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Abstract Introduction: Our current understanding of CLL biology and optimal therapies is based on clinical trials that have largely enrolled White patient populations. Whether these findings are applicable to Hispanic or non-White patients is unknown but critical to evaluate given the growing numbers of minority patients in the U.S. While retrospective studies have suggested inferior outcomes in non-White patients, prospective data on these disparities are lacking. Patients and Methods: We utilized the Mayo Clinic CLL database to better understand variations in disease characteristics, molecular differences, and treatment selections between non-Hispanic White patients and those not in this category (referred to hereafter as “other race”). After excluding 49 patients with missing diagnosis date and another 490 patients with missing race and/or ethnicity, the remaining 4104 cases within the registry were included in the analysis. Comparisons by race were made using Chi-square or Wilcoxon rank sum tests for qualitative or quantitative variables, respectively. Overall survival (OS) was calculated from diagnosis until death or last known alive date; time to first treatment (TTFT) was calculated from diagnosis until treatment date or date last known to be untreated. OS and TTFT were displayed via Kaplan-Meier plots, and differences in OS and TTFT by race were calculated using log-rank statistics. Individuals who were enrolled in early intervention trials (n=61) were excluded from analyses involving treatment. Results: In total, 4104 CLL patients were identified: 4011 (97.7%) non-Hispanic Whites and 93 (2.3%) other races. Median age at CLL diagnosis was younger for other race patients (59 vs. 63 years; p=0.002), while gender and stage distributions were comparable between both groups. Serum β-2-microglobulin and absolute lymphocyte counts at diagnosis were similar but more other race patients had elevated LDH (29.5% vs. 16.1%; p=0.02). Cytogenetic data by FISH were available for 1884 non-Hispanic Whites and 46 other race patients; poor-risk cytogenetics (17p and/or 11q deletions) were present in 23.9% of other race and 13.5% of non-Hispanic White patients, respectively (p=0.04). No statistically significant differences were observed for CD38, ZAP-70, or IGHV by race TTFT was similar for both non-Hispanic Whites and other race patients (4.6 vs. 5.5 years, respectively; p=0.12). First-line treatment selection was similar for both groups (Table). . With a median follow-up of 12.1 years, median overall survival was 9.3 years for other race and 12.2 years in non-Hispanic White patients (P=0.15; Figure). Conclusions: This prospective data supports the notion that CLL might be biologically different between non-Hispanic White and other race patients with less favorable FISH present in the latter group. Despite the observation that TTFT and type of first therapy are similar between both groups, the trend towards shorter OS in other race patients argues that other factors might contribute to outcome differences, such as disease biology, access to care, co-morbidities, and socioeconomic considerations. Further studies with larger cohorts are warranted. Table: First-line treatment selection among treated individual by racial First-line Treatment Non-Hispanic Whites(n=1875) Other race(n=41) Chemoimmunotherapy Purine analogue-based Alkylator-based 675 (36.0) 495 (26.4) 180 (9.6) 15 (35.6) 9 (22.0) 6 (14.6) Purine analogue -monotherapy 157 (8.4) 4 (9.8) Alkylator monotherapy 610 (32.5) 9 (22.0) PA+Alkylator 41 (2.2) 1 (2.4) Antibody alone 190 (10.1) 6 (14.6) Other 202 (10.8) 6 (14.6) Figure: Overall survival in CLL cases stratified by race Figure:. Overall survival in CLL cases stratified by race Disclosures Nabhan: Genentech: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Speakers Bureau. Kay:Celgene: Research Funding. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding.

Abstract Introduction: Several B-cell malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are known to have a familial component to disease risk. Although several common inherited genetic variants have been found to be associated with risk of these malignancies, a large proportion of the heritability remains unexplained. Here we set out to identify rare high-penetrance susceptibility variants for CLL and MM using the established Mayo Clinic family study of B-cell malignancies. Methods: We performed exome sequencing on germ line DNA of 106 families with either two or more members with CLL (n=93 families) or two or more members with MM or monoclonal gammopathy of undetermined significance (MGUS) (n=13 families) using Aglient capture kits. All samples were sequenced on an Illumina HiSeq2000. Bioinformatics analyses leveraged the following software packages: Novoalign, Picard, The Genome Analysis Toolkit (GATK), and the Biological Reference Repository (bioR). Quality control filters were implemented; subjects with mis-specified relationships were removed as were variants with <95% call rate or <20x coverage, and those variants identified as sequencing artifacts. To identify potentially functional single nucleotide variants, the following was required a) uncommon in public databases, b) cosegregating variants in multiple families, c) variants to be highly conserved and in coding regions, and d) functional prediction status of deleterious (SIFT score), damaging (PolyPhen score), and a moderate, or high variant impact (SNPEffect). The association of these variants with risk was then evaluated in large samples of cases and controls in genome-wide association studies (GWAS). Results: In the CLL familial cohort, there were 443 individuals (160 with CLL, 73 with monoclonal B cell lymphocytosis (MBL), and 210 relatives without CLL or MBL) sequenced; and in the MM cohort, there were 39 individuals (13 with MM, 13 with MGUS and 13 without MM or MGUS) sequenced. A total of 61,992 single nucleotide variants passed quality control filters in the CLL cohort and 39,456 single nucleotide variants in the MM cohort. From these, we identified 32 variants in the CLL cohort and 39 variants in the MM cohort that were cosegregating in multiple CLL or MM families, respectively, and were predicted to affect protein structure or function. Of these, one CLL variant was within 1 Mb of a known GWAS-discovered locus. Moreover, there were seven CLL and four MM variants that were within 1 Mb of single nucleotide polymorphisms (SNPs) that had statistical significance between p<10-4 and p<10-6 in GWAS. Finally, there were 3 variants found to be common between CLL and MM families (see Table). Conclusions: Through whole exome sequencing, we identified a number of rare, highly penetrant predisposition variants for CLL and MM. Several of these variants were common to both B-cell malignancies providing evidence of shared genetic components; one variant may be a functional driver of the GWAS-discovered loci. These identified variants provide a list for future validation studies. Table: Exome variants common in both Chronic Lymphocytic Leukemia and Multiple Myeloma SNP ID rs41314099 rs61754114 rs143025033 Chromosome 17 6 7 Gene WSCD1 BACH2 TNS3 Penetrance (CLL) 0.70 0.75 0.80 Penetrance (MM) 1.00 0.80 0.75 Allele Frequency (CLL) 0.015 0.025 0.017 Allele Frequency (MM) 0.077 0.077 0.058 1000 Genome Frequency (European) 0.010 0.020 0.010 SIFT Term Deleterious Deleterious Deleterious PolyPhen Term Probably Damaging Probably Damaging Probably Damaging SNP: Single Nucleotide Polymorphism; CLL: Chronic Lymphocytic Leukemia; MM: Multiple Myeloma; Penetrance is defined as proportion of individuals carrying the alternate allele that are also affected; SIFT: Sorting Intolerant From Tolerant, predicts whether an amino acid substitution affects protein function; PolyPhen: Polymorphism Phenotyping, predicts possible impact of an amino acid substitution on the structure of a human protein Disclosures No relevant conflicts of interest to declare.

Abstract Although multiple myeloma is clinically defined as the accumulation of clonal, malignant plasma cells in the bone marrow, the disease shows significant heterogeneity with regard to morphology, progression and therapeutic response. Our hypothesis is that this heterogeneity likely is due, in part, to germline genetic polymorphisms that contribute significantly to an individual’s disease course and response. In particular, individuals deficient in the repair of DNA damage are not only at high risk for developing cancer, but also could show DNA repair dependent responses associated with DNA damaging therapeutic agents. In this study we examined the association of functional genetic polymorphisms in the DNA repair genes, XRCC1 (SNP positions 280, 399) and ERCC2 (SNP positions 312, 751), with toxicities and clinical outcomes in the ECOG, 3 arm chemotherapeutic trial, E9486 [A-VBMCP, B-VBMCP+Cylophosphamide, C- VBMCP+interferon]. DNA from 359 patients was genotyped and examined for association with response, toxicities, blood counts, bone pathology, and survival parameters. Notably, in the interferon arm of the trial significant associations were observed in progression free survival and polymorphisms of XRCC399 (AA median survival 51 months versus AG/GG median survival of 33 months; p=,008), ERCC751 (AA/AC median survival 33 months versus CC median survival of 48 months; p=.05), and ERCC312 (AA median survival 49 months versus AG/GG median survival of 34 months; p=.02). Hazard ratios ranged form 1.7 to 2.05 for survival differences associated with DNA repair genes in this arm. Interestingly, T cell counts are known to be impacted by interferon; and DNA repair polymorphisms in this arm were also associated with CD8+ T cell counts. While this represents a single arm of one clinical trial, it is intriguing to consider the impact of genetic polymorphisms on DNA repair in light of chemotherapeutic agents that may affect cells of the immune system. Previous studies from the ECOG Myeloma Committee of the E9486 trial show highly significant associations of survival and immune status in myeloma patients. Our results suggest genetic polymorphisms in DNA repair genes may influence clinical outcome in certain therapeutic regimens. Genetic polymorphisms in these DNA repair genes are currently being completed in 800 patients of the SW9321 SWOG/ECOG intergroup trial and additional association studies will be included in the presentation.

AbstractRecent analyses of circulating blood B cells in myeloma have generated controversy concerning the exact levels of these cells and whether they may represent circulating clonal tumor B cells. Previous reports suggested that CD19+ B cells are markedly increased in myeloma patients and that this population shares clonotypic rearrangements with the malignant plasma cell. We studied the numbers of CD19+ B cells by flow cytometry in previously untreated newly diagnosed myeloma patients in Eastern Cooperative Oncology Group (ECOG) phase III trial E9486. There were 628 patients who were eligible for the clinical protocol E9486, but of these 521 were also entered on the companion laboratory study (E9487) and had CD19 data. In comparison with normal controls, the myeloma patients exhibited a marked heterogeneity in the number of circulating CD19+ B cells as detected by flow cytometry. Approximately 20% of patients had significantly increased levels of circulating CD19+ B cells. However, the total CD19+ blood population from myeloma was not significantly different from the median of age-matched, normal controls. Analysis of CD19+ blood cells in relationship to circulating clonal cells was done in 13 myeloma patients using a clonotypic, quantitative allele-specific oligonucleotide-polymerase chain reaction (PCR) assay. No correlation was found between the numbers of CD19+ B cells (range, 5% to 51%) and PCR estimates of the number of clonal cells in the peripheral blood (range, .009% to 3.6%). Low CD19+ B-cell level (&lt;125 μL) was associated with clinical stage III (P = .033). A significant relationship exists between higher levels (≥125/μL) of CD19 cells and longer overall survival (P &lt; .0001). In addition, high CD19 levels also predicted a clinical response and longer event-free survival. There was a strong inverse association between the level of CD19 values at diagnosis and infections within the first 2 months of diagnosis. Importantly, the number of deaths related to infections was significantly greater in the low versus high CD19 group (P &lt; .0202). Also, CD19 is an independent prognostic factor in addition to plasma cell labeling indices, β2 -microglobulin, hemoglobin, and plasmablastic morphology. Patients with infections were more likely to have low levels of CD19+ cells. In summary, higher CD19+ cell levels are a favorable prognostic sign with no apparent relationship to circulating tumor cells. In addition, this analysis strongly suggests that low peripheral blood levels of CD19+ cells are an adverse prognostic sign in myeloma. The CD19+ cell levels in myeloma patients is an important parameter in the overall assessment of these patients.

Abstract The majority of pts with newly diagnosed CLL are classified with Rai stage 0 or 1 disease and typically experience an indolent clinical course. However, little is known regarding the reasons for initiating therapy in this population or patterns of initial treatment in the contemporary era of our practicing community. We utilized data from the Connect CLL registry, a US-based multi-center, prospective observational cohort study that is aimed at understanding patterns of CLL management without a study-specific intervention. Eligible pts were adults with a clinical diagnosis of CLL who initiated therapy ≤2 months prior to enrollment, and were enrolled from academic (n=155), community (n=1311), or government (n=28) sites between 2010-2014. Descriptive statistics were applied to examine data on demographics, baseline characteristics, treatment selection, early treatment outcomes, and reasons for initiating therapy that were reported for pts with Rai 0/1 CLL receiving first-line therapy. Multivariable logistic regression models were constructed to evaluate predictors of complete response (CR) and event-free survival (EFS) defined as the occurrence of the first of disease progression, relapse or death within 24 months of enrollment. Among 684 pts who received first-line therapy at enrollment and for whom Rai stage was available, 363 had Rai 0/1 CLL. This group had a median age of 68 years (range 41-90); 94% were white, 60% male, 98% insured, and 93% were treated in community practices; 47% had Rai stage 0 disease (54% Binet A); 33% had ≥1 constitutional symptoms; 96% had ECOG performance status of 0 or 1; 35% had conventional cytogenetics and 53% had FISH analysis performed. Among pts for whom cytogenetic or FISH analysis were performed, 159 (72%) were abnormal, of them 37 pts (23%) had del(11q), 104 pts (65%) had del(13q), 33 pts (21%) had trisomy 12, and 20 pts (13%) had del(17p) by conventional cytogenetics and/or FISH, including 8% with del(17p) by FISH. The median time from diagnosis to initiation of therapy was 2.5 years (range 0-20 years) for Rai 0/1 pts compared with 0.5 years for pts enrolled with Rai stage ≥2 (range 0-32). The most common reasons for initiating therapy (with >1 reason possible) in Rai 0/1 CLL were: bulky or progressive or lymphadenopathy (40%), progressive lymphocytosis (39%), any disease-related symptom (weight loss, fatigue, fever, night sweats; 28%), anemia (20%), and thrombocytopenia (15%); 42% of pts had >1 reason for initiation. The most common first-line regimens used in this cohort were: fludarabine/cyclophosphamide/rituximab (FCR; 29%), bendamustine/rituximab (BR; 20%), and rituximab alone (12%). Of the 317 pts who either responded to treatment during the first 24 months of enrollment or were followed ≥24 months, 143 pts (45%) achieved CR (investigator assessed as per protocol) and 264 (83%) achieved CR or partial response. Female gender (odds ratio [OR] = 2.22), treatment with BR or FCR (OR = 3.41), and time from diagnosis to therapy (OR = 0.92; with each year increase in time from diagnosis to initiation of therapy, relative odds of CR decreases by 8%), were independent predictors of CR. Of the 311 evaluable pts, 82 pts (26%) experienced an EFS event during the 24 months on study, of which 19 (23%) were deaths and 48 (59%) were progressions. Age ≥75 years (OR = 4.46), lymphocytosis as a reason for therapy initiation (OR = 1.89), and male gender (OR = 1.73) were independent predictors of an EFS event within 24 months. The Connect CLL registry represents the largest comprehensive prospective cohort of CLL pts collected from multiple practices across the US. In concordance with other studies, pts with Rai 0/1 initiated therapy approximately 2.5 years following CLL diagnosis and included a cohort of pts with poor prognostic FISH characteristics. Although chemoimmunotherapy was associated with a significantly higher likelihood of CR, progressive lymphocytosis as a reason for initiating therapy (and not any form of treatment) was more likely to predict an early EFS event. Approaches using novel agents or combination therapies aimed towards addressing pts with progressive lymphocytosis should be considered in future studies. Disclosures Flowers: Seattle Genetics: Consultancy; OptumRx: Consultancy; AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead Sciences: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; Celegene: Other: Unpaid consultant, Research Funding; Spectrum: Research Funding; Onyx Pharmaceuticals: Research Funding; Spectrum: Research Funding; Acerta: Research Funding; Gilead Sciences: Research Funding; Janssen: Research Funding; Infinity Pharmaceuticals: Research Funding; Infinity Pharmaceuticals: Research Funding; Janssen: Research Funding; Millennium/Takeda: Research Funding; Onyx Pharmaceuticals: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Millennium/Takeda: Research Funding. Nabhan:Celgene Corporation: Honoraria, Research Funding. Kay:Pharmacyclics: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharma: Research Funding. Mato:AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Celgene Corporation: Consultancy, Research Funding; Pronai Pharmaceuticals: Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Research Funding; Gilead: Consultancy, Research Funding; Genentech: Consultancy. Lamanna:Genentech-Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Grinblatt:Celgene Corporation: Consultancy, Honoraria, Speakers Bureau. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor; Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor. Kozloff:Roche: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; AbbVie: Consultancy. Sullivan:Celgene Corporation: Employment, Equity Ownership. Flick:Celgene Corporation: Employment, Equity Ownership. Kiselev:Celgene Corporation: Consultancy. Bhushan:Celgene Corporation: Employment, Equity Ownership. Swern:Celgene Corporation: Employment, Equity Ownership. Sharman:TG Therapeutics, Inc.: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding; Calistoga: Honoraria; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Roche: Research Funding.

Abstract Introduction: We demonstrated that elevated levels ofcirculating microvesicles (MV) in CLL activate CLL bone marrow stromal cells (BMSCs) with resultant enhanced vascular endothelial growth factor (VEGF) production, a pro-survival factor for CLL B-cells (Ghosh 2009 Blood 113:5568-74). We also detected a phenotypic shift from predominantly platelet/megakaryocyte-derived MV (CD61+) in early stages towards a more leukemic B-cell derived MV (CD19+) in advanced Rai stages (Ghosh 2010 Blood 115:1755-64). These observations suggest that the MV parameters of their levels and surface phenotypes may predict or be associated with the clinical outcome and/or therapeutic response in a given CLL patient. In this study we therefore designed an approach to being to assess these latter possibilities in regard to MV in CLL. Methods: In vitro stimulation – Purified CLL B-cells were stimulated in vitro using CpG oligonucleotide (n=10) or anti-IgM antibody (n=10) for 72 hours. MV were isolated from the used media, levels measured and then phenotyped by flow cytometry (BD Canto-I) using standard TRUCount beads (BD Biosciences). In vivo (plasma) work – MV were isolated from normal plasma (n=5) or plasma of 17 CLL patients entered onto a previously reported Pentostatin, Cyclophosphamide, Ofatumumab (PCO) clinical trial at their entry to the trial and at 6-months after therapy and then sequentially over a period of 18 months (Shanafelt 2013 Cancer 119:3788-96). We determined the phenotypes and plasma levels of MV as described above. Results Phenotypes of in vitro generated MV: We found that, for the most part, CLL B-cells shed MV upon in vitro stimulation however; some CLL B-cells of unmutated IGHV status, but not mutated status, spontaneously generated MV in culture (Fig. 1). Most interestingly, we found that the surface phenotype of MV generated from CLL B-cells carrying predominantly the CD52 surface marker, not the CD19 or CD5 marker. Interestingly, none of the in vitro generated MV was CD20+, another known marker of CLL B-cells. We found similar phenotypes of the in vitro generated MV by stimulating two different signaling pathways, Toll-like receptor and BCR, in CLL B-cells. Together these results suggest that CLL B-cells generate predominantly CD52+ MV, but not CD19 or CD5, upon in vitro stimulation. MV phenotypes in pre- and post-therapy CLL plasma: Here we focused on the kinetics of MV levels and phenotype in relation to disease outcome for PCO treated CLL. Thus, we found total MV levels were elevated in all patients compared to normal plasma at entry to the PCO trial and then dropped substantially after therapy when compared to the trial entry. Also most CLL patients (n=11) continued to maintain low plasma MV levels until the end of the follow up period (18 months). Interestingly, in a few cases (n=6) total plasma MV levels decreased initially after therapy but then started to rise. These patients are now being followed for disease status in order to determine if rising MV levels are related to a disease relapse. Of interest on flow phenotyping the plasma MV obtained at entry to the PCO trial we detected predominantly CD52+ MV but not CD19+ MV, similar to the findings of the in vitro MV generated from CLL B-cells. Normal plasma (n=5) does not contain significant levels CD52+ MV or CD19+ MV. Together these results suggest that the malignant B-cells shed preferentially CD52+ MV, but not CD19+ MV. We are currently analyzing the MV levels and phenotypes pre- and post-therapy response levels in relation to the patients’ response to therapy in a larger cohort of treated CLL patients (n=60). Conclusion: Current understanding about the phenotypes of MV is based on the concept that MV carry the unique surface marker of the originator cells, e.g., CD19 on B-cell derived MV. However, findings from our in vitro stimulation studies demonstrate that the CD52 but not CD19 is preferentially expressed on CLL B-cell derived MV. We are currently studying the mechanism for such a preferential surface expression on these leukemic derived MV. To our knowledge, this is the first report describing that CLL B-cells generate preferentially CD52+ MV. Our studies are now committed to a more extensive analysis of MV parameters in association with CLL disease progression, depth of clinical response and if the elevation of plasma MV heralds a relapse in responding patients. Figure 1 Figure 1. Disclosures Shanafelt: Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 873 Introduction: Clonal chromosomal anomalies have been well-characterized in the blood cells of patients with Chronic Lymphocytic Leukemia (CLL) and other hematological malignancies. However, the frequencies and types of chromosomal anomalies found in “normal” people (i.e. no history of hematological cancer) are much less well known. Here we compare chromosomal anomalies in untreated CLL patients with those found in a large sample of people (n>50,000) recruited for genome-wide association studies in the Gene Environment Association Studies (GENEVA) consortium. This comparison provides information about the extent to which people without a prior history of hematological cancer have chromosomal anomalies that are characteristic of CLL. Methods and Results: The anomaly detection method focused on acquired, rather than inherited, chromosomal anomalies identified in whole blood samples. The method and results for the GENEVA subjects were published recently (Laurie et al. 2012, Nat. Gen. 44:642–650). The same method was applied to pre-treatment samples from 220 CLL patients from US Intergroup Trial E2997 of the Eastern Cooperative Oncology Group (ECOG). Acquired anomalies were detected as mosaic mixtures of normal disomic cells along with cells containing autosomal anomalies consisting of large (generally > 1 Mb) deletions, duplications and/or acquired uniparental disomy (UPD). Detection requires a large fraction of cells with the same abnormal karyotype (>5–10%), which is presumably of clonal origin. The subjects (of all ages) came from 12 different GENEVA studies, which addressed a variety of health conditions not directly related to hematological cancer (e.g. addiction and glaucoma). DNA was extracted mainly from blood samples (or, in 8% of subjects, from buccal/saliva samples) and genotyped on Illumina SNP mircroarrays. Clonal mosaic anomalies were detected from change points in intensity and genotype data (B Allele Frequency and Log R Ratio). We found 514 anomalies in 404 GENEVA subjects out of 50,222 examined (0.8% of subjects), although the prevalence increased with age. Clinical data from about 75% of subjects with a clonal chromosomal abnormality revealed that only a small fraction (2.8%, 95% CI=1.0 – 4.7%) had a record of prior hematological cancer. In the ECOG study, blood samples from 220 CLL patients enrolled in protocol E2997 were genotyped on the Illumina HumanOmni1-Quad_v1 array and clonal mosaic chromosome anomalies were detected using the GENEVA method. Chromosomal anomalies typical of CLL also were identified using interphase FISH (i.e. 6q23.3-, 11q22-, 12q15+, 13q14-, 17p13-) as previously reported (Grever, JCO 25:799-804). We found 489 anomalies in 148 patients of the 220 examined (67% of patients). The anomalies detected by FISH and SNP microarray were highly concordant (>98%). Although, the most common recurrent anomalies occurred on chromosomes 6, 11, 12, 13 and 17 in regions that span the FISH probes (except for chromosome 6), a majority of the anomalies detected by SNP data (74% of 489) were not detected by the CLL FISH panel. The frequency of large chromosomal anomalies was much higher in CLL patients than in GENEVA (67% versus 0.8% of subjects) and the overall composition of anomaly types found was also different. The fraction of anomalies consisting of acquired UPD was substantially higher in GENEVA subjects than in ECOG (34.0% versus 10.6%), while deletions were less common in GENEVA (50.1% versus 72.4%) and duplications were similar (15.6% versus 17.0%) (Chi-squared test p-value=3 × 10−18). We compared the most commonly deleted regions (CDR) on each chromosome across studies. In the ECOG CLL patients, four chromosomes each had 10 or more overlapping deletions in their top CDR (6, 11, 13 and 17), which span the FISH probes (except for chromosome 6). The GENEVA subjects also had CDR on chromosomes 11, 13 and 17, which overlap those in ECOG. In addition, GENEVA had CDR spanning genes or regions implicated in myeloid malignancies, including DNMT3a on 2p, TET2 on 4q, PRAME on 22q, and a region on 20q13. Conclusions: These genotyping results of >50,000 subjects suggest clonal chromosomal anomalies may represent precursor states, or perhaps undiagnosed or unrecorded cases, of a variety of hematological malignancies. Further studies to validate these findings and explore the natural history of these genetic defects in individuals without hematologic malignancy are warranted. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Abstract 4252 Background: Multiple recent studies have demonstrated the survival benefits of reduced intensity allogeneic stem cell transplant for high risk CLL patients. These transplant series, however, represent highly selected cases where age, co-morbidities and performance status preclude transplant in many patients with CLL. The portion of newly diagnosed CLL patients that are eventually considered for transplant is unknown, as well as the proportion that are referred for transplant evaluation; the proportion that actually receive stem cell transplant, and the reasons for not pursuing transplant in referred patients. We conducted a cohort study of newly diagnosed CLL patients to address this gap in knowledge. Methods: We used the Mayo Clinic database to identify all patients with newly diagnosed CLL seen at Mayo Clinic within 1 year of diagnosis between July of 2003 and May of 2011 (time interval during which reduced intensity allogeneic stem cell transplantation was part of routine care at Mayo Clinic). Electronic medical record and laboratory studies were reviewed to identify all patients referred for stem cell transplant evaluation (either allogeneic transplant for progressive CLL or autologous stem cell transplant for Richter's transformation). Patients referred for transplant were analyzed to determine what proportion completed transplant and, for those who were not transplanted, the reason they were not transplanted (e.g. lack of insurance approval, co-morbidity, lack of a donor, inadequate disease control, death prior to transplant or patient preference). Results: A total of 1250 patients with newly diagnosed CLL between July 2003 and May 2011 who were seen at Mayo Clinic within 1 year of diagnosis were identified. The mean age at diagnosis was 63.6 yrs and 34.2% were women. With respect to disease stage, 48.7% were Rai stage 0, 45.5% Rai stage I/II, and 5.8% Rai stage III/IV. CLL FISH analysis revealed 126 (13.6%) patients who had either del (17p) or del (11q) and 801 (86.4%) patients who had other FISH abnormalities or normal FISH results. After a median follow-up of 41 months, 425 (34%) of patients have received treatment. A total of 54 patients (4.3%) were referred for a transplant evaluation during follow-up. Of these 54 patients, 48 (3.8% overall cohort) were referred for consideration of allogenic transplant for treatment of progressive CLL and 6 (0.5%) were referred for autologous transplant for treatment of Richter's transformation. Baseline characteristics associated with subsequent transplant referral included age at diagnosis (median: 56 yrs) (p &lt; 0.0001, Odds Ratio (OR) for each year older = 0.91, 95% Confidence Interval (CI)=(0.88, 0.95)), Rai stage (III/IV vs. 0) (p = 0.0002, OR = 22.7, 95% CI=(5.1, 102.4)) and FISH (del(11q), del(17p) vs Normal, del(13q) or Trisomy 12) (p &lt; 0.0001, OR = 7.3, 95% CI=(3.3, 16.1)). Gender, Rai stage (I/II vs. 0), and patient residence (local vs. not local) were not significant. Among the 54 patients referred for transplant, 28 (51.9%) completed a transplant. This included 22 (45.8%) patients referred for allogenic transplant and 6 (100%) patients referred for autologous transplant. Among the 26 patients referred who did not undergo transplant, the principal reasons were patient preference (n = 6, 23.1%), patient still being evaluated for transplant as of the study end date (n = 6, 23.1%), alternative therapy chosen (n = 5, 19.2%), inadequate disease control (n = 3, 11.5%), death prior to transplant (n = 3, 11.5%), lack of insurance approval (n = 2, 7.7%), and lack of a donor (n = 1, 3.8%). Conclusion: 4.3% of the newly diagnosed CLL patients in this series were considered for stem cell transplant at some point during follow-up. Young age, high Rai stage and high risk FISH abnormalities are principal reasons for transplant referral. Only 51.9% of those considered for transplant completed transplant. The principal reasons for referral patients not completing transplant who had adequate time to follow were patient preference, alternative therapy, inadequate disease control and death prior to transplant. The remaining 6 patients (23%) are in process of transplant evaluation with longer follow required. These analyses provide first incidence data for CLL transplant referral in a cohort of newly diagnosed patients. Disclosures: Zent: Biothera: Research Funding; Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; GlaxoSmithKlime: Research Funding. Shanafelt:Celgene: Research Funding; Teva/Cephalon: Research Funding; GlaxoSmith Klein: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Polyphenon E International: Research Funding.

Abstract Abstract 3865 ZAP-70 expression is a prognostic marker differentiating chronic lymphocytic leukemia (CLL) patients with indolent disease from those rapidly progressing after diagnosis and requiring treatment. We previously demonstrated in four independent clinical cohorts that DNA methylation at a single CpG dinucleotide in the ZAP-70 promoter region impacts on ZAP-70 transcriptional regulation and is prognostic in CLL (Claus et al. J Clin Oncol. 2012;30(20):2483–91). Here, we confirm and extend our previous findings by investigating the significance of ZAP-70 methylation relative to established prognostic biomarkers in CLL: IGHV mutation status or expression of ZAP-70 protein or CD38. DNA methylation analysis of the ZAP-70 core promoter region was conducted in mononuclear cells of 295 untreated CLL patients, previously reported by the CRC (Rassenti et al. Blood 2008;112:1923–30) using MALDI-TOF mass spectrometry (MassARRAY, Sequenom). The 295 patients had a median age of 55 years (range 26–82), 69% were male, and 46% were Rai stage 0, 47% stage I/II, and 7% stage III/IV at diagnosis. ZAP-70 protein and CD38 expression were measured by four-color flow cytometry and IGHV mutational status was assessed, as previously described. By hierarchical clustering, the previously identified single CpG dinucleotide (“CpG unit 1”, position +223 relative to the transcription start) separated patients with high methylation levels across the entire region from those with lower methylation levels at this specific site. With a median follow-up of 3.9 years in 50 patients who had not started treatment and 5.6 years among those still alive (n=196), patients with lower methylation levels at CpG unit 1 had shortened time to first treatment (TTT) and overall survival (OS) (both p<0.0001), confirming the prognostic relevance of this site. In our previous study, an optimal cut-off for CpG unit 1 methylation across four data sets was between 10% and 20%. In this cohort, the optimal cut-off across the two endpoints was between 15% and 25%. A natural gap in CpG unit 1 methylation levels occurred between 20% and 25%, adding potential biological significance, as lower values may represent patient cell samples in which both alleles are unmethylated. Applying a 20% cut-off, TTT and OS were significantly different for those with lower and higher methylation levels (both p<0.0001). The 5-year TTT estimates were 22% (95 % CI: 0.16–0.28) and 64% (95% CI: 0.52–0.74), respectively, for patients with low versus high methylation levels; 5-year OS estimates were 80% (95% CI: 0.73–0.85) and 90% (95% CI: 0.81–0.95). Low ZAP-70 methylation was significantly associated with CLL-cell expression of ZAP-70, CD38, or unmutated IGHV status (each p<0.0001). In a model of TTT, expression of ZAP-70 modified the strong effect of ZAP-70 methylation. In patients with CLL cells lacking ZAP-70 expression, high methylation was protective (hazard ratio [HR]=0.37, 95% CI: 0.23–0.58). In contrast, methylation had no significant effect in ZAP-70+ CLL (HR=1.47, 95% CI: 0.81–2.68); TTT was short regardless of methylation levels (Figure 1). IGHV mutation status and expression of CD38 did not provide significant additional prognostic information. The effect modification described for TTT was not observed for OS (p=0.57). Here, ZAP-70 methylation was the strongest risk factor (hazard ratio=0.32, 95% CI: 0.19–0.54; p<0.0001), and expression of ZAP-70, CD38 or unmutated IGHV did not significantly improve the ability to explain differences in OS. Hence, the prognostic value of ZAP-70 CpG unit 1 methylation was confirmed in a large patient cohort with extensive follow-up. We defined a clinically applicable threshold for ZAP-70 methylation assessment and determined its relative value to current prognostic markers in CLL. ZAP-70 methylation predicted longer TTT among patients with CLL cells lacking ZAP-70 expression, but not among those with ZAP-70+ CLL cells, although this could reflect sampling bias due to the small number of patients (n=16) in our cohort with both high methylation and expression of ZAP-70. Lastly, methylation was the strongest risk factor for OS, and neither IGHV mutation status nor expression of ZAP-70 or CD38 significantly improved the ability to predict prognosis. In conclusion, ZAP-70 DNA methylation represents a clinically valuable biomarker for predicting TTT and OS in previously untreated patients with CLL. Disclosures: Gribben: Gilead: Honoraria; Mundipharma: Honoraria; GSK: Honoraria; Pharmacyclics: Honoraria; Roche: Honoraria; Celgene: Honoraria. Kay:Celgene: Research Funding.

Abstract Abstract 3927 PURPOSE: CLL B-cells interact with their microenvironment in a variety of ways that influence leukemia cell survival. Contact mediated interactions are the most widely studied. More recently, bidirectional cytokine mediated interactions have been found to modulate spontaneous and drug induced CLL B-cell death. Specifically, upregulation of CCL2, CCL3, and CCL4 are found when CLL B-cells are co-cultured with various accessory cells. We set out to more comprehensively profile the effect of CLL-stromal cell co-culture on immune cytokine regulation and to evaluate the effect of novel therapeutic agents in clinical testing for patients with CLL on these interactions. METHODS: Freshly isolated CLL B-cells (n=24) were cultured in AIM V media for 24 hours alone or in direct contact with HS-5 stromal cells. HS-5 cells were cultured alone in AIM V to determine basal level of immune cytokine secretion. Immune cytokine levels in the media under these 3 conditions (CLL alone; HS5 alone; co-culture) were assessed using multiplex cytokine arrays (Invitrogen). The protective effects of the 6 most highly upregulated cytokines on spontaneous apoptosis of CLL B-cells after 7 day culture were then examined by culturing leukemic cells with and without recombinant cytokines. The effect of sub-lethal doses of the PI3K inhibitor CAL-101, the AKT inhibitor MK2206 and the Bruton's Tyrosine Kinase inhibitor PCI-32765 were examined. To compare the effect of these drugs to a more conventional chemotherapeutic agent, we evaluated fludarabine (F-Ara-A) under the same conditions. To explore the ability of these novel agents to overcome stromal rescue, CLL B-cells pre-exposed to bendamustine (BEN) were cultured in media alone, or in direct contact with stroma with and without sub-lethal doses of CAL-101, MK2206, or PCI-32756. Leukemic cell viability was assessed by flow cytometry using annexin/PI staining. RESULTS: Six cytokines increased ≥4 fold (e.g. >400%, all p<0.0001) on coculture relative to the expected additive value of individual cultures. Consistent with previous reports, (Schulz 2011; Burger 2009) CCL3 and CCL4 were found to have the greatest upregulation (both >20 fold the additive value). The other highly upregulated cytokines detected have not been previously reported to be influenced by coculture: IL12 (15 fold additive value), IL1β (11 fold additive value), TNF-α (11 fold additive value), and IL-1RA (4 fold additive value). Although transwell experiments (n=6) suggested upregulation of these cytokines was largely contact dependent, substantial upregulation of several cytokines still occurred without direct contact. Each of the 6 most upregulated cytokines reduced spontaneous apoptosis when CLL B-cells were cultured for 7 days in media plus recombinant cytokine. When CLL B-cells and HS-5 cells were co-cultured with CAL-101, MK-2206, or PCI-32765, the effects on cytokine secreted upregulation were cytokine and drug specific (e.g. one drug affected individual cytokines differently; different drugs affected different cytokines). For example, PCI-3276 and CAL-101 had similar affects on the upregulation of CCL3 and CCL4 on co-culture but opposite effects on TNF-α and IL-1β. In contrast, F-Ara-A had minimal effect on upregulation of any of these 6 cytokines. Finally, we evaluated the ability of these novel agents to overcome stromal rescue of CLL B-cells treated with conventional chemotherapeutic agents. At sublethal doses CAL-101, MK2206, PCI-32765 each significantly overcame stromal rescue of BEN exposed CLL B-cells. (Fig.1) CONCLUSIONS: Direct physical interaction of CLL B-cells with bone marrow stromal cells resulted in profound upregulation of multiple immune cytokines. The 6 most upregulated cytokines identified in these experiments all reduced spontaneous apoptosis of CLL B-cells on extended culture. Sublethal doses of the PI3K inhibitor CAL-101, the AKT inhibitor MK2206, and the Bruton's Tyrosine Kinase inhibitor PCI-32765 had distinct effects on stromal cell mediated cytokine upregulation. Importantly, each of these 3 drugs also overcame stromal mediated rescue of BEN exposed CLL B-cells. These in vitro findings support the ongoing clinical trials of BEN in combination with CAL-101 (ASH 2011), PCI-32765 (ASCO 2012) or MK2206 (Ding et. al., unpublished observation). Disclosures: Kay: Celgene: Research Funding. Shanafelt:Celgene: Research Funding; Hospira: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Polyphenon E International: Research Funding.