Cerhan, James R.

Abstract Background Next Generation sequencing (NGS) is a powerful tool to identify somatic mutations associated with tumor onset and drug response. While it is well suited for high quality fresh/frozen samples, NGS is not proven for FFPE tissue which is the most common type of clinical specimen. Since the nucleic acids can be readily extracted from FFPE samples for a variety of genomic analyses, a comparative mutational analysis of paired frozen and FFPE tissues is urgently needed. Our long term goal is to establish a lab protocol to detect mutations in FFPE tumors using a targeted capture and sequencing approach for genes of interest. This pilot study focuses on the comparison of FFPE and frozen samples to test the validity of using FFPE tissues in such application. Methods Gene Selection: 128 genes associated with known pathogenic mutations in lymphoma Sample Selection: 9 diffuse large B-cell lymphoma (DLBCL) cases with FFPE, frozen and germline samples, as well as 10 frozen normal lymphatic tissues as references for CNV detections Capture Probe Design: We targeted coding exons and UTR, as well as the evolutionarily conserved intronic regions. The capture probes were designed using the Agilent eArray tool. The titling density of the probes was set to 3 probes overlapping with every base in the target region to improve the capture efficiency in FFPE samples. The least stringent masking of the repeat regions was allowed to include regions with small repeats that are shorter than the length of the sequencing reads (100-bp). In addition, boosting parameters were picked to set various levels of probe replication in different regions in order to minimize the local coverage differences (e.g. between regions of different GC contents) Sequencing and Bioinformatics: The target capture and sequencing were performed by the Mayo Clinic Medical Genome Facility. The reads were mapped to Human Reference Genome Build 37 using Novalign, and SNVs were called using GATK. The CNVs were identified using an in-house developed algorithm, patternCNV. Results The designed probes covered 99.65937% of the target regions. We generated 2.2-6.7 Gbp of reads per sample, 57.4-71.5% of which were on target. This equalled an average coverage of 2100-6700 folds which is 10-30 times higher than the minimal coverage recommended by Agilent. Due to this high coverage, we observed duplicate reads that accounted for 7.7-73.5% of the total reads. When we analysed the data with and without the duplicated reads, the concordance of the called SNVs was between 84-93% out of 207-249 mutated positions per trio-sample. There were 7.8-8.9% and 1.1-2.2% unique SNVs per sample by excluding or including duplicate reads, respectively. The dis-concordances were mostly missed calls, where a SNV was observed in only 1 or 2 of the trio samples. The missed calls from frozen samples ranged from 0-10.4% compared to 1.4-10.4% from the FFPE tissues, with 0.88-2.4% more SNVs missed in FFPE. Further analyses showed that all of the missing calls came from the lack of or low coverage of the corresponding positions. There were also differences of the called SNVs between the trio samples. However, this was extremely rare. Only 2 out of the 9 trio samples at a total of 3 positions had disagreements in called SNVs between FFPE and frozen tissues, all due to the allelic imbalance where the percentage of reads supporting the alternative alleles were below 20%. Therefore, this dis-concordance can be removed by back-filling of the read-level information for each position. Unfortunately only 11.9-47.4% of the CNVs called in frozen tissues were identified in FFPE samples, due to the widely various coverage in FFPE samples. The consequent large noises of the log ratio values between the FFPEs and normal references significantly reduced the sensitivity for CNV calling. Conclusions This pilot study compared the performance of SNV and CNV detection in FFPE and paired frozen tissues using a target capture and sequencing approach. With a capture probe design strategized to benefit FFPE samples, we observed SNV detection rates in FFPE that were only slightly lower (0.88-2.4%) than those of frozen tissues due to poor coverage of some positions in FFPE samples. With a proper back-filling step, there was no dis-concordance of the called SNVs between FFPE and frozen samples. However, CNV detections in FFPE were more problematic due to the un-predictable regional coverage in FFPE samples. Disclosures: No relevant conflicts of interest to declare.

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 Abstract 2443 Background: There is strong and consistent evidence that a genetic component contributes to the etiology of chronic lymphocytic leukemia (CLL). A recent genome-wide association (GWA) study of CLL identified genetic variants located on chromosomes 2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13 that increased the risk of CLL within a European population. We replicated 5 of these 6 loci in an independent sample of CLL cases and controls from the United States. We now investigate whether these loci also influences MBL, a reported precursor condition of CLL. In addition, a follow-up analysis of the initial GWA study identified four more CLL-susceptibility loci on 2q37.3, 8q24.21, 15q21.3, and 16q24.1. Herein, we also evaluate the association of these four loci with risk of CLL. Methods: Peripheral blood samples were obtained from three ongoing studies: the Genetic Epidemiology CLL (GEC) Consortium, the Mayo Clinic non-Hodgkin lymphoma (NHL)/ CLL study, and the Mayo Clinic Biobank. We implemented rigorous genotyping quality-control measures, and successfully genotyped a total of 407 CLL patients, 965 controls, and 60 MBLs from these studies. Within each locus, the previously reported single nucleotide variants (SNPs) or variants in high linkage disequilibrium (LD) with the previously reported SNPs were evaluated with risk of MBL or CLL. Tests for association was done using the Cochran-Armitage trend test, and unconditional logistic regression was used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for CLL or MBL risk Results: In our evaluation of the six initially reported CLL-susceptibility loci (2q13, 2q37.1, 6p25, 11q24, 15q23, and 19q13) with MBL risk, we found three of the six had suggestive associations (p-value < 0.20) with ORs comparable to and in the same direction as those observed from CLL risk. Our strongest finding was with rs13397985 at locus 2q37.1 (OR= 1.56; 95% CI: 1.05, 2.31; p-trend = 0.041), followed by rs17483466 at locus 2q13 (OR= 1.49; 95% CI: 0.99, 2.24; p-trend = 0.074). As expected given our previously reported findings with CLL risk, the association between rs11083846 on chromosome 19q13 and MBL risk was not significant (p-trend = 0.70). Of the four recently reported CLL-susceptibility loci SNPs located on 2q37.3, 8q24.21, 15q21.3, and 16q24.1, we found all to be associated with CLL risk but one. Specifically, the strongest association was seen for locus 8q24.21 (best tagged SNP rs1021955; OR = 1.37; 95% CI: 1.10, 1.70; p-trend = 0.005), followed by locus 16q24.1 (best tagged SNP rs305065; OR= 0.77; 95% CI: 0.61, 0.97; p-trend = 0.024). However, we found no associations for locus 15q21.3 for the previously reported SNP nor for any SNPs in LD with the previously reported SNP. Conclusions: Our MBL results provide additional robust genetic evidence that MBL is a precursor to CLL and that it shares similar underlying genetic predisposition. Also our results confirm three of the four recently reported CLL-susceptibility loci and further support the role of a genetic basis in the etiology of CLL. More research is needed to elucidate the potential manner in which these genetic loci function in CLL or MBL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4610 Introduction Infections are a major cause of morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). Up to 50% of CLL patients develop infectious complications, and 30–50% of all deaths in CLL patients are attributed to infections. Despite these data, the available research on infectious complications in CLL is primarily limited to patients participating in clinical trials of chemotherapy, obscuring the distinction between the risk of infection due to the disease’s natural history relative to the risk associated with therapy related immune suppression. METHODS: The Mayo Clinic Rochester (MCR), a tertiary referral center in rural southeastern Minnesota, is also the primary hematology center for southeastern Minnesota, northern Iowa, and western Wisconsin. There are no metropolitan areas or hematology specialty centers within a ∼50 mile radius. To explore the natural history of infection in a community-based cohort of CLL patients, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed CLL who resided within 50 miles of MCR and diagnosed between January, 1999 and December, 2009. The comparison cohort consisted of 689 adult patients who also resided within 50 miles of MCR who were seen for a general medical examination between September, 2002 and December, 2009 and who were enrolled as controls in a case-control study of non-Hodgkin lymphoma. All hospitalizations at MCR among both cohorts were audited to document hospitalization with infection and cross-referenced with the Mayo Clinic infection database, which includes all culture results obtained from hospitalized MCR patients. Serologies and viral assays were also reviewed. Patients whose cultures were negative but who were given a clinical diagnosis of infection (e.g. pneumonia) and who were treated with a full course of antibiotic therapy were considered to have a culture negative infection. RESULTS: In May 2010, there were 2022 CLL patients in the Mayo Clinic CLL Database diagnosed between 1999 and 2009. Of these, 174 (9%) were local CLL patients residing within 50 miles of MCR. Median age at diagnosis for these non-referred CLL patients was 69 years (range 27–97). Most had early stage disease (60% stage 0; 32% stage I-II). On prognostic evaluation, 121 (75%) patients were CD38 negative, 66 (61%) ZAP-70 negative, 47 (50%) IGHV mutated, and 56 (62%) had either no abnormality or 13q14- as a sole abnormality on FISH analysis. 64 (37%) patients progressed to require treatment for CLL during follow-up. After median follow-up of 4.0 years (range 0–11), 32 (18.4%) CLL patients were hospitalized with infection at least once as compared to 20/689 (2.9%) individuals in the control cohort (odds ratio=7.5; p<0.0001) Figure 1A. Even prior to receiving treatment for CLL, the rate of hospitalization for infection remained higher than the control cohort (p=0.002; Figure 1B). The 32 CLL patients hospitalized with infection were admitted with infection a total of 89 times (median 1 hospitalization for infection per patient). A specific infectious organism was identified by cultures or serologies in 60 (63%) of the 89 infections in CLL cases and 10/27 (37%) infections among controls. Hospitalization for an opportunistic infection (e.g. cytomegalovirus, cryptococcus, filamentous fungi, mycobacteria, Pneumocystis) occurred in 5/174 (3%) CLL patients during follow-up compared to 0/689 controls (p<0.001). Finally, we performed a pooled multivariable analysis of all patients (n=863) to identify factors (age, sex, CLL diagnosis, CLL treatment) associated with infection risk. Most factors were independently associated with hospitalization for infection [Age (per year, OR=1.03, p=0.02), sex (male, OR=4.0, p<0.001), diagnosis of CLL (OR=2.1, p=0.09), and treatment for CLL (OR=5.9, p<0.001)]. In univariate analysis of CLL patients [sex (OR=4.2, p=0.01), higher Rai stage (p=0.046), unfavorable (del 17p13; del 11q23) FISH [OR=4.6, p=0.02] and unmutated IGHV [OR=2.8, p=0.07]) were associated with risk of infection. CONCLUSIONS: In this cohort study, patients with newly diagnosed CLL had a 7.5-fold risk of hospitalization for infection relative to the control cohort. After 4 year follow-up, ∼1 in 5 CLL patients required hospitalization for infection. Although CLL treatment was a substantial risk factor for infection, the risk of infection among untreated patients remained higher than controls. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity’s Board of Directors or advisory committees. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

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.

Abstract Background: Obesity has been associated with risk of non-Hodgkin lymphoma (NHL) in several recent studies. However, the effect of weight gain during adulthood, a potentially modifiable risk factor, on the incidence of NHL is not clear. We therefore examined the association of weight gain since age 18 years with the risk of NHL among older women. Methods: We examined this association in the Iowa Women’s Health Study, a prospective study of 37,762 Iowa women aged 55 to 69 years at enrollment in 1986. Lifestyle and other risk factors, including weight at age 18 and at enrollment, were self-reported on a mailed questionnaire. We defined stable weight as no increase in weight or up to a 10% weight loss from age 18 to enrollment, while percent weight gain was divided into tertiles based on the distribution observed in the cohort. NHL incidence through 18 years of follow-up was ascertained by linkage with the Iowa SEER Cancer Registry. Cox proportional hazards models were used to estimate relative risks (RRs) and 95% confidence intervals (CIs) of percent weight gain with NHL incidence, adjusting for age (Age-adjusted model) and age, marital status, farm residence, transfusion history, diabetes, use of hormone replacement therapy, alcohol use, smoking, and intake of red meat and fruits (Multivariate model). Results: The mean age of the women in the cohort in 1986 was 62.1 years. During 593,474 person-years of follow-up, 379 incident cases of NHL were observed. The mean age at diagnosis was 72.6 years (range 58–87 years). The main results are reported in the table. Similar results were found for absolute weight gain. Stratification of these results on body mass index at age 18 (using a cutpoint of 21 kg/m2) showed similar results, with the exception of >10% weight loss for women with a BMI of <21 at age 18 where we could not estimate a RR (no cases observed). Conclusions: Weight gain or large weight loss (>10% of body weight) during adulthood were both associated with increased risk of NHL, although the RRs for percent weight gain were not statistically significant and there was no dose-response. This study suggests another potential benefit of avoiding adult weight gain, although further studies are needed to confirm these novel findings. Association of percent weight change during adulthood (age 18 to age 55–69 years) and risk of NHL % Weight Change NHL Cases Person-years Age-Adjusted RR(95% CI) Multivariate RR (95% CI) >10% weight loss 15 17053 2.06 (1.05–4.02) 2.02 (1.03–3.95) Stable 20 45973 1 (reference) 1 (reference) 0–18.2% gain 115 175883 1.50 (0.94–2.42) 1.50 (0.93–2.41) 18.3–34.8% gain 119 176258 1.54 (0.96–2.47) 1.53 (0.95–2.47) >34.8% gain 110 178308 1.40 (0.87–2.25) 1.32 (0.82–2.14).

Abstract DLBCL is a curable subtype of non-Hodgkin lymphoma, although a significant number of patients do not achieve a remission or they relapse with conventional chemotherapy. While clinical variables (e.g., IPI), tumor (somatic) genetic alterations, and gene expression profiling have all been shown to predict outcome, there remains a need for additional prognostic biomarkers. One understudied class of biomarkers is host genetic background. We evaluated the hypothesis that germline variability in 73 SNPs from 44 candidate immune genes was associated with overall survival in DLBCL. We addressed this hypothesis in 365 DLBCL patients aged 20–70 years who participated in a population-based case-control study conducted from 1998–2000 (prior to the use of R-CHOP) through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles. Germline DNA was extracted from a venous blood sample or mouthwash buccal cell sample, which was collected a median of 4.8 months after diagnosis in this population-based study. All genotyping was conducted at the National Cancer Institute Core Genotyping Facility using the Taqman platform, and was successful in over 95% of the DNA samples for the SNPs evaluated. 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 evaluate the association between individual SNPs, adjusted for age, demographic and clinical factors. Parallel modeling strategies were used to identify the best summary multi-SNP risk score to predict survival. At a median follow-up of 56 months (range, 27-78 months) for surviving patients, there were 96 deaths in 365 patients (26%). In multivariate modeling, SNPs in IL1A (rs1800587; HRCT/TT=1.90, 1.26–2.87), IL6 (rs1800795; HRGG=1.48, 0.99–2.23), IL-10 (rs1800896; HRAG/GG=1.48, 0.91–2.38), and IFNGR2 (rs2070385; HRAG/GG=1.35, 0.86–2.11) were the strongest and most robust predictors of overall survival. A summary score of the number of deleterious genotypes from these four genes in combination with clinical and demographic variables was strongly associated with survival (p=9.3 x 10−12); Kaplan-Meier 5-year survival estimates for low, intermediate, and high risk patients were 89%, 68%, and 47% respectively. In conclusion, host genetic background as measured by germline polymorphisms in immune genes including IL1A, IL6, IL10, and IFNGR2 were associated with overall survival in DLBCL after accounting for clinical and demographic factors. These promising results require confirmation and need further evaluation in patients treated with R-CHOP in conjunction with tumor and other prognostic biomarkers.

Abstract Abstract 1540 Background: Diffuse Large B-cell Lymphoma (DLBCL) is the most common lymphoma in the western world. Outcomes have improved with the standard initial therapy of rituximab and anthracyline based chemotherapy (immunochemotherapy). However, 20–40% of patients will either fail to achieve remission or relapse following initial immunochemotherapy. Most relapses occur early, and long-term outcome of relapsed/refractory patients is generally poor despite salvage regimens and stem cell transplant. Traditionally, studies for DLBCL have been evaluated using progression-free and/or overall survival. However, the event rate in DLBCL slows significantly approximately 12–18 months after diagnosis, and late events are often due to competing (non-DLBCL related) risks, especially in older patients. Here we examine outcome of DLBCL patients based on their event status at 12 months. Methods: Newly diagnosed DLBCL patients were prospectively enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource (MER). Clinical data were abstracted from medical records using a standard protocol. Patients were actively followed for events (progression, re-treatment, or death due to any cause) and overall survival (OS). Event-free status at 12 months after diagnosis (EFS12) was assessed as a dichotomous variable. Cause of death was determined by medical record and death certificate review using prospectively determined definitions. Expected survival was based on age and sex matched survival using the Minnesota population death rates. Confirmation cohorts were from a Lyon, France hospital based registry and NCCTG clinical trial N0489. Results: 680 patients with newly diagnosed DLBCL and treated with rituximab + anthracycline based chemotherapy were enrolled in the MER from 2002 to 2009. The median age was 62 years (range 18–92). 53% were male. At a median follow-up of 59 months (range 8–116), 266 patients (39%) had an event and 188 patients (28%) died. 162 patients (24%) had an event in the first 12 months after diagnosis, comprising 60% of all events. Patients with an event in the first 12 months had poor survival with death almost exclusively due to disease (Figures 1a, 2a). In contrast, patients who were event-free at 12 months had comparable survival to the age and sex matched general population (Figure 2b) and were more likely to die of other causes than DLBCL (Figure 1b). Overall survival rates by EFS12 status were validated in patients from a French hospital-based registry (N=265) and the NCCTG N0489 clinical trial (N=87) with additional replication studies underway. Conclusions: DLBCL patients who are event-free at 12 months after diagnosis have an excellent prognosis with an overall survival similar to that of an age- and sex- matched general population and are more likely to die of other causes than DLBCL. This finding has implications for clinical management, new clinical trials, and follow-up testing in this patient population. In contrast, patients with an event within the first 12 months after diagnosis have a poor prognosis with almost all deaths secondary to DLBCL. EFS status at 12 months identifies patients with poor outcomes due to disease. This can be utilized as an endpoint in biologic studies and clinical trials to address early treatment failures. EFS status at 12 months should be incorporated into prognostic models to identify high risk patients in newly diagnosed DLBCL. Disclosures: No relevant conflicts of interest to declare.