Zent, Clive S.

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 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 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.

Abstract Abstract 3899 BACKGROUND: Although chemoimmunotherapy (CIT) has substantially improved response rates, treatment free survival, and overall survival in patients with chronic lymphocytic leukemia (CLL), only 40–50% of patients achieve a complete remission and the majority have residual disease when evaluated using sensitive assays for minimal residual disease (MRD). While these facts generated interest in consolidation strategies for patients with residual disease, previous trials of alemtuzumab-based consolidation found excess morbidity/mortality. In 2008 we began a trial of lenalidomide-based consolidation for patients with previously untreated CLL who had received 6 cycles of CIT induction. Preliminary analysis suggested that lenalidomide consolidation may improve the quality of response and prolong time to retreatment (Shanafelt; ASH 2010), although follow-up was short (median 16 months). METHODS: Eligible patients were previously untreated and had CLL in need of treatment according to the NCI-WG criteria (Blood 111:5446). Treatment schema consisted of 6 cycles of pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 21 days (Blood 109:405). All patients completing 6 cycles of PCR induction, underwent complete restaging including evaluation for MRD using sensitive flow cytometry. Patients subsequently received 6 months of lenalidomide consolidation by daily administration with a starting lenalidomide dose of 5 mg/day and escalation to 10 mg/day after the first cycle as tolerated. Patients again underwent complete restaging including MRD evaluation after 6 months of lenalidomide. At the time of restaging, MRD negative patients entered observation. Those with residual disease continued on lenalidomide and underwent repeat MRD assessment every 3 months until an MRD negative state was achieved. RESULTS: 45 patients were enrolled at Mayo Clinic between 3/2008 and 12/2009 of whom 44 were eligible. Median age was 65 (range: 44–78) and 71% of patients were male. 61% of patients had intermediate Rai risk and 39% high Rai risk. On prognostic testing 32% were CD38+, 52% Zap-70+, 52% IGHV unmutated, and 16% had high risk FISH (del 17p13; del 11q22). Of the 38 eligible patients who completed 6 cycles of PCR induction, 34 (89%) initiated lenalidomide consolidation. Thus by intent to treat analysis, 34/44 (77%) patients starting CIT with PCR were able to receive consolidation therapy. Among the 34 patients who initiated consolidation, the median number of cycles of lenalidomide received was 6 (range: 1–29). Nine of 34 (26%) patients had the lenalidomide dose increased to 10 mg daily while 18 (53%) required a reduction to 5 mg every other day. Five patients are still on lenalidomide at the time of this report. No cases of tumor lysis syndrome or tumor flare reaction were observed. Adverse events deemed at least possibly related to lenalidomide consolidation included 23 (68%) patients with grade 3+ hematologic toxicity and 5 (15%) with grade 3+ non-hematologic toxicity. Among the 34 patients who received at least 1 cycle of lendalidomide, 6 patients have improved the quality of their response including 3 patients who converted from having residual disease to a MRD- state. After a median follow-up of 29 months, 39/44 patients are alive and the median duration of response has not been reached. To date 8/44 (18%) patients have progressed to require additional treatment. Finally, since the eligibility criteria were nearly identical to our historic trial of PCR without lenalidomide consolidation, we conducted an intent to treat analysis that compared all 44 patients in the present trial (i.e. regardless of whether they received lenalidomide consolidation) to the 64 patients previously treated on our PCR trial (Blood 109:405). The demographic and prognostic characteristics of patients in the two studies were similar. With the caveat it represents a comparison across phase II trials, the proportion of patients free of retreatment at 30 months was 79% (95% CI:67–93) for PCR followed by lenalidomide consolidation vs 66% (95% CI: 54–80) for PCR alone (Figure 1). CONCLUSION: Lenalidomide-based consolidation for CLL patients receiving first-line CIT induction appears to improve the quality of response and prolong time to retreatment. Randomized clinical testing of lenalidomide-based consolidation appears warranted. Disclosures: Shanafelt: Celgene: Research Funding; Hospira: Research Funding; Genentech: Research Funding. Off Label Use: PCR-Lenalidomide (off label use of pentostatin and lenalidomide). 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 1790 CLL is incurable with conventional therapies and prognosis is poor in patients with relapsed or treatment refractory disease. Patients with purine analogue refractory disease and defective TP53 function have limited therapeutic options. Based on: 1) The ability of purine analogues to decrease tumor burden; 2) Higher response rates in patients treated with alemtuzumab and rituximab vs. alemtuzumab alone; and 3) Data showing that more frequent lower doses of rituximab could be more effective than standard therapy, we hypothesized that combination therapy with pentostatin, alemtuzumab and low dose rituximab (PAR) would be effective and tolerable therapy for patients with relapsed/refractory CLL and those with previously untreated progressive CLL who had 17p13-. This two stage phase II University of Iowa/Mayo Clinic Lymphoma SPORE clinical trial (NCT00669318) was approved by Mayo Clinic and the University of Iowa IRBs according to the principles of the Declaration of Helsinki. The aims are to assess: 1) Complete (CR) and overall responses; and 2) Progression-free survival (PFS), duration of response, and time to next treatment (TTT). Patients with progressive CLL (including the SLL variant) defined by standard criteria are eligible for this study if they had either previously treated CLL or previously untreated CLL with 17p13-. Cycle 1 of therapy is 5 weeks with rituximab 20 mg/m2/d IV Mon-Wed-Fri starting day 1, subQ alemtuzumab beginning days 3–5 with a dose escalation (3 mg–10 mg– 30 mg/d) and then 30 mg/d Mon-Wed-Fri, and pentostatin 2mg/m2/dose/IV days 8 & 22. Cycles 2–3 are each of 4 weeks duration and are the same as weeks 2–5 of cycle 1. Patients who achieve CR after cycle 2 with a negative CT scan and bone marrow study with no evidence of residual CLL cells after immunohistochemical staining, do not receive cycle 3 of therapy. All patients receive granulocyte growth factor support after pentostatin therapy as well as PCP and herpes virus prophylaxis. Patients are tested for CMV reactivation by PCR weekly during therapy and treated with valganciclovir if reactivation is detected. We report the results of the planned interim analysis performed after completion of therapy for the first 19 patients (July 2008 - October 2010). The median patient age was 63 years (range 47–78) with 74% males. Two patients with 17p13- were previously untreated. Of the 17 previously treated patients (median 2 prior regimens, range 1–6), 8 (47%) had purine analogue refractory CLL (disease progression < 6 months of treatment). Nine (47%) patients had advanced clinical stage (Rai III-IV). Adverse molecular prognostic factors were 17p13- (n=8, 42%, 2 also had 11q22-), 11q22- (n=1), unmutated IGHV (12/17, 71%), ZAP70+ (≥20%, 12/17, 71%) and CD38+ (≥30%, 6/18, 33%). The median beta-2-microglobulin level was 4.3 (range 2.4 – 12.6). Grade 3–4 treatment related toxicities were 9 non-hematological and 20 hematological events. Grade 3–4 infections occurred in 3 patients including one patient requiring hospitalization for CMV re-activation. Fourteen (74%) patients responded to treatment with 6 (32%) CR (includes 2 CRi) and 8 (42%) partial responses (PR). Five (26%) patients had stable disease (SD). Eleven (58%) patients completed 3 cycles of therapy. One patient achieved a CR with no residual disease after 2 cycles of therapy and received no further treatment per protocol. Median duration of response is 7 months (95% CI: 4–not reached (NR)). Among the 8 patients with 17p13- there were 3 CR/CRi, 4 PR, and 1 SD. Four patients proceeded to allogeneic transplant with reduced intensity conditioning (RIC) and were censored for TTT at the initiation of transplant related therapy. With a median of 16 (range 6 – 35) months of follow up, median PFS is 7 months (95% CI: 5–NR), median TTT is 27 months (95% CI: 5-NR) and the calculated median overall survival is 23 months (95% CI: 14–NR). We report that PAR is effective and tolerable therapy in this population of relapsed refractory patients with high to very-high risk CLL. In addition, the responses in patients with 17p13- were similar to that of the patients who did not have 17p13-. The treatment regimen was useful for disease control in 3 patients who proceeded to reduced intensity conditioning allogeneic transplantation. This study shows that PAR could play an important role in the treatment of recurrent and high risk CLL. Supported by the University of Iowa/Mayo Clinic Lymphoma SPORE CA097274 Disclosures: Zent: GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract Abstract 2486 In patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL), deletion of 17p13 (17p-) resulting in loss of one allele of TP53 and 11q22 (11q-) resulting in loss of one allele of ATM predicts a significantly poorer prognosis. These lesions are nearly always monoallelic in patients with CLL and their consequences are considerably influenced by the integrity of the remaining allele. A dysfunctional mutation in the remaining allele, resulting in more aggressive disease, ultimately occurs in most patients with 17p- (> 80%) but fewer patients with 11q- (30%). Because ATM and TP53 are integral components of the p53 pathway, we hypothesize that patients with clonal 17p- and 11q- in the same CLL cell would have a worse prognosis than patients with only one of these deletions. To test this hypothesis, we compared the clinical outcomes (overall survival (OS) and time to treatment (TTT)) of patients with 17p- and 11q- in the same cell to patients with either 17p- or 11q- or neither defect. Methods: We used the Mayo Clinic CLL Database and the Cytogenetics laboratory database to identify all CLL patients seen in the Division of Hematology from 1/1/1995-3/29/2012 who had FISH analysis. Data was extracted on demographics, date of first (sentinel) occurrence of 11q- and 17p-, treatment, and vital status. FISH studies were performed on uncultured blood or bone marrow by standard methods using the Mayo Clinic CLL FISH panel of centromere specific control probes and probes for 6q (MYB), 11q (ATM), 13q14, and 17p (TP53) deletions, trisomy 12 and IGH. Patients with both 11q- and 17p- were further tested with the Abbott Molecular (Des Plaines, IL) ATM/TP53 combination probe set, to determine if the 11q and 17p deletions were present in the same cells. Patients were grouped into 4 cohorts: 1) clonal 17p- and 11q-; 2) 17p- only; 3) 11q- only; and 4) neither 17p- nor 11q-. OS and TTT were calculated and Kaplan-Meier curves were drawn from sentinel FISH date until their last known alive date or death date for OS and until treatment or last known untreated date for TTT. We censored all data on 3/29/2012 and analyzed using log-rank tests from date of sentinel FISH. Sanger sequencing of exons 4–9 of TP53 was done in samples from 15 patients with clonal 17p- and 11q- with an available sample collected within 1 year of the sentinel FISH test and without treatment in that interval. All mutations were confirmed by a repeat assay and analyzed for their predicted effect using the UMD p53 database. Results: We studied 2234 CLL patients with a median follow-up of 4.8 years (range 0 – 29.6 years). Twenty (1%) patients had clonal 17p- and 11q-, 159 (7%) had 17p-, 251 (11%) had 11q-, and 1804 (81%) had neither 17p- nor 11q-. Median OS from sentinel FISH study (Figure 1) was significantly shorter in the clonal 17p- and 11q- group (1.9 years), 17p- group (3.2 y), and 11q- group (4.7 y) compared to the patients with neither 17p- nor 11q- (9.2 y) (p < 0.0001). OS was significantly shorter in the patients with clonal 17p- and 11q- cells vs those with 17p- (p=0.048). TTT was also significantly different among the 4 groups (p<0.0001) with median time to treatment of 0.07 y in the clonal 17p- and 11q-, 0.2 y in 17p-, 0.7 y in 11q-, and 5.6 y in the neither 17p- nor 11q- group. TP53 analysis performed in samples from 15 patients with clonal 17p- and 11q- showed that 10 patients had mutations in the remaining TP3 allele predicted to be deleterious to normal p53 protein function. Median OS was 2.1 years for those with TP53 mutations and 0.9 years for those without mutations (p = 0.03) and median TTT was 0.25 years for those with TP53 mutations and 0.05 years for those without mutations (p = 0.049). Conclusion: “Double hit” clonal deletions of 17p and 11q occurred in 1% of this large cohort of CLL patients and were associated with a significantly poorer prognosis. We recommend that testing for this “double hit” phenomenon, by use of an ATM/TP53 combination probe set, should thus be performed in all patients with 17p- and 11q- on FISH analysis because these patients have a poorer prognosis than those with either 17p- or 11q- and could be candidates for novel therapies. The finding that dysfunctional TP53 mutations in the remaining allele did not worsen prognosis in patients with clonal 17p- and 11q- suggests that monoallelic deletion of both ATM and TP53 in a clone of CLL cells is sufficient to disable p53 pathway function in these patients. Future study of clonal 17p- and 11q- CLL could thus be informative in this regard. Disclosures: Kay: Celgene: Research Funding. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Biothera: Research Funding; Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding.

Abstract Abstract 3575 Unconjugated monoclonal antibodies (MoAb) have proven value in the therapy of CLL, and alemtuzumab (ALEM) has efficacy in patients with purine analog refractory/p53 defective disease. Although the mechanisms of action of ALEM in vivo are not fully defined, complement dependant cytotoxicity (CDC) appears to play an important role. ALEM CDC resistance in a subpopulation of CLL cells in vivo could thus contribute to treatment failure. We hypothesized that the CD20 targeted complement activating human MoAb ofatumumab (OFA) would increase complement activation (as manifested by C3b deposition) as well as CDC in CLL cells treated with ALEM in vitro. Methods: We have completed experiments on 4 of 15 specimens collected from patients with progressive untreated CLL. CLL cells were isolated from blood by density gradient centrifugation and negative selection with magnetic beads. CD52 and CD20 expression were measured with fluorescent beads (Quantum MESF, Bangs Laboratories, IN). CLL cells were treated at a concentration of 2 × 106/ml with 10 mcg/ml of ALEM (Genzyme) and/or OFA (G.S.K.) in AIM V medium (Invitrogen CA) and 10% standard normal human serum (NHS) (Sigma, MO) as a source of complement for 1 hour at 37C. Absolute viable cell counts were assayed with counting beads (TruCOUNT, BD, CA) and propidium iodide (PI) staining (Sigma) using a FACSCalibur (BD) and CellQuest Pro software (BD). CDC was determined by comparing counts to CLL cells treated with NHS alone. Membrane levels of CD59, bound MoAb, and deposited C3b on the CLL cells membranes were measured by flow cytometry using using the anti-CD59 antibody FITC-CD59 (BD), mouse anti-human Fc antibody FITC-HB43, and the anti-C3b antibody FITC-7C12. To study ALEM CDC resistant cells, CLL cells were first treated with ALEM and NHS as detailed above. Surviving viable cells were isolated using density gradient centrifugation and then retreated. C5 deficient serum (Sigma) was used in experiments to determine MoAb binding and C3b deposition in the entire CLL cell population. Results: Binding of either ALEM or OFA to the CLL cells in NHS resulted in activation of complement and deposition of large amounts of membrane C3b on the cells. However, ALEM induced higher levels of C3b deposition (delta MFI 220) and caused more CDC (73% vs. 18%) compared to OFA. This correlated well with the higher levels of expression of CD52 vs. CD20 expression in CLL cells (143 ×103, range 131 ×103 – 159 ×103 vs. mean 20 ×103 molecules/cell, range 10 ×103 – 28 ×103). Studies with the anti-Fc antibody showed that only a small subpopulation of CLL cells had weak MoAb binding. Addition of OFA to ALEM increased complement-mediated C3b deposition. In three patients with high initial ALEM CDC, addition of OFA did not appreciably increase CDC (81% to 87%). However, in one patient with cells that were less sensitive to ALEM CDC, addition of OFA did appreciably increase CDC (51% to 86%). There was no relationship between levels of CD59 expression and CDC. All four CLL specimens studied contained a subpopulation of cells that were resistant to CDC (mean 13%, range 9–18%) despite robust C3b deposition. Conclusions: These data suggests that there are multiple mechanisms of resistance to MoAb-mediated CDC including: 1. Low MoAb binding because of low target antigen expression; 2. Insufficient C3b deposition after MoAb ligation and; 3. Intrinsic resistance in some cells in cells in which high levels of C3b deposition were clearly demonstrable. OFA can increase MoAb binding, C3b deposition, and CDC in CLL cells treated with ALEM. However, addition of OFA does not overcome the intrinsic resistance of a small subpopulation of CLL cells to CDC. Future studies will be needed to examine these resistance mechanisms and to develop interventions to disrupt them. In addition, membrane C3b could be a target for complement dependent cellular cytotoxity in these CDC resistant cells. Disclosures: Taylor: G.S.K.: Research Funding; Genmab: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 3585 Relapsed/refractory CLL is often resistant to antibody-based therapy. Understanding the dynamics of interaction between complement and cell-based antibody-antigen complexes that promote complement dependent cytotoxicity (CDC) of targeted cells could lead to improved therapies. We are conducting a clinical trial of chemoimmunotherapy for relapsed/refractory CLL using pentostatin, alemtuzumab (ALEM), and low-dose rituximab (RTX). Correlative studies were designed to determine if circulating CLL cells developed resistance to CDC. Peripheral blood CLL cells were obtained from 12 patients (median age 66 years, 11 male, 17p13- in 4) including 7 (58%) who responded (4 CR, 3 PR) to treatment. Blood samples were obtained before initiating therapy (day 1), 48 hr after the 1st RTX dose (20 mg/m2 IV MWF), one hour after the 2nd dose of RTX and 1st dose of ALEM (3 mg SC) (day 3), and 72 hr after the 3rd doses of RTX (cumulative dose 60 mg/m2) and ALEM (cumulative dose 43 mg) (day 8). All blood samples were taken prior to the 1st dose of pentostatin. B cells were examined for expression of several markers including complement control proteins (CCP), CD20 and CD52. By treatment day 8, ALC decreased by 70 % or more for 7 patients. The other 5 patients had ALC that were 50 – 100% of pre-treatment levels. In agreement with previous studies from our laboratories, low-dose RTX rapidly decreased CD20 levels on circulating CLL cells; on day 3 after the 2nd RTX dose (and 1st ALEM dose), CD20 was reduced by > 75% in 10 subjects. CD20 levels recovered by day 8 in 6 of these subjects. One hour after completion of mAb infusions on day 3, fragments of complement breakdown product C3dg were demonstrable on circulating CLL cells in 7 subjects, suggesting that infusion of RTX had promoted complement activation on circulating cells. After the RTX and ALEM infusions on day 3, circulating CLL cells with bound C3dg fragments had considerably reduced levels of CD20 but were not cleared from the circulation, suggesting that RTX had bound to the cells, activated complement and promoted shaving (CD20 loss) without inducing bloodstream clearance of these cells. CD52 levels on circulating CLL cells showed modest decreases by day 8 (~ 50% of pre-treatment values) in 4 subjects. To study CDC in vitro, washed blood samples were reconstituted with ABO-matched normal human serum, ALEM was added and complement activation (C3 fragment deposition) and CDC of targeted CLL cells were measured by flow cytometry. Alternatively, mononuclear cells were isolated by ficoll-hypaque centrifugation and subjected to the same analyses, with very similar results. ALEM-mediated in vitro CDC of CLL cells varied from 10 to 90 % for pre-treatment blood samples. Measures of ALEM-mediated CDC and C3b fragment deposition in vitro correlated with the density of CD52 on CLL cells (P<0.001). The initial burden of circulating cells was not predictive of a drop in ALC, and pre-therapy levels of CDC were not correlated with cell clearance after therapy. On day 8, ALEM-mediated in vitro CDC of circulating CLL cells had decreased to <50% of the values measured on pre-treatment samples in 6 subjects, suggesting that cells of these patients might be developing resistance to ALEM-mediated CDC. This decrease in CDC was not correlated with decreases in levels of CD52 expression on patients’ CLL cells or with increases in expression of CCP (CD35, CD46, CD55, CD59). Our findings demonstrate that even after treatment with both RTX and ALEM, CLL cells can persist in the bloodstream in a subset of patients, suggesting that some patients have circulating CLL cells that are resistant to mAb-mediated clearance and CDC due to mechanism(s) that may include loss of antigen and possibly intrinsic resistance. Disclosures: Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding. Weiner:Genentech: Consultancy.