ALBERT

Description: Introduction Adult patients (pts) diagnosed with acute lymphocytic leukemia (ALL) are known to have a poor clinical outcome as compared to children. Studies report a 2 year event free survival of 30-40% for Philadelphia chromosome negative (Ph-) patients age 〉30 yrs and 17% for age 〉50 yrs. In order to improve outcome for adult ALL, agents that are effective, safe and associated with a low morbidity are needed. Clofarabine, a second generation purine nucleoside analog, has clinical activity as a single agent and in combination with cytosine arabinoside (ara-C) against refractory and relapsed ALL. Clofarabine exerts its cytotoxicity through multiple mechanisms of action, with major effects via inhibition of ribonucleotide reductase (RR) and DNA polymerase-alpha, and incorporation into DNA leading to DNA damage and activation of apoptotic pathways. Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing of tumor suppressor genes. HDAC inhibitors are shown to be apoptogenic in vitro for ALL cell lines and have received FDA approval for the treatment of CTCL and peripheral T cell lymphoma. Pre-treatment with entinostat has been shown to enhance the cytotoxic activity of fludarabine in leukemia cell lines in vitro (Maggio et al, Cancer Research 2004). Given the similarity of clofarabine to fludarabine, and its FDA approval for children with refractory ALL, the combination of entinostat with clofarabine was pursued. Methods A Phase I window of opportunity study using overlapping schedule of entinostat and clofarabine was used in adult pts with ALL (B precursor) or Acute Bilineage Leukemia (ABL). Pts were enrolled onto one of two arms; arm “A” received repeated cycles of entinostat-clofarabine every 21 days as long as there was evidence of response (CR, CRi, or PR following cycle 1) and pts on arm “B” received one cycle of entinostat-clofarabine prior to standard multi-agent chemotherapy. Entinostat was administered orally on day 1 and day 8 (with dose escalation from flat dosing of 4mg to 6mg to 8mg from cohort 1 to 3). Clofarabine was administered intravenously at a fixed dose for all dose cohorts at 10mg/m2 for 5 days (day 3-7). Adults 〉40 yrs with newly diagnosed, Ph- B-lineage ALL or ABL were eligible. Additionally, adults 〉 21 yrs with relapsed and refractory, Ph- ALL or ABL were eligible. Eligibility criteria included serum creatinine 〈 2.0 mg/dl, hepatic enzymes 〈 or = 2.5 ULN and bilirubin

Description: Key Points Hundreds of lineage-specific lncRNAs are expressed during mouse and human erythropoiesis. Most mouse erythroid lncRNAs are not expressed in human erythroblasts and vice versa, yet some appear to be functional.

Description: Abstract 4013 Background: In recent years, azacytidine (AZA) has become the standard of treatment for high risk myelodysplasia (MDS). The approved schedule of AZA uses a 75mg/m2/d s.c. regimen for 7 days based on the CALGB-9221 (Silverman, JCO 2002) and the AZA-001 studies (Fenaux, Lancet Oncol 2009). The clinical response rates after AZA have been extensively presented but there is only limited data on the rates of cytogenetic response (CyR). Based on the review of the literature, there are no specific cytogenetic data published on prospective trials. Methods: We based our analysis on a randomized phase 2 study from the US Leukemia Intergroup (E1905 study, NCT00313586) testing 10 days of AZA (50mg/m2/d s.c.) vs 10 days of AZA+ the histone deacetylase inhibitor entinostat (4 mg/m2/d PO days 3 and day 10). MDS, CMML, and AML with myelodysplasia-related changes were included. This analysis includes all patients with cytogenetic abnormalities (at baseline or acquired following treatment) with available cytogenetic follow-up (cycle 6). Of 150 patients, 70 demonstrated baseline cytogenetic abnormalities. To date, forty patients (27 MDS and 13 AML) were evaluable for both time points. Karyotypes were performed at local laboratories, and reviewed centrally (RPK and GH). Cytogenetic response was assessed using IWG 2000 (Cheson et al, Blood 2000) criteria. Results: The clinical response rate (CR+PR+ trilineage HI) according to IPSS cytogenetic risk stratification were of 20%, 33%, and 35% for favorable, intermediate and poor cytogenetic risk groups respectively (p=NS). Patients with Chr 7 abnormalities (i.e. -7 or -7q, n=18) had a response rate of 28% including 17% CR. Of patients with complete cytogenetic data, the rate of overall CyR was 52% (n=21): 22% (n=9) complete CyR, 30% (n=12) partial CyR. This represents a complete CyR of 13% and a partial CyR of 23% as a proportion of all treated patients with initial cytogenetic abnormalities (including those who did not receive six cycles of therapy). To date, confirmatory FISH analyses were available for 4 patients with CyR (2 CCyR and 2 PCyR). All four had complete clearance of their cytogenetic clone. Among the cytogenetic responders, 15 had MDS and 6 had AML (p=NS). CyR did not differ between the two treatment arms. CyR and clinical response were highly correlated (p

Description: Abstract 4456 Dasatinib is a highly effective oral TKI for CML at various stages of the disease. The most common non-hematologic side-effect is pleural effusion. The exact mechanism of this pathology remains to be fully elicited, although studies report it to be an immunological phenomenon. Current literature shows dasatinib alone to be the cause of such effusions. More recently, pulmonary hypertensions have been reported on dasatinib therapy. However, in our cohort of CML patients receiving dasatinib as a first or second line therapy, other pathologies were documented. Grade 2/3 unilateral pleural effusions were detected in 3 patients. Case 1. A 31 yr old male with CML-CP was on first line dasatinib 100 mg OD and CCyR was documented at 3 months. At 6 month, he was detected to have grade 1/2 bilateral pleural effusion and was managed with drug interruption and oral dexamethasone with no significant reduction of fluid. CBC was normal. Dasatinib was continued. At one year, he lost cytognetic response with emergence of complex karyotype and medullary lymphoid blast crisis was confirmed. Pleural fluid showed lymphoblasts proven by cytology and immunophenotype. He received vincristine and prednisone followed by a change to imatinib. His disease progressed further and died at 2 years from diagnosis. Case 2. A 39 yr old male with molecular relapse and M244V Abl Kd mutation at 8 years since diagnosis and 6.5 yrs on imatinib 400–800 mg daily was offered dasatinib 100 mg OD. After 24 months, he developed grade2 left pleural effusion which responded poorly to drug interruption, therapeutic tapping and prednisone. A few months later he developed constitutional symptoms and CT chest, bronchoscopy/BAL proved to have left lung tuberculosis. Currently, he is responding favorably to anti-tubercular therapy. He was switched back to imatinib and remains in cytogenetic remission. Case 3. A 21 yr old male with cytogenetic non-responder to imatinib 800 mg daily was put on dasatinib 100 mg OD. An MMR was achieved. He developed left pleural effusion after 4 years of dasatinib. Extensive investigation for any specific pathology was not productive. He was put on empiric anti-tubercular therapy with no response. It is being treated as a dasatinib induced effusion. Conclusions. During dasatinib therapy, the causes of pleural effusions could be varied – drug related, extramedullary blast crisis or tubercular. Radiological, cytological and microbiological investigations could be indicated after careful clinical evaluation. Disclosures: Saikia: Bristol -Myers Squibb: Research Funding, Speakers Bureau.

Description: Abstract 1337 Background: Oral azacitidine (CC-486) is bioavailable, biologically and clinically active, and well tolerated in patients (pts) with myelodysplastic syndromes (MDS) and acute myeloid leukemia (Garcia-Manero, J Clin Oncol, 2011). Because DNA methyltransferase (DNMT) inhibitors require active cell cycling to effect methylation reversal, prolonged administration of lower doses of azacitidine may provide more extensive reversal of DNA methylation compared with shorter administration. Purpose: We conducted analyses 1) to determine the pharmacokinetic (PK) and pharmacodynamic (PD; ie, DNA demethylating activity) profiles following subcutaneous (SC) AZA and various dosing schedules of oral azacitidine; 2) to assess the correlation between the PK and PD profiles of oral azacitidine administered in extended dosing schedules; and 3) to compare PD effects observed at different time points in the 28-day (d) treatment cycle with SC AZA or oral azacitidine administered for 7 days vs. the PD effects of 300mg QD oral azacitidine administered in extended dosing schedules in pts with MDS. Methods: This multicenter, phase 1 study had 2 parts. In Part 1, 41 pts with MDS, CMML, or AML received SC AZA (75mg/m2 QDx7d of a 28d cycle) for 1 cycle, then oral azacitidine doses ranging from 120 to 600mg (QDx7d of a 28d cycle) in subsequent cycles. In Part 2, 86 pts received oral azacitidine in 1 of 4 extended dosing schedules: 300mg QD or 200mg BID, each for 14d or 21d of repeated 28d cycles. PK parameters were derived from plasma concentrations. The correlation between PK and PD was determined using data from pts who had received oral azacitidine 200mg BID or 300mg QD for 14d or 21d for whom PD data were available at day 15 of the first 28d cycle (C1D15). The PD endpoint was reduction in percentage of highly methylated (≥70%) loci of DNA in whole blood, assessed using Illumina's Infinium Methylation27 Bead Array. Results: SC AZA or oral azacitidine were rapidly absorbed and reached Tmax (median [min, max]) within 0.5 hr [0.2, 1.1] and 1.0 hr [0.3, 3.6] post-dose, respectively. Mean elimination half-life was 1.5 +/− 0.7 hr and 0.6 +/− 0.2 hr for SC and oral azacitidine, respectively. No drug accumulation was noted following multiple dose administration. Compared with SC AZA, 300mg oral azacitidine QDx14d and QDx21d provided mean cumulative exposures (AUC) per cycle of 38% and 56%, respectively. A PK/PD correlation (AUC C1D1 vs. change in methylation on C1D15 compared with baseline) was observed with oral azacitidine 300mg QD or 200mg BID administered for 14d or 21d (r2=0.659, p

Description: Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.

Description: The t(1;19)(q23;p13) is one of the most common chromosome translocations in ALL. In 90–95% of ALL cases with a t(1;19), the 19p13.3 gene E2A is fused to PBX1 located at 1q23, producing E2A-PBX1 chimeric proteins that possess transforming properties. The molecular abnormalities present in the 5–10% of ALL cases with a t(1;19) but no E2A-PBX1 fusion are unknown. TS-2 is an ALL cell line with a t(1;19)(q23;p13.3) but no E2A-PBX1 fusion. We used fluoresence in situ hybridization to localize the chromosome 19 breakpoint in TS-2 to a region approximately 400 kilobases telomeric to E2A and found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 (deleted in azoospermia associated protein 1) to the 1q23 gene MEF2D (myocte enhancer factor 2D). We cloned and sequenced the fusion genes and found they encode for reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 fusion transcripts, both of which are expressed in TS-2. MEF2D is a member of the MEF2 family of DNA binding proteins, which were originally characterized as muscle-specific transcription factors that regulated transcription of genes involved in myogenic differentiation. MEF2 proteins are now recognized to have more diverse functions: they are transcriptional effectors of mitogenic signaling pathways and inflammation, play critical roles in calcium-regulated signaling pathways that mediate survival of neurons and T-lympocytes, and participate in neuronal plasticity. DAZAP1 is a protein with novel RNA binding properties that is expressed most abundantly in testis and to a lesser extent in thymus. MEF2D-DAZAP1 includes the MEF2D MADS (MCM1, agamous, deficiens, and serum response factor) box and adjacent MEF2D domain that mediate sequence-specific DNA binding and protein-protein interactions, as well as one of two MEF2D transcriptional activation domains (TAD) fused to the C-terminus of DAZAP1. The DAZAP1-MEF2D chimera includes an intact first and truncated second RNA recognition motif from DAZAP1 joined to the C-terminus of MEF2D that includes its second TAD. We performed electrophoretic mobility shift assays using cognate and mutant MEF2D DNA recognition sites and found that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable from that of native MEF2D. We found that MEF2D/DAZAP1 activated transcription of a luciferase reporter gene under control of MEF2D recognition elements with substantially more potency than did wild type MEF2D. We also show that DAZAP1/MEF2D proteins bind RNA in a sequence specific manner analogous to that of wild type DAZAP1. MEF2D has been identified as a candidate oncogene involved in development of leukemia/lymphoma via murine retroviral insertional mutagenesis studies. Our data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contributes to leukemogenesis by altering signaling pathways normally regulated by wild type MEF2D and DAZAP1.

Description: Despite its clinical activity in myelodysplastic syndrome (MDS), the relationship between the inhibition of DNA methyltransferase 1 (DNMT) by 5-azacitidine (5AC) and hematologic improvement has not been well-explored. Optimal in vitro re-expression of genes silenced through promoter methylation requires sequential exposure to a DNMT inhibitor (i) and an histone deacetylase (HDAC)i. In a dose/schedule exploration study, patients with MDS (including MDS-AML) were treated with various regimens of 5AC SQ, followed by a 7 day infusion of the HDACi sodium phenylbutyrate (PB). Patients received 5AC at the following doses: 50 mg/m2/day * 10 doses (cohort A, n = 8), 50 mg/m2/day * 14 doses (cohort B, n = 3), and 25 mg/m2/day * 14 doses (cohort C, n = 5). Patients received a minimum of 4 cycles (q28 days). Clinical responses were graded according to IWG criteria. Changes in promoter methylation of p15INK4B and E-Cadherin (E-CAD), the most commonly methylated genes in MDS and AML, were monitored using a real time-PCR modification of methylation specific-PCR (rtMSP). Changes in gene expression were monitored using real time rtPCR. Dose-limiting hematologic toxicity (myelosuppression 〉 14 days) occurred in 2/3 patients in cohort B. The other two dose schedules were well-tolerated. Clinical responses developed in 4/8 patients in cohort A (3 CR, 1 PR), 2/3 patients in cohort B (1 hematologic improvement (HI)-P, N, major, 1 HI-P-major), and 3/3 currently evaluable patients in cohort C (2 HI-N-major, 1 HI-P, major). p15 was methylated in 10/10 evaluable samples pre-treatment; E-CAD was methylated in 5/7. Treatment with 5AC decreased p15 methylation in 50% of patients studied. p15 expression increased in patients in whom methylation was decreased; this included 3/4 clinical responders studied. In several cases, reversal of methylation was confirmed by bisulfite sequencing (BSQ) of serial samples. BSQ suggested that 5AC treatment led to gradual demethylation of the clone, rather than replacement of a methylated clone with a normal unmethylated clone. Surprisingly, treatment with 5AC increased global acetylation of histones H3 and/or H4 (Western analysis) in 7/8 patients in cohort A, and 2/2 evaluable patients in cohort B, (cohort C data pending). Acetylation was further increased following PB administration in 4/8 patients in cohort A and 1/2 in cohort B. These data confirm for the first time that clinical administration of 5AC leads to substantial reversal of promoter methylation associated with gene re-expression. Administration of 5AC is also associated with induction of global histone acetylation; the mechanism underlying histone acetylation in response to 5AC is unclear. While administration of 5AC and PB is associated with re-expression of p15, the relative contributions of the DNMT and HDAC inhibitors cannot be determined from the present study. Gene methylation data obtained using rtMSP correlates well with BSQ. The use of this semi-quantitative technique to monitor larger studies of 5AC with and without HDAC inhibitors will facilitate determination of the relationship between reversal of promoter methylation of p15 and other genes and clinical response to DNMTis.

Description: Introduction Children and young adults with relapsed acute lymphoblastic leukemia (ALL) are at high risk for infectious complications, particularly invasive fungal infections, during their intensive re-induction therapy. We report results of a Pilot study investigating decitabine and vorinostat in combination with intensive re-induction chemotherapy for children and young adults with relapsed or refractory ALL. This study is currently open through the TACL Consortium [NCT01483690] and all patients and/or their parents or guardians signed informed consent to participate in this institutional review board approved therapeutic trial in accordance to the Declaration of Helsinki. Methods Patients 1-25 years of age with 2nd or greater relapse or refractory ALL are eligible. Seventeen patients have enrolled with a median age of 12 years (range, 19 months - 21 years). Patients received one course of therapy which included decitabine (15mg/m2 days 1-7; 15-21), vorinostat (180mg/m2 days 3-10; 17-24), vincristine (1.5mg/m2 days 10, 17, 24, 31), dexamethasone (10mg/m2/dose BID days 8-12; 22-26), mitoxantrone (10mg/m2 days 8, 9), PEG-asparaginase (2500 IU/m2 days 10, 24) and intrathecal methotrexate (dosed according to age and CNS status). Nine patients (53%) relapsed after a prior allogeneic hematopoietic cell transplant and 5 patients had refractory disease (29%) prior to enrolling on this study. Initial infection prophylaxis guidelines did not require anti-bacterial or anti-fungal therapy. Results The study was suspended after 5 patients enrolled due to 2 patients reporting a DLT [Grade 3 cholestasis/steatosis, Grade 4 bilirubin (n=1); Grade 3 delirium, Grade 4 seizure, Grade 4 somnolence (n=1)] and 4 of the 5 patients (80%) reporting non-albicans Candidemia [C. kruseii (n=2), C. lusitaniae (n=1), C. guillermondii (n=1)]. Based on this significant rate of fungal infection, despite all patients receiving prophylactic anti-fungal therapy (micafungin n=2, fluconazole n=1, amphotericin n=1), the study was amended to decrease the decitabine dose (15mg/m2 to 10mg/m2) and duration of decitabine (days 1-7; 15-21 to 1-5; 15-19) as well as require treatment dose non-azole class anti-fungal therapy (echinocandin or amphotericin) to be given to all patients on study. Twelve patients enrolled post-amendment; only 1 of 12 (8.3%) experienced a fungal infection (C. guillermondii). Of note, this infection occurred in a patient using fluconazole for prophylaxis in place of a protocol-specified agent. There have been no fungal infections reported to date for the 11 patients on study who have received echinocandin or amphotericin therapy at treatment doses. Treatment responses include 9 patients achieving a complete remission (CR) (53%), 5 with stable disease (29%), two treatment related deaths and 1 patient removed from protocol therapy on day 6 based on the physician’s decision. The median minimal residual disease (MRD) response in patients who achieved a CR and submitted bone marrow samples for end of therapy testing (n=5) was 0.056% (range, 0.00%-5%). Conclusions We report a substantial initial incidence of invasive Candida infections in patients treated with decitabine and vorinostat in combination with intensive chemotherapy despite anti-fungal prophylaxis which appears to be abrogated once the fungal prophylaxis was required at treatment doses using either an echinocandin or amphotericin class agent. As well, the lower dose and duration of decitabine may have contributed to this improvement, resulting in potentially shorter periods of neutropenia which may contribute to the risk of fungal disease. The mechanism for the unique propensity for non-albicans Candida infections in this study remains unclear. The initial response to this regimen incorporating epigenetic therapy is promising with correlative biology studies investigating methylation, acetylation and gene expression changes pending study completion. The study continues to accrue at a dose expansion cohort. Disclosures Off Label Use: Decitabine in relapse ALL Vorinostat in relapse ALL.

Description: Abstract 3815 ESAs are recommended by the National Comprehensive Cancer Network for low-risk MDS patients with symptomatic anemia who have low serum erythropoietin (Epo) levels and limited transfusion burden. Because ESAs are costly and involve repeated physician office visits, socioeconomically vulnerable patients may be less likely to receive them. The advent of reporting MDS to the Surveillance Epidemiology and End Results (SEER) registries as a cancer beginning in 2001 affords the ability to examine population-based utilization of therapies for this group of disorders. Medicare patients diagnosed with MDS from 2001–2005 were identified in SEER registries. Medicare claims provided detailed treatment-related data on ESA use. Bivariate analyses and multivariate logistic regressions examined the effects of patient demographic, socioeconomic, geographic and health status characteristics, measured during 12 months prior to MDS diagnosis, on the probability of receiving ESAs between the year prior to SEER-reported diagnosis and either death or censoring. Analyses examined all MDS patients, and a subset with lower-risk MDS (modified French-American-British categories of refractory anemia (RA), RA with sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), or del(5q) syndrome). The MDS sample included 7,385 patients, with 2,568, or 35%, identified as lower-risk. 66% of MDS patients (70% of lower-risk) received ESAs at some point during the observation period. Multivariate estimates indicated that ESA use rates were higher among patients with an FAB classification of RARS relative to RA (Odds ratio [OR] 1.27; 95% Confidence interval [CI] 1.02–1.57), a history of transfusions prior to MDS diagnosis (OR 1.82; CI 1.58–2.09), diagnosis in 2004 versus 2001 (OR 1.21; CI 1.02–1.43), and residence in the South (OR 1.36; CI 1.12–1.65) or West (OR 1.28; CI 1.06–1.56) compared to the Northeast. Lower ESA use rates were also associated with baseline health status measures, including a diagnosis of dementia (OR 0.62; CI 0.51–0.76), other severe mental illness (OR 0.54; CI 0.36–0.83), use of a wheelchair (OR 0.71; CI 0.58–0.88) or nursing home stay (OR 0.40; CI 0.30–0.53), and demographic and socioeconomic characteristics including age 85+ compared to age 65–69 years (OR 0.76; CI 0.61–0.95), black versus white (OR 0.77; CI 0.62–0.97), prior-year enrollment in Medicaid or Medicare Savings Programs (MSP) (OR 0.63; CI 0.53–0.75), and area percent of adults without any college education (OR 0.99; CI 0.98–1.00, p