Kay, Neil E.

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 Background: High risk CLL patients treated with novel targeted therapies continue to experience disease progression (PD), and more than 50% of these PD patients evolve into Richter's syndrome (RS), (Maddocks, Jama Oncol 2015). Standard chemotherapy for large cell lymphoma has limited efficacy (median overall survival <12 months) in RS. Novel immunotherapy using a PD-1 antibody to block immune checkpoint signals to activate the cytotoxic T -cell has significant efficacy in relapsed Hodgkin lymphoma (Ansell, NEJM 2015). We conducted a phase 2 trial to test the safety and clinical efficacy of the anti-PD-1 antibody pembrolizumab in patients with relapsed/refractory CLL and relapsed low grade B non-Hodgkin lymphoma (B-NHL) (MC1485 trial). Methods: MC1485includes two arms: patients with relapsed/refractory CLL (including RS) and patients with relapsed B-NHL. The main end points of this study are safety and overall response rate. The first 6 patients enrolled in either arm were included in a designed safety interim analysis. Here we report the initial interim analysis from the CLL arm. Relapsed/refractory CLL including RS who had ECOG performance 0-2 and normal organ function were included. Patients with active autoimmune disease or interstitial lung disease were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks until progression, excessive toxicity, or completion of 2 years of therapy. Soluble plasma PD-L1 levels were measured in these 6 patients using an established ELISA assay (Frigola, Clin Cancer Res, 2011). CD8+ T cell subsets were assessed for their functional status using an established analysis (Liu, Oncoimmunology. 2013) at baseline and after 2 cycles of therapies. Results: As of July 27th, 2015, 16 relapsed/refractory CLL patients including 5 RS patients were enrolled. The median age was 71 years (58-81). The median number of prior therapies was 3 (1-6). All CLL patients had received chemoimmunotherapy (FCR/PCR). 4 out of 5 RS patients had received anthracycline-containing chemotherapy. 5 out of 8 patients who had received prior ibrutinib therapy progressed on ibrutinib. 8 patients had 17p-/TP53 mutation. Herein, we report preliminary results from the interim analysis of CLL arm including 4 RS and 2 CLL as well as the 5th RS who had response evaluation by PET. The median cycles of therapy administered was 4 (3-7). One patient experienced grade 2 cytokine release syndrome with transient febrile neutropenia, grade 3 headache and skin rashes. These symptoms resolved with steroid therapy. One patient experienced grade 3 myalgia and was also treated with steroids. The other patients in this safety cohort tolerated pembrolizumab well. The most common drug-related adverse events were grade 1 dyspnea (33%, 2/6) and grade 1 anemia (33%, 2/6). Based on investigator assessment using the Cheson 2007 International Working Group Criteria, 4 out of 5 RS patients had responded to therapy. One patient had a complete remission after 2 cycles and remains in remission. One patient had almost complete PET response after two cycles and is classified as PR as her bone marrow have not been re-evaluated yet. Two patients had notable responses with nodal and skin lymphoma improvements before they showed potential evidence of progressive diseases. The remaining 1 RS and 2 CLL had stable disease and continued on therapy at the last follow-up. Soluble PD-L1 levels (sPD-L1, n=6) were measured at baseline and after 2 cycles of therapies. 3 RS patients who had responded to therapy had decreased or stable sPD-L1 levels. The 4th RS and two CLL had increased sPD-L1 levels and had not demonstrated clinical response. CD8 T-cell subset analyses performed blindly (n=4) showed increased CD8 T cell function in one of the 4 patients tested. This patient who demonstrated CD8 T cell functional enhancement is the only patient in the 4 tested patients who had notable response to pembrolizumab. Conclusion: Pembrolizumab is tolerated in relapsed CLL and RS patients. Early efficacy observed in heavily pretreated RS patients including patients progressed on both anthracycline-based treatment and ibrutinib indicate PD-1 inhibition is a promising novel approach in patients with RS. Our CD8 T cell subset analysis may be used as a biomarker assay to predict clinical response in the era of novel immunotherapy. This trial and further correlative analysis is ongoing and will be presented at the ASH meeting. Disclosures Ding: Merek: Research Funding. Shanafelt:Cephalon: Research Funding; Pharmactckucs: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding; Celgene: Research Funding; Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding. Kay:Tolero Pharma: Research Funding; Hospira: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding.

Abstract Background: Using fluorescent in-situ hybridization (FISH), a number of investigators have identified specific cytogenetic abnormalities that identify CLL patients with a more aggressive (17p-, 11q-) or indolent (13q-) disease course. Some have suggested patients who initially have a normal karyotype may acquire new chromosome abnormalities during the course of their disease. Since patients with specific cytogenetic abnormalities (17p-, 11q-) are less likely to respond to purine nucleoside analogues, such clonal evolution has potential implications for treatment as well as prognosis. No study has prospectively investigated the frequency of clonal evolution in a cohort of patients with newly diagnosed untreated CLL. Methods: Between 1994 and 2000, we enrolled 167 patients with previously untreated CLL seen at Mayo Clinic in a prospective trial evaluating the prognostic importance of cytogenetic abnormalities and clonal evolution detected by FISH. All patients provided a baseline blood specimen for FISH testing and follow-up specimens over the following 24 months. Other research samples from later timepoints were tested where available. Study participants were contacted by mail in 2004 to update vital and treatment status. Of 83 living responders, 70 (84%) indicated they would be willing to provide an additional follow-up sample for cytogenetic analysis of whom 48 have returned a sample to date. Results of clinical FISH testing during the follow-up interval were also abstracted. FISH was performed on interphase nuclei from blood as we have previously described (BJH 121:287). Results: Median age at diagnosis was 64. Median time from diagnosis to study enrollment was 3.3 months. 94% of patients had early stage disease at enrollment (88 Rai 0; 48 Rai I, 18 Rai II, 2 Rai III; 8 Rai IV). Median follow-up time from diagnosis for all 164 eligible study participants was 8.5 years (range: 0.33–22.9 yrs). As of last follow-up, 48% of patients have received treatment and 57 (35%) have died. 75% of patients had chromosome abnormalities on FISH testing at baseline. The frequency of individual cytogenetic abnormalities on baseline FISH analysis along with overall survival by hierarchical FISH risk category are shown in Table I. 106 patients had sequential samples for FISH analysis at least 2 years apart, 61 had samples at least 5 years apart, and 22 had samples at least 10 years apart. 15 patients had evidence of clonal evolution during follow up as evidenced by a new FISH anomaly not present on the baseline specimen. No clonal evolution was observed in the first 2 years of follow-up (n=106), however of 61 patients with samples at least 5 years apart, 14 (23%) had evidence of clonal evolution. Median time for development of a new cytogenetic abnormality among these patients was 9.3 years. Conclusions: Clonal evolution occurs during the course of disease for approximately 25% of patients with early stage CLL. Clonal evolution appears to occur at low frequency during the first 2 years of follow-up but increases in frequency after 5 years. This finding has potentially significant implications for prognosis and treatment of patients with CLL. FISH Risk Category* N (Baseline) Median Overall Survival (Years) * Difference between groups significant p=0.0038 13q- x 1 37 14.4 13q- x2 35 17 Normal Karyotype 40 13.2 12+ 24 11.1 11q- 12 8.6 17p- 10 10.5 6q- 2 4.1 Other 2 Not reached

Abstract Abstract 3605 CLL is a malignant B-cell disorder characterized by the accumulation of small B lymphocytes with a mature appearance in blood, marrow and lymph nodes. Despite effective treatment options, all patients with CLL will eventually relapse after therapy. This could be due in part to the presence of subclones of the CLL cell population that harbor genetic abnormalities, which confer resistance to treatment. The aims of this study were to investigate the clonal evolution in longitudinal samples of CLL patients and to identify genetic alterations associated with disease progression and resistance to therapy. Sequential analyses were performed in 51 samples from 23 patients who were included in a previously reported clinical trial of pentostatin, cyclophosphamide and rituximab (PCR) given every 3 weeks for 6 cycles in previously untreated CLL (Blood 109:2007). In all cases the first sample analyzed was prior to therapy. In 5 of 23 patients, three time points were analyzed: >6 months prior to entry onto PCR trial (time point A), just before starting with the PCR regimen (time point B), and the time of relapse after PCR trial (time point C). Seven patients were analyzed at time points A and B; 9 at time points B and C and 2 at time points A and C. The median time between points A and B was 17.5 months (range 8–48 months) and between points B and C was 20.5 months (7–60 m). All samples were examined by array-based comparative genomic hybridization (aCGH) using the Agilent Sureprint G3 (1 million probe) array. aCGH findings were confirmed by interphase FISH using probes for D13S319 (MIR16–1/MIR15A), RB1, MDM2, CEP12, CEP6, MYB, TP53, NFKBIA, PERP and FGFR1 loci. Overall, we observed a small increase in the number of copy-number abnormalities (CNA) with disease progression. Twenty-two of the 23 patients with paired samples harbored at least one CNA that persisted in all samples, indicating clonal relationship between the sequential samples. In 15 of the 23 patients the tumor clone was stable and no CNA differences between time points were identified. Conversely, genomic evolution was found in 8 patients. In 3 cases the genetic differences were observed pre treatment (between time points A and B) and in the other 5 cases, the observed changes were found after therapy (between time points A and C or between B and C). One remarkable case with genome evolution exhibited two subclones sharing trisomies 12 and 19, but with several unique CNA confined to each subclone. The first subclone was characterized by deletions of 6q, RB1, MIR16-1/MIR15A and 3 other losses, while the second subclone showed homozygous deletion of MIR16–1/MIR15A and 5 other monoallelic deletions. The first subclone was predominant at time points A and B (60–70% of cells), but was present in only 10–20% of cells at time point C as confirmed by FISH. Conversely, the second subclone was observed in ~20% of cells at time points A and B and became predominant after therapy, found in ~80% of cells at time point C. Another case was characterized by deletion 11q32 (including ATM and others) as the sole abnormality at time point B. Significant genomic complexity was observed at time point C, including deletions of 11q32, 9p21 (CDKN2A), 9q12-q33, 14q13.2 (NFKBIA) and 17p (TP53), and gains of 2p16 (REL) and 9q34. Interestingly, the deletion 11q32 from both time points arose independently at each time point, as they exhibited different chromosomal breakpoints and copy number variants. Moreover, the other CNA found at relapse were not identified at diagnosis (confirmed by aCGH and FISH). For evolution of specific CNA, trisomy 12 was found in 5 cases at the first sample analyzed and was stable with no changes between time points. The frequency of deletions 13q14.3 (MIR16-1/MIR15A) and 17p increased at the later time points. Conversely, –6q decreased in frequency across time points (3 cases in time points A–B and 1 case in time point C). In summary, at least 35% of CLL patients exhibited clonal evolution and at least 9% showed evidence of multiple subclones. This subgroup of CLL patients provides an exceptional framework for comprehensive analysis of genome evolution during disease progression before and after therapy. Our observations also support the hypothesis of a common CLL progenitor cell can give rise to clonally related, but genetically evolving subpopulations of tumor cells. Finally, this study may bring novel information regarding the drug resistance pathways utilized by CLL B cell clones post therapy. Disclosures: Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding. Fonseca:Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

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 Introduction: Ibrutinib has shown remarkable safety and efficacy in CLL. Due to CYP3A4 (3A4) metabolism, the concurrent use of 3A4 inhibitors/inducers with ibrutinib should be avoided. Ibrutinib is also associated with bleeding complications, in part due to effects on collagen-mediated platelet aggregation, thus caution is advised with the simultaneous use of anticoagulant (AC) and antiplatelet agents. It is unknown what proportion of CLL patients starting ibrutinib therapy in routine practice are on these medications. We conducted a retrospective study to evaluate these aspects in a large cohort of CLL pts treated with ibrutinib outside the context of clinical trials. Methods: Following IRB approval, consecutive CLL pts who initiated ibrutinib off-protocol at Mayo Clinic, Rochester, MN from November 2013 through July 2015 were evaluated. A medication review for drug-drug interactions was performed by a pharmacist on all pts. Baseline characteristics, concomitant medications, and toxicity were recorded. Time to toxicity and ibrutinib discontinuation were analyzed by Kaplan Meier and cumulative incidence methods accounting for competing risk of death, respectively. Results: Ninety-six CLL pts started ibrutinib. Median age was 66 years and 64 (67%) were male. Ibrutinib indications included: relapsed/refractory CLL (n=84), treatment-naïve CLL with del17p (n=8), and CLL with Richter's transformation (n=4). Approximately 80% had unmutated IGHV and 45% had del17p or del11q on FISH. Upon initial pharmacy consult prior to ibrutinib, the median number of concomitant medications was 9 (range, 1-31). Sixty (63%) pts were taking concurrent medications increasing their risk of ibrutinib toxicity and 4 (4%) pts were on drugs potentially reducing ibrutinib efficacy. At ibrutinib initiation, 16 (17%) pts were on concomitant 3A4 inhibitors. This included 9 (9%) on moderate 3A4 inhibitors (fluconazole, diltiazem, imatinib, cyclosporine) and 7 (7%) on strong 3A4 inhibitors (voriconazole, posaconazole, clarithromycin, itraconazole). In 4 pts, the 3A4 inhibitor was switched to another agent or discontinued to allow standard 420 mg daily dosing. Ibrutinib was initiated at 140 mg daily in 7 pts on moderate 3A4 inhibitors and at 140 mg every 48 hours in 5 pts on strong 3A4 inhibitors. Four (4%) pts were on strong 3A4 inducers (carbamazepine, rifampin, rifabutin) at the initiation of ibrutinib. The 3A4 inducer was discontinued prior to ibrutinib start in 3 pts, while the initial ibrutinib dose was decreased to 140 mg every 48 hours in the remaining patient (also on 2 strong 3A4 inhibitors). During the course of ibrutinib, an additional 8 (8%) pts were started on 3A4 inhibitors/inducers which necessitated dose modifications. Upon commencing ibrutinib, 9 (9%) pts were on concomitant AC (6 warfarin, 3 enoxaparin). Due to significant bleeding risk, warfarin was switched to enoxaparin in 2 pts, to aspirin (ASA) in 1 and warfarin was discontinued in 1. In 2 pts, an alternative AC could not be used so ibrutinib was begun at 140 mg daily and titrated upward. Twenty-nine (30%) pts were on ASA (3 [3%] also on clopidogrel) at ibrutinib initiation. Nineteen (20%) pts were on selective serotonin release inhibitors (SSRIs), and 9 (9%) were on NSAIDs. Thirteen (13%) pts were on fish oil and 24 (25%) were on herbal medications; all of which were discontinued prior to starting ibrutinib. Ten (10%) pts had clinically significant bleeding on ibrutinib including 4 (4%) requiring hospitalization (1 subdural hematoma). Of these 4 pts, 1 was on enoxaparin, 2 were on a SSRI and 1 a NSAID. Six pts had minor bleeding necessitating a dose reduction of ibrutinib to 140-280 mg daily. After 16 months follow-up, the risk of bleeding was 21% (95% CI: 1-37%, Figure). After a median follow-up of 7.6 months, 73 (76%) pts remain on ibrutinib. There was no difference in rates of discontinuation of ibrutinib between patients who were on 3A4 inhibitors/inducers versus those who were not. Conclusion: In the "real-world" setting, 2 out of 3 CLL patients commencing ibrutinib therapy is on a concomitant medication with a potentially clinically significant drug-drug interaction with ibrutinib. These findings have implications for the practicing hematologist who must maintain a high degree of vigilance when prescribing ibrutinib to CLL pts. We highly recommend a formal medication review by a clinical pharmacist in all patients initiating ibrutinib. Figure 1. Figure 1. Disclosures Ding: Merck: Research Funding. Kay:Hospira: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shanafelt:Glaxo-Smith_Kline: Research Funding; Pharmactckucs: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Cephalon: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding.

Motexafin gadolinium (MGd, Xcytrin®) is a novel anti-cancer drug that selectively concentrates in cancer cells and disrupts redox dependent pathways by targeting oxidative stress related proteins such as thioredoxin reductase and metallothioneins. MGd has been shown to generate reactive oxygen species and induce apoptosis in tumor cells. We initiated a study to evaluate the safety and efficacy of MGd in two B-cell malignancies, relapsed/refractory LG and relapsed/refractory CLL or CLL/small lymphocytic lymphoma (CLL/SLL). MGd was given intravenously (IV) at 6mg/kg/day for 3 days q 2 weeks for LG and 5mg/kg/day for 10 days q 3 weeks for CLL/SLL. Ten patients (pts) with LG (2 grade 1, 5 grade 2, 1 grade 3 follicular, 2 marginal zone), 2 pts with CLL/SLL and 1 pt with CLL have been enrolled. Median age was 64.5 yrs (range 44–77), median prior treatment regimens was 3.5 (range 1–6) and median time from diagnosis to treatment was 52 mo. Many LG pts had failed prior aggressive treatment regimens including: CHOP (4), ICE (1), HyperCVAD(1), auto-bone marrow transplant (1); all failed rituxamab and 6 failed Zevalin. CLL/SLL and CLL pts had failed rituxamab (3), fludarabine (2), and R-CHOP (1). MGd related adverse events included skin and urine discoloration (6 and 4 pts), diarrhea, nausea, vesiculobullous rash, peripheral neuropathy (3 pts each). MGd related ≥gr 3 adverse events included skin rash (3 pts), fatigue, neuropathy, photosensitivity (1 pt each). Importantly, no MGd-related myelosuppression was observed. In 12 evaluable pts, there have been three PRs of 2+, 5+ and 8 months duration (2 follicular, 1 CLL). Two pts had SD (follicular, SLL) one of these had resolution of lymphoma related autoimmune hemolytic anemia. Responses occurred after ≤2 cycles of therapy and were seen in patients after extensive prior treatment (mean 4.3 prior regimens, range 3–7). MGd is a non-myelosuppressive drug with single agent activity in LG and CLL/SLL.

Abstract Abstract 3606 Background: Cytogenetic abnormalities are important prognostic indicators in CLL. At Mayo Clinic, CLL FISH analyses are derived from buffy coat samples not purified for lymphocytes. Scoring is therefore performed on consecutive qualifying nuclei regardless of cell size, shape or morphology. This scoring may not reflect the percent of abnormal nuclei of the affected cell type, but rather the entire population of cells present in the sample. We sought to determine whether the percent abnormal nuclei in only the affected cell type (e.g. lymphocytes) of patients with CLL differed from that of the general cell population. Scoring only B-lymphocytes could increase the sensitivity of the test in patients with low B-cell counts, either early in their disease or after treatment. Cell sorting techniques could be used to reach this goal but can be expensive, labor intensive and add to completion time. We propose a “Poor Man's Cell Sorting” technique based on cell morphology when stained with 4',6-diamidino-2-phenylindole (DAPI). In CLL samples, both round and so called “lobed” cells are seen by DAPI staining but we hypothesize that the CLL B-cells typically present as perfectly round in shape (Fig. 1). Thus our hypothesis: if only the round cells are scored for a genetic defect, would this more accurately represent the malignant leukemic B-cell population and allow for enhanced disease status by FISH? Methods: After IRB approval, we identified 87 CLL patients (75% male; 25% female, mean age 62.4 y, range 43–89 y) in the Mayo Clinic CLL Database, who were seen at Mayo Clinic between March, 2002 - July, 2010. We selected patients known to have low level FISH abnormalities and who therefore should have a significant population of both lymphocytes and neutrophils. Selection criteria included <20×106 B-cells per microliter (by flow cytometry) of peripheral blood and ≤40% of interphase nuclei expressing a specific FISH abnormality. Although most of the 87 patients exhibited more than one FISH abnormality, we focused on only one FISH defect per patient for this study. The original specimen (slide or fixed cell pellet) was rescored for % abnormal nuclei among 100 consecutive round and 100 consecutive lobed (or multilobular) nuclei, using DAPI to identify nuclear architecture. These scores were compared to each other and to the original clinical FISH analysis (scored for 200 consecutive nuclei and not selected for nuclear morphology). Results: Among 87 cases, FISH signals were scored for 6q-(1), 11q-(9), +12(15), 13q-(58), 17p-(2) and 2 IGH rearrangements. In 86/87 cases, the abnormal lobed nuclei did not have FISH defects. One patient exhibited 35% +12 by clinical assay, 52% +12 in the round nuclei, and 11% by lobed nuclei. For all cases, the mean percentage of abnormal nuclei was greater in the round cells (46%) vs original scoring (23.6%). In 79/87 patients (91%), the % of abnormal nuclei was greater in the round cells vs original FISH (mean increase 24.3%; range 1.5–57%). One patient's score was the same in round cells vs original, and 7 patients exhibited fewer abnormal nuclei in the round cells vs original. For these 7 cases, the mean % abnormal nuclei was 5.6% for round nuclei vs 8.4% for original score (score differences ranged from 1 – 5.5%). Overall, by univariate regression analysis, round cells (p=0.0043) have a better correlation with % B-cells, ascertained by flow cytometry, than either the current clinical approach (p=0.0462) or the lobed-cell approach (p=0.4058). Discussion: In virtually all cases (99%), the abnormal FISH patterns were confined to the round nuclei, and the lobed nuclei were virtually always normal by FISH. For patients with <20×106 lymphocytes, selecting for round nuclei uniformly resulted in identification of a higher percentage of abnormal cells. Further studies, including comparison of our round nucleus approach to CLL FISH analysis to actual sorted B-lymphocyte cell selection and the association of this leukemic restricted estimation of abnormal FISH levels to clinical outcome are necessary. Given our findings, we believe that estimating FISH defects restricted to the leukemic B-cell population will become an important adjunct to cytogenetic analysis for patients with low lymphocyte counts, including those in clinical remission and those with minimal residual disease. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Over the last two decades several phenotypic, molecular, and chromosomal markers have been identified that are significantly associated with the prognosis of CLL patients. Therefore, clinicians managing CLL patients would benefit from a simplified prognostic index. Methods: We analyzed prospectively collected data from 337 Binet A CLL patients enrolled in Italian the O-CLL1-GISL protocol with the aim of developing scores capable of predicting treatment free survival (TFS). Factors independently associated with TFS were included in the prognostic indexes. To account for differences in the magnitude of the association between the individual independent factors and TFS, we assigned a weighted risk score to each factor based on ranges of their corresponding hazard ratios (HRs) (i.e., 1 point for HR 1.1-1.9; 2 points for HR 2.0-2.9, etc.). The total risk score was then calculated by the sum of the ratings of each factor on individual basis. Risk groups were identified combining risk categories with a non-statistically different TFS. Results: We developed two scores based on weighted multivariable models: the first included clinical and laboratory parameters [clinical score (c-score)], while the second was based on biological markers [biological score (b-score)] (Table 1). The c-score allowed to predict the TFS of patients through the combination of Rai stage, b2-microglobulin and absolute lymphocyte count (ALC), while the b-score predicted TFS by IGHV mutational status and CD38 expression. The c-score showed a C-statistic of 0.72, while the b-score was 0.67, although cases stratified according to the b-score showed a more specific mRNA/microRNA profile. When the two scores were forced in a multivariate analysis, both showed an independent predictive value on TFS with a similar HR, demonstrating their complementarity. Thus, we attempted to integrate the two scores performing a further multivariate analysis in which all parameters, significantly associated with TFS at univariate analysis, were tested (Table 1). ALC, Rai stage, b2-microglobulin together with IGHV mutational status, resulted independently associated with TFS. We constructed a weighted score [comprehensive score (co-score)], including all the above 4 variables, which allowed the identification of 3 different risk groups with significantly different TFS (Figure 1). The C-statistic of the g-score was 0.75, showing a better concordance than the other two scores. Moreover, its validity was externally validated in a series of 297 newly diagnosed Binet A CLL patients from the Mayo Clinic, USA. Conclusions: Using this multistep process and external validation, we developed a score with high discriminatory power and predictive significance on the individual patient level. Table 1. Univariate and multivariate Cox proportional Hazards Models Variable Univariate analysis Multivariate analysis Clinical model Biological model Comprehensive model HR (95% CI) P HR (95% CI) P score HR (95% CI) P score HR (95% CI) P score Age (years) <60/>60 1.12 (0.73-1.74) 0.59 - - - - - - - - - Sex Male/Female 0.93 (0.6-1.44) 0.93 - - - - - - - - - Rai stage 0/I-II 2.30 (1.47-3.50) <0.0001 2.13 (1.24-3.03) 0.004 0/2 - - - 1.76 (1.11-2.78) 0.015 0/1 ALC (109/L) <10/>10 3.43 (1.99-5.92) <0.0001 2.91 (1.85-5.20) <0.0001 0/2 - - - 2.70 (1.54-4.72) 0.001 0/2 b-2 microglobulin normal/elevated 3.04 (1.96-4.70) <0.0001 2.78 (1.79-4.30) <0.0001 0/2 - - - 2.65 (1.66-4.21) <0.0001 0/2 LDH normal/elevated 1.25 (0.57-2.71) 0.57 - - - - - - - - - CD38 negative/positive 3.22 (2.06-5.02) <0.0001 - - - 2.11 (1.15-3.16) 0.02 0/2 1.40 (0.80-2.42) 0.24 - ZAP-70 negative/positive 2.34 (1.51-3.61) <0.0001 - - - 1.21 (0.70-2.16) 0.485 - 1.0 (0.98-1.01) 0.72 - IGHV mutated/unmutated 3.57 (2.32-5.50) <0.0001 - - - 2.10 (1.12-3.90) 0.019 0/2 2.39 (1.27-4.50) 0.007 0/2 FISH risk low+int/high 2.93 (1.46-5.90) 0.002 - - - 1.65 (0.76-3.34) 0.216 - 1.80 (0.84-3.88) 0.13 - Abbreviations: ALC: absolute lymphocyte count; CI: confidential interval; HR: hazard ratio. Figure 1. TFS according to comprehensive progression risk score. Figure 1. TFS according to comprehensive progression risk score. Disclosures Shanafelt: Cephalon: Research Funding; Glaxo-Smith-Kline: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Jannsen: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding. Kay:Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Tolero Pharma: Research Funding; Pharmacyclics: Research Funding.