ALBERT

Description: BACKGROUND: Despite recent advances, outcomes remain poor in secondary and relapsed/refractory (R/R) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We previously demonstrated that combination thioguanine (6TG) and decitabine restores therapeutic efficacy in vitro in R/R AML (O'Dwyer K et al. Blood 2010;114:2657). Based on these data, we completed a Phase I dose-escalation study of 6TG with decitabine in advanced myeloid malignancies with an overall response rate (ORR) of 67% and median progression-free survival (PFS) of 42 weeks in responding patients (Lee DJ, et al. Blood 2016;128:2816). Moreover, 5 of the 6 study participants who had previously received a hypomethylating agent (HMA) responded, suggesting that 6TG in combination with decitabine can overcome HMA resistance. Because of these results, a cohort of patients with high-risk and R/R AML and MDS were treated with combination 6TG and decitabine at Columbia University Irving Medical Center, and we now report our clinical experience with this regimen. METHODS: A retrospective chart review was performed of all adult patients at our institution with advanced myeloid malignancies who had received at least one cycle of 6TG and decitabine between 2013-2017. The treatment regimen utilized the maximum tolerated dose of 6TG identified in the phase I trial. Up to 2 cycles of induction with 6TG 80 mg/m2/day PO in 2 divided doses was given on Days 1-12. Decitabine 20 mg/m2 IV was given on Days 3-12. Following this, maintenance cycles consisted of 6TG on Days 1-7 and decitabine on Days 3-7 at the same doses. Treatment was continued until the time of hematopoietic stem cell transplant (HSCT), disease progression, or toxicity. The primary objective of this retrospective analysis was to determine the ORR. Secondary objectives were to evaluate PFS and overall survival (OS) in this population. RESULTS: Forty-two patients were identified, 55% of whom were female, with a median age of 68 years (range, 23-83 years). Thirty-three percent of the patients had secondary AML; 62% had R/R AML; 2% had R/R MDS; and 2% had chronic myelomonocytic leukemia. By ELN genetic risk stratification, 38% were adverse-risk, 43% were intermediate, and 17% were favorable, while 2% were unknown. However, 36 (86%) patients failed prior therapy, suggesting that nearly all of the treated patients were poor-risk. Nineteen (45%) patients had received prior HMA therapy, and 6 (14%) had undergone previous HSCT. Patients received a median of 2 cycles of therapy (range, 1-10). Five (12%) patients achieved complete remission (CR), 7 (16%) obtained a CR with incomplete count recovery (CRi/p), and 2 (5%) had morphologic leukemia-free state (MLFS), for an ORR of 33% (CR+CRi/p+MLFS). One notable patient who achieved CR was a 73-year-old with secondary AML, complex cytogenetics, and a TP53 mutation who subsequently went on to HSCT. Another 5 (36%) responders had complex karyotypes or poor-risk genetics. Ten of the 14 responders (71%) failed prior therapy. Of the 19 who had received prior HMA therapy, 6 (32%) responded. Six of 14 (43%) responders proceeded to HSCT. Two additional patients had significant blast reduction and underwent HSCT. Responses and maximum blast reduction were obtained after two cycles of therapy. For the responders, the median PFS was 38 weeks (range, 11-not reached), while the median OS was 43 weeks (range, 11-not reached). CONCLUSIONS: Combined 6TG and decitabine is a highly active regimen in advanced myeloid malignancies, with an ORR of 33% in this cohort, and median PFS and OS of 38 and 43 weeks, respectively, in responding patients. This compares favorably to the expected response rate of 10-20% with decitabine monotherapy in this poor-risk population consisting of mostly secondary and R/R AML. These data also confirm our prior phase I study findings that the addition of 6TG to decitabine can overcome prior HMA resistance, with 32% of those who had previously failed HMA therapy responding. This combination remains a viable therapeutic option for elderly and unfit patients with high-risk and R/R AML who cannot tolerate intensive chemotherapy. Disclosures Heaney: BMS: Research Funding; Partner Therapeutics: Consultancy; Roche: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; AbbVie: Consultancy; Deciphera: Research Funding; Constellation: Research Funding; Blueprint: Research Funding; CTI: Research Funding. Lamanna:Ming: Research Funding; Celgene: Consultancy; Oncternal: Research Funding; TG Therapeutics: Research Funding; Infinity/ Verastem: Research Funding. Jurcic:Syros Pharmaceuticals: Research Funding; Astellas: Research Funding; AbbVie Inc: Consultancy; Daiichi Sankyo: Research Funding; Celgene: Consultancy, Research Funding; Incyte: Consultancy; Kura Oncology: Research Funding; Forma Therapeutics: Research Funding; Genentech: Research Funding; Novartis: Consultancy; Actinium Pharmaceuticals: Research Funding. Frattini:Celgene: Employment, Equity Ownership; Lin Bioscience: Consultancy. Lee:Abbvie: Research Funding; Roche: Research Funding; Tolero: Research Funding; Forty Seven, Inc.: Research Funding; Bayer: Research Funding.

Description: Background: Disease-specific measures of quality of life (QOL) can allow for improved assessment of disease-specific symptomatology and psychosocial factors. We recently reported on the development of the QUALMS, a 33-item QOL assessment tool for patients with myelodysplastic syndromes (MDS). We now report preliminary internal consistency and validity results from an international prospective study. Methods: From December of 2013 to July of 2014,an international cohort of MDS patients completed the QUALMS as well as the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and the Functional Assessment of Cancer Therapy Anemia Scale (FACT-An). Eligible patients were 18 or older, and had to have biopsy-proven MDS; those who had undergone stem cell transplantation were excluded. Baseline medical record review was performed at the time of enrollment to document key clinical and laboratory data, including bone marrow pathology and treatments; a second QUALMS administration and medical record review is planned for each patient to assess responsiveness. Participants were recruited from MDS centers across the United States, Canada and Italy. Individual QUALMS items were scored on a 5-point scale ranging from “Never” to “Always”. Overall mean score was calculated by transforming the raw mean to a 100 point scale, with higher scores indicating better MDS-related QOL. Baseline QUALMS scores were compared to clinical factors such as hemoglobin (Hg) and transfusion dependence as well as with scores on the other QOL scales. Preliminary exploratory factor analysis was also undertaken to identify candidate subscales. Results: As of this analysis, 201 patients had enrolled. The mean age was 71.7 years; there were 55% men, and the IPSS distribution was 44% LO, 43% INT-1, 10% INT-2, 1% HI and 2% unclassifiable. The majority of patients (53%) were receiving an erythropoiesis-stimulating agent, hypomethylating agent or lenalidomide, and 29% of the overall cohort was transfusion-dependent. The geographical distribution was as follows: 20% from the Dana-Farber Cancer Institute (Boston, MA); 9% from Columbia University (New York, NY); 15% from the Moffitt Cancer Center (Tampa, FL); 25% from the Odette Cancer Center (Toronto, Canada); and 31% from two GIMEMA hospitals (Rome and Sardegna, Italy). Scores on the QUALMS ranged from 19 to 78; the mean and median scores were 49.6 and 50.0 respectively. The measure had excellent internal consistency (α=.91), and was moderately correlated with the EORTC QLQ-C30: correlations (r’s) with the global health status scale, functional scales (i.e., physical, role, emotional, cognitive and social) as well as fatigue, nausea and pain scales ranged from 0.33 to 0.63 (all p’s

Description: Abstract 3556 Introduction: Posaconazole has been shown to be effective in reducing the incidence of invasive fungal infections (IFI) in high-risk, neutropenic leukemia patients following induction chemotherapy. Its clinical utilization, however, may be limited by decreased oral absorption secondary to mucositis and other adverse events that occur during therapy. Micafungin is a broad spectrum antifungal agent approved for prophylaxis in stem cell transplant recipients. Nonetheless, its safety and efficacy in neutropenic leukemia patients has not been established. We therefore designed a prospective randomized study comparing micafungin to posaconazole as anti-fungal prophylaxis in patients with acute leukemia undergoing induction chemotherapy. The aim of this interim analysis is to compare (1) the total time on study drug between the micafungin and posaconazole treatment groups and (2) the percentage of patients completing the planned treatment course between the two groups. Methods: Patients 〉 18 years of age were eligible for study with newly diagnosed acute leukemia (either AML or ALL) or MDS who were to receive myeloablative therapy with an anticipated neutropenic period of at least 7 days following anthracycline-based chemotherapy. Patients with first relapse of AML and ALL were also eligible. Patients who were pregnant, unable to swallow, hypersensitive to azoles, taking medications known to interact with posaconazole, and those receiving dasatinib were excluded from the study. Patients with a history of invasive fungal infection requiring therapy within 30 days of randomization, prolonged QTc interval on EKG, and moderate or severe liver dysfunction were also excluded. Participants were randomized to receive micafungin (100 mg IV daily) or posaconazole (400 mg orally twice daily). The absolute neutrophil count (ANC) was monitored daily. Neutrophil recovery was defined as ANC ≥500 cells/μl for two consecutive days. Prophylaxis completion was defined as continuation of treatment until neutrophil recovery or 28 days after treatment initiation, whichever was earlier. Prophylaxis failure was defined as discontinuation of study drug due to adverse event, persistent neutropenic fever requiring change to an alternative antifungal agent, or withdrawal from the study before 28 days. Fungal markers (Galactomannan, BD glucans) were followed weekly from randomization to neutrophil recovery. Continuous and categorical variables were tested with Wilcoxon rank-sum test and chi-square tests, respectively. Kaplan-Mayer method was used to measure the completion rates of anti-fungal prophylaxis, and the log-rank test was used to compare the completion between two groups. Results: From March 2011 to May 2012, 46 patients were randomized to prophylaxis with either micafungin (n=23) or posaconazole (n=23). The two groups did not differ in age, sex, race, neutropenic duration, or liver dysfunction (p=0.35, 0.56, 0.36, 0.07 and 0.4366, respectively). Nineteen patients (83%) completed micafungin therapy while only eight (35%) completed posaconazole (p=0.001). Median number of days on prophylaxis was 18.2 days for the micafungin group and 14.8 days for the posaconazole group (p=0.0584). Eight patients (34.8%) were removed from study in the posaconazole group after developing colitis or being made NPO by the treating physician. There were no positive fungal markers during the study period. Nine patients (20%) underwent bronchoscopy because of new chest CT abnormalities while on study drug. Bronchoalveolar lavage was sent for routine cultures, cytology, Galactomannan, and pan-fungal PCR (University of Washington). Two patients (one from each treatment group) had IFI identified by PCR (Rhodotorula nothofagi and Cryptococcus species). Conclusions: For patients undergoing myeloablative therapy for the treatment of acute leukemia or MDS, micafungin prophylaxis is associated with a superior completion rate and longer time to failure of prophylaxis compared to posaconazole. In addition, there is no difference in breakthrough fungal infections between the two treatment groups. Micafungin as antifungal prophylaxis is well tolerated and appears as effective as posaconazole prophylaxis for leukemia patients with prolonged neutropenia from induction chemotherapy. Our ongoing clinical study will continue to assess the efficacy of micafungin in this patient population. Disclosures: No relevant conflicts of interest to declare.

Description: HuM195, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells and has activity against minimal residual disease. To enhance the potency of native HuM195 and avoid nonspecific cytotoxicity seen with β-emitting radioimmunoconjugates, the α-emitting radiometal 213Bi was conjugated to HuM195. The feasibility, safety, and antileukemic activity of therapy with 213Bi-HuM195 were shown in a phase I trial (Jurcic et al. Blood 2002). Because of the short-range (50–80 μm) and high linear energy transfer (8400 keV) of α particles, radioimmunotherapy with 213Bi is ideally suited for the treatment of residual disease. To determine the effects of 213Bi-HuM195 after partial cytoreduction with chemotherapy, we treated 25 patients (median age, 67 years; range, 49–80) with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-HuM195 in a phase I/II trial. Fourteen patients had relapsed or primary refractory AML; 5 patients had previously untreated de novo AML, and 6 patients had untreated secondary AML. Sixteen patients had intermediate-risk cytogenetics, and 9 had poor-risk cytogenetics. During the phase I portion of the study, cohorts of 3–6 patients were treated with 0.5, 0.75, 1, and 1.25 mCi/kg. At the 1.25 mCi/kg dose level, 2 of the 4 patients had dose-limiting myelosuppression (grade 4 leukopenia lasting ≥ 35 days) and 1 patient died of progressive pneumonia. Therefore, the maximum tolerated dose was determined to be 1 mCi/kg. No responses were seen at the first two dose levels. Seven of the 19 patients (37%) who received 1 mCi/kg (n=15) or 1.25 mCi/kg (n=4) responded. There were 2 CRs lasting 9 and 12 months; 3 CRp (CR with incomplete platelet recovery) lasting 1, 2, and 6 months and 2 PRs lasting 3 and 8 months. The median time from initiation of chemotherapy to recovery of leukocyte counts was 34 days (range, 21–59 days). Delayed count recovery was attributed to persistent leukemia in 6 patients. Neutropenic fever occurred in all patients, and 19 patients had documented infections. At the phase II dose level, two of the 15 patients died of progressive infections, and one had grade 4 hyperbilirubinemia. The most common extramedullary toxicities were transient, low-grade elevations in liver function tests (n=19) and serum creatinine (n=11). Thirteen patients had infusion-related reactions following the first injection of 213Bi-HuM195, typically characterized by reversible grade 1 or 2 fever and/or chills. One patient had orthostatic hypotension and syncope after the first antibody infusion associated with concomitant bacteremia. Sequential administration of cytarabine and 213Bi-HuM195 is tolerable and can produce complete remissions in patients with AML.

Description: HuM195, a humanized anti-CD33 antibody, targets myeloid leukemia cells and has modest activity alone against AML. To increase the antibody’s potency and allow single cell killing, but avoid the nonspecific cytotoxicity associated with β-emitting isotopes, the α-emitter bismuth-213 (213Bi) was initially conjugated to HuM195. In phase I and II trials, 213Bi-HuM195 was capable of inducing remissions in AML after partial cytoreduction with cytarabine. Therapeutic applications of 213Bi, however, are limited by its 46-minute half-life. The isotope generator, 225Ac, a radiometal which yields 4 α-emitting isotopes and has a 10-day half life, can be conjugated to a variety of antibodies using the bifunctional chelate DOTA-SCN. 225Ac-containing immunoconjugates can kill in vitro at radioactivity doses 1000 times lower than 213Bi analogs and prolong survival of animals in several xenograft models (McDevitt et al. Science 2001). We are conducting a first-in-man phase I dose escalation trial to determine the safety, pharmacology, and biological activity of such an in vivo isotope generator using 225Ac-HuM195. Seven patients (median age, 61 years; range, 46–77) with relapsed (n=3) or refractory (n=4) AML were treated to date. Three had poor-risk cytogenetics. Patients received a single infusion of 225Ac-HuM195 at doses of 0.5 (n=3), 1 (n=3), or 2 μCi/kg (n=1). Total administered activities of 225Ac ranged from 23–170 μCi, and HuM195 doses ranged from 1–1.9 mg. No acute toxicities were seen. One of 2 patients evaluable for neutropenia developed an ANC 33% of BM blasts in 4 patients at 4 weeks following treatment. One patient had 3% bone marrow blasts after therapy. This is the first study to show that targeted therapy with an in vivo α-particle generator is feasible in humans. 225Ac-HuM195 appears safe and has antileukemic activity. Accrual to this trial continues.

Description: The t(15;17) translocation in acute promyelocytic leukemia (APL) yields a PML/RAR-α fusion messenger RNA species that can be detected by reverse transcription–polymerase chain reaction (RT-PCR) amplification. Breakpoints within intron 3 of PML produce a short PML/RAR-α isoform, whereas breakpoints within intron 6 result in a longer form. Using RT-PCR, serial evaluations were performed on the bone marrow of 82 patients with APL (median follow-up, 〉 63 months) who received retinoic acid (RA) induction followed by postremission treatment with chemotherapy, RA, and biologic agents. Sixty-four patients attained a clinical complete remission and had at least 2 RT-PCR assays performed after completing therapy. Forty of 47 patients (85%) with newly diagnosed APL who were induced using RA had residual disease detectable by RT-PCR before additional therapy. After 3 cycles of consolidation therapy, residual disease was found in only 4 of 40 evaluable patients (10%). Among newly diagnosed patients who had 2 or more negative RT-PCR assays, only 3 of 41 (7%) had a relapse, whereas all 4 patients (100%) who had 2 or more positive results had a relapse. Among 63 newly diagnosed patients, those who expressed the short isoform appeared to have shorter disease-free and overall survival durations than patients who expressed the long isoform. These data indicate that 2 or more negative RT-PCR assays on bone marrow, performed at least 1 month apart after completing therapy, are strongly associated with long-term remissions. Conversely, a confirmed positive test is highly predictive of relapse.

Description: Introduction: Gilteritinib is a novel, potent, highly-selective oral fms-like tyrosine kinase 3 (FLT3)/AXL inhibitor. Once-daily single-agent gilteritinib doses of ≥80 mg/day elicited antileukemic responses in FLT3 mutation-positive (FLT3mut+) subjects with relapsed/refractory AML (Perl AE, et al. Lancet Oncol. 2017). We examined the safety/tolerability and antitumor activity of gilteritinib plus front-line intensive chemotherapy in newly diagnosed AML patients. Methods: This ongoing open-label, dose-escalation/expansion phase 1 study (NCT02236013) assesses the safety/tolerability and antitumor effects of gilteritinib combined with 7+3 induction and high-dose cytarabine consolidation, and as single-agent maintenance therapy in subjects aged ≥18 years with newly diagnosed AML (excluding core-binding factor translocations). Dose escalation followed a 3+3 design; successive cohorts of 3-6 subjects received 40, 80, 120, or 200 mg/day gilteritinib. Subjects received ≤2 cycles of a 7+3 induction regimen (cytarabine 100 mg/m2/day, Days 1-7 plus idarubicin 12 mg/m2/day, Days 1-3 [dose-escalation and dose-expansion cohorts], and once-daily gilteritinib on Days 4-17 [Schedule 1]). After completion of the dose-expansion cohort using Schedule 1, a new cohort of patients were enrolled. In this cohort of six patients, gilteritinib administration was changed to Days 8-21 (Schedule 2) in preparation for phase 3 studies and daunorubicin 90 mg/m2/day, administered on Days 1-3, was used as an alternative anthracycline to idarubicin. During consolidation, subjects received cytarabine (1.5 g/m2 every 12 hours, Days 1, 3, and 5) and once-daily gilteritinib (Days 1-14) at the induction dose for ≤3 cycles. Subjects in the dose-expansion cohort received gilteritinib at the recommended expansion dose established during dose escalation. Transplantation was allowed for responding subjects. After consolidation or transplantation with stable engraftment, subjects received maintenance therapy with once-daily gilteritinib (28-day cycles; ≤26 cycles). Results: As of July 2, 2018, 62 subjects have been enrolled; 60 are included in the safety analysis set. Most subjects were male (66.7%; median age, 59.5 years [range, 23-77]) and 32 (53.3%) had FLT3 mutations (FLT3-ITD, n=23). During dose-escalation, two subjects in the 40 mg/day cohort who had received gilteritinib on Days 1-14 experienced dose-limiting toxicities (DLTs; neutropenia, thrombocytopenia, decreased ejection fraction). After the gilteritinib induction schedule change, no more DLTs occurred at this dose. Two subjects in the 200 mg/day cohort experienced DLTs (neutropenia, neutropenic enterocolitis). The maximum tolerated dose and the recommended expansion dose were established at 120 mg/day. Grade ≥3 adverse events (AEs) in ≥10% of patients were febrile neutropenia (63.3%), thrombocytopenia (18.3%), decreased platelet count (16.7%), neutropenia (15.0%), bacteremia (10.0%), sepsis (10.0%), and decreased white blood cell count (10.0%). Serious drug-related AEs in 〉1 subject were febrile neutropenia (n=9), small intestinal obstruction, lung infection, sepsis, and decreased ejection fraction (all n=2). The end-of-treatment investigator-reported rate of composite complete remission (CRc) for response evaluable FLT3mut+ subjects receiving gilteritinib 120 mg on Schedule 1 (n=17) was 100%. The CRc rate in FLT3mut+ subjects receiving Schedule 2 induction with daunorubicin was also 100% (Table). Enrollment in the Schedule 2 cohort receiving idarubicin is ongoing; the two subjects in this cohort have not been assessed for response. Among subjects who received ≥80 mg/day gilteritinib (n=47), CRc rates for FLT3mut+ subjects were 88.9% (n=24/27). Median overall survival has not been reached. Median disease-free survival was 297 days (95% CI: 112, not reached). Assessment of minimum residual disease in FLT3-ITD patients using a next-generation sequencing-based assay is ongoing; results will be available at the time of presentation. Conclusions: Gilteritinib can be safely combined with intensive chemotherapy, and given as single-agent maintenance therapy in subjects with newly diagnosed AML. Treatment was well tolerated. High response rates were observed in FLT3mut+ subjects after treatment with either idarubicin or daunorubicin in combination with two different gilteritinib administration schedules. Disclosures Pratz: Millenium/Takeda: Research Funding; AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy. Altman:Astellas Pharma: Other; Agios: Other: Payment to the institution to conduct the trial ; Epizyme: Other: payment to the institution to conduct clinical trial work; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work; Cyclacel: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Genetech: Other: Payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Incyte: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; GSK: Other: payment to the institution to conduct clinical trial work; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Cruz:Takeda: Speakers Bureau. Jurcic:Kura Oncology: Research Funding; Forma Therapeutics: Research Funding; Celgene: Research Funding; Daiichi-Sankyo: Research Funding; Incyte: Consultancy; Actinium Pharmaceuticals, Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Research Funding; Genetech: Research Funding; AbbVie: Consultancy, Research Funding; Astellas: Research Funding. Lin:Jazz Pharmaceuticals: Honoraria. Perl:AbbVie: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy. Podoltsev:Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Research Funding; LAM Therapeutics: Research Funding; Astex Pharmaceuticals: Research Funding; Genentech: Research Funding; Boehringer Ingelheim: Research Funding; Sunesis Pharmaceuticals: Research Funding; Daiichi Snakyo: Research Funding; Celator: Research Funding; Celgene: Research Funding; Pfizer: Research Funding. Schiller:bluebird bio: Research Funding; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding. Liu:Astellas Pharma: Employment. Bahceci:Astellas Pharma: Employment.

Description: Abstract 3595 Background: Cytokines are posited to play a critical regulatory role on the survival of the B-cell neoplastic clone in Chronic Lymphocytic Leukemia (CLL). AIM: The primary goals of this study were 1) to define additional relevant cytokines, growth factors and chemokines in CLL pathophysiology and 2) to correlate abnormal cytokine levels with disease stage, relevant hematological data and multiple prognostic factors. METHODS: A novel bead-based protein array system was employed to simultaneously measure 38 proinflammatory cytokines in the sera of CLL patients (N=116) and healthy age and sex matched controls (N=30). These results were correlated with Rai stage, β2-microglobulin level, LDH, CD38 expression and cytogenetic abnormalities. RESULTS: We first confirmed previous observations that TNFα, IL-1α, IL-1RA, IL-10, sIL-2Rα, VEGF and sCD40 ligand are significantly elevated in patients with CLL as compared with healthy controls. Expanding on the current literature, we demonstrated perturbations in an additional 15 serum cytokines in affected individuals. Compared to healthy controls, CLL patients had an increase in serum levels of IL-3 (p=0.002), IL-7 (p=0.008), INF-2α (p

Description: Abstract 1992 The myeloproliferative neoplasms (MPN) can have a variable natural history. Polycythemia vera and essential thrombocythemia, in particular, are conditions that can extend over decades, but some patients have clinical progression to myelofibrosis or acute myeloid leukemia. As first articulated by Warburg, cancers are metabolically distinguished from normal tissues by the use of glycolysis under aerobic conditions. To metabolically characterize the blood cells of patients with myeloproliferative neoplasms, we measured the mitochondrial membrane potential using the cyanine dye, JC-1. In examining cells derived from the blood and/or marrow of 159 patients with primary myelofibrosis, polycythemia vera and essential thrombocythemia, we found that the mitochondrial membrane potential (FL2/FL1=electrochemical potential/mitochondrial mass) was elevated compared to the blood cells of normal individuals. Thirty five percent of patients with polycythemia vera and essential thrombocythemia had normal MMP. In contrast, 97% of patients with primary myelofibrosis, post-polycythemia myelofibrosis, post-essential thrombocythemia myelofibrosis and acute myeloid leukemia following an MPN had evidence of cell populations with higher mitochondrial membrane potential. Cells with distinctly higher mitochondrial membrane potential could be indentified in platelets and polymorphonuclear leukocytes; however the MMP of lymphocytes was normal, indicating that the alteration in metabolic state likely occurred in a multipotential myeloid stem cell. Cell populations were confirmed by co-staining with anti-CD19, -CD45, -GlycophorinA and -β3-integrin antibodies. Sequential analysis of patient samples found that the acquisition of higher mitochondrial membrane potential was stable and persistent over 2 years or more of follow up and that elevated membrane potential predisposed patients to disease progression. The balance of patients (65%) with ET had evidence of increased MMP suggesting the possibility of disease in an early state of evolution to a more aggressive condition. The increased MMP did not correlate with the presence of mutation in JAK2. These results indicate that clinically advanced MPN can be characterized by changes in mitochondrial physiology that might be identified non-invasively by flow cytometric staining with JC-1. In addition, the early nature of these changes may help to identify therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2482 Despite the advent of prognostic factors such as cytogenetics and IGH mutational status, the natural history of chronic lymphocytic leukemia (CLL) remains heterogenous and difficult to predict in individual patients. We used the carbocyanine dye, JC-1, to investigate mitochondrial membrane potential in patients with untreated, predominantly early stage CLL. We found that JC-1 staining separated CD5+/CD19+ CLL cells into two discrete populations, Low Red (R)/Green (G) and High R/G, which mainly differed with regard to JC-1 green fluorescence (530 nm) in all patients (Fig. 1). In evaluating CLL patients, we found substantial variation in the proportion of cells in the two populations, with a range of 1.1% to 96.7% (median=79.9%) in the High R/G population. CD19+ B-cells from normal individuals typically had few cells with high JC-1 red fluorescence and had a JC-1 staining pattern that was distinct from CLL patients. Electron microscopy revealed disorganized mitochondrial christae, particularly in patients with elevated High R/G cell populations, compared with mononuclear cells from normal donors. Consistent with the morphologic disorganization, we found that the two populations of CLL cells had differential survival in culture with pyruvate and glucose as primary energy sources; cells in the High R/G population had inferior survival (p=0.016), while cells in the Low R/G population had equal survival in pyruvate compared with glucose (Fig. 2). This result indicates that the CLL clonal cell population can be subdivided into cells able to use oxidative phosphorylation and those dependent on aerobic glycolysis fro metabolic energy needs. Interestingly, patient samples with High R/G cell populations 〉90% had better cell survival in glucose-containing medium than samples derived from patients with 〈 75% High R/G cells (p=0.0085). We found that the mononuclear cells from patients with CLL expressed the M2 isoform of pyruvate kinase, which has been linked to the regulated utilization of aerobic glycolysis. We assessed the outcome of 100 patients with untreated CLL with more than 1 month of follow up (median 36 months) after measuring the proportion of cells in the mononuclear fraction of the peripheral blood with in the High R/G population. The clinical characteristics of the patients at the time of JC-1 staining are characteristic of patients with early stage, untreated CLL. The population was divided into approximate quartiles based on the percentage of mononuclear cells in the High R/G population, and progression-free survival (PFS) was estimated within each quartile. Patients with 50% or fewer cells in the High R/G gate appeared to have an indolent natural history; an estimated 88% (95% CI: 73%, 99%) of the patients had no evidence of significant clinical progression at two years. In contrast, patients with more than 90% of cells in the gate with High R/G cells had rapid progression with an estimated PFS of 50% (95% CI: 31%–82%) at two years (Fig. 3). Patients with 50–90% cells in the High R/G gate had an intermediate disease course. There was strong evidence of a difference in survival distributions across the four JC-1 categories (log-rank p-value 〈 0.001). The percentage of cells in the High R/G gate provided prognostic information that was independent of good risk or poor risk cytogenetic changes. In summary, we found that the clonal, neoplastic cells in patients with CLL comprise discrete populations separated on their metabolic dependence on aerobic glycolysis and aerobic glycolysis. We also show that the percentage of cells utilizing glycolysis has a significant adverse impact on the natural history of patients with CLL. Aerobic glycolysis, the so-called Warburg effect, is a common and incompletely understood aspect of cancer biology. These data tie the acquisition of aerobic glycolysis to more aggressive cancer biology, identify an important new prognostic measure for CLL and could lead to the development of therapies that target metabolic differences in cancer cells. Disclosures: No relevant conflicts of interest to declare.