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Abstract Abstract 3642 Purpose: The reactive pathologic background in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) consists of lymphocytes and histocytes. Lymphopenia is a negative prognostic factor in NLPHL as it is also in Classical Hodgkin Lymphoma (cHL). In cHL, tumor-associated macrophages recruited from circulating monocytes are a negative prognostic factor for survival. Thus, we studied if the peripheral blood absolute lymphocyte count/absolute monocyte count ratio at diagnosis (ALC/AMC-DX), as a surrogate biomarker of the host response against the neoplastic lymphocytic and/or histiocytic (L&H) cells, affects survival in NLPHL. Patients and Methods: We performed a retrospective analysis of the association between ALC/AMC-DX and survival in 103 consecutive NLPHL patients that were followed at Mayo Clinic from 1974 to 2010. Receiver operating characteristic (ROC) and area under the curve (AUC) were used for ALC/AMC-DX cut-off value analysis and proportional-hazards models were used to compare survival based on the ALC/AMC-DX ratio. Results: The cohort included 67 (65%) males and 36 (35%) females. Eighty-three percent of patients presented with stage I/II and 17% with stage III/IV. Sixty-five (63%) patients were treated with radiation alone; 20 (20%) patients with chemotherapy alone; and 18 (17%) patients with chemotherapy and radiation. The median follow-up period was 8.9 years (range: 0.3–31 years). An ALC/AMC-DX ≥ 2.1 was the best cut-off value for survival with an empirical AUC of 0.82 (95% confidence interval [CI], 0.78 to 0.88), a sensitivity of 70% (95% CI, 61% to 76%) and specificity of 84% (95%CI, 59% to 82%). The cut-off value for ALC/AMC-DX ≥ 2.1 was validated by the k-fold cross validation method, showing a cross validation ROC with an AUC of 0.82 (95% CI, 0.72 to 0.93) for an ALC/AMC-DX ≥ 2.1. Using Kaplan-Meier analysis, we studied overall survival (OS), lymphoma-specific survival (LSS), progression-free survival (PFS), and time to progression (TTP) based on ALC/AMC-DX ≥ 2.1. Patients with an ALC/AMC-DX ≥ 2.1 experienced an superior OS, LSS, PFS, and TTP compared with patients with an ALC/AMC-DX < 2.1: [OS: median was 22.1 years vs 6.3 years, 10-years OS rates of 89% (95%CI, 80% to 98%) vs 44% (95%CI, 27% to 65%), p < 0.0001, respectively; LSS: median was not reached vs 7.3 years, 10-years LSS rates of 99% (95%CI, 90% to 100%) vs 45% (95%CI, 29% to 67%), p < 0.0001, respectively; PFS: median was 20.6 years vs 4.6 years, 10-years PFS rates of 84% (95%CI, 80% to 97%) vs 26% (95%CI, 18% to 55%), p < 0.0001, respectively; and TTP: median was not reached vs 5.5 years, 10-years TTP rates of 93% (95%CI, 85% to 99%) vs 29% (95%CI, 19% to 57%), p < 0.0001, respectively]. After adjusting for the International Prognostic Factors and the International Prognostic Score, ALC/AMC-DX remained an independent prognostic factor for OS {hazard ratio (HR), 0.42, 95%CI, 0.23 to 0.64, p< 0.004]; LSS [HR, 0.14; 95%CI, 0.04 to 0.23, p <0.0004]; PFS [HR, 0.14; 95%CI, 0.03 to 0.34, p < 0.001], and TTP [HR, 0.16, 95%CI, 0.04–0.34, p <0.002]. Conclusion: ALC/AMC-DX is an independent prognostic factor for survival and provides a single biomarker to predict clinical outcomes in NLPHL. Disclosures: No relevant conflicts of interest to declare.

Abstract Background IL-10 polarized macrophages promote tumor growth and survival. The T-cell transcription factor GATA binding protein-3 (GATA3) epigenetically regulates IL10 expression. Therefore, we investigated the extent to which GATA-3 regulates IL-10 production and macrophage polarization in T-cell lymphomas. Methods Monocyte-derived macrophages were generated and polarized with T-cell lymphoma conditioned media ((TCL-CM). Immunophenotypic and functional characteristics associated with IL10-polarized macrophages were examined. GATA3-dependent cytokine production was evaluated in T-cell lymphoma lines by shRNA knockdown. Alternatively polarized macrophages and GATA3 expression were examined in a cohort of patients with peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) (n=76). Comparisons among groups were evaluated using a Student's t test. Progression-free and overall survival (PFS & OS) was estimated using the Kaplan-Meier method and two-tailed log-rank test based on Cox proportional hazards model. Results To investigate the ability of T-cell lymphomas to alternatively polarize macrophages, monocytes from healthy donors were cocultured with TCL-CM. Macrophages were shown to have immunophenotypic characteristics of IL10-polarized macrophages (pSTAT3+/CD16hi/CD163hi/HLA-DR-/lo) by flow cytometry, which was abrogated by IL10, but not IL6, neutralization. TCL-CM polarized macrophages produced abundant IL-10 and were impaired in their ability to stimulate T-cell proliferation. Collectively, these results demonstrate that T-cell lymphomas promote alternative macrophage polarization in an IL-10 dependent manner, and both CD163 and pSTAT3 may aid in their identification in clinical T-cell lymphoma specimens. Therefore, CD163+/pSTAT3+ macrophages were visualized by immunofluorescence in PTCL, NOS specimens. IL-10 polarized macrophages were not identified in normal tissues, but were abundant constituents of the tumor microenvironment in 68% of the PTCL, NOS specimens examined (n=31). STAT3 phosphorylation in response to IL-10 is dependent upon the Janus kinases (JAK) JAK1 and TYK2, both of which are inhibited at nanomolar (and clinically achievable) concentrations by the “JAK2” inhibitor ruxolitinib. Therefore, we sought to determine whether ruxolitinib may prevent IL-10-induced macrophage polarization. As expected, ruxolitinib inhibited IL-10-induced STAT3 phosphorylation. More importantly, macrophages polarized in the presence of ruxolitinib lost the immunophenotype characteristics of IL-10 polarized macrophages, and were CD16-/loCD163-/loHLA-DRhi. Furthermore, IL-10 production was significantly impaired in macrophages polarized in the presence of ruxolitinib, while their ability to stimulate T-cell proliferation was significantly increased. In a separate initiative, we identified GATA3 as the key regulator of IL-10 and Th2 cytokine (IL-4, IL-13 and IL-5) expression in T-cell lymphoma lines by shRNA knockdown assays. Therefore, we examined GATA3 expression by immunohistochemistry in PTCL, NOS specimens. GATA-3 expression was observed in most of the T-cell lymphoma lines and in 47% of PTCL, NOS specimens (n=76). In the cohort of the 76 PTCL, NOS patients, the median PFS and OS observed in GATA3+ PTCL, NOS was 5 months (4-8 months, 95% CI) and 8 months (5-14 months, 95% CI), respectively. In contrast, the median PFS and OS in GATA3- PTCL, NOS was 1.3 years (0.6-1.6, 95% CI) and 1.6 years (0.7-6.9 95% CI), respectively. Conclusions Alternatively polarized macrophages are abundant in T-cell lymphomas and are generated in an IL-10- and STAT3-dependent manner. GATA-3 regulates IL-10 production in these lymphomas, and its expression identified a high-risk subset of PTCL, NOS with distinct clinicopathological features. As IL-10-dependent STAT3 phosphorylation and macrophage polarization were impaired by ruxolitinib, the JAK2 inhibitors may warrant closer scrutiny as immunomodulatory agents. Disclosures: Off Label Use: Ruxolitinib, a JAK2 inhibitor, may warrant closer scrutiny as an immunomodulatory agent in TCL therapeutics.

Abstract Background Relatively few studies have evaluated the role of host (germline) genetics in predicting outcome in diffuse large B-cell lymphoma (DLBCL), and of these, most were small candidate gene studies without replication and/or from the pre-R-CHOP era. We report the first genome-wide association study (GWAS) of outcome in DLBCL patients treated with rituximab and anthracycline-based chemotherapy. We used two independent studies for discovery, and validated the results in two additional independent studies. Methods In the discovery phase, we conducted a meta-analysis of two GWAS studies of DLBCL patients treated with rituximab and anthracycline-based chemotherapy, the first study as part of the prospective clinical trials of the LNH2003B program (N=540) of the Lymphoma Study Association (LYSA) and the second as part of the Molecular Epidemiology Resource (N=316), a prospective observational study from the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence (SPORE). Genotyping for the GWAS was performed using the Illumina 610 (LYSA) and 660 Quad (SPORE) BeadArrays (Illumina, San Diego, USA). HapMap2 was used to impute additional SNPs. Association testing was conducted assuming a log-additive genetic model and using Cox regression analyses. We calculated the hazard ratios (HRs) and 95% CIs for event-free survival (EFS), with adjustment for age, sex and age-adjusted IPI. The two studies were then combined using the fixed effects inverse variance method based on the beta estimates and standard errors from each study. From the meta-analysis, 89 SNPs were selected for validation, based on a minor allele frequency (MAF) >0.05 and LD pattern for multiple SNPs from the same region. Validation cohorts were an independent set of DLBCL patients from the SPORE (SPORE-2, N=366) and DLBCL patients from the prospective GOELAMS 075 phase III study (N=294), and validation genotyping was conducted using the Illumina VeraCode platform. We then conducted a meta-analysis of the 4 studies. We report SNPs with both a statistical significance of p<5x10-6 and effect sizes in the same direction across the 4 studies in the meta-analysis. For the latter SNPs, we also report the results for overall survival (OS), adjusted for age, sex and age-adjusted IPI. Results In the final meta-analysis of all four studies for the prediction of EFS, the top SNP was rs7765004 (MAF=0.30; HR=1.45; p=9.7 x 10-8) in the 6q21 region, which is near the MARCKS (myristoylated alanine-rich protein kinase C substrate) and HDAC2 genes. MARCKS encodes for a protein involved in cell mobility, while HDAC2 belongs to the histone deacetylase family. Additional SNPs from this region included rs6918103 (MAF=0.25; HR=1.38; p=7.6 x 10-6), which marks an EZH2 histone binding site, and rs11969684 (MAF=0.12; HR=1.43; p=3.3 x 10-5). The next hit was rs7712513 (MAF=0.31; HR=1.40; p=3.2 x 10-7), which is near arginine-fifty homeobox pseudogene 1 (ARGFXP1) on chromosome 5q23.2. This was followed by a region on chromosome 8q13.3, with the SNP rs9298183 (MAF=0.29; HR=1.41; p=1.4 x 10-6) from LOC100132891 showing the strongest association, along with 3 other SNPs from this region (rs2035376, rs6472637, and rs1600797; all p<5 x 10-6). These SNPs are near the musculin gene (MSC) a transcriptional repressor of E2A frequently silenced by promoter methylation in DLBCL. Finally, rs2253986 (MAF=0.27; HR=1.39; p=3.4 x 10-6) at chromosome 16q12.2, along with rs2253971 and rs6499810, were associated with EFS. These SNPs are located in CES5A (carboxylesterase 5A), which encodes a member of the carboxylesterase family. The family members are responsible for the hydrolysis or transesterification of various xenobiotics. The top SNPs associated with EFS also predicted OS, including rs7765004 (HR=1.41; p=1.2 x 10-5), rs7712513 (HR=1.39; p=8.4 x 10-6), rs9298183 (HR=1.36; p=3.5 x 10-5), and rs2253986 (HR=1.35; p=5.2 x 10-5). Conclusions Although our top SNPs on did not reach genome-wide significance (p<5x10-8), they showed clear consistency across the four large prospective studies and predicted both EFS and OS independently of IPI. The strongest findings were from loci on 6q21, 8q13.3, 5q23.2, and 16q12.2, and several genes from these regions are involved in methylation and xenobiotic metabolism, supporting a role for host genetic variation in these pathways in DLBCL prognosis. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2642 BAFF is essential for B cell maturation, and a lack of either BAFF or its primary receptor, BAFF-R, results in a severe depletion of T2 marginal zone and follicular B cells. Elevated serum BAFF levels have been correlated with an increased risk of developing non-Hodgkin's lymphoma (NHL), along with a more aggressive phenotype. A growing body of genetic evidence points toward an association between the development of human disease and variation in genes encoding BAFF and its receptors. Recently, we characterized a novel lymphoma-associated mutation in TNFRSF13C, the gene encoding BAFF-R. This mutation (BAFF-R H159Y) encodes a His159Tyr substitution in the C-terminus of BAFF-R adjacent to the TRAF3 binding motif. Signaling through BAFF-R H159Y results in increased NF-κB activity, elevated immunoglobulin production and increased association with TRAF2, TRAF3 and TRAF6 compared to wild type (WT) BAFF-R. We have detected this mutation in 6% of total NHL cases (n=129), and in 10% of follicular lymphoma (FL) cases (n=41) evaluated thus far. We previously reported that BAFF-R H159Y expressing mouse B cells exhibited significantly more resistance to Fas ligand (FasL) induced apoptosis compared to their cells expressing BAFF-R WT, and we propose here that BAFF-R H159Y mediated increases in PI3K activity may explain such an enhanced anti-apoptotic response. In this study we now show that BAFF stimulated HEK 293 cells stably expressing BAFF-R H159Y not only display significantly increased Akt phosphorylation when compared to BAFF-R WT expressing cells, but also demonstrate robust Akt phosphorylation in the absence of BAFF. BAFF-R H159Y-dependent Akt activation also led to activation of the downstream Akt targets mTOR and GSK3β and their phosphorylation was inhibited following treatment with the PI3- kinase inhibitor wortmannin. We next examined the impact of the BAFF-R H159Y mutation on expression of BAFF-target genes. Quantitative PCR analyses revealed that BAFF-R H159Y cells exhibited a pattern of gene expression indicative of promoting cell survival, displaying significantly higher levels of BCL2, BCL2L1 and PIN1, while down-regulating expression of the pro-apoptotic gene BIM. We recently reported that TRAF6 associates with BAFF-R, and that such binding is more pronounced in cells expressing BAFF-R H159Y. In order to investigate the role TRAF6 plays in mediating BAFF-R-dependent PI3K activity, we silenced TRAF6 expression in HEK 293 and Karpas 422 lymphoma cells using TRAF6 shRNA. Reduced TRAF6 protein expression resulted in a parallel decrease in BAFF-R WT mediated phosphorylation of mTOR in Karpas 422 cells and phosphorylation of both Akt and GSK3β was markedly reduced in BAFF-R H159Y expressing HEK 293 cells. Interestingly, TRAF6 knock-down did not affect NF-kB2 activation in either Karpas 422 or HEK BAFF-R expressing cells suggesting that Akt does not play a role in BAFF-R mediated activation of non-canonical NF-kB. Finally, preliminary co-precipitation studies indicate that Akt can be recruited to BAFF-R itself, and our initial observations suggest that such an association is significantly reduced in cells expressing BAFF-R H159Y. Taken together, these studies suggest that the BAFF-R H159Y mutation confers enhanced BAFF-R-dependent PI3K signaling and pro-survival gene expression independent of BAFF. Moreover, such enhanced P13K activation is partly dependent upon TRAF6, and decreased recruitment of Akt to BAFF-R H159Y may function to increase the amount of this PI3K target for activation. Thus, BAFF-R H159Y likely contributes to BAFF signaling irregularities in NHL patients harboring this mutation, and may predispose individuals to developing lymphoma regardless of their serum BAFF concentration. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 996 Background: Treatment with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) has improved survival in diffuse large B-cell lymphoma (DLBCL) lymphoma, although a significant percentage of patients do not achieve a remission or they relapse. One potential explanation for this observation is that host pharmacogenetic background may impact metabolism, detoxification and transport of R-CHOP. However, there are few prospectively collected data that address pharmacogenetics in DLBCL, particularly for event-free and overall survival in the rituximab era. We therefore tested the hypothesis that functional candidate single nucleotide polymorphisms (SNPs) from genes involved in the metabolism of CHOP (CYP2C19, CYP3A5, ABCB1, ABCC2, ABCG2, RAC2, CYBA, NR3C1, GSTA1, GSTP1, TP53) and rituximab (FCGR2A, FCGR3A) influence prognosis in DLBCL. Methods: We genotyped 19 SNPs from 13 genes in a prospective cohort of newly diagnosed DLBCL patients with germline DNA enrolled at the Mayo Clinic and University of Iowa from 2002–2008 as part of the Molecular Epidemiology Resource of the Iowa/Mayo Lymphoma SPORE. All patients were systematically followed through 2009 for overall survival (OS) and event-free survival (EFS), defined as disease progression, retreatment or death due to any cause. All SNPs were genotyped on the Illumina Golden-Gate platform. Cox regression was used to estimate Hazard Ratios (HRs) and 95% confidence intervals (CI) for individual SNPs with OS and EFS. Each SNP was modeled with the most prevalent homozygous genotype used as the reference group. An ordinal test was used to assess the trend across genotypes, and a p<0.05 was considered statistically significant. All Cox models adjusted for International Prognostic Index (IPI) and treatment. Results: The median age at diagnosis of the 439 DLBCL patients was 62 years (range 18–92). The IPI was distributed as follows: 0–1 (34%), 2 (26%), 3 (24%) and 4–5 (16%). All patients were treated with chemotherapy, and 91% received R-CHOP. Through 2009, there were 171 events (39%) and 122 (28%) deaths, with a median follow-up for living patients of 49 months (range 1–98). The SNP rs1045642 in the ATP-binding cassette (ABC) protein gene ABCB1 was associated with OS (p=0.012). The variant T allele for ABCB1 rs1045642 (minor allele frequency (MAF)=0.47) is associated with a silent I→I substitution, and the T allele predicts low enzyme expression. Compared to CC homozygotes, patients with CT (HR=0.66, 95% CI 0.44–0.99) or TT (HR=0.53, 95% CI 0.31–0.90) genotypes had superior OS. We also observed an association of the GSTP1 SNP rs1695 with OS (p-trend=0.018). The variant G allele (MAF of 0.31) is associated with an A→V substitution, and this change predicts low enzyme expression. Compared to AA homozygotes, patients with the AG (HR=0.78, 95% CI 0.54–1.14) or GG (HR=0.39, 95% CI 0.17–0.90) genotypes had superior OS. Nearly identical associations for these two SNPS were observed with EFS. There were no significant associations with any of the other SNPs that we evaluated. Conclusions: In a large series of newly diagnosed DLBCL patients treated in the rituximab era, we found evidence that genetic variation in ABCB1 and GSTP1 is associated with improved OS and EFS after adjusting for IPI and treatment. The variant allele for ABCB1 is associated with slower enzyme activity, favorably impacting doxorubicin and vincristine pharmacodynamics, and the variant allele for GSTP1 is associated with slower detoxification of doxorubicin and vincristine, increasing bioavailability of these drugs. A comprehensive evaluation of the role of pharmacogenetics in DLBCL is warranted. Support: P50 CA97274, R01 CA129539. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1640 Background: The introduction of radioimmunotherapy (RIT) into clinical practice for the treatment of follicular lymphoma (FL) occurred at a time at which many patients did not have prior exposure to rituximab. Clinical trials in the relapsed/refractory setting, as well as consolidation in the upfront setting have included a small number of patients that had been previously treated with rituximab or a rituximab containing treatment regimen. We evaluated the effect of prior rituximab therapy on the efficacy of RIT. METHODS: The prospective lymphoma database was queried for all patients having received a single dose of yttrium-90 ibritumomab tiuxetan for relapsed/refractory FL which had previously been treated with systemic therapy (patients receiving only prior external beam radiation therapy were excluded). The database includes all consecutive adult patients diagnosed with lymphoma seen at Mayo Clinic Rochester. The diagnosis of follicular lymphoma was as by WHO classification and centrally reviewed. Patients with evidence of histologic transformation to diffuse large B cell lymphoma prior to RIT were excluded. Time to treatment failure (TTF) was defined as the time from RIT to next anti-lymphoma therapy. RESULTS: 111 patients were identified. 57 (51%) were male. Median age at time of RIT was 58 (19–88). Median time from diagnosis to RIT was 35 (6–281) months in patients having received prior treatment with rituximab and 47 (12–272) months in those who had not (p=0.19). 63 (57%) of patients received prior treatment with rituximab or rituximab containing chemotherapy. Median number of prior therapies was 2 (1–10), which was not different between the rituximab treated or untreated groups [2(1–6) vs 2(1–10) p=0.12]. Median TTF was 10.0 months in the patients having received prior rituximab and 10.1 months in those that had not (p=0.29). Figure 1. The median overall survival from RIT was not reached, but appeared similar (Kaplan-Meier estimate 125 months without prior rituximab exposure vs 95 months with prior rituximab exposure; p=0.33). CONCLUSIONS: Prior rituximab exposure does not seem to adversely impact the time to treatment failure or overall survival in those patients treated with RIT. While the data leading to the approval of RIT were accrued prior to the routine clinical use of rituximab, our data demonstrate similar clinical benefit of RIT in patients with prior exposure to rituximab or rituximab containing therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 991 Background: The outcome of patients with DLBCL has significantly improved with the addition of rituximab to CHOP chemotherapy. While standard clinical prognostic factors such as those included in the International Prognostic Index (IPI) still predict outcome in DLBCL in R-CHOP treated patients, predicting the outcome of patients might be further refined using biological factors. We therefore tested whether pretreatment serum cytokines, that measure the biological activity of the tumor as well as the host response, could provide additional prognostic information in DLBCL patients. Methods: Newly diagnosed DLBCL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum cytokines were measured from pre-treatment blood draws using a multiplex ELISA (Upstate Cell Signaling Solutions, Lake Placid, NY). Thirty cytokines, including pro-inflammatory, Th1 and Th2 associated cytokines, were analyzed using the Luminex-100 system Version 1.7 (Luminex, Austin, TX). Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). The assay was also performed on research serum draws from 400 non-lymphoma epidemiology controls enrolled on a corresponding lymphoma etiology case-control study. The upper limit of normal for each cytokine was defined as the 95th percentile from the control distribution. 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 association between cytokines and outcome. Principal components analysis (PCA) was performed on the combined DLBCL and control cytokine dataset, agnostic to clinical characteristics and outcome, to identify cytokine profiles. Results: 206 DLBCL patients diagnosed between September 2002 and February 2008 with available serum were included. The median age was 64 years (range 20–92) and 52% were male. At a medium follow-up of 47 months (range 5–94), 75 patients (36%) had relapsed and 63 patients had died (31%). A cytokine profile was determined based on the first two principal components (PC) of the PCA analyses with PC1 corresponding to a general (host) cytokine elevation (also seen in normal controls) and PC2 corresponding to an increase predominantly in cytokines that were commonly elevated in DLBCL patients. 75 DLBCL patients (36%) had a normal cytokine profile similar to the general population of controls; 114 DLBCL patients (55%) had elevation in tumor specific cytokines (CXCL10, CXCL9, IL-2R, IL-12, IL-8, and IL-10) and 17 (8%) had a general cytokine elevation. Patients with any elevation of cytokines (tumor specific or general elevation) had a significantly worse event-free (HR=3.96, 95% CI: 2.13–7.37, p<0.00001) and overall survival (HR=3.75, 95% CI: 1.99–7.09, p<0.0001) than those with a normal cytokine profile (see figure). While the cytokine elevations were associated with advanced stage, poor performance score and elevated LDH, they were not associated with advanced age or extranodal disease and the results remained significant after adjustment for the IPI and treatment in the Cox model (EFS HR=3.11, 95% CI: 1.66–5.85, p<0.0001; OS HR=2.93, 95% CI: 1.53–5.63, p=0.001). Elevated serum cytokines also identified patients who were unlikely to complete initial therapy or who relapsed early. In low-risk patients (IPI 0–2), only 3% of patients with normal serum cytokines relapsed in the first 12 months compared to 23% of patients with elevated cytokines. Similarly in the high-risk group (IPI 3–5), none of the patients with normal cytokines relapsed in the first year compared to 43% with elevated cytokines. Conclusion: Elevated pretreatment serum cytokines, predominantly related to the malignancy, are associated with an increased likelihood of disease relapse and an inferior survival in patients with DLBCL. Furthermore, the pretreatment cytokine profile may also identify DLBCL patients at high risk for early disease relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1570 B-cell non-Hodgkin lymphoma encompasses a heterogeneous group of B-lymphocyte-derived malignancies that are characterized by chromosomal translocations involving the immunoglobulin (IG) gene loci and specific histological subtypes of disease are associated with a different spectrum of IG translocations. Marginal zone derived B cell lymphomas encompass three distinct entities: extranodal marginal zone B lymphoma (MZL) of mucosa associated lymphoid tissue (MALT), nodal MZL (NMZL), and splenic MZL (SMZL). MALT lymphoma is genetically unique and four mutually exclusive chromosomal translocations have been identified in this disease thus far. However, the known translocations are only present in a subset of cases suggesting that additional uncharacterized translocations or other genetic events may exist that contribute to disease development. In previous studies we characterized the novel t(X;14)(p11.4;q32) translocation in a patient with MALT lymphoma and found that GPR34, an orphan G-protein coupled receptor (GPCR), was highly expressed due to its juxtaposition to the IGHSA2 switch region. We then measured GPR34 mRNA expression in tissue biopsies from a panel of MALT, NMZL, SMZL, lymphoplasmacytic (LPL), diffuse large B cell (DLBCL), follicular (FL), and mantle cell (MCL) lymphomas. Expression of GPR34 was detected in all tissues examined, but was significantly increased in MALT (37 fold), LPL (23 fold), NMZL (18 fold), and SMZL (21 fold) compared to normal CD19+ B cells. GPR34 mRNA expression was also analyzed by gene expression profiling (GEP) of a panel of lymphomas or control lymphoid tissue and we confirmed elevated expression of GPR34 in the t(X;14) case and detected elevated GPR34 mRNA expression in MALT, LPL, NMZL, SMZL, and ABC DLBCL. When we grouped specimens by high and low expression of GPR34, a clear molecular subtype of SMZL could be identified and suggests that GPR34 high expressing tumors have a unique GEP. To confirm the qPCR and GEP analysis of GPR34 expression, we analyzed surface expression of GPR34 by flow cytometry of normal and malignant tissues and found that expression of GPR34 was detected on all MALT and SMZL B cells with an average DMFI of 2.08 (DMFI range 1.26–3.18, n=12) and on the JeKo-1 lymphoma B cell line (DMFI= 4.75). Analysis of CD19+ cells from normal controls (n=3) revealed a low level of GPR34 on (average DMFI =1.12). Little is known about the function of GPR34 and to further characterize the impact of GPR34 overexpression on cell signaling, OCI-LY19 lymphoma B cells and HeLa cells were transduced with GPR34 or a vector control. To determine if MAPK activity, a pathway regulated by GPCRs, was upregulated, the phosphorylation status of ERK1/2 in GPR34 or control cells was analyzed and we found that ERK1/2 was constitutively phosphorylated at significantly higher levels in GRP34 expressing cells. To further explore which signaling pathways were affected by GPR34 overexpression, GPR34 or vector control cells were transfected with an AP-1, CRE, NF-kB, E2F, SRE, NFAT, or ISRE- luciferase reporter plasmid and GPR34 expressing cells had significantly increased luciferase activity driven by AP-1 (5.35-fold), CRE (4.7-fold), NF-kB (2.6-fold), and E2F (2.1) when compared to vector control cells. To determine the biologic impact of GRP34 overexpression, the proliferation rates of vector control and GPR34 cells were compared and we found that proliferation of GPR34 WT cells was significantly higher than that seen in the control. We next tested the effect of the MEK inhibitor on proliferation and saw a dose dependent decrease in proliferation of GPR34 expressing cells. We then tested the ability of GPR34 cells to generate colony formation in a soft-agar colony formation assay. After 3 weeks of culture, the GPR34 cells had formed significantly more colonies than the vector control suggesting that overexpression of GPR34 can result in increased anchorage-independent cell growth. Taken together, our results identify a novel translocation in MALT lymphoma, emphasize a novel role for GPR34 in regulation of gene expression and tumor cell growth, and suggest that MEK inhibitors may be useful in a subset of translocation–harboring or GRP34 high-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

The tumor microenvironment plays a central role in lymphoproliferative disorders and constitution of the microenvironment is associated with patient outcome. Although malignant lymphocytes predominate, cells other than tumor cells are commonly present in malignant lymph nodes. These cells include T lymphocytes, NK cells, dendritic cells and monocytes that seem to be more than simple residual elements from the normal lymph node structure. It is thought that these infiltrating immune cells are part of an antitumor immune response, yet they appear unable to eradicate the malignant clone. Previous studies have shown however that multiple factors present in the tumor microenvironment oppose an effective antitumor immune response. Cells with suppressive function, including T regulatory cells, myeloid derived suppressor cells and suppressive monocytes, are abundant in lymphoma tissue. Suppressive cytokines such as TGFβ and IL-10 are highly expressed in tumors. Furthermore, intratumoral T-cell exhibit an exhausted phenotype with limited proliferation, cytokine production or cytolytic function. Recent therapeutic approaches have focused on overcoming T-cell suppression by activating T-cells or blocking inhibitory signals, thereby re-educating the suppressive tumor microenvironment. Clinical trial results with PD-1 and CTLA-4 directed antibodies in both non-Hodgkin lymphoma and Hodgkin lymphoma have been very promising. Overall response rates particularly in Hodgkin lymphoma patients treated with anti-PD-1 antibodies have been remarkable, although complete responses have been uncommon. Current studies are in progress to confirm the initial results, and further trials will assess the efficacy of immune checkpoint blockade in combination with standard therapies. Disclosures Ansell: Bristol-Myers Squibb: Research Funding; Celldex: Research Funding.

Abstract Background: Primary Mediastinal Large B-cell Lymphoma (PMLBCL) is a biologically and pathologically distinct subset of large B-cell lymphoma of thymic origin. The natural history of PMLBCL is not well defined. While earlier reports suggested that PMLBCL is an aggressive disease with a poorer prognosis when compared to other large cell B cell lymphomas, recent studies report more favorable results. Some of the outcome variability reported in previous studies may be related to the inclusion of other pathological lymphoma subtypes involving mediastinum. Thus, we reviewed the pathology and clinical course of patients with PMLBCL seen at our institution. Methods: We reviewed the biopsies on all patients diagnosed with mediastinal B-cell non-Hodgkin lymphoma (NHL) between 10/1983 and 10/2004. The pathological criteria for diagnosis of PMLBCL was a diffuse infiltrate of neoplastic CD20+ large B-cells within the mediastinum that frequently had "clear" cytoplasm and were often associated with fibrosis. We then reviewed the clinical data for patients with a pathological diagnosis of PMLBCL. Results: 121 patients were diagnosed with mediastinal B cell NHL and had tissue available for review. Of these, 88 fulfilled the pathological diagnostic criteria for PMLBCL. The median age at presentation was 37 years (range 18–83). The male to female ratio was 1.06. Age adjusted IPI was: 0, 1, 2, 3 in 18%, 56%, 18% and 8% of the patients respectively. The most commonly used treatments were: CHOP (70%), R-CHOP (22%) and ProMACE-CYTABOM (6%). 62% of the patients received adjuvant radiotherapy. 24% of the patients received high dose chemotherapy with autologous stem cell transplantation (2 patients in first remission). The median follow up was 66 months. The 2 and 5 year survival was 72% and 68% respectively. On univariate analysis, age >60 (p=0.012) and presence of B symptoms (p=0.0094) were associated with an adverse outcome. On multivariate analysis, only the presence of B symptoms (p=0.039) was an independent predictor of shortened overall survival. The 2 year survival of patients with age adjusted IPI of 0–1 or 2–3 was 86% and 68% respectively (p=0.023). Patients treated with adjuvant radiation therapy had a 5 year survival of 74% versus 53% in patients who did not receive adjuvant radiation (p=0.042). However, there was a higher percentage of Ann Arbor stage 3 and 4 disease patients found in the group who did not receive radiotherapy treatment (66% vs 17% respectively). Conclusions: The overall survival rates seen in patients with a confirmed pathological diagnosis of PMLBCL were superior to that reported in earlier studies and similar to that reported with the use of more intensive chemotherapy regimens. While many authors report a high proportion of female patients with PMLBCL, we did not observe any gender differences. While patients receiving adjuvant radiation therapy had a better outcome than those that did not, the retrospective nature of this study and differences in stage distribution preclude conclusions regarding the role of adjuvant radiation therapy. Randomized studies will be necessary to assess the optimal treatment for PMLBCL.