Kay, Neil E.

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 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.

Abstract Abstract 2368 Poster Board II-345 Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. A novel therapeutic agent, Bosutinib, was initially developed as an inhibitor of Src and Abl kinases and is currently in phase II clinical trials for the treatment of several human malignancies. Recently, Bosutinib has been shown to inhibit phosphorylation of a novel receptor tyrosine kinase, Axl which has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Previously, Dasatinib, another Src/Abl kinase inhibitor, showed cytotoxic effects on CLL B cells by decreasing levels of activated, phosphorylated forms of Akt, Erk1/2 and p38 and reducing expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in CLL B cells. Here, we wished to examine receptor or non-receptor tyrosine kinases active in primary CLL B-cells and determine their status after exposure to Bosutinib as well as the latter drug's effect on CLL B-cell viability. Methods: We used freshly isolated CLL B cells after obtaining written consent from patients. Bosutinib was used at various doses (2.5, 5.0, 10.0 and 20.0 μM) to treat CLL B cells in vitro for 24/48 hrs. Induction of apoptosis was assessed by annexin/propidium staining. To examine the impact of stroma on Bosutinib induced CLL B-cell death, primary CLL-bone marrow stromal cells (BMSC) were cocultured with CLL B cells at a cell density ratio of 1:20 and treated with various doses of Bosutinib for 24 hrs. Expression status of various kinases and downstream targets were analyzed in CLL B cell lysates with or without Bosutinib-treatment by Western blot using specific antibodies. Results: Treatment of CLL B-cells with Bosutinib induces a massive apoptotic cell death in a dose- and time-dependent manner (IC50 for 24 h; ∼10 μM and IC50 for 48 h: 5-10 μM) which involves PARP cleavage as demonstrated by Western blot analysis. Moreover, Bosutinib-treatment reduced expression of several key anti-apoptotic proteins, Mcl-1, XIAP and Bcl-2 reported to be overexpressed in CLL B cells. Interestingly, we detected that the majority of CLL B-cells express constitutively active Axl. Importantly, Bosutinib treatment inhibited phosphorylation of Axl in CLL B cells resulting in inhibition of AKT-activation, one of its downstream signaling pathways. Previous studies have suggested a possible physical association between Axl and Src kinase. We observed that expression of constitutively active Axl was associated with the presence of highly phosphorylated Src kinase when compared with that in CLL B-cells with low or unphosphorylated Axl. These observations suggest that phosphorylation of Axl may be an upstream event for Src activation in CLL. We found inhibition of constitutively active Axl resulted in subsequent inhibition of Src kinase activation in CLL B-cells following Bosutinib-treatment. We also detected inhibition of ZAP70/Syk-phosphorylation in CLL B cells upon Bosutinib-treatment. Finally, we found Bosutinib was able to overcome stomal protection of CLL B cells at a dose of 10 μM in an in vitro coculture system suggesting its potential as a therapeutic agent against CLL. Conclusion: Together, these observations suggest that Bosutinib induces apoptosis in CLL B-cells, even in the presence of stromal cells, in association with the down regulation of multiple kinases including the novel receptor-tyrosine kinase, Axl, and reduces expression of the anti-apoptotic proteins critical to CLL B-cell survival. In total, these findings for the first time indicate that Bosutinib has the potential to be a very potent therapeutic agent for CLL patients. Disclosures: Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Abstract Abstract 3926 Introduction. Chronic lymphocytic leukemia (CLL) patients will have significant variation in signs and symptoms at initial presentation and across lines of therapy, with concomitant effect on patient health-related quality of life (HRQOL). HRQOL and other patient-reported outcomes, together with clinical outcomes, provide a more complete perspective on the burden of disease and facilitate a broader view of the impact of treatment regimens. This analysis evaluates whether the HRQOL of patients with CLL in the United States (US) varies at the time the patients are about to embark on various lines of therapy, and offers a baseline report from which subsequent longitudinal analysis post-treatment will be possible. Methods. Clinical and HRQOL data were collected in Connect®CLL, a prospective observational registry initiated in March 2010 involving centers in the US. Physicians provided data on the demographics and clinical characteristics of patients receiving therapy. HRQOL was self-reported by patients at enrollment using the Brief Fatigue Inventory (BFI, a symptom assessment tool), the EQ-5D (a non-disease-specific HRQOL instrument), and the Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu, a leukemia-specific HRQOL instrument). We characterized patients who had initial (First Line), second (Second Line) or subsequent (Higher Line) treatment regimens prior to initiation of regimens. Reported mean overall and/or domain-specific BFI, EQ-5D and FACT-Leu scores were analyzed by line of therapy. Statistical significance of score differences among sub-cohorts was ascertained by ANOVA using SAS 9.1. Results. Evaluable data were reported on 1005 patients, enrolled from 161 centers (93% community, 7% academic). Patients were predominantly male (62%) and white (89%) with mean age at 69 (standard deviation [SD] 11) years. HRQOL scores by line of therapy are presented: The total FACT-Leu and FACT-G results, and the total EQ-5D Index and Visual Analogue Scale (VAS) results, consistently suggest that patients initiating first line therapy have somewhat better HRQOL compared with those initiating subsequent lines of therapy. FACT-Leu total scores of patients initiating first line therapy were associated with better physical and leukemia-specific considerations, and the EQ-5D total score was associated with better mobility and pain/discomfort. Conclusions. Initial results from the Connect® CLL Registry indicate that HRQOL prior to treatment is better among patients initiating first line therapy compared to patients initiating later lines of treatment. Future analyses should be conducted to determine what clinical or other factors may be associated with the HRQOL deterioration in patients initiating subsequent lines of therapy, so as to inform clinician decision making. Also, subsequent longitudinal analyses should be undertaken to determine how HRQOL might be affected by the different lines of therapy and the specific treatment regimens, as well as by their initial HRQOL and other patient factors. Disclosures: Kay: Celgene: Research Funding. Flowers:Celgene: Consultancy; Prescription Solutions: Consultancy; Seattle Genetics: Consultancy; Millennium: Research Funding, Unpaid consultancy, Unpaid consultancy Other; Genentech: Unpaid consultancy, Unpaid consultancy Other; Gilead: Research Funding; Spectrum: Research Funding; Janssen lymphoma research foundation: Membership on an entity's Board of Directors or advisory committees. Weiss:Celgene: Consultancy. Lamanna:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Flinn:Celgene: Research Funding. Grinblatt:Celgene: Honoraria, Speakers Bureau. Kipps:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kozloff:Celgene: Consultancy. Lerner:Celgene Connect: Membership on an entity's Board of Directors or advisory committees. Sharman:Celgene: Consultancy; Pharmacyclics: Honoraria; Calistoga: Honoraria; Portola pharmaceuticals: Consultancy. Khan:Celgene Corporation: Employment, Equity Ownership. Street:Celgene: Employment, Equity Ownership. Swern:Celgene: Employment. Sullivan:Celgene: Employment. Pashos:Celgene: Membership on an entity's Board of Directors or advisory committees, 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.