Ansell, Stephen M.

Abstract Abstract 3637 Background: A recent large genome-wide association study confirmed the important role of the major histocompatibility complex in classical Hodgkin Lymphoma (cHL) susceptibility (rs6903608), but also identified significant associations with genetic variants outside of the HLA region at chromosomal regions 2p16.1 (REL, rs1432295), 8q24.21 (PVT1, rs2608053, rs2019960), and 10p14 (GATA3, rs501764, rs485411). These single nucleotide polymorphisms (SNPs) are localized in or near important genes involved in lymphoma pathogenesis: REL, which encodes for c-rel, is a member of NFkB pathway; PVT1, which is downstream of the MYC locus, encodes for non-coding microRNAs, some of which have oncogenic properties; and GATA3, which is a critical transcriptional factor for priming lymphoid T-cells for Th2 phenotype, is aberrantly expressed in HL. We investigated whether these six SNPs described in cHL etiology also influence the outcome of newly diagnosed cHL patients in two independent cohorts. Patients and Methods: The first cohort consisted of 342 cHL patients from the Groupe d'Etude des Lymphomes de l'Adulte (GELA) who were prospectively enrolled between 1998 and 2002. The second cohort consisted of 259 patients prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). DNA for genotyping was extracted from a blood samples collected at diagnosis, and genotyping was conducted using Taqman technology (ABI Prism 7000, Applied Biosystems) for HLA-DRA, PVT1, GATA3 and PCR-based restriction fragment length polymorphism for REL in GELA cohort and Illumina iSelect for the 6 SNPs in MER cohort. Both cohorts were followed for disease progression, re-treatment and death. In the GELA series, the median age was 32 years (range, 15–93), 57% were male, and 72% had an Ann Arbor stage I–II. Histology was nodular sclerosis in 84%, and 29% of available cases (n=40/140) were EBV positive. In the MER series, the median age was 38 years (range, 18–89), 53% were male, and 53% had an Ann Arbor stage I-II. Nodular sclerosis was the most common subtype (62%) and 23 of 115 available cases were EBV positive (20%). The International Prognostic Score (IPS) was 0–1 in 46% and 52% of GELA and MER patients, respectively. ABVD regimen was used for 60% and 88% of the GELA and MER patients, respectively. The 5-year progression free survival (PFS) was 83% and 75% in GELA and MER cohorts, respectively. We estimated the prognostic value of each individual SNP for PFS for allelic and genotypic (dominant and recessive) modes of transmission in each series. In order to increase power, we then performed a meta-analysis of the two studies. Results: Consistent with prior reports, the minor allele frequencies for REL (rs1432295), HLA-DRA (rs6903608), PVT1 (rs2608053), PVT1 (rs2019960), GATA3 (rs501764) and GATA3 (rs485411) were 0.44, 0.50, 0.43, 0.29, 0.22, 0.28 in the GELA and 0.46, 0.41, 0.46, 0.27, 0.25, 0.31 in the MER series, respectively. In GELA, GATA3 (rs501767) was associated with PFS in the ordinal model (HR=1.66, 95%CI 1.11–2.49, P=.01), while there were suggestive associations for PVT1 (rs2608053) (HR=1.76, 95%CI 0.95–3.26, P=.07) and REL (rs1432295) (HR=0.63, 95%CI 0.37–1.06, P=.08) in the dominant model. In the MER, PVT1 (rs2608053) was associated with PFS in the ordinal model (HR=1.42, 95%CI 0.99–2.03, P=.06), while there was a suggestive association with HLA-DRA (rs6903608) in the dominant model (HR=0.64, 95%CI 0.39–1.05, P=.08) for whole cohort but also for EBV negative cHL (HR=0.41, 95%CI 0.17–0.99, P=.05). In a meta-analysis of the two studies, PVT1 (rs2608053) was significantly associated with PFS in the ordinal (HR=1.34, 95%CI 1.04–1.73, P=.02) and the dominant (HR=1.88, 95%CI 1.20–2.95, P=.006) models. Further adjustment for IPS had minimal impact on these associations (ordinal HR=1.30, 95%CI 1.00–1.70; dominant HR=1.78, 95%CI 1.13–2.83). Patients with PVT1(rs2608053) GG, GA, AA genotypes had a 5-year PFS rate of 83%, 74% and 73% in GELA and 86%, 70%, and 73% in the MER, respectively. Conclusions: The non-HLA cHL susceptibility locus PVT1 (rs2608053) was associated with PFS in two independent cohorts of cHL cases, and after adjustment for the IPS. While associations with GATA3 and REL were only observed in one of the studies, their evaluation in other studies is warranted. Functional studies of PVT1 in cHL pathogenesis should be pursued. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1665 Poster Board I-691 Mantle cell lymphoma (MCL) is an aggressive, incurable B-cell malignancy that represents approximately 6% of lymphoma cases. Patients with MCL who relapse after conventional therapy or stem cell transplantation have a poor prognosis and are candidates for treatment with novel agents. The characteristic feature of MCL is overexpression of cyclin D1. Cyclin D1 is under the control of the phosphatidylinositol-3 kinase signal-transduction pathway and is downstream of the mammalian target of rapamycin kinase (mTOR). We have previously shown that temsirolimus (CCI-779), a dihydroester of the selective mTOR inhibitor rapamycin, is an active antitumor agent in MCL. In previous phase 2 trials of temsirolimus as a single agent, we observed a 40% overall response rate (ORR) with a median duration of response (DR) of 6 months in patients with relapsed or refractory disease (J Clin Oncol 23:5347-56, 2005; Cancer 113:508-514, 2008). Due to the fact that rituximab has improved the response rate and overall outcome of lymphoma patients when combined with chemotherapy, we tested the efficacy and toxicity of temsirolimus in combination with rituximab in patients with MCL. Eligible patients with confirmed relapsed or refractory MCL based on morphologic and phenotypic features received temsirolimus 25 mg intravenously every week and four weekly doses of rituximab with the first cycle and then one dose of rituximab every other cycle. Patients with a tumor response after six cycles were eligible to continue treatment for a total of 12 cycles or two cycles after complete remission, and were then observed without maintenance. Seventy-one patients were enrolled between May 2005 and March 2009. The median age was 67 years (range, 44 to 86 years), with 51 males and 20 females enrolled. Patients had received a median of two prior therapies (range, 1-9), 31% had received a prior stem cell transplant and 28% were rituximab refractory. The ORR was 48% (34/71 patients) with 20% (14/71) complete responses and 28% (20/71) partial responses. The median DR was 9.5 months (95% CI, 4.5 to 11.3 months). The median DR in the rituximab sensitive patients was 9.5 months (CI: 4.5-11.6) and 7.15 months (CI: 4.1-11.3) in the rituximab refractory patients (p= 0.73). While the combination was generally well tolerated, 12 patients experienced grade 4 toxicity and 2 patients died while on therapy – both from progressive disease. Hematologic toxicities were the most common, with 5 patients having grade 4 thrombocytopenia and 3 having grade 4 neutropenia. This study demonstrates that temsirolimus in combination with rituximab is well tolerated and has substantial antitumor activity in relapsed MCL. Further comparative studies of this combination in MCL are therefore warranted. Disclosures Witzig: Wyeth: Patents & Royalties.

Abstract Abstract 143 IL-12 induces IFN-g production and contributes to anti-tumor immunity. However, administration of IL-12 in cancer patients has resulted in a limited clinical benefit. In follicular B-cell lymphoma (FL), a clinical trial of IL-12 in combination with rituximab showed a lower response rate in patients treated with the combination than in patients treated with rituximab alone (Clin Cancer Res 2006), suggesting that, in contrast to the observations in vitro or in vivo in mice, IL-12 actually plays a detrimental role in FL patients. Recently, a type of immune response, termed “ T cell exhaustion” describing a condition in which T cells exhibit reduced differentiation, proliferation, and effector function, has been shown to impair anti-tumor immunity and result in disease progression. TIM-3, a family member of T-cell immunoglobulin and mucin domian-containing proteins, inhibits TH1-mediated immune response and promotes immunological tolerance. A recent study has suggested that TIM-3 may play an important role in mediating T cell exhaustion. However, the biological and clinical relevance of TIM-3 in cancers remains completely unknown. In this study, we determined whether T cell exhaustion exists in the tumor microenvironment, whether IL-12 contributes to T cell exhaustion, and whether TIM-3-mediated T cell exhaustion impacts patient outcome in FL. We found that serum IL-12 levels were elevated in FL patients compared to healthy individuals (median: 0.50 ng/ml, n=30 vs 0.32 ng/ml, n=22; p= 0.03) and that elevated serum IL-12 levels were associated with a poor outcome in these patients when treated with rituximab alone as initial therapy. Using 0.56 ng/ml as a cutoff, patients with serum IL-12 levels of greater than 0.56 ng/ml had a significantly shorter time to progression than patients with IL-12 levels less than 0.56 ng/ml (12 months versus 40 months; p=0.001). Both lymphoma B cells and monocytes were able to produce IL-12 and contributed to elevated serum levels of IL-12 in FL. Importantly, we found that IL-12 strongly induced TIM-3 expression in a dose-dependent manner. Endogenous production of IL-12 by lymphoma B cells and monocytes was capable of regulating TIM-3 expression on T cells from FL. As a consequence, TIM-3 was highly expressed on a subset of T cells from PBMCs or lymph nodes from FL patients while its expression was negligible or moderate on T cells from normal PBMCs or benign lymph nodes, respectively. The number of TIM-3-expressing T cells accounted for approximately 32% and 39% of CD4+ or CD8+ T cells in biopsy specimens and 6.2% and 6.7% in peripheral blood of FL patients. TIM-3+ T cells, co-expressed with PD-1, exhibited a reduced ability to proliferate and decreased cytokine production compared to TIM-3- T cells. Similarly, IL-12-induced TIM-3+ T cells gradually lost the capacity to produce cytokines over a period of time. These results suggest that TIM-3-expressing T cells are functionally exhausted. In addition, TIM-3+ T cells were prone to apoptotic induction by its ligand galectin (Gal) -9. The phosphorylation of p38 was higher in TIM-3+ T cells compared to TIM-3- T cells when exposed to Gal-9, suggesting MAPK pathway was involved in Gal-9-mediated apoptosis of TIM-3+ T cells. Finally, increased numbers of intratumoral TIM-3-expressing cells were associated with a higher histological grade, higher LDH levels and a poor survival in FL patients. Taken together, these results indicate that IL-12, in contrast to its role in augmenting immune response through IFN-g, induces T cell exhaustion by upregulating TIM-3 expression. We further demonstrated that lymphoma B cells produce IL-12 thereby contributing to T cell exhaustion by promoting TIM-3 expression on intratumoral T cells. Impairment of anti-tumor immunity due to T cell exhaustion induced by the IL-12-TIM-3 pathway may account for the observation that high levels of serum IL-12 and increased number of TIM-3+CD4+ T cells correlate with a worse outcome in FL patients. These findings not only reveal a novel IL-12-TIM-3 pathway that plays an important role in impairing tumor immunity and detrimentally affecting prognosis in FL patients, but may have therapeutic potential for cancer patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1691 Poster Board I-717 Waldenström macroglobulinemia (WM) is a B-cell malignancy that is characterized by the production of a monoclonal IgM protein and a lymphoplasmacytic infiltrate in the bone marrow. The aberrant production of the monoclonal IgM can result in serum hyperviscosity that can cause significant morbidity in patients with this disease. In previous work, we have shown that IL-6 significantly upregulates IgM secretion by WM cells and that IL-6 secretion is regulated by CCL5 (Rantes). We have also shown that IL-6 mediated IgM secretion in WM requires phosphorylation of Stat1 and Stat3. Because IL-6 induced signaling involves the Jak/Stat pathway, we tested whether the use of a Jak/Stat inhibitor, TG101348, would result in down regulation of CCL5, IL-6 and IgM production and inhibit cell proliferation and viability in WM. First, we determined whether TG101348 could inhibit the production of CCL5 because other Jak inhibitors have been shown to inhibit cytokine production. Using the BCWM.1 cell line as well CD19+ malignant cells from bone marrow specimens from WM patients, we measured CCL5 by ELISA in the culture supernatant 24 hours after treatment with increasing concentrations of the inhibitor. We found that CCL5 secretion was decreased by 50% at a concentration of TG101348 of 250nM and was completely inhibited at 2μM. Next, we measured IL-6 production after treatment with TG101348. We had previously shown that stromal cells are the primary source of IL-6 and therefore used the stromal cell line HS-5 to measure IL-6 by ELISA after treatment with the inhibitor. Our previous work had also shown that IL-6 secretion was mediated by GLI (a member of the Hedgehog pathway) rather than the Jak/Stat pathway. Interestingly, we found that IL-6 secretion was inhibited in a dose dependent fashion but required higher doses for complete suppression (8μM). We then measured IgM production by malignant B-cells 24 hours after treatment with TG101348. Our previous work had shown that IL-6 mediated IgM secretion was dependent on the Jak/Stat pathway. We found that IgM production was inhibited by 50% at 500nM and completely suppressed at 2μM. Finally, we measured the effect of TG101348 on cell proliferation and survival. Using the BCWM.1 cell line, we found that cell proliferation as determined by tritiated thymidine uptake was inhibited in a dose dependent fashion with 50% inhibition at 1μM. Inhibition of cell viability as measured by Annexin V/propidium iodide staining, however, required higher concentrations and cell viability was inhibited with an IC50 of 8μM. These data confirm the role of Jak/Stat signaling in the CCL5-IL-6-IgM axis in WM. We found that TG101348 generally suppressed the signaling and growth of WM cells but that pathways that were known to be Jak/Stat dependent required significantly lower doses to be completely inhibited. These data provide a strong rationale for the use of inhibitors of this pathway, such as TG101348, in the treatment of patients with WM. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 440 Background: Complement plays a central role in innate immunity, and also functions as a regulator of the overall immune response, including T-cell responses. We recently reported that germline genetic variation in C2, C5, C7 and C9 was associated with risk of developing non-Hodgkin lymphoma (Br J Haematol 2009;145:614). Here we evaluate whether complement genes are associated with event-free survival (EFS) in follicular lymphoma. Methods: We genotyped 166 single nucleotide polymorphisms (SNPs) from 32 complement pathway genes in a prospective cohort study of 115 newly diagnosed follicular lymphoma patients enrolled at the Mayo Clinic from 2002-2005 as part of the Lymphoma SPORE. All patients were systematically followed through 2008 for EFS (defined as disease progression, retreatment, or death due to any cause). Tagging and nsSNPs were selected from HapMap for the genes encoding complement proteins and their key regulators (C1QB, C1QG, C1QR1, C1QTNF7, C1RL, C1S, C2, C3, C3AR1, C4BPA, C4BPB, C5, C5R1, C6, C7, C8B, C9, BF, CFH, CFHL5, CFHR1, CGHR4, CLU, CR1 (CD35), CR2 (CD21), DF, MASP2, MBL2, MCP (CD46), DAF (CD55), CD59 and SERPING1). Cox regression was used to estimate Hazard Ratios (HRs) for individual SNPs with EFS. The most prevalent homozygous genotype was used as the reference group, and each polymorphism was modeled individually as having a log-additive effect in the regression model. For gene-level analyses, we used a principal components based gene-level test. All models were adjusted for sex, FLIPI, follicular grade III, and initial treatment. Results. The median age at diagnosis was 61 years (range, 25-85). 57 (50%) of the patients had an event, at a median follow-up of 59 months (range, 33-73) for living patients. In gene-level analyses, CFH (p=0.004), CD55 (p=0.02), CFHR5 (p=0.02) and CFHR1 (p=0.02) were significant at p<0.05, and these genes had noteworthy q-values after consideration of multiple testing (q=0.04 for CFH and q=0.08 for CD55, CFHR5, and CFHR1). For CFH, 2 of the 11 tagSNPs (rs1329423, HR=0.46, p=0.001; and rs3766404, HR=2.0, p=0.003) were significantly associated with EFS, as was the nsSNP rs1065489 (amino acid change E936D: HR=0.39, p=0.004). For CD55, both tagSNPs (rs4844591, HR=0.58, p=0.03; and rs2564978, HR=0.58, p=0.03) were associated with EFS, as were 3 of 5 tagSNPs from CFHR5 (rs3748557, HR=0.52, p=0.03; rs12092294, HR=1.9, p=0.03; and rs6694672, HR=1.9, p=0.03) and the only tagSNP for CFHR1 (rs436719, HR=0.54, p=0.03). Conclusions. Genetic variation in CFH, CD55, CFHR5, and CFHR1 was associated with EFS in follicular lymphoma after adjustment for clinical variables. These genes are part of a gene cluster at 1q32-q32.1 involved in the regulation of C3. CD55 encodes a membrane complement regulatory protein which can allow tumor cells to evade complement attack. These results are being validated in a second set of SPORE patients. Support: P50 CA97274, R01 CA92153. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 140 Background: Peripheral T-cell lymphomas (PTCLs) have poor outcomes, and there is a lack of prognostic biomarkers and therapeutic targets to guide treatment. We recently proposed that the transcription factor, interferon regulatory factor-4 (IRF4, also called multiple myeloma oncogene-1 [MUM1]), might be oncogenic in PTCLs based on its expression in association with translocations between IRF4 and the T-cell receptor gene, TRA@. IRF4 is a therapeutic target in multiple myeloma, where high expression is a poor prognostic factor. In addition, at least two germline IRF4 single nucleotide polymorphisms (SNPs), rs12203592 and rs872071, are associated with IRF4 expression and disease risk and progression in various lymphoid neoplasms. However, the prognostic effects of IRF4/MUM1 expression and IRF4 SNPs in PTCLs are unknown. Methods: Forty seven newly diagnosed PTCL patients with available tissue were identified from the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource. There were 5 anaplastic large cell lymphomas, 14 cutaneous T-cell lymphomas (CTCLs), 6 cytotoxic T-cell lymphomas (cytTCLs: 4 extranodal NK/T-cell and 2 enteropathy type), and 22 PTCLs, not otherwise specified (NOS). Patients were diagnosed between September 2002 and February 2008 and systematically followed through March 2010 for overall survival (OS). Tumor cell IRF4/MUM1 expression was examined by immunohistochemistry on paraffin tissue sections (MUM1p clone; Dako). Positivity was defined as >30% of tumor cells with nuclear staining, as in previous studies. rs12203592 and rs872071 were genotyped in peripheral blood DNA. Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for both IRF4/MUM1 expression and SNPs with OS. Chi-squared tests were used to assess the relationship between IRF4/MUM1 expression and IRF4 SNPs. Results: The median age at diagnosis was 60 years (range, 24–88). At a median follow-up of 62 months (range, 29–89), 22 (47%) of the patients had died. Fifteen (32%) of the 47 T-cell patients had IRF4/MUM1 positive tumors. IRF4/MUM1 positivity was associated with poorer overall survival (HR=4.3; 95% CI: 1.8–10.2; p=0.0008). This association was seen across PTCL subtypes, including PTCL, NOS (HR= 6.5; 95% CI: 1.5–27.7; p=0.01), CTCL (HR=13.4; 95% CI: 1.2–150.1; p=0.03), and cytTCL (HR=5.8; CI: 0.5–65.9; p=0.15). The minor allele (T) in SNP rs12203592 was positively associated with IRF4/MUM1 positivity in a dominant model, with IRF4/MUM1 expression in 60% of patients with CT or TT genotypes compared to 19% of patients with CC genotype (p=0.01). Patients with the CT/TT genotype at rs12203592 also had inferior overall survival (HR=3.7; 95% CI: 1.4–9.5; p=0.007). The rs872071 SNP showed no significant association with either IRF4/MUM1 expression (p=0.38) or overall survival (p=0.71). Conclusions: This study is the first to demonstrate that IRF4/MUM1 expression is poor prognostic factor in PTCLs. This association was observed across PTCL subtypes, including the most common subtype, PTCL, NOS. IRF4/MUM1 expression and poor survival in our patients also were associated with the minor allele (T) in the IRF4 SNP rs12203592; these findings are consistent with previous in vitro data showing the major allele (C) represses IRF4 promoter activity. Interestingly, rs872071 was not associated with IRF4/MUM1 expression or prognosis, similar to findings in multiple myeloma. In contrast, rs872071 is a risk and prognostic factor in chronic lymphocytic leukemia and classical Hodgkin lymphoma, diseases in which IRF4/MUM1 expression has been associated with favorable prognosis. IRF4/MUM1 expression is a poor prognostic factor in PTCLs. Disclosures: No relevant conflicts of interest to declare.

AbstractBackgroundHuman pegivirus (HPgV) is a single-strand RNA virus belonging to the Flaviviridae. Although no definitive association between HPgV infection and disease has been identified, previous studies have suggested an association of HPgV viremia with risk of lymphomas.MethodsWe conducted a systematic review and meta-analysis, including 1 cohort study and 14 case-control studies, assessing the association of HPgV viremia with adult lymphomas. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model, overall and by geographic region and lymphoma subtype.ResultsThe overall OR for lymphoma was 2.85 (95% CI, 1.98–4.11), with statistically significantly elevated ORs observed in 8 of 15 studies. There was a small amount of heterogeneity among studies (I2 = 28.9%; Q = 18.27, P = .16), and the funnel plot provided no evidence for publication bias. The strongest association with lymphoma risk was observed for studies from Southern Europe (OR, 5.68 [95% CI, 1.98–16.3]), whereas weaker ORs (with 95% CIs) were observed for studies from North America (2.24 [1.76–2.85]), Northern Europe (2.90 [.45–18.7), and the Middle East (2.51 [.87–7.27]), but all of similar magnitude. Participants with HPgV viremia had statistically significantly increased risks (OR [95% CI]) for developing diffuse large B-cell (3.29 [1.63–6.62]), follicular (3.01 [1.95–4.63]), marginal zone (1.90 [1.13–3.18]), and T-cell (2.11 [1.17–3.89]) lymphomas, while the risk for Hodgkin lymphoma (3.53 [.48–25.9]) and chronic lymphocytic leukemia (1.45 [.45–4.66]) were increased but did not achieve statistical significance.ConclusionsThis meta-analysis supports a positive association of HPgV viremia with lymphoma risk, overall and for the major lymphoma subtypes.