Kay, Neil E.

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 Abstract 3872 Background: Mesenchymal stromal cells are one of the components in the CLL microenvironment known to support leukemic cells survival and growth. Crosstalk between CLL B-cells and marrow-derived mesenchymal stromal cells (MSC) can modulate the activation of both cell types. Indeed we have shown that soluble factors secreted from CLL B cells can activate MSC with increased production of angiogenic cytokines (Blood. 2010. 116:2984). We hypothesize that MSC derived from CLL patients are modified by their interaction with leukemic cells in their gene expression profiles (GEP) and cytokine production (CP) capabilities. Given this, intrinsic differences should be present amongst MSC derived from CLL patients versus normal subjects. Method: MSC collected from CLL patients with Rai stage 1–4 before any therapy or who have not had therapies for at least 2 years as well as normal MSC from healthy controls were cultured and expanded one time in vitro. Subsequently, MSC after one in vitro passage were sorted with CD45 negative selection to eliminate any contaminating non-stromal cells. Approximately 100,000 to 300,000 cells were then stored at −70C and used for RNA purification. Nine CLL MSC and five normal MSC cell samples had RNA purified from each sample and then the RNA preparations were used for comparative analysis of GEP using affymetrix U133A plus B chip. Data were transformed using two different methods: MAS5 or Plier and only probes that were present in at least 50% of cases were included in the analysis. We have recently observed that MSC derived from bone marrow of CLL patients promotes Th17 cell differentiation (Blood. 2010. 116: Abstract 2414). Since IL-17 is the signature cytokine produced by TH17 cells, we decided to test if IL-17 could alter the cytokine production from CLL and normal MSC. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) spontaneously or with recombinant IL-17 during a 72h-culture were tested using a commercial cytokine array (R&D). Result: Both MAS5 and Plier method of transformation yielded similar results for GEPs of the MSC. The preliminary data from these analyses showed that multiple transcription factors including PITX2, HOXB7, SIM1, ZFY, EMX2, HOXB3, HOXB4, and IRF6 were significantly up-regulated (more than 2 fold) in CLL MSC compared to normal MSC. In contrast, the genes up-regulated in normal MSC compared to CLL MSC include genes involved in the immune response (IGH, IGK, IGL, BST2, PPBP, RGS1, etc.), antigen processing and presentation (HLA genes, IFI30, etc.) and inflammatory response (CD163, LYZ, TNFAIP6, etc.). We are currently in the process of performing further analysis using additional MSC samples to validate these findings with further GEP assays and also by validating the dysregulated genes detected by our GEP studies using real-time PCR analysis. We performed the analysis of multiple cytokines produced by CLL (n=16) and normal MSC (n=12) with and without stimulation with recombinant IL-17 (8 ng/ml) over a 72 hour culture period. Without stimulation, there is approximately two-fold increase of MCP-1 production in CLL MSC compared to normal MSC (CLL vs. normal, 164.2 ± 26.1 vs. 82.7 ± 15.4 pg/ml, p = 0.02). After IL-17 stimulation, an approximately four-fold increase of MCP-1 production (CLL vs. normal, 1200.1 ± 258.6 vs. 334.1 ± 68.2 pg/ml, p = 0.005), three-fold increase of IL-8 production (CLL vs. normal, 2073.1 ± 504.1 vs. 708.7 ± 218.9 pg/ml, p = 0.02), two-fold increase of IL-6 production (CLL vs. normal, 1400.6 ± 214.0 vs. 705.6 ± 98.2 pg/ml, p = 0.008) were detected in CLL MSC compared to normal MSC. Conclusion: We have detected the presence of intrinsic mRNA expression differences for both transcription factors and immune response genes between CLL and normal MSC. The expression differences of these genes could be facilitating the differential cytokine responses we observed in CLL MSC compared to normal MSC. Given these findings it is possible that the dynamic interactions between the CLL leukemic cell and MSC influence the transcription profiles and cytokine production from MSC. This is of biologic and clinical interest in that the GEP dysregulation in CLL MSC may be a primary mechanism for the differential cytokine production seen in CLL MSC. This outcome in turn has the potential to modify the immune and non-immune environment in favor of tumor proliferation and progression. 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 Abstract 2085 Introduction. Although advanced patient age is commonly used as a factor in selecting therapy for patients with chronic lymphocytic leukemia (CLL), based on presumed associations with functional status, limited data exist regarding the relationships between age and physical, emotional, social, and functional well being. We examined the relationships between age and these domains of health-related quality of life (HRQOL) for CLL patients treated in US community practices. Methods. Baseline data were collected as part of Connect CLL®, a prospective observational registry initiated in March 2010 involving centers in the US. Data on patient demographics and clinical characteristics were provided by clinicians. HRQOL was self-reported by patients in the clinic at enrollment. Patients completed 3 psychometrically validated instruments: the Brief Fatigue Inventory (BFI), EQ-5D, and Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu). Standard analyses were conducted of each instrument given clinical characteristics at that time. Reported mean BFI, EQ-5D and FACT-Leu scores were analyzed by age group (<65, 65–74, >74). Statistical significance of score differences among sub-cohorts was ascertained by ANOVA using SAS 9.1. Results. Baseline HRQOL data were reported by 604 patients, enrolled from 161 centers. Patients were predominantly male (62%) and white (90%) with mean age at 69.9 (standard deviation [SD] 11.2) yrs. HRQOL scores by age group are presented: There were no significant differences between the age groups in fatigue as measured by the BFI, or differences in overall HRQOL as measured by the EQ-5D Visual Analogue Scale (VAS) or the FACT-G. Anxiety/depression and self care are EQ-5D domains that also did not vary by age. Although mobility was most impaired in the oldest age group compared to the two younger groups, usual activities and pain/discomfort were worse in both the younger and older cohorts compared to those 65–74 years of age. FACT-Leu results indicated that the social/family domain scores did not vary by age, but that physical, emotional, and functional domains did vary statistically with the oldest typically doing better than the 65–74 year olds, but not necessarily better than those <65. Conclusions. Initial results from the Connect CLL® Registry indicate that HRQOL does not worsen monotonically with older age. In this cohort, both the youngest and oldest age groups had worse HRQOL in certain domains, presenting an inverted v-shaped relationship. Future analyses should be conducted on: (1) how HRQOL may be affected over time with changes in disease; and, (2) how HRQOL may be influenced by alternative therapies. Results reported here should serve as a useful baseline reference. Disclosures: Pashos: Celgene: Membership on an entity's Board of Directors or advisory committees. Flowers:Genentech/Roche (unpaid): Consultancy; Celgene: Consultancy; Millennium/Takeda: Research Funding; Wyeth: Research Funding; Novartis: Research Funding. Weiss:Celgene: Membership on an entity's Board of Directors or advisory committees. Lamanna:Celgene: Membership on an entity's Board of Directors or advisory committees. Farber:Celgene: Membership on an entity's Board of Directors or advisory committees. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding. Lerner:Celgene: Membership on an entity's Board of Directors or advisory committees. Kay:Celgene: Membership on an entity's Board of Directors or advisory committees. Sharman:Celgene: Membership on an entity's Board of Directors or advisory committees. Grinblatt:Celgene: Membership on an entity's Board of Directors or advisory committees. Flinn:Celgene: Membership on an entity's Board of Directors or advisory committees. Kozloff:Celgene: Membership on an entity's Board of Directors or advisory committees. Swern:Celgene Corporation: Employment, Equity Ownership. Kahn:Celgene Corporation: Employment, Equity Ownership. Street:Celgene: Employment, Equity Ownership. Sullivan:Celgene: Employment, Equity Ownership. Keating:Celgene: Membership on an entity's Board of Directors or advisory committees.

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.

Abstract Abstract 3538 Background: Cytogenetics detection of common recurring genetic defects by FISH is an important clinical prognostication tool in CLL and is frequently used by clinicians for counseling and risk stratification. Thus, any improvement in FISH technology is a welcome advance in our care for CLL patients. Mayo Clinic CLL FISH analyses are prepared directly from buffy coat samples rather than mononuclear cells or lymphocytes. FISH probes (deletion 6q, 11q, 13q, 17p, +12 & IGH/CCND1) are scored among consecutive nuclei without selection for nuclear morphology (so called clinical scoring). CLL B-cells are observed, microscopically, primarily as round lymphocytes with round nuclei, and we found (ASH 2010, #3606) that the % abnormal nuclei in the CLL round cell population is significantly higher than that of the non-round nuclei population. In 79 of 87 (91%) patients, the % abnormal nuclei was greater in round vs unselected nuclei (mean difference 24%; range 1.5 – 57%). Round nuclei (p = 0.004) correlated better with % B-cells by flow cytometry than either consecutive unselected nuclei (p = 0.046) or the lobed (non-round) nuclei (p = 0.40). Utilizing this “Poor Man's” sorting technique, we proposed that scoring consecutive round nuclei would improve sensitivity of detection of CLL FISH defects in patients, especially those with low B-cell counts. In a retrospective study of Mayo Clinic patients with normal baseline FISH who had a FISH defect in follow-up, we showed that by re-scoring 28 baseline cases for round nuclei exclusively, 12 originally normal samples remained normal but 16 (57%) were abnormal using the “Poor Man's” scoring technique. In the present study, we prospectively compared FISH scoring on unsorted samples using the “Poor Man's” technique vs the clinical technique. Methods: After IRB approval, we collected 63 consecutive unsorted Mayo Clinic patient blood samples referred for CLL FISH testing with an absolute lymphocyte count (ALC)< 30K × 109/L, and scored each sample using both the “Poor Man's” round nuclei method and the standard clinical method. The kappa statistic was used to adjust for chance agreement. Bland Altman plots of difference (“Poor Man's” result minus clinical result) vs the average of the two methods were generated. Results: Median age of the 40 males (64%) and 23 females (36%) was 66 years (range 36–87), and median ALC was 6.803 (range 0.980–29.750). The most common FISH defects were 11q-, trisomy 12 & 13q- by clinical technique (14%, 22% & 46% respectively) as well as by “Poor Man's” method (16%, 24% & 49% respectively). A high degree of agreement was observed between the two methods when positive/negative comparisons were made (p<0.0001). Additional FISH defects were observed in 12 of 63 (19%) patients (one 11q-, one +12; six 13q-, and four 17p-) when we utilized the “Poor Man's” method that were not observed by the clinical method. Discussion: The “Poor Man's” sorting method compares well with the clinical method for the presence and absence of FISH defects. The abnormal CLL FISH patterns are truly confined to round nuclei. The “Poor Man's” method is more sensitive than the clinical method, resulting in higher reported abnormal cells and detection of more defects. Importantly, additional FISH defects were identified in 12 patients via “Poor Man's” method that were not observed by clinical technique and 6 of these involved a change in the patients' hierarchical (Döhner) classification due to detection of the more unfavorable prognostic markers of trisomy 12 or deletion 11q or 17p. Thus, selectively scoring FISH in the round nuclei increases the sensitivity for detection of a genetic defect in CLL and provides a practical framework for prognostication in CLL patients with low lymphocyte counts and, we would speculate, including those in clinical remission or with minimal residual disease. 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 We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

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

Abstract Abstract 3903 INTRODUCTION: Monoclonal B-Cell Lymphocytosis is an asymptomatic hematologic condition characterized by the presence of small populations of clonal B-cells in the blood or bone marrow occurring in 3–5% of the general population over age 40. Although MBL is considered the precursor state to CLL and other sub-types of indolent non-Hodgkin Lymphoma, most individuals with MBL will not develop a B-cell malignancy. It is unknown, however, whether individuals with MBL may be at risk for other adverse outcomes associated with CLL, such as increased risk of infection. To explore this aspect, we evaluated the risk of hospitalization for infection among patients with clinically recognized MBL and compared this to a cohort of general medicine patients and a cohort of CLL patients. METHODS: The Mayo Clinic Rochester (MCR), a tertiary referral center in rural southeastern Minnesota, is also the primary hematology center for southeastern Minnesota, northern Iowa, and western Wisconsin. There are no metropolitan areas or hematology specialty centers within a 50 mile radius. To explore the natural history of infection in a local, community dwelling cohort of individuals with clinically identified, CLL-phenotype MBL, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed MBL seen in the MCR Hematology Division between January 1999 and December 2009. Analysis was limited to MBL cases who resided within 50 miles of MCR. The comparison cohort consisted of 689 adult patients also resided within 50 miles of MCR who were seen for a general medical examination between September 2002 and December 2009 and who were enrolled as controls in a case-control study of non-Hodgkin lymphoma. Rates of infection among individuals with MBL were also compared to a cohort of 174 patients with newly diagnosed CLL diagnosed in the same time interval and who resided within 50 miles of MCR. All hospitalizations in these 3 groups were audited to document hospitalization with infection including cross-referenced with the electronic Mayo Clinic infection database, which contains all culture results obtained from hospitalized patients at MCR. Serologies and viral assays (e.g. CMV viral load) were also reviewed. Patients whose cultures were negative but who were given a clinical diagnosis of infection (e.g. pneumonia) and who were treated with a full course of antibiotics were considered to have a culture negative infection. Among the cases, only hospitalizations after CLL or MBL diagnosis were considered. RESULTS: In May 2010, there were 524 individuals in the Mayo Clinic MBL/CLL Database with the diagnosis of CLL-phenotype MBL made between January 1999 – December 2009. Of these, 154 (29%) patients resided within a 50 mile radius of Mayo Clinic and were considered a local, community-based cohort of MBL patients. Median age at MBL diagnosis for these 154 patients was 70 years (range 45–94). After median follow-up of 4.2 years (range 0.3–10.6 years), 25 (16%) individuals with MBL were hospitalized with infection as compared to 20/689 (3%) individuals in the control population (odds ratio MBL relative to control=6.5; p<0.001) and 32/174 (18%) in the cohort of patients with newly diagnosed CLL (OR MBL relative to CLL=0.9; p=0.61) (Figure 1). The 25 MBL patients hospitalized for infection were hospitalized a total of 39 times (median 1 hospitalization for infection per individual). A specific infectious organism was identified by cultures or serologies in 8/39 (21%) hospitalizations among individuals with MBL, 10/27 (37%) hospitalizations for infection among controls, and 60 (63%) of the 89 hospitalizations for infection in CLL cases. Finally we performed a pooled multivariable analysis of all patients (n=1017) adjusting for age, sex, presence of MBL, diagnosis of CLL, and whether patients with CLL or MBL received treatment for CLL during follow-up. All factors were independently associated with risk of hospitalization for infection [Age (per year, OR=1.02, p=0.02), sex (male, OR=2.6, p=0.001), presence of MBL (OR=3.3, p<0.001), diagnosis of CLL (OR=3.0, p=0.002), and treatment for CLL (OR=3.3, p<0.001)]. CONCLUSIONS: In this cohort study, patients with newly diagnosed clinical-MBL had a 6.5 fold risk of hospitalization for infection relative to the control cohort. MBL was an independent risk factor for hospitalization for infection after controlling for age, sex, and treatment. Disclosures: Zent: GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: 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.