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Abstract Abstract 136 Background: The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. This assay has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and AL amyloidoisis, and has been incorporated in response criteria for both MM and AL amyloidosis. A percentage of non-Hodgkin's lymphoma (NHL) patients also present with abnormal FLC concentrations and recent data suggest that AIDS patients with elevated serum FLC are at higher risk of developing NHL. Here we assess the impact of FLC in a cohort of patients with diffuse large B-cell lymphoma (DLBCL) enrolled on a cooperative group clinical trial. Methods: Newly diagnosed DLBCL patients were treated with epratuzumab + R-CHOP (ER-CHOP) on NCCTG clinical trial N0489. Serum FLC was quantitated prior to treatment and after cycles 2 and 6 using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Patients were followed per clinical trial protocol for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively). Results: 107 patients were enrolled on the trial. 80 patients had a pre-treatment serum draw and a central pathology confirmed diagnosis of DLBCL, of which 47 had a post-cycle 2 sample (C2) and a 54 had a post-cycle 6 sample (C6). At a medium follow-up of 25 months (range 7-39), 22 patients (28%) had an event and 17 patients died (21%). Prior to treatment 23 patients (29%) had abnormal kappa concentrations (all elevated), 16 (20%) had abnormal lambda concentrations (11 elevated, 5 decreased), and 9 (11%) had an abnormal kappa/lambda ratio. FLC concentrations were significantly reduced in patients during treatment, with a median reduction in kappa (C2: 46%, C6: 64%) and lambda (C2: 44%, C6: 49%), all p<0.0001. Elevated FLC prior to treatment was highly associated with both OS (logrank p=0.009) and EFS (logrank p=0.006); these associations remained significant after adjusting for IPI (both p = 0.01). Conclusions: A significant percentage of DLBCL patients present with abnormal FLC concentrations. Kappa and lambda are significantly reduced for most patients during treatment. Elevated pre-treatment FLC concentrations are associated with inferior EFS and OS in DLBCL and this association is independent of the IPI. A follow-up study of FLC and prognosis is currently underway in a cohort of 1500 NHL patients from the Mayo Clinic and University of Iowa SPORE. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1952 Poster Board I-975 Background: Vitamin D is a naturally occurring steroid hormone. In addition to its well-established role in maintaining serum calcium homeostasis, vitamin D has effects on cellular differentiation, proliferation, apoptosis, and angiogenesis. Vitamin D3 is naturally produced in skin in response ultraviolet-B (UVB) radiation from the sun. Several recent studies, however, have shown that a high proportion of community-dwelling subjects in both tropical and temperate climates are deficient in vitamin D, and that subjects in northern latitudes often require dietary supplementation to maintain vitamin D sufficiency. Further, several reports now suggest that vitamin D sufficiency is protective against the development of several cancers, including non-Hodgkin lymphoma (NHL). However, it is not known whether vitamin D impacts prognosis in diffuse large B-cell lymphoma (DLBCL), the most common NHL subtype. Methods: We evaluated serum vitamin D concentrations in two cohorts of DLBCL patients. The first cohort consisted of 374 patients newly diagnosed from September 2002-February 2008 who were prospectively enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource, an observational study in which neither therapeutic nor diagnostic management is prescribed. All serum was obtained within 120 days of diagnosis, and was prior to treatment in 221 (59%). In the second study, 62 patients newly diagnosed from February 2006-August 2007 were enrolled on NCCTG clinical trial N0489, and all serum was obtained before the first course of therapy. Central pathology review was performed on all patients to confirm the diagnosis of DLBCL. Serum 25-hydroxyvitamin (25-OH-VitD) levels were measured by the gold standard method for vitamin D determination: liquid chromatography tandem mass spectroscopy (LC-MS/MS). Vitamin D deficiency was defined as total serum 25-OH-VitD < 25 ng/mL (62.5 nmol/L). SPORE patients were followed per a standard protocol for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively); N0489 patients were followed in a similar way per the clinical trial protocol. We used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CI). Results: SPORE patients were primarily treated with immunochemotherapy (83%); all N0489 patients were treated with epratuzumab + R-CHOP21. The median age at diagnosis was 62 years (range, 19-93) for SPORE patients and 61 years (range, 21-82) for N0489 patients. Median follow-up for SPORE patients was 36 months (range, 1-77) with 95 deaths and 131 events; median follow-up for N0489 patients was 24 months (range, 7-39) with 13 deaths and 18 events. Vitamin D deficiency was identified in 188 SPORE patients (50%) and 15 N0489 patients (24%). There were no differences in the prevalence of vitamin D deficiency by age, IPI, or whether serum was obtained prior to starting treatment (all p > 0.28). Vitamin D deficiency was associated with inferior overall (HR=2.33, 95% CI 1.53-3.57, logrank p = 0.0001) and event-free survival (HR=1.71, 95% CI 1.20-2.44, logrank p = 0.003) in the SPORE cohort. These associations remained significant after adjusting for IPI and treatment: OS HR=1.97, 95% CI 1.27-3.07; EFS HR=1.47, 95% CI 1.02-2.21. These results were similar for the subset of SPORE patients who were treated with R-CHOP. The HRs for vitamin D deficiency and event-free survival were consistent in the N0489 patients (HR=1.63, 95% CI 0.61-4.35, IPI adjusted HR=1.43, 95% CI 0.53-3.85), although these results lacked precision due to a smaller sample size. We were unable to evaluate OS in N0489 due to the small number of deaths. Conclusions: Approximately 50% of all DLBCL patients in this northern US latitude population are vitamin D deficient at the time of diagnosis and treatment. Vitamin D deficient patients have an inferior event-free and overall survival compared to patients with vitamin D levels within the normal range. Vitamin D supplementation during treatment of DLBCL patients with vitamin D deficiency should be evaluated in a clinical trial setting. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 137 Background: FDG-PET/CT after 2–4 cycles of chemotherapy has been shown to be predictive of outcome in patients with aggressive lymphoma. This multicenter NCCTG phase II study was carried out to assess efficacy of ER-CHOP in newly diagnosed DLBCL; functional response as measured by FDG-PET/CT was done after 2 cycles and after 6 cycles of therapy. Methods: Patients received immunochemotherapy on the following schedule: epratuzumab 360 mg/m2, rituximab 375 mg/m2, and standard dose CHOP every 3 weeks for 6 cycles. FDG-PET/CT was done after 2 cycles and again after 6 cycles of therapy. All PET scans were centrally reviewed by an expert in nuclear medicine who was blinded to the outcome of the patients. Results: 107 patients were accrued from Feb 2006 to Aug 2007. 27 patients were ineligible resulting in 80 eligible patients. Baseline patient characteristics for the eligible patients included median age 60 (range 21–82); 56% were male. 80% had advanced stage; 73% had an elevated LDH. Performance score was 0–1 in 71 pts and 2–3 in 9 pts. IPI was 0–1 in 18 pts (23%), 2 in 22 pts (28%), 3 in 29 pts (36%) and 4–5 in 11 pts (14%). PET scans were available on 76 patients (95%); 3 patients completed treatment but scans were unavailable and 1 died prior to cycle 2 PET. The PET CR rate after 2 cycles was 79%. The PET CR rate after 6 cycles was 90%. Using an intent to treat (ITT) analysis, 67 patients (87%) achieved PET CR of which 61 (79%) also completed all 6 cycles of treatment. Based on interim PET after 2 cycles, the EFS at 24 months (EFS24) was 73% for the PET negative patients vs 60% for the PET positive patients (p=0.25); OS at 24 months (OS24) was 83% vs 73% for the PET negative and positive pts, respectively (p=0.17). In comparison, EFS24 for patients with a negative PET after 6 cycles was 80% vs 57% for those with a positive PET (p=0.11); OS24 was 92% vs 57%, respectively (p=0.01). In intent to treat (ITT) analysis, patients that achieved a functional CR had improved overall survival (OS24 = 84%) and event free survival (EFS24 = 74%) compared to patients that failed to achieve a functional CR (OS24 = 50%, EFS24 = 40%, p=0.01 and p=0.006 respectively). Conclusions: ER-CHOP every 21 days × 6 cycles results in a functional CR rate of 87%. Early PET scan during therapy does not significantly predict outcome. Achievement of PET negativity by completion of therapy is associated with a good outcome Disclosures: No relevant conflicts of interest to declare.

Abstract Background Performance status (PS) is a measurement of a patient’s functional capabilities and has been validated as a prognostic indicator in patients with cancer. This study was conducted to compare patient and physician-rated Eastern Cooperative Oncology Group (ECOG) PS to determine the incidence of inter-observer variability and identify determinants of PS disagreement among a cohort of patients with hematologic malignancy. Methods Newly diagnosed patients with leukemia and lymphoma were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) from 2002-2008. At diagnosis, patients and their physicians were asked to independently rate ECOG PS. Those who reported PS within 1 month of diagnosis were included in this analysis. Chi-squared and Wilcoxon rank-sum tests were used to assess the association between PS disagreement and prognostic or demographic factors; Cox proportional hazards models and c-statistics were used to evaluate the association between PS and overall survival. Results 1269 patients were included with a median age of 61 years (range 18-91). Of these, 58% were male; 275 had chronic lymphocytic leukemia (CLL), 127 had Hodgkin lymphoma (HL), and 867 had non-Hodgkin lymphoma (NHL). Overall, 829 (65%) patients and physicians rated PS the same. Age greater than 60 was a significant predictor of disagreement overall (p<0.001), as well as in CLL (p=0.014) and NHL (p=0.004), but not in HL (p=0.65). We found that across disease subtypes, the level of disagreement increased with more aggressive disease. This finding was significant in those with HL (p=0.027) and NHL (p<0.0001) as International Prognostic Score (IPS) and International Prognostic Index (IPI) increased respectively. In CLL, the trend for Rai stage was suggestive but not statistically significant (p=0.27). The incidence of disagreement was highest among patients with NHL (37%), with a higher percentage of disagreement among those with aggressive (45%) vs. non-aggressive (25%) subtypes (p<0.001). Disagreement was lower in patients with CLL (27%) and HL (33%). There were no significant associations between patient gender or education level and PS rating among subtypes or overall. Patient and physician-rated PS were both significant predictors of overall survival in univariate models and also adjusted for subtype and subtype specific risk score. The prognostic ability for PS was similar for both patient rated and physician-rated assessment overall (c-statistic=0.76 for both patient and physician rated PS) and in those with HL (c-statistic=0.85 for both patient and physician rated PS) and NHL (c-statistic=0.76 for both patient and physician rated PS). However, patient-rated PS was better for prognostication in CLL (c-statistic=0.75; p<0.0001), compared to physician-rated PS (c-statistic=0.67; p=0.002). Conclusions Patients with hematological malignancies and their physicians do not always rate PS the same, particularly in patients who are older or have more advanced or aggressive disease. These findings suggest the need for physicians to communicate with patients when determining PS, as PS is a strong predictor of survival. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction Relapse in the central nervous system is an uncommon complication of diffuse large B cell lymphoma (DLBCL) associated with a poor prognosis. The addition of rituximab to chemotherapy has improved outcomes in patients with DLBCL but its effect on the outcome of patients with CNS relapse is not well characterized. Here we present the natural history of patients with CNS relapse in a large cohort of patients with DLBCL who were treated in the immunochemotherapy (IC) era. Methods Newly diagnosed patients with DLBCL or primary mediastinal B-cell lymphoma (PMBCL) and treated with primary anthracycline based IC were prospectively enrolled on the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) within 9 months of diagnosis and followed for relapse, retreatment, and death. Clinical management at diagnosis and subsequent therapies were per treating physician. Patients with documented CNS involvement at diagnosis, primary CNS lymphoma, or post-transplant lymphoproliferative disorder at diagnosis were excluded. All CNS relapse and retreatment details were verified by medical record review. Results 1017 patients with newly diagnosed DLBCL or PMBCL, no documented CNS disease at diagnosis, and treated with IC were enrolled in the MER between 2002 and 2012. At a median follow-up of 59 months (range 1-148), 36 patients had CNS relapse. The cumulative incidence of CNS relapse was 3.1% (95% CI: 2.2%-4.4%) at 2 years and 3.7% (95% CI: 2.7%-5.0%) at 5 years after diagnosis. CNS involvement was identified after first line IC in 25 patients, and after salvage therapy in 11 patients. 22 patients had an isolated CNS relapse, while 14 had both systemic and CNS disease at relapse. The incidence of isolated CNS relapse at two years from diagnosis was 1.9% (95% CI: 1.2%-3.0%, figure 1). CNS involvement was parenchymal in 22 patients, leptomeningeal in 11, and parenchymal and leptomeningeal in 3 patients. At diagnosis, this subset of 36 patients had a median age of 61 years (range 20-86); 25 (69%) were male. IPI was 0-1 in 6 patients, 2 in 13 patients, 3 in 11 patients and 4-5 in 6 patients. Cell of origin per Hans algorithm was available in 17 patients, 8 were GCB and 9 were non-GCB. Extranodal sites of disease at diagnosis included testicular (2), renal (5), bone (10) and bone marrow (6). 7 patients had no sites of extranodal disease. LDH was elevated in 71%. The German High-Grade Non-Hodgkin Lymphoma Study Group CNS risk score was low risk in 6 patients, intermediate risk in 22 patients, and high risk in 8 patients. 6 patients received CNS prophylaxis with their initial immunochemotherapy, including 3 of the 21 patients who subsequently developed isolated CNS disease. First line therapy after CNS relapse included high dose methotrexate based chemotherapy in 22 patients and other systemic regimens in 6, with intrathecal (IT) chemotherapy in 5. Overall survival in patients after CNS relapse was poor with a median survival of 6.3 months (95% CI: 2.9-15.5) and 12-month survival rate of 29% (95% CI: 17%-49%). Patients who had CNS relapse following initial IC had better subsequent survival (median OS = 7.6 months) compared to patients with first presentation of CNS relapse following salvage therapy (OS = 2.1 months, p=0.005, see figure 2). 10 patients (28%) proceeded to SCT after CNS relapse, of which 6 remain alive at a median follow-up of 39 months. There were no differences in post-CNS relapse survival by age, sex, CNS risk score, cell of origin, extranodal site involvement at diagnosis, or site of CNS relapse (all p>0.10), though power was limited due to small numbers. Conclusions The incidence of CNS relapse in DLBCL is approximately 4% in the immunochemotherapy era. The vast majority of CNS relapses occur within 2 years of diagnosis. Isolated relapse in patients without CNS involvement at diagnosis is a relatively rare event with an incidence of approximately 2% at 2 years of diagnosis. Outcomes remain poor, and standard clinical variables do not predict survival after CNS relapse. Novel therapeutic approaches are needed in this population, with consideration to autologous SCT, which produces durable remission in a subset of patients. Disclosures Maurer: Kite Pharma: Research Funding. Farooq:Kite Pharma: Research Funding. Thompson:Kite Pharma: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding. Cerhan:Kite Pharma: Research Funding. Link:Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding.

Abstract Background Immune editing is a major mechanism used by tumors to promote their survival. We have previously reported the presence of immunosuppressive monocytes (CD14+HLA-DR low/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. Here we report an in vitro model to examine tumor monocyte cross talk and demonstrate that these monocytes may directly promote tumor cells’ resistance to chemotherapy. Method Monocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, Su-DHL2, Su-DHL6, Jeko-1, Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability, proliferation, and induced protein expression. Changes in vitro were confirmed in primary tissue by gene expression profile from 48 primary diffuse large B cell (DLBCL) lymphoma tumors. Results Monocytes converted CD14+HLA-DR+ to the CD14+HLA-DRlow/neg phenotype by co-culture with 5 of the 8 B cell lymphoma cell lines. The HLA-DR suppression was not due to the known regulatory effects of IL-10 nor was the effect seen in co-culture using B cells from healthy donors. Thus, the magnitude of DR loss is mediated by a yet undetermined mechanism. DOX incubation induced apoptosis in all of the cell lines. Co-culture with monocytes improved lymphoma cell survival in 3 of the 4 lymphoma cell lines. For example, untreated OCI-Ly3 had a 1.3±0.2 fold expansion that was reduced to 0.6±0.1 when treated with DOX (p<0.0001, n=13). However, when the cells were treated with DOX and co-cultured with monocytes the survival improved to 0.8±0.2 (p<0.0001, n=13). Having previously shown that CD14+HLA-DRlow/neg were capable of suppressing patient immunity, partially through arginase I, we determined if co-culture of monocytes with tumor cells increased arginase I expression in either monocytes or tumor cells. Not only did we see arginase I expression increases during co-culture(n=10, p<0.01), but surprisingly, the greatest increases were seen during co-culture and DOX treatment (n=10, p=0.01). To determine if our in vitro observations reflect human tumors, we used gene expression data obtained from 48 DLBCL tumors and found that increased CD14 gene expression was positively correlated with increased arginase I expression (n=48, p=0.02). Conclusions We have demonstrated that certain tumor cells induced phenotype changes in monocytes in an IL-10 independent and a tumor specific way. In those cells where tumors factors mediate monocyte phenotype, the monocytes can promote tumor cell resistance to cytotoxic killing by DOX, thus demonstrating a cross-talk between monocyte and tumor function. We found that in an environment of monocyte/tumor cross talk, arginase I expression is further increased in a cytotoxic environment. Together, this data demonstrates an active cross talk between monocytes and tumors resulting in multiple mechanisms of tumor resistance to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2409 Background: Serum free light chain (FLC) quantitation has prognostic value in MGUS, multiple myeloma (MM), solitary plasmacytoma, and amyloid light chain (AL) amyloidosis, and it has been incorporated in response criteria for both MM and AL amyloidosis. Two recent studies have shown FLC abnormalities are present in 39–54% of CLL patients and are associated with shorter time to treatment (TTT) and inferior overall survival (OS) in CLL. Here we report the largest study to date of FLC abnormalities in a prospective cohort of CLL patients and explore the effects of both monoclonal and polyclonal FLC elevations. Methods: Newly diagnosed CLL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum FLC was quantitated from stored research serum samples using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Elevated FLC was defined as either kappa or lambda above the reference range (k>19.4 mg/L, l>26.3 mg/L). Monoclonal FLC elevation was defined as elevated FLC with an abnormal FLC ratio. Polyclonal FLC elevation was defined as elevated FLC with a normal FLC ratio (0.26–1.65). Patients with normal FLC but abnormal FLC ratio were evaluated as a distinct group (Abnormal FLC Ratio). Clinical data was abstracted using a standard protocol for the MER and patients were followed systematically for TTT and OS. Cox proportional hazards models were used to assess the association of FLC with TTT and OS. Results: 385 CLL patients diagnosed between September 2002 and February 2008 with available serum were assayed. At a median follow-up of 47 months (range 1–94), 135 patients (35%) have been treated and 43 patients have died (11%). Abnormalities in FLC were present in nearly half of the CLL patients (48.8%). Although 32.4% had an elevated kappa or lambda, only 14.0% had a monoclonal FLC elevation while the remaining 18.4% had a polyclonal FLC elevation. An additional 16.4% had an abnormal ratio without elevated FLC. Monoclonal FLC elevation was associated with high risk features of CD38+, CD49D+, ZAP70+, IGHV unmutated and having high risk FISH (del 17p13; del 11q22). Monoclonal FLC elevation was highly associated with shorter TTT (HR=3.24, p=0.0007) and poor overall survival (HR=4.14, p=0.0006). These associations remained statistically significant after adjusting for stage and prognostic factors. Abnormal FLC ratio without elevated FLC was also associated with shorter TTT (HR=1.74, p=0.03), though this association did not hold after adjustment for stage and prognostic factors. Polyclonal FLC elevations were associated high risk FISH and poor overall survival (HR=6.41, p<0.0001, adjusted HR = 4.82, p=0.0002) but not with TTT. Conclusions: In the largest series of newly diagnosed CLL patients reported to date, nearly half of CLL patients have abnormal FLC concentrations. Monoclonal FLC elevation is an independent predictor of shorter TTT and inferior overall survival in CLL, is associated with high risk markers, and may be a marker of an aggressive tumor. Polyclonal FLC elevation without abnormal FLC ratio is independently associated with poor overall survival but not TTT suggesting it may be a non-specific marker of host frailty/comorbidity rather than disease specific outcomes. Regardless of cause, CLL patients with elevated FLC have poor outcome and FLC may have utility as a biomarker in CLL. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Background: Patients (pts) with aggressive NHL who relapse after conventional chemotherapy or stem cell transplant have a poor prognosis. Novel agents are needed for this important subset. Tipifarnib is an orally available methylquinolone derivative that is an inhibitor of the farnesyl transferase enzyme that transfers the farnesyl group from farnesyl pyrophosphate to specific polypeptides including small GTPases of the Ras, Rho-B, and Rheb families, as well as nuclear lamins and kinetochore proteins. Tipifarnib has documented activity in acute leukemia and produces stable disease in relapsed multiple myeloma. The goal of this study was to evaluate the activity of single-agent tipifarnib in patients with relapsed aggressive NHL in the context of a phase II trial. Methods: Pts with relapsed diffuse large, follicular grade III, or mantle cell lymphoma were eligible for this study. Pts were treated with oral tipifarnib 300 mg twice daily d1–21 q 28 days until progression. Pts were restaged after 2 cycles, 6 cycles, and every 3 cycles thereafter. Tumor was sampled prior to tipifarnib and at day 8 in selected patients with accessible tumors. These paired samples were assayed for accumulation of unprocessed HDJ-2 and prelamin A, two polypeptides that require farnesylation for processing. Results: Forty-two pts were accrued between March 2004 and June 2006. Thirty-seven pts had diffuse large cell, 1 follicular grade III, and 4 had mantle cell. Of the 38 patients evaluable for response, 7 (18%) had a partial response and 8 (21%) had stable disease. The 7 responders all had diffuse large cell histology. Median progression-free survival (PFS) for all pts was 2 months (range, 0–25+). The PFS of the 7 responders was noteworthy at 4.2, 4.9+, 6.7, 9.6, 14.9+, 21.6+, and 25.1+ months, respectively. The primary toxicity was myelosuppression and 33% of pts required dose reductions. The median dose received in all pts was 300 mg twice daily (mean 275, range, 100–400). The median dose received by the 7 responders was 200 mg twice daily (range, 100–400). The dose of 300 mg twice daily resulted in accumulation of unfarnesylated forms of one or both markers in all pts studied to date. Conclusion: Oral tipifarnib has antitumor activity as a single agent with an excellent toxicity profile in pts with relapsed/refractory aggressive lymphoma. When given in this dose and schedule, tipifarnib is well-tolerated and can be taken for months without cumulative toxicity. This drug warrants further investigation in combination with conventional chemotherapy and other novel agents for the treatment of this important subset of patients.

Abstract Small lymphocytic lymphoma (SLL) is an incurable indolent lymphoma, and there are relatively few prognostic biomarkers for this important NHL subtype. We evaluated the hypothesis that inherited variability in cytokine and immune-related genes was associated with overall survival in SLL. We genotyped 73 SNPs in 44 candidate genes in 140 SLL patients aged 20–70 years who participated in a population-based case-control study conducted from 1998–2000 through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles. DNA was extracted from a venous blood sample or mouthwash buccal cell sample. All genotyping was conducted at the National Cancer Institute Core Genotyping Facility using the Taqman platform. Histology, stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site in the spring of 2005. Cox proportional hazards analysis was used to estimate the hazard ratio (HR) and 95% confidence interval for the association between individual SNPs and overall survival, adjusted for age, demographic and clinical factors. Multiple simultaneous modeling strategies were used to identify the best summary multi-SNP risk score to predict survival. At last follow-up, there were 45 deaths in 140 patients (32%). The median follow-up of the 95 surviving patients was 58 months (range 24–75 months). In multivariate modeling, SNPs in IL13 (rs1800925; HRCC=2.36, 1.24–4.49), IL7R (rs1494555; HRGG =2.20, 0.84–5.76), and TNF-alpha (rs1800630; HRAC/AA=1.73, 0.95–3.17) were the strongest and most robust predictors of survival. Two or more deleterious genotypes from these three SNPs increased the risk of death (HR=2.2, 1.2–4.1) compared with one or fewer deleterious genotypes (p=0.009). Three groups (low, intermediate, and high risk) were defined by combining the SNP score and a clinical/demographic score. The 5-year Kaplan-Meier survival estimates for these groups were 85%, 65%, and 11%, respectively. Compared to patients with a low risk score, patients with intermediate (HR=2.5, 1.2–5.1) or high (HR=11, 4.3–27.7) risk scores had poorer overall survival (p=3.9 x 10−8). Our preliminary results suggest that SNPs in IL13, IL7R, and TNF-alpha alone and in combination predict overall survival in SLL, lending support to the hypothesis that host genetic background is a promising class of prognostic biomarkers in SLL.

Abstract Tumors arise and evolve by iterative steps of mutation, subclonal selection, and clonal expansion due to growth advantage of the fittest subclones and external mutation induction and selection pressure from radiation and drug therapies. Various studies have shown that greater clonal complexities in primary tumors are correlated with poor clinical outcome of tumor progression and/or drug resistance. Recently, we have reported that very few of the driver mutations, all of which were clonal, detected in primary diffuse large B-cell lymphoma (DLBCL) were associated with DLBCL outcome as measured by 24-month event-free survival (EFS24). In the current project, we identified and studied subclonal mutations in primary DLBCL tumors and assessed their associations with EFS24, as well as the role of activation-induced cytidine deaminase (AID) in genomic mutations occurred in DLBCL tumors. The detection of subclonal mutations is still a significant bioinformatics challenge. In addition, identification of clonal mutations in samples with lower tumor purities faces similar challenges due to the low concentration of reads supporting the mutant alleles. The current methods of somatic mutation calling are not sufficiently sensitive to identify the low concentration mutations in tumor DNA sequencing data. We implemented a bioinformatics workflow for low-concentration and subclonal mutation detection which is based on the positional read pile-up data and a back-fill approach (PUB). The pile-up files were first generated using the re-aligned and re-calibrated BAM files after read alignment using Burrows-Wheeler Aligner (BWA). The variant positions with alternative bases were annotated by attributes defined in the Variant Quality Score Recalibration (VQSR). PUB then used a boosting method and a generalized linear model (GLM) to train a model of 'good quality' variants using common variants from HapMap, and prioritized and called clonal and subclonal variants based on the trained model. The VQSR attributes related to alternative allele depth were less-weighed in order to call subclonal mutations. The somatic mutations were then identified by Fisher's Exact Test using sequencing depths of the reference and alternative alleles from paired tumor and germline sequencing data. The exome sequencing data of paired tumor and peripheral blood from 48 newly diagnosed DLBCL patients were analyzed using PUB. Thirty-six of the 48 patients achieved EFS24 with the other 12 patients experienced primary treatment failure. Most of the tumors studied had tumor purities between 40-70%. PUB identified substantially higher number of somatic mutations, both clonal and subclonal, compared to those detected using existing somatic callers. We observed that the prevalence of mutations in previously reported driver genes were higher using thresholds of mutation concentration ≥ 5% and Fisher's Exact Test p ≤ 0.05, including EZH2 (mutated in 26% of DLBCL tumors analyzed by PUB, compared to 12.7% as previously reported), MLL2 (72% vs. 31%), CD79B (22% vs. 14%), TNFRSF14 (36% vs. 20%), MEF2B (22% vs. 16.4%), CARD11 (46% vs. 21.8%), and MYD88 (18% vs. 11%). In addition, other genes involved tumorigenesis that have not been previously linked to DLBCL also harbored both clonal and subclonal mutations with substantial prevalence, including FGFR3 (12%), KIT (24%), and ATM (28%). Furthermore, association of genes displaying clonal and subclonal mutations with EFS24 identified potential biomarkers for DLBCL outcome. Among these, two genes EPGN and ASTE are involved in epithelial growth factor receptor signaling and had association p values of 0.0001 and 0.0016, respectively. The oncogene MAFB was also associated with EFS24 (p= 0.0016). We searched for AID induced mutations among all identified variant positions and concluded that there was no evidence of AID site enrichments compared to a simulated data set. In summary, we developed and applied a sensitive bioinformatics pipeline for the identification of both clonal and low concentration somatic mutations in primary DLBCL exome sequencing data which further revealed the clonal complexity of the primary DLBCL tumors. Several of the genes were identified as potential biomarkers for DLBCL outcome. Disclosures Maurer: Kite Pharma: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding. Link:Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding. Cerhan:Kite Pharma: Research Funding.