+ blasts or lymphocytes expressed PD-L2 (n = 13 MDS and n = 7 AML) but was expressed in a subset of patients with MM on plasma cells (median, 18%; n = 5) but not lymphocytes (< 2%). Across all indications on both tumor cells and lymphocytes, PD-L1 was expressed by a larger fraction of cells than PD-L2 (AML: CD34+ blasts, P < .01 and lymphocytes, P = .005 [n = 7]; MDS: CD34+ blasts, P < .01 and lymphocytes, P = .0001 [n = 13]; MM: plasma cells, P = .03 and lymphocytes, P = .04 [n = 5]). These results were confirmed in a distinct set of 16 cases of primary AML analyzed independently, with detectable PD-L1 expression on myeloid blasts in 14 of 16 cases (88%) without co-expression of PD-L2 (0 of 16 cases). Comparisons of flow cytometry and IHC revealed that some IHC methods may underestimate the prevalence of PD-L1/PD-L2 in marrow-based hematologic malignancies, and results comparing different methods for detecting PD-L1/PD-L2 in the bone marrow will be presented. Conclusions:PD-L1 is highly prevalent in MM, MDS and AML, with significant expression by non-tumor hematopoietic cells, particularly CD8+ T cells. PD-L2 expression was largely absent in myeloid diseases but detectable in MM. Interestingly, PD-L1 expression was most common on tumor cells in MM and on non-tumor hematopoietic cells in MDS, whereas expression on non-tumor and tumor cells in AML was comparable. These data support clinical development of anti-PD-L1/PD-1 therapies in MM, MDS and AML. Future analyses will determine whether different patterns of PD-L1 expression are associated with clinical efficacy. Authors M Dail, L Yang, S Rodig, and J Venstrom contributed equally to this work. Disclosures Dail: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Kadel:Genentech, Inc.: Employment. Koeppen:Roche: Employment, Equity Ownership. Adamkewicz:Genentech, Inc.: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Venstrom:Genentech: Employment.

Background: Primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL) are rare and aggressive extranodal non-Hodgkin lymphomas with shared molecular features and a poor prognosis. PCNSL is currently treated with high-dose methotrexate (HD-MTX) based induction chemotherapy. Younger patients who are healthy and eligible for consolidation treatment are considered for whole brain radiation (WBRT) or high-dose chemotherapy with autologous stem-cell transplant (ASCT). Despite this, nearly 50% of patients with PCNSL relapse within 2 years of diagnosis and one third of patients have primary refractory disease. PTL is treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). However, almost 50% of patients with PTL progress after induction treatment, frequently with CNS or contralateral testicular involvement. Treatment options are limited for patients with recurrent and refractory PCNSL or PTL, and the majority of patients ultimately die of their disease. We recently identified frequent 9p24.1/ PD-L1(CD274)/ PD-L2 (PDCD1LG2) copy number alterations and additional translocations in PCNSL and PTL (Chapuy and Roemer et al Blood 2016; 127:869-81). These genetic alterations are associated with increased expression of the programmed cell death protein 1 (PD-1) ligands, PD-L1 and PD-L2. Activation of PD-1 signaling by PD-L1 or PD-L2 binding leads to a reversible state of T-cell "exhaustion" characterized by decreased T-cell receptor signaling, reduced T-cell proliferation and survival, perturbed metabolism and altered transcription factor expression. The activity of PD-1 blockade in other lymphomas with 9p24.1 alterations such as classical Hodgkin's lymphoma prompted us to test the efficacy of this approach in PCNSL and PTL. Patients and Methods: We treated 5 patients with recurrent/refractory PCNSL and PTL with off-label Nivolumab, a humanized IgG4 monoclonal antibody checkpoint inhibitor that targets PD-1 and blocks binding by PD-L1 or PD-L2. Median age was 64 (range, 54-85) years with a median Karnofsky Performance Score (KPS) of 70 (40-80) %. Four patients had multiply recurrent disease (3 patients with PCNSL and 1 patient with PTL and CNS relapse); 1 patient had primary refractory PCNSL. All patients had been treated with standard of care treatments and had no other available options. Prior therapies included HD-MTX based chemotherapy, pemetrexed, high-dose cytarabine, WBRT and thiotepa-based chemotherapy followed by ASCT. All patients signed informed consent and were treated with nivolumab at 3mg/kg intravenously every 2 weeks. Only 1 patient received corticosteroids (dexamethasone 2mg daily) during initial nivolumab treatment; steroids were rapidly tapered and discontinued in 4 weeks. Additionally, 1 patient received 3 doses of rituximab and 2 others received WBRT. All patients had objective radiographic responses to treatment with nivolumab, including 4 complete responses and 1 partial response. The patient with CNS relapse from PTL had complete radiographic resolution of CNS abnormalities with persistent intraocular disease that required vitrectomy. All responses were confirmed at repeat restaging. The median number of nivolumab treatments to objective radiographic response was 3 (range, 2-4). The 4 patients who were symptomatic at the initiation of nivolumab therapy had complete or near-complete resolution of neurologic signs/symptoms and a marked improvement in KPS. All patients are alive and remain progression-free at this time. Four patients continue to receive treatment. The median progression-free survival is 9 (range, 7-11) months. Conclusion: Our pilot series indicates that nivolumab is active in primary refractory and relapsed/refractory PCNSL and PTL with CNS relapse. Based on our preclinical molecular data and these pilot clinical results, a multi-institutional phase 2 open-label, single-arm trial of nivolumab in recurrent and refractory PCNSL and PTL patients (CA209-647) is scheduled to open shortly. Disclosures LaCasce: Forty Seven: Consultancy; Seattle Genetics: Consultancy. Armand:Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Pfizer: Research Funding; Roche: Research Funding; Sequenta Inc: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Shipp:Bayer: Research Funding; Merck, Gilead, Takeda: Other: Scientific Advisory Board; Cell Signaling: Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding.

Background: Double-hit lymphomas (DHL) - diffuse large B-cell lymphomas (DLBCL) with concurrent rearrangements of MYC and BCL2 and/or BCL6, and double-expressor lymphomas (DEL) - DLBCL with co-expression of MYC and BCL2 by immunohistochemistry (IHC), are associated with poor outcomes after standard chemoimmunotherapy. We have previously demonstrated that patients with relapsed or refractory (rel/ref) DHL and DEL have inferior outcomes after autologous stem cell transplantation (autoSCT) compared to patients with neither DEL nor DHL [Herrera et al, ASH 2015]. Although patients with DEL and DHL have inferior outcomes after chemotherapy-based treatment modalities, we hypothesized that allogeneic SCT (alloSCT) could potentially abrogate that negative prognostic impact. Data are extremely limited regarding the outcome of patients with DHL who undergo alloSCT, and no study has examined alloSCT outcomes in patients with DEL. We studied alloSCT outcomes in a multicenter cohort of rel/ref DLBCL patients and evaluated the prognostic impact of DEL and DHL status. Methods: We retrospectively studied patients with rel/ref DLBCL, transformed indolent lymphoma (TIL), or high-grade B-cell lymphoma unclassified (BCLU) who had available tumor tissue and underwent alloSCT at Dana-Farber Cancer Institute, Massachusetts General Hospital, or City of Hope between 1/2000 and 5/2014. IHC for MYC, BCL2, and BCL6 were performed. DEL was defined as MYC expression in ≥ 40% tumor cells and BCL2 expression in ≥ 50% tumor cells. FISH for MYC was performed using dual-color break-apart probes. Cases with MYC-rearrangement had FISH performed for BCL2 and BCL6 using break-apart probes. Rearrangement was defined as ≥ 10% nuclei with break-apart signals. DHL was defined as concurrent rearrangement of MYC and BCL2 and/or BCL6. Results: Tumor tissue was available in 103 patients, among whom we could obtain complete IHC and FISH data in 74. In these 74 patients, the median age was 54 years (range 24-69); 69% had DLBCL/BCLU whereas 31% had TIL; the median number of prior therapies was 4 (range 2-9); 58% had prior autoSCT; 73% were in complete or partial remission (CR/PR) at alloSCT; 77% had reduced intensity conditioning (RIC); 78% had a matched related or unrelated donor. 4y progression-free survival (PFS), overall survival (OS), cumulative incidence of relapse (CIR), and non-relapse mortality (NRM) in the overall cohort were 34%, 40%, 44% and 22%, respectively, with a median follow-up of 46 months for survivors. 47% of patients had DEL and 14% had DHL. The proportion of patients with a history of primary refractory disease was higher among DHL (60%) and DEL (52%) patients compared to nonDHL/nonDEL patients (37%), although the difference was not significant (p=0.3). Overall, there were no significant differences in clinical characteristics between patients with DHL, DEL, and nonDHL/nonDEL. Neither DEL nor DHL were significantly associated with outcome (Figure). The 4y PFS in DEL v non-DEL patients was 29% v 39% (p=0.2), 4y OS 30% v 49% (p=0.11), 4y CIR 50% v 40% (p=0.3), and 4y NRM 21% v 22% (p=1.0). The 4y PFS in DHL v non-DHL patients was 40% v 33% (p=0.6), OS 50% v 37% (p=0.4), CIR 40% v 45% (p=0.9), and NRM 20% v 22% (p=0.8). In multivariable Cox models for PFS and OS, age ≥ 55 (PFS: HR 0.4, p=0.002; OS: HR 0.4, p=0.005), refractory disease (not CR/PR) at alloSCT (PFS: HR 2.4, p=0.009; OS HR 2.6, p=0.007), and TIL (PFS HR 0.4, p=0.018; OS HR 0.4, p=0.028) were associated with PFS and OS, but DEL (PFS HR 1.2, p=0.5; OS HR 1.6, p=0.12) and DHL (PFS HR 0.8, p=0.7; OS HR 0.8, p=0.7) were not. We also constructed multivariable competing risk regression models for CIR and NRM. Age, remission status, histology, and conditioning intensity were associated with relapse, while no factor was significantly associated with NRM. Neither DEL (CIR HR 1.2, p=0.7, NRM HR 0.8, p=0.7) nor DHL (CIR HR 1.1, p=0.9, NRM HR 0.8, p=0.8) were associated with either outcome in those models. Conclusions: AlloSCT produced durable remissions in heavily treated rel/ref DLBCL patients, regardless of DEL and DHL status. In our cohort, DEL and DHL status did not have a significant prognostic impact. Although patients with DEL or DHL have poorer outcomes after chemoimmunotherapy and autoSCT, our results suggest that alloSCT may overcome the chemoresistance of double-hit/double-expressor tumors. Figure Progression-Free Survival After AlloSCT in DEL, DHL, and nonDEL/nonDHL Patients Figure. Progression-Free Survival After AlloSCT in DEL, DHL, and nonDEL/nonDHL Patients Disclosures Herrera: Adaptive Biotechnologies: Research Funding; Genentech: Research Funding; Immune Design: Research Funding; Merck: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Research Funding. Song:Seattle Genetics: Consultancy. Chen:Genentech: Consultancy, Speakers Bureau; Millenium: Consultancy, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Consultancy, Research Funding. Chen:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Koreth:LLS: Research Funding; amgen inc: Consultancy; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; kadmon corp: Membership on an entity's Board of Directors or advisory committees; prometheus labs inc: Research Funding; millennium pharmaceuticals: Research Funding. Pillai:Trillium Therapeutics: Research Funding. Siddiqi:Janssen Biotech: Research Funding, Speakers Bureau; Seattle Genetics: Speakers Bureau; Juno Therapeutics: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Research Funding; MedImmune: Research Funding; Genentech: Research Funding; TG Therapeutics: Research Funding. Zain:Seattle Genetics: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Kwak:XEME BioPharma: Consultancy, Equity Ownership; Antigenics: Equity Ownership; Celltrion: Consultancy; Sella Life Sciences: Consultancy. Nademanee:Celgene: Consultancy; Seattle Genetics: Consultancy, Research Funding. Weinstock:Novartis: Consultancy, Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Armand:Roche: Research Funding; Pfizer: Research Funding; Sequenta Inc: Research Funding; Merck: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.

The development of B-cells is a complex process that proceeds through multiple stages and is regulated by a hierarchy of transcription factors and other physiologic signals. Each unique B-cell malignancy can be aligned with a 'normal counterpart' at one or more of these discrete developmental stages. However, with the exception of translocations of transcription factor genes, the genetic basis for this is not well defined. We performed an analysis of high-resolution single nucleotide polymorphism (SNP) microarrays from 694 diffuse large B-cell (DLBCL) tumors to identify significant somatic copy number alterations (SCNA). Through integrative analysis of 249 tumors with matched gene expression profiling (GEP) data, we identified the likely targets of these alterations and found that genes that were targeted by DNA copy number gain were significantly enriched for DNA binding activity and transcription factor function. We extended upon this observation by analyzing SNP microarray data of a further 2,716 tumors from 7 additional subtypes of B-cell malignancy. Through this analysis, we identified patterns of transcription factor alterations that aligned with the differentiation state of the 'normal B-cell counterpart' of each malignancy. This provides evidence that SCNA of B-cell transcription factors may underlie the differentiation state of B-cell malignancies. DLBCL can be divided into two subtypes based upon gene expression profiles that align with either the germinal center B-cell differentiation state (GCB-like) or a post-GCB activated B-cell state (ABC-like). Having observed an enrichment for transcription factor SCNAs in DLBCL, and an alignment between transcription factor alterations and differentiation states in other B-cell malignancies, we hypothesized that SCNAs of transcription factors may also underlie the etiology of these molecular subtypes. By testing for associations between SCNAs and cell of origin subtype, we identified three co-segregating DNA copy number gains that were significantly enriched in the ABC-like subtype. These included gains of the BCL6 and SPIB genes that have been previously observed to be associated with the ABC-like subtype. In addition, we found gains of the TCF4 (E2-2) gene to be significantly enriched in ABC-like tumors. In line with this, TCF4 alterations were significantly associated with reduced overall survival in cohorts of patients treated with either CHOP (n=232, P=0.009) or R-CHOP (n=197, P=0.041). B-cell receptor (BCR) signaling is a key survival pathway in ABC-like DLBCL, and the TCF4 gene has a defined role in promoting the expression of immunoglobulin (Ig) genes that encode the B-cell receptor (BCR). The analysis of paired SCNA and GEP data revealed a significantly higher expression of Ig genes in tumors with TCF4 DNA copy number gain compared to those without, suggesting that normal BCR expression may be deregulated by this genetic alteration. In addition, chromatin-immunoprecipitation sequencing (ChIP-seq) for TCF4 in ABC-like DLBCL cell lines also revealed binding of TCF4 to an Ig gene enhancer region. As BCR signaling can be altered by somatic mutations in the CARD11, CD79B and MYD88 genes, we evaluated the relative representation of these mutations and TCF4 DNA copy number gains using targeted deep sequencing of 124 DLBCL tumors. This revealed that TCF4 DNA copy number gains largely mutually excluded CARD11 mutations, but significantly co-segregated with both MYD88 (FDR=0.005) and CD79B (FDR=0.053) mutations. In addition, we observed significant co-segregation between CD79B and MYD88 mutations (FDR Together these data highlight an association between SCNA of B-cell transcription factors and the differentiation state of the 'normal counterpart' of the respective malignant B-cell. In line with this, we show that DNA copy number gains of the TCF4 transcription factor are associated with the ABC-like subtype of DLBCL, significantly worse overall survival, and increased Ig expression. These characteristics, in addition to the co-association between TCF4 DNA copy number gains and somatic mutations of CD79B and MYD88, suggest that TCF4 may be an important modifier of BCR signaling and contribute to the etiology of ABC-like DLBCL. Disclosures Rosenquist: Gilead Sciences: Speakers Bureau. Lunning:TG Therapeutics: Consultancy; AbbVie: Consultancy; Gilead: Consultancy; Bristol-Myer-Squibb: Consultancy; Juno: Consultancy; Genentech: Consultancy; Spectrum: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

Large B-cell lymphomas (LBCL) exhibit multiple genetic alterations, often in specific combinations. However, little is known about the biologic consequences of these concurrent genetic features. We recently characterized the comprehensive molecular signatures of two LBCL subtypes that exclusively involve extranodal disease sites, primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL). In contrast to systemic diffuse large B-cell lymphomas (DLBCLs) or most activated B-cell (ABC)-type DLBCLs, the majority of PCNSLs and PTLs exhibit oncogenic Toll-like receptor (TLR) signaling (MYD88L265P activating mutations), often with concurrent B-cell receptor (BCR) activation (CD79BY196mut) and/or additional mutations or inactivating rearrangements of TBL1XR1 (Chapuy and Roemer et al, Blood 2016; 127:869-81). TBL1XR1 is a component of the NCoR/SMRT co-repressor complex that modulates TLR/MYD88 signaling by increasing the clearance of NCoR/SMRT transcriptional co-repressors from certain TLR/MYD88 target genes. However, the functional consequences of somatic mutations in TBL1XR1 (TBL1XR1mut) or reduced TBL1XR1wt expression in LBCLs remain to be defined. For these reasons, we performed in silico modeling of the identified TBL1XR1mut and functionally characterized TBL1XR1mut and decreased TBL1XR1wt expression in model LBCL systems. First, we evaluated the frequency and types of TBL1XR1mut in our local series of PCNSLs and 151 primary DLBCLs (102 local and 49 previously published) for which we had whole exome sequenceing data. Thirty-six percent of PCNSLs and 6% of DLBCLs in this series exhibit TBL1XR1mut, over half of which occur in the context of MYD88 and/or CD79B mutations. In these PCNSLs and DLBCLs, we identifed 13 different non-synonymous TBL1XR1 mutations. All identified non-overlapping TBL1XR1 mutations were in the WD40 propellar domains, which are critical for protein-protein interactions. To gain insights into the structural consequences of TBL1XR1mut, we overlayed these alterations onto the TBL1XR1 protein crystal structure, modeled the corresponding mutations in silico and assessed protein stability changes. Eighty percent of the TBL1XR1mut resulted in significant destabilization of the mutant protein (ΔΔGibbsFreeEnergy median: 9.78 kcal/mol, range: 1.91 - 28.31). We found that mutations often perturbed the 3D protein structure by abrogating critical polar interactions (representative example TBL1XR1wt vs. TBL1XR1H390R in Figure 1). These in silico modeling data suggest that genetic perturbations of TBL1XR1mut disrupt the function of the encoded protein. For that reason, we modeled reduced TBL1XR1wt expression in informative DLBCL cell lines with or without concurrent CD79B/MYD88 mutations (TMD8 and OCI-Ly1, respectively) by genetically depleting endogenous TBL1XR1 alleles with CRISPR/Cas9. All of the resulting LBCL cell lines with loss of either one or two TBL1XR1 alleles exhibited significantly increased proliferation in comparison to the parental controls. We next assessed the role of TBL1XR1mut by generating HA-tagged versions of each identified TBL1XR1 mutation with site-directed mutagenesis. Viral particles of TBL1XR1mut constructs and the TBL1XR1wt control were used to reconstitute mutant and wild type protein in the TBL1XR1-/- Ly1 cell line. Reconstitution with each TBL1XR1mut significantly enhanced LBCL proliferation, in comparison to TBL1XR1wt. Taken together, these in silico and experimental data suggest that TBL1XR1mut in PCNSL and DLBCL are inactivating events and establish TBL1XR1 as a tumor suppressor in these diseases. Additional analysis of potential interactions between perturbed TBL1XR1 and MYD88/CD79B are underway. Disclosures Rodig: Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees.

Introduction: Nivolumab, a fully human IgG4 monoclonal antibody targeting programmed death receptor-1, has recently been FDA approved for classical Hodgkin lymphoma (cHL) that has relapsed or progressed after autologous stem cell transplantation (ASCT) and post-transplantation brentuximab vedotin (BV). CheckMate 205 (NCT02181738) is a phase 2, international, multicohort study evaluating nivolumab monotherapy in patients with cHL after failure of ASCT. At minimum 6 months' follow-up of Cohort B (205B) which included patients who had received BV after disease recurrence following ASCT, objective response rate (ORR) per independent radiologic review committee (IRRC) was 66% and 6-month progression-free survival (PFS) was 77%, with an acceptable safety profile (Younes et al. Lancet Oncol 2016; Jul 20 [Epub ahead of print]). Here we report outcomes with longer follow-up (minimum 12-month follow-up) in 205B. Primary results from Cohort A, which enrolled patients who relapsed after ASCT but were BV-naïve, will also be presented for the first time as data were not available at time of abstract submission. Methods: CheckMate 205 was designed to evaluate the efficacy and safety of nivolumab monotherapy in patients with cHL who relapsed after ASCT in 3 cohorts: 205A (BV-naive patients; n = 63); 205B (BV after failed ASCT; n = 80); 205C (BV at any time prior to study drug; n = 100). Nivolumab was given at 3 mg/kg intravenously every 2 weeks until disease progression or unacceptable toxicity in 205A and 205B. The primary endpoint was IRRC-assessed ORR. Results: 80 patients were treated in Cohort 205B; the median age was 37 years (range 18-72). Patients had received a median of 4 prior regimens (range 3-15). At data cut-off (April 2016) with median duration of follow-up of 15.4 months (range 1.9-18.5), 43 patients (54%) still remained on therapy. IRRC-assessed ORR for patients in 205B was 68% (95% confidence interval [CI], 56%, 78%). CR and partial response (PR) rates per IRRC were 8% (3%, 16%) and 60% (48%, 71%), respectively. With longer follow-up, the median duration of response was prolonged to 13.1 months (95% CI, 8.7, not reached; range, 0.0+, 14.2+). The median duration of CR (DOCR) was not reached (95% CI, 4.6, not available [NA]; range, 0.7+, 10.4+) and the median duration of PR was 13.1 months (95% CI, 7.79, NA; range, 0.0+, 13.4+) (Table 1). IRRC median PFS was 14.8 months (95% CI, 11.3 months, NA); 12-month PFS was 54.6% (95% CI, 40.9%, 66.4%), and 12-month overall survival (OS) was 94.9% (median OS not reached). Of 37 patients (46%) who discontinued nivolumab, the most common reasons were disease progression (n = 19 [24%]), allogeneic stem cell transplant (n = 7 [9%]) and adverse events (n = 5 [6%]). Seventy-four patients (93%) had drug-related adverse events (AEs) of any grade. The most common drug-related AEs were fatigue (28%), infusion reaction (20%), arthralgia (15%), and rash (15%). 29% of patients had Grade 3-4 drug-related AEs; the most common were increased lipase (8%), neutropenia (5%), and increased aspartate aminotransferase (4%). The most common serious AEs were pyrexia, pneumonia, tumor progression, arrhythmia, infusion reaction, and meningitis (≤4% each). Conclusions: In this report with extended 12-month follow-up, longer remissions are noted following nivolumab monotherapy in relapsed cHL, including durable PRs in heavily pretreated patients. Nivolumab has an acceptable safety profile, similar to previously reported. Primary results following nivolumab monotherapy in BV-naïve patients with cHL who progressed after ASCT (Cohort 205A) will also be presented at the meeting. Study funding: Study funded by Bristol-Myers Squibb. Professional writing assistance was provided by K. Jesien of Caudex and funded by Bristol-Myers Squibb. Disclosures Timmerman: Seattle Genetics, Genmab, Celgene: Consultancy, Honoraria; Bristol-Myers Squibb, Kite Pharma, Valor Biopharmaceuticals, Janssen: Research Funding. Engert:Takeda, BMS: Consultancy, Honoraria, Research Funding. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Research Funding; Roche: Research Funding; Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Sequenta Inc: Research Funding. Collins:Takeda: Consultancy, Honoraria, Speakers Bureau. Kuruvilla:Roche Canada: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Honoraria; Merck: Honoraria; BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Lundbeck: Honoraria. Cohen:Millennium/Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Trneny:Roche, Celgene, Takeda, Janssen, Gilead, Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche, Celgene: Research Funding. De Boer:NKI-AVL: Employment. Shipp:Cell Signaling: Honoraria; Merck, Gilead, Takeda: Other: Scientific Advisory Board; Bayer: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Kato:Bristol-Myers Squibb: Employment. Sumbul:Bristol-Myers Squibb: Employment. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Mantle cell lymphoma (MCL) carries an unfavorable prognosis and requires new treatment strategies. The associated t(11:14) translocation results in enhanced cyclin D1 expression and cyclin D1–dependent kinase activity to promote cell-cycle progression. A pharmacodynamic study of the selective CDK4/6 inhibitor PD0332991 was conducted in 17 patients with relapsed disease, using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and 3-deoxy-3[18F]fluorothymidine (FLT) positron emission tomography (PET) to study tumor metabolism and proliferation, respectively, in concert with pre- and on-treatment lymph node biopsies to assess retinoblastoma protein (Rb) phosphorylation and markers of proliferation and apoptosis. Substantial reductions in the summed FLT-PET maximal standard uptake value (SUVmax), as well as in Rb phosphorylation and Ki-67 expression, occurred after 3 weeks in most patients, with significant correlations among these end points. Five patients achieved progression-free survival time of > 1 year (range, 14.9-30.1+ months), with 1 complete and 2 partial responses (18% objective response rate; 90% confidence interval, 5%-40%). These patients demonstrated > 70%, > 90%, and ≥ 87.5% reductions in summed FLT SUVmax and expression of phospho-Rb and Ki67, respectively, parameters necessary but not sufficient for long-term disease control. The results of the present study confirm CDK4/6 inhibition by PD0332991 at a well-tolerated dose and schedule and suggest clinical benefit in a subset of MCL patients. This study is registered at www.clinicaltrials.gov under identifier NCT00420056.

Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.

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.

BACKGROUND: The programmed cell death-1 ligands (PD-Ls; PD-L1 and PD-L2) act as negative regulators of anti-tumor immunity by binding their cognate receptor, PD-1, on cytotoxic T-cells and inducing T-cell "exhaustion", a phenotype that is reversible with PD-1 blockade. Human antibodies that block PD-1 induce objective clinical responses in the majority of patients with relapsed / refractory classical Hodgkin Lymphoma (CHL). CHLs include small numbers of malignant Hodgkin Reed-Sternberg (HRS) cells (~5% of total cellularity) within an extensive but ineffective inflammatory and immune cell infiltrate. Over 85% of CHLs express PD-Ls on both the HRS cells and additional non-malignant cells within the tumor microenvironment (Chen et al., CCR 2010). PD-L expression in HRS cells is attributable, in part, to copy gain of chromosome 9p24.1, a region that includes PD-L1, PD-L2, and JAK2 (Green et al., Blood, 2010). However, the contribution of non-malignant cells to the overall PD-L expression within the tumor micro-environment of CHL is poorly defined. METHODS: We analyzed select CHLs (12 EBV+, 8 EBV-) by multiplex immunofluorescence using formalin-fixed, paraffin embedded tissue sections, with successive labeling by primary antibodies (PD-L1, CD30, CD68, pSTAT3, CD163), followed by secondary amplification and tyramide-conjugated fluorophores. For each case 2 large representative areas of tissue, totaling eight 20x fields of view were selected and imaged using a multispectral imaging platform. Two specific image analysis algorithms were designed to accurately identify CD30+ HRS cells and CD68+ macrophages simultaneously, then to threshold PD-L1 by relative fluorescent units (RFU) in each phenotype. Cartesian coordinates for all cells were exported and distance calculations were generated between PD-L1+ and PD-L1- macrophages and their Ônearest neighborÕ CD30+ PD-L1+ HRS cell. RESULTS: The percentages of CD30+ HRS cells and CD68+ macrophages expressing PD-L1 was highly variable across cases (range 9 - 94%, median 46.6% for HRS cells; range 6 - 91.3%, median 48.2% for macrophages). In all cases the majority of PD-L1 protein within the tumor micro-environment was contributed by macrophages (median 77.9%, range 50.4 - 98.5%), although the mean relative intensity of PD-L1 per cell was higher for HRS cells than for macrophages (3.13 +/-0.02 RFU vs 2.85 +/- 0.01 RFU; p < 0.0001 by Welch t-test). Further analysis revealed that the percentage of HRS cells and macrophages expressing PD-L1 was highly correlated (Pearson r = 0.67; 95% CI 0.32 - 0.85; p=0.001) and, in 18/20 tumors, PD-L1+ macrophages were in greater proximity to PD-L1+ HRS cells than PD-L1- macrophages (across 20 cases mean distance of 32.6 µm (SE 5 µm) versus 51.2 µm (SE 6.8 µm), respectively; p < 0.05). CONCLUSIONS: CD68+ tumor-associated macrophages (TAMs) express the majority of PD-L1 in CHLs, which contain rare tumor cells. Image analysis of the distribution of PD-L1 in the tumor microenvironment indicates that PD-L1+ TAMs are significantly enriched in proximity to PD-L1+ HRS cells. These data implicate HRS cells in coordinating PD-L1 induction among TAMs to limit anti-tumor immunity. Figure 1. Figure 1. Disclosures Shipp: Merck: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) have characteristic cellular, phenotypic, and genetic characteristics that reflect their distinctive biology. These distinctions are attributable, in part, to differential activity of defined oncogenic signaling pathways, including TCF3/ID3, NFkB, MYC, and BCL2 (Carey et al, JMD, 2015). B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCL-U) encompasses a group of tumors with ambiguous features and likely heterogeneous biology. We used targeted gene expression profiling and a molecular classifier based on the major signaling networks active in DLBCL and/or BL to address the biological heterogeneity of BCL-U. METHODS: We analyzed RNA isolated from paraffin-embedded tissue sections of 72 aggressive B-cell lymphomas (BCLs) selected according to original pathological diagnoses of BL (12 cases), DLBCL (20 cases), BCL-U (22 cases), and 'large B-cell lymphoma with high grade features' (18 cases). Sixteen genetic double hit lymphomas (DHLs) that combine a MYC -rearrangement with a BCL2 - and / or BCL6 -rearrangement were included in the series. Five hematopathologists reviewed each case to separate those with a consensus diagnosis from those where there was no consensus. We profiled the RNA in 2 laboratories and used a molecular diagnostic classifier to provide a "probability score" for the diagnosis of BL or DLBCL. Cases with a diagnostic score of >0.75 were categorized as "molecular BL" (mBL), between 0.25 and 0.75 as intermediate between BL and DBLCL (intBCL), and ≤0.25 as "molecular DLBCL" (mDLBCL). Finally, we compared the results of expert review with the results of molecular classification. RESULTS: Eight of 8 consensus diagnoses of BL classified molecularly as mBL and 13/13 consensus diagnoses of DLBCL classified as mDLBCL (100%). Seventy of 72 cases (97.2%) were molecularly classified with consensus upon profiling in 2 laboratories. Consensus pathological diagnoses that agreed with the original diagnoses were reached in 6/9 BL (66.7%), 6/12 DLBCL (50%) and 2/22 BCL-U (9.1%). Cases classified as BCL-U by two or more expert pathologists (29) were assigned to all molecular diagnostic categories, including 11 (15.7%) to mBL, 12 (41.4%) to intBCL, and 6 (20.7%) to mDLBCL. Similarly, subsets of DHLs with molecular consensus (14) classified as mBL (21.4%), intBCL(64.3%) and mDLBCL (14.3%). Among cases without a consensus pathological diagnosis, 9 (34.6%) were classified as mBL with high probability, 14 (53.8%) were classified as mDLBCL with high probability, and 3 (11.5%) were classified as intBCL. CONCLUSIONS: We find that the quantitative analysis of oncogenic signaling pathways using targeted expression profiling accurately and reproducibly 1) segregates BL from DLBCL, 2) assigns a subset of BCLs that are not diagnosed with consensus by traditional methods to mBL and mDLBCL and 3) identifies subsets of DHLs with molecular signatures of mBL, intBCL, and mDLBCL. Targeted molecular profiling and molecular classification of aggressive B-cell lymphomas is a useful new ancillary test in the diagnostic evaluation of aggressive B-cell lymphomas that provides a biological framework for designing rational treatment strategies. Figure 1. Figure 1. Disclosures Rodig: Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease defined by different transcriptional classifications, associated signaling and survival pathways and additional recurrent genetic alterations. In the cell-of-origin (COO) scheme, DLBCLs subtypes share certain features with normal germinal center B-cells (GCB) and activated B-cells (ABC). In comparison to GCB DLBCLs, ABC tumors have increased baseline NFκB activity and more frequent genetic alterations of NFκB pathway components. DLBCLs with shared functional features are also defined by the consensus clustering classification (CCC) which delineates B-cell receptor (BCR), Oxidative Phosphorylation (OxPhos) and Host Response (HR) tumors. BCR DLBCLs have increased reliance on BCR-signaling and survival pathways and aerobic glycolysis. BCR-dependent DLBCLs with high or low baseline NFκB activity (which largely correspond to ABC or GCB tumors, respectively) have distinct SYK-PI3K-dependent survival pathways and shared sensitivity to proximal BCR pathway inhibitors. Although DLBCLs have infrequent inactivating somatic mutations of TP53, these tumors commonly have copy number alterations (CNAs) of TP53 and genes encoding cell cycle pathway components. Given the clinical and molecular heterogeneity of DLBCL, we sought to develop faithful subtype-specific model systems to assess targeted therapies. Fresh tumor biopsies from 27 primary LBCLs were implanted under the renal capsule of immune compromised NSG mice. Nine of 27 tumors were successfully expanded in vivo, serially propagated for > 5 generations and considered stable LBCL PDX models. All models were EBV- and had clonal IgH rearrangements. Morphological and immunohistochemical signatures defined 8 PDX models as DLBCL and 1 as EBV- plasmablastic lymphoma (PBL). All LBCL PDX models were subjected to RNA-Seq and classified with respect to COO and CCC subtypes. Models were also evaluated by whole exome sequencing with a modified bait set which captured coding mutations and selected chromosomal rearrangements. Six of 9 DLBCL PDX models were ABC type. These models exhibited mutations of MYD88 alone or in association with PIM1 or CD79B with other alterations, as reported in primary ABC DLBCLs. The remaining 2 DLBCL PDX models were GCB type, with characteristic alterations of GNA13 and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Of note, BCL2 and MYC translocations are known adverse prognostic features of primary GCB DLBCL. Certain PDX models had additional mutations including B2M, MLL2, TNFAIP3, MEF2B and TP53. Only 25% (2/8) of the DLBCL PDX models harbored inactivating TP53 mutations whereas 75% (6/8) of tumors exhibited CNAs of TP53 or its upstream modifier, CDKN2A. These data are consistent with the reported incidence and type of TP53 pathway alterations in primary DLBCLs and contrast sharply with the near-uniform presence of TP53 mutations in DLBCL cell lines. Using the CCC classification, 6/8 DLBCL PDX models (both GCBs and 4 of 6 ABCs) were defined as BCR-subtype and 2 models as non-BCR type. To assess the utility of the DLBCL PDX models for functional analysis of BCR signaling, we first assessed cell surface immunoglobulin (sIg) expression by flow cytometry. All 6 BCR-type DLBCLs expressed sIgM whereas the 2 non-BCR DLBCL models and the PBL model lacked sIg. Next, we treated viable tumor cell suspensions with a selective SYK inhibitor, entospletinib (GS-9973). SYK inhibition significantly decreased the proliferation of all 6 BCR-type DLBCLs, but had no effect on the non-BCR-type DLBCLs or the PBL PDX. Given the distinctive SYK/PI3K-dependent signaling and survival pathways in DLBCLs with low or high baseline NFκB, we also assessed selective apoptotic pathway readouts in entospletinib-treated PDX cell suspensions. SYK inhibition selectively upregulated the pro-apoptotic BH3 family member, HRK, in BCR-dependent GCB-type DLBCL PDX samples and significantly downregulated the anti-apoptotic BCL2 family member, BCL2A1, in BCR- dependent ABC-type DLBCL PDX tumors, effects consistent with those previously observed in primary DLBCL samples. In summary, we have established and molecularly characterized faithful PDX models of DLBCL and PBL and demonstrated their usefulness in evaluating novel BCR pathway inhibitors. Disclosures Rodig: Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:Gilead: Consultancy; Sanofi: 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; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: High dose chemotherapy followed by autologous stem cell transplantation (ASCT) cures a subset of patients with chemosensitive relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL). Several factors associated with post-ASCT outcome have been identified, including pre-ASCT PET status, but better biomarkers are needed in order to optimally select candidates for the procedure. In other lymphoma subtypes with defining chromosomal translocations, PCR detection of pre- and post-ASCT minimal residual disease (MRD) in peripheral blood and of tumor contamination in the stem cell product is associated with inferior outcome. Until recently, MRD detection in DLBCL was limited by the rarity of detectable circulating disease using conventional techniques. The immunosequencing platform (Adaptive Biotechnologies, Corp.) is a next-generation-sequencing (NGS)-based MRD assay that detects small amounts of circulating tumor DNA (CTD) in patients with lymphoid malignancies. The assay detects CTD at diagnosis in most DLBCL patients and CTD levels track with response to induction therapy (Armand, 2013). Persistence of CTD or recurrence of CTD after completion of therapy is highly associated with DLBCL relapse (Kurtz, 2015; Roschewski, 2015). We evaluated whether CTD in autologous stem cell grafts was predictive of outcome in patients with rel/ref DLBCL undergoing ASCT. Methods: We retrospectively studied patients with rel/ref DLBCL, including transformed indolent lymphoma (TIL), who had paired archival tumor and autologous stem cell specimens and underwent ASCT at Brigham and Women's Hospital/Dana-Farber Cancer Institute from 2003-2013. Genomic tumor DNA was extracted from archival formalin-fixed paraffin-embedded (FFPE) tissue and analyzed using the NGS-based MRD assay. PCR amplification of IGH-VDJ, IGH-DJ,and IGK regions using universal consensus primers was performed followed by NGS to determine the tumor clonotype(s), defined as having a frequency > 5% in the tumor specimen. DNA from all available autologous peripheral blood or bone marrow stem cell specimens from each patient was amplified using universal consensus primers and sequenced to determine the level of CTD, defined as the number of lymphoma molecules per diploid genome. Results: We identified 98 eligible patients with rel/ref DLBCL/TIL. The median age was 60 (range 22-77) years; 63% were male; 65% had DLBCL, 29% had TIL, and 5% had primary mediastinal DLBCL; the median number of prior lines of therapy was 2 (range 2-5); all had received prior rituximab; 38% had primary refractory disease; 60% were in complete remission at ASCT; 96% received CBV conditioning. Median follow-up was 56 (range 19-123) months. The 4y progression-free survival (PFS) and overall survival (OS) in the entire cohort were 46% and 64%, respectively. Among 83 patients (85%) with sufficient DNA for clonotype determination, a clonotype was identified in 59 (71%). CTD data was complete in 53 patients (52 received peripheral blood stem cells (PBSC) and 1 received bone marrow). Eight patients (15%) had detectable CTD (CTD+) in the stem cell autografts (all PBSC) and 6/8 relapsed after ASCT. One CTD+ patient had early non-relapse mortality less than 1 month after ASCT and was never restaged. Seven of 8 CTD+ patients had TIL histology, 5 of whom relapsed (4 with aggressive lymphoma). The 4y PFS and OS in CTD+ v CTD- patients were 13% v 48% (p=0.01), and 38% v 67% (p=0.013), respectively [Figure 1]. In multivariable models including CTD status and pre-ASCT characteristics, CTD+ was the only factor associated with OS (HR 3.1, p=0.018), but was not significantly associated with PFS. Discussion: In patients with rel/ref DLBCL undergoing ASCT, the presence of CTD in the autologous stem cell graft is associated with inferior survival. CTD detection in the autograft may be more common in patients with TIL. In studies evaluating CTD detection in DLBCL, the plasma compartment has been more sensitive for detecting CTD than mononuclear cells. The use of concentrated cell specimens in this study may have decreased the sensitivity of the assay. Nevertheless, if the present findings are confirmed in a larger population, CTD detection may permit the identification of a subgroup of patients with a particularly poor outcome after ASCT, for whom alternative approaches could be considered. Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 2. Figure 2. Disclosures Herrera: Sequenta, Inc.: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding. Kong:Adaptive Biotechnologies, Corp.: Employment, Other: Stockholder. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Armand:BMS: Research Funding; Infinity: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Sequenta, Inc.: Research Funding.

Outcomes among persons with lymphoma in sub-Saharan African (SSA) remain poor, largely due to resource limitations in diagnostics, therapeutics and supportive care. We hypothesized that subclassification of lymphoma specimens from SSA could improve diagnostic accuracy, thereby altering treatment recommendations and identifying cases responsive to less toxic, targeted agents. The Queen Elizabeth Central Hospital (QECH) in Malawi serves as the teaching hospital for the University of Malawi College of Medicine (UOMCOM) in Blantyre, Malawi's largest city. Pathologic diagnosis of lymphoma at QECH/UOMCOM is based solely on HE 25% £ 16); 274 males, 189 females, 35 unknown. HIV status was available for 10% (36 reactive; 12 non-reactive). At Brigham and Women's Hospital, we performed immunohistochemistry (IHC) for 28 markers and classified each case per the 2008 WHO Classification. 170 cases (34%) could not be classified, largely due to poor tissue antigenicity or lack of lesional tissue on TMA. The remaining 328 cases (66%) are classified in the Table . 37% of the 133 DLBCL were germinal center B cell-like (GCB), 59% non-GCB, and 4% could not be characterized (Hans criteria). We also performed targeted expression profiling with a previously-published, NanoString-based diagnostic molecular classifier of aggressive B-cell lymphomas. This classifier distinguishes ~85% of pathological BL cases from DLBCL in Western cohorts (Carey CD, J Mol Diagn 2015). Evaluable RNA was available in 64/133 (48%) DLCBL, 38/73 (52%) BL, and 8/9 (89%) BCL-U cases. Unsupervised clustering of transcriptional profiles identified 2 groups (n = 54, 56). All 38 BL cases were classified into Group I (100%; exact binomial 95% CI: 91-100), along with 14 DLBCL (22%; 13-34%) and 4 BCL-U (50%; 16-84%). Group II combined 50 of 64 DLBCL cases (78%; 66-87%), 4 of 8 BCL-U cases (50%; 16-84%), and no BL (0%; 0-9%). Concordance between the classifier and pathologic diagnosis was 84%. Overall, incorporation of phenotypic and transcriptional data allowed reclassification of 83 cases into categories that may be responsive to ibrutinib or idelalisib (MCL; non-GCB DLBCL; CLL/SLL); 75 into categories that could benefit from CHOP (ALCL; DLBCL; PBL; PTCL-NOS); 8 into T-ALL; and 30 additional cases of BL by IHC alone. 18 cases of DLBCL or BCL-U were also found to have a similar transcriptional profile to BL, suggesting that they may benefit from BL-directed therapy. Thus, our findings demonstrate that ancillary phenotypic and transcriptional characterization can reclassify a large fraction of lymphomas in SSA and guide potentially beneficial therapies. Implementation of these tools and enhanced drug access and supportive care are desperately needed to improve patient outcomes across the developing world. | | | Original Pathologic Classification (Malawi) | | --------------------------------- | --------------- | ------------------------------------------- | ---------------------------------------------------- | | | | DLBCL/high grade NHL (n=118) | NHL/low grade NHL/lymphoma NOS/low grade BCL (n=101) | BL (n=70) | Suspicious for NHL (n=21) | CLL/SLL (n=7) | FL (n=3) | ALCL (n=2) | LBL (n=1) | MZL (n=3) | PBL (n=2) | | Refined Diagnosis Using IHC n (%) | DLBCL | 70 (59) | 46 (45) | 8 (12) | 7 (32) | | 1 (33) | 1 (50) | | | | | | BL | 12 (10) | 14 (14) | 43 (62) | 4 (19) | | | | | | | | | PBL | 11 (9) | 2 (2) | 5 (7) | 1 (5) | | | | 1 (100) | | | | | BCL-U | 4 (3) | | 3 (4) | | 1 (14) | | | | | 1 (50) | | | CD5-/CD10- BCL | 10 (9) | 13 (13) | 2 (3) | | 2 (29) | 2 (67) | | | | | | | CLL/SLL | | 2 (2) | | | 4 (57) | | | | | | | | MCL | | 2 (2) | | | | | | | | | | | FL | | 1 (1) | | | | | | | | | | | MZL | | | | 2 (10) | | | | | 2 (67) | | | | ALK+ ALCL | | 1 (1) | | 1 (5) | | | | | | | | | PTCL, NOS | | | 1 (1) | | | | | | | | | | T-ALL | 2 (2) | 3 (3) | 1 (1) | 2 (10) | | | | | | | | | Myeloid sarcoma | | 2 (2) | | | | | | | 1 (33) | | | | cHL | 1 (1) | 1 (1) | 1 (1) | | | | 1 (50) | | | | | | Plasmacytoma | | 1 (1) | | | | | | | | 1 (50) | | | Reactive | | 1 (1) | | | | | | | | | | | Carcinoma NOS | 2 (2) | 6 (6) | | | | | | | | | | | Neuroendocrine | 6 (5) | 6 (6) | 6 (9) | 4 (19) | | | | | | | * CLL/SLL,chronic lymphocytic leukemia/small lymphocytic lymphoma;FL,follicular lymphoma;ALCL,anaplastic large cell lymphoma;LBL,lymphoblastic lymphoma;MZL,marginal zone lymphoma;PBL,plasmablastic lymphoma;BCL-U,BCL,unclassifiable,with features intermediate between DLBCL & BL;MCL,mantle cell lymphoma;PTCL-NOS,peirpheral T-cell lymphoma,not otherwise specified;T-ALL,T-lymphoblastic lymphoma;cHL,classical Hodgkin lymphoma Table 1. Disclosures Rodig: Bristol Myers Squibb: Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees.

Background: Double-hit lymphomas (DHL) are a subset of diffuse large B-cell lymphoma (DLBCL) with concurrent chromosomal rearrangements involving the MYC and BCL2 or BCL6 genes, and are associated with dismal outcomes with standard upfront therapy. Double expressing lymphomas (DEL) are a subset of DLBCL with co-expression of MYC and BCL2 by immunohistochemistry (IHC), and also have a poor prognosis with standard therapy. While DHL status may be associated with inferior outcomes in patients with relapsed or refractory (rel/ref) disease (Cuccuini, Blood, 2012), little is known about the outcome of DHL patients with rel/ref disease who proceed to autologous stem cell transplantation (ASCT), and no study to date has examined the outcome of patients with DEL following ASCT. We evaluated the prognostic impact of DHL and DEL status in patients with rel/ref DLBCL who underwent ASCT at 2 centers. Methods: We retrospectively studied patients with rel/ref DLBCL, including transformed indolent lymphoma (TIL), who underwent ASCT at Brigham and Women's Hospital/Dana-Farber Cancer Institute (DFCI) and City of Hope (COH) between 1/2000 and 7/2013. The most recent biopsy prior to ASCT was used for testing when possible. IHC for MYC and BCL2 were performed using the Ventana (MYC: DFCI) and Leica BOND III (MYC: COH; BCL2: DFCI, COH) platforms according to standard protocols. For DEL, IHC cutoffs of ≥ 40% MYC-positive and ≥ 50% BCL2-positive cells were used (Johnson, JCO, 2012). Fluorescence in situ hybridization (FISH) for MYC was performed using LSI MYC dual-color break-apart probes (Abbott Molecular, Des Plaines, IL). MYC -rearranged cases had FISH for BCL2 and BCL6 performed using LSI BCL2 and BCL6 dual-color break-apart probes. DHL was defined as > 20% nuclei with break-apart signals for MYC and BCL-2 and/or BCL-6. Results: 201 patients with available archival tissue and clinical data were included. The median age was 60 (range 30-77) years; 60% were male; 26% had TIL; the median number of prior lines of therapy was 2 (range 2-5); 99% had prior rituximab; 53% had primary refractory disease or early (< 6 mo) relapse; 60% were in CR by PET at ASCT; and conditioning regimens were: 66% CBV v 16% BEAM v 10% rituximab and/or ibritumomab tiuxetan-BEAM v 8% other. Overall, the 4y progression-free survival (PFS) and overall survival (OS) were 44% and 61%, respectively. Among 185 patients with complete IHC data, 38% were DEL. The 4y PFS and OS in patients with DEL compared to non-DEL patients were 37% v 52% (p =0.001), and 51% v 69% (p =0.005), respectively [Figure 1]. Results were similar using other reported IHC cutoffs for DEL (e.g. MYC ≥ 40%/BCL2 ≥ 70%, Green, JCO, 2012). Among 93 patients with complete FISH and IHC data available, 13% had MYC rearrangement: 4% were MYC/BCL2 DHL, 3% were MYC/BCL6 DHL, and 2% had rearrangements of all 3 loci. The 4y PFS and OS in DHL v non-DHL were 30% v 42% (p =0.042), and 40% v 57% (p =0.026), respectively. Patients with DEL (excluding DHL) and patients with DHL had similar PFS, which was inferior to non-DEL/non-DHL patients (4y PFS 35% v 30% v 45%, respectively, p =0.026) [Figure 2]. In multivariable models testing pre-ASCT variables, including PET response to salvage, DEL (HR 2.1, p=0.0002), TIL histology (HR 1.8, p=0.009), and SD/PD at ASCT (HR 2.9, p=0.025) were associated with poorer PFS, while DEL (HR 2.0, p=0.004) and SD/PD (HR 3.1, p=0.021) were associated with poorer OS. Neither MYC (≥ 40%) nor BCL2 (≥ 50%) expression alone was independently associated with PFS or OS. When analysis was restricted to the subset of patients with complete IHC and FISH data, DEL (HR 1.9, p=0.023), DHL (HR 2.4, p =0.048), and SD/PD at ASCT (HR 7.6, p =0.009) were associated with inferior PFS. No center effect was observed. Conclusions: DEL and DHL status are both associated with inferior PFS in patients with rel/ref DLBCL who undergo ASCT, regardless of remission status. Although ASCT remains a potentially curative approach, these patients should be targeted for study of pre- or post-ASCT relapse risk reduction strategies. Figure 1. (A) Overall survival and (B) Progression-Free Survival after ASCT in DEL vs non-DEL Patients Figure 1. (A) Overall survival and (B) Progression-Free Survival after ASCT in DEL vs non-DEL Patients Figure 2. Progression-Free Survival after ASCT in Patients with DEL vs DHL vs non-DEL/DHL Figure 2. Progression-Free Survival after ASCT in Patients with DEL vs DHL vs non-DEL/DHL Disclosures Herrera: Genentech: Research Funding; Pharmacyclics: Research Funding; Sequenta, Inc.: Research Funding. Budde:Atara Biotherapeutics: Consultancy; Seattle Genetics, Inc.: Research Funding; Merck: Research Funding; Ikara Inc: Patents & Royalties. Chen:Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau. Davids:Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Other: ad board. Nademanee:Seattle Genetics Inc.: Research Funding; Celgene: Consultancy; Gilead: Consultancy; Spectrum: Research Funding. Siddiqi:Pharmacyclics: Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Forman:Mustang: Research Funding; Amgen: Consultancy. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding. Krishnan:Onyx: Speakers Bureau; Janssen: Consultancy; Millenium: Speakers Bureau; BMS: Consultancy; Jazz: Consultancy; Celgene: Consultancy, Speakers Bureau. Armand:Merck: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; BMS: Research Funding; Sequenta, Inc.: Research Funding.

Introduction. Classical Hodgkin Lymphomas (cHL) include small numbers of malignant Reed-Sternberg (RS) cells within an extensive but ineffective inflammatory/immune cell infiltrate. In cHL, chromosome 9p24.1 alterations increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and their further induction via JAK2-STAT signaling. PD-1 ligands engage the PD-1 receptor on T-cells and induce PD-1 signaling and T-cell exhaustion. Tumor cells expressing PD-1 ligands on their surface utilize the PD-1 pathway to evade an effective immune response. In recent pilot studies, PD-1 blockade was associated with high response rates and durable remissions in relapsed/refractory cHL. The unique composition of cHL limits its analysis with high throughput genomic assays. Therefore, the precise incidence, nature and prognostic significance of PD-L1 and PD-L2 alterations in cHL remain undefined. Herein, we utilize a recently developed fluorescence in situ hybridization (FISH) assay to characterize 9p24.1/PD-L1/PD-L2 alterations in a cohort of 108 newly diagnosed cHL patients (pts) who were uniformly treated with StanfordV (a combined modality therapy regimen) and have longterm followup. Methods. Pts were characterized as Ann Arbor early stage I/II favorable risk (ES-F), early stage unfavorable risk (bulk ≥ 10cm or ≥ .33 mediastinal dimension and/or B symptoms) (ES-U) or advanced stage III/IV (AS). ES-F pts received 8 weeks of Stanford V and 30 Gy involved field radiation (IFR); ES-U and AS pts received 12 weeks of Stanford V and 36 Gy IFR to initial sites > 5 cm. FISH was performed on formalin-fixed paraffin-embedded diagnostic biopsy specimens using bacterial artificial chromosome probes which covered CD274/PD-L1 (labeled with spectrum orange) and PDCD1LG2/PD-L2 (labeled with spectrum green) and a control centromeric probe (spectrum aqua-labeled CEP9, from 9p11-q11). Malignant RS cells were identified by their nuclear morphologic features and 50 RS cells/case were analyzed. Nuclei with a target:control probe ratio of at least 3:1 were defined as amplified (amp), those with a probe ratio of more than 1:1 but less than 3:1 were classified as relative copy gain, and those with a probe ratio of 1:1 but more than 2 copies of each probe were defined as polysomic for chromosome 9p. In each case, the percent and magnitude of disomy, polysomy, copy gain and amp were noted. In accordance with clinically approved diagnostic criteria, cases were classified by the highest observed level of 9p24.1 alteration. Specifically, cases with polysomy lacked copy gain or amp and cases with copy gain lacked amp. Immunohistochemical staining for PD-L1/PAX5 was performed as previously described and PD-L1 expression in PAX5 dim+ malignant RS cells and PAX5- infiltrating normal cells was assessed separately. Results. Almost all newly diagnosed cHL pts in this series had concordant alterations of the PD-L1 and PD-L2 loci; disomy was found in only 1% (1/108), polysomy in 5% (5/108), copy gain in 56% (61/108) and amp in 36% (39/108) of study pts. There was a correlation between intensity of PD-L1 protein expression and relative genetic alterations in this series. Two additional pts had translocations of PD-L1 or PD-L2 (2%, 2/108). We next assessed the association between specific types of PD-L1/PD-L2 alterations, clinical risk factors and outcome. Overall, the progression-free survival (PFS) was significantly lower for AS pts compared to ES-F/U pts (p=0.017). A model of PFS for the cHL pts by genetic alteration indicated that PFS was also significantly lower for pts with amp (p=0.02). Consistent with these findings, the incidence of 9p24.1 amp increased by clinical risk group: ES-F, 24%; ES-U, 34%; and AS, 50% (p=0.024, Kruskal-Wallis test). Therefore, we fit a full model of clinical and genetic factors including B-symptoms, bulk, stage and amp. Despite the association of amp with increased clinical risk groups, the genetic alteration further delineated PFS in the multivariate model (p=0.075). Conclusions. PD-L1/PD-L2 alterations are a defining feature of cHL with rare polysomy and more frequent copy gain and amp. There is an increased incidence of amp in pts with AS disease and a highly significant association of PD-L1/PD-L2 amp with PFS. These findings underscore the importance of genetically defined PD-1 mediated immune evasion in cHL and provide a rationale for the efficacy of PD-1 blockade in this disease. Disclosures Rodig: Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Gilead: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees.

Introduction Programmed cell death-1 (PD-1) is an immune checkpoint receptor that inhibits T cell activation upon interaction with its ligands PD-L1 or PD-L2. Increased PD-L1 expression has been reported in various lymphoid malignancies, and may allow these tumors to circumvent host anti-tumor immunity. Nivolumab, a fully human IgG4 monoclonal PD-1 receptor blocking antibody, potentiates T cell activity, and has clinical efficacy in various solid tumors. We hypothesized that nivolumab might also have clinically important anti-tumor activity in patients with lymphoid malignancies. Methods This open-label study enrolled patients with relapsed or refractory lymphoid malignancies including B-cell non-Hodgkin lymphoma (B-NHL), T-cell NHL (T-NHL), multiple myeloma (MM), and classical Hodgkin lymphoma (cHL). Patients were treated using a dose escalation design (1 mg/kg and 3 mg/kg) of nivolumab administered every two weeks for up to two years. Responses were assessed using standard criteria. The primary endpoint was safety; key secondary endpoints included anti-tumor activity and expression of immunomodulatory proteins in tumor biopsies. The preliminary results for the cHL patients will be reported separately. Results Twenty-nine patients with B-NHL, 2 patients with primary mediastinal B-cell lymphoma; 23 patients with T-NHL; 27 patients with MM; and 1 with chronic myelogenous leukemia were enrolled. Patients were heavily pretreated with 67%, 69%, and 78% of MM, B-NHL, and T-NHL patients, respectively, having received ≥ 3 prior treatment regimens. Previous autologous stem cell transplantation was reported for 56% of MM, 14% of B-NHL, and 9% of T-NHL patients. Prior brentuximab treatment was reported in 7% of B-NHL and 26% of T-NHL patients. When this pre-planned interim analysis was performed, six patients had been treated at the 1 mg/kg dose with 2 dose-limiting toxicities (DLTs) occurring in the same patient: grade 3 pneumonia and pneumonitis. At the 3mg/kg dose, seven patients were treated with one patient experiencing two DLTs: grade 3 eosinophilia and diplopia. Additional patients were enrolled in the cohort expansion at 3 mg/kg. Drug-related adverse events (AEs) occurred in 72%, 65%, and 52% of B-NHL, T-NHL, and MM patients, respectively. Serious AEs in B-NHL patients were pneumonitis (7%), acute respiratory distress syndrome, dermatitis, diplopia, enteritis, eosinophilia, mucosal inflammation, pyrexia and vomiting, each occurring in 3%. In the T-NHL patients, serious AEs were pneumonitis, rash, and sepsis, each occurring in 4%, and in MM patients, serious AEs were pneumonitis, myositis, and increased creatine phosphokinase, each occurring in 4%. The incidence and severity of drug related AEs were similar across tumor types. Efficacy results are shown for each tumor type in the table. The overall response rate (ORR) and complete response (CR) rate in patients with B-NHL were 28% and 7%, respectively, including an ORR of 36% in patients with diffuse large B-cell lymphoma (DLBCL), and 40% in patients with follicular lymphoma (FL). In patients with T-NHL, ORR was 17% (no CR), including an ORR of 40% in the 5 patients with peripheral T cell lymphoma. No objective responses were observed in MM. Analysis of PD-L1 expression and correlation to clinical outcome is being performed and will be presented. Conclusions Nivolumab administered at a dose of 3 mg/kg every two weeks was tolerable and the safety profile was similar to that of the agent in solid tumors. Objective responses were observed in DLBCL, FL, mycosis fungoides (MF), and peripheral T cell lymphoma (PTCL). Durable stable disease was observed in relapsed MM. The results of this phase 1 study have led to phase 2 studies in DLBCL and FL, which are ongoing. Table: Efficacy Results Tumor N Complete Response n (%) Partial Response n (%) Stable Disease (SD) n (%) Progression Free Survival Rate at 24 Weeks (%) Diffuse Large B Cell Lymphoma (DLBCL) 11 1 (9) 3 (27) 3 (27) (24) Follicular Lymphoma (FL) 10 1 (10) 3 (30) 6 (60) (68) Other B Cell Lymphoma 8 0 0 5 (63) (38) Primary Mediastinal B Cell Lymphoma 2 0 0 2 (100) (0) Mycosis Fungoides (MF) 13 0 2 (15) 9 (69) (39) Peripheral T Cell Lymphoma (PTCL) 5 0 2 (40) 0 (30) Other T Cell Lymphoma 5 0 0 1 (20) (0) Multiple Myeloma (MM) 27 0 0 18 (67) (15) Chronic Myelogenous Leukemia 1 0 0 1 (100) (100) Disclosures Lesokhin: Bristol-Myers Squibb: Consultancy, Research Funding. Ansell:Bristol-Myers Sqibb: Research Funding. Armand:Merck: Consultancy. Cohen:Celgene: Member, Independent Response Adjudication Committee Other; Onyx: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other. Lebovic:Genentech, Allos, Celgene, Onyx, Millennium: Consultancy, Research Funding, Speakers Bureau. Rodig:Bristol-Myers Squibb: Research Funding. Zhu:Bristol-Myers Squibb: Employment. Grosso:Bristol-Myers Squibb: Employment, Equity Ownership. Kim:Bristol-Myers Squibb: Employment. Shipp:Merck: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; Janssen R&D: Membership on an entity's Board of Directors or advisory committees. Borrello:Bristol-Myers Squibb: Research Funding. Timmerman:Bristol-Myers Squibb: Research Funding.