Kelly, Jennifer L.

Abstract Abstract 3933 Poster Board III-869 Background Non-Hodgkin Lymphoma (NHL) is a malignancy of lymphocytes with few known risk factors identified to date. Members of the BCL2 and caspase gene families are known regulators of programmed cell death in these cells, and the t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in NHL, a somatic event which results in constitutive BCL2 expression and inhibition of apoptosis. Further, recent pooled analyses of three case-control studies reported gene level associations for both BCL2L11 (Cancer Epidemiol Biomarkers Prev 2009;18:1259) and CASP9 (Blood 2009;114:264) with NHL risk. We therefore evaluated the hypothesis that germline variation in genes from the apoptosis pathway is associated with risk of developing NHL. Methods We genotyped 226 single nucleotide polymorphisms (SNPs) within 36 candidate BCL2-family and capsase-family member genes within the apoptosis pathway in a clinic-based study of 441 newly diagnosed NHL cases (within 9 months of first NHL diagnosis) and 475 frequency matched controls from Minnesota, Iowa, and Wisconsin who were seen at the Mayo Clinic from 2002-2005. Tagging SNPs were selected from HapMap for the following candidate genes: BCL2; BCLAF1; BCL2LA1; BCL2L1, 2, 10, 11, 12, 13, 14; BAD; BAX; BAG1, 3, 4, 5; BAK1; BID; BIK; BNIP2, 3; HRK; CASP1 – CASP10; APAF1; BIRC3; AIF1; and DFFB. Genotyping was completed using the ParAllele (now Affymetrix) Immune and Inflammation SNP panel, supplemented with 9 SNPs from a custom Illumina OPA. SNP call rate and per person call rate were >95% for all SNPs evaluated. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect in a logistic regression model adjusted for age, sex, and residence. We used principal components analysis to assess the association with NHL risk at the gene level. Genes at p<0.05 were declared statistically significant, as were SNPs at p<0.05 from significant genes. Results The mean age at diagnosis for cases was 60.1 years, and 58% were male; among controls, the mean age at enrollment into the study was 61.7 years, and 55% were male. The most common NHL subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 29%), follicular lymphoma (FL, 27%), and diffuse large B-cell lymphoma (DLBCL, 16%). In gene level analyses, BCL2L11 (also know as BIM) (p=0.0019), BLCAF1 (p=0.0097), BAG5 (p=0.026), and CASP9 (p=0.0022) were statistically significant at p<0.05. For BCL2L11, 2 of the 5 tagSNPs (both intronic) were significant at p<0.05 (rs6746608 and rs12613243). Both genotyped BCLAF1 tagSNPs (rs797558 and rs703193, both intronic) were significant, as was the single genotyped BAG5 tagSNP (rs7042474) from the non-coding region interval of an mRNA transcript. Three of the 7 genotyped CASP9 tagSNPs were significant (rs6685648, rs2020902, rs2042370, all intronic). Similar associations (as assessed by ordinal odds ratios) were observed for significant SNPs from BCLAF1, BAG5, BCL2L11 and CASP9 within the subtypes of CLL/SLL, FL, and DLBCL; however, caution is warranted in interpreting the subtype results due to small sample sizes. Conclusions Three genes from the BCL2 family (BCL2L11, BCLAF1, and BAG5) and one gene from the caspase family (CASP9) were associated with risk of NHL. This confirms gene level results for BCL2L11 and CASP9 in NHL overall and suggests a role for additional BCL2 family members in NHL etiology. In addition to extending these results to other populations, investigation of the functional significance of these genes and consideration of their upstream regulators and downstream targets should assist in clarifying the role of this pathway in lymphomagenesis. Furthermore, in the era of targeted therapeutics, identification of genetic variation in the apoptotic pathway may also assist in predicting prognosis as therapies targeting this pathway are developed. 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 684 Background. Obesity has been associated with an increased risk of developing of lymphoma, particularly DLBCL. Genetic variation in several energy balance genes, including leptin (LEP), its receptor LEPR, ghrelin (GHRL) and neuropeptide Y (NPY) have been shown to modulate risk of lymphoma, although their impact on survival is not known. In contrast, recent studies suggest that patients either overweight (Body Mass Index (BMI), 25 to<30 kg/m2) or obese (≥30 kg/m2) may have a superior prognosis in DLBCL, although its impact in FL and in the current treatment era is not known. We evaluated the association of BMI at the time of diagnosis, 2 years prior to diagnosis and at ages 18, 35 and 50 years, along with germline genetic variation in selected energy balance genes with event-free (EFS) and overall (OS) survival in DLBCL and FL. Methods. We evaluated these associations in a prospective cohort of newly diagnosed DLBCL (N=764) and FL (N=649) patients enrolled at the Mayo Clinic and University of Iowa from 2002–2009 as part of the Molecular Epidemiology Resource. Peripheral blood DNA, clinical data and self-reported historical weights were collected at enrollment. Patients received standard disease treatment strategies, and were systematically followed for disease progression, re-treatment, and death. Single nucleotide polymorphisms (SNPs) were genotyped as part of a custom Illumina iSelect panel. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled as having a log-additive effect in the regression model. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CI) for the association of the obesity measures and SNPs with outcome, adjusted for treatment class and subtype specific prognostic factors (IPI, FLIPI). Results. For DLBCL patients (median age 63 years, range 18–92) the median follow-up was 47 months (range 0–101); 58% had an IPI 0–1 risk disease; and 87% received immunochemotherapy. For FL patients (median age 60 years, range 25–94), the median follow-up was 60 months (range 11–110); 24% had FLIPI score 3–5; and main initial treatments were observation (32%), rituximab (R)-monotherapy (12%), CVP +/−R (19%), and anthracycline +/−R (22%). The main results for BMI and prognosis are reported in the Table. For DLBCL, obese patients at diagnosis had better EFS and OS; this association was weaker for BMI at age 50 and 35 years. In contrast, for FL, overweight and obese patients at diagnosis had a poorer EFS and OS, and this was also observed for BMIs two years before diagnosis and at ages 50 and 35 years. In FL, the LEP SNP rs2167270 was associated with inferior EFS (HR=1.25; 95%CI 1.05–1.49) and OS (HR=1.28; 95%CI 0.90–1.83). The NPYSNP rs16147 was associated with inferior FL OS (HR=1.50; 95%CI 1.03–2.17) but was not associated with EFS. None of the energy balance and metabolism SNPs were associated with DLBCL EFS or OS. Conclusions. Higher BMI prior to and at diagnosis, as well as SNPs in LEP and NPY,were significantly associated with FL prognosis, with inferior outcomes in overweight and obese patients. These novel findings in the modern treatment era suggest an important role of obesity pathways in FL prognosis. In DLBCL, we confirm the recent findings of improved survival in DLBCL in obese patients, and extend this to the modern treatment era and BMI earlier in life. Disclosures: No relevant conflicts of interest to declare.

Abstract Background. Several recent studies have reported an inverse relationship between sun exposure and non-Hodgkin (NHL) risk. One hypothesis is that vitamin D mediates this effect. We evaluated the association of sun exposure at various ages and common variants in vitamin D related genes with risk of NHL and its major subtypes. Methods. Newly diagnosed NHL cases (N=1023) and frequency matched controls (N=1254) were enrolled in a clinic-based case-control study conducted at the Mayo Clinic. Thirty-eight single nucleotide polymorphisms (SNPs) from 5 candidate genes relevant to vitamin D metabolism and sun sensitivity (IRF4, RXR, VDR, CYP24A1, CYP27B1) were genotyped. Participants also completed a detailed questionnaire, including self-reported sun exposure at ages 13-21, 22-40, and 41+ years. Odds ratios (OR), 95% confidence intervals (CI), and tests for trend and effect modification were estimated using unconditional logistic regression adjusted for age and gender. ORs are reported for the highest vs. lowest exposure category or as the ordinal OR for SNPs. Results. The mean age of diagnosis/enrollment was 61 years for cases and 60 years for controls. The most common subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; 34%), follicular (FL; 24%), and diffuse large B-cell (DLBCL; 18%) lymphomas. There was a significant decrease in NHL risk with increased sun exposure at ages 13-21 years (OR=0.66, 95% CI 0.44-1.00; ptrend=0.04), which attenuated with increasing age at exposure. Exploratory analysis by NHL subtype indicated a trend toward a protective effect of sun exposure at ages 13-21 years for each of the major subtypes, though these estimates did not reach statistical significance. Of the 38 SNPs evaluated, there was a significant association for 3 SNPs in VDR and 1 SNP in CYP24A1: rs3819545 (OR=1.24, 95% CI 1.10-1.40, p=0.0004), rs2239186 (OR=1.22, 95% CI 1.05-1.41, p=0.009), and rs886441 (OR=0.82, 95% CI 0.70-0.96, p=0.02) for VDR and rs2762939 (OR=0.85, 95% CI 0.75-0.98, p=0.02) for CYP24A1. Both the VDR and CYP24A1 associations were observed for CLL/SLL, FL, but were attenuated for DLBCL. There was no evidence of any significant effect modification of sun exposure at ages 13-21 years by genotype in these four SNPs for NHL overall, or for NHL subtypes. Conclusions. We observed an inverse association between the frequency of sun exposure at ages 13-21 years and NHL risk, which attenuated for sun exposure at older ages, suggesting that early life exposure may be more relevant. While genetic variation in VDR and CYP24A1 were associated with NHL risk, there was no evidence for an interaction of sun exposure with these genes, suggesting that a sun association may not act through the vitamin D metabolism pathway. This would be consistent with a lack of association of circulating vitamin D with NHL risk recently published from the Cohort Consortium pooling project. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2011-1894

Abstract Background: Obesity has been linked to NHL risk in some studies, and may be stronger for obesity in early adulthood (ages 18-20). There has only been limited evaluation of physical activity and risk of NHL, particularly physical activity earlier in life. The purpose of this study was to evaluate the association of body mass index (BMI), physical activity (PA), and NHL risk during adulthood and at age 18. Methods: Self-reported anthropometrics and PA were collected in a clinic-based study of 954 newly diagnosed NHL cases and 1146 controls enrolled at the Mayo Clinic from 2002-2008. PA variables included duration and frequency of mild, moderate, and strenuous PA two years prior to case diagnosis or control selection, strenuous activity at age 18 (any vs. none), and height and weight at both time periods. An overall PA index was estimated by weighting the reported duration and frequency of each PA intensity by the average energy requirement (defined in METs) to obtain an average MET-minutes/week for each participant. BMI was calculated as weight (kg) divided by height (m) squared. Odds Ratios (OR), 95% confidence intervals (CI), and test for trend were estimated using unconditional logistic regression adjusted for the age, gender, and residence. Continuous variables were divided into quartiles according to the distribution among the controls; results are reported for the highest vs. lowest category or quartile unless otherwise specified. Results: The mean age at diagnosis/enrollment was 61 for both cases and controls; 47% and 43% of the cases and controls, respectively, were female. The most common NHL subtypes were CLL/SLL (32%), follicular lymphoma (FL; 26%), and diffuse large B-cell lymphoma (DLBCL; 21%). BMI 2 years prior to diagnosis was not associated with NHL risk (OR=1.21, p-trend=0.13), while there was a positive association with BMI at age 18 (OR=1.40, p-trend=0.001). There was no association of the PA index with risk (OR=0.91, p-trend=0.29). Furthermore, frequency of walking (OR=0.71, p-trend 0.43), mild (OR=1.30, p-trend=0.64), moderate (OR=0.99, p-trend=0.75), and strenuous (OR=0.77, p-trend=0.20) PA were not associated with NHL risk when evaluated individually. However, there was a suggestive weak inverse association between strenuous activity at age 18 and NHL risk (OR=0.85, 95% CI 0.71-1.05). On exploratory analysis, the association of BMI at age 18 with NHL risk was specific to DLBCL (OR=1.84, p-trend=0.002) and CLL/SLL (OR=1.25, p-trend=0.044) but not FL; all other results were similar across these subtypes. Conclusions: We did not observe an association between adult BMI and NHL risk; however a higher BMI in early adulthood was associated with NHL risk, consistent with limited prior reports. We found little evidence to support an association between PA in either early adulthood or later in life and NHL risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1833.

Abstract Background: The t(14;18)(q32;q21) translocation is the most commonly observed chromosomal translocation in non–Hodgkin's lymphoma (NHL), resulting in constitutive Bcl-2 expression and apoptosis inhibition. In addition, germline variation in both BCL2L11 (BIM) and CASP9, known regulators of apoptosis, has recently been linked to NHL risk. We conducted a comprehensive evaluation of 36 apoptosis pathway genes with risk of NHL. Methods: We genotyped 226 single-nucleotide polymorphisms (SNP) from 36 candidate genes in a clinic-based study of 441 newly diagnosed NHL cases and 475 frequency-matched controls. We used principal components analysis to assess gene-level associations, and logistic regression to assess SNP-level associations. MACH was used for imputation of SNPs in BCL2L11 and CASP9. Results: In gene-level analyses, BCL2L11 (P = 0.0019), BCLAF1 (P = 0.0097), BAG5 (P = 0.026), and CASP9 (P = 0.0022) were associated with NHL risk after accounting for multiple testing (tail strength, 0.38; 95% confidence interval, 0.05-0.70). Two of the five BCL2L11 tagSNPs (rs6746608 and rs12613243), both genotyped BCLAF1 tagSNPs (rs797558 and rs703193), the single genotyped BAG5 tagSNP (rs7693), and three of the seven genotyped CASP9 tagSNPs (rs6685648, rs2020902, and rs2042370) were significant at P &lt; 0.05. We successfully imputed BCL2L11 and CASP9 SNPs previously linked to NHL, and replicated all four BCL2L11 and two of three CASP9 SNPs. Conclusion: We replicated the association of BCL2L11 and CASP9 with NHL risk at the gene and SNP level, and identified novel associations with BCLAF1 and BAG5. Impact: Closer evaluation of germline variation of genes in the apoptosis pathway with risk of NHL and its subtypes is warranted. Cancer Epidemiol Biomarkers Prev; 19(11); 2847–58. ©2010 AACR.

Purpose Vitamin D insufficiency is common in the United States, with low levels linked in some studies to higher cancer incidence, including non-Hodgkin's lymphoma (NHL). Recent data also suggest that vitamin D insufficiency is related to inferior prognosis in some cancers, although there are no data for NHL. Patients and Methods We tested the hypothesis that circulating 25-hydroxyvitamin D [25(OH)D] levels are predictive of event-free survival (EFS) and overall survival (OS) in a prospective cohort of 983 newly diagnosed patients with NHL. 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels were measured by liquid chromatography-tandem mass spectrometry. Results Mean age at diagnosis was 62 years (range, 19 to 94 years); 44% of patients had insufficient 25(OH)D levels (< 25 ng/mL) within 120 days of diagnosis. Median follow-up was 34.8 months; 404 events and 193 deaths (168 from lymphoma) occurred. After adjusting for known prognostic factors and treatment, 25(OH)D insufficient patients with diffuse large B-cell lymphoma (DLBCL) had inferior EFS (hazard ratio [HR], 1.41; 95% CI, 0.98 to 2.04) and OS (HR, 1.99; 95% CI, 1.27 to 3.13); 25(OH)D insufficient patients with T-cell lymphoma also had inferior EFS (HR, 1.94; 95% CI, 1.04 to 3.61) and OS (HR, 2.38; 95% CI, 1.04 to 5.41). There were no associations with EFS for the other NHL subtypes. Among patients with DLBCL and T-cell lymphoma, higher 1,25(OH)2D levels were associated with better EFS and OS, suggesting that any putative tumor 1-α-hydroxylase activity did not explain the 25(OH)D associations. Conclusion 25(OH)D insufficiency was associated with inferior EFS and OS in DLBCL and T-cell lymphoma. Whether normalizing vitamin D levels in these patients improves outcomes will require testing in future trials.