ALBERT

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: Introduction: Real-world survival for pts with MDS treated with HMAs is substantially inferior than the landmark AZA-001 trial (median OS, 11-16 and 24.5 months, respectively). The reasons behind this gap remain unclear. Clinical use of HMAs differs from traditional chemotherapy in that multiple HMA cycles are generally required before responses are observed, therapy is continued even in the presence of significant cytopenias, and therapy must be continued to sustain response. We hypothesized that practice at academic hospital setting or prior experience of providers in treating MDS pts (more specifically the number of pts initiated on HMAs) are positively associated with persistence in HMA therapy and improved OS. Methods: Using SEER-Medicare linked data, we identified MDS pts diagnosed in 2004-13. Inclusion criteria included being ≥66 years at diagnosis, continuous Medicare Parts A&B coverage, and ≥ 1 full HMA cycle (defined by 3-10 days of HMA use within 28 days). Pts were followed until death or December 31, 2014. We identified the physician associated with HMA initiation, and assessed that provider's prior experience in caring for MDS pts or in initiating HMAs in a 2-year lookback period. Only pts diagnosed with MDS and initiated HMA in 2006-2013 were included for analysis to allow 2-year lookback for volume calculation. MDS volume and HMA volume were categorized into dichotomous variables given their skewed distribution (0-14 vs. 15 or more for MDS volume, 0 vs. 1 or more for HMA volume). Provider type was defined based on claim type (hospital outpatient [HOPD] or community provider) and linked information on hospital characteristics as 1) HOPD, teaching or cancer center (academic) or 2) HOPD, neither teaching hospital nor cancer center (HOPD-nonacademic) and 3) community-based practice. We assessed HMA duration as number of cycles of HMA therapy, with cycles separated by a gap of 〉= 2 weeks without therapy. We calculated OS from the first day of HMA. Chi-squared tests were used to assess differences in proportion of pts receiving ≥4 HMA cycles, and proportion with 2-year survival across different levels of HMA volume, MDS volume, and provider types. Wilcoxon rank sum test and Kaplan-Meier (KM) log-rank test was used to examine distribution of HMA cycles and OS across different levels of volume and provider types. Finally, we used multivariate Cox proportional hazards model to analyze OS, and similarly multivariate logistic regression for duration of HMA treatment, both of which were adjusted for relevant covariates (see footnote of Figure 1 for covariates). Two-sided statistical tests were used with alpha=0.05. Results: We identified 2,128 eligible pts under the care of 1,157 providers with 2-year lookback period. Median age at diagnosis was 77 (interquartile range 72-82) years, and 90.2% of patients were white. Median number of HMA cycles was 4, and median OS was 10 months (95% CI: 10-11). Median MDS volume was 8 and 32.2% of pts' providers had at least 1 HMA initiation in the past 2 years. Pts treated by provider with MDS volume ≥15 or with HMA initiation volume ≥1 are more likely to receive ≥4 HMA cycles in unadjusted analyses (P=.03 and P=.002 respectively). Community providers had significantly higher percentage of ≥ 1 HMA initiation than either group of HOPD hospital providers (P

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: Disease-free survival (DFS) is short in patients ≥ age 60 or with secondary AML, adverse cytogenetics, or leukostasis at presentation. In such patients, median CR duration is ≤ 8 mos and only 20% achieve 1 yr DFS. Historically, maintenance therapy with low doses of cytotoxic chemotherapy has not prolonged DFS. We tested the hypothesis that Tipifarnib (T) might be active as maintenance therapy for adults with poor-risk AML in first CR after induction and consolidation therapies. Oral T 400 mg bid for 2/3 wks was begun median 2.4 mos (range 1.0–4.1) after start of final consolidation cycle and given for up to 48 wks (16 cycles) to 36 adults with median age 63 (range 27–82), secondary AML 31%, adverse cytogenetics 47%, leukostasis 17%, ≥ 2 risk factors 40%. T was well-tolerated, with only 4 of 36 unable to complete 2 cycles because of constitutional symptoms (1 rash, 3 non-compliant). To date, 256 cycles have been administered (median 8 per pt, range 1–16), with hospitalization required during only 4 (1.5%) cycles (infection 3, bowel obstruction 1). Dose reductions for myelosuppression (400 mg bid to 300 mg bid) occurred in 17/32 (53%) by cycle 3, and 2 (6%) needed platelet transfusions. A total of 9 patients completed all planned 16 cycles of T with a median CR duration of 24 mos (range 15–36+). Five of the 9 remain in continuous CR (CCR) 19+-36+ mos, median 26+), compared with 4 who relapsed after CR of 15–24 mos (median 22). There are 8 additional pts in CCR and still receiving T (2–12 cycles) with CCR 4+-12+ mos. A total of 15 patients progressed while on T at median 6.5 mos CR (range 3.5–12). Median CR duration for all patients is 10+ mos (range 3.5–36+), with 88% ≥ 6 mos and 48% ≥ 12 mos. In 13 “comparable” poor-risk pts (age ≥ 60 46%, secondary AML 25%, adverse cytogenetics 46%, leukostasis 23%) who were eligible for but declined T, 4 are in unmaintained CCR at 14+-25+ mos, 9 have relapsed at median 7.5 mos (5–13 mos). Treatment with T did not have a negative impact on reinduction chemotherapy at relapse, as 6 of 9 patients achieved second CR. Administration of T in CR after induction and consolidation therapy has low toxicity and is associated with prolonged DFS in some adults with poor-risk AML. Phase 3 studies are warranted in this patient population.

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: Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15INK4B, CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34+ population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage–associated variant histone γ-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination “epigenetic” therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.

Description: Background: Loss of the long arm of chromosome (Ch) 5 or complete loss of Ch 5 is frequent in de novo MDS and AML. Epigenetic modifications of tumor suppressor genes, including aberrant DNA methylation, may play an important role in the progression of MDS and AML. Cell signaling is regulated by a-catenin, which forms a trimolecular complex with E-cadherin (ECAD) and b-catenin that links with actin-containing filaments of the cytoskeleton. Studies have reported reduced expression of a-catenin in MDS and AML patients with 5q deletion as compared to those without 5q deletion. Whether promoter methylation of the a-catenin gene is responsible for this decreased expression is controversial. To explore the potential role of a-catenin in the pathogenesis of AML transformation, we (a) determined the tumor specificity of methylation in AML and non- myeloid malignancies and (b) frequency of methylation in patients with −5/del(5q) MDS/AML and those with normal cytogenetics; (c) performed bioinformatics and experimental analysis of the a-catenin adhesion complex and local 5q31.1 genes to investigate mechanisms of epigenetic silencing; and (d) performed a detailed analysis of primary AML samples correlating promoter methylation, a-catenin expression, and chromatin conformation. Methods and Results: Using methylation sensitive PCR, we found that methylation of the a-catenin promoter gene was specific for myeloid malignancy. No methylation was observed in 19 acute lymphocytic leukemia cases, 20 chronic myelogenous leukemia cases, or in 99 primary gastric and esophageal samples where a-catenin has been implicated as a tumor suppressor gene. In those patients with AML and an associated 5q deletion the frequency was 31% (8/26) as compared to those without a 5q deletion with 13% (16/120). Bioinformatics analyses of the Valk et al., 2004 leukemia database provide supportive data for under expression of a-catenin in non-5/del (5q) AML cases. We quantitated a-catenin mRNA expression by Q-PCR in our cohort. Expression was lowest in AML patients with a-catenin methylation (n=9), but also in a subset of patients without promoter methylation (n=17), suggesting alternative mechanisms of inactivation. In contrast to AML, methylation of a-catenin was rare in myelodysplastic syndrome (MDS). Although p15 was methylated in over 50% of these cases as a positive control, only 2/18 MDS cases with 5q deletion (11%) and 1/13 MDS cases with 5q intact (8%) were methylated at the a-catenin promoter. The three positive cases were RAEB-2 (1) or RAEB-t (2), suggesting that a-catenin methylation may be most important in promoting transformation from MDS to AML. To explain a potential lack of correlation of methylation with decreased a-catenin in MDS and AML, we investigated a-catenin chromatin in a myeloid stem cell progression model and in primary AML samples. We performed chromatin immunoprecipitation on the CTNNA1 promoter using two active chromatin histone marks, H3K9Ac and H3K4me2 and two inactive marks, H3K9me2 and H3K27me3. In cell lines and primary leukemia samples where a-catenin was highly expressed (N=4), activation marks were present and repression marks absent. In cell lines and primary samples with low a-catenin expression and methylation of the promoter (N=4), the opposite pattern was observed. So called “bivalent” chromatin with mixed marks, no a-catenin methylation, and intermediated mRNA expression levels were observed with 7 additional cases (2 cell lines, 5 primary AML). Conclusions: Our data indicate that methylation of a-catenin is common in AML patients with 5q deletion but also observed in cases with normal chromosome 5 copy number. We propose a model of progressive inactivation of the a-catenin locus with AML transformation, with methylation representing a late stage event. The tissue-specificity of our results and in vivo chromatin observations in primary AML samples have implications for the timing and combinatorial therapy of MDS/AML with HDAC inhibitors and methylation inhibitors. Additionally, it appears that inactivation of adhesion molecules (ECAD, a-catenin) are frequent events overall in AML, suggesting a new pathway of transformation from MDS to AML.

Description: Bone marrow (BM) is the accepted source for monitoring post-therapy minimal residual disease (MRD) in APL. PB is easier and less painful to obtain but variable, sometimes contradictory evidence has been presented for its efficacy in MRD monitoring. In this study, PB vs BM monitoring, as well as conventional, qualitative RT-PCR (C-PCR) vs Q-PCR were directly and prospectively compared for their effectiveness as part of an intensive MRD monitoring schedule applied as a safety measure to an investigative Phase II trial (J0422) designed to test the efficacy of minimizing chemotherapy exposure and treatment duration. This trial consisted of one cycle of induction with all-trans retinoic acid (ATRA) and daunorubicin, followed by consolidation with single-agent arsenic trioxide (ATO), followed by a maintenance phase of intermittent ATRA alone or with 6-mercaptopurine and methotrexate for patients (pts) with a presenting white blood cell count (WBC) of 〉10,000 WBC/uL. The MRD monitoring schedule was as follows: BM and PB after the induction and consolidation treatment modules (modules 1 & 2), then, PB every month and BM every 3 months during 2 years of maintenance therapy. C-PCR and Q-PCR were performed according to published procedures for monitoring the APL-specific fusion gene PML-RARα by the BIOMED-1 Concerted Action and the North American Cooperative Oncology Groups, respectively. Criteria for positive assays were: C-PCR, confirmed visualization of an appropriate-sized gel band after conventional, double-nested PCR amplification; Q-PCR, demonstration of a CT value 12 mo, suggesting continued reduction of MRD during first 12 mo of maintenance therapy. No C-PCR assays were positive beyond module-1. In 1 exceptional pt, excluded from the above maintenance analysis, the Q-PCR assays became recurrently positive in BM and/or PB after 6 mo maintenance at a level below the criterion for molecular relapse (normalized quotient relative to the housekeeping gene GAPDH, NQGAPDH, ≥10−5). After 18 mo, the C-PCR became repeatedly positive in PB but not BM with Q-PCRs positive (2 BM & PB, 1 PB-only at shared checkpoints) in the NQGAPDH 4×10−7 to 4×10−6 range, which was associated with relapse in the central nervous system but not the BM. These results indicate that molecular monitoring of PB or BM was equally effective in detecting MRD and that Q-PCR was a more critical measure of MRD than C-PCR on protocol J0422 after single-cycle ATO-based consolidation therapy. The results further suggest that PB monitoring may be more effective in detecting extramedullary relapse, a relatively increasing cause of disease relapse with improved overall therapy for APL.