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Abstract Background WM remains an incurable cancer despite recent advances. While a phase II trial has demonstrated efficacy of dexamethasone, rituximab and cyclophosphamide (DRC) in the treatment-naïve (TN) patients, this regimen has not been studied systematically in the relapsed/refractory (R/R) setting. Recent data has suggested the MYD88mutation status in WM patients can serve as a predictive marker with therapies such as ibrutinib. We examined the efficacy and tolerability of up to 6 cycles of DRC, with focus on R/R population and activity of this regimen with respect to the patients' MYD88L265Pstatus. Methods Records of WM patients seen consecutively at Mayo Clinic campuses in Rochester, Arizona and Florida from 01/2007 to 12/2014 were reviewed. MYD88L265P status, as assessed by AS-PCR, was recorded when available. Data obtained from symptomatic patients treated with DRC (dexamethasone 20 mg intravenously followed by rituximab 375 mg/m2 intravenously on day 1 and cyclophosphamide 100 mg/m2 orally bid on days 1 to 5) were analyzed. Time-to-event analyses were performed from DRC therapy using the Kaplan-Meier method. We used the Consensus response criteria (6th International Workshop). Results Of 100 symptomatic patients who received DRC, 50 had R/R WM (40% refractory) and 50 were TN. Table 1 shows the patients' baseline characteristics. In the R/R population, DRC was 2nd line (range 2-8) therapy in 58% of patients. The median IgM levels declined from 3,870 mg/dL to 1,846 mg/dL (p=0.0001) at best response. Median follow-up from DRC was 51 months (95% CI: 38-55). The median time-to-best response was 6.8 (0.5-28) months. Overall response rate (ORR) was 87% [VGPR 4%, PR 64%, MR 19%]. Four patients (9%) achieved SD and 2 patients (4%) had PD. Of the sixteen deaths (32%) noted at time of analysis, 11 were related to progressive disease. The median disease-specific survival (DSS) from DRC was not reached (NR) (95% CI: NR-NR). The median progression-free survival (PFS) and time-to-next-therapy (TTNT) were 32 months (95% CI: 15-51) and 50 months (95% CI: 35-60), respectively. In contrast, in the TN patients the median IgM levels declined from 4,130 mg/dL to 1,250 mg/dL (p=0.0001) at best response; median time to best response was 11 (0.6-47) months; median follow-up from DRC was 30 months (95% CI: 21-36). ORR was 96% [VGPR 17% (n=8), PR 70% (n=32), MR 9% (n=4)]. Of the 7 deaths (14%) at time of analysis, 4 were related to WM progression. Median DSS from DRC was NR (95% CI: NR-NR); median PFS and TTNT were 34 months (95% CI: 23-NR) and NR (95% CI: 37-NR), respectively. Among 29 genotyped patients in whom MYD88 mutation status was available, MYD88L265P was present in 25 patients (8 of 10 TN and 17 of 19 R/R patients). The response rates and the time-to-event outcomes were similar in the MYD88L265P and wild-type patients. Grade ≥3 adverse effects included neutropenia (20%), thrombocytopenia (7%) and infections (3%). Conclusions Similar to the frontline setting, DRC is an active and well-tolerated salvage regimen. In contrast to ibrutinib, DRC offers a less expensive, limited-duration treatment option, with preliminary data suggesting activity independent of patients' MYD88 mutation status. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Kumar:Glycomimetics: Consultancy; Sanofi: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Array BioPharma: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Dispenzieri:Jannsen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; pfizer: Research Funding; Celgene: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Kapoor:Amgen: Research Funding; Celgene: Research Funding; Takeda: Research Funding.

Abstract Abstract 4136 Background: The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. This assay has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and amyloid light chain (AL) amyloidosis, and has been incorporated in response criteria for both MM and AL amyloidosis. We previously showed elevated FLC was associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) patients from a phase II cooperative group clinical trial (Blood (ASH Annual Meeting Abstracts) 2009 November 20, 2009; 114(22): 136). Here we replicate those findings in a large clinic based epidemiology cohort, as well as examine FLC abnormalities in other lymphoma subtypes. Methods: Newly diagnosed lymphoma patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum FLC was quantitated from enrollment research serum draws within 9 months of diagnosis using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Elevated FLC was defined as either kappa or lambda above the published reference range. Monoclonal elevated FLC was defined as elevated FLC with an abnormal FLC ratio; polyclonal elevated FLC was defined as elevated FLC with an FLC ratio in the normal reference range. Clinical data was abstracted using a standard protocol for the MER and patients were followed systematically for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively). Cox proportional hazards models were used to evaluate the unadjusted association between FLC and outcome. Results: 1118 lymphoma patients diagnosed between September 2002 and February 2008 with available serum were assayed. At a medium follow-up of 49 months (range 1–94), 484 patients (43%) had an event and 249 patients died (22%). Elevated FLC was present in 27% of lymphomas, with prevalence ranging from 13% in follicular lymphoma (FL) to 64% for mantle cell lymphoma (MCL) and 77% for lymphoplasmacytic lymphoma. Elevated FLC was present in 27% of DLBCL and associated with poor EFS (monoclonal hazard ratio (HR)=2.56, polyclonal HR=1.99, all p<0.001) and OS (monoclonal HR=3.22, polyclonal HR=2.91, all p<0.0001), replicating our previous findings. Elevated FLC was generally polyclonal (FLC ratio normal) across lymphoma subtypes, though monoclonal elevated FLC (FLC ratio abnormal) was common in MCL and the post-follicular lymphomas. Elevated FLC was associated with inferior EFS in all lymphomas combined (monoclonal HR=1.92, 95% CI:1.44-5.55, polyclonal HR=1.38, 95% CI:1.09-1.74); this association remained consistent in most subtypes (see table). In addition, elevated FLC was associated with poor EFS in MCL (monoclonal) and Hodgkin lymphoma (HL; polyclonal). Elevated FLC was also significantly associated with OS in HL (polyclonal HR=6.24, 95% CI 2.44–15.9), FL (polyclonal HR=5.85, 95% CI 2.62–13.0), post follicular lymphomas (polyclonal HR=4.16, 95% CI 1.17–14.8), as well as for all lymphomas combined (monoclonal HR=2.24, 95% CI 1.52–3.29, polyclonal HR=2.33, 95% CI 1.74–3.11). Conclusions: A significant percentage of lymphoma patients have elevated FLC. Our previous discovery of elevated FLC predicting poor outcome in DLBCL was confirmed in a large, independent dataset. Prevalence and type of elevated FLC (monoclonal vs. polyclonal) varies by lymphoma subtype. Elevated monoclonal and polyclonal FLC is associated with poor outcome broadly in lymphoma, as well as specifically for a number of lymphoma subtypes, supporting FLC as a potentially useful prognostic biomarker in many lymphoma subtypes. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Aberrant B-cell receptor (BCR) and MYD88 signaling cooperatively drive Waldenstrom macroglobulinemia (WM) tumor cell survival. To sustain BCR-driven growth and maintain optimal cellular homeostasis, WM cells upregulate proteasome function. We have recently described the role of two distinct 19S proteasome-associated deubiquitinating enzymes (DUBs), USP14 and UCHL5, in maintaining WM-cell viability. Pharmacologic inhibition with b-AP15 (commercial compound) results in WM-cell death and accumulation of high molecular weight ubiquitinated protein conjugates; an event that is phenocopied by siRNA knockdown of USP14 and UCHL5. VLX1570 is a lead-optimized clinical grade analog of b-AP15 with enhanced solubility, stability and target residence time. We investigated the effect of VLX1570 on BCR components, WM cell survival proteins and its in vivo activity in a human xenograft model of WM. Materials: VLX1570 was obtained from Vivolux AB and bortezomib from Sellekchem. Human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1) and their drug-resistant derivatives were used (total n=12). Bone marrow aspirates from WM-patients and from healthy donors were obtained from the Mayo Clinic biospecimens core. PBMCs from healthy donors were used as comparators where needed. NOD/SCID mice were obtained from Taconic Labs and used for profiling the in vivo properties of VLX1570. Results: We first examined for USP14/UCHL5 protein expression by immunohistochemical analysis in primary tumor tissue from WM patients and compared it to healthy donor cells. WM patient-derived cells exhibited greater staining intensity of both DUBs. Preferential binding of VLX1570 to USP14 and UCHL5 in WM cells was probed in a WM cell using an enzyme-based competition assay (HA-Ub-Vs), which demonstrated time-dependent decrease in labeled USP14 and UCHL5 after treatment with VLX1570. In drug-resistant (bortezomib or ibrutinib-resistant) WM cells, VLX1570 induced >50% tumor-specific apoptosis and displayed synergistic activity with ibrutinib (median CI: 0.5). In addition to ER stress markers, we had previously observed that gene expression of BCR-signalosome components were reduced in VLX1570-treated WM cells (NanoString platform) and this was substantiated at the protein level where p-SYK, SYK and BTK decreased in a time-dependent manner. BCR end-effectors were probed in response to VLX1570 and a decrease in nuclear translocation of NFkB (p65) was noted. USP14 has been shown to control the availability of the chemokine receptor CXCR4, which regulates homing of WM cells to bone marrow. After VLX1570 treatment, we noted a significant decrease in CXCR4 surface receptor expression in all WM cell lines tested and primary WM cells. Lastly, to determine the in vivo anti-WM effects of VLX1570, we injected NOD/SCID mice (xenotransplanted with RPCI-WM1-Luc cells) with VLX1570 (I.P. route, 4.4mg/kg every other day) and measured tumor burden along with pharmacodynamic indicators. VLX1570-treated mice (n=7) demonstrated significantly lower tumor volumes by caliper measurement and bioluminescent signal (p=0.01 - 0.003) as well as reduced serum human-IgM, compared to vehicle-treated mice (n=7). Importantly, mice treated with VLX1570 survived longer vs. vehicle-treated mice (median survival, 62 vs. 44 days, respectively, p=0.0008). No unforeseen toxicities were noted and weight of the mice in vehicle vs. VLX1570-treated mice did not significantly change. IHC analysis in mice tumor tissues demonstrated reduced CXCR4, human-IgM, Ki67 and increased cleaved caspase-3 and ubiquitinated (k48-linked) protein level, the latter serving as a more specific indicator of USP14/UCHL5 inhibition. Conclusions: We provide substantial in vitro and in vivo evidence on the anti-WM effects of a novel first-in-class clinical grade USP14/UCHL5 inhibitor, VLX1570. In addition to anticipated induction of ER stress machinery, we describe for the first time VLX1570-mediated modulation of BCR components and WM cell survival proteins as well as demonstrate its robust activity in an aggressive human xenograft model of WM. These data provide the rationale for a clinical trial of single agent VLX1570 in patients with symptomatic WM. Disclosures Martin: Janssen: Consultancy, Honoraria; Acerta: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bayer: Consultancy. Gullbo:Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

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 T-cell exhaustion plays an important role in attenuating the function of immune cells in B-cell non-Hodgkin's lymphoma (NHL) and PD-1 expression is typically used to identify exhausted T-cells. We have however previously shown that not all PD-1+ cells are exhausted and that PD-1 is differentially expressed on two distinct T-cell subpopulations, with high expression on T follicular helper cells and dim expression on exhausted T cells. Other markers are therefore needed to more clearly identify exhausted intratumoral T cells. To further define exhaustion of intratumoral T cells, we determined the co-expression, regulation and function of PD-1, TIM-3 and LAG-3 on CD4+ or CD8+ T cells by flow cytometry. Using biopsy specimens from follicular B-cell NHL, we found that the percentages of PD-1+ and TIM-3+ T cells were 53.1% (range: 17.2-81.2%, n=32) and 34.5% (range: 14.9-62.6%, n=34) in CD4+ T cells and 46.8% (range: 12.8-81.7%, n=32) and 40.4% (range: 15.0-78.4%, n=34) in CD8+ T cells, respectively. We observed that TIM-3 was predominantly expressed on PD-1dim T cells and TIM-3+ cells accounted for 40% of CD4+ PD-1dim or 45% of CD8+ PD-1dim T cells. Similarly, LAG-3 was variably expressed on intratumoral T cells from B-cell NHL. A median of 9.54% (range: 3.01-15.46, n=6) of CD4+ or 20.48% (7.93-33.9, n=8) of CD8+ T cells express LAG-3. We found that LAG-3+ T cells almost exclusively came from PD-1+ TIM-3+ cells, forming a defined population of intratumoral PD-1+ TIM-3+ LAG-3+ CD4+ or CD8+ T cells. While the majority of LAG-3+ T cells were effector memory T cells (CD45RA- CCR7-), some LAG-3-expressing T cells displayed a phenotype of terminally-differentiated T cells (CD45RA+ CCR7-). Functionally, the intratumoral TIM-3+ LAG-3+ T cells exhibited reduced capacity to produce cytokines (IL-2, IFN-γ) and granules (perforin, granzyme B). Similar to TIM-3, LAG-3 expression was strongly up-regulated on CD4+ or CD8+ T cells by IL-12, a cytokine that has been shown to induce T-cell exhaustion. Interestingly, we observed that while expression of TIM-3 on CD8+ T cells was upregulated by IL-12 at an early time point (day 1), LAG-3 was only induced after TIM-3 up-regulation (day 3) and almost exclusively on TIM-3+ T cells. Furthermore, we found that blockade of both TIM-3 and LAG-3 signaling was able to reverse the exhausted phenotype of CD8+ T cells resulting in increased IFN-γ and IL-2 production. This effect was further enhanced when CD8+ T cells were treated with both anti-TIM-3 and anti-LAG-3 Abs. Taken together, these results suggest that PD-1, TIM-3 and LAG-3 were involved in the induction of exhaustion of T cells in B-cell NHL. We find that PD-1, TIM-3 and LAG-3 are expressed on the same T cells and that blocking TIM-3 and LAG-3 can reverse T-cell exhaustion signaling. These results suggest that PD-1, TIM-3 and LAG-3 play a synergistic role in the development of T cell exhaustion in NHL. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 283 Background: Hodgkin lymphoma (HL) is characterized by the presence of CD30-positive Hodgkin Reed-Sternberg cells. The antibody-drug conjugate (ADC) brentuximab vedotin (SGN-35) delivers the highly potent antimicrotubule agent monomethyl auristatin E (MMAE) to CD30-positive malignant cells by binding specifically to CD30 on the cell surface and releasing MMAE inside the cell via lysosomal degradation. Binding of MMAE to tubulin disrupts the microtubule network within the cell, induces cell cycle arrest, and results in apoptotic death of the CD30-expressing tumor cell. In a previous phase 1 study with brentuximab vedotin dosing every third week, 11 of 12 patients (92%) treated at the maximum tolerated dose of 1.8 mg/kg had tumor reductions and 6 of 12 patients (50%) achieved an objective response (complete remission [CR] + partial remission [PR]). Methods: A pivotal, phase 2, single-arm, multicenter study was conducted to evaluate the efficacy and safety of brentuximab vedotin in patients with relapsed or refractory HL post autologous stem cell transplant (ASCT). Brentuximab vedotin 1.8 mg/kg was administered every 3 weeks as a 30-minute outpatient IV infusion for up to 16 cycles of treatment. The primary endpoint was the overall objective response rate (ORR) as assessed by an independent review facility according to the Revised Response Criteria for Malignant Lymphoma (Cheson 2007). Results: A total of 102 patients with relapsed or refractory HL were treated at 26 study centers. Median age was 31 years (range 15–77) and approximately half of the patients were female (53%). Eastern Cooperative Oncology Group (ECOG) performance status at baseline was 0 (41%) or 1 (59%). All patients had previously received an ASCT and the median number of prior chemotherapy regimens was 4 (range 1–13). More than 70% of the patients had primary refractory disease, defined as failure to achieve a CR or progression within 3 months of completing frontline therapy. In addition, 39% of patients had lymphoma that was refractory to the most recent salvage therapy excluding ASCT. The median duration of brentuximab vedotin treatment on this study was 27 weeks (range 3–54) and the median number of cycles was 9 (range 1–16). The most common (>15%) treatment-related adverse events (AEs) of any grade were peripheral sensory neuropathy (43%), fatigue (40%), nausea (35%), neutropenia (19%), diarrhea (18%), and pyrexia (16%); most events were Grade 1 or 2. Grade 3 treatment-related AEs reported in >1 patient were neutropenia (14%), peripheral sensory neuropathy (5%), thrombocytopenia and hyperglycemia (3% each), and fatigue (2%). The only Grade 4 treatment-related events were neutropenia (4%), and thrombocytopenia, abdominal pain, and pulmonary embolism (1% each). No related Grade 5 events were observed. 18 patients discontinued treatment due to an AE. Consistent with findings from the phase 1 study, 97 patients (95%) had a reduction in tumor size per investigator assessment. Of the 35 patients who had B symptoms at baseline, 29 (83%) experienced resolution of these symptoms. The median time to B symptom resolution was 3 weeks (range <1–16 weeks). To date, 5 patients have received an allogeneic stem cell transplant as their first therapy after discontinuing treatment in the study; all of these patients remain in follow up. Per protocol, no interim analyses were planned for the primary endpoint of ORR by independent review. Independent assessment of ORR is being conducted and final efficacy results will be presented at the meeting. Conclusions: Brentuximab vedotin was associated with manageable adverse events and, based on investigator assessment, demonstrated encouraging activity in heavily pretreated patients with relapsed or refractory HL. Tumor shrinkage was observed in 95% of patients and the B symptom resolution rate was 83%. Final results of the independent assessment of ORR, as well as duration of response, progression-free survival, and updated safety data, will be presented at the meeting. Disclosures: Chen: Seattle Genetics, Inc.: Research Funding. Off Label Use: This clinical trial uses the investigational drug, brentuximab vedotin (SGN-35). Gopal:Seattle Genetics, Inc.: Research Funding. Smith:Seattle Genetics, Inc.: Research Funding. Ansell:Seattle Genetics, Inc.: Research Funding. Rosenblatt:Seattle Genetics, Inc.: Research Funding. Klasa:Seattle Genetics, Inc.: Research Funding. Connors:Seattle Genetics, Inc.: Research Funding. Engert:Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Larsen:Seattle Genetics, Inc.: Employment, Equity Ownership. Kennedy:Seattle Genetics, Inc.: Employment, Equity Ownership. Sievers:Seattle Genetics, Inc.: Employment, Equity Ownership. Younes:Seattle Genetics, Inc.: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; SBIO: Honoraria, Research Funding.

Abstract Recent next generation sequencing of paired normal and tumor lymphoma tissue highlights a significant role for the MYD88L265P mutations in Waldenstrom Macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL). In recent work we have found that MYD88L265P signaling is constitutively active in both WM and DLBCL cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and TAK1 and NF-kB activation. However, little else is know about the full impact of the mutation, especially in combination with other genetic events. Recent mouse models suggest that MYD88L265P alone may not be sufficient to induce tumor formation and that the pathway requires additional genetic hits. Interplay between MYD88L265P and other oncogenic events are further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88L265P. These data, combined with the finding that loss of TNFAIP3 promotes accumulation of MYD88L265PB-cells in a mouse model, suggest a potential relationship between these genetic events, and more importantly, a possible clinical impact. The aim of this study was to generate a human cell line model which mimics the effect of MYD88L265P andTNFAIP3 loss to further understand the cellular consequence of these genomic alterations in lymphoma. To mimic the TNFAIP3 loss in combination with the MYD88L265P mutation,we used the WM cell line MWCL1, which harbors a heterozygous mutation in MYD88. For the TNFAIP3 specific knock out, we used the transcription activator-like effector nucleases (TALENs) genome editing approach to induce a double stranded break in exon 5 of the TNFAIP3 gene. TALENs were expressed in MWCL1 cells by transient transfection, and single-cell clones were isolated. Sanger sequencing of the TNFAIP3 gene was performed to identify clones that carried the deletion and all clones were screened by Western blot to confirm knock down of A20. We successfully generated a panel of MWCL1 cell lines that harbor a heterozygous deletion in the TNFAIP3 gene. Western blot analysis confirm a 2.4 fold reduction in A20 expression in the heterozygous cell line (MWCL1A20-/A20+), Because A20 has been shown the be a central negative regulator of NF-κB activation, we first measured the impact of A20 loss on phosphorylation of NF-κB p65 and found that MWCL1A20-/A20+ cells had higher base line levels of pNF-κB compared to the wild type (WT) cell line (1.77 fold). Furthermore, time course stimulation experiments with the TLR ligand lipopolysaccharide (LPS) showed that MWCL1A20-/A20+ cells had a higher amount of pNF-κB after 30 (3 fold) and 60 (1.5 fold) minutes of stimulation. We next looked at expression of NF-κB target genes by quantitative real time PCR and found that IL-6 (2.5 fold; p≤ 0.05) and CXCL10 (IP10) (4 fold; p≤ 0.05) were upregulated in MWCL1A20-/A20+cells compared to WT. In summary, we have established a new WM cell line model which mimics the effect of the MYD88L265P mutation in combination with a heterozygous loss of the TNFAIP3 gene. We show that loss of TNFAIP3 results in a higher baseline and TLR-stimulated phosphorylation of NF-κB as well as in higher expression IL-6 and IP10. Overall, this cell line will be a useful tool to study biological consequences of TNFAIP3loss in WM patients as well as novel therapeutic strategies. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Abstract Abstract 4984 Background: Waldenström macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration, immunoglobulin M (IgM) monoclonal gammopathy and increased production of interleukin-6 (IL-6). Anemia occurs in a significant number of these patients and it is postulated that this occurs independently of tumor burden. Hepcidin is a hepatic peptide hormone that plays a central role in iron homeostasis and has been implicated in anemia of inflammation. Hepcidin binds to the cell-membrane iron exporter-ferroportin, causing its internalization and degradation, leading to iron deficiency anemia. The role of hepcidin in WM patients with anemia is not clearly understood. In this study we investigated the association between anemia, inflammation and hepcidin in WM patients with an eventual goal of providing improved treatment strategies for anemia in this complex set of patients. Patients and Methods: Fifty-nine WM patients [Female N=25, Median Age 70 (49-89), Hemoglobin (Hg) 11.1gm/dl, IgM 3420mg/dl, Beta-2 Microglobulin (β2M) 3.105mcg/ml and Bone Marrow Involvement 30%] and fifteen healthy patients [Female N=6, Median Age 36 (18-51)] were analyzed. The patients were divided into anemics (Hg <12, N=38) and non-anemics (Hg >12, N=21). The serum level of IL-6 was measured using Human IL-6 ELISA Kit II (BD Biosciences) and hepcidin using Hepcidin ELISA (DRG Instruments GmbH, Germany) with protocols supplied by the manufacturers. Tumor cells from patients with WM were sorted by either CD19 or CD138 expression, RNA extracted and hybridized to Affymetrix U133A 2.0 expression arrays. Gene expression analysis was performed using Agilent Genespring GX 11.0 software. Gene expression data was summarized using GCRMA and median normalized per gene. Statistical analyses were performed by utilizing student t tests and One-way ANOVA using PRISM software. Results: WM patients with anemia had significantly higher levels of IgM (4121 vs. 2845mg/dl, p=0.021), beta-2 microglobulin (3.77 vs. 2.72mcg/ml, p=0.009) and bone marrow involvement (41.45 vs. 27.14%) as opposed to non-anemics. IL-6 levels were significantly higher in anemics when compared to non-anemics and controls (6.486 vs. 1.952 vs. 1.716 pg/ml, p=0.0433). Further, in WM patients with anemia serum hepcidin levels were significantly elevated when compared to non-anemics (13.03 vs. 6.08ng/ml, p=0.0123). An additional comparison was made with severe anemics (Hg <10g/dl) to non-anemics and higher mean hepcidin levels were noted in the former (16.21 vs. 6.08 gm/dl, p=0.0057). Gene expression analysis was then performed on 12 patients identified as severe anemics (Hg < 10g/dl) or non-anemics (Hg > 12g/dl). Unsupervised clustering identified 2 major groups that separated anemics versus non-anemics. Further, unsupervised clustering segregated anemic patients with the largest IgM values, suggesting that the expression and subsequent clustering of genes was driven by the level of tumor burden in this patient cohort. Also, up-regulation of genes involved in the cell cycle and proliferation such as cyclin A2, cyclin B1, PCNA and BUB1B were identified in anemic WM patients. Conclusion: A statistically significant increase in tumor burden (increased IgM, β2M and bone marrow involvement) was observed in WM patients with anemia as compared to patients without. A statistically significant increase in serum IL-6 was noted in WM patients with anemia. Interestingly, the WM patients with anemia were also noted to have higher serum hepcidin levels. Taken together, our data suggests that increased tumor burden may promote increased IL-6 and subsequently results in increased hepcidin, which correlates with anemia in WM patients. Additional studies are required to determine the specific role of hepcidin in anemia of WM. Anemia causes significant morbidity in WM patients; therefore, understanding the mechanism of anemia is important in providing superior patient care. Disclosures: Fonseca: Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Onyx: Research Funding; Otsuka: Consultancy; Medtronic: Consultancy.

Abstract Background Waldenstrom macroglobulinemia (WM) is an IgM-associated lymphoplasmacytic lymphoma, first described over seven decades ago. Although WM is typically a disease of the elderly, with a median age at diagnosis of ~ 67 years, approximately 10% of patients are ≤50 years (y) of age at diagnosis. Data for young patients are sparse and the few available studies demonstrate inconsistent findings, with potential overestimation of survival owing to inclusion of patients with smoldering WM. In this case-control study, we evaluate outcomes, prognostic features and impact of changing therapies in a large cohort of young symptomatic WM patients compared to matched older patients seen over the course of the past five decades. Methods The medical records of all WM patients seen consecutively at Mayo Clinic from 01/1960 to 10/2013 were reviewed. Of 1181 patients, 140 (11.8%) were ≤ 50 y of age at diagnosis. A cohort of patients 65 y or older at diagnosis, matched 1:1 by the time of diagnosis, served as the control population. The patients were divided into 3 groups based on the timing of initiation of therapy: Group 1 (1960-1977), Group 2 (1978-1995) and Group 3 (1996-2013). The baseline characteristics were compared. Initiation of frontline therapy was used for all time-to-event analysis using the Kaplan Meier method. Results Younger patients were more likely to present with adenopathy and splenomegaly, have higher IgM levels and hyperviscosity symptoms (Table 1). The median follow-up from the frontline therapy was similar (10.7 y vs. 10 y for the control population). At the time of analysis, 91% of the deaths for the younger cases were WM-related compared to 58% in the control arm (p=0.0001). Younger patients had a better OS with a median disease-specific survival (DSS) of 15.6 y (95% CI: 13-21; 10-y OS of 77%) vs. 11 y (95% CI: 7.8-12; 10-y OS of 51%) for the older patient (p=0.0003). Among the young patients, there was no difference in the median DSS across the 3 groups (p=0.42). However, the median DSS for the control group incrementally improved (p=0.02) over the 3 time periods (Table 1). In the younger patients, no improvement in DSS was noted with the use of either frontline rituximab-based therapy compared to non-rituximab based regimens [median NR (95% CI: 7.6-NR) vs. 15.8 y (95% CI: 13-22) with other regimens, p=0.30], or frontline chlorambucil-based compared to non-chlorambucil based regimens [median 16 y (95% CI: 12-22) vs 15.6 y (95% CI: 12-NR) with other regimens, p=0.73]. In the control group, however, there was significant difference in DSS among patients who received frontline rituximab-based compared to non-rituximab based regimens [median NR (95% CI: 8.3-NR) vs 9.1 y (95% CI: 5.6-12) with other regimens, p=0.04], or frontline chlorambucil-based vs non-chlorambucil based regimens [8 y (95% CI: 5-12) vs 12.3 y (95% CI: 11-NR) in other regimens; p=0.001]. Conclusion Striking differences in presentation are evident in young WM patients compared to their older counterparts. The incorporation of rituximab to the previously existing anti-WM regimens and the transition to non-chlorambucil based regimens has resulted in substantial survival gains in the older WM population over the past five decades. However, such improvement in outcome has not yet been observed in the young patients. The majority of younger patients, despite a protracted disease course, succumb to their disease. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Reeder:Novartis: Research Funding; Celgene: Research Funding; BMS: Research Funding; Millennium: Research Funding. Kumar:Kesios: Consultancy; Janssen: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; AbbVie: Research Funding; BMS: Consultancy; Onyx: Consultancy, Research Funding; Glycomimetics: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dispenzieri:Alnylam: Research Funding; Celgene: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; pfizer: Research Funding; Jannsen: Research Funding. Kapoor:Celgene: Research Funding; Takeda: Research Funding; Amgen: Research Funding.

Abstract Background Bendamustine/rituximab (BR) and dexamethasone/rituximab/cyclophosphamide (DRC) combination therapy are acceptable options for the treatment of WM, both in the frontline or salvage setting. Prospective data, however, are only available in treatment-naïve (TN) WM patients, and no direct comparison between BR and DRC has been reported. We compared outcomes of BR and DRC in relapsed/refractory (R/R) and TN WM patients seen at a single institution. Methods Records of WM patients seen consecutively at Mayo Clinic from 01/2007 to 12/2014 were reviewed. The MYD88L265P status, as assessed by AS-PCR, was recorded when available. The data obtained from symptomatic patients treated with DRC or BR were analyzed. The time-to-event analyses were performed from DRC or BR therapy using the Kaplan-Meier method. We used the Consensus criteria (6th International Workshop) for response assessment. Results Of 160 WM patients, 60 patients received BR (73% in R/R setting) and 100 patients received DRC (50% in R/R setting). In the R/R population, BR was the 2nd line (range 2-11) therapy in 47% of patients, while DRC was 2nd line (range 2-8) in 58% of patients. Rituximab monotherapy was the only prior line of therapy in 8 (20%) patients in the BR group and 20 (40%) in the DRC group (p=0.66). Baseline characteristics were similar in patients treated with BR or DRC (Table 1). Six patients received both BR and DRC during their disease course and overlapped between the 2 cohorts. Among the previous untreated patients, the median IgM levels decreased from 3,785 mg/dL to 724 mg/dL (p=0.0001) at best response with BR, while it decreased from 4,130 mg/dL to 1,250 mg/dL (p=0.001) with DRC. The median time to best response was 6.1 (1-25) months in the BR group and 11 (0.5-47) months in the DRC group (p=0.13). For patients treated with BR, the overall response rate (ORR) was 93% [VGPR 29% (n=4), PR 57% (n=8), MR 7% (n=1)]. One patient (7%) had progressive disease. For patients treated with DRC, ORR was 96% [VGPR 17% (n=8), PR 70% (n=32), MR 9% (n=4)]. One patient (4%) achieved SD. Median follow up in the BR and DRC groups were similar (30 months). The 2-year progression-free survival (PFS) was 88% and 61% in the BR and DRC groups, respectively (p=0.08). The 2-year time-to-next therapy (TTNT) was 88% and 76% in the BR and DRC groups, respectively (p=0.35). Median PFS, TTNT and disease specific survival (DSS) were not reached with either regimen. In the R/R setting, the median IgM levels decreased from 3,880 mg/dL to 659 mg/dL (p=0.0001) at best response with BR, and from 3,870 mg/dL to 1,846 mg/dL (p=0.001) with DRC. The median time to best response was similar [7 (1-39) months with BR and 7 (0.5-28) months with DRC (p=0.77)]. For patients treated with BR, ORR was 95% [CR 3% (n=1), VGPR 38% (n=14), PR 41% (n=15), MR 13% (n=5)]. Two patients (5%) had progressive disease. With DRC, the ORR was 87% [VGPR 4% (n=2), PR 64% (n=30), MR 19% (n=9)]. Four patients (9%) achieved SD and 2 patients (4%) had progressive disease. Median follow up from BR was 32 months and 51 months from DRC (p=0.24). The 2-year PFS was 66% and 53% in the BR and DRC groups, respectively (p=0.08). The 2-year TTNT was 75% and 68% in the BR and DRC groups, respectively (p=0.24). The median DSS in the BR group was 69 months (95% CI: 65-69) and NR (95% CI: NR-NR) in the DRC group (p=0.57). The proportion of patients with R/R disease was significantly higher in the BR cohort (Table 1). In a bivariate analysis of the entire cohort for PFS, incorporating the setting (TN vs. RR) and the regimen involved (BR vs. DRC), the latter emerged as a significant factor (hazard ratio 0.52, p=0.019) in favor of BR. Time-to-event outcomes and response rates were similar in the MYD88L265P and MYD88WT patients. Grade ≥ 3 adverse events were neutropenia (12%), infections (7%) and nausea/vomiting (2%) with BR, and neutropenia (20%), thrombocytopenia (7%) and infections (3%) with DRC. Conclusions Although both DRC and BR regimens show activity and comparable toxicities, BR regimen shows a trend for superior PFS in WM patients, both in the TN and R/R setting. No difference was seen in TTNT. MYD88 L265P mutation status does not appear to impact activity of BR or DRC. Randomized controlled trial(s) are needed to confirm our findings. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Reeder:Millennium: Research Funding; BMS: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Dispenzieri:Jannsen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; pfizer: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Alnylam: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees. Kumar:Sanofi: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; AbbVie: Research Funding; Array BioPharma: Consultancy, Research Funding; BMS: Consultancy; Kesios: Consultancy; Glycomimetics: Consultancy; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Noxxon Pharma: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kapoor:Celgene: Research Funding; Takeda: Research Funding; Amgen: Research Funding.