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Abstract Background: Non-Hodgkin lymphomas (NHL) are increasing in incidence and are now the fifth most common tumor diagnosed each year in the United States. Most NHLs are of B-cell origin but the tumor tissue is variably infiltrated with T-cells. Our group has shown in diffuse B-cell large cell lymphoma, that a high number of intratumoral CD4+ T-cells predicts a better overall survival. It has been shown that recently-characterized CD4+CD25+ regulatory T-cells (Treg cells) played an important role in the mediation of anti-tumor immunity. However, there is no data on the role of intratumoral Treg cells in suppression of autologous infiltrating CD4+ T-cells in B-cell NHL. Goal: To investigate the effect of intratumoral Treg cells on the proliferation of tumor-infiltrating CD4+CD25- T-cells, to determine the underlying mechanism of the T-cell suppression, and evaluate the role that malignant B-cells may play in the recruitment of Treg cells to the site of B-cell NHL. Results: We identified a subset of CD4+CD25+ T-cells over-represented in biopsy specimens of B-cell NHL (these cells comprise 17% of cells in lymphoma biopsies, compared 7% of peripheral blood mononuclear cells, 12% of cells in inflammatory tonsil and 6% of cells in tumor free lymph nodes; p-value =0.001). These CD4+CD25+ T-cells are memory-like T-cells (CD45RO+ and CD45RA−) and express high levels of CTLA-4 and Foxp3 when compared to autologous tumor-infiltrating CD4+CD25- T-cells. Importantly, these CD4+CD25+ T-cells displayed the ability to suppress the proliferation and cytokine (IFN-g and IL-4) production of tumor-infiltrating CD4+CD25- T-cells in response to PHA stimulation. Treatment with anti-B7-H1 antibody or PD-1 fusion protein enhanced the proliferation of infiltrating CD4+CD25- T-cells when co-cultured with intratumoral CD4+CD25+ T-cells. Our results suggest that interaction between B7-H1 and PD-1 accounts for about 30% of intratumoral Treg cell-mediated inhibition of autologous infiltrating CD4+CD25- T-cells in tumor sites of B-cell NHL. Lastly, we found that CCL22 secreted by lymphoma B-cells is involved in the chemotaxis and migration of intratumoral CD4+CD25+ T-cells which express chemokine receptor CCR4, but not CCR8. Conclusion: Our results suggest that tumor microenvironmental CD4+CD25+ regulatory T-cells are important regulators of tumor immunity and that these cells are recruited to the area of lymphoma involvement by the malignant B-cells.

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DRlow monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68+ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14+ and CD14- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function. METHODS Human mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry. RESULTS Using a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14+ mo/mΦ were larger than CD14- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αdim and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αdim cells typically constituted the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. CD14-SIRP-α- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14+SIRP-αhigh and CD14-SIRP-αdim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14-SIRP-α- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues. CONCLUSIONS We have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14-SIRP-α- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Peripheral T-cell lymphomas (PTCLs) comprise a group of non-Hodgkin lymphomas (NHLs) of mature T-cell origin with generally aggressive clinical behavior. Most systemic PTCLs are treated with anthracycline-based combination chemotherapy; however, outcomes remain relatively poor for most subtypes except ALK-positive anaplastic large cell lymphoma (ALCL). We have used landmark analyses based on event-free survival (EFS) to identify clinically useful endpoints in B-cell NHLs, with EFS at 24 (EFS24) months identified to stratify overall survival (OS) in aggressive B-cell NHL. Here we examined the ability of EFS24 to predict subsequent OS in a large, multinational PTCL cohort. Methods: A cohort of newly diagnosed PTCL patients treated with curative intent combination chemotherapy regimens was assembled from the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), Swedish Lymphoma Registry (SWE), and British Columbia Cancer Agency (BCCA). Subtypes included ALK-negative ALCL, angioimmunoblastic T-cell lymphoma (AITL), PTCL, not otherwise specified (NOS), enteropathy-associated T-cell lymphoma (EATL), extranodal NK/T-cell lymphoma, nasal type (ENKTL), and hepatosplenic T-cell lymphoma (HSTCL). Patients were followed based on local institution guidelines, and EFS was defined as time from the date of pathologic diagnosis to progression, re-treatment, or death due to any cause. EFS24 was defined as being alive and event-free 24 months from diagnosis. Subsequent OS was defined as time from achieving EFS24 (24 months from diagnosis) or time from progression in patients failing to achieve EFS24 (progression within 24 months of diagnosis). OS was compared to the age-, sex-, and country-matched general population using United States, Sweden, and British Columbia rate tables via standardized mortality ratios (SMR) and expected survival. Results: 775 patients diagnosed from 2000-2012 were included in the combined analysis with diagnosis by the WHO classification at the respective institutions (MER=138, SWE=422, BCCA=215). Median age at diagnosis was 64 years (range 18-89) and 63% were male. Patient characteristics are summarized in the table. 736 (95%) received anthracyline-based therapy at diagnosis. At a median follow-up of 77 months (range 1-185), 516 patients (67%) had died. The percentage of patients achieving EFS24 was similar across the 3 cohorts (MER=39%, SWE=35%, BCCA= 36%, combined=36%). Median survival after progression within the first 24 months was only 4.9 months (95% CI: 3.8-5.9), with a 5-year OS of 11% and SMR of 46.4 (95% CI: 41.8-51.3). In contrast, median survival after achieving EFS24 was not reached, with a 5-year OS of 78% (95% CI: 73%-84%). This was inferior to the expected survival of 92% in the age-, sex-, and country matched population (SMR=3.16; 95% CI: 2.48-3.98). In EFS24 subgroup analyses, more favorable outcomes were observed in younger patients (age <=60 years, N=137, 5-year survival of 91% vs. 98% expected) and in patients receiving autologous stem cell transplant in first remission (N=72, 5-year survival of 88% vs. 96% expected). Patients achieving EFS24 who were not transplanted in first remission (N=189) had a 5-year survival of 74% vs. 90% expected. Conclusions: Assessment of EFS24 stratifies subsequent outcome in PTCL. Patients with early relapse from PTCL have extremely poor outcomes. However, over one-third of patients with PTCL remain in remission two years from diagnosis after initial chemotherapy and have encouraging OS rates, although survival remains significantly worse than the matched general population. Subset analysis suggests that younger patients and patients who receive autologous stem-cell transplant in first CR have 5-year survival rates that approach 90%, though still below the expected survival of the background population. The marked differences in OS after failing or achieving EFS24 in PTCL patients suggest that this endpoint may be useful for patient counseling, as a clinical trial endpoint, and as an endpoint to assess novel biomarkers for risk stratification. Table Table. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Maurer: Celgene: Research Funding; Kite Pharma: Research Funding. Jerkeman:Mundipharma: Research Funding; Amgen: Research Funding; Janssen: Research Funding; Gilead: Research Funding; Celgene: Research Funding. Connors:Seattle Genetics: Research Funding; Millennium Takeda: Research Funding; NanoString Technologies: Research Funding; F Hoffmann-La Roche: Research Funding; Bristol Myers Squib: Research Funding. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Abstract Background: Numerous studies have shown that lymphoma B-cells are resistant to CTL-mediated death, however the underlying mechanism for this resistance is not clear. In previous work, we have identified a subset of CD4+CD25+ T-cells overrepresented in the tumor sites of B-cell NHL that display a phenotype compatible with regulatory T cells (Treg cells). These cells express high levels of Foxp3 and CTLA-4, and are capable of suppressing the proliferation and cytokine production of autologous infiltrating CD4+CD25- T cells in B-cell NHL. Goal: To explore whether Treg cells exert a suppressive effect on the induction and function of autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. Results: Using fresh specimens obtained from patients with B-cell lymphoma, we found that intratumoral Treg cells had a significantly suppressive effect on autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. When activated CD8+ T-cells were cocultured with infiltrating CD4+CD25- T-cells, they displayed less proliferation than CD8+ T-cells cultured alone. However, we found that with a 1:4 ratio of stimulating cells (Treg cells) to responding cells (CD8+ T-cells), intratumoral Treg cells completely inhibited the proliferation of autologous tumor-infiltrating CD8+ T-cells activated with PHA. Furthermore, we have observed that 20% of infiltrating CD8+ T-cells in fresh isolated samples from B-cell NHL coexpress perforin and granzyme B. When intratumoral Treg cells were cocultured with CD8+ T-cells, the Treg cells inhibited the activation-induced production of perforin and granzyme B of autologous tumor-infiltrating CD8+ T-cells in a dose-dependent fashion. We also found that cytokine treatment (IL-2 and IL-12) or PHA activation induced the capability of tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells, which was accompanied by upregulation of expression of CD107a on the surface of cytotoxic CD8+ T-cells. Intratumoral Treg cells significantly inhibit cytotoxicity of activated infiltrating CD8+ T-cells to lymphoma B-cells. Finally, we found that there was an inverse correlation of cell frequencies between intratumoral Treg cells and tumor-infiltrating CD8+ T cells in patients with B-cell NHL, suggesting that Treg cells may inhibit the migration of cytotoxic T-cells to the sites of B-cell lymphoma. Conclusion: Intratumoral Treg cells dramatically suppress the proliferation and activation-induced production of granules in infiltrating CD8+ T-cells in B-cell NHL. These Treg cells also inhibit the ability of activated tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells in vitro, and may prevent the migration of CD8+ cells to the sites of lymphoma. Taken together, these data indicate an important role for intratumoral Treg cells in CTL-mediated anti-tumor immunity in B-cell NHL.

The tumor microenvironment plays an important role in regulating malignant cell growth and mechanisms to enhance anti-tumor immune function have been shown to improve patient outcome. Interactions between programmed death 1 (PD-1) and its ligands (PD-L1 and PD-L2) have been shown to be an important checkpoint in immune regulation. While it is well known that PD-1 is expressed on normal T cells and signaling through PD-1 inhibits T cell function, PD-1 is also expressed on a subset of B-cells but little is known about PD-1 signaling in B-cells. The goal of this study was to determine if the PD-1 is expressed on malignant B cells in Waldenstrom macroglobulinemia (WM) and whether this pathway plays a role in the survival and growth of malignant B cells in this B cell lymphoma. Using flow cytometry, we found that the cell lines MWCL-1, BCWM.1 and RPCI, all derived from patients with Waldenstrom macroglobulinemia, expressed PD-1 to varying degrees on their cell surface. PD-1 expression in the cell lines was further confirmed by RT PCR analysis. Using flow cytometry and immunohistochemistry to examine bone marrow specimens from WM patients, we further confirmed PD-1 expression on CD19+ CD138+ malignant B-cells. Furthermore, intense staining for the ligands PD-L1 and PD-L2 was found by in bone marrows of WM patients when compared to normal bone marrow specimens. When WM cell lines are co-cultured with stromal cells engineered to express PD-L1 or PD-L2, there was a consistent increase in cell viability compared to controls. When malignant B cells from WM patients were co-cultured with stromal cells expressing the ligands, viability was unchanged but there was an increase in cell proliferation, most noticeably when cocultured with cells expressing PD-L2. To determine potential mechanisms that account for upregulation of PD-1 on malignant B-cells, we tested whether cytokines that promote WM cell growth and survival, including IL-6, IL-21 and BAFF, increased PD-1 expression. We found that WM cell lines and patient derived CD19+CD138+WM B-cells (n=4) treated with IL-21 demonstrated an increase in PD-1 expression compared to untreated controls. We conclude that PD-1 is expressed on malignant B-cells in WM and that signaling through PD-1 may promote WM cell growth and survival. Blocking PD-1/PD ligand interactions may therefore be a potential therapeutic strategy in patients with Waldenstrom macroglobulinemia. Disclosures No relevant conflicts of interest to declare.

Abstract Waldenströms Macroglobulinemia (WM) is a unique form of lymphoplasmacytic lymphoma, in which the tumor compartment is comprised of B-lymphocytes, plasmacytoid lymphocytes and plasma cells. The majority of WM cases are immunophenotypically characterized by expression of surface antigens that are present on both B-cells (CD19/20) and plasma cells (CD38/138). However, in advanced stage disease, which has been treated with various chemoimmunotherapeutics the molecular profile of WM cells can drastically shift resulting in a majority of the WM tumor compartment exhibiting loss of CD20 and concurrent upregulation of atypical surface antigens (Barakat et al, Am J Clin Pathol 2011; Morice et al Mod Pathol 2009). These external immunophenotypic changes are associated with underlying epigenetic and genomic alterations, whose integrated study may provide further insight into the biology of advanced stage WM. Moreover, such an analysis holds clinical potential by uncovering of therapeutic vulnerabilities, which can direct the selection of appropriate therapeutics for patients with CD20- WM. With this in mind, we conducted comprehensive molecular testing coupled with functional studies to understand if underlying genomic/epigenetic changes correlated with drug sensitivity using appropriate preclinical models of CD20+ WM (BCWM.1 cell line) and CD20- WM (RPCI-WM1 cell line). Importantly, the index patient from whom the CD20- cells were developed had received a total of 6 lines of treatment, two of which contained rituximab and was documented as being refractory to rituximab. Immunophenotyping of CD20- cells showed differential downregulation of 19 and upregulation of 7 CD antigens relative to CD20+ WM cells (cutoff at 1.5 fold; >20% gated expression). Upregulated antigens were associated with a phenotype reminiscent of a memory B-cell with plasmacytoid features. Next, we conducted RNA-Seq analysis (Illumina HiSeq), which revealed downregulation of 6,673 and upregulation of 4,594 genes (>1.5 fold cutoff) in CD20- WM cells compared with CD20+ cells. Gene pathway enrichment analysis (NextBio, Illumina Inc.) of mRNA differentially expressed in CD20- cells showed significant enrichment of upregulated genes corresponding to Protein Transport (p<1.8E-98) and Response to Unfolded Protein Gene Ontology groups (p<5.9E-20). Interestingly, pathway analysis of downregulated genes in CD20- WM cells showed enrichment within the B-cell receptor-signaling (BCR) pathway (Broad MSigDB) (p<6.1E-14) relative to CD20+ WM cells. BCR signaling has been linked to dysregulated expression of several micro-RNAs (miRs). Consistent with pathway analysis results, we observed decreased levels of numerous miRs (Exiqon qPCR) in CD20- vs. CD20+ WM cells, which target and regulate components involved in BCR activation. To examine further epigenetic changes that may account for decreased BCR signaling, we examined the global methylation profile (Infinium HumanMethylation 450K array) of CD20- and CD20+ WM cells and noted hypermethylation of the promoter sites (200 base pairs upstream) for a number of transcription factors involved in the BCR pathway. We therefore examined sensitivity of CD20- WM cells to the BTK inhibitor ibrutinib, which abrogates BCR signaling. As anticipated, CD20- WM cells retained >90% viability and demonstrated significantly less apoptosis in presence of ibrutinib (4uM) compared to CD20+ cells, suggesting decreased reliance on canonical BCR signaling for survival. In parallel and guided by pathway analysis results, we exposed both CD20- and CD20+ WM cells to a novel 19S proteasome inhibitor (VLX1570), which caused profound proteotoxicity and ER stress at concentrations ranging between 250 – 500nM. We noted CD20- cells were significantly more sensitive (p<0.01) to 19S proteasome inhibition than CD20+ WM cells indicating the formers critical dependence on tightly regulated protein homeostasis. In summary we describe for the first time, a comprehensive molecular and immunophenotypic investigation of CD20- WM cells compared to CD20+ WM cells, using validated preclinical models of the two disease phenotypes. Overall our data suggests significant architectural differences between CD20+ and CD20- WM, which can potentially impact the behavior of the cells and their sensitivity towards novel therapeutics being developed for treatment of advanced stage WM. Disclosures Martin: Janssen: Honoraria.

Abstract 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.

Abstract Waldenstrom’s macroglobulinemia (WM) is a serious and frequently fatal disorder characterized by the production of a monoclonal IgM protein, a lymphoplasmacytic infiltrate in the bone marrow, and associated symptoms including anemia, lymphadenopathy and hyperviscosity. Many of the mechanisms leading to this disease are not yet known. It is clear, however, that there is dysregulation of the balance between cell proliferation and programmed cell death. BLyS (B-lymphocyte stimulator) is a TNF family member expressed by monocytes, neutrophils, macrophages, and dendritic cells. BLyS has been shown to be critical for maintenance of normal B cell development and homeostasis, and has been found to stimulate lymphocyte growth. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B-cells. Studies of the effects of BLyS on B cell physiology have shown that it also regulates immunoglobulin secretion. In previous work, we have shown that malignant B cells from patients with WM are able to bind soluble BLyS and variably express the BLyS receptors, BAFF-R, TACI and BCMA. We also found expression of BLyS in bone marrow specimens by immunohistochemistry and elevated serum BLyS levels in patients with WM. The goal of this study was to determine the functional role of BLyS-receptor ligand system in Waldenstrom’s macroglobulinemia and its relevance to the increased immunoglobulin production seen in this disease. Using cells from WM patients, we first examined the ability of BLyS to increase the secretion of IgM by malignant B cells. BLyS, alone or in combination with cytokines that induce plasmacytic differentiation and immunoglobulin production (IL-2, IL-6, IL-10 and IL-12), was found to increase IgM secretion by malignant B cells. Mean baseline IgM levels significantly increased in cells treated with BLyS (p=0.03), cytokines (p=0.0002) and a combination of BLyS and cytokines (p<0.0001). We then determined the effect of BLyS on the survival of malignant B cells using Annexin-V/PI staining. Compared to cells cultured in media alone, BLyS was found to increase viability of malignant B cells from WM patients. Cell viability was normalized relative to the media-alone control and the median relative viability increased significantly compared to controls (median increase 41.2%; range 8 – 46%). Next, we examined the ability of BLyS to modulate cell proliferation using thymidine incorporation. Using WM patient samples, BLyS was found to significantly enhance the proliferation of malignant B cells (p=0.0002). Furthermore, addition of anti-Ig antibody further enhanced the ability of BLyS to promote the proliferation of malignant B cells (p<0.0001). In summary, we have demonstrated that BLyS enhances IgM secretion by malignant B cells from patients with Waldenstrom’s macroglobulinemia. We have also demonstrated the ability of BLyS to enhance the survival and proliferation of malignant B cells. Strategies to inhibit BLyS may potentially have therapeutic efficacy in Waldenstrom’s macroglobulinemia.

Abstract Background: Adult T-cell acute lymphoblastic leukemia / lymphoma (T-ALL) is a rare and aggressive hematological malignancy. Unlike B-cell ALL, apart from age and presenting WBC, additional prognostic factors remain largely unknown. Additionally, the prognostic impact of a monosomal karyotype, which has a negative impact on survival in myeloid neoplasms, remains to be determined in T-ALL (Kenderian et al, BCJ 2013) Aim: To study predictors of survival including the role of a monosomal karyotype in T-ALL. Methods: After due IRB approval, adults diagnosed with T-ALL from 1990-2014 at Mayo Clinic Rochester were identified. All clinical and pathologic data including FISH studies and cytogenetics were retrospectively reviewed. Chi-square test was used to compare variables. Survival was estimated and compared using the Kaplan-Meier Method and log-rank test. Univariate and multivariate analysis was performed using the Cox regression model. Results: Between 1990 and 2014, a total of 92 consecutive patients (pts) with T-ALL were identified, 41 had acute lymphoblastic lymphoma (LBL), and 51 (55%) acute lymphoblastic leukemia (ALL). Median age at diagnosis was 33 years (range; 18-88 years) with 72%males. Median follow up of our cohort was 25 months (range; 0.9-260 months) during which time 42 deaths (45%) were documented. A. Baseline characteristics: Fifty six (67%) had constitutional symptoms, and 63 (77%) had lymphadenopathy at diagnosis. Mediastinal mass was identified in 49 (55%) pts with palpable splenomegaly in 16 (20%) pts. Fifty one (58%) pts had extranodal disease, and 17 (19%) pts had CNS involvement. Pleural and pericardial effusion were evident in 16 (19%), and 6 (7%) pts, respectively. Median laboratory values included; LDH 288 U/L (115-6590), hemoglobin (Hb) 12.8 g/dl (4.5-18.2), WBC 8.65 x109 (1.4-406), platelets 169 x109 (8-485), bone marrow blasts 29% (0-99). B. Treatments: Induction therapies were variable and included; 11 (12%) CHOP like regimens, 7 (8%) modified NHL regimens (Asparginase+CHOP), 54 (58%) ALL like regimens (CALGB 9111, E2993, or HyperCVAD), 13 (14%) Pediatric regimens (ALL 10403, CCG 1961, BFM, Augmented BFM, GRAALL 2003, or Hoelzer regimen), with 7 (8%) patients' receiving palliative care only. Seventy-one (77%) pts achieved complete remission (CR1) during induction therapy while eight pts had refractory disease (8%). Thirty-three (35%) pts underwent stem cell transplant; 7 (21%) autologous and 26 (79%) allogeneic. Forty one (44%) pts had disease relapse and median time to relapse was 10.8 months (range; 8.2-16 months) C. Predictors of survival: In univariate analysis, that included age > 60 years, leukocyte count Hb, Plt, Blast percentage, mediastinal mass, LDH and CNS involvement, only age >60 and CNS involvement were predictors of inferior survival and both retained significance in the multivariable analysis with HR 3.9 (CI 1.5 - 9; p=0.0055) and 2.8 (1.3 - 5.7; p =0.0085), respectively (Figure 1 and 2). In this cohort age >60 was a better stratification that the conventionally used age above 40 (p = 0.1). Among 56 pts with evaluable cytogenetics data, 7 (13%) pts had MK which did not have an impact on survival with HR 1.9 (CI 0.4-5.6; p=0.33). Conclusion: In this large cohort of adult T-ALL patients, we observed that age >60 years and CNS involvement were independent predictors of inferior survival. MK was infrequent, but unlike in myeloid neoplasms, does not seem to impact overall survival. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Celgene: Research Funding. Thompson:Kite Pharma: Research Funding. Witzig:Novartis: Research Funding; Celgene: Research Funding; Amgen: Honoraria; Spectrum: Research Funding; Valeant Pharma: Equity Ownership.

Abstract Background: Acute T-cell lymphoblastic leukemia (T-ALL)/ lymphoma (T-LBL) are immature T-cell malignancies with very poor outcomes. In patients with relapsed disease, survival is often < 12 months (Marks et al, Blood, 2009). Prognostic correlates and treatment outcomes after relapse remain largely unknown. Aims: To evaluate prognostic correlates and treatment outcomes in patients with relapsed T-ALL/LBL. Methods: After IRB approval, 92 consecutive patients with T-cell ALL/LBL were identified. 41 patients with relapsed disease after achieving a first complete remission (CR1) were included in the study group. Features at the time of relapse were retrospectively abstracted and analyzed. Survival time after relapse was calculated from the time of relapse until death or last follow-up. Conventional methods were used for statistical analysis. Results: A. Baseline characteristics of all patients 92 patients were identified [41 LBL, 51 ALL]. Median age was 33 years (range; 18-88) and 66 (72%) were males. Median follow-up was 25 months (range 0.9-260). 13(14%) received palliative care or were lost to follow-up before treatment initiation. At last follow-up, there were 41 (52%, n = 79) relapses and 42 (46%, n = 92) deaths. Of 79 patients, 71 (90%) achieved a CR1 and 8 (10%) had primary refractory disease. Comparisons between ALL and LBL revealed expected differences in presenting features such as frequency of primary mediastinal mass at presentation in LBL and high leukocyte count in ALL. Significant differences in treatment were seen. Asparaginase-based and ALL-directed therapy were more common in the ALL group (71% vs. 45%, p = 0.009; 88% vs. 71%, p = 0.04). 13 (81%) LBL and 14 (27%) ALL underwent allogeneic SCT. The remaining three in each group underwent autologous SCT in CR1. B. Characteristics of relapsed patients 41 relapsed after achieving CR1 [23 LBL, 18 ALL]. Median time to relapse was 8.0 months (range 2.3-130), with no difference between ALL and LBL groups. Median age was 34 years (range 19-71) and 29 (71%) were males. Median follow-up after relapse was 7.2 months (range 0.3-207). At time of last follow-up, there were 30 (73%) deaths, 25 (61%) achieved a second complete remission (CR2) and 14 (34%) had persistent disease. 12 (48%) had relapse following CR2. 34 (83%) relapsed following CR1 after induction chemotherapy alone. 6 (15%) and 1 (2%) relapsed following allogeneic and autologous SCT respectively. 27 (66%) had BM involvement, 5 (12%) had CNS involvement, 13 (32%) had mediastinal involvement, and 3 (7%) had isolated extramedullary relapse. Ten (43%) patients with LBL relapsed with ALL. Of 27 patients with available data, 9 (33%) had high-risk cytogenetics at relapse, defined as ≥ 5 chromosomal abnormalities and/or markers known to confer an unfavorable prognosis. 16 of these 27 patients had follow-up cytogenetic studies performed, of which 8 (50%) had evidence of clonal evolution. Patients were treated with up to five regimens. 12 (31%) received asparaginase-based therapy and 13 (33%) received nelarabine with CR2 rates of 75% and 25% respectively. Median number of regimens needed to achieve CR2 was one. 18 (46%) went onto SCT following CR2: 14 (allogeneic), 3 (autologous), and 1 (donor lymphocyte infusion). The majority received myeloablative conditioning (92%). C. Outcomes and prognostic correlates for survival after relapse Median survival following relapse was 8.2 months (IQR 4.1-21.3) and did not differ between LBL and ALL. Nine (36%) who achieved CR2 are alive at time of last follow-up. 25 (61%) achieved CR2, but 12 (48%) relapsed following CR2, with median time to second relapse of 7.6 months (IQR 4.7-14). Nine of 18 who underwent SCT following CR2 had disease relapse (8 allogeneic, 1 autologous). Only BM involvement at relapse significantly correlated with post-relapse survival (HR 2.4 [1.1, 5.1], p = 0.03). Clonal evolution, high-risk cytogenetics, nelarabine therapy, achieving CR2, and SCT were not independent predictors of survival in our small subset of patients. Conclusion: Patients with relapsed T-ALL/LBL have dismal outcomes, in spite of advances in therapy with newer agents such as nelarabine and the use of allogeneic SCT. Regardless of T-ALL/LBL subtype, BM involvement at relapse appears to be a significant factor negatively impacting post-relapse survival. Further studies are needed to validate these findings. Disclosures Al-Kali: Celgene: Research Funding. Thompson:Kite Pharma: Research Funding.