Ansell, Stephen M.

Abstract Background: POEMS syndrome is a devastating syndrome, characterized by peripheral neuropathy, organomegaly, endocrinopathy, monoclonal plasma cells, skin changes, papilledema, volume overload, sclerotic bone lesions, thrombocytosis, & high VEGF. We noted an unexpectedly high transplant related morbidity, which we have since postulated to be ES. Methods: 30 patients with POEMS were treated with PBSCT at Mayo Clinic Rochester. We retrospectively studied outcomes, with an emphasis on treatment related morbidity. Two definitions of ES were used: Spitzer (BMT 2001) and Maiolino (BMT 2003). Results: Two-thirds were male. Median age was 48, range 20–70. Time from first symptoms and diagnosis was 26 and 4 months, respectively. To mobilize stem cells, CTX/G-CSF was used in 5 & G-CSF in the remainder. Conditioning was Mel200 (n=19), Mel140 (n=10), and BEAM (n=1). Post-transplant 15 had GM-CSF begun day+6. Only 10% remained outpatient, and median time to discharge from hospital was transplant day 17 (range 0–175). Factors predicting for later dismissal included age (p=0.04), abnormal CXR 7 to 17 days post transplant (p<0.0001), & bolus corticosteroids (CS) beyond day 12 post-transplant (p=0.006). Ninety-three percent had fever, although only 9 had bacteremia. Eight satisfied criteria for ES according to Spitzer and 14 according to Maiolino. Another 3 patients received steroid bolus days 8, 12, and 11 for presumed ES but did not meet criteria because of delayed ANC recovery (days 16, 18, and 20, respectively). Of the 5 patients requiring endotracheal intubation, 3 satisfied Maiolino’s criteria for ES. Although these 3 received bolus CS during their course, administration was delayed at 18, 14, and 18 days. The patient whose CS bolus antedated intubation only received prednisone 30 mg/day. In toto, fourteen patients received bolus CS (daily doses between 20 and 1200 mg prednisone equivalents) commencing day 8 to 59. Those 7 patients who received CS before day 13 did better than the 7 who received them day 13 or later (Table). Conclusions: It is essential to recognize that nearly 50% of patients satisfied formal criteria for ES as defined by Maiolino. In these patients ES may run a self-limited course or lead to catastrophe. No Steroid (n=16) Steroid ≤ D12 (n=7) Steroid > D12 (n=7) P *Engraftment syndrome according to definition of Maiolino (M) or of Spitzer (S). ES M / S, n* 5 / 2 4 / 4 5 / 2 NA Wt change, % 0.6 (−.4.2–6.7) 6.7 (3.6–27.2) 11.2 (–2.1–23.2) 0.005 Rash, % 27 71 43 0.13 Diarrhea, % 73 86 86 0.71 Tmax, C 39 (37.8–41) 40.1 (39–41.1) 38.9 (38.7–40.8) 0.08 1st fever, day 10 (6–15) 8 (7–9) 12 (8–146) 0.007 Abn CXR1, % 13 71 71 0.03 Ventilator, % 0 14 71 0.004 First WBC, day 12 (8–21) 14 (12–14) 14 (12–17) 0.03 ANC500, day 15 (12–29) 16 (14–115) 18 (15–45) 0.08 PLT20, day 12 (8–41) 20 (11–115) 24 (9–170) 0.05 PLT50, day 15 (11–192) 32 (16–115) 56 (13–551) 0.03 RBCs, units 3 (2–8) 6 (4–31) 11 (6–64) 0.0008 PLTS, aph. units 2 (1–9) 9 (4–51) 18 (4–60) 0.0004 Hosp dsm, day 15 (13–36) 21 (15–69) 41 (16–175) 0.009

Abstract Peripheral blood absolute lymphocyte count (ALC) at diagnosis is a predictor of survival in B-cell lymphomas. The role of ALC at diagnosis on survival in T-cell lymphoma has not been studied. Thus, we studied the role of ALC at diagnosis on clinical outcome in adult patients with primary anaplastic large cell lymphoma (PALCL) that were diagnosed, treated, and followed at the Mayo Clinic, Rochester. Between 1985 and 2006, 50 patients with PALCL qualified for the study. ALC was identified to be a strong predictor for complete response (CR), area under the curve (AUC = 0.83, p < 0.002). The median follow-up was 31.8 months (range: 1–212.6 months). ALC, as a continuous variable was a predictor for overall survival (OS) (HR = 0.143; 95%CI = 0.042–0.416; p < 0.0001) and progression-free survival (PFS) (HR = 0.150; 95%CI = 0.047–0.415; p < 0.0001) .Superior OS and PFS (Figure 1) were observed with an ALC ≥ 1.0 x 109/L (N = 31) versus an ALC < 1.0 x 109/L (N=19) (median OS: not reached vs 7.5 months, OS rates at 5 years, 81% vs 36%, p < 0.0006, respectively; and median PFS: not reached vs 6.3 months; PFS rates at 5 years, 77% vs 37%, p < 0.0007, respectively). Multivariate analysis demonstrated ALC to be an independent prognostic indicator for OS (HR = 0.196; 95%CI = 0.125–0.693; p < 0.002) and PFS (HR = 0.191; 95%CI = 0.053–0.559; p < 0.0008) when compared to anaplastic lymphoma kinase, international prognostic index, and cutaneous versus systemic presentation. Figure 1 Figure 1.

Abstract Abstract 633 Background: To date, the only well characterized chromosomal translocations among peripheral T-cell lymphomas (PTCLs) are those involving ALK, which occur in about half of anaplastic large cell lymphomas (ALCLs). ALK-negative ALCLs remain poorly understood, despite morphology and phenotype similar to ALK-positive ALCLs. We recently described translocations near IRF4 on 6p25.3 in cutaneous ALK-negative ALCLs. In the present study, we utilized massively parallel (Next Generation) sequencing to answer three questions about these translocations: (1) Were they recurrent in the more lethal systemic form of ALK-negative ALCL? (2) What was the translocation partner? (3) What were the likely genetic consequences? Methods: We used a mate pair library approach to juxtapose DNA sequences originally lying ~5 kb apart: these paired sequences map to distinct areas of the genome if a translocation breakpoint lies between them. Because only the ends of the original DNA fragments are sequenced, this approach is more resource efficient than whole genome sequencing. Genomic DNA from a systemic ALK-negative ALCL with an uncharacterized 6p25.3 rearrangement was fragmented to 2–5 kb, biotinylated, and separated on an agarose gel. Fragments ~5-5.5 kb were excised, purified, circularized, and further fragmented to ~300-600 bp. Fragments containing biotin (representing the ends of the original 5 kb fragments) were immobilized on streptavidin beads and amplified using the Illumina library protocol. Paired end sequencing was performed on one lane of a flow cell on an Illumina GAIIx. Sequencing data were mapped to the genome using a binary indexing algorithm. Non-mapping and duplicate sequences were discarded. Candidate translocations suggested by >4 unique mate pairs were examined by BLAT to exclude false positives explained by mapping to homologous regions. PCR and Sanger sequencing of tumor DNA was used for validation. Fluorescence in situ hybridization (FISH) was performed on additional PTCLs. mRNA and miRNA expression was quantified using real-time PCR. Results: Of 28.90×106 paired sequences, 8.61×106 were non-duplicate pairs in which both ends mapped to the human genome (bridged coverage, ~7x). Only one candidate translocation involved 6p25.3, represented by 10 unique mate pairs. PCR and Sanger sequencing confirmed a balanced translocation: one breakpoint disrupted the dual-specificity phosphatase gene, DUSP22, telomeric to IRF4 on 6p25.3; the other was telomeric to the FRAH7 fragile site on 7q32.3. Additional ALK-negative ALCLs with known 6p25.3 rearrangements were tested using a 7q32.3 breakapart FISH probe, which showed a 7q32.3 rearrangement in 13/29 (45%; 7 systemic and 6 cutaneous). Dual fusion FISH confirmed t(6;7)(p25.3;q32.3) in 11/11 cases tested. None of 120 PCTLs (including 39 ALCLs) lacking 6p25.3 rearrangements had a 7q32.3 rearrangement. Levels of 5′ and 3′ DUSP22 mRNA in cases with 6p25.3 rearrangements were decreased relative to those in non-rearranged cases (5′: 0.02±0.01 vs 1.00±0.74, p=.002; 3′: 0.09±0.08 vs 1.00±0.55, p=.0002; mean±SD, t test). IRF4 mRNA levels were similar in both groups. Levels of MIR29A and MIR29B1 within FRAH7 in cases with 7q32.3 rearrangements were increased relative to those in non-rearranged cases (MIR29A: 2.44±2.20 vs 1.00±1.34, p=.15; MIR29B: 4.93±4.68 vs 1.00±1.05, p=.007). Conclusions: The t(6;7)(p25.3;q32.3) is the first recurrent translocation characterized in systemic and cutaneous ALK-negative ALCLs. This translocation was entirely specific for ALK-negative ALCLs, and was present in 45% of cases with 6p25.3 rearrangements. The t(6;7)(p25.3;q32.3) was associated with down-regulation of DUSP22 and up-regulation of MIR29, but not dysregulation of IRF4. DUSP22 inhibits mitogen activated protein kinase activity in T cells, and may represent a novel tumor suppressor in ALCLs. Increased MIR29 levels may additionally contribute to tumorigenesis, as recently shown in acute myeloid leukemias. Finally, clinical testing for t(6;7)(p25.3;q32.3) may aid in differentiating ALK-negative ALCL from other PTCLs. Our findings highlight the utility of mate-pair library sequencing to discover novel translocations in lymphoma and other cancers. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2682 Background: The PI3K-Akt pathway is critical to tumor growth in lymphoid malignancies and mTOR inhibitors have shown promising activity in lymphoid malignancies. However, alternate signaling pathways allow cells to escape the effect of mTOR inhibition. Pre-clinical studies have shown that the combination of LBH589, an HDAC inhibitor, with the mTOR inhibitor RAD001 results in potent synergy. We then explored this combination in a phase I/II clinical trial. Patients and Methods: Patients with relapsed multiple myeloma (MM), non-Hodgkin lymphoma (NHL) or Hodgkin Lymphoma (HL) were enrolled into a phase I trial of escalating doses of LBH589 and RAD001. The doses are shown in the table. The study utilized a 3+ 3 design with MTD defined as the maximum dose resulting in less than 2 dose limiting toxicities (DLT) during the first cycle among 6 patients treated at that dose. DLT was defined as a grade 4 neutropenia or thrombocytopenia lasting >= 7 days, febrile neutropenia or any other grade >=3 non-hematologic toxicity considered possibly related to the drugs. Once MTD was determined, patients were enrolled into two separate phase 2 studies, one for relapsed MM and another for relapsed NHL or HL. Results: Twenty-six patients were enrolled to the phase 1 study; 23 were eligible for MTD analysis. There were 16 patients with NHL, 4 with HL and 3 with MM. Median age was 61 (range, 24–73) and 17 were male. The median number of prior therapies was 4 (range 2–11), including a prior stem cell transplant in 11 (48%). No DLT was observed at any of the doses tested in the trial (See below). Hematological toxicities were the most common AEs observed, with a grade 3 or 4 hematologic AE in 15 patients and grade 3 or 4 non-hematologic AEs in 6 patients across all cycles administered. Grade 3 and 4 hematologic AEs included 10 patients with neutropenia and 8 patients with thrombocytopenia. Grade 3 and 4 non-hematologic AEs included hyperglycemia, hypertriglyceridemia, hypophosphatemia, diarrhea, pneumonia and mucositis in one patient each. Overall, 10 responses (43%) were seen (Table). Conclusions: The combination of LBH589 and RAD0001 is tolerated at the doses tested, with hematological toxicities being the most common adverse events. The recommended phase II dose is LBH 40 mg MWF every other week along with 5 mg RAD001 every day in 4-week cycles. Clinical efficacy was seen in all diseases tested with partial responses seen in NHL and HL as well as a minor response in a patient with refractory myeloma, who had previously failed single agent RAD001. Phase 2 trials are ongoing in both disease groups. Disclosures: Kumar: Merck: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Genzyme: Research Funding; Novartis: Research Funding. Off Label Use: Use of the investigational agent MLN9708 for the treatment of previously untreated multiple myeloma. Lacy:Celgene: Research Funding. Reeder:Celgene: Research Funding; Millennium Pharmaceuticals Inc.: Research Funding; Novartis: Research Funding.

Abstract Abstract 3642 Purpose: The reactive pathologic background in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) consists of lymphocytes and histocytes. Lymphopenia is a negative prognostic factor in NLPHL as it is also in Classical Hodgkin Lymphoma (cHL). In cHL, tumor-associated macrophages recruited from circulating monocytes are a negative prognostic factor for survival. Thus, we studied if the peripheral blood absolute lymphocyte count/absolute monocyte count ratio at diagnosis (ALC/AMC-DX), as a surrogate biomarker of the host response against the neoplastic lymphocytic and/or histiocytic (L&H) cells, affects survival in NLPHL. Patients and Methods: We performed a retrospective analysis of the association between ALC/AMC-DX and survival in 103 consecutive NLPHL patients that were followed at Mayo Clinic from 1974 to 2010. Receiver operating characteristic (ROC) and area under the curve (AUC) were used for ALC/AMC-DX cut-off value analysis and proportional-hazards models were used to compare survival based on the ALC/AMC-DX ratio. Results: The cohort included 67 (65%) males and 36 (35%) females. Eighty-three percent of patients presented with stage I/II and 17% with stage III/IV. Sixty-five (63%) patients were treated with radiation alone; 20 (20%) patients with chemotherapy alone; and 18 (17%) patients with chemotherapy and radiation. The median follow-up period was 8.9 years (range: 0.3–31 years). An ALC/AMC-DX ≥ 2.1 was the best cut-off value for survival with an empirical AUC of 0.82 (95% confidence interval [CI], 0.78 to 0.88), a sensitivity of 70% (95% CI, 61% to 76%) and specificity of 84% (95%CI, 59% to 82%). The cut-off value for ALC/AMC-DX ≥ 2.1 was validated by the k-fold cross validation method, showing a cross validation ROC with an AUC of 0.82 (95% CI, 0.72 to 0.93) for an ALC/AMC-DX ≥ 2.1. Using Kaplan-Meier analysis, we studied overall survival (OS), lymphoma-specific survival (LSS), progression-free survival (PFS), and time to progression (TTP) based on ALC/AMC-DX ≥ 2.1. Patients with an ALC/AMC-DX ≥ 2.1 experienced an superior OS, LSS, PFS, and TTP compared with patients with an ALC/AMC-DX < 2.1: [OS: median was 22.1 years vs 6.3 years, 10-years OS rates of 89% (95%CI, 80% to 98%) vs 44% (95%CI, 27% to 65%), p < 0.0001, respectively; LSS: median was not reached vs 7.3 years, 10-years LSS rates of 99% (95%CI, 90% to 100%) vs 45% (95%CI, 29% to 67%), p < 0.0001, respectively; PFS: median was 20.6 years vs 4.6 years, 10-years PFS rates of 84% (95%CI, 80% to 97%) vs 26% (95%CI, 18% to 55%), p < 0.0001, respectively; and TTP: median was not reached vs 5.5 years, 10-years TTP rates of 93% (95%CI, 85% to 99%) vs 29% (95%CI, 19% to 57%), p < 0.0001, respectively]. After adjusting for the International Prognostic Factors and the International Prognostic Score, ALC/AMC-DX remained an independent prognostic factor for OS {hazard ratio (HR), 0.42, 95%CI, 0.23 to 0.64, p< 0.004]; LSS [HR, 0.14; 95%CI, 0.04 to 0.23, p <0.0004]; PFS [HR, 0.14; 95%CI, 0.03 to 0.34, p < 0.001], and TTP [HR, 0.16, 95%CI, 0.04–0.34, p <0.002]. Conclusion: ALC/AMC-DX is an independent prognostic factor for survival and provides a single biomarker to predict clinical outcomes in NLPHL. Disclosures: No relevant conflicts of interest to declare.

Abstract Background IL-10 polarized macrophages promote tumor growth and survival. The T-cell transcription factor GATA binding protein-3 (GATA3) epigenetically regulates IL10 expression. Therefore, we investigated the extent to which GATA-3 regulates IL-10 production and macrophage polarization in T-cell lymphomas. Methods Monocyte-derived macrophages were generated and polarized with T-cell lymphoma conditioned media ((TCL-CM). Immunophenotypic and functional characteristics associated with IL10-polarized macrophages were examined. GATA3-dependent cytokine production was evaluated in T-cell lymphoma lines by shRNA knockdown. Alternatively polarized macrophages and GATA3 expression were examined in a cohort of patients with peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) (n=76). Comparisons among groups were evaluated using a Student's t test. Progression-free and overall survival (PFS & OS) was estimated using the Kaplan-Meier method and two-tailed log-rank test based on Cox proportional hazards model. Results To investigate the ability of T-cell lymphomas to alternatively polarize macrophages, monocytes from healthy donors were cocultured with TCL-CM. Macrophages were shown to have immunophenotypic characteristics of IL10-polarized macrophages (pSTAT3+/CD16hi/CD163hi/HLA-DR-/lo) by flow cytometry, which was abrogated by IL10, but not IL6, neutralization. TCL-CM polarized macrophages produced abundant IL-10 and were impaired in their ability to stimulate T-cell proliferation. Collectively, these results demonstrate that T-cell lymphomas promote alternative macrophage polarization in an IL-10 dependent manner, and both CD163 and pSTAT3 may aid in their identification in clinical T-cell lymphoma specimens. Therefore, CD163+/pSTAT3+ macrophages were visualized by immunofluorescence in PTCL, NOS specimens. IL-10 polarized macrophages were not identified in normal tissues, but were abundant constituents of the tumor microenvironment in 68% of the PTCL, NOS specimens examined (n=31). STAT3 phosphorylation in response to IL-10 is dependent upon the Janus kinases (JAK) JAK1 and TYK2, both of which are inhibited at nanomolar (and clinically achievable) concentrations by the “JAK2” inhibitor ruxolitinib. Therefore, we sought to determine whether ruxolitinib may prevent IL-10-induced macrophage polarization. As expected, ruxolitinib inhibited IL-10-induced STAT3 phosphorylation. More importantly, macrophages polarized in the presence of ruxolitinib lost the immunophenotype characteristics of IL-10 polarized macrophages, and were CD16-/loCD163-/loHLA-DRhi. Furthermore, IL-10 production was significantly impaired in macrophages polarized in the presence of ruxolitinib, while their ability to stimulate T-cell proliferation was significantly increased. In a separate initiative, we identified GATA3 as the key regulator of IL-10 and Th2 cytokine (IL-4, IL-13 and IL-5) expression in T-cell lymphoma lines by shRNA knockdown assays. Therefore, we examined GATA3 expression by immunohistochemistry in PTCL, NOS specimens. GATA-3 expression was observed in most of the T-cell lymphoma lines and in 47% of PTCL, NOS specimens (n=76). In the cohort of the 76 PTCL, NOS patients, the median PFS and OS observed in GATA3+ PTCL, NOS was 5 months (4-8 months, 95% CI) and 8 months (5-14 months, 95% CI), respectively. In contrast, the median PFS and OS in GATA3- PTCL, NOS was 1.3 years (0.6-1.6, 95% CI) and 1.6 years (0.7-6.9 95% CI), respectively. Conclusions Alternatively polarized macrophages are abundant in T-cell lymphomas and are generated in an IL-10- and STAT3-dependent manner. GATA-3 regulates IL-10 production in these lymphomas, and its expression identified a high-risk subset of PTCL, NOS with distinct clinicopathological features. As IL-10-dependent STAT3 phosphorylation and macrophage polarization were impaired by ruxolitinib, the JAK2 inhibitors may warrant closer scrutiny as immunomodulatory agents. Disclosures: Off Label Use: Ruxolitinib, a JAK2 inhibitor, may warrant closer scrutiny as an immunomodulatory agent in TCL therapeutics.

Abstract Background Relatively few studies have evaluated the role of host (germline) genetics in predicting outcome in diffuse large B-cell lymphoma (DLBCL), and of these, most were small candidate gene studies without replication and/or from the pre-R-CHOP era. We report the first genome-wide association study (GWAS) of outcome in DLBCL patients treated with rituximab and anthracycline-based chemotherapy. We used two independent studies for discovery, and validated the results in two additional independent studies. Methods In the discovery phase, we conducted a meta-analysis of two GWAS studies of DLBCL patients treated with rituximab and anthracycline-based chemotherapy, the first study as part of the prospective clinical trials of the LNH2003B program (N=540) of the Lymphoma Study Association (LYSA) and the second as part of the Molecular Epidemiology Resource (N=316), a prospective observational study from the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence (SPORE). Genotyping for the GWAS was performed using the Illumina 610 (LYSA) and 660 Quad (SPORE) BeadArrays (Illumina, San Diego, USA). HapMap2 was used to impute additional SNPs. Association testing was conducted assuming a log-additive genetic model and using Cox regression analyses. We calculated the hazard ratios (HRs) and 95% CIs for event-free survival (EFS), with adjustment for age, sex and age-adjusted IPI. The two studies were then combined using the fixed effects inverse variance method based on the beta estimates and standard errors from each study. From the meta-analysis, 89 SNPs were selected for validation, based on a minor allele frequency (MAF) >0.05 and LD pattern for multiple SNPs from the same region. Validation cohorts were an independent set of DLBCL patients from the SPORE (SPORE-2, N=366) and DLBCL patients from the prospective GOELAMS 075 phase III study (N=294), and validation genotyping was conducted using the Illumina VeraCode platform. We then conducted a meta-analysis of the 4 studies. We report SNPs with both a statistical significance of p<5x10-6 and effect sizes in the same direction across the 4 studies in the meta-analysis. For the latter SNPs, we also report the results for overall survival (OS), adjusted for age, sex and age-adjusted IPI. Results In the final meta-analysis of all four studies for the prediction of EFS, the top SNP was rs7765004 (MAF=0.30; HR=1.45; p=9.7 x 10-8) in the 6q21 region, which is near the MARCKS (myristoylated alanine-rich protein kinase C substrate) and HDAC2 genes. MARCKS encodes for a protein involved in cell mobility, while HDAC2 belongs to the histone deacetylase family. Additional SNPs from this region included rs6918103 (MAF=0.25; HR=1.38; p=7.6 x 10-6), which marks an EZH2 histone binding site, and rs11969684 (MAF=0.12; HR=1.43; p=3.3 x 10-5). The next hit was rs7712513 (MAF=0.31; HR=1.40; p=3.2 x 10-7), which is near arginine-fifty homeobox pseudogene 1 (ARGFXP1) on chromosome 5q23.2. This was followed by a region on chromosome 8q13.3, with the SNP rs9298183 (MAF=0.29; HR=1.41; p=1.4 x 10-6) from LOC100132891 showing the strongest association, along with 3 other SNPs from this region (rs2035376, rs6472637, and rs1600797; all p<5 x 10-6). These SNPs are near the musculin gene (MSC) a transcriptional repressor of E2A frequently silenced by promoter methylation in DLBCL. Finally, rs2253986 (MAF=0.27; HR=1.39; p=3.4 x 10-6) at chromosome 16q12.2, along with rs2253971 and rs6499810, were associated with EFS. These SNPs are located in CES5A (carboxylesterase 5A), which encodes a member of the carboxylesterase family. The family members are responsible for the hydrolysis or transesterification of various xenobiotics. The top SNPs associated with EFS also predicted OS, including rs7765004 (HR=1.41; p=1.2 x 10-5), rs7712513 (HR=1.39; p=8.4 x 10-6), rs9298183 (HR=1.36; p=3.5 x 10-5), and rs2253986 (HR=1.35; p=5.2 x 10-5). Conclusions Although our top SNPs on did not reach genome-wide significance (p<5x10-8), they showed clear consistency across the four large prospective studies and predicted both EFS and OS independently of IPI. The strongest findings were from loci on 6q21, 8q13.3, 5q23.2, and 16q12.2, and several genes from these regions are involved in methylation and xenobiotic metabolism, supporting a role for host genetic variation in these pathways in DLBCL prognosis. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2642 BAFF is essential for B cell maturation, and a lack of either BAFF or its primary receptor, BAFF-R, results in a severe depletion of T2 marginal zone and follicular B cells. Elevated serum BAFF levels have been correlated with an increased risk of developing non-Hodgkin's lymphoma (NHL), along with a more aggressive phenotype. A growing body of genetic evidence points toward an association between the development of human disease and variation in genes encoding BAFF and its receptors. Recently, we characterized a novel lymphoma-associated mutation in TNFRSF13C, the gene encoding BAFF-R. This mutation (BAFF-R H159Y) encodes a His159Tyr substitution in the C-terminus of BAFF-R adjacent to the TRAF3 binding motif. Signaling through BAFF-R H159Y results in increased NF-κB activity, elevated immunoglobulin production and increased association with TRAF2, TRAF3 and TRAF6 compared to wild type (WT) BAFF-R. We have detected this mutation in 6% of total NHL cases (n=129), and in 10% of follicular lymphoma (FL) cases (n=41) evaluated thus far. We previously reported that BAFF-R H159Y expressing mouse B cells exhibited significantly more resistance to Fas ligand (FasL) induced apoptosis compared to their cells expressing BAFF-R WT, and we propose here that BAFF-R H159Y mediated increases in PI3K activity may explain such an enhanced anti-apoptotic response. In this study we now show that BAFF stimulated HEK 293 cells stably expressing BAFF-R H159Y not only display significantly increased Akt phosphorylation when compared to BAFF-R WT expressing cells, but also demonstrate robust Akt phosphorylation in the absence of BAFF. BAFF-R H159Y-dependent Akt activation also led to activation of the downstream Akt targets mTOR and GSK3β and their phosphorylation was inhibited following treatment with the PI3- kinase inhibitor wortmannin. We next examined the impact of the BAFF-R H159Y mutation on expression of BAFF-target genes. Quantitative PCR analyses revealed that BAFF-R H159Y cells exhibited a pattern of gene expression indicative of promoting cell survival, displaying significantly higher levels of BCL2, BCL2L1 and PIN1, while down-regulating expression of the pro-apoptotic gene BIM. We recently reported that TRAF6 associates with BAFF-R, and that such binding is more pronounced in cells expressing BAFF-R H159Y. In order to investigate the role TRAF6 plays in mediating BAFF-R-dependent PI3K activity, we silenced TRAF6 expression in HEK 293 and Karpas 422 lymphoma cells using TRAF6 shRNA. Reduced TRAF6 protein expression resulted in a parallel decrease in BAFF-R WT mediated phosphorylation of mTOR in Karpas 422 cells and phosphorylation of both Akt and GSK3β was markedly reduced in BAFF-R H159Y expressing HEK 293 cells. Interestingly, TRAF6 knock-down did not affect NF-kB2 activation in either Karpas 422 or HEK BAFF-R expressing cells suggesting that Akt does not play a role in BAFF-R mediated activation of non-canonical NF-kB. Finally, preliminary co-precipitation studies indicate that Akt can be recruited to BAFF-R itself, and our initial observations suggest that such an association is significantly reduced in cells expressing BAFF-R H159Y. Taken together, these studies suggest that the BAFF-R H159Y mutation confers enhanced BAFF-R-dependent PI3K signaling and pro-survival gene expression independent of BAFF. Moreover, such enhanced P13K activation is partly dependent upon TRAF6, and decreased recruitment of Akt to BAFF-R H159Y may function to increase the amount of this PI3K target for activation. Thus, BAFF-R H159Y likely contributes to BAFF signaling irregularities in NHL patients harboring this mutation, and may predispose individuals to developing lymphoma regardless of their serum BAFF concentration. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 996 Background: Treatment with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) has improved survival in diffuse large B-cell lymphoma (DLBCL) lymphoma, although a significant percentage of patients do not achieve a remission or they relapse. One potential explanation for this observation is that host pharmacogenetic background may impact metabolism, detoxification and transport of R-CHOP. However, there are few prospectively collected data that address pharmacogenetics in DLBCL, particularly for event-free and overall survival in the rituximab era. We therefore tested the hypothesis that functional candidate single nucleotide polymorphisms (SNPs) from genes involved in the metabolism of CHOP (CYP2C19, CYP3A5, ABCB1, ABCC2, ABCG2, RAC2, CYBA, NR3C1, GSTA1, GSTP1, TP53) and rituximab (FCGR2A, FCGR3A) influence prognosis in DLBCL. Methods: We genotyped 19 SNPs from 13 genes in a prospective cohort of newly diagnosed DLBCL patients with germline DNA enrolled at the Mayo Clinic and University of Iowa from 2002–2008 as part of the Molecular Epidemiology Resource of the Iowa/Mayo Lymphoma SPORE. All patients were systematically followed through 2009 for overall survival (OS) and event-free survival (EFS), defined as disease progression, retreatment or death due to any cause. All SNPs were genotyped on the Illumina Golden-Gate platform. Cox regression was used to estimate Hazard Ratios (HRs) and 95% confidence intervals (CI) for individual SNPs with OS and EFS. Each SNP was modeled with the most prevalent homozygous genotype used as the reference group. An ordinal test was used to assess the trend across genotypes, and a p<0.05 was considered statistically significant. All Cox models adjusted for International Prognostic Index (IPI) and treatment. Results: The median age at diagnosis of the 439 DLBCL patients was 62 years (range 18–92). The IPI was distributed as follows: 0–1 (34%), 2 (26%), 3 (24%) and 4–5 (16%). All patients were treated with chemotherapy, and 91% received R-CHOP. Through 2009, there were 171 events (39%) and 122 (28%) deaths, with a median follow-up for living patients of 49 months (range 1–98). The SNP rs1045642 in the ATP-binding cassette (ABC) protein gene ABCB1 was associated with OS (p=0.012). The variant T allele for ABCB1 rs1045642 (minor allele frequency (MAF)=0.47) is associated with a silent I→I substitution, and the T allele predicts low enzyme expression. Compared to CC homozygotes, patients with CT (HR=0.66, 95% CI 0.44–0.99) or TT (HR=0.53, 95% CI 0.31–0.90) genotypes had superior OS. We also observed an association of the GSTP1 SNP rs1695 with OS (p-trend=0.018). The variant G allele (MAF of 0.31) is associated with an A→V substitution, and this change predicts low enzyme expression. Compared to AA homozygotes, patients with the AG (HR=0.78, 95% CI 0.54–1.14) or GG (HR=0.39, 95% CI 0.17–0.90) genotypes had superior OS. Nearly identical associations for these two SNPS were observed with EFS. There were no significant associations with any of the other SNPs that we evaluated. Conclusions: In a large series of newly diagnosed DLBCL patients treated in the rituximab era, we found evidence that genetic variation in ABCB1 and GSTP1 is associated with improved OS and EFS after adjusting for IPI and treatment. The variant allele for ABCB1 is associated with slower enzyme activity, favorably impacting doxorubicin and vincristine pharmacodynamics, and the variant allele for GSTP1 is associated with slower detoxification of doxorubicin and vincristine, increasing bioavailability of these drugs. A comprehensive evaluation of the role of pharmacogenetics in DLBCL is warranted. Support: P50 CA97274, R01 CA129539. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1640 Background: The introduction of radioimmunotherapy (RIT) into clinical practice for the treatment of follicular lymphoma (FL) occurred at a time at which many patients did not have prior exposure to rituximab. Clinical trials in the relapsed/refractory setting, as well as consolidation in the upfront setting have included a small number of patients that had been previously treated with rituximab or a rituximab containing treatment regimen. We evaluated the effect of prior rituximab therapy on the efficacy of RIT. METHODS: The prospective lymphoma database was queried for all patients having received a single dose of yttrium-90 ibritumomab tiuxetan for relapsed/refractory FL which had previously been treated with systemic therapy (patients receiving only prior external beam radiation therapy were excluded). The database includes all consecutive adult patients diagnosed with lymphoma seen at Mayo Clinic Rochester. The diagnosis of follicular lymphoma was as by WHO classification and centrally reviewed. Patients with evidence of histologic transformation to diffuse large B cell lymphoma prior to RIT were excluded. Time to treatment failure (TTF) was defined as the time from RIT to next anti-lymphoma therapy. RESULTS: 111 patients were identified. 57 (51%) were male. Median age at time of RIT was 58 (19–88). Median time from diagnosis to RIT was 35 (6–281) months in patients having received prior treatment with rituximab and 47 (12–272) months in those who had not (p=0.19). 63 (57%) of patients received prior treatment with rituximab or rituximab containing chemotherapy. Median number of prior therapies was 2 (1–10), which was not different between the rituximab treated or untreated groups [2(1–6) vs 2(1–10) p=0.12]. Median TTF was 10.0 months in the patients having received prior rituximab and 10.1 months in those that had not (p=0.29). Figure 1. The median overall survival from RIT was not reached, but appeared similar (Kaplan-Meier estimate 125 months without prior rituximab exposure vs 95 months with prior rituximab exposure; p=0.33). CONCLUSIONS: Prior rituximab exposure does not seem to adversely impact the time to treatment failure or overall survival in those patients treated with RIT. While the data leading to the approval of RIT were accrued prior to the routine clinical use of rituximab, our data demonstrate similar clinical benefit of RIT in patients with prior exposure to rituximab or rituximab containing therapy. Disclosures: No relevant conflicts of interest to declare.