Slager, Susan L.

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 Abstract 4152 BACKGROUND: Patients with biologically aggressive chronic lymphocytic leukemia (CLL) characterized by being refractory to purine nucleoside analogues and/or possessing the cytogenetic abnormality del (17p13) have poor clinical outcomes with a median survival of <3 years. Several groups have reported that nonmyeloablative allogeneic stem cell transplant (alloSCT) provides long-term disease control in a subset of high-risk CLL patients. The Seattle group also recently reported that alemtuzumab use prior to alloSCT may increase the risk of early relapse/progression (Sorror, ASH 2010). This is a potentially critical observation since alemtuzumab is one of the few active agents in patients with del(17p13) and is frequently used for pre-transplant debulking in patients with this genetic defect. METHODS: We performed retrospective analysis of 38 CLL patients who underwent nonmyeloablative alloSCT for CLL at the Mayo Clinic between 2003 and 2011. The relationship between transplant outcome and debulking regimen used immediately prior to alloSCT, disease status at the time of alloSCT, post transplant engraftment, GVHD development was explored. The degree of marrow involvement prior to alloSCT was assessed using the leukemia index calculated by multiplying the % involvement by CLL cells and the % cellularity. RESULTS: Median age at the time of alloSCT was 55.5 (range, 29–69) and 13 patients (34%) were female. The median duration from CLL diagnosis to alloSCT was 4.7 years. Thirty-two patients (84%) had received more than 3 prior therapies prior to alloSCT. Thirty-seven patients (97%) had received purine analog-based therapies, 18 (47%) patients had received alkylating agents and 25 (66%) had received alemtuzumab. 33 (87%) patients were purine analog refractory. On cytogenetic evaluation by FISH, 17 (45%) patients had del(17p13), 10 (26%) had del(11q23) and 1 (3%) had del(6q23). Nine patients were in complete remission (CR) by IWCLL 2008 criteria at the time of alloSCT and 7 patients were in complete remission with incomplete marrow recovery (CRi). Of the remaining 22 patients, 20 had either a partial remission or stable disease at the time of alloSCT and 2 had progressive disease. Two (5%) patients had bulky lymphadenopathy (≥ 5cm by CT imaging), 11 (29%) patients had splenomegaly, and 8 (21%) patients had more than 30% CLL involvement by the leukemia index. Fludarabine (F) and 200cGy total body irradiation (TBI) was used as the conditioning regimen for 35 patients (92%). 22 patients received unrelated donor grafts (15 matched, 7 mismatched), and 16 received related donor grafts (all matched). Patients received mycophenolate (MMF) and either tacrolimus (unrelated grafts) or cyclosporin (related grafts) for graft versus host disease (GVHD) prophylaxis. 31 out of 37 evaluable patients (84%) achieved initial 100% donor chimerism (22 within 100 days) and 24 (63%) developed ≥grade 2 GVHD. After median follow-up of 1 year (range = 0–5.4 years), 26 of 38 patients (68%) are alive, and the median overall survival is 47 months. To date, 13 (34%) have experienced CLL progression/relapse requiring accelerated taper of GVHD prophylaxis (n = 2), donor lymphocyte infusion (n = 5), or chemo/immunotherapies (n= 6). For the 12 deceased patients, causes of death was CLL relapse/progression (n = 5), GVHD (n = 2) and infectious or other complications without CLL relapse/progression (n = 5). Pre-transplant use of alemtuzumab (A) within 3 months of transplant was the only pre-transplant characteristics associated with overall survival (OS). Median OS of patients receiving A within 3 months of alloSCT was 1 year compared to not reached for those who did not receive A within 3 months of alloSCT (p = 0.011; Figure 1a). A trend was also observed between alemtuzumab use within 12 months of alloSCT; however, it did not reach statistical significance (p=0.069; Figure 1b). No statistically significant associations between residual marrow involvement at time of alloSCT (p = 0.93), type of graft (p = 0.65), or presence of del (17p13) (p = 0.13), number of prior treatments (p = 0.45), patient age (p = 0.80) or disease status at time of alloSCT (p = 0.21) were observed. CONCLUSION: In this cohort of patients with high risk CLL, alemtuzumab use prior to transplant was the greatest predictor of OS after nonmyeloablative alloSCT. Non-alemtuzumab based debulking strategies need to be developed for patients with del (17p13). Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3933 Poster Board III-869 Background Non-Hodgkin Lymphoma (NHL) is a malignancy of lymphocytes with few known risk factors identified to date. Members of the BCL2 and caspase gene families are known regulators of programmed cell death in these cells, and the t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in NHL, a somatic event which results in constitutive BCL2 expression and inhibition of apoptosis. Further, recent pooled analyses of three case-control studies reported gene level associations for both BCL2L11 (Cancer Epidemiol Biomarkers Prev 2009;18:1259) and CASP9 (Blood 2009;114:264) with NHL risk. We therefore evaluated the hypothesis that germline variation in genes from the apoptosis pathway is associated with risk of developing NHL. Methods We genotyped 226 single nucleotide polymorphisms (SNPs) within 36 candidate BCL2-family and capsase-family member genes within the apoptosis pathway in a clinic-based study of 441 newly diagnosed NHL cases (within 9 months of first NHL diagnosis) and 475 frequency matched controls from Minnesota, Iowa, and Wisconsin who were seen at the Mayo Clinic from 2002-2005. Tagging SNPs were selected from HapMap for the following candidate genes: BCL2; BCLAF1; BCL2LA1; BCL2L1, 2, 10, 11, 12, 13, 14; BAD; BAX; BAG1, 3, 4, 5; BAK1; BID; BIK; BNIP2, 3; HRK; CASP1 – CASP10; APAF1; BIRC3; AIF1; and DFFB. Genotyping was completed using the ParAllele (now Affymetrix) Immune and Inflammation SNP panel, supplemented with 9 SNPs from a custom Illumina OPA. SNP call rate and per person call rate were >95% for all SNPs evaluated. The most prevalent homozygous genotype was used as the reference group, and each SNP was modeled individually as having a log-additive effect in a logistic regression model adjusted for age, sex, and residence. We used principal components analysis to assess the association with NHL risk at the gene level. Genes at p<0.05 were declared statistically significant, as were SNPs at p<0.05 from significant genes. Results The mean age at diagnosis for cases was 60.1 years, and 58% were male; among controls, the mean age at enrollment into the study was 61.7 years, and 55% were male. The most common NHL subtypes were chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, 29%), follicular lymphoma (FL, 27%), and diffuse large B-cell lymphoma (DLBCL, 16%). In gene level analyses, BCL2L11 (also know as BIM) (p=0.0019), BLCAF1 (p=0.0097), BAG5 (p=0.026), and CASP9 (p=0.0022) were statistically significant at p<0.05. For BCL2L11, 2 of the 5 tagSNPs (both intronic) were significant at p<0.05 (rs6746608 and rs12613243). Both genotyped BCLAF1 tagSNPs (rs797558 and rs703193, both intronic) were significant, as was the single genotyped BAG5 tagSNP (rs7042474) from the non-coding region interval of an mRNA transcript. Three of the 7 genotyped CASP9 tagSNPs were significant (rs6685648, rs2020902, rs2042370, all intronic). Similar associations (as assessed by ordinal odds ratios) were observed for significant SNPs from BCLAF1, BAG5, BCL2L11 and CASP9 within the subtypes of CLL/SLL, FL, and DLBCL; however, caution is warranted in interpreting the subtype results due to small sample sizes. Conclusions Three genes from the BCL2 family (BCL2L11, BCLAF1, and BAG5) and one gene from the caspase family (CASP9) were associated with risk of NHL. This confirms gene level results for BCL2L11 and CASP9 in NHL overall and suggests a role for additional BCL2 family members in NHL etiology. In addition to extending these results to other populations, investigation of the functional significance of these genes and consideration of their upstream regulators and downstream targets should assist in clarifying the role of this pathway in lymphomagenesis. Furthermore, in the era of targeted therapeutics, identification of genetic variation in the apoptotic pathway may also assist in predicting prognosis as therapies targeting this pathway are developed. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1785 Background: Chronic lymphocytic leukemia (CLL) is a malignant lymphoproliferative disorder characterized by the progressive clonal expansion of CD5+ B cells. Approximately 8–10% of CLL patients have a first degree relative with CLL, indicating that genetic factors can contribute to disease susceptibility. Genome wide association (GWA) studies have identified 16 “common variants,” single-nucleotide polymorphisms (SNPs) with a population prevalence of >5%, that confer risk for CLL. Among patients with CLL, the clinical course can be highly heterogeneous. Studies have defined disease-characteristics that vary among patients that can be used as markers to help predict clinical outcome. African American (AA) CLL patients have a poorer overall survival than Caucasians. However, there is little information on whether there are distinctive, ethnic-associated disease-characteristics that contribute to differences in clinical outcome. Moreover, studies of the genetics of CLL have involved primarily Caucasian patients. For these reasons, we examined for ethnic-related differences in the distribution and prevalence of CLL-associated SNPs and adverse disease-associated characteristics among AA with CLL relative to those of Caucasian patients with this disease. Methods: The clinical and biologic characteristics of 43 AA CLL patients ascertained at Duke University and the Durham VA Medical Center were tabulated and compared to Caucasian CLL patients at the same centers. Fifteen SNPs were genotyped in this “discovery” cohort of 43 AA CLL patients using TaqMan SNP genotyping assays. As a “validation” cohort, 70 AA CLL patients ascertained by the CLL Research Consortium (CRC) were assayed using the same platform. Three control groups were considered in analysis: (1) previously published Caucasian CLL patients, (2) control AA genotypes reported in the HapMap database and (3) control AA with no history of malignancy from other GWA studies. The 3' untranslated region of CPEB1 was sequenced in AA CLL patients using conventional Sanger sequencing. Results: Among the Duke/Durham VA CLL patients, no significant differences were observed in age, WBC count, platelet count, or hemoglobin at diagnosis; lymphocyte doubling time; Rai stage; ZAP70 expression; or CD38 expression between AA and Caucasian patients. AA had a larger proportion of trisomy 12 and fewer 13q.14 deletions than Caucasians: 40.0% of AA had trisomy 12 and 15.0% had del 13q, compared to 14.6% and 40.7% of Caucasians, respectively. IGHV mutation status also varied between races, with 62.2% of AA having unmutated IGHV, compared to 38.4% of Caucasians. Disparities in clinical outcomes were similar to that reported in the SEER database: overall survival was 8.8 years in AA patients and 17.8 years in Caucasians, and time to first treatment was 2.8 years among AAs and 4.5 years for Caucasians. Risk allele frequencies among 15 genotyped SNPs showed that 14 SNPs had risk allele frequencies that statistically differed between AA patients and Caucasian CLL patients. In 9 of 14 SNPs, the risk allele was less common in AA than in Caucasians but similar to that AA controls. In the other 5 SNPs, the risk allele was more common in AA CLL than Caucasian CLL, and similar to that of AA controls in 4 of the SNPs. A single SNP, rs783540 (CPEB1) was found to have an increased frequency in AA patients with CLL when compared to both Caucasian CLL and control AA. As an exploratory analysis to identify possible pathogenic variants, the 3' UTR of CPEB1 was sequenced as there are no common non-synonymous SNPs in CPEB1. Preliminary review of the sequencing data revealed that a G insertion SNP (rs34816185) that alters a predicted miRNA binding site is in linkage disequilibrium with rs783540 (D'= 0.978). Conclusions: Most SNPs associated with CLL risk are less frequent in AA than in Caucasian CLL patients. However, rs783540 (intron 2, CPEB1) risk allele was more frequent in AA CLL than that previously reported in Caucasian CLL or AA controls. Initial data suggests that rs783540 is linked to a SNP in the 3' UTR of CPEB1, which may have functional significance. Studies to better delineate the functional and clinical implications of these germline variants are underway. Our data provide the first evidence of differential genetic risk as a determinant of CLL biology among different ethnic groups. Disclosures: No relevant conflicts of interest to declare.

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 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 We have recently reported that event-free survival at 24 months from diagnosis (EFS24) is a robust endpoint for assessment of disease related outcome for newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) treated with anthracycline-based immunochemotherapy (IC). Patients who achieve EFS24 following IC have a subsequent overall survival that is equivalent to the age and sex matched general population. The IPI was developed in the pre-rituximab era using OS/EFS endpoints. While the IPI is associated with outcome in the IC era, the original survival estimates for the IPI risk groups are outdated. The IPI is also a discrete risk classifier, with only 6 possible groups. Here we remodel the IPI using EFS24 as the endpoint in a large international cohort of patients treated with IC. This model provides an individual level 0-100% risk of failing to achieve EFS24 for each patient and can be used for patient prognosis, treatment stratification, and risk assessment. Methods Newly diagnosed DLBCL patients treated with immunochemotherapy from the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource, NCCTG N0489 clinical trial, GELA LNH2003B program, and a Lyon, France based hospital registry were included in the original EFS24 study. Patients with a relapse, progression, unplanned re-treatment, or death due to any cause within 730 days of diagnosis were considered a failure for the EFS24 endpoint. Patients alive and progression/relapse free at 730 days from diagnosis with no subsequent treatment following initial IC were considered a success for the EFS24 endpoint. Variables selected for potential model inclusion were the IPI components (age, stage, ECOG PS, number of extranodal sites, and LDH), as well as standard clinical variables typically reported in studies of DLBCL: presence of B-symptoms, bone marrow involvement, bulky disease (>10 cm), and sex. The IPI24, a prognostic model for EFS24, was developed using logistic regression models. Model building began by fitting the classic IPI score (0-5), then allowing individual IPI variables to carry different model weights and number of categories, followed by dropping insignificant variables, finally adding any additional significant variables. Model performance was assessed using bias corrected c-statistics, AIC, and R2 values. Model selection was done using the complete case set with multiple imputation on the full dataset used to refine parameter estimates for the final model. Results 1596 total patients were available for modeling. The median age was 62 (range 18-93) and 53% were male; 33% were IPI 0-1, 25% were IPI 2, 25% were IPI 3, and 17% were IPI of 4-5. The failure rate for achieving EFS24 was 29%. All variables but sex (p=0.33) were univariately associated with EFS24 (p<6.4x10-5). The original IPI had good prognostic ability for EFS24 (c=0.696, p=2.2x10-32). Allowing multiple categories for ECOG PS, stage, and age (continuous) improved model fit and prognostic ability (c=0.727). The number of extranodal sites was not significant when IPI variables were modeled separately and was removed from the model. Sex and bulky disease were additional significant variables when added to the model. Thus, the final IPI24 model contained the following risk predictors: male sex, ECOG PS (0 vs. 1 vs 2 vs 3-4), stage (I vs. II, vs. III vs. IV), LDH (elevated vs. normal range), bulky disease > 10cm, and age (continuous). This model resulted in improved fit and prognostic ability (c=0.749) over the standard IPI (c=0.696, bootstrap p<0.001). Model validation was performed on an additional 158 patients from the SPORE MER and showed good concordance (c=0.697). Additional validation is planned in a larger set of independent patients. Conclusion The IPI24, generated using standard clinical variables, yields an individual risk prediction for failing to achieve EFS24 following IC for treatment of DLBCL. The IPI24 has superior prognostic ability to the standard IPI and can be implemented in the clinic using a nomogram or as a simple electronic application. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 700 Studies conducted over the last 10 years suggest that 3–5% of the general population over age 40 harbor a clonal population of lymphocytes of CLL phenotype when evaluated using sensitive flow cytometry assays (Rawstron Blood 100:635, 2002; Ghia Blood 103:2337, 2003). This discovery led to creation of the new diagnostic entity, monoclonal B-cell lymphocytosis (MBL; Marti BJH 130:325, 2005), which is considered the precursor state to chronic lymphocytic leukemia (CLL) (Landgren NEJM 360:659, 2009). The current diagnostic criteria for CLL-like MBL specify patients have a clonal B-cell population with appropriate phenotype, an absolute B-cell count <5 × 10^9/L, and no characteristics of a lymphoproliferative disorder (e.g. lymphadenopathy, organomegaly, B-symptoms) while individuals with a clone of CLL phenotype and B-cell count >5 × 10^9/L or who have lymphadenopathy are classified as having CLL (Hallek Blood 111:5446, 2008). The natural history of MBL appears to differ based on the absolute B-cell count. From an epidemiologic perspective, the majority of individuals with MBL have very small clonal lymphocyte populations, B-cell counts within the normal range, and appear to be at low likelihood of developing CLL (Rawstron ASH 2009). Such individuals with “low count” MBL do not appear to be at increased risk of death (Rawstron ASH 2009). From a clinical perspective, most patients with MBL are identified due to lymphocytosis and have B-cell counts between 3–5 × 10^9/L. While such individuals with “high count” MBL progress to require treatment for CLL at a rate of ~1.5% per year (Shanafelt JCO 27:3959, 2009), whether their survival differs from that of age- and sex-matched individuals is unknown. Conceptually, patients with the precursor state to malignancy should have a risk of death similar to those without the precursor state unless they progress to develop malignancy. To determine the impact of clinically “high count” MBL on survival, we compared the survival of 300 patients with clinically recognized MBL to the age- and sex-matched population. In addition, to explore whether the current B-cell threshold used to segregate MBL from CLL optimally classifies risk, we also evaluated the survival of 94 patients with newly diagnosed Rai 0 CLL and a B-cell count between 5–10 × 10^9/L and 218 newly diagnosed Rai 0 CLL patients with an ALC > 10 × 10^9/L to the age- and sex-matched population. Although it appeared slightly lower, the survival of patients with clinical recognized MBL (n=300, median age 68.6) was not statistically different than age- and sex-matched individuals (p=0.14; Figure 1A). In contrast, the survival of patients with newly diagnosed Rai 0 CLL and a B-cell count between 5–10 × 10^9/L (p=0.03) and newly diagnosed Rai 0 CLL patients with an ALC > 10 × 10^9/L (p=0.00018) were both significantly shorter than expected for the age- and sex-matched population. While these data are consistent with the designation of clinically recognized MBL as a premalignant condition, a more nuanced story emerged when the individuals with clinically recognized MBL were stratified by the CD38 status of the B-cell clone. Although the survival of individuals with a CD38- clone (n=213; 78%) was not different than age- and sex-matched individuals (p=0.14), the survival for those with a CD38+ clone (n=59; 22%) was substantially shorter than the age- and sex-matched population (p=0.0002; Figure 1B). Collectively, these data provide additional clinical evidence in support of the 5 × 10^9/L B-cell threshold to distinguish between MBL and CLL as patients with a B-cell count between 5–10 × 10^9/L or higher are at increased risk of death compared to the age- and sex-matched population. However they also suggest that, in addition to absolute B-cell count, biologic characteristics of the B-cell clone influence survival in patients with clinically recognized MBL. Additional studies are needed to determine whether such characteristics should be considered when classifying individuals with B-cell clones <5 × 10^9/L as having a premalignant or malignant condition. Figure 1A: Survival of patients with clinically recognized MBL (n=300) compared to the age- and sex-matched general population Figure 1A:. Survival of patients with clinically recognized MBL (n=300) compared to the age- and sex-matched general population Figure 1B: Survival of patients with clinically recognized MBL by CD38 status compared to the age- and sex-matched general population. CD38- p=0.14, CD38+ p=0.0002 Figure 1B:. Survival of patients with clinically recognized MBL by CD38 status compared to the age- and sex-matched general population. CD38- p=0.14, CD38+ p=0.0002 Disclosures: Shanafelt: Celgene: Research Funding; Hospira: Research Funding; Genentech: Research Funding. Off Label Use: Lenalidomide. Kay:Celgene: Research Funding; Hospira: Research Funding; Genentech: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 991 Background: The outcome of patients with DLBCL has significantly improved with the addition of rituximab to CHOP chemotherapy. While standard clinical prognostic factors such as those included in the International Prognostic Index (IPI) still predict outcome in DLBCL in R-CHOP treated 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 DLBCL patients. Methods: Newly diagnosed DLBCL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). Serum cytokines were measured from pre-treatment blood draws using a multiplex ELISA (Upstate Cell Signaling Solutions, Lake Placid, NY). 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 400 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 was abstracted using a standard protocol for the MER and 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. Principal components analysis (PCA) was performed on the combined DLBCL and control cytokine dataset, agnostic to clinical characteristics and outcome, to identify cytokine profiles. Results: 206 DLBCL patients diagnosed between September 2002 and February 2008 with available serum were included. The median age was 64 years (range 20–92) and 52% were male. At a medium follow-up of 47 months (range 5–94), 75 patients (36%) had relapsed and 63 patients had died (31%). A cytokine profile was determined based on the first two principal components (PC) of the PCA analyses with PC1 corresponding to a general (host) cytokine elevation (also seen in normal controls) and PC2 corresponding to an increase predominantly in cytokines that were commonly elevated in DLBCL patients. 75 DLBCL patients (36%) had a normal cytokine profile similar to the general population of controls; 114 DLBCL patients (55%) had elevation in tumor specific cytokines (CXCL10, CXCL9, IL-2R, IL-12, IL-8, and IL-10) and 17 (8%) had a general cytokine elevation. Patients with any elevation of cytokines (tumor specific or general elevation) had a significantly worse event-free (HR=3.96, 95% CI: 2.13–7.37, p<0.00001) and overall survival (HR=3.75, 95% CI: 1.99–7.09, p<0.0001) than those with a normal cytokine profile (see figure). While the cytokine elevations were associated with advanced stage, poor performance score and elevated LDH, they were not associated with advanced age or extranodal disease and the results remained significant after adjustment for the IPI and treatment in the Cox model (EFS HR=3.11, 95% CI: 1.66–5.85, p<0.0001; OS HR=2.93, 95% CI: 1.53–5.63, p=0.001). Elevated serum cytokines also identified patients who were unlikely to complete initial therapy or who relapsed early. In low-risk patients (IPI 0–2), only 3% of patients with normal serum cytokines relapsed in the first 12 months compared to 23% of patients with elevated cytokines. Similarly in the high-risk group (IPI 3–5), none of the patients with normal cytokines relapsed in the first year compared to 43% with elevated cytokines. Conclusion: Elevated pretreatment serum cytokines, predominantly related to the malignancy, are associated with an increased likelihood of disease relapse and an inferior survival in patients with DLBCL. Furthermore, the pretreatment cytokine profile may also identify DLBCL patients at high risk for early disease relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3881 Background: Chronic lymphocytic leukemia (CLL) is largely incurable neoplasm of the blood and has one of the highest familial risks of disease among cancers. Monoclonal B-cell lymphocytosis (MBL) with a CLL immunophenotypic profile (i.e., CLL-like MBL) is a precursor state to CLL. In the general adult population, the prevalence rate of MBL is 5–9% and increases with age. This is in contrast to the 13–18% rate of MBL in first-degree relatives of CLL patients from high-risk families (defined here as families with at least two confirmed living cases of CLL). Few longitudinal studies of MBL incidence exists. One study evaluated MBL status over time in a cohort of individuals with MBL from a rural valley in Northern Italy. Our work herein will be the first to evaluate the status of MBL over time in relatives of CLL patients from high-risk families. Methods: Using a cohort of 41 high-risk CLL families, we evaluated the MBL status and clonal blood B-cell counts in 94 relatives of CLL patients at two time points at least 1 year a part. High sensitivity, multi-color flow cytometry was performed on fresh or previously frozen blood samples in three laboratories: Duke University, the US Food and Drug Administration, and Mayo Clinic. The number of cell events collected for flow cytometry evaluation varied from 1 to 5 x105 cells. To assure concordance across flow cytometry laboratories, 15 samples were evaluated at multiple labs. The concordance of MBL calling was a 100%. We defined three monotypic B-cell subsets as CLL-like MBL (CD5+ CD20dim), atypical MBL (CD5+ CD20+), and non-CLL like MBL (CD5−CD20+). Results: Of the 94 relatives, the median age at consent was 57 years (range 42–93) and 39% were male. At the initial MBL screening, 70 (74%) relatives had normal immunophenotype (NIPT) and 24 (26%) had MBL (1 atypical MBL, 3 non-CLL like MBL, and 20 CLL-like MBL). Seven of the 24 individuals with MBL had monoclonal B-cell counts available at baseline screening; median = 147.4 cells/uL (range 22.7–1663). The 94 relatives underwent repeat evaluation with a median follow up time of 1.8 years (range 1–6). At the second evaluation, we found an increase in the prevalence of MBL, with 11 of the 70 (12%) initially NIPT relatives developing a new MBL clone (10 CLL-like, 1 non-CLL like). These 11 incident MBL cases were from 9 families with 2 families each having 2 relatives changing MBL status. The median number of monoclonal B-cells in these 11 incident MBL cases was 0.58 cells/uL (range 0.38–60.3). The 23 out of the 24 relatives with baseline MBL (20 CLL-like MBL and 3 non-CLL like MBL) had a persistent MBL clone. Only 6 of these 23 individuals had monoclonal B-cell counts available at both time points, and among these 6, the clonal B-cell counts decreased at follow-up screening; median difference −64.1 cells/uL (range −1204.3– 10.5). The second evaluation of the initial atypical MBL relative had too few clonal cells for flow assessment. Conclusion: We found that our CLL-like MBL clones persisted over the period of observation in relatives from high-risk CLL families; this finding is similar to that previously reported using a population-based study conducted in Northern Italy. Notably, 12% of relatives from high-risk CLL families developed incident MBL during just 2 years of follow up. Although these MBL clones are all low count (< 1500 cells/uL), these data provide further evidence that MBL is a marker of inherited predisposition to CLL. It will be important to see how our rate of change of MBL status compares to that of the general population as we continue to follow our cohort of CLL families. Disclosures: No relevant conflicts of interest to declare.