ALBERT

Beschreibung: Mutations in the ligand binding domain (LBD) of the RARα-region of PML-RARα were detected in 33% of tested patients who relapsed after treatment with all-trans retinoic acid (ATRA) on the first North American intergroup APL trial INT0129 (E2491). We hypothesized that the incidence of these mutations at the time of first morphological relapse would be reduced on the successor trial C9710, since the new treatment regimen (concurrent ATRA and chemotherapy for induction, first consolidation with two cycles of arsenic trioxide [ATO] in 50% of patients, and 2 subsequent cycles of consolidation with ATRA and daunorubicin) might prevent the early selection of ATRA-resistant PML-RARα mutant subclones. Procedures for making this assessment from low-density bone marrow (BM) and peripheral blood (PB) cells are well-established. In patients found to have a PML-RARα mutation, pre-relapse BM and PB samples collected for monitoring minimal residual disease (MRD) are also being tested for the presence of the mutation, using a mutation-specific real-time PCR assay, in order to assess the dynamics of mutant subclone emergence. Reconstruction experiments in which first-round, allele-specific PCR products from relapse mutant cells were serially diluted in PCR product from non-mutant APL cells documented a capacity to detect 1 mutant template among 103 to 104 non-mutant templates. 9/18 (50%) relapse patients, representing about half of all C9710 relapses to date, tested positive for PML-RARα LBD mutations by DNA sequence analysis, usually with virtual replacement of non-mutant PML-RARα. 9/10 mutations (1 patient had a double mutation) were missense, 2 repeats of previously reported mutations (Arg276Trp, Gly289Arg) and 5 novel mutations (Leu224Pro, Lys238Glu, Ile273Phe, Arg276Gln x2, Gly289Glu x2); 1 was an in-frame 3-codon deletion (Δ412–414). Serial monitoring of post-consolidation samples for the relapse mutation in 3 patients did not detect mutations until or just prior to clinical relapse. The median time (months) to relapse from post-consolidation therapy assessment for MRD was 9.5 (range, 4–36) for mutant patients and 9 (range, 1.5–16) for non-mutant patients. Two of the serially monitored patients relapsed 7 and 24 months after the termination of 12-month maintenance therapy with ATRA ± methotrexate (MTX) and 6-mercaptopurine (6MP). This, together with the lack of a detectable mutant subclone at the conclusion of maintenance therapy, indicates that proximate ATRA selection was not involved in the emergence of the predominant PML-RARα mutant subclone in these 2 patients. Overall, these results support two conclusions: contrary to our pre-study hypothesis, the incidence of PML-RARα mutations was not reduced in patients who relapsed on protocol C9710, but, since the patient assignment to consolidation therapy with ATO and/or maintenance therapy with MTX/6MP has not been disclosed, a difference between the randomized treatment groups is possible and the late emergence of PML-RARα mutant subclones suggests that the mutations provide an intrinsic APL cell growth/survival advantage that likely contributes to the probability of disease recurrence.

Beschreibung: 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.

Beschreibung: We recently tested the feasibility of incorporating Alemtuzumab, a humanized anti-CD52 monoclonal antibody, into frontline therapy of adult ALL (CALGB 10102) in an attempt to eradicate minimal residual disease (MRD). We have previously reported that CD52 expression occurs in 68% of newly diagnosed ALL. This assessment was based upon the use of a qualitative flow cytometric assay performed on all pre-treatment cases in a central CALGB reference laboratory. Cases with 〉 10% CD52 expression on lymphoblasts relative to an isotype control were considered CD52 positive and eligible to receive Alemtuzumab during post-remission therapy. To better characterize the CD52 expression level on lymphoblasts and to obtain insights for the design of future subset specific therapies in adult ALL, we developed a quantitative assay to measure CD52 antigen density on lymphoblasts using custom PE conjugated clinical grade Alemtuzumab on 29 cases of precursor B-cell (pre-B) ALL and 9 cases of precursor T-cell (pre-T) ALL. In this assay, CD52 is expressed in arbitrary units of antibody bound per cell (ABC). We also measured CD52 levels on residual normal B and T lymphocytes in every specimen to calculate a normalized ratio (NR) of lymphoblast CD52ABC/ normal lymphocytes CD52 ABC. The results are tabulated below: CD52 Expression Cell Type Precursor B-Cell ALL (n=29) median (range) Precursor T Cell ALL (n=9) median (range) CD52 ABC units Blasts 27658 (2042–206952) 10222 (3784–35337) CD52 ABC units Normal B Lymphocytes 125475 (59872–282245) 146478 (68098–291878) CD52 ABC units Normal T Lymphocytes 64142 (27406–141635) 78720 (22412–144350) CD52 NR Blasts/Normal B Lymphocytes 0.19 (0.02–0.88) 0.06 (0.02–0.16) CD52 NR Blasts/Normal T Lymphocytes 0.38 (0.04–2.16) 0.12 (0.07–0.26) Pre-B lymphoblasts express significantly lower levels of CD52 antigen than normal B lymphocytes (p

Beschreibung: Background: AraC is considered to be the most effective single drug in the treatment of AML. For initial treatment of AML, araC is typically administered by intravenous continuous infusion for 5–7 days at doses of 100–200 mg/m2/day, usually in combination with an anthracycline. In the relapsed setting, araC remains an option for treatment and is generally administered at higher doses either alone or in combination with other agents and in a variety of schedules. Cloretazine (VNP40101M) is a novel alkylating agent that preferentially targets the O6 position of guanine. A Phase I trial of Cloretazine with araC in advanced hematologic malignancies demonstrated significant anti-leukemic activity with minimal extramedullary toxicity (Giles et al, 2005). The purpose of the current double blind randomized Phase III study is to determine if araC with Cloretazine improves outcome in AML patients (pts) in first relapse. Methods: Eligible pts must be ≥18 years old, PS 0–2, and have AML in first relapse following a CR or CRp of 3–24 months duration. Pts are randomized using a 2:1 scheme to receive either araC 1.5 gm/m2 (d 1–3) + Cloretazine 600mg/m2 or placebo on d 2. Pts are stratified by both age and remission duration. Pts achieving CR or CRp are consolidated with araC + Cloretazine 400mg/m2, or araC + placebo according to original treatment assignment. Pts with partial response or bone marrow improvement may receive a second induction cycle. The study will accrue 420 pts, with an interim analysis for safety and efficacy at 210 pts. The primary endpoint is overall response (CR and CRp) rate. Secondary endpoints include time-to-progression, duration of response, and survival. Results: A data safety monitoring board review of the first 32 pts was performed in 12/05. Differential toxicity between the two arms was not observed. From 03/05 to 07/06, 164 pts were enrolled by 47 sites. Median age=59 yrs (range 22–83), and 52%= male. Distribution by stratum: I = 66 (

Beschreibung: XK469R is a quinoxaline phenoxypropionic acid derivative which possesses broad activity against murine and human tumors (including leukemia) and high activity against multidrug-resistant tumors. COMPARE analysis of cytotoxicity data from the NCI cell line screen suggested a unique mechanism. Phase I studies in patients with advanced solid tumors indicated that the dose-limiting toxicity (DLT) was myelosuppression, without other significant toxicities noted, at a fixed dose of 1400 mg/dose when given on a day 1,3,5 schedule every 21 days. Therefore, we conducted a phase I study to establish the DLT and maximally tolerated dose (MTD) of XK469R in patients with refractory hematologic malignancies, as well as to study the pharmacokinetics of XK469R in this patient population. XK469R was given as a straight dose as an IV infusion over 30 minutes-1 hour on days 1, 3, and 5 of a 21 day cycle. Because significant interpatient variability in drug clearance (associated with toxicity) was noted in prior studies, each dose cohort included a minimum of six patients. The dose levels studied were 1400 mg (n=6), 1750 mg (n=12), 2200 mg (n=14), and 2750mg (n=14). A total of 46 patients with relapsed/refractory leukemia have been treated and are evaluable for toxicity; 41 patients with AML, 4 ALL, and 1 CML-BC. The group consists of 26 males and 20 females with a median age of 53 (range 20–85). ECOG PS included 0 (n=19), 1 (n=21), and 2 (n=6). Median number of cycles received was 1; 10 patients received 2 cycles and 2 patients received 3 cycles. DLT was defined as any clinically significant grade 3 or 4 adverse nonhematologic toxicity other than prolonged myelosuppression, as defined by NCI criteria specific for leukemia. DLTs of colitis and mucositis were observed at the 2200 mg dose level, and mucositis and elevated bilirubin at the 2750 mg dose level. Other possibly related grade 1 and 2 toxicities noted were SGOT/PT elevations, nausea/vomiting, diarrhea, anorexia, indigestion, rash, and alopecia. The MTD, defined as the dose level at which

Beschreibung: 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%)

Beschreibung: 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