ALBERT

Description: Abstract 770 Background: Waldenstrom's macroglobulinemia (WM) is a B-cell lymphoma characterized by high serum monoclonal IgM and infiltration of lymphoplasmacytic cells into the bone marrow. As with many hematologic malignancies, cytokines within the tumor microenvironment play an important role in supporting the growth and survival of malignant WM cells. IL-21 is a pleiotropic cytokine involved in the differentiation of B cells into plasma cells. During malignancy, IL-21 has demonstrated diverse effects promoting the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in WM. Our objective here was to assess the expression of IL-21 and its receptor in WM cells and to examine whether IL-21 contributes to the biology of WM. Results: When compared to normal bone marrows, immunohistochemistry revealed significant IL-21 staining in the bone marrow of patients with WM (n=5). To determine whether WM cells are susceptible to IL-21 in the microenvironment, expression of the IL-21 receptor (IL-21R) was assessed via PCR in CD19+CD138+ cells isolated by positive selection from patients with WM (n=8) and in the newly characterized WM cell line, MWCL-1. Nearly all (7/8) CD19+CD138+ WM cells expressed IL-21R, as did MWCL-1 cells. Using flow cytometry we detected expression of IL-21R protein on the surface of WM cells as well. The contribution of microenvironmental IL-21 to the biology of WM tumors was then examined. In MWCL-1 cells, IL-21 (100 ng/mL) increased proliferation by 37% (p=0.005) over untreated controls as determined by thymidine incorporation at 72 hr, and in primary WM cells, proliferation increased by nearly 50% (p=0.003). Interestingly, the immortalized B cell line, IM-9, responded to IL-21 with a significant decrease in proliferation, consistent with previous data indicating differential effects of IL-21 depending on the pathological status of the B-cell in question. IL-21 also significantly induced (p

Description: Abstract 2699 Myeloid differentiation factor 88 (MYD88) is an adaptor protein that plays a key role in mediating innate immune response. Upon activation of toll-like (TLRs) or IL-1b receptors, MYD88 activates a signaling cascade that culminates in activation of NF-kB and expression of proinflammatory genes. Activating mutations in MYD88 have been reported in non-Hodgkin's lymphoma (NHL) tumors, including diffuse large B cell and marginal zones lymphoma. The most common MYD88 mutation described thus far is a single base pair mismatch resulting in an amino acid switch from lysine to proline at position 265 (MYD88L265P). MYD88L265P functions as a constitutively active MYD88 protein resulting in dysregulated NF-kB and STAT3 signaling. This mutation has recently been reported to be present in lymphomoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM) at a high frequency, ∼ 90% (Xu et al, ASH, #261, 2011). The discovery of MYD88L265P in a high percentage of LPL/WM tumors, and other subtypes of NHL, provides the basis for the identification and targeting of MYD88 dependent pathways. Additionally, the ability of this mutation to drive NF-kB-mediated cytokine expression combined with our finding that LPL/WM patients have elevated serum cytokines suggests that the MYD88 mutation may play a significant role in LPL/WM pathogenesis. We first wanted to confirm the incidence of the MYD88L265P mutation in LPL/WM and other subtypes of indolent lymphoma. Using Sanger sequencing we found that 57% (n=58) of LPL, 4% of MALT (n=23), 5% of nodal marginal zone (n=34), and 6% of splenic marginal zone (n=34) lymphomas harbor the MYD88L265P mutation, with all but 1 case being heterozygous. When we specifically looked at the LPL cases with WM, we found that 70% of WM tumors have the MYD88L265P mutation. In a validation cohort of WM (n=41), we found a similar mutation rate of 68%. Additionally, the WM cell line MWCL1 and the IgM secreting cell line BCWM.1 also harbor the MYD88L265P mutation. The prevalence of the MYD88L265P mutation in NHL, specifically WM and LPL, suggests that it may be a novel driver of lymphoma pathogenesis and a potential therapeutic target. However, it is not yet clear if this mutation correlates with activation of signaling pathways, or cell growth and survival in LPL/WM. To address these questions we first explored the possibility that the MYD88 signaling pathway is constitutively active in cells that express MYD88L265P. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodiamer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-kB. Using three cell lines that express MYD88L265P (BCWM, MWCL1, and OCI-Ly3), we looked for complex formation of MYD88, IRAK1/4, TRAF6, and TAK1. Immunoprecipitation of either endogenous IRAK1 or TAK1 revealed constitutive association with both TRAF6 and MYD88. We also detected phosphorylation of TAK1, and taken together, these data suggest that the MYD88 pathway is activated in cells that harbor the MYD88L265P mutation. We next wanted to confirm the significance of the MYD88 pathway on lymphoma cell growth. Using our cell lines we tested the effect of IRAK1/4, TAK1, and NF-kB inhibitors on cell proliferation. All MYD88L265P expressing cell lines were sensitive to the TAK1 and NF-kB inhibitors in a dose dependent manner (0–10 uM), however, we only saw inhibition of cell proliferation with the IRAK1/4 inhibitor at the highest dose (10 uM) in the BCWM cell line. These data suggest that cell proliferation is dependent on the MYD88 signaling pathway, with some variation being detected between cell line models and inhibitors. Because the MYD88 pathway has also been shown to drive cytokine secretion we next tested the impact of the IRAK1/4, TAK1, and NF-kB inhibitors on expression of IL-10. Using drug doses that were shown to inhibit cell proliferation, but not impact survival, we found that all three inhibitors reduced the level of IL-10 secreted by each of cell lines. In summary, our data confirm the high incidence on the MYD88L265P mutation in LPL/WM, we find that the MYD88 signaling pathway is constitutively activated in cells the harbor the mutation, and we show that cell proliferation and cytokine secretion are dependent on MYD88 signaling targets. These studies highlight the biologic significance of the MYD88L265P mutation in LPL/WM and suggest that the MYD88 signaling pathway may be a novel therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points STAT5 is constitutively phosphorylated in malignant B cells obtained from patients with Waldenström's macroglobulinemia. Inhibition of STAT5 signaling significantly decreases IgM production and may be useful therapeutically for patients with high IgM levels.

Description: True long-term nonprogressors (LTNPs)/elite controllers (ECs) maintain durable control over HIV replication without antiretroviral therapy. Herein we describe 4 unique persons who were distinct from conventional LTNPs/ECs in that they had extraordinarily low HIV burdens and comparatively weak immune responses. As a group, typical LTNPs/ECs have unequivocally reactive HIV-1 Western blots, viral loads below the lower threshold of clinical assays, low levels of persistent viral reservoirs, an over-representation of protective HLA alleles, and robust HIV-specific CD8+ T-cell responses. The 4 unique cases were distinguished from typical LTNPs/ECs based on weakly reactive Western blots, undetectable plasma viremia by a single copy assay, extremely low to undetectable HIV DNA levels, and difficult to isolate replication-competent virus. All 4 had at least one protective HLA allele and CD8+ T-cell responses that were disproportionately high for the low antigen levels but comparatively lower than those of typical LTNPs/ECs. These unique persons exhibit extraordinary suppression over HIV replication, therefore, higher-level control than has been demonstrated in previous studies of LTNPs/ECs. Additional insight into the full spectrum of immune-mediated suppression over HIV replication may enhance our understanding of the associated mechanisms, which should inform the design of efficacious HIV vaccines and immunotherapies.

Description: Abstract 9 Introduction: The rate of complications after surgery is increased in patients with Sickle Cell Disease (SCD) and pre-operative blood transfusion has historically been used to decrease this risk. Observational studies and one limited Randomised Controlled Trial (RCT) have suggested that in some patients, transfusion can safely be omitted. Since transfusion is associated with complications including alloimmunisation and increased post-operative infections, we performed a RCT to address whether overall peri-operative complications in SCD are reduced by pre-operative transfusion. Methods: TAPS was a Phase III multicentre, pragmatic, randomised controlled trial with a parallel group sequential superiority design, carried out between November 2007 and March 2011 at 22 sites in the UK, Netherlands and Canada. Eligible patients had HbSS or HbSβ°thal, were aged one year or more and were having low risk (eg adenoidectomy, dental surgery) or medium risk (eg joint replacement, cholecystectomy, tonsillectomy) elective surgery. Patients were excluded if they had a haemoglobin (Hb)

Description: While extensive, the genetic studies of DLBCL tumors to date have primarily focused on somatic mutations that are associated with development of lymphoma, i.e., driver mutations. Identification of key genetic events that impact and/or predict response to therapy and clinical outcome of lymphoma patients has not been accomplished. In an exploratory study, we used whole-exome sequencing to identify novel biomarkers that can predict which patients have an aggressive form of DLBCL, and therefore would likely benefit from a different and more aggressive treatment plan. Using whole-exome sequencing data from 54 newly diagnosed DLBCL patients, we evaluated the association of somatic coding single nucleotide (cSNV) and copy number (CNV) variants with aggressive DLBCL. All patients were treated with R-CHOP or immunochemotherapy, and disease aggressiveness was based on relapse, with patients classified as having aggressive disease (AD) (n=13, defined as treatment failure, relapse, or progression within 24 months of diagnosis) versus non-aggressive disease (n=41, defined as achieving at least 24 months of relapse-free survival). An in-house algorithm, patternCNV was used to detect somatic CNVs. Logistic regression was used to assess statistical significance. In the cSNV analysis, 24 genes were found to be significantly (p ≤ 0.05) associated with AD. Of these genes, CIITA (p=0.01) was previously identified as a mutational target in DLBCL and is a recurrent gene fusion partner in lymphoid cancers. Metacore analyses showed that seven of these genes, including CIITA, can be placed in the same regulatory network centered around CREB1. Interestingly, the CREB binding protein (CREBBP) is a known target of pathogenic mutations in DLBCL. In the CNV analyses, we identified 245 gene amplifications and 209 gene deletions associated with AD (p ≤ 0.05). Deleted genes were localized in three major chromosomal regions, 6p22.3-6p22.1 (26 genes), 6q13-6q16.3 (31 genes), and 6q21-6q24.2 (86 genes). In the CNV amplification analysis, the genes were localized in two major chromosomal regions, 3q12.3-3q29 (90 genes) and 19q13.12-19q13.43 (122 genes). We also quantified the association of each regional deletion/amplification with patient outcome. As expected, each deleted region was univariately associated with time to relapse: 6p22.3-6p22.1 (HR = 7.19, 95% CI: 2.66-19.46, p=1.33x10-5), 6q13-6q16.3 (HR = 4.44, 95% CI: 1.74-11.32, p=6.84x10-4), and 6q21-6q24.2 (HR = 5.42, 95% CI: 2.12-13.87, p=9.23x10-5) as well as the two amplification regions: 3q12.3-3q29 (HR = 4.60, 95% CI: 1.49-14.24, p=4.27x10-3) and 19q13.12 -19q13 (HR = 13.79, 95% CI: 4.39-43.34, p=2.81x10-8). Deletions at 6q21-6q24.2 have been previously reported in DLBCL and two tumor suppressor genes associated with lymphoma pathogenesis are localized in the region, TNFAIP3 (p=0.14) and PRDM1 (p=0.02), the latter of which was significantly associated with AD. However, the genes in the 6p21 locus that were most significant (p=0.002) were SLC22A16, GPR6, SOBP, MATTL24, DDO, and REV3L. SLC22A16, which also had cSNVs in two tumors, is an organic cation transporter that has been shown to transport chemotherapeutic drugs including doxorubicin. Successful drug response has been correlated with the level of activity and expression of this transporter in tumor cells. To determine if expression of SLC22A16 would have an impact on doxorubicin transport, and may therefore impact response to R-CHOP we overexpressed either an empty vector control or SLC22A16 in OCI-Ly7 DLBCL cells, which do not express endogenous SLC22A16 mRNA. We found that OCI-Ly7 cells expressing SLC22A16 have increased 14C-doxorubicin uptake and are more sensitive to doxorubicin-induced cell death when compared to the empty vector control cells. These data suggest that loss of SLC22A16 expression through gene deletion could impact the ability of DLBCL cells to respond to therapy and indicates an aggressive form of the disease. In summary, our data are the first to use whole exome sequencing to identify genetic variants associated with aggressive DLBCL, which will require replication in additional samples. Nevertheless, our data highlight the role for somatic CNVs in patient outcome and we biologically validate the potential impact of deletions in SLC22A16 at 6p21. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2688 Members of the signal transducers and activators of transcription (STAT) family of proteins function as secondary messengers mediating cellular responses to various cytokines. Aberrant activation of STAT5 protein has been implicated in the pathogenesis of hematologic malignancies due to the ability of these transcription factors to regulate genes involved in cellular proliferation and survival. The two highly homologous transcription factors collectively known as STAT5, namely STAT5A and STAT5B, display both redundant and distinct physiologic functions in non-malignant B cells. Yet, despite evidence suggesting independent functions for STAT5A and STAT5B in the pathophysiology of solid tumors, the precise roles of these isoforms in hematologic malignancies such as Waldenstrom's macroglobulinemia (WM) are not known. Initial immunohistochemical staining of WM bone marrow biopsies (n=6) revealed higher levels of STAT5 phosphorylation compared to bone marrows from normal controls (n=6). Additionally, phosphorylated STAT5 was highly expressed in CD19/CD138+ sorted cells obtained from the bone marrow of patients with WM (average ΔMFI compared to isotype control, 4.64 +/− 1.7, n=6) as determined by FACS analysis. Furthermore, western blotting demonstrated baseline STAT5 phosphorylation in both WM and IgM-secreting cell lines (MWCL-1 and BCWM-1, respectfully). The expression of other phosphorylated STAT proteins (1, 3, 4, and 6) was not detected. To better understand the overall role of STAT5 biologically in WM, MWCL-1 and BCWM.1 cells were treated with a specific inhibitor of STAT5, N′-((4-Oxo-4H-chromen-3-yl)methylene) nicotinohydrazide. Following 72 hours in culture, this inhibitor significantly decreased IgM secretion at lower doses when compared to controls (10 μM, MWCL-1: 9413 +/− 511 ng/mL vs. 6166 +/− 160 ng/mL, p

Description: Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p

Description: Programmed death 1 (PD-1) is a T cell co-inhibitory receptor with two ligands, PD-L1 and PD-L2. In B-cell malignancies, this pathway plays a major role in immune resistance in the tumor environment by promoting the differentiation of regulatory T-cells and inducing T-cell exhaustion. Recent data have shown that blockade of this pathway can enhance antitumor immune responses. While interactions between PD-1 and its ligands have been studied in various lymphoid tissues, little data exist regarding these interactions in the bone marrow, particularly in patients with Waldenstrom macroglobulinemia (WM). The goal of this study was therefore to determine the expression of PD-1 and its ligands in the bone marrow of patients with WM and to assess the functional role of PD-1 signaling in this disease. Using immunohistochemistry, the expression of PD-1, PD-L1 and PD-L2 in bone marrow (BM) specimens from patients with WM (n=6) was compared to normal control BM. PD-1 expression was modest and the pattern of staining in WM was similar to normal BM. In contrast however, intense staining for PD-L1 and PD-L2 was seen in WM compared to controls. When analyzed by flow cytometry, PD-L1 and PD-L2 expression was seen on malignant B-cells and also on dendritic cells, monocytes and macrophages isolated from the bone marrow. To determine the functional effect of PD-1 signaling in WM, we stably transfected PD-L1 and PD-L2 into the HEK-293 cell line. The WM cell lines, MWCL-1, BCWM.1 and RPCI-WM1 as well as patient specimens, were then cocultured with either the PD-L1 or PD-L2 expressing HEK-293 cells. When compared to cells expressing an empty vector control, proliferation and viability of WM cells was increased when they were cocultured with cells expressing PD-L1 or PD-L2. Addition of blocking antibodies abrogated the increase in proliferation. These results show that PD-L1 and PD-L2 are highly expressed on both malignant B-cells and non-malignant cells within the BM microenvironment in WM and promote malignant B-cell growth. Inhibition of PD-1 signaling may therefore be a promising therapeutic strategy in patients with WM. Disclosures: No relevant conflicts of interest to declare.