Citations

Abstract The human IGHV4-34 gene encodes antibodies which are intrinsically autoreactive when the VH domain is unmutated. Therefore, B cells expressing IGHV4-34 B-cell receptor immunoglobulins (BcR IG) are normally under close scrutiny in order to avoid unwanted autoreactivity, especially against DNA. The IGHV4-34 gene is frequently utilized in chronic lymphocytic leukemia (CLL), where, typically, it shows a high load of somatic hypermutation (SHM). We have previously reported distinctive SHM patterns amongst IGHV4-34 CLL, especially for subsets with stereotyped BcR IG. However, although a large number of cases (~2000) was previously studied, since even the largest subsets account for only ~3% of CLL, meaningful conclusions could not be reached for smaller subsets. Here we revisit this issue in a series of 16,528 CLL cases and focus on IGHV4-34 expressing subsets: #4 (IGHV4-34/IGHD5-18/IGHJ6 | 156 cases, 0.9%); #11 (IGHV4-34/IGHD3-10/IGHJ4 | 16 cases, 0.1%); #16 (IGHV4-34/IGHD2-15/IGHJ6 | 41 cases, 0.25%); #29 (IGHV4-34/IGHD: unassignable/IGHJ3 | 39 cases, 0.24%); and #201 (IGHV4-34/IGHD: unassignable/IGHJ3 | 43 cases 0.26%). Focusing on codons 27-104 within the VH domain (from CDR1-IMGT to FR3-IMGT), we calculated the sequence distance between subsets and the corresponding IGHV4-34 germline sequence based on a pairwise qualitative and quantitative comparison of the respective amino acid composition. The minimum distance calculated, and hence the greatest identity, was observed between subsets #4 and #16, both concerning IgG-switched cases (IgG-CLL), which is notable given the overall rarity of IgG-CLL. In contrast, the maximum distance, implying the least identity, was between subsets #16 and #201, the latter concerning IgM/D-CLL. Extreme variations between subsets were noted in codons spanning the entire VH domain. This result is consistent with our finding of a subset-biased distribution of mutations over the VH domain. More specifically, while subsets #11, #16, #29 and #201 had a lower frequency of mutations within VH CDR1 compared to VH CDR2, the exact opposite was seen in subset #4, with 40% of mutations in VH CDR1 versus 27% in VH CDR2. In addition, subsets #4, #11, #16 and #29 had a similar distribution of mutations in VH FR2 and VH FR3, in contrast to subset #201 that showed a preference for VH FR3 over VH FR2. Consequently, we noted that certain positions were targeted in a subset-specific manner e.g. codon 28 in VH CDR1 was heavily targeted in subsets #4 (68.6%) and #16 (87.8%), with most cases carrying an acidic amino acid (AA) introduced by SHM, glycine to glutamic acid, G>E: 51.3% for subset #4 and 78% for subset #16. The high prevalence of acidic AA introduced by SHM in these subsets is notable considering the electropositive nature of their VH CDR3 (especially of subset #4), strongly recalling edited anti-DNA antibodies. Interestingly, the G>E change was identified at a much lower frequency in other IGHV4-34 subsets: 18.75% for subset #11; 2.6% for subset #29; 7% for subset #201, all of which carried electronegative VH CDR3. Further, we noted that certain positions were heavily targeted in all subsets e.g. 56-86% targeting for SHM at codon 92 in VH FR3 where serine is encoded by the agc triplet, the ”hottest of hotspots”. This result could be viewed as sequence- rather than subset-dependent and linked to the molecular features of this codon, which is supported by the low targeting of codon 93 (0-6%), also encoding serine by the tct triplet. Other positions were targeted in all subsets but at vastly different frequencies e.g. codon 64 was targeted in 37.8% in subset #4 rising to 100% in subset #29. Finally, positions heavily targeted by SHM in certain subsets were unmutated in other subsets e.g. codon 36 in VH CDR1 remained unmutated in subset #16, in contrast 76.9% of subset #29 were mutated at this position resulting in an AA change. In conclusion, we document different spectra of SHM and AA changes between stereotyped IGHV4-34 CLL subsets. The finding of subset-biased, recurrent AA changes at certain codons indicates that the respective progenitor cells may have responded in a specific manner to the selecting antigen(s), despite expressing the same IGHV gene, indicating a functional purpose for these modifications. This is exemplified by the molecular characteristics of the recurrent AA changes in subset #4, thereby offering interesting pathogenetic hints. Disclosures No relevant conflicts of interest to declare.

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 < 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 < 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 3871 Background. A number of investigators have provided data supporting the value of CD49d expression on B leukemic cells in chronic lymphocytic leukemia (CLL) as independent prognostic variable. These studies used a variety of clinical end points including overall survival (OS), time to treatment (TTT) or treatment free survival (TFS). The factors included in multivariate analyses of these studies also differed. Unresolved issues regarding the prognostic value of CD49d assessment in CLL included the choice of the optimal cut-off to define positivity and CD49d prognostic value in patients subsets defined by other standard prognostic factors. Aim. To perform meta-analysis using individual patient level data from studies of CD49d expression to evaluate its ability to predict OS (primary end-point) and TTT/TFS (secondary end-point). Methods. Studies published by 30 April 2011, reporting an association of high CD49d expression on B CLL cells, measured by flow cytometry, and end-points were identified by Medline search. Additionally, we performed a manual review of abstracts presented at the congress of the American Society of Hematology from 2006 to 2010. CD49d was used as a categorical variable, as coded in original studies. Results. We identified 6 published studies and one abstract for inclusion. All authors agreed to provide individual data on the patients in these publications as well as unpublished data on additional patients to be used for replication analysis. Four authors also submitted updated follow-up data. We collected the following variables: date of diagnosis, OS, TTT/TFS, CD49d, CD38, ZAP-70, immunoglobulin mutational (IGHV) status, del17p and del11q chromosomal aberrations, age, stage, ALC, and β2 microglobulin concentration. Data from 3146 individual patients was available with 261 subsequently excluded due to missing end point and/or CD49d data. Of 2771 patients with valid data, 1405 (51%) were included in the previous publications and 1366 (49%) were unpublished. Before starting analyses a decision was made to perform meta-analysis on published data and use data from previously unpublished data as a validation cohort. Pooled CD49d hazard ratio for OS was 2.62 (1.88–3.64). In bivariate analysis, the prognostic value of CD49d was confirmed and of comparable value in patients subsets defined by CD38 and ZAP-70 expression, IGHV status, unfavorable chromosomal aberrations. Finally, we performed a multivariate analysis including CD49d, CD38, ZAP-70, IGHV status, del17p and del11q. CD49d was significantly associated with shorter overall survival in this adjusted model, with a hazard ratio of 2.47(1.17–5.21). Inspection of Martingale residuals plots of CD49d in each study failed to show a recognizable cut-point. CD49d cut-points previously adopted in published studies (30% and 45%) were subjected to validation in the cohort of 1366 unpublished patients. The Cox model with 45% cut-off had a slight greater hazard ratio (2.73 vs 2.65, log-rank p<0.0001). Only 4% of patients changed prognostic category after swapping the cut-off from 45% to 30%. Conclusions. Preliminary results of a meta-analysis using individual patient data from >1400 patients confirm the association of high CD49d expression with short OS independent of other prognostic parameters. While the 45% cut-off has a marginal better prognostic power, the 30% cut-off has the advantage of being already in use for CD38 and generally adopted for other surface antigens in chronic leukemia immunophenotyping. These findings may have implication for patient's stratification in future prospective studies and potential therapeutic efforts to target CD49d or CD49d signaling. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Burger:Pharmacyclics: Consultancy, 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.

Abstract Abstract 669 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world but its pathogenesis remains largely unknown. More recently, a widely prevalent premalignant condition termed monoclonal B cell lymphocytosis (MBL) has been defined and similarly involves expansion of a CD19+CD5+ population of B cells. MBL has been the focus of a wide number of recent studies in hopes it can provide insight into the early pathological events that lead to clonal expansion of a pre-leukemic CLL-like clone. Previously, we and others identified the transcription factor lymphocyte enhancer-binding factor-1 (LEF-1) as one of several genes significantly over expressed in CLL B cells relative to blood CD19+ B cells from healthy adults. LEF-1 is crucial for the proliferation and survival of pro-B cells during B cell development and deregulated LEF-1 activation has been directly linked with leukemogenesis in a transgenic murine model. In the present study, we addressed three critical questions concerning LEF-1 and CLL: 1) do CD5+ normal B cells express LEF-1; 2) at what stage of transformation does LEF-1 expression first appear; and 3) what mechanism(s) underlies LEF-1 expression in leukemic B cells. Methodology: The goals of our study were to: 1) determine the LEF-1 expression status of normal CD 19+/CD5+ human B cells; 2) determine the expression status of LEF-1 in clonal B cells present in patients with MBL; and 3) identify regulatory mechanisms that control aberrant expression of LEF-1 in CLL B cells. In order to achieve the first two goals, a 3-color flow cytometry assay for CD19, CD5, and intracellular LEF-1 was developed. To achieve the third goal, we performed in silico analysis of the LEF-1 locus and correlated this with publicly available genome wide methylated CpG island recovery assay (MIRA) data describing the methylome of normal human B cells (Rauch, T. A. Proc Natl Acad Sci U S A. 2009; 106(3): 671-8). Results: Analysis of human umbilical cord blood B cells, a rich source of the CD19+/CD5+ B cell subset, revealed that all normal CD 19+/CD5+ human B cells are negative for LEF-1 protein expression. These data demonstrate LEF-1 expression by CLL B cells is truly aberrant and does not simply reflect the phenotype of CD5+ lineage B cells. We next tested if the CD19+/CD5+ cells obtained from patients with MBL express LEF-1 protein. In these analyses, we restricted our study to MBL patients with absolute B cell counts of less than 2.5 ×10 9 cells/(L) (range: 0.756 - 2.44 × 10 9 cells/(L)), which is significantly below the upper limit that defines the MBL to CLL transition. Of interest, each MBL sample analyzed (n=6) revealed the presence of two populations of cells: 1) CD19+/CD5+ B cells expressing LEF-1; and 2) CD19+/CD5- B cells lacking expression of LEF-1. To confirm the clonal nature of the CD19+/CD5+ cells from MBL patients we demonstrated that the cells were light chain restricted. These data clearly indicated that LEF-1 expression becomes deregulated in the premalignant state of MBL and may therefore represent an early event in CLL leukemogenesis. In order to identify differential pathways of LEF-1 regulation that may be altered in CLL and MBL B cells, in silico promoter analysis of LEF-1 was performed. We identified a number of potential transcription factor binding sites as well as a putative CpG island in the 5' promoter region of LEF-1. Using data from a human B cell genome wide methylation array, we were able to determine that this same putative LEF-1 promoter CpG island was highly methylated in normal human B cells. Methylation of promoter region CpG islands is known to play an important role in developmental regulation of gene expression and may be the operative mechanism underlying silencing of LEF-1 expression in normal B cells. Conclusions: LEF-1 expression is deregulated in MBL and appears to be an early and possibly key event in the transition of normal B cells into a premalignant/malignant state. Our data suggest that loss of epigenetic regulation of this developmentally important locus may play a role in aberrant LEF-1 expression in MBL and CLL B cells. Ongoing studies are aimed at determining the methylation status of the LEF-1 promoter in CLL B cells and the functional role of this protein in transcriptional regulation and survival of CLL and MBL cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2342 Poster Board II-319 PURPOSE: Numerous clinical and biologic parameters are able to predict time to first treatment (TFT) and overall survival (OS) for patients with CLL. The utility of prognostic testing in CLL patients may vary based on age at diagnosis given the higher mortality from other causes among older individuals. The goals of the present study were to i) evaluate differences in natural history of CLL patients based on age at diagnosis, ii) compare survival to age-matched individuals in the general population, and iii) determine the age-stratified utility of prognostic testing. METHODS: All 2487 CLL/SLL patients diagnosed between January 1995 and June 2008 and cared for in the Mayo Clinic Division of Hematology were categorized by age at diagnosis (<55 years, 55-64 years, 65-74 years, or ≥75 years) and evaluated for differences in clinical characteristics, TFT, and OS using chi-square tests. IGHV gene mutation analysis, ZAP-70 status, CD38 status, and cytogenetics abnormalities by interphase FISH testing were performed as part of clinical and/or research studies using methods previously described by our group. Cox regression was used to evaluate the ability of prognostic parameters to predict clinical outcome stratified by age at diagnosis. Patient survival was compared to that of the age matched Minnesota population. RESULTS: When grouped by age, 593 patients were age <55, 713 age 55-64, 748 age 65-74, and 433 age ≥75 at diagnosis. As of last follow-up, 268, 282, 296, and 108 patients in these age groups have been treated, respectively and 112, 171, 254, and 199 patients have died. Survival decreased as the age at diagnosis increased (p<0.001). OS of CLL patients was significantly shorter than that of the age-matched general population for patients age <55 years (p<0.001), 55-64 years (p<0.001), and 65-74 years (p<0.001) at diagnosis but not those ≥75 years (p=0.136). Among Rai stage 0 patients, survival was also shorter than that of the age-matched general population for CLL patients age <55 years (p<0.001), 55-64 years (p<0.001), and 65-74 years (p<0.001) but not those ≥ age 75 at diagnosis (p=0.07). No statistically significant differences were observed in CD38 status, IGHV mutation status, ZAP-70 status or the frequency of common cytogenetic abnormalities on FISH based on age at diagnosis category. Consistent with prior reports, all of these parameters were powerful predictors of TFT and OS for the overall cohort on univariate analysis (all p<0.001 for both TFT and OS). Given the variation in TFT and OS based on age at diagnosis, we next evaluated the utility of these prognostic parameters for predicting TFT and OS in each age category after adjusting for stage. CD38, IGHV mutation, and ZAP-70 each predicted TFT independent of stage for all age groups (all p <0.05). FISH results predicted TFT independent of stage for patients age 55-64 and 65-74 however did not reach statistical significance for those age <55 (p=0.18) or ≥75 (p=0.07). The utility of these parameters for predicting OS independent of stage was less consistent and varied by age. IGHV predicted OS independent of stage for patients <age 55 (p<0.001), 55-64 (p=0.007), and 65-74 (p'0.001) but was not a significant predictor of OS among those ≥75 (p=0.15). CD38, ZAP-70, and FISH each predicted survival independent of stage for only 2 of the 4 age categories (ZAP-70 for age <55 and 55-64; FISH for age <55 and 65-74; CD38 for 55-64 and ≥75). CONCLUSIONS: Survival of CLL patients is shorter than that of the age-matched general population for individuals age <75 at diagnosis regardless of disease stage. In contrast, survival does not differ from the age-matched general population among CLL patients ≥age 75 at diagnosis. Prognostic testing using CD38, IGHV mutation, and ZAP-70 is useful for predicting TFT independent of stage for CLL patients of all ages (including those ≥age 75) but has less value for predicting OS particularly as the age at diagnosis increases. IGHV appeared to be the best predictor of OS independent of stage among all patients age <75. Unexpectedly, prognostic testing had little utility for predicting OS independent of stage among patients age ≥75. These findings have important implications regarding the use of prognostic tests in patients with CLL as well as the use of test results to select patients for clinical trials testing early treatment for asymptomatic individuals. Disclosures: Shanafelt: Cephalon: Research Funding; Celgene: Research Funding; Polyphenon Pharma : Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. 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. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding.

Abstract Abstract 4410 BACKGROUND Autoimmune cytopenia occurs in 5%-10% of patients with CLL. Treatment for patients with both autoimmune cytopenia and progressive CLL is challenging due to poor tolerance of myelosuppression, and the risk of exacerbation of autoimmune cytopenia by purine analogues. METHODS We identified 20 patients from our CLL database of approximately 3000 patients who were treated with a chemoimmunotherapy regimen of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP) for both autoimmune cytopenia and progressive CLL from January 2000-December 2008 at Mayo Clinic Rochester. RESULTS Autoimmune cytopenia responded in 19 patients (14 CR and 5 PR) with a median time to progression (TTP) of 13.6 months (range 5 – 29). Five patients remain in sustained remission at last follow up (range 15 – 30 months) and 4 patients have had recurrent autoimmune cytopenia requiring re-treatment. Six patients required maintenance corticosteroid therapy after completing R-CVP. Progressive CLL responded to treatment in 17 patients (9 CR/CCR, 8 PR by NCI-WG 1996 criteria) with a median TTP of 6.3 months (range 1-40 months). Fourteen patients have required additional treatment for progressive CLL at a median of 5.4 months (range 4-22 months). Five patients have not required additional treatment for either CLL or autoimmune cytopenia at 15 -30 months after completing treatment. R-CVP treatment toxicity included 3 patients with grade 3-4 infections, 2 with grade 1 peripheral neuropathy, and one with grade 3 drug-induced pneumonitis. No patient required blood cell transfusions after the completion of cycle 1 of therapy. CONCLUSIONS Although R-CVP is an effective and tolerable therapy for patients with simultaneous autoimmune cytopenia and progressive CLL requiring therapy, the duration of response is suboptimal. Clinical trials testing alternative treatment strategies are needed for this patient population. Disclosures: Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Cephalon: Research Funding; Celgene: Research Funding; Polyphenon Pharma : Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. 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 Abstract 3928 Background: Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods: Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results: MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion: MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments: This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures: Shanafelt: Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.