Rodig, Scott J

Background Pancreatic ductal adenocarcinoma (PDAC) is a challenging target for immunotherapy because it has an immunosuppressive tumor microenvironment. Neoadjuvant chemoradiotherapy can increase tumor-infiltrating lymphocyte (TIL) density, which may predict overall survival (OS). We hypothesized that adding programmed cell death protein 1 (PD-1) blockade to chemoradiotherapy would be well tolerated and increase TILs among patients with localized PDAC. Methods Patients were randomized 2:1 to Arm A (receiving pembrolizumab plus chemoradiotherapy (capecitabine and external beam radiation)) or Arm B (receiving chemoradiotherapy alone) before anticipated pancreatectomy. Primary endpoints were (1) incidence and severity of adverse events during neoadjuvant therapy and (2) density of TILs in resected tumor specimens. TIL density was assessed using multiplexed immunofluorescence histologic examination. Results Thirty-seven patients were randomized to Arms A (n=24) and B (n=13). Grade ≥3 adverse events related to neoadjuvant treatment were experienced by 9 (38%) and 4 (31%) patients in Arms A and B, respectively, with one patient experiencing dose-limiting toxicity in Arm A. Seventeen (71%) and 7 (54%) patients in Arms A and B, respectively, underwent pancreatectomy. Median CD8+ T-cell densities in Arms A and B were 67.4 (IQR: 39.2–141.8) and 37.9 (IQR: 22.9–173.4) cells/mm2, respectively. Arms showed no noticeable differences in density of CD8+Ki67+, CD4+, or CD4+FOXP3+ regulatory T cells; M1-like and M2-like macrophages; or granulocytes. Median OS durations were 27.8 (95% CI: 17.1 to NR) and 24.3 (95% CI: 12.6 to NR) months for Arms A and B, respectively. Conclusions Adding pembrolizumab to neoadjuvant chemoradiotherapy was safe. However, no convincing effect on CD8+ TILs was observed.

Abstract 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.

4128 Background: Pancreatic cancer (PC) is a challenging target for immunotherapy due to suppressive immune-microenvironment. Neoadjuvant chemoradiation (CRT) can increase the presence of tumor-infiltrating lymphocytes (TILs). We hypothesized that the combination of CRT and pembrolizumab can lead to further increase in TILs and their activation. Methods: Patients with resectable or borderline resectable PC were randomized 2:1 to the investigational treatment (Arm A) of pembrolizumab 200mg IV every 3 weeks concurrently with CRT (capecitabine 825 mg/m2 orally twice daily and radiation 50.4 Gy in 28 fractions over 28 days) or CRT only (Arm B) prior to surgical resection. The primary endpoints were treatment safety and density of TILs with the objective to estimate differences in TILs density between the investigational and the control arms. Immune cell densities were assessed using multiplexed immunofluorescence on resected tumor specimens. Densities of CD8+TILs were measured in 2-10 representative regions containing residual cancer per case and then averaged to obtain overall densities. The study was amended after enrollment of 37 patients to allow FOLFIRINOX prior to CRT, given changes in standard of care. Results: 37 patients were enrolled (24 Arm A and 13 Arm B). Post-neoadjuvant therapy, 13 patients had unresectable disease (9 on A and 4 on B), and 24 patients underwent surgery and were evaluable for the TILs primary endpoint (17 arm A and 7 arm B). The mean difference (A-B) in CD8+ cell density was 36 cells/mm2 (95% CI -85 to 157, stdev 130) (p 0.48). Additional analysis did not show significant differences in CD8+Ki67+ (activated cytotoxic T-cells), CD4+, and CD4+FOXP3+ (regulatory T cells), M1- or M2-like polarized macrophages, or granulocytes. The median recurrence free survival (RFS) was 18.2 months on Arm A and 14.1 on Arm B (p 0.41) and Overall Survival was 27.8 months on Arm A and 24.3 on Arm B (p 0.68) with a median follow up of 2.2 years. The most common grade 3 treatment-related toxicities were lymphopenia reported in 29% on Arm A and 31% on Arm B followed by diarrhea in 8% on Arm A attributed to CRT. There was only 1 DLT of increased ALT attributed to the combination on Arm A that resolved after holding the treatment and receiving steroids. There were no major surgical complications reported within 30 days post-surgery. Conclusions: The combination of CRT and pembrolizumab is safe. Preliminary analysis shows that the addition of pembrolizumab to CRT has minimal effects on several immune cell populations including CD8+TILs in the PC microenvironment. The study is currently enrolling 25 more patients who receive FOLFIRINOX prior to randomization to CRT+/- Pembrolizumab, which will help to dissect the immune modulatory effect of chemotherapy followed by CRT. Clinical trial information: NCT02305186.

Abstract 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.

Abstract Introduction: Classical Hodgkin lymphoma (cHL) is characterized by Reed Sternberg (RS) cells surrounded by an extensive but ineffective inflammatory/immune cell infiltrate. Recent studies suggest that Hodgkin RS cells have developed mechanisms that exploit the programmed cell death-1 (PD-1) pathway to evade immune detection. In cHL, chromosome 9p24.1 gain is a frequent structural alteration that increases the gene dosage of the PD-1 ligands, PD-L1 and PD-L2, and their induction via JAK/STAT signaling. Epstein-Barr Virus (EBV) infection also increases the expression of the PD-1 ligands in EBV-positive cHL. Ligand binding to PD-1 receptor-positive activated T cells induces “T cell exhaustion,” a reversible inhibition of T cell activation and proliferation. Nivolumab (BMS-936558, Bristol-Myers Squibb) is a fully human IgG4 monoclonal PD-1 blocking antibody that potentiates anti-tumor T cell activity, and exhibits clinical efficacy in several solid tumors. We hypothesized that nivolumab may augment anti-tumor activity in patients with relapsed or refractory (R/R) cHL, including those who had failed brentuximab vedotin (BV), and evaluated the PD-1 blocking antibody. These are the first analyses being reported with PD-1 antibody for the treatment of cHL. Methods: Patients with R/R cHL were included as an independent cohort in a dose escalation and cohort expansion phase I study of nivolumab in lymphoma and multiple myeloma (MM). Results for patients with non-Hodgkin lymphoma and MM are reported separately. Patients with cHL received nivolumab 3 mg/kg every 2 weeks until confirmed tumor progression or excessive toxicity. Responses were evaluated using standard criteria. The primary endpoint was safety; key secondary endpoints included anti-tumor activity and expression of immunomodulatory proteins in tumor biopsies. Results: Twenty-three patients were enrolled with R/R cHL. Patients were heavily pre-treated, 87% had received ≥ 3 prior treatment regimens, 78% had prior autologous stem cell transplant (ASCT), and 78% had prior BV treatment. Drug-related adverse events (AEs) of any grade occurred in 78% of patients, the most common of which were rash (22%), decreased platelet count (17%), diarrhea, nausea, pruritus, fatigue, and pyrexia (each at 13%). Drug-related grade 3/4 AEs occurred in 22% of patients. Three patients experienced 1 serious AE each (grade 3 myelodysplastic syndrome [MDS], grade 3 pancreatitis, and grade 2 lymph node pain). The objective response rate (ORR) was 87% (20/23), with 4 patients (17%) achieving a complete response (CR) and 16 (70%) obtaining a partial response (PR). The remaining 3 patients (13%) had stable disease (SD). All 23 patients had a reduction in tumor burden at 1 or more efficacy assessments during treatment with nivolumab (Figure). Among the 18 patients who had previously failed BV, the ORR was 89% (16/18), with 6% (1/18) achieving CR and 83% (15/18) PR. Progression-free survival (PFS) rate at 24 weeks was 86% (95% CI, 62-95%). The median overall survival (OS) has not been reached (range, 21+ to 75+ weeks). Six of 23 patients elected to discontinue study treatment to undergo stem cell transplantation, 2 patients discontinued due to toxicities (MDS and thrombocytopenia in 1 patient; pancreatitis in 1 patient), 4 patients discontinued due to disease progression, and 11 patients continue on study as of June 16, 2014. In a subset of study patients (10/23), PD-L1 and PD-L2 copy numbers in RS cells were assessed using fixed tumor biopsy specimens and a 3-probe fluorescence in situ assay (PD-L1,PD-L2, and control centromeric probe). In all tumors, RS cells had copy gains of PD-L1 and PD-L2, as a result of either polysomy or amplification; these tumors also exhibited increased protein expression of PD-L1. RS cells were also largely positive for pSTAT3, indicative of active JAK/STAT signaling. Conclusions In patients with R/R cHL, nivolumab-mediated PD-1 blockade is safe and tolerable with a safety profile similar to that in solid tumors. The frequent and long-lasting responses in heavily pretreated, R/R patients, including those who have failed BV, highlight the importance of the PD-1 pathway in cHL, and the genetically defined sensitivity to PD-1 blockade in this disease. Based on these results, the FDA granted nivolumab breakthrough status in relapsed cHL and a large, multinational, phase II trial of this therapy is underway. Figure 1 Figure 1. Disclosures Armand: Merck: Consultancy. Off Label Use: Nivolumab was tested for the treatment of hematologic malignancies.. Ansell:Bristol-Myers Squibb: Research Funding. Lesokhin:Bristol-Myers Squibb: Consultancy, Research Funding. Timmerman:Bristol-Myers Squibb: Research Funding. Borrello:Bristol-Myers Squibb: Research Funding. 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.

Abstract 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.

Abstract B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell tumors including diffuse large B-cell lymphoma (DLBCL). BCR signaling activates proximal pathway components such as the spleen tyrosine kinase (SYK) and downstream effectors including PI3K/AKT and NF-κB. In recent studies, we characterized distinct SYK/PI3K/AKT-dependent viability pathways in BCR DLBCL cell lines and primary tumors with high- or low-baseline NF-κB activity and defined additional SYK/PI3K/AKT-dependent processes essential for BCR DLBCLs with low- or high-baseline NF-κB (Chen et al Cancer Cell 2013; 23:826-838). For example, SYK/PI3K/AKT-dependent cholesterol biosynthesis was identified as a feed-forward mechanism of preserving the integrity of BCRs in lipid rafts in all BCR-dependent DLBCLs. In the current study, we sought to identify biomarkers of intact BCR signaling in DLBCLs with low or high baseline NF-κB. We found highly significant transcriptional upregulation of CXCR4 in all BCR-dependent DLBCLs treated with a chemical SYK inhibitor (R406). These results were of particular interest because BCR signaling inhibits stromal cell-derived factor (SDF)-1α chemotaxis and promotes intermobilization of the SDF-1α receptor, CXCR4. To expand on our findings, we analyzed CXCR4 transcript abundance in a large panel of cell lines including BCR-dependent/ low NF-κB (GC type) and BCR-dependent/ high NF-κB (ABC type) and BCR-independent/ OxPhos DLBCLs. CXCR4 transcript abundance was significantly and selectively upregulated in all BCR-dependent DLBCL lines assessed by qRT-PCR following chemical SYK inhibition. Consistent with the qRT-PCR results, SYK blockade also selectively upregulated cell surface CXCR4 expression in all BCR-dependent DLBCLs. Similar results were obtained following SYK knockdown with multiple independent shRNAs; CXCR4 transcript abundance was significantly and selectively upregulated in all BCR-dependent/ low NF-κB (GCB) DLBCLs and BCR-dependent/ high NF-κB (ABC) DLBCLs but was not altered in BCR-independent/ OxPhos tumors. Following SYK blockade, we also observed significant CXCR4 induction (by qRT-PCR) in multiple primary BCR-dependent DLBCLs (NF-κB low and NF-κB high) and unchanged CXCR4 levels in primary BCR-independent tumors. These data confirm that the observations regarding SYK blockade and CXCR4 induction in BCR-dependent DLBCL cell lines translated to primary tumors. Our recent studies indicate that all BCR-dependent DLBCLs exhibit SYK/PI3K/AKT signaling and modulation of FOXO1 regardless of baseline NF-κB levels. Given the recent identification of CXCR4 as a FOXO1 target, we asked whether modulation of CXCR4 in DLBCLs was via a PI3K/AKT pathway. In BCR-dependent DLBCLs with low or high baseline NF-κB, chemical PI3K inhibition (LY294002) phenocopied chemical SYK blockade (R406) with highly significant transcriptional upregulation of CXCR4. Moreover, in BCR-dependent DLBCL cell lines, enforced expression of a constitutively active form of AKT (myrAKT) abrogated CXCR4 upregulation resulting from SYK blockade. These data indicate that in BCR-dependent DLBCLs, CXCR4 is modulated by a SYK/PI3K/AKT-dependent pathway. To assess the functional significance of CXCR4 upregulation in BCR-dependent DLBCLs, we performed a transwell chemotaxis assay and evaluated migration toward a SDF-1α gradient. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased migration toward the CXCR4 ligand, SDF-1α, whereas the BCR-independent/ OxPhos DLBCLs were unaffected. The enhanced SDF-1α dependent migration was abrogated when the chemotaxis assay was performed in the presence of the specific CXCR4 inhibitor, AMD3100, confirming the specificity of the observed effect. Therefore, in BCR-dependent DLBCLs with low or high baseline NF-kB, SYK/PI3K inhibition is associated with significant and functionally relevant upregulation of CXCR4. For these reasons, CXCR4 may serve as a robust biomarker to assess the integrity of the BCR pathway and evaluate the efficacy of BCR inhibition in DLBCL. Disclosures: Off Label Use: R406 is not get approved, but is being evaluated in DLBCL and CLL.

Abstract 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.

Abstract Abstract 1545 Introduction: The diagnosis of peripheral T-cell lymphoma (PTCL) is difficult and accurate diagnosis and subclassification relies on correlating histologic, immunophenotypic, molecular genetic, and clinical data. Evidence-based guidelines for the appropriate diagnostic work-up of PTCL are lacking. The objective of this study was to evaluate a large series of PTCLs by experienced hematopathologists with a tiered approach to immunohistochemistry (IHC) and molecular genetic characterization to document overall diagnostic accuracy and clinical relevance using this approach. Methods: 7 experienced hematopathologists from 5 institutions reviewed 374 cases of peripheral T and NK cell lymphomas (referred to collectively as PTCL). 6 cases of cutaneous T-cell lymphoma were excluded after final review in addition to 29 non-PTCL cases submitted as control cases to mimic diagnostic practice during the review. Cases received tier 0, 1 and 2 diagnoses by 3 independent pathologists, based on review of hematoxylin and eosin (HE) stain with basic demographic data, panel 1 IHC (CD3, CD5, CD10, CD20, CD21, CD30, CD45, PAX5), and panel 2 IHC (CD2, CD4, CD7, CD8, CD23, PD1, CD56, EBER, ALK, TIA1, TCRg, TCRbF1), respectively. A tier 2b diagnosis was then rendered after gene rearrangement data were available. A final consensus diagnosis was rendered after discussion of each case by the 3 reviewers with all available clinical data. Overall survival (OS) was assessed using Kaplan-Meier (KM) curves and Cox proportional hazards models. Results: 1122 individual diagnoses leading to 339 final consensus PTCL diagnoses were rendered. 341 (91%) cases had complete phenotypic data and 241 had gene rearrangement data. There was no bias of missing data according to final diagnosis subtype. Reviewer diagnoses using specific WHO subclassification were 16.3%, 36.9%, 82.7%, and 85.9% for tier 0, 1, 2, and 2b, demonstrating a significant increase in diagnostic certainty after a complete IHC panel. Gene rearrangement only contributed to a change in diagnosis in 51/650 (8%) individual reviews. Across all 374 cases, a small number of cases (n=28, 7.5%) showed no agreement among the 3 independent reviewers after tier 2b and required debate. These generally represented refinement of the subclassification of a PTCL. The most common disagreements were between PTCL, not otherwise specified (nos) vs. unclassifiable T-cell lymphoma, and PTCL, nos vs. angioimmunoblastic T-cell lymphoma (AITL). Currently, OS data was available in 198 cases and 52% have died (median age follow-up of 15 months for those still alive, range 0–160 mo). Figure 1 shows the KM survival curves for types with more than 10 cases. Of note, unclassifiable PTCL cases had poor OS, comparable to PTCL nos; a trend was seen for CD30+ ALK- ALCL to have a longer OS compared to PTCL nos (HR=0.5, 95% CI: 0.21–1.19, P=.09). EBER positivity in tumor cells was associated with poor OS in the cohort of all PTCL patients (HR=2.32, 95% CI: 1.28–4.23, p=0.006, Figure 2); the association was similar when nasal NK/T-cell lymphoma cases were excluded (HR=2.50, 95% CI: 1.00–6.26, p=0.05). Trends for poor prognosis were also seen for TIA-1 expression in PTCL, nos (HR=1.9, 95% CI 0.91–3.96, P=.09) and PD1 expression in AITL (HR=6.25, 95% CI 0.85–46.09, P=.07). Clinical data collection is still ongoing and will be updated. Conclusions: We demonstrate the diagnostic accuracy among experienced hematopathologists of a defined IHC panel, showing an overall ability to reach consensus diagnosis of 93% in PTCL cases. The resulting consensus diagnostic subtypes showed expected outcomes relative to other large series of PTCLs, and EBER positivity is a poor prognostic marker among T and NK cell lymphomas. A tiered approach to IHC is recommended when a PTCL is under differential diagnostic consideration since the first tier can often resolve the main question of whether a lesion is reactive or lymphoma. This can lead to more efficient use of the expanded tier 2 panel that will enable diagnosis and specific subclassification of PTCL. Gene rearrangement studies are not required in the great majority of cases. This evidence-based approach to the diagnosis of PTCL should inform practicing pathologists, clinical trial design, and policy makers regarding required ancillary studies in this group of diseases. Disclosures: Hsi: Allos: Research Funding. Said:Allos: Research Funding. Macon:Allos: Research Funding. Rodig:Allos: Research Funding. Gascoyne:Seattle Genetics: Research Funding. Ondrejka:Allos: Research Funding. Dorfman:Allos: Research Funding. Maurer:Allos: Research Funding. Dogan:Allos: Research Funding.

Abstract Abstract 1534 + equal contributions Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate and infrequent somatic mutations of TP53 and genes encoding cell cycle pathway components. Despite recent advances in the molecular understanding of DLBCL pathogenesis, clinical models such as the International Prognostic Index (IPI) are still used to identify high-risk patients. By integrating high-resolution high-density SNP array data with transcriptional profiles and performing pathway analysis in 180 primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary DLBCLs with single copy loss of 17p13.1 (TP53/RPL26/KDM6B) often had CNAs of an additional p53 modifier – 9p21.3 (CDKN2A/ARF), 19q13.42 (BCL2L12), 12q15 (MDM2) or 1q23.3 (MDM4/RFWD2). CNAs of the respective p53 modifiers, CDKN2A (ARF, 9p21.3), MDM2 (12q15) and MDM4/RFWD2 (1q23.3), occurred in largely separate groups of tumors. DLBCLs with CNAs of p53 pathway members frequently exhibited concurrent alterations of additional cell cycle components such as CCND3 (6p21.32), CDK6 (7q22.1), CDK2/CDK4 (12q15) and/or RB1 (13q14.2) or RBL2 (16q12.2). When the primary DLBCLs were clustered in the space of CNAs that perturb p53 pathway and cell cycle components, 66% of tumors had multiple alterations (“complex” pattern) whereas the remaining 34% of tumors lacked these lesions (“clean” signature). Tumors with “complex” alterations of p53 pathway and cell cycle components also had more total CNAs and more frequent TP53 mutations, highlighting the association between p53 deficiency, cell cycle deregulation and increased genomic instability in DLBCL. The patterns of “complex” vs. “clean” CNAs of p53 pathway and cell cycle components and the association between the “complex” signature and total CNAs were confirmed in an independent series of primary DLBCLs. To further characterize DLBCLs with “complex” vs. “clean” CNA patterns, we performed gene set enrichment analyses with publicly available series of p53 target genes and a RB-deficiency gene set which included multiple E2F targets. The p53 target transcripts were significantly less abundant in “complex” DLBCLs, directly linking their genetic signature of p53 deficiency with decreased p53 activity. Furthermore, the RB-deficiency gene set was significantly enriched in “complex” DLBCLs suggesting that these tumors had increased E2F-mediated cell cycle progression. Consistent with these observations, DLBCLs with “complex” CNA patterns also had significantly higher proliferation indices as determined by Ki67 immunostaining. We next assessed the prognostic significance of the “complex” CNA pattern in the subset of patients who were treated with R-CHOP (rituxan, cyclophosphamide, adriamycin, oncovin, prednisone) and had long-term follow-up. R-CHOP treated patients with “complex” CNA patterns had a 5-year overall survival of only 62%; in contrast, those with “clean” CNA signatures had an 100% overall survival (p =.001). The association between CN complexity and outcome was independent of transcriptionally defined categories and additive to the clinical IPI risk model. Taken together, these data provide a structural basis for deregulated cell cycle, increased cellular proliferation and unfavorable outcome in DLBCL and suggest targeted treatment strategies. Disclosures: No relevant conflicts of interest to declare.