Cerhan, James R.

Abstract Background: Peripheral T-Cell lymphomas (PTCLs) constitute approximately 10% of lymphoid malignancies and consist of several distinct entities based on pathologic and clinical characteristics. With the exception of a few subtypes (e.g., ALK-positive anaplastic large cell lymphoma (ALCL) and some primary cutaneous or leukemic forms of PTCL), a majority of PTCLs are aggressive as characterized by poor treatment response, rapid disease progression and poor overall survival. We have shown that landmark timepoints of event-free survival after diagnosis can stratify subsequent overall survival (OS) in diffuse large B-cell and follicular lymphoma. Here we evaluate this approach in newly diagnosed aggressive PTCLs treated with anthracyline-based or related chemotherapy. Methods. Newly diagnosed PTCL patients were prospectively enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource (MER) from 2002-2012. Clinical data were abstracted from medical records using a standard protocol. For this analysis, we included patients receiving anthracycline-based or other multiagent chemotherapy for the following PTCL subtypes: ALK-negative ALCL (N=24); angioimmunoblastic T-cell lymphoma (AITL, N=34); PTCL, not otherwise specified (NOS; N=60); enteropathy-associated T-cell lymphoma (EATL, N=8); extranodal NK/T-cell lymphoma, nasal type (ENKTL, N=11); and hepatosplenic T-cell lymphoma (HSTCL, N=1). Patients were prospectively followed, and event-free survival (EFS) was defined as time from diagnosis to progression, re-treatment, or death due to any cause. Landmark EFS timepoints were assessed at 12 (EFS12) and 24 (EFS24) months after the date of diagnosis. Subsequent OS was defined as time from a specific endpoint (diagnosis, event or EFS landmark). Replication was performed in a population-based cohort of T-cell lymphomas diagnosed from 2000-2009 from the Swedish Lymphoma Registry. Results. 138 eligible patients were enrolled in the MER from 2002-2012, the median age at diagnosis was 58 years (range, 19-88), 66% were male, 73% had Stage III-IV disease, and 33% had IPI 0-1. At a median follow-up of 47 months (range 11-120), 87 patients (63%) had an event and 70 patients (51%) had died. From diagnosis, only 60 patients were event-free at 12 months (EFS12 45%). Patients who failed to achieve EFS12 had a poor subsequent OS from event (median OS = 6.8 months, 95% CI: 5.3-14.0, figure 1). In contrast, patients who achieved EFS12 had a favorable subsequent OS (median unreached, figure 2). Of the 427 eligible patients in the Swedish registry, the median age at diagnosis was 66 years (range, 18-88), 63% were male, 68% had Stage III-IV disease, and 25% had IPI 0-1. PTCL subtypes were: ALK-negative ALCL (N=89); AITL (N=80); PTCL, NOS (N=183); EATL (N=44); ENKTL (N=24); and HSTCL (N=7). At a median follow-up of 86 months (range 40-158), 333 patients (79%) had an event and 316 patients (74%) had died. From diagnosis, 183 patients were event-free at 12 months (EFS12 44%). Similar to the MER cohort, Swedish patients failing EFS12 had poor subsequent survival (median OS = 3.7 months, 95% CI: 2.9-5.3, figure 1). Swedish patients achieving EFS12 had a favorable subsequent OS (median OS = 89 months, figure 2). Similar results were obtained when conducting landmark analysis at 24 months after diagnosis (EFS24). Conclusion. Relapse and re-treatment events within the first 12 months of diagnosis are associated with very poor OS in PTCL treated with anthracyclines or related chemotherapy, while patients achieving EFS12 have encouraging subsequent OS. Stratifying patients into prognostically distinct subsets using EFS12 may help focus biologic and biomarker studies. EFS12 has potential as an early endpoint for studies of newly diagnosed PTCL. Further investigation of determinants related to post-EFS12 survival is needed. Disclosures Maurer: Kite Pharma: Research Funding. Cerhan:Kite Pharma: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding. Link:Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding. Thompson:Kite Pharma: Research Funding. Relander:Respiratorius: Patents & Royalties: valproate for DLBCL.

Abstract Abstract 2702 Background: CXCR5 [chemokine (C-X-C motif) receptor 5; also known as Burkitt lymphoma receptor 1 (BCR1)] is expressed on mature B-cells, subsets of CD4+ and CD8+ T-cells, and skin-derived migratory dendritic cells. CXCL13 is the ligand for CXCR5, and together they are involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. CXCR5 is also expressed in malignant cells in NHL patients. Single nucleotide polymorphisms (SNPs) in CXCR5 have not been extensively studied, although two studies reported that SNPs from this chromosome 11 gene were associated with risk of primary biliary cirrhosis in a European population and with risk of NHL in an Asian population. This study evaluated the role of common germline genetic variation in the risk and prognosis of the common NHL subtypes of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and peripheral T-cell lymphoma (PTCL). Methods: Ten SNPs in CXCR5 were genotyped using a clinic-based study of 2694 NHL cases (including 586 DLBCL, 588 FL, 137 MCL, 230 MZL, and 158 PTCL) and 1521 controls enrolled from 2002–2009 in the Molecular Epidemiology Resource of the University of Iowa/Mayo Clinic Lymphoma SPORE. Clinical data were abstracted for all NHL patients and all were prospectively followed for disease progression, retreatment, transformation and death. SNPs were selected from HapMap using a standard tagging approach (r2≥0.80 and minor allele frequency (MAF) ≥0.05) and included SNPs 5kb upstream and downstream from the gene. Genotyping was conducted using a custom Illumina iSelect panel. All SNPs were in Hardy-Weinberg equilibrium in the control group. 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. For risk of NHL, odds ratios (ORs) and 95% confidence intervals were calculated using unconditional logistic regression, adjusted for age and gender. For prognosis, hazard ratios (HR) were calculated for both event-free (EFS) and overall (OS) survival using Cox proportional hazards regression, adjusting for age, treatment type, and IPI (DLBCL, PTCL), FLIPI (FL), or MIPI (MCL). To determine risk of FL transformation to DLBCL, a competing risk Cox model was used. A p<0.05 was declared as statistically significant. Results: Of the 10 CXCR5 tag SNPs in our study, 5 were associated with risk of NHL, with rs1790192 (MAF=0.39 among controls) having the strongest association (OR=1.19, 95%CI 1.08–1.30; p=0.0003). This SNP was strongly associated with FL (OR=1.44, 95%CI 1.25–1.66; p=3.1×10−7), and was also associated with DLBCL (OR=1.16, 95%CI 1.01–1.33; p=0.038) and PTCL (OR=1.29, 95%CI 1.02–1.64; p=0.037) but not MCL or MZL. For FL, none of the SNPs were associated with EFS or OS, although rs1790192 was associated with better outcome among FL patients that were observed (HR=0.64; 95%CI 0.47–0.87; p=0.0039). For DLBCL, none of the SNPs were associated with EFS, while three SNPs were associated with OS: rs523604 (HR=0.79; 95%CI 0.64–0.97; p=0.028), rs12363277 (HR=0.57; 95%CI 0.33–0.99; p=0.046), and rs3922 (HR=0.79; 95%CI 0.65–0.97; p=0.027). The latter SNP was also associated with risk of transformation from FL to DLBCL (HR=0.61; 95%CI 0.40–0.91; p=0.016). For MCL, MZL and PTCL, none of the SNPs were associated with either EFS or OS. Conclusions: Several germline CXCR5 polymorphisms were associated with NHL risk, particularly rs1790192 with risk of FL as well as DLBCL and PTCL. CXCR5 SNPs did not appear to be important in prognosis for most NHL subtypes, although rs1790192 was associated with a better outcome in untreated FL and rs3922 was associated with OS in DLBCL patients and with risk of FL transformation. These results provide additional evidence for a role of host genetic variation in CXCR5 in lymphomagenesis, mainly in risk of disease development, although there may also be a role for CXCR5 in FL and DLBCL prognosis. Replications of these findings in additional studies are needed. Disclosures: Ansell: Seattle Genetics, Inc.: Research Funding.

Abstract Abstract 4127 Introduction: Mantle cell lymphoma (MCL) is an aggressive lymphoma. NF-κB has been shown to be constitutively activated in MCL cell lines and patient biopsy samples and may play a key role in the growth and survival of MCL cells. We previously reported in a pilot study of 39 MCL patients from the NCI-SEER Survival Study that host genetic variation in candidate TNF and NF-κB genes was associated with overall survival after accounting for clinical variables (Blood 2007;110(11):472a). Here, we attempt to replicate the top 8 genes (NFKB1, IRF4, TNFSF13B, TNFRSF25, NFKBIA, LTA/TNF, TRAF5, RELB) and associated single nucleotide polymorphisms (SNPs) in an independent sample of MCL patients. Methods: We genotyped 71 SNPs from 8 genes in a prospective cohort of newly diagnosed MCL 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). Genotyping was performed on an 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 genotype. A principal components methodology was used for gene-level analyses. All Cox models were adjusted for MIPI and treatment. We compared our results to the previously reported NCI-SEER study that included MCL patients diagnosed from 1998–2000. The NCI-SEER study had a median age at diagnosis of 64 years (range 38–74), 27 deaths (69%), and a median follow-up for living patients of 47 months (23-85 months). Replication was declared based on a p-trend<0.10 in the gene-level test or p-trend<0.10 in the SNP-level ordinal trend test and a HR of similar direction and magnitude to the NCI-SEER study result. Results: The median age at diagnosis of the 101 patients in the SPORE was 64 years (range 42–88), and by simplified MIPI score there were 38% low risk, 37% intermediate risk, and 26% high risk patients. The most common initial therapy was anthracycline-based chemotherapy ± rituximab (59%) with or without stem cell transplantation. Through 2009, there were 61 events (60%) and 31 (31%) deaths, with a median follow-up for living patients of 59 months (range 29–90). At the gene-level, none of the 8 genes replicated at p<0.10. However, at the SNP level, the genes with SNPs that replicated for OS included TRAF5 (rs3738199, rs6684874, rs12569232), TNFRSF25 (rs3138156), and RELB (rs10424046, rs1560725). The strongest TRAF5 SNP was rs3738199 which had a minor allele frequency (MAF) of 0.35. Compared to patients with the AA genotype, those with the AG (HR=2.14, 95% CI 0.91–5.01) or GG (HR=3.72, 95% CI 1.34–10.3) genotypes had inferior survival (p-trend=0.0092). The TNFRSF25 SNP rs3138156 had a MAF of 0.054; compared to patients with the AA genotype, those with the AG or GG genotype (HR=2.46, 95% CI 0.88–6.89) had inferior survival (p=0.087). Finally, the strongest RELB SNP was rs10424046, and had a MAF of 0.54. Compared to patients with the GG genotype, those with the GC (HR=0.57, 95% CI 0.25–1.28) or CC (HR=0.39, 95% CI 0.14–1.12) genotypes had superior survival (p-trend=0.065). Similar results for EFS were observed for the TRAF5 and RELB SNPs. Discussion: Germline genetic variation in the TNF and NF-κB pathway genes TRAF5, RELB, and TNFRSF25 were associated with prognosis in MCL after adjustment for clinical and treatment factors, and this result replicates findings from an independent dataset. TRAF5 is one of the components of a multiple protein complex which binds to TNF receptor cytoplasmic domains and mediates TNF-induced activation; RELB is part of the NF-κB complex; and TNF super family receptor member 25 stimulates NF-κB activity and regulates apoptosis through signal transduction that is mediated by various death domain containing adaptor proteins. In summary, genetic variation in TNF and NF-κB pathway genes may play a role in disease progression and overall survival in MCL, supporting further targeting of this pathway for therapy. Support: Lymphoma Research Foundation, P50 CA97274, R01 CA129539. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2699 Myeloid differentiation factor 88 (MYD88) is an adaptor protein that plays a key role in mediating innate immune response. Upon activation of toll-like (TLRs) or IL-1b receptors, MYD88 activates a signaling cascade that culminates in activation of NF-kB and expression of proinflammatory genes. Activating mutations in MYD88 have been reported in non-Hodgkin's lymphoma (NHL) tumors, including diffuse large B cell and marginal zones lymphoma. The most common MYD88 mutation described thus far is a single base pair mismatch resulting in an amino acid switch from lysine to proline at position 265 (MYD88L265P). MYD88L265P functions as a constitutively active MYD88 protein resulting in dysregulated NF-kB and STAT3 signaling. This mutation has recently been reported to be present in lymphomoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM) at a high frequency, ∼ 90% (Xu et al, ASH, #261, 2011). The discovery of MYD88L265P in a high percentage of LPL/WM tumors, and other subtypes of NHL, provides the basis for the identification and targeting of MYD88 dependent pathways. Additionally, the ability of this mutation to drive NF-kB-mediated cytokine expression combined with our finding that LPL/WM patients have elevated serum cytokines suggests that the MYD88 mutation may play a significant role in LPL/WM pathogenesis. We first wanted to confirm the incidence of the MYD88L265P mutation in LPL/WM and other subtypes of indolent lymphoma. Using Sanger sequencing we found that 57% (n=58) of LPL, 4% of MALT (n=23), 5% of nodal marginal zone (n=34), and 6% of splenic marginal zone (n=34) lymphomas harbor the MYD88L265P mutation, with all but 1 case being heterozygous. When we specifically looked at the LPL cases with WM, we found that 70% of WM tumors have the MYD88L265P mutation. In a validation cohort of WM (n=41), we found a similar mutation rate of 68%. Additionally, the WM cell line MWCL1 and the IgM secreting cell line BCWM.1 also harbor the MYD88L265P mutation. The prevalence of the MYD88L265P mutation in NHL, specifically WM and LPL, suggests that it may be a novel driver of lymphoma pathogenesis and a potential therapeutic target. However, it is not yet clear if this mutation correlates with activation of signaling pathways, or cell growth and survival in LPL/WM. To address these questions we first explored the possibility that the MYD88 signaling pathway is constitutively active in cells that express MYD88L265P. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodiamer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-kB. Using three cell lines that express MYD88L265P (BCWM, MWCL1, and OCI-Ly3), we looked for complex formation of MYD88, IRAK1/4, TRAF6, and TAK1. Immunoprecipitation of either endogenous IRAK1 or TAK1 revealed constitutive association with both TRAF6 and MYD88. We also detected phosphorylation of TAK1, and taken together, these data suggest that the MYD88 pathway is activated in cells that harbor the MYD88L265P mutation. We next wanted to confirm the significance of the MYD88 pathway on lymphoma cell growth. Using our cell lines we tested the effect of IRAK1/4, TAK1, and NF-kB inhibitors on cell proliferation. All MYD88L265P expressing cell lines were sensitive to the TAK1 and NF-kB inhibitors in a dose dependent manner (0–10 uM), however, we only saw inhibition of cell proliferation with the IRAK1/4 inhibitor at the highest dose (10 uM) in the BCWM cell line. These data suggest that cell proliferation is dependent on the MYD88 signaling pathway, with some variation being detected between cell line models and inhibitors. Because the MYD88 pathway has also been shown to drive cytokine secretion we next tested the impact of the IRAK1/4, TAK1, and NF-kB inhibitors on expression of IL-10. Using drug doses that were shown to inhibit cell proliferation, but not impact survival, we found that all three inhibitors reduced the level of IL-10 secreted by each of cell lines. In summary, our data confirm the high incidence on the MYD88L265P mutation in LPL/WM, we find that the MYD88 signaling pathway is constitutively activated in cells the harbor the mutation, and we show that cell proliferation and cytokine secretion are dependent on MYD88 signaling targets. These studies highlight the biologic significance of the MYD88L265P mutation in LPL/WM and suggest that the MYD88 signaling pathway may be a novel therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 468 The requirement for BAFF and BAFF-R in normal human and murine B cells is well studied, but there is also significant evidence to suggest that BAFF plays an important role in malignant B cell proliferation and survival. Serum BAFF levels are elevated in patients with non-Hodgkin lymphoma (NHL) and high BAFF levels correlate with aggressive disease and a poor response to therapy. There is also increasing genetic evidence suggesting an association between the development of human disease and genetic variation in genes encoding BAFF and its receptors. Mutations in TNFRSF13B (TACI) were identified in patients with familial common variable immunodeficiency (CVID) and IgA deficiency and we have found that single nucleotide polymorphisms (SNP) in TNFSF13B (BAFF) are associated with elevated BAFF levels and risk for developing NHL. To build upon these findings we sequenced BAFF and its receptors; TNFSF13B, TNFRSF13B, TNFRSF17(BCMA), and TNFRSF13C (BAFF-R) in NHL patients to identify novel genetic variants that may be associated with NHL risk. Among 40 individual samples (20 controls and 20 follicular lymphoma (FL) cases) that were bi-directionally sequenced we identified a heterozygous cytosine to thymidine transition in 1 patient specimen at position 475 (C475T) of TNFRSF13C. The C475T transition encodes a missense substitution of tyrosine for histidine in codon 159 (H159Y) in the highly conserved cytoplasmic tail of BAFF-R, adjacent to the TRAF3 binding motif PVPAT. We next expanded our analysis of BAFF-R H159Y and analyzed NHL tumor biopsies for the presence of the mutation. 4/41 (10%) follicular lymphomas (FL), 2/42 (5%) diffuse large B cell lymphomas, 1/22 (5%) lymphoplasmacytic lymphomas (LPL), and 1/24 (4%) mucosal associate lymphoid tissue lymphomas carried the heterozygous mutation. The BAFF-R H159Y mutation was not detected in any of the normal control DNA from healthy donors (n=100). Given its close proximity to the TRAF3 binding site in the cytoplasmic domain of BAFF-R we first wanted to determine if the H159Y mutation altered BAFF induced signaling. We generated cell lines that express HA-tagged wildtype BAFF-R, BAFF-R with the H159Y mutation, or BAFF-R with an ablated TRAF3 binding site as a negative control. Analysis of cells expressing H159Y BAFF-R demonstrates that this mutation results in increased BAFF-R-mediated NFκB1 and NF-κB2 activation. The enhanced signal activated by BAFF-R H159Y is coupled with a several fold increase in TRAF3, TRAF2, and TRAF6 recruitment to BAFF-R and increased IgM production. We further demonstrate that recruitment of TRAF6 to BAFF-R is not unique to the mutant H159Y BAFF-R, but is also an important and necessary feature of BAFF-R signaling in normal B cells. Collectively, our data identify a novel lymphoma-associated mutation in BAFF-R and describe exciting new aspects of BAFF-R signaling that are important for understanding normal B cell homeostasis and function, as well as pathogenic BAFF-R contributions to human disease. 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 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

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

Abstract Abstract 1232 Poster Board I-254 B-Chronic Lymphocytic Leukemia (CLL) shows a strong familial risk and also co-aggregates with other indolent lymphomas. Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic hematologic condition characterized by small B-cell clones, most with a surface phenotype similar to that of chronic lymphocytic leukemia (CLL). These clones are detectable at low absolute lymphocyte cell (ALC) numbers in otherwise healthy individuals using sensitive 6 or 8 color flow cytometry analysis and can be a precursor to CLL. In the general population, MBL increases with age with a prevalence of 5% in individuals over age 65. In contrast, the rate of MBL is 13-18% in first degree relatives of CLL patients in high risk families. In the largest study to date, we report the characteristics of MBL among 430 first-degree relatives of CLL patients (with no associated lymphoproliferative diseases [LPD]) in 132 high risk families. Patients and Methods Individuals studied came from “high risk” families (defined as those with two or more confirmed cases of CLL) that are participating in the Genetic Epidemiology of CLL Consortium. Multi-parameter flow cytometry analysis with a detection sensitivity of 0.02% was performed on either fresh or cryopreserved PBMC and MBL was classified according to previously published criteria (Marti et al, Br. J Haematol 130:325, 2005). Both the ALC and the absolute B cell count (B-ALC) were calculated for each individual. Survival analysis was used to compute the probability of developing MBL with age using the life table method. Results The overall rate of MBL was 17% (73/430) among first–degree relatives but the probability for developing MBL by age 90 was 51%. Males had a slightly higher (but non-significant) risk for MBL than females. MBL patients had significantly higher ALC and B-ALC than did those with normal immunophenotype. The mean ALC was 2.5×109/L among MBL patients, and 20% had an ALC greater than 3 ×109/L. The B-ALC count averaged 0.51 ×109/L. Ninety percent of the MBL cases had a CLL-like phenotype (CD20dim, CD5+, CD23+). Conclusions MBL is found at a very high rate in families selected for having 2 or more patients with CLL suggesting that MBL reflects inherited predisposition to CLL. Although most of the MBL cases in these families had low cell counts and thus have a presumed low likelihood of progressing to CLL or other LPD, a higher proportion of them had lymphocytosis compared to those with normal immunophenotype. We hypothesize that if MBL is an early step in the process of development of CLL, then germ line genes are likely to be acting early in carcinogenesis with more “hits” required before CLL develops. By looking for genes associated with MBL or CLL in families or in the population, we could substantially increase our power to identify germ line genes predisposing to CLL. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

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