Shanafelt, Tait D.

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by defects in the level of apoptosis rather than accelerated leukemic cell proliferation. We have been interested in identification of naturally occurring substances that induce apoptosis in CLL B cells with potential for therapeutic application with a favorable toxicity profile. Two such agents, epigallocatechin-3-gallate (EGCG) in green tea and curcumin from the spice turmeric have recently been studied by our laboratory. We have shown previously that in vitro treatment of CLL B cells with EGCG induces apoptosis [Blood, 2004, 104:788] and is now being tested in a Phase I/II clinical trial. In this study, we explored the possibility of using curcumin in the induction of CLL B cell apoptosis either alone or in combination with EGCG. We also tested their ability to induce CLL B cell apoptosis in the presence of human stromal cells. Results: In vitro treatment of CLL B cells with curcumin induces apoptosis in a dose (5–20 μM) and time dependent manner. Further analysis suggested that curcumin treatment inhibited the effects of the constitutively active proteins STAT3, AKT and NF-κB and suppressed expression of XIAP in CLL B cells. Interestingly, we observed that curcumin/EGCG combination was more effective in the induction of apoptosis in CLL B cells as compared to the corresponding individual dose of the drug alone. We next evaluated the effects of curcumin when CLL B cells were co-cultured with the human stromal cell line HS-5. Curcumin-induced apoptosis was significantly inhibited when CLL B cells were co-cultured in physical contact with HS-5 cells. We also found protection against curcumin-induced cell death, albeit less, when CLL B cells were co-cultured with HS-5 separated by transwells. However, higher doses of curcumin can overcome HS-5 mediated apoptosis-protection of CLL B cells in transwells but, did not overcome the protective effect of physical contact with HS-5. Further analysis of CLL B cells co-cultured with HS-5 in transwells demonstrated elevated levels of STAT3 with increased phosphorylation at serine-727 and increased expression of Mcl-1 and XIAP. At this stage, we employed combined therapeutic approach to evaluate whether combining curcumin with EGCG could overcome stromal cell-mediated protection. Interestingly, combined treatment of CLL B cells could significantly overcome stromal cell-mediated protection against apoptotic cell death, although the effect was more pronounced when CLL B cells were separated by transwells (see figure below). Conclusion: Together, these results suggest that curcumin induces apoptosis in CLL B cells in vitro and may mediate its effects by suppressing the constitutively active signaling pathways that are known to be important in apoptosis resistance This work also demonstrates that stromal cells and their soluble products can resist drug-induced apoptosis of leukemic CLL B cells. Importantly, the apoptosis-inducing activity of curcumin and EGCG is not impaired completely in the presence of stromal cells, suggesting that the combination of curcumin/EGCG may warrant clinical testing in patients with CLL. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars.

Abstract It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.

Abstract Background: Patients with CLL have a variable clinical course. Current novel prognostic indicators (ZAP-70, CD38, IgVH gene mutation status, FISH) are able to identify early-stage CLL patients at high risk of rapid disease progression. These prognostic factors focus on characteristics of the malignant B cell clone and are not currently changeable factors. Identification of modifiable characteristics of the host unrelated to the leukemic B-cell but that relate to CLL-specific survival may provide opportunities for therapeutic intervention. Since the immune system is likely to modulate disease progression in CLL patients, we evaluated critical features of that system in relation to the size of the circulating clonal B-cell population in a CLL cohort. Methods: 186 consecutive patients with CLL who were evaluated at Mayo Clinic within 2 months of diagnosis between 2000 and 2002 were identified. The primary end-point of the study was to assess if host immunity affected time to treatment (TTT). Baseline flow cytometry analysis was available for 166 patients (88%) and was used to calculate the absolute number of T-cell and natural kill (NK)-cells present at diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was then evaluated by calculating the NK:MBC ratio and T:MBC ratio. Relationships with other prognostic parameters and with TTT were evaluated. Results: Patients exhibited substantial variation in the absolute number of T-cells and NK cells as well as T:MBC and NK:MBC ratios at the time of diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early Rai stage and mutated IgVH genes (all P≤0.0002). As continuous variables, both the T:MBC ratio (p value=0.03) and NK:MBC ratio (p value=0.02) were associated with TTT. Since they correlated with TTT as continuous variables, thresholds to classify MBC, T:MBC ratio, and NK:MBC were identified using the method of Contal and O’Quigly. On multivariate Cox modeling including stage, CD38, absolute MBC count, T:MB ratio, and NK:MB ratio, the independent predictors of TTT were disease stage, T:MB ratio, and NK:MB ratio (Table 1). Conclusion: Measurable characteristics of the host immune system appear to relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies, and cellular therapies to delay/prevent disease progression are warranted. Additional studies exploring how interactions between the host immune system and the leukemic clone influence clinical outcomes are needed.

Abstract Background: CpG 7909 is a synthetic 24 mer oligodeoxynucleotide designed to provide optimal activation of immune mechanisms through the TLR9 receptors on B-cells and plasmacytoid dendritic cells. In vitro CpG can upregulate CD20 expression as well as stimulate the immune system. Radioimmunotherapy (RIT) with anti-CD20 radioimmunoconjugates such as Zevalin produces responses in approximately 80% of patients with relapsed indolent lymphoma; however, only 30% obtain a complete response and 20% have long-term disease free survival. Objectives: To combine CpG 7909 with rituximab and Zevalin to enhance the response to RIT. Patients and Methods: Patients were eligible if they had biopsy proven relapsed, refractory, or residual CD20+ NHL. Disease types included relapsed indolent and aggressive B-cell NHL. Patients were required to have measurable disease, a platelet count >150,000, and an absolute neutrophil count >1,500. This was a phase I study where all patients received a standard dose of Zevalin (0.4mCi/kg with a maximum of 32mCi for patients over 80kg) along with a phase I dose of CpG 7909. Four dose levels were studied—0.08mg/kg; 0.16mg/kg; 0.32mg/kg; and 0.48mg/kg. CpG was dosed on Day 6, 13, 20, and 27; rituximab was provided Day 1, 8, and 15; indium-111 Zevalin on Day 1 and 8 (followed by indium-111 Zevalin imaging); and yttrium-90 Zevalin on Day 15. Response was assessed at week 4 and week 12. Patients are also being assayed pre- and post-CpG and at multiple time points up to 1 year following treatment for changes in T-cell, NK-cell, and cytokine levels. Results: A total of 30 patients have now been enrolled—6 at each of the first 3 dose levels and 12 at dose level 4. Three pts had relapsed diffuse large cell (DLC) and 27 pts were relapsed indolent. One of the first 6 patients at dose level 4 (0.48mg/kg) had a dose limiting toxicity (DLT - hematologic with full recovery). The other 5 patients at dose level 4 did not have DLT; and thus, 0.48mg/kg was determined to be the dose for further studies. An additional 6 patients at dose level 0.48mg/kg were accrued. Response information is available on the first 26 pts and the median follow-up is 12.4 months (range, 0–30.8). The overall response rate, to date, is 92% (24/26) – 66% (2/3) for the DLC pts and 96% (22/23) for the indolent pts. The complete remission rate is 58% (15/26). After follow-up, 7 of the 26 patients have progressed and one has died of relapsed NHL; therefore, 73% (19/26) remain in response and overall survival is 97% (29/30). As part of the study, we performed indium-111 Zevalin imaging before CpG and after 1 dose of CpG (prior to the yttrium-90 Zevalin) to evaluate the effect of CpG on the biodistribution of the radioimmunoconjugate. In the first 24 patients treated to date, review of the paired gamma camera images does not show any significant effects of CpG on tumor or normal organ biodistribution. To date, there has been no significant differences in T-cell, NK-cell, and cytokine activation by CpG dose level. Summary: CpG 7909 can be safely combined with Zevalin RIT in patients with advanced stage relapsed B-cell NHL.

Abstract Abstract 1772 Background: B-Chronic Lymphocytic Leukemia (CLL) is notorious for its complex and heterogeneous clinical behavior pattern in a given patient. Panels of prognostic predictors in CLL can be complex but in general have been found to be useful in counseling a given patient. The current most powerful prognostic parameters for disease progression potential are related to leukemic B cell genetic features. Using fluorescent in situ hybridization (FISH) panels, we and others have found a hierarchy of clinical outcome with 17p- and 11q- most aggressive. The limitation of the FISH panel is that while it detects the most common recurring defects, it does not evaluate the global genomic status of the leukemia in terms of less common genetic changes or in terms of genetic complexity. Array comparative genomic hybridization (CGH) has shown that CLL B cells frequently do exhibit greater genetic complexity than is detected by the FISH panel. To further assess the relationship of genomic defects in CLL to disease outcome, we used CGH for genetic analysis of the leukemic cells at or close to the diagnosis in two cohorts of CLL patients who had either stable or progressive disease. Methods: We obtained leukemic B cells from patients retrospectively identified as “progressive” (n=47) or “stable” (n=21) CLL patients. The CLL patients had given informed consent to have tissue stored and had robust long term clinical outcome available on our Mayo Clinic CLL Tissue Bank and clinical database. Patients were selected based on whether they were stable as defined by no need for therapy after 5 years from diagnosis, or progressive as defined by requiring therapy within 2 years of the actual CLL diagnosis. All progressive patients had leukemic blood cells sampled within 12 months of diagnosis and prior to treatment. All stable patients had blood cells sampled within 24 months of diagnosis. DNA was extracted and then used for global genomic defect detection using a custom high density 2 × 400k oligonucleotide CGH chip (Agilent). Copy number abnormalities (CNA) and copy number variants (CNV) were assessed. CNA were studied for relationship to clinical outcome for the two cohorts of CLL patients.and CNV were excluded from further analysis. Results: The median age was 61 (range 42–81) years in progressive and 67 (range 42–80) in stable patients. Males comprised 77% of the progressive and 57% of the stable patients. CGH was successful in all 68 patients. FISH and CGH were discordant in 8 cases, all with <20% abnormal cells by FISH. Total number of CNA (excluding known benign variants and immunoglobulin rearrangements) was 816, with 597 in progressive (mean 12.7) and 219 in stable patients (mean 10.4). Two stable patients had a normal CGH result. Progressive patients included 15 of 16 trisomy 12, all 4 deletion 17p, and 10 of 11 deletion 11q abnormalities by CGH. Deletion 13q was observed in 39 (83%) progressive and 15 (71%) stable patients, with Rb1 deletion in 11 (23%) progressive and 4 (19%) stable patients. Remarkably, trisomy 18 (2 patients), trisomy 19 (4 patients), and trisomy 21 (1of 2 patients) were observed by CGH in association with trisomy 12. Other recurrent CNA were del(3)(q22q22) in 2 progressive and 1 stable patient and dup(8)(q24.3q24.3) plus dup(11)(p15.4p15.5) in 3 progressive patients; one progressive patient had the dup(8q) without dup (11p). None of the dup(8q) or dup(11p) patients exhibited 11q-, 17p- or +12 defects. Cytogenetic complexity (measured by number of CNA) was greater in progressive than stable patients (p=0.03; odds ratio 1.196), although there was substantial overlap between the groups (range 6–28 CNA in progressive and 5–18 in stable patients). The number of CNA tended to be higher in the presence of 11q- (8–16 CNA) and trisomy 12 (7–21 CNA), and especially 17p- (13–28 CNA). Conclusions: CGH and FISH were comparable for the FISH probes tested. CGH revealed that deletion breakpoints were highly variable around the regions targeted by the FISH probes. That trisomy 18, 19, and 21 were confined to the trisomy 12 patients suggests a different mechanism of genetic instability underlying trisomy 12 in CLL. Trisomy 12, deletion 11q and deleton 17p were confined almost exclusively to patients with progressive disease. Likewise, 3 other recurrent CNA (3q-, 8p dup and 11p dup) were over-represented in progressive disease, may comprise high risk genetic defects, and offer new targets for understanding genetic pathways in CLL. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Abstract The interactions between leukemic cells and the bone marrow microenvironment play a critical role in angiogenesis, disease progression and cell adhesion mediated drug resistance (CAM-DR). VLA-4 (CD49d) is one of the most important adhesion receptors involved in these interactions. The binding of VLA-4 to fibronectin was shown to protect CLL B-cell from apoptosis (de la Fuente, 1999). The introduction of monoclonal antibodies targeting VLA-4 provides the opportunity for the development of novel treatment strategies. We examined the ability of VLA-4 blocking antibodies to induce apoptosis and overcome CAM-DR of primary B cells from CLL patients. Methods: Blood samples were obtained from consenting CLL patients. Mononuclear cells were isolated by density gradient centrifugation. Only samples with over 80% CLL B cells as assessed by flow cytometry (minimum 80% CD19+/CD5+ cells) were used. HS-5 human stromal cell line was cultured in 24 well plates (10% FCS DMEM media) and grown until they reached confluence. The confluent cells were then washed with PBS and incubated with AIM-V media 24 hours before CLL cells were co-cultured. CLL B cells were incubated in AIM-V media with and without fludarabine (1 mmol/L) for 24 hours, spun and resuspended in AIM-V. CLL B cells were then incubated with or without anti VLA-4 antibodies (BD Biosciences) at 10 μg/106 CLL cells for 2 hours and directly added into a co-culture with HS-5 cells. The CLL B cells remained in the co-culture for 24 hours, and were then collected and analyzed by flow cytometry for apoptosis/cell death by annexin/propidium iodide assay (i.e., total culture time 48 hours). Paired T-test was used to compare B cell viability results. Results: After 48 hour culture, the viability cells cultured in media alone or fludarabine for the first 24 hours were 40.6% (18.2%–73.7%) and 8.5% (0.2%–17.9%) respectively. Significant rescue from fludarabine induced cell death was seen if CLL B cells were co-cultured with HS-5 stromal cells for the second 24 hours rather than media alone (86.5% vs. 8.5%; p<0.001). The addition of anti VLA-4 antibodies, reduced stromal cell rescue from fludarabine induced apoptosis by 30% (61.5% vs. 86.5%; p= 0.0053). The addition of VLA-4 antibodies to untreated CLL B cells cultured with stromal cells had no effect on cell viability (90.2% vs. 91.2 p=0.59). Conclusion: Stromal cell adhesion rescues CLL B-cells from fludarabine induced apoptosis. Stromal cell rescue of CLL B-cells from drug induced apoptosis can be significantly reduced by blocking VLA-4 mediated cell adhesion of CLL B-cells to stromal cells. Blocking VLA-4 has no effect on the survival of untreated CLL B cells cultured with stromal cells suggesting the interaction mediated by VLA-4 protects against drug induced cell death rather than directly inducing apoptosis. These findings suggest anti VLA-4 antibodies are unlikely to be useful as a single agent therapy for treatment of CLL. However, blocking VLA-4 may enhance the efficacy of other agents used to treat CLL by overcoming adhesion mediated protection against drug induced cell death.

Abstract Purpose Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multi-center analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as overall survival (OS) and treatment free survival (TFS) predictor. Patients and Methods A training/validation strategy was chosen to find the optimal CD49d cut-off. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLL, and Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (CD38, ZAP-70) was ranked by recursive partitioning. Results Patients with ≥30% of neoplastic cells expressing CD49d were considered CD49d+. The decrease in OS at 5 and 10-years among CD49d+ cases was 7% and 23% (decrease in TFS 26% and 25% respectively). The pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR=2.0) in a Cox model adjusted for clinical and biological prognosticators. Hierarchical trees including all cases, or restricted to early stage or patients ≤65 years, always selected CD49d as the most important flow-cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patients' subsets with poorer outcome independent of CD38 and ZAP-70. Conclusions In this analysis of ∼3000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL. 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: Research Funding.

Abstract Abstract 3882 PURPOSE: Nurturing of leukemic B-cells by the microenvironment appears to be an important mechanism of chemoresistance. Recently, several groups have shown that immune cytokines such as MIP-1a (CCL3) and MIP-1b (CCL4) are upregulated when CLL B-cells interact with stroma or nurse-like cells. It appears that stroma may nurture CLL B-cells by providing critical growth factors and signals enhancing survival. Disruption of these signals could be a strategy to overcome stromal cell mediated chemoresistance. METHODS: We cultured CLL B-cells alone or with human stromal cells (HS-5) and evaluated levels of immune cytokines in the media using multiplex cytokine arrays (Invitrogen). To explore the effect of different drugs on cytokine release, CLL B-cells were treated with sub-lethal doses of fludarabine (F) and chlorambucil (C) in co-culture with the stromal cell line HS-5 cells. Based on our previous observation that pharmacologically achievable doses of EGCG are able to overcome stromal rescue of CLL B-cells exposed to purine nucleoside analogues and alkylating agents (ASH 2010), we examined the effects of EGCG on stromal cell mediated cytokine release in co-cultures. In addition, plasma samples collected prior to and after 1 month of treatment from Rai stage 0-II CLL patients participating in a phase II trial of EGCG were used to explore whether there was a clinical correlation of the in vitro effects of EGCG. RESULTS: Co-culture of CLL B-cells with HS-5 cells resulted in upregulation of multiple cytokines. In addition to the previously reported upregulation in MIP-1a and MIP-1b, increases in IL-1RA (5 fold) and MCP-1 (4 fold) were also observed. Upregulation of these cytokines in co-culture was largely contact dependent. When CLL B-cells and HS5 cells were co-cultured with sub-lethal doses of F, modest reductions in the upregulation of IL-1RA (12%) and MCP-1 (18%) were observed compared to co-culture without drug exposure. Similar experiments using sub-lethal doses of C resulted in more substantial reductions in IL-1RA (68%) and MCP-1 (48%) upregulation. Interestingly, the effect of sub-lethal doses of EGCG was similar to C with respect to reducing upregulation of IL-1RA (69%) and greater than both F and C with respect to upregulation of MCP-1 (76%; Figures 1A and B). To explore the potential clinical correlations of these in vitro observations, we assessed the effects of EGCG on plasma IL-1RA and MCP-1 levels in 42 CLL patients participating in a phase II trial evaluating the efficacy of EGCG (2000 mg BID). Of these 42 patients, 13 (31%) were Rai stage 0 and 29 (69%) Rai stage I-II at enrollment. As previously reported (Shanafelt, ASCO 2010), clinical activity was observed with 13 (31%) patients experiencing a sustained ≥20% reduction in absolute lymphocyte count (ALC) and 20 of 29 (69%) patients with palpable adenopathy experiencing at least a 50% reduction in the sum of nodal products with treatment. Median baseline plasma MCP-1 levels prior to treatment were 687 pg/ml and median IL-1RA levels were 468 pg/ml. At the cohort level, no change in median MCP-1 (p=0.21) or IL1-RA (p=0.95) levels were observed after 1 month of EGCG treatment. We next explored whether changes in the MCP-1 or IL1-RA levels of individual patients correlated with response. Although no correlation between reductions these cytokines and declines in ALC were observed, a significant association between reductions of both MCP-1 (Spearman coefficient: 0.51; p=0.02) and IL1-RA (Spearman co-efficient: 0.60; p=0.006) and reductions in lymphadenopathy was found suggesting EGCG effects on these cytokines may relate to clinical efficacy. CONCLUSIONS: Co-culture of CLL B-cells with marrow stromal cells resulted in contact dependent up-regulation of multiple immune cytokines including IL-1Ra and MCP-1 which we speculate may partially facilitate stromal cell mediated chemoresistance. Several drugs, including EGCG appear to modulate this up-regulation. An association between changes in these cytokine levels and reductions in lymphadenopathy in a phase II trial of EGCG suggests that interrupting signaling by these cytokines may be a potential therapeutic target. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees. Shanafelt:Polyphenon E International: Research Funding.

Abstract Abstract 3898 Background: Although chemoimmunotherapy (CIT) has substantially improved response rates, treatment free survival, and overall survival in patients with chronic lymphocytic leukemia (CLL), only 4050 of patients achieve a complete remission and the majority have residual disease when evaluated using sensitive assays. There remains interest in identifying new approaches to improve the effectiveness of CIT. Ofatumumab is a humanized anti-CD20 monoclonal antibody that is approved for patients with relapsed CLL refractory to fludarabine and alemtuzumab. The single agent activity of ofatumumab in patients with refractory CLL suggests it may have greater efficacy than rituximab in patients with CLL. To explore the efficacy of ofatumumab based CIT, we initiated a clinical trial studying the effect of ofatumumab in combination with pentostatin and cyclophosphamide (PCO) for patients with previously untreated CLL. METHODS: Eligible patients were previously untreated and had CLL in need of treatment according to the NCI-WG criteria (Blood 111:5446). The treatment schema included 6 cycles of ofatumuamb (300 mg on day 1 of cycle 1;1000 mg on day 2 of cycle 1 and day 1 of cycles 26) followed by pentostatin (2 mg/m2) and cyclophosphamide (600 mg/m2) administered every 21 days. All patients underwent complete response evaluation including evaluation for MRD using sensitive flow cytometry (Leukemia 21:956) at the completion of treatment. RESULTS: 33 patients were enrolled at Mayo Clinic and Duke University between 3/2010 and 9/2010. All patients were eligible for treatment and have completed active treatment. Clinical characteristics included: 76 male, median age 63 (range: 5083); intermediate Rai risk 27; and high Rai risk 73. On prognostic testing 33 were CD38, 48 Zap-70 , 58 IGHV unmutated, and 6 had high risk FISH (del 17p13; del 11q22). 26 of 33 eligible patients (79) completed all 6 cycles of PCO induction. Seven patients went off treatment early (toxicity: n5; refusal: n1; progression: n1). Adverse events deemed at least possibly related to PCO induction included 8 (24) patients with grade 3 hematologic toxicity and 5 (15) with grade 3 non-hematologic toxicity. The overall response rate to PCO induction was 94 (31/33) with 15 (45) CR/CRi, 5 (15) CCR, 9 (27) nPR, and 2 (6) PR. 2/10 (20) patients with CR's tested for MRD were also MRD negative by 6 color flow cytometry analysis. Finally, we compared this cohort of patients treated with ofatumuamb-based CIT to our prior 108 patient cohort of patients treated with rituximab-based CIT both of which used an identical chemotherapy backbone (pentostatin and cyclophosphamide). Although the age, gender, ALC, and prognostic profile (CD38, ZAP-70, and IGHV mutation status) of treated patients were similar (all p0.3), the ofatumumab cohort included more advanced stage patients (Rai stage III/IV: 73 vs. 41 p0.002). Ofatumumab-based CIT appeared to be better tolerated than our previous experience with rituximab-based CIT (grade 3 hematologic toxicity ofatumumab-CIT 24 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{8$}\!\left/ \!\raisebox{-1ex}{$33$}\right.}\]\) \end{document} vs. rituximab-CIT51 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{55$}\!\left/ \!\raisebox{-1ex}{$108$}\right.}\]\) \end{document}, p0.009; grade 3 non-hematologic toxicity ofatumumab-CIT 15 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{5$}\!\left/ \!\raisebox{-1ex}{$33$}\right.}\]\) \end{document} vs. rituximab-CIT 24 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\scriptstyle \raisebox{1ex}{26$}\!\left/ \!\raisebox{-1ex}{$108$}\right.}\]\) \end{document}, p0.34). Despite a higher proportion of Rai Stage III/IV patients in the ofatumumab cohort, the efficacy of ofatumumab-based CIT also compared favorably to our previous experience with rituximab based CIT (Table). CONCLUSION: Front-line ofatumumab-based CIT appears to be well-tolerated in patients with CLL. Compared to our historic experience with rituximab based CIT, ofatumumab-based CIT appeared to have less hematologic toxicity and improved efficacy. This trial has been expanded to provide a larger experience with ofatumumab-based CIT. Disclosures: Shanafelt: Hospira: Research Funding; Glaxo-Smith-Kine: Research Funding; Genentech: Research Funding. Off Label Use: Off label use of pentostatin and Ofatumumab. Lanasa:GlaxoSmithKline: Consultancy, Speakers Bureau. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding. Tun:Celgene: Research Funding. Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

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