Cerhan, James R.

Abstract Background: The frequency of preventive health practices (PHP), including age-appropriate screening for malignancies and immunizations, and compliance with PHP guidelines in lymphoma survivors is not known. We hypothesized that lymphoma survivors are more compliant with PHP than the general population but PHP rates may differ based on patient, disease and treatment characteristics. Methods: We identified eligible lymphoma patients from the SPORE Molecular Epidemiology Resource (MER) cohort, in which they were prospectively enrolled within <9 months of diagnosis. Survivors self-reported their PHP (colon, breast, and prostate cancer screening and influenza immunization) in the 3-year follow-up questionnaire (FU3). Per US Preventative Task Force guidelines, survivors were considered eligible for colon cancer screening if they were >50 years and compliant if they had fecal occult blood test <1 year or sigmoidoscopy/colonoscopy <10 years of FU3. Eligible female survivors age 50-75 were considered compliant if they had a mammogram after lymphoma diagnosis. Since USPTF recommended against PSA for prostate cancer screening in 2012, males aged 55-69 prior to 2012 were eligible and considered compliant if PSA test was <1 years of FU3. Survivors who received influenza vaccine <1 year of FU3 were considered compliant. Chi-square test was used to compare characteristics of compliant vs non-compliant patients; p value <0.05 was considered statistically significant. Results: Of 4248 lymphoma patients enrolled in the MER from 2002 to 2012, 2038 participants completed FU3. 1105 (54%) were male. The median age at diagnosis was 61 (IQR 51-69) years. There were equal numbers of indolent (N=1002) and aggressive (N=995) histologies with follicular (N=515, 25%) and diffuse large B-cell (N=427, 21%) being the most common. Majority (N=1221, 60%) had advanced stage (Ann Arbor III-IV) disease. 469 (23%) had events prior to FU3 and majority 1777 (88%) had received lymphoma treatment prior to FU3. Of 2038 survivors who completed FU3, 1576 (79%) reported having colon cancer screening in their lifetime. 1328 (91%) of 1570 eligible survivors were compliant, 124 (9%) were not and 118 had missing information. Survivors of age >60 years (93% vs 88%, p<0.001), those with indolent lymphomas (93% vs 90%, p=0.046), or diagnosed between 2010-2012 (94% vs 90% 2002-2009, p=0.007) were more likely to be compliant. There was no difference in colon cancer screening by sex, events prior to FU3 or treatment prior to FU3. 872 (96%) of 933 female survivors reported having a breast exam and 836 (91%) had a mammogram at least once in their lifetime. 495 (96%) of 561 eligible female survivors were compliant, 23 (4%) were not and 43 had missing information. There was no difference in mammographic screening based on age (<=60 vs >60), histology (indolent vs aggressive), events prior to FU3, treatment prior to FU3 or year of diagnosis (2010-2012 vs 2002-2009). 737 (78%) of 1105 male survivors had a PSA test at least once in their lifetime. 115 (71%) of 216 eligible men were compliant, 47 (29%) were not and 54 had missing information. There was no difference in PSA screening by age (<=60 vs >60), histology (indolent vs aggressive), events prior to FU3, and treatment prior to FU3. 1818/2038 (90%) survivors had received an influenza vaccine in their lifetime. 1567 (81%) were compliant, 373 (19%) were not and 98 had missing information. Survivors >60 years of age (87% vs 75%, p<0.001) and those with indolent lymphoma (83% vs 78%, p=0.015) were more likely to be compliant. There was no difference in influenza vaccination by sex, events prior to FU3, treatment prior to FU3, and year of diagnosis (2010-2012 vs 2002-2009). As a comparison in the US general population [Figure], between 2002-2015 the highest rates published by the Center for Disease Control are 62% for colon cancer screening (2015), 75% for mammography (insured, 2003) and influenza vaccination 42% (2015). Per the American Cancer Society, the highest rate of PSA screening was 54% (2010). Conclusion: Lymphoma survivors have high compliance with PHP recommendations compared to the general population. Although older individuals and those with indolent histologies were more likely to receive influenza vaccination and colon cancer screening, this difference was not seen in breast and prostate cancer screening practices. Lymphoma treatment and events did not seem to affect the frequency of PHP among survivors. Figure. Figure. Disclosures Cerhan: Nanostring: Research Funding; Jannsen: Other: Scientific Advisory Board; Celgene: Research Funding.

Abstract Background: B-cell non-Hodgkin lymphomas (NHL) are common lymphoid malignancies in which malignant B-cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. The microenvironment that supports the growth and survival of NHL B cells consists of a complex network of immune cells and cytokines and its specific composition has been shown to have significant clinical implications. The intratumoral immune cells include effector and regulatory T (Treg) lymphocytes that are more than simple residual elements from the normal lymph node structure. We previously explored whether the extent of T cell infiltration played a role in the clinical outcome of patients with B-cell NHL and found that an increased percentage of CD4+ T cells in the diagnostic biopsy of lymphoma patients significantly correlated with an improved 5-year overall survival. We have also shown that intratumoral Treg cells significantly suppress the anti-tumor response. Based on the fact that intratumoral T-cells are important in B-cell NHL, we evaluated whether genetic variation in genes that regulate the T-cell response may be associated with lymphoma risk. Methods: We genotyped 257 single nucleotide polymorphisms (SNPs) from 50 candidate genes related to T-cell differentiation and function in a clinic-based study of 441 Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. Tagging and nsSNPs were selected from HapMap. The most prevalent homozygous genotype was used as the reference group and each SNP was modeled individually as having a log-additive effect, expressed as ordinal odds ratios (OR) per variant allele and 95% confidence intervals, in an age- and sex-adjusted logistic regression analysis. We also evaluated the consistency of the findings for the most common types of B-cell NHL - diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and CLL/SLL. For gene-level analyses, we used principal components (PC) analysis using all SNPs from a gene; those PCs that explained > 90% of the variability were then modeled in a logistic regression analysis and significance was determined using a likelihood ratio test. We also evaluated the association of haplotypes from each gene with risk of NHL using the score test implemented from HAPLO.SCORE. Results. The mean age at diagnosis was 60.1 years for cases and 58% were male; among controls, the mean age at enrollment was 61.7 years and 55% were men. In the PC gene analyses, PRF1 (perforin gene - involved in cytotoxicity; p=0.004), CD276 (B7-H3 gene - involved in co-stimulation; p=0.01), TBX21 (T-bet gene – regulates Th1 cell development; p=0.02), and IL6 (p=0.02) were significant at p<0.05; haplotype analysis showed similar results, with the addition of CARD15 (NOD2 gene – regulates Th1 responses; p=0.02). There were 2 SNPs in PRF1, and both the intronic SNP rs3758562 (OR=1.38; 1.13–1.69) and the synonymous coding SNP rs885821 (OR=0.80; 0.63–1.02) were associated with NHL risk overall and for each subtype. There were 2 SNPs in CD276, and both the intronic SNP rs7176654 (OR=1.29; 1.07–1.56) and the mrna-utr SNP rs3816661 (OR=0.82; 0.68–0.99) were associated with NHL risk overall and with the CLL/SLL and follicular NHL subtypes specifically. There were 2 SNPs in TBX21, and only the mrna-utr SNP rs7502875 (OR=0.73; 0.59–0.92) was associated with NHL risk overall and for each subtype. There were 11 SNPs in IL6, and the intronic SNP rs2069835 (OR=1.88; 1.24–2.84) and the locus-region SNP rs2069824 (OR=1.73; 1.20–2.50) were each associated with NHL risk overall and each subtype. Finally, there were 9 SNPs in CARD15, with only the nonsynonymous coding SNP rs5743291 (OR=1.43; 1.02–1.98) being associated with overall NHL risk, and risk was specific to FL. Conclusions. Genetic variation in several genes that play critical roles in T-cell maturation and function was associated with lymphoma risk. These genes appeared to be associated with the differentiation and function of effector T-cells particularly Th1 and Th17 cells. These results provide potentially important insights into the role of T-cells in lymphomagenesis.

Abstract Background: Reactive oxygen species cause DNA damage and altered immunologic responses, and have been linked to the development of NHL. Diets rich in fruits and vegetables are excellent sources of antioxidants, and vitamins C, E, polyphenols, and carotenoids, along with selected micronutrients such as zinc are thought to be responsible for most of the antioxidant activity in foods. We evaluated the hypothesis that vegetables, fruits and nutrients involved in antioxidant activity would protect against developing NHL. In a secondary analysis, we also assessed heterogeneity of this hypothesis for the most common subtypes of NHL: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma, and diffuse large B cell lymphoma (DLBCL). Methods: We evaluated dietary antioxidant intake and NHL risk in a clinic-based study of 416 newly diagnosed NHL cases and 926 frequency-matched controls enrolled at the Mayo Clinic from 2002–2007. Usual diet two years before diagnosis/enrollment was assessed using a self-administered, 128-item food frequency questionnaire. Dietary intake of antioxidants was estimated using the Food Processor SQL system, further supplemented with data from US Department of Agriculture nutrient databases. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusted for age, sex, residence, and total energy. NHL subtypes were centrally reviewed, and subtype-specific risks were estimated using polychotomous logistic regression. Results: The mean age at diagnosis was 60.8 years for cases and 57% were male; for controls, the mean age at enrollment was 60.8 years and 54% were men. NHL risk was inversely associated with intake of total vegetables (OR for highest compared to lowest quartile, 0.46; 95% CI 0.32–0.68; p-trend<0.001), and specifically green leafy (OR=0.54; 95% CI 0.39–0.76; p-trend<0.001) and cruciferous (OR=0.62; 95% CI 0.43–0.88; p-trend=0.02) vegetables, but not legumes or red/yellow/orange vegetables. There was no association with total fruit intake or intake of citrus fruits in particular. When modeled together, the association for green leafy vegetables was unchanged while the association for cruciferous vegetables attenuated (OR=0.75; 95% CI 0.50–1.13; p-trend=0.3). Higher intake of total vitamin A (OR=0.60; 95% CI 0.41–0.88; p-trend<0.001) and vitamin E (OR=0.53; 95% CI 0.34–0.82; p-trend<0.001), but not vitamin C, were associated with lower NHL risk. For vitamin A, there was no association with preformed Vitamin A, but there was an inverse association with beta-carotene (OR=0.56; 95% CI 0.39–0.81; p-trend<0.001); only weak inverse associations were observed for other carotenoids (lutein/zeaxanthin, lycopene, and cryptoxanthin). When modeled together, the inverse association for vitamin E was unchanged while the association for beta-carotene attenuated. For micronutrients, there were inverse associations for manganese (OR=0.60; 95% CI 0.40–0.90; p-trend=0.02), selenium (OR=0.47; 95% CI 0.27–0.80; p-trend=0.01), and zinc (OR=0.46; 95% CI 0.27–0.80; p-trend<0.001) but not copper; when modeled simultaneously, both manganese (p-trend=0.03) and zinc (p-trend=0.02) remained significantly and inversely associated with NHL risk. Further adjustment for education, family history of NHL, smoking, alcohol use, and body mass index did not alter these results. There was no NHL subtype heterogeneity for the associations with total vegetables, green leafy vegetables or vitamin E, while inverse associations were not observed for manganese and follicular lymphoma or zinc and CLL/SLL. Conclusion: Higher intakes of vegetables, particularly green leafy vegetables, vitamin E, manganese, and zinc were associated with lower risk of NHL. These finding extend a growing literature from case-control and cohort studies that support a protective role of foods and nutrients that function in antioxidant related pathways against development of NHL, and therefore may represent one of the few known modifiable risk factors for this cancer.

Abstract Abstract 775 Introduction: WW has been an initial strategy for advanced stage FL because randomized trials showed no clear benefit for immediate initiation of therapy with single agent alkylators when compared to deferring therapy (Brice, JCO 1997; Ardeshna, Lancet 2003), although a recent trial raises questions about WW in the era of rituximab (R) based frontline therapies (Ardeshna, ASH Meeting 2010). Methods: The NLCS is a prospective, multicenter, observational study collecting data on 2,738 newly diagnosed pts with FL diagnosed from 2004–2007 at 265 US sites (80% non-academic). Initial management decisions were made by the treating physician and were categorized as WW (for pts who did not receive therapy in the 90 period following diagnoses and were coded by physicians as WW) or active therapy (AT) including: R alone, R-chemotherapy (RChemo), or other. Baseline clinical factors and treatment setting were compared for WW and AT pts using multivariable logistic regression. Kaplan-Meier (KM) estimation and Cox models were used to evaluate progression-free survival (PFS), time to 2nd and 3rd line management strategy (TT2, TT3), transformation-free survival (TFS), PFS following 1st AT, and overall survival (OS). Due to differences between WW and AT groups, adjusted Cox regression models are presented for the primary analysis of study endpoints controlling for sex, community vs. academic practice site, FL grade, and FLIPI. All time to event analyses were calculated from the 90 day period following diagnosis to allow enrollment in WW, and PFS was calculated from the starting point to the clinician's recording of progressive disease (PD) or death. Results: Among 1,737 pts presenting with stage III/IV FL in the analysis population, 237 underwent WW and 1,500 received AT immediately following diagnosis including: 241 pts treated with R, 1046 with RChemo, 26 with radiation, and 187 other therapies. Multivariable logistic regression demonstrated that the following pt characteristics were predictive of selection of WW rather than initial AT: age > 60 years, no B-symptoms, FL grade 1 or 2, ECOG PS = 0, ≤1 extranodal sites involved, and LDH ≤ULN (all p < 0.05). Median PFS and TT2 were 27 and 35 months for WW, and 64 months and not reached for AT. With a median follow-up of 55 months, 17% of WW pts and 19% of AT pts have died, with no significant difference in OS between WW and AT (p = 0.31). Compared with WW, RChemo improved PFS: adjusted Hazard Ratio (HR) = 0.36, TFS: HR=0.65, TT2: HR=0.48, and TT3: HR=0.53, all p<0.01. For most of the 145 WW pts that later initiated an AT, PD was recorded as the reason for treatment (83%). ATs following WW were: R (51, 35%), RCVP (21, 14%), RCHOP (16, 11%), R + other chemo (23, 16%), investigational (15, 10%), chemo alone (9, 6%), radiation (8, 6%), BMT (1, 1%), or other therapy (1, 1%). The median PFS for R was 42 months when given at diagnosis and 55 months when given following WW. The median PFS for RChemo was 71 months when given at diagnosis and 37 months when given following WW. After adjustment for FLIPI, FL grade, sex, practice setting, and type of treatment, WW adversely affected PFS following 1st AT, H=2.02, p <0.0001. Conclusions: FL pts in the US who undergo initial WW are treated at a median of nearly 3 years. Median PFS following RChemo, but not R is shorter when utilized after period of WW than at diagnosis – albeit in different clinical circumstances. Additional follow-up is needed to assess the tradeoff of this time without therapy with as yet no observable differences in OS. Disclosures: Sinha: CELGENE: Research Funding. Friedberg:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Link:Genentech: Consultancy. Flowers:Spectrum: Consultancy, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech/Roche (unpaid): Consultancy; Novartis: Research Funding; Seattle Genetics: Consultancy.

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.

Abstract Background: Elevated serum BLyS levels have been found in patients with both malignant and autoimmune diseases when compared to those of healthy donors, suggesting that BLyS may play a pathogenic role. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the mechanistic details underlying control of BLyS expression remain to be defined. In previous work we found that a single nucleotide polymorphisms (SNP) in the BLyS promoter (−871C→T) resulted in increased BLyS transcription suggesting that genetic variation in the BLyS gene may influence its expression. Because elevated BLyS levels have been detected in patients with NHL we wanted to determine if additional SNPs in the BLyS gene were associated with high serum BLyS levels and risk of developing of NHL. Methods: We genotyped 9 tagSNPs within the BLyS gene in a clinic-based study of 441 incident Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. We evaluated the association of individual SNPs as well as haplotypes from the BLyS gene with risk of NHL. We also jointly tested the main effects for all independent (r2<0.25) SNPs using a multivariate logistic regression (MLR) model. As a secondary analysis, for those SNPs showing significant results (< 5% significance), we evaluated associations between those who had all high risk alleles at the SNPs of interest compared to those who had all low risk alleles at the SNPs of interest. Additionally, we evaluated serum BLyS levels by ELISA in these same subjects. Serum BLyS levels were detemined by ELISA. Results: In the individual single SNP logistic regression analysis, 3 of the 9 tagSNPs were significant at p<0.05. Haplotype and MLR results were nonsignificant. However, when we categorized participants into low and high risk groups based on risk alleles at the three statistically significant SNPs, we found the high risk variant had an odds ratio (OR) of 2.086 (p=0.0001) for risk of B-cell NHL. When the analysis was restricted by histologic subtype we found that diffuse large B-cell lymphoma or follilcular lymphoma grade 3 had an OR of 3.163 (p=0.01), follicular lymphoma grade I/II had an OR= 2.547 (p=0.046), and chronic lymphocytic leukemia (CLL) had an OR=1.238 (p=0.13). Because there was not a significant correlation of the high risk variant with CLL, we performed an additional analysis in which we included all B cell NHL cases excluding CLL and the OR was 2.799 (p=0.0001). We next wanted to determine if serum BLyS levels correlated with either the high or low risk variant. The mean serum BLyS level in those individuals (untreated cases and controls) who carried the low risk variant at all three SNPs was significantly lower (p=0.0061) at 1.3 ng/ml (n=25, range: undetectable-4.4 ng/ml) compared to 4.3 ng/ml in those with the high risk variant (n=74, range: undetectable- 66.8 ng/ml). Conclusions: In summary, we have found that genetic variation in the BLyS gene is significantly associated with an increased risk of developing B-cell NHL, particularly follicular and large B-cell lymphoma. Further, we found that genetic variation is also associated with an increase in serum BLyS levels.Taken together, our data further highlight the role of the BLyS gene in B cell malignancies and characterization of the precise genetic and environmental mechanisms that regulate BLyS may have significant clinical impact.

Abstract Abstract 97 Background: There is no consensus or standard of care for front-line therapy for FL as demonstrated by our initial publication of the National LymphoCare Study (NLCS) showing that strategies used in newly diagnosed FL patients (pts) in the US included: watchful waiting (17.7%), rituximab (R) with chemotherapy (51.9%), R alone (13.9%), radiation therapy (5.6%), chemotherapy (3.2%), or a clinical trial (6.1%). Given the lack of observational data comparing the effectiveness of front-line R-chemotherapy regimens, we examined the outcomes for patients with stage III/IV FL receiving R with cyclophosphamide, vincristine, and prednisone (RCVP), R with cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP), or R with a fludarabine-based regimen (RFlu) as first-line therapy. Methods: The NLCS is a prospective, multicenter, observational study collecting data on 2,738 previously untreated patients (pts) with FL diagnosed from 2004 to 2007 at 265 sites in the US. Initial management decisions were made by the treating physician without protocol-specified treatment assignments. Descriptive statistics for the baseline characteristics of pts who received RCVP, RCHOP, and RFlu were compared using Pearson Chi-square tests. A generalized logistic model was used to identify baseline factors of clinical interest (FLIPI, sex, practice setting [academic vs. community]) that were correlated with treatment. Kaplan–Meier estimation was used to evaluate progression-free survival (PFS) and overall survival (OS) for the treatment regimens; comparisons for the 2-year landmark estimates were obtained using a Wald statistic. PFS was defined as the number of days from diagnosis up to and including the date of progressive disease (PD) as assessed by the treating physician or death from any cause. Pts who had not yet experienced PD at the time of analysis were censored at the date of the most recent response assessment. To evaluate the effects of treatment on PFS and OS, Cox proportional hazards models were used controlling for FLIPI, sex, community or academic practice setting, and maintenance R or observation following treatment. Results: Of the 2,738 pts enrolled in NLCS, 926 pts were identified as having stage III/IV FL and having received the regimens of interest. Within this cohort 60% received RCHOP (n=558), 27% received RCVP (n=247) and the remaining 13% received RFlu (n=121). Baseline characteristics were correlated with induction treatment and whether a patient received subsequent R maintenance. Women (odds ratio [OR]=1.50, 95%CI 1.05–2.13) and pts aged >60 years (OR=2.36, 95%CI 1.64–3.38) more commonly received RCVP than RCHOP, and pts with LDH > upper limit of normal (OR=1.75, 95%CI 1.02–3.03) more commonly received RCHOP than RFlu. A greater % of pts receiving RCVP (61%) had poor-risk FLIPI (3–5) vs. RCHOP (49%) or RFlu (44%). In this cohort, 60% of RCVP, 43% of RCHOP, and 46% of RFlu pts received maintenance R. The overall response rates for RCVP, RCHOP, and RFlu were 88%, 94%, and 96%, respectively, and 2-year PFS was 72% (95% CI 66–78%) for RCVP, 78% (95%CI 74–81%) for RCHOP, and 76% (95%CI 67–-83%) for RFlu. There were no significant differences in PFS between treatments after adjustment (p= 0.07). However, with a median follow-up of 58 months there were significant differences in unadjusted OS (p=0.03), with 2-year OS probabilities of 88% (95%CI 84–92%) for RCVP, and 91% (84–95%) for RFlu, and 94% (92–96%) for RCHOP (Figure A). After adjustment for sex, FLIPI, practice setting, and maintenance use, the OS benefits of RCHOP persisted over RCVP HR=1.67 (95%CI 1.15–2.42) but not RFlu HR=0.94 (0.53–1.68). The benefits of RCHOP were even more pronounced in poor risk FLIPI pts (Figure B). Conclusions: First-line R-chemotherapy regimens in clinical practice have high overall response rates and high 2-year PFS and OS. With 4.75 years of follow-up in this large pt cohort, these regimens appear similar in response rates and early PFS and OS with suggestions that RCHOP may provide benefits over RCVP particularly for poor risk patients. Disclosures: Sinha: Celgene: Research Funding. Friedberg:Genentech: Consultancy; astellas: ; Lilly:; Abbott/Trubion:; Seattle Genetics: Honoraria; Cephalon: Consultancy. Hirata:Roche: Equity Ownership; Genentech Inc/Roche: Employment. Flowers:Seattle Genetics: Consultancy; Millennium/Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech/Roche (unpaid): Consultancy; Novartis: Research Funding; Spectrum: Consultancy, Research Funding.

Abstract Abstract 429 BACKGROUND: While standard clinical prognostic factors such as those included in the International Prognostic Score (IPS) predict outcome in cHL patients, predicting the outcome of patients might be further refined using biological factors. We therefore tested whether pretreatment serum cytokines, that measure the biological activity of the tumor as well as the host response, could provide additional prognostic information in cHL patients. METHODS: Newly diagnosed cHL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum cytokines were measured from pretreatment blood draws using a multiplex ELISA (Invitrogen, Camarillo, CA). Thirty cytokines, including pro-inflammatory, Th1 and Th2 associated cytokines, were analyzed using the Luminex-100 system Version 1.7 (Luminex, Austin, TX). Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). The assay was also performed on research serum draws from 50 non-lymphoma epidemiology controls enrolled on a corresponding lymphoma etiology case-control study. The upper limit of normal for each cytokine was defined as the 95th percentile from the control distribution. Clinical data were abstracted using a standard protocol and all patients were followed systematically for event-free (progression, retreatment, or death due to any cause) and overall survival (EFS and OS, respectively). Cox proportional hazards models were used to evaluate the association between cytokines and outcome. RESULTS: 140 HL patients with cHL diagnosed between September 2002 and February 2008 with available pretreatment serum were included. The median age was 40 years (range 18–89) and 53% were male. Seventy-seven patients (55%) had limited stage disease and 63 (45%) had advanced disease. One hundred and twenty one patients (86%) were treated with ABVD chemotherapy, while the remainder received the Stanford V regimen (9%), BEACOPP (4%) or MOPP chemotherapy (2%). At a median follow-up of 47 months (range 0–87), 25 patients (18%) had an event and 16 patients had died (11%). The serum levels of 12 cytokines (EGF, FGFb, GCSF, HGF, IL-6, IL-8, IL-12, IL-2R, IP-10, MIG, TNFa and VEGF) were significantly higher in cHL patients than controls. When correlated with clinical factors, IL-2R, IP-10, MIG, IL-6 and HGF were associated with an increased sedimentation rate, B-symptoms, advanced stage and splenic involvement. Elevated IL-8 levels were associated with bulky disease. Elevated levels of HGF, IL-6, IL-8, IL-2R, IP-10 and MIG were all associated with a poorer EFS, however only IL-2R (p=0.002) and IL-6 (p<0.001) were independently prognostic for both EFS and OS. Furthermore, using IL-6 and IL-2R in a 2-cytokine model (see Figure), patients with elevation of both cytokines had a dramatically greater likelihood of early relapse and death than those with normal levels or elevations of only one of the cytokines (1-year EFS 40% compared to 97% and 93% respectively, p<0.0001; 2-year OS 49% compared to 98% and 95%, p<0.0001). While elevated IL-6 and IL-2R levels correlated with the IPS (p=0.002), this 2-cytokine model remained independently predictive of prognosis. In fact, when only good risk patients (IPS 0–3) were considered, the 2-cytokine model remained predictive of patients at risk for early relapse (1-year EFS 54% versus 95% and 97%, p<0.0001). CONCLUSION: Elevated pretreatment serum cytokines are associated with an increased likelihood of disease relapse and an inferior survival in patients with cHL. The pretreatment cytokine profile, particularly serum levels of IL-6 and IL-2R, may identify cHL patients at high risk for early disease relapse who may benefit from novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Patients with newly diagnosed, advanced-stage, follicular B-cell non-Hodgkin’s lymphomas (NHL) commonly receive rituximab as initial therapy. While antibody dependent cytotoxicity (ADCC) is felt to be the predominant mechanism of action for rituximab, the immunologic effects of rituximab are largely unknown. We have previously shown that genetic polymorphisms in IL-8, IL-2, IL-12 and IL-1 receptor antagonist (IL-1RN) have prognostic significance in follicular lymphoma. The goal of this study was to measure changes in the abovementioned and other serum cytokine levels in patients receiving single agent rituximab as initial therapy for follicular NHL and to identify cytokines that are associated with an increased risk of disease relapse. Methods: Patients with untreated follicular grade 1 NHL, and measurable stage III/IV disease were treated with rituximab 375 mg/m2 intravenous weekly for 4 doses and were then followed for response and time to progression (TTP); no maintenance therapy was provided. Thirty-seven patients were enrolled in this clinical trial between July 1999 and May 2001. As previously reported, the overall response rate in the 36 eligible patients was 72%, with 36% complete remissions. Now, with a median follow up of 5.1 years, the median TTP is 2.2 years (95% CI, 1.2–3.3 years) and the 5-year overall survival is 77% (95% CI: 64–92%). We measured the serum levels of a variety of inflammatory cytokines by ELISA at baseline and 1 month after completing treatment. Serum specimens were available in 30 patients. Thirty two cytokines or receptors including IL-1β, IL-1RN, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, GCSF, EGF, FGF-basic, HGF, BLyS and APRIL were analyzed. Results: A significant increase was seen in the serum levels of BLyS (p=0.0001) after rituximab therapy, while IL-12 (p=0.0002), IL-1RN (p=0.0001), IL-2R (p=0.0001), IL-1β (p=0.02), IL-7 (p=0.02), IL-10 (p=0.02), and MIG (p=0.05) all significantly decreased. All other cytokines/receptors remained unchanged. Higher pretreatment serum levels of IL-2R (p=0.008) and IL-12 (p=0.001) correlated with a shorter TTP. Furthermore, elevated serum IL-2R levels pretreatment correlated with a lack of response to rituximab (p=0.03). Differences in overall survival could not be determined due to the fact that only 8 patients enrolled in the study have died. Because serum IL-2R levels correlated with response and TTP, we measured the cell surface expression of IL-2R in the tumor by flow cytometry. IL-2R was detected on a subpopulation of CD4+ T-cells in all specimens tested (n=14), and on malignant B-cells in 5 cases (36%). Conclusions: Pretreatment serum levels of IL-2R and IL-12 are important prognostic factors for response to therapy or TTP in follicular lymphoma patients treated with rituximab. Furthermore, serum levels of BLyS, IL-1RN, IL-7, IL-10, IL-1β and MIG also change after rituximab treatment. Targeting these cytokines may therefore provide additional clinical benefit to patients treated with rituximab.

Abstract Serum BLyS levels have been found to be elevated in a number of immune disease models and there is increasing evidence that this may correlate with pathogenesis of various B cell related disorders including B cell malignancies. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in these scenarios and definition of the mechanisms that control BLyS expression remain to be fully elucidated. Serum BLyS levels are elevated in a number of B cell malignancies known to have a familial incidence. Therefore, we sought to determine if there was any correlation between serum BLyS levels and family history of B cell cancers. In our initial studies we examined the serum BLyS levels in patients with sporadic vs. familial B-cell chronic lymphocytic leukemia (B-CLL; near relative with B-CLL, multiple myeloma, or non-Hodgkin lymphoma), and normal age-matched controls. In the normal controls (n=50), the mean serum BLyS level was 12.7 ng/ml, and we found that 4/50 (8%) had BLyS levels exceeding 20 ng/ml. In the sporadic CLL cohort (n=52), the mean serum BLyS level was 18.3 ng/ml, and we found that 5/52 (10%) had BLyS levels exceeding 20 ng/ml. In striking contrast, in the familial CLL cohort (n=24), the mean serum BLyS levels was 33.4 ng/ml, and we found that 11/24 (46%) had BLyS levels exceeding 20 ng/ml. The percentage of patients with elevated BLyS levels, as well as the mean BLyS levels in the familial CLL cohort compared to the normal controls, was significantly higher (mean, p=0.002) and suggests that elevated BLyS levels may correlate with familial CLL. Because of the correlation between BLyS levels and family history B cell malignancies, we next wanted to determine if there was a common underlying genetic event influencing BLyS expression in this group of patients. We began by sequencing the BLyS promoter in 19 patients with B-CLL and 11 normal controls. We identified 2 sites that were polymorphic, −661 A/G and −871 C/T. The −871 T/T genotype has been previously reported to be expressed at increased frequency in SLE patients and is associated with elevated BLyS mRNA levels. Using RFLP analysis we examined the presence of the two polymorphisms in our control, sporadic, and familial CLL cohorts. In the control group (n=50) we found that at −871, C/C (wildtype) was expressed in 24%, C/T in 54%, and T/T in 22% of patients. Similarily, in the sporadic CLL group (n=35) we found that at −871, C/C was expressed in 29%, C/T in 57%, and T/T in 14%. In the familial group (n=24) we found that at −871, C/C was expressed in only 8%, C/T in 67%, and T/T in 25%. These data suggest a decreased representation of −871 C/C in the familial group. No significant changes were observed between the three groups at the −661 A/G locus. Additionally, we found that none of the patients with BLyS levels exceeding 20 ng/ml expresses C/C at −871. In summary, our data suggest that BLyS levels are elevated in patients with familial CLL relative to both normal controls and individuals with sporadic CLL and that elevated BLyS levels may correlate with the presence of a T at −871 in the BLyS promoter.