ALBERT

Description: Flavopiridol has in vitro activity in CLL and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol administered using two different schedules has activity in CLL. Patients with previously treated CLL were enrolled on two sequentially performed phase II studies. Patients in the first trial received flavopiridol (50 mg/m2 daily) as a continuous infusion (CI) over 72-hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m2 as a 1-hour intravenous bolus (IVB) daily for three days repeated every 3 weeks. Patients received up to 6 (CI cohort) or 8 (IVB cohort) cycles of therapy. Fifteen patients enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate (Rai stage I or II) and 9 (60%) high (Rai stage III and IV) risk stages. No responses were noted in this group with 27% having stable disease (SD) and 73% progressive disease (PD). Thirty-six patients enrolled in the IVB study, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) SD, and 13 (36%) PD. The progression-free survivals for responders in the IVB study were 2.9, 3.2, 8.7, and 19.3 months. The median progression-free survival was 2.1 months (95% confidence interval [CI] 1.8 – 3.8) for patients in the CI study and 3.2 months (95% CI [2.5 – 7.4]) for the IVB study. The median overall survival was 27 months (95% CI [20–42]) for the CI study and 24 months (95% CI [18–31]) for the IVB study. Toxicity was manageable and included mainly myelosuppression (granulocytopenia and thrombocytopenia), infections, diarrhea and fatigue. Grade 3 and 4 toxicities were 20% and 27%, respectively, on the CI study and 39% and 33% on the IVB study. One patient on the IVB study had tumor lysis syndrome that was managed medically and did not require dialysis. There was one on-study death following a myocardial infarction on the IVB study. We conclude that flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.

Description: Recruitment of histone deacetylases and DNA hypermethylation of promoter regions of specific genes are two mechanisms of transcriptional repression and gene silencing which have been linked, and are implicated in differentiation block in AML. We hypothesized that the histone deacetylase inhibitor (HDI) depsipeptide could result in transcriptional de-repression, upregulation of specific target genes and differentiation of the leukemic clone in AML. Eighteen patients (pts), median age 60 years (range 25–77) with relapsed or refractory AML were enrolled on a multicenter Phase II study of depsipeptide in AML. Patients were stratified into 2 groups on study entry: Group A (n=14) included patients without specific chromosomal abnormalities known to recruit histone deacetylases. Group B (n=4) included patients with chromosomal aberrations such as the t(8;21), inv 16 and t(15;17) known to recruit histone deacetylases. Depsipeptide was administered intravenously at a dose of 18mg/m2/d on days 1, 8 and 15 of a 28 day cycle. Peripheral blood mononuclear cells were obtained prior to (hour 0), and after 4 (hr 4) and 24 hrs (hr 24), on days 1 and 8 of the first cycle of therapy for evaluation of histone acetylation by flow cytometry, and gene re-expression by REAL-time RT-PCR. Target genes of interest include MDR1, a target of HDI mediated upregulation, and p15INK4B (p15), a target of DNA hypermethylation in AML. MDR1 and p15 copy numbers are expressed as a normalized quotient of MDR1 and p15, respectively, to the housekeeping gene ABL. The drug has been well tolerated. The most common adverse effects noted included grade 1/2 nausea, vomiting and fatigue. No objective evidence of response (CR or PR) or other evidence of antileukemic activity has been seen in group A. In contrast, 2 of 4 pts (50%) in Group B, have had a disappearance of bone marrow blasts (blast percentage 〈 5%) in the setting of a normocellular marrow, with concomitant recovery of near-normal hematopoiesis following 1 and 2 cycles of therapy respectively. This anti-leukemic effect was short-lived, with both pts developing an increase in bone marrow blasts within 30 days of the initial response. Both of these patients also had translocations involving the AML1 gene {1 had t(8;21) and the other had a novel translocation t(4;21)}. Interestingly both of these responding pts and one other pt (75%) in cohort B demonstrated an increase in H3 acetylation at 4 and/or 24 hrs, in contrast to 4 of 14 pts (28%) in cohort A. There was an overall mean increase of 41% in MDR1 expression at hr 4 on days 1 and 8 (p=0.04). p15 expression was also upregulated at hr 4 on days 1 and 8 (91% mean increase, p=0.01). We conclude that the HDI, depsipeptide, may have anti-leukemic activity in specific cytogenetic subsets of AML known to recruit histone deacetylases, and this is associated with a concomitant increase in histone acetylation. In addition, upregulation of specific target genes occurred in patient derived mononuclear cells, following depsipeptide treatment. The study remains open to accrual for pts with specific chromosomal abnormalities known to recruit histone deacetylases.

Description: DNA hypermethylation of promoter-specific CpG islands is a well known mechanism of epigenetic silencing, and has been implicated in the pathogenesis and progression of disease in MMM. We are evaluating 5-aza-2′deoxycytidine (Decitabine), a potent DNA methyltransferase inhibitor in a Phase II trial in patients (pts) with MMM. Decitabine was administered subcutaneously at a dose of 0.3mg/kg/d on days 1–5, and days 8–12 and cycles were repeated every 6 weeks, in the absence of dose limiting toxicities. Response was determined every 12 weeks, and defined as an improvement in cytopenias and/or splenomegaly. Pts who had no response after 2 cycles were eligible for dose escalation to 0.4mg/kg/d. Elevated levels of circulating CD34+ cells are associated with advanced stage and evolution to the blast phase of the disease in MMM. Therefore, CD34+ cells were measured in peripheral blood at baseline, and at days 1, 5 and 12 of the first 2 cycles of therapy as a surrogate marker of disease activity. Seven pts (5 males, 2 females) have been enrolled, median age 71 (range 42–89), median baseline absolute CD34+ cell count 27× 106/L (11–4959), Dupriez score of 2, 1 and 0 in 72%, 14% and 14% respectively. Median number of cycles administered was 4 (range 1–7). Median WBC and platelet (plt) count at baseline were 3.6K/uL (range 1.5–29), and 188K/uL (range 62–446K/uL) and 3 pts were red cell transfusion dependent. Grade 4 neutropenia (ANC) occurred in all pts, and grade3/4 thrombocytopenia in 5 pts. Nadir ANC and plts occurred at a median of 31 days (range 24–44) and 23 days (range 17–31) respectively. Recovery to ANC 〉0.5K/uL and plts 〉50K/uL occurred at a median of 43 (range 35–58) and 26 days (23–36) respectively. Two pts required a dose reduction for prolonged myelosuppression, and in 1 pt the dose was escalated to 0.4mg/kg/d for lack of a response after 2 cycles. Two pts have developed febrile neutropenia; one of these pts had a documented infection. Grade 3–4 non-hematologic toxicities were rare and include a variceal bleed in a patient with baseline portal hypertension, occurring in the setting of a platelet count of 486K/uL. There have been no injection site reactions. Five pts are evaluable for response. Of these, two pts have had a response including 1 pt with a CR (normalization of blood counts including transfusion independence). One pt in the blast phase of the disease has had a hematological improvement as evidenced by a normalization in platelet counts (from 62K/uL to 200K/uL) associated with a significant decrease (from 2.58K/uL to 0.03K/uL) in peripheral circulating blasts. The other 3 pts have had stable disease; 2 of these remain on treatment and have received 4 and 7 cycles of treatment, respectively. A trend towards a decrease in spleen size has been observed in 3 of 4 patients with palpable splenomegaly at baseline. Overall, there was a significant reduction in circulating CD34+ levels, with a mean decrease of approximately 70% at day 12 of cycles 1 and 2 (p=0.01) We conclude that low dose decitabine administered subcutaneously is feasible in MMM and is associated with minimal non-hematologic toxicity. Myelosupression is significant, though reversible and requires close monitoring. To our knowledge this is the first report demonstrating the potential clinical activity of decitabine in MMM. This observation requires confirmation in a larger group of patients, and accrual is ongoing to this multi-center Phase II study.

Description: There are limited data regarding the incidence or prognostic value of cytogenetic abnormalities in pts with leukemic relapse after allogeneic hematopoietic cell transplantation (HCT). Between 2002 and 2005, 70 consecutive pts with high risk AML or MDS were transplanted with a reduced intensity preparative regimen of fludarabine 30 mg/m2/day IV (150 mg/m2 total), alemtuzumab SC 20 mg/day IV (100 mg total) D-7 to D-3, and melphalan 140 mg/m2 IV D-2, with tacrolimus given for post-transplantation immunosuppression. Twenty-five pts relapsed or progressed; 21 had AML, 3 had MDS and 1 had mast cell leukemia. Twenty-two pts had cytogenetic analysis available prior to HCT and at relapse. Cytogenetic abnormalities were present in 12/22 (55%) pts prior to HCT. The median OS was 184 days (95% CI: 81 – 300) after relapse. Four pts with cytogenetic abnormalities prior to HCT reverted to a normal karyotype at relapse. Ten pts had no changes in their cytogenetics from HCT to relapse; they either remained normal or retained the same abnormality. Eight pts developed a new clonal abnormality at relapse, and had a median OS of 106 days (95% CI: 30 – 322). There was a non-significant trend toward inferior OS among pts with new abnormalities compared to the other groups (HR = 1.74, 95% CI 0.69 – 4.44, P = 0.24). The higher than previously reported rate of clonal evolution (8/22, 36%) may be due to the high prevalence of refractory disease at HCT in this cohort, more refined cytogenetic analysis, or regimen related factors (e.g. reduced intensity conditioning). The same clonal abnormality with or without new changes occurred in 7/22 pts. Thus, minimal residual disease monitoring in the subset of pts harboring pre-HCT karyotypic derangements may be a viable strategy for early detection and intervention. Our data suggest that clonal evolution at relapse of AML and MDS after HCT is relatively frequent, and in this small series, a trend toward worse outcomes exists for pts who develop new cytogenetic abnormalities. Larger studies are warranted to more completely characterize the prognostic value of cytogenetics and karyotypic evolution at relapse after HCT. Cytogenetic abnormalities for AML/MDS relapsing after HCT (N = 22) Pre HCT* Relapse *History of cytogenetic abnormality any time before HCT **Clonal evolution in 8/22 (36%) No 10 (45%) No 7 (32%) Yes (New)** 3 (14%) Yes 12 (55%) No 4 (18%) Yes (Same) 3 (14%) Yes (New)** 5 (27%)

Description: Recruitment of histone deacetylases and CpG island methylation at promoter regions of specific genes are two mechanisms of epigenetic silencing which have been linked and are implicated in differentiation block in AML. We hypothesized that the HDI depsipeptide could cause transcriptional de-repression and upregulation of specific target genes in AML, with subsequent differentiation of the leukemic clone. Twenty-one patients (pts), median age 60 years (range 25–77) were enrolled on a multicenter Phase II study of depsipeptide in AML. Pts were stratified into 2 groups on study entry: Group A (n= 14) included pts without specific chromosomal abnormalities known to recruit histone deacetylases. Group B (n=7) included pts with chromosomal aberrations such as the t(8;21) and inv 16 known to recruit histone deacetylases. All 7 pts in cohort B had translocations involving CBFα or AML1 (n=6) or CBFβ (n=1). Depsipeptide was administered intravenously at a dose of 13mg/m2/d on days 1, 8 and 15 of a 28 day cycle. Peripheral blood mononuclear cells were obtained prior to, and after 4 and 24 hrs, on days 1 and 8 of the first cycle of therapy for evaluation of histone (H3) acetylation by flow cytometry, and gene re-expression by Quantitatative real-time RT-PCR (RQ-PCR). Target genes of interest include MDR1, a target of HDI mediated upregulation, p15INK4B(p15) a target of DNA hypermethylation in AML, and p14ARF (p14), a target of AML1-ETO mediated transcriptional repression. H3 acetylation at the p15 and MDR1 promoters was analyzed by chromatin immunoprecipitation, followed by Q- PCR. The most common adverse effects noted included grade 1/2 nausea, vomiting and fatigue. No objective evidence of response (CR or PR) or other evidence of antileukemic activity has been seen in group A. In contrast, in group B, antileukemic activity has been observed in 4 of 7 (57%) of pts. These include 2 pts with clearance of bone marrow (BM) blasts in the setting of a normocellular marrow, and 2 other pts with a significant decrease (〉50% decrease) BM blasts. This effect was short-lived, with all 4 pts developing evidence of disease progression within 30 days of the initial response. Interestingly 5 of 7 pts (including all 4 pts with evidence of an antileukemic response) in cohort B demonstrated an increase in global H3 acetylation at 4 and/or 24 hrs, in contrast to 4 of 14 pts (28%) in cohort A. Furthermore, in cohort B, at 24hrs, there was a 75% mean increase in MDR1 expression (p=0.005), a 162% mean increase in p15 (p=0.01) and a 106% mean increase in p14 (p

Description: DNA hypermethylation of promoter-specific CpG islands has been implicated in the pathogenesis and progression of myelofibrosis (MF). We have evaluated Decitabine, a DNA methyltransferase inhibitor in patients (pts) with MF, including primary MF (PMF), and MF arising after polycythemia vera or essential thrombocythemia (post-PV or post-ET MF). This was a multicenter, single stage Phase II trial, with a planned accrual of 20 patients. The regimen would be deemed worthy of further investigation if ≥ 5 pts responded. Eligibility criteria included myelofibrosis associated with anemia (hemoglobin 50% increase in platelet levels) have also been observed. Median time to response was 2 cycles (range 1–6); median duration of response was 5 months (range 2–15). Two pts are maintaining their responses at 2 and 14 months. All responders who developed disease progression did so while off therapy. Analysis of the effects of decitabine on CD34+ cells revealed a 61% reduction (p1%) than non-responders at baseline and post-therapy (p

Description: Reduced intensity conditioning regimens and improvements in supportive care have allowed for the expansion of allogeneic stem cell transplantation to those previously ineligble. However, such patients remain at significant risk for regimen related toxicity. We examined whether clinical predictors that influence toxicity after standard chemotherapy, such as age, comorbidity and functional status similarly predict increased transplant related mortality (TRM) and decreased survival after a RIST with in-vivo T-cell depletion. We analyzed 81 consecutive patients on a single protocol with high-risk and refractory hematologic malignancies transplanted at our institution from 2002 to 2004. The conditioning regimen consisted of fludarabine 30 mg/m2/d (D-7 to D-3), Campath-1H 20 mg/d (D-7 to D-3), and melphalan 140 mg/m2 (D-2) with tacrolimus given for post-transplant immunosuppression. Comorbidity was scored from a retrospective chart review using the Charlson Comorbidity Index (CC) and the Kaplan-Feinstein scale (KF). Eastern Cooperative Oncology Group performance status (PS) and age at transplant were tabulated. TRM was defined as any death occurring without disease progression. Median age was 51 years (range 17–68). Fifty-five percent had HLA-identical sibling donors, 5% had mismatched related donors, 35% had matched unrelated donors and 5% had mismatched unrelated donors. KF served as a more sensitive indicator of comorbidity than CC. Fifty-three percent of patients scored at least one point for a comorbid condition by KF, as opposed to 25% by CC (P0 0.53 Comorbidity** CC, no comorbidity 0.76 2.3 0.042 0.089 2.25 0.014 0.03 CC, ≥1 comorbidity 0.52 KF, no comorbidity 0.85 2.7 0.027 0.183 2.74 0.004 0.03 KF, ≥1 comorbidity 0.56 Age Age50 0.63

Description: Patients (pts) with leukemia who relapse after myeloablative allogeneic stem cell transplantation have a poor prognosis due to reduced tolerance and suboptimal responses to salvage therapies. Select pts who respond to salvage and achieve prolonged remissions may benefit from second transplantations. We hypothesized that less toxic conditioning, as well as advancements in supportive care and salvage therapies, may improve outcomes when pts relapse after transplantation. Among 72 pts with high risk and refractory leukemias consecutively enrolled to receive a RIST from 2002–05, we describe outcomes for the 31 pts who relapsed. The preparative regimen consisted of fludarabine 30 mg/m2/d IV (D-7 to D-3), Campath-1H 20 mg/d IV (D-7 to D-3), melphalan 140 mg/mg2 IV (D-2) and tacrolimus for post-transplantation immunosuppression. Of the 31 pts who relapsed after transplantation, 23 had AML/MDS, 3 had CML, 2 had ALL, 2 had leukemic phase of mantle cell lymphoma and 1 had mast cell leukemia (MCL). Only 4 pts had achieved a complete remission (CR) at initial transplantation. 16 pts (52%) had HLA-identical sibling donors, 1 (3%) had a mismatched related donor and 15 (47%) had unrelated donors. The median age was 51 years (range 20–68), and the median time from initial transplantation to relapse was 4 months (range 1–14). After relapse the median follow-up for survivors was 6.6 months (range 2–28) and the median overall survival (OS) was 7.8 months (95% CI, 4.2–13.5) (Figure 1). The median whole blood or marrow donor chimerism at relapse was 71% (range 0–99%). The median marrow blast percentage at relapse was 21% (range 0–99%); 8 pts had 100 days after transplantation (n=9, P=.02) and marrow blast count