MDS AML Research Articles

Cbl and TET2 Mutations Are Present in Refractory Ph+ Disorders Including Accelerated and Blast Crisis CML and ALL.

Abstract 2173Poster Board II-150Loss of heterozygosity (LOH) due to acquired uniparental disomy (UPD) is a commonly observed chromosomal lesion in myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including chronic myelomonocytic leukemia (CMML). Most recurrent areas of LOH point towards genes harboring mutations. For example, UPD11q23.3 and UPD4q24 were found to be associated with c-Cbl and TET2 mutations, respectively. Cbl family mutations (c-Cbl and Cbl-b) have been associated with atypical MDS/MPN including CMML and juvenile myelomonocytic leukemia (JMML) as well as more advanced forms of MDS and secondary AML (sAML). Ring finger mutants of Cbl abrogate ubiquitination and thereby tumor suppressor function related to inactivation of phosphorylated receptor tyrosine kinases, Src and other phosphoproteins. TET2 mutations are present in a similar disease spectrum. The TET family of proteins is involved in conversion of methylcytosine to methylhydroxycytosine which cannot be recognized by DNMT1. Thereby, the proteins seem to counteract maintenance hypermethylation. In our screen of MDS/MPN, we found c-Cbl and Cbl-b ring finger mutations in 5/58 (9%) of CMML and AML derived from CMML, 2/39 (5%) MDS/MPNu, 4/21 (19%) JMML and 14/148 (9%) RAEB/sAML. In the same cohort, TET2 mutations were present in 37% and 14% of patients with MDS/MPN and MDS, respectively. Of note we did not find any TET2 mutations in JMML. We and others have also noted that TET2 and c-Cbl mutations were also detected in atypical chronic myeloid leukemia. While translocations resulting in BCR/ABL fusion characterize CML, we stipulated that in analogy to other chronic myeloproliferative diseases, TET2 and c-Cbl mutations may be also present in CML and contribute to phenotypic heterogeneity within BCR/ABL associated disorders. In particular, progression of CML to accelerated phase (AP) or blast crisis (BC) could be associated with acquisition of additional lesions. When 22 patients with CML chronic phase (CP) were screened, no TET2 and c-Cbl mutations were found. However, we identified 1 c-Cbl, 2 Cbl-b (6%) and 6 TET2 (12%) mutations in 51 patients with CML-AP (N=18) and CML-BC (N=33) with myeloid and lymphoid/mix 24 and 9 phenotype, respectively. These mutations were mutually exclusive. We also noted that TET2 mutations were present in 1/9 CML in BC with lymphoid phenotype. We subsequently screened Ph+ ALL cases (N=9) and found a TET2 mutation in 1 case but no Cbl family mutations. In contrast when 9 Ph- ALL cases were screened as controls, neither TET2 or Cbl mutations were found. SNP-A analysis revealed 2 cases of LOH involving chromosome 4 (UPD4q24 and del4) in a patient with lymphoid blast crisis and Ph+ ALL, respectively. However, UPD was not found in Cbl family gene regions (11q23.3 or 3q13.11). A homozygous deletion of Cbl-b region was seen in a CP patient. Cbl family mutations were associated with a more complex karyotype than TET2 mutations (67% vs. 17% cases with abnormal phenotype). Patients with Cbl family mutations were resistant to imatinib which was effective in only 2 out of 6 patients with TET2mutations. Dasatinib was effective in 2 patients with TET2 mutation. Median over all survival of progressed CML was 47, 49 and 48 months in patients with Cbl, TET2 or no mutations, respectively. In conclusion, our results indicate that Cbl family mutations can occur as secondary lesions in myeloid type aggressive CML (AP and myeloid BC), but not in lymphoid types. TET2 mutations were identified in both lymphoid BC and Ph1+ALL, as well as myeloid BC and AP. In contrast to CMML or JMML in which a vast majority of mutations are homozygous, all Cbl family mutations were heterozygous (no LOH). Similarly, all but two TET2 mutations were heterozygous (1 hemizygous in del4 and 1 homozygous case in UPD4q), suggesting that additional cooperating lesions affecting corresponding pathways may be present. These mutations likely represent secondary lesions which contribute to more either progression (CML) or more aggressive features (Ph+ ALL) and characterize disease refractory to therapy with imatinib. Disclosures:No relevant conflicts of interest to declare.

P035 Improvement in cytogenetic diagnosis and clinical prognostication using SNP-A karyotyping in combination with metaphase cytogenetics in MDS, MDS/MPD and secondary AML

P035 Improvement in cytogenetic diagnosis and clinical prognostication using SNP-A karyotyping in combination with metaphase cytogenetics in MDS, MDS/MPD and secondary AML

Maintenance Treatment with 5-Azacitidine for Patients with High Risk Myelodysplastic Syndr–ome (MDS) or Acute Myeloid Leukemia Following MDS (MDS-AML) in Complete Remission (CR) after Induction Chemotherapy

Around 50% of patients with high-risk MDS or MDS-AML may enter CR after induction chemotherapy, but CR duration, as well as overall survival is usually short. To address this clinical problem the Nordic MDS Group designed a prospective multicenter phase II study, which assessed the clinical feasibility and utility of long-term maintenance treatment with azaciditine. Sixty patients with high-risk MDS (IPSS intermediate-2 or high) (n=23) or AML following a previous known MDS (n=37) were enrolled between 2004 and 2006. The mean age was 68 (54–83) and patients should not be eligible for stem cell transplantation. Induction treatment consisted of standard doses of daunorubicin and ara-C. Patients in CR received low dose azacitidine subcutaneously 5/28 days until relapse, unless unacceptable toxicity developed. Methylation status of the P15ink4b (P15), E-cadherine (CDH) and Hypermethylated in Cancer 1 (HIC) gene was analysed at study start, in CR and in some patients during follow up. Last follow up was on August 1 2008, 24 months after the last CR was reported. Twenty-four patients (40%) reached CR and 23 of these started maintenance treatment with azacitidine. The initial dose of azacitidine was 75 mg/m2 but as four of the first five enrolled patients developed grade 4 cytopenia, the starting dose was lowered to 60 mg/m2, and was allowed to be reduced to 45 or 30 mg/m2 to avoid severe cytopenias. The mean dose of azacitidine was 54.3 mg/m2. Azacitidine was well tolerated. In 52% of the cases no side effects at all were reported. The most commonly reported side effect was mild rashes at the injection site (35%). Twenty-two percent developed fever or some kind of infection, mostly mild. Myelosuppression (grade 1–3) was seen in 22% of the cases. As previously reported, the probability of reaching CR was negatively correlated to promoter hypermethylation of CDH (p=0.008) and none of the 6 patients hypermethylated on all 3 genes reached CR (p=0.03) and hence only four patients hypermethylated on other genes than P15 received demethylating therapy. The median CR duration for the azacididine treated group was 13.5 months (2–49+) and median survival time from time of inclusion in the study for the same group was 20 months (4–52+). Four of 23 patients (17%) had a CR exceeding 24 months (32–52+). The two patients hypermethylated on CDH pre-induction had CR durations of only 2 and 5 months respectively. By last follow up 3 patients were still in CR. Of 10 patients without any methylation pre-treatment, all but one maintained this pattern in CR. Of the nine patients with pre treatment methylation of at least one gene, only one remained hypermethylated in CR. This patient had a CR duration of only 5 months. One patient showed development of P15 hypermethylation in the bone marrow sampled at 12 months and relapsed at 15 months. These findings support previous reports on P15 hypermethylation as a marker for minimal residual disease (MRD) and threatening relapse. In the whole group, survival was significantly shorter in patients with CDH methylation (3 vs 9 months, p=0.005), while pre-treatment p15 methylation status did not affect CR duration or overall survival. In conclusion, we show for the first time that maintenance treatment with azacytidine is feasible and associated with a median CR duration of 13.5 months, and very mild side effects. However azacytidine does not seem to prevent relapse in the majority of patients, including those with hypermethylation pre-treatment and/or in CR. Hypermethylation of multiple genes is a strong negative factor for survival, probability of CR, and CR duration. We observe a subset of patients, 17%, with a CR duration of >24 months; but no persistent pattern regarding cytogenetics, methylation or morphology could be identified in this group. The strong negative impact of E-Cadherin methylation, a gene involved in adhesion, warrants further investigation.

Investigations of Genetic Risk Factors in MDS and AML Using High- Density 6.0 Affymetrix Arrays

Genetic predisposition to MDS and AML is likely polygenic and may involve several low penetrance alleles which in concert with exogenous factors result in highly variable presentation, not easily amenable to genetic studies. With the advent of whole genome scanning (WGS) technologies utilizing various SNP array (SNP-A) platforms, large scale investigations in various disorders have been conducted. In hematological malignancies to date no systematic disease-association studies using SNP-A have been reported, likely due to lower prevalence of these conditions and a highly variable phenotype. We have applied SNP-A to conduct the first GWS in MDS and MDS-derived AML with the goal to identify possible low prevalence genetic variants that contribute to the pathogenesis of these conditions and explain individual disease risk. We have studied 189 patients with MDS and secondary AML as well 119 internal controls using SNP-A. Affymetrix GeneChip 6.0 (924644 SNP probes covering most of the known LD blocks) is designed to capture 67%-89% of SNP variation among Caucasians. Following exclusion of SNP's with a call rate of <95%, and those with serious violation of Hardy Weinberg equilibrium, single allele X2 statistics for all autosomal markers was performed. For the purpose of this study, SNP's with minor allele frequency (MAF) <10% and p<0.001 after false discovery rate correction, were selected. Top 11 polymorphisms were chosen pointing directly to 4 genes or indirectly to informative loci through LD, informative genes include e.g., LAMC2, SGCE, FRAP1 and PTPRT. Remarkably, several informative LD blocks were also identified represented by multiple markers pointing to the presence of an informative polymorphisms in the corresponding regions. For example, 5/30 markers (all p<8×10−4) including, rs2477436, rs503243, rs3768593, rs4651151 and rs549191 are part of an LD block spanning NMAT2 and LAMC2 loci. The corresponding minor variant frequencies were 6.6% and 37.6% in homozygous and heterozygous constellation, respectively (controls: 0% and 21.6%). Second potential locus identified in our study consisted of 4 markers, all of them located on SGCE gene (rs1357318, rs2037496, rs4330611, rs13225971; p<1.9×10−4) with frequencies of homozygous variant in patients at 0.8% and 28.9% with heterozygous variant (controls 0% and 15.2%), respectively. FRAP1 (MTOR) gene was represented by singular rs3730380 marker (p=2.7×10−6), occurring at the heterozygous frequency of 17.8% vs. allelic frequency of 0% in controls. FRAP1 is a critical downstream effector of Akt involved in cell cycle regulation and angiogenesis being central regulator in PI3K/Akt/mTOR pathway. Genetic alterations of the pathway are frequent events in preneoplastic lesions and advanced cancers. Similarly, increased frequency of minor alleles of rs6030469 in PTPRT locus was found in homozygous and heterozygous constellation at 1.4% vs. 0% and 27.3% vs. 8.5% (p=4.80 × 10-5) in patients and controls, respectively. PTPRT gene was also found to be frequently mutated in cancer and is involved in growth regulation. For example, overexpression of PTPRT may lead to reduced expression of STAT3 target genes. In sum, our study constituting the first systematic approach of WGS to identify genetic risk factors in AMS and AML, suggests that several informative loci can be selected for delineation of the causative polymorphisms.

SNP-Array Based Karyotyping Complements Routine Cytogenetics in Diagnosis and Risk Stratification Schemes of MDS

While current pathomorphologic criteria distinguish MDS, MDS/MPD and MDSderived AML (sAML), these conditions share common unbalanced chromosomal abnormalities highly predictive of prognosis. Reflecting their clinical importance, IPSS is highly influenced by metaphase cytogenetics (MC), the standard for detection of chromosomal abnormalities. Due to its low resolution and need for cell divisions, MC can only detect abnormalities in 50% of patients with MDS, novel technologies have been developed that could improve the diagnostic yield. Among these methods, single nucleotide polymorphism arrays (SNP-A) have been adopted as a cytogenetic platform. SNP-A karyotyping can be performed on interphase cells and allows for detection of smaller lesions as well as for copy-neutral loss of heterozygosity (LOH). As a result, some reference laboratories offer this test for routine cytogenetic diagnostics. However, the clinical relevance of lesions detected by SNP-A remains unclear and, consequently, we designed this comprehensive study to clarify the clinical impact of cytogenetic results generated through a combined application of SNP-A and MC. A cohort of 352 patients (218 MDS, 59 MDS/MPD, 75 sAML) was studied using Affymetrix 250K (N=160), Affymetrix 6.0 (N=190) and both platforms (N=95). Controls included 362 and 118 samples analyzed by 250k and 6.0 arrays, respectively. CNAGv3 or GCv2.1 software was used for analysis. To avoid biological and technical artifacts, known copy number variants as well as areas of LOH<2.5Mba were excluded. All other lesions, if not present on MC, were confirmed in germ line DNA if possible. The median follow-up time was 29 months (95% CI; 21-38 mo) and for many patients serial samples were studied, showing sequential acquisition of chromosomal defects. Overall, the detection rate of chromosomal abnormalities was improved compared to MC (57% vs. 44%; p=.0096), MDS/MPD (66% vs. 39%, p=.0055) and sAML (63% vs. 45%, p=.048). Somatic uniparental disomy (UPD) accounted for a significant proportion of newly detected defects (26% of new lesions). Overall, UPD was detected in 100 patients (78 as the sole and 22 as an additional lesion). Chromosomal lesions were identified in 98/180 with normal MC. In 61% of patients with non-informative MC (N=18), SNP-A demonstrated an abnormal karyotype; those with newly detected lesions showed inferior outcomes in overall survival (OS) [16 vs. 36 mo, p=.03]. Analysis of OS showed that patients with new defects detected by SNP-A and previously normal MC have worse outcomes compared to those in whom normal chromosomes were confirmed (39 vs. 73 mo, p=.03). Moreover, additional lesions also conferred worse prognosis for patients with abnormal karyotype by MC (17 vs. 38 mo, p=.01). This trend was even more pronounced when specific subgroups were analyzed based on OS and event free survival (EFS). For example, detection of new lesions in MDS (OS p=.02), MDS/MPD (OS p=.009, EFS p=.01) or sAML (OS p=.02, EFS p=.008) results in significant worsening of OS and EFS as compared to patients with truly normal cytogenetics. Newly detected lesions allowed for more precise prognostication within established IPSS strata of MDS and MDS/MPD patients. The detection of SNP-A defects negatively impacted OS/EFS in patients with low IPSS (NR vs. 69 mo, p=<.0001; 42 vs. 112 mo, p=<.0001), decreased EFS in Int-1/2 (24 vs. 29 mo; p=<.0001) and decreased OS/ EFS in high IPSS categories (11 vs. 26 mo, p=<.0001; 7 vs. 14 mo, p=<.0001). In sAML, patients with SNP-A lesions, regardless of MC, have worse OS (6 vs. 21 mo, p=.009) and EFS (5 vs. 13 mo, p=.01). In summary, SNP-A karyotyping facilitates detection of lesions which can complement MC and contribute to a better molecular diagnosis. SNPdetected lesions have impact on prognostic parameters which will likely affect future risk stratification schemes and have prognostic significance regardless of MC in sAML. As a result, new molecular subtypes of MDS may be identified and better-defined recurrent lesions will contribute to identification of causative genes.

Treatment of Progression of Myeloproliferative Disorders(MPD) to MDS/AML by Azacytidine (AZA) : A Preliminary Report on 17 Patients (pts)

Background: MDS or AML occurring during the course of MPD carry a very poor prognosis and current treatments, with the exception of allogeneic SCT, have very limited efficacy. AZA significantly improves survival in higher risk MDS (ASH 2007, abstr n° 817) and in some AML, but its use has not been extensively reported so far in MDS and AML post MPD.Methods: The French health agency (AFSSAPS) designed a still ongoing pt named program (ATU) of AZA in higher risk MDS and poor risk AML. 17 pts with MDS or AML post MPD, included in this program in 5 centers (Paris-Avicenne, Paris St Louis, Paris-Cochin, Tours and Toulouse), before April 08 and having completed ≥ 1 cycle of AZA (75 mg/m2/d during 7 days per 28 d cycle) are analysed here. Response was evaluated according to IWG 2006 criteria for MDS and IWG-AML 2003 criteria for AML.Results: median age was 68y (range 38–83), M/F: 7/10. The initial MPD was Polycythemia Vera (PV) in 6 pts, Essential Thrombocytopenia (ET) in 7 pts, primary myelofibrosis (MF) in 2 pts and unclassified in 2 pts. Median interval from diagnosis to progression was 10 years (range 7 months-22 years). 10/16 (62%) pts had JAK2-V617F mutation. Treatment of MPD had consisted of HU in 7 pts, pipobroman in 4 pts, both in 4 pts, imatinib in 1 pt and Interferon Alfa in 1 pt. At the time of inclusion, WHO classification was AML in 8 pts, and MDS in 9 pts (RAEB-2 in 8pts, RAEB-1 in 1pt) Karyotype was normal (n=2), isolated −7/7q- (n=3), isolated del 5q (n=1), + 8 (n=2), isolated −17 (n=1), t[1;7] (n=2), complex (n=3) including −7 (3), −5(2) and +8(1), and a failure (n=3). Before AZA, 2 pts had received intensive chemotherapy (1 CR, 1 failure) followed by allo SCT in 1 of them (who subsequently relapsed).The median number of cycles of AZA administered was 4 (range 1–9). 2 pts received only 1 cycle due to early death (from aspergillosis and unknown cause). The overall response rate (ORR, including CR, PR, CRi) was 10/17 (59%). In the 15 pts who received more than 1 cycle, 4/8 MDS achieved CR (including one cytogenetic CR) and 2/8 achieved PR. RBC and platelet transfusion independence were achieved in 4/8 and 6/8 respectively (resp). In AML, 1/7 pts achieved CR and 3/7 achieved CRi. 2 of the 10 responders relapsed (after 5 and 12 months resp) and the other 8 remained responders after 1+ to 12 + months (median 3.5). 9/10 responders were still alive after 2 to 16 months (median 5.5). Interestingly, in 3 pts with a very long history of MPD (15, 16 and 22 years) treated with HU or pipobroman, who at progression had MDS and cytopenias, response to AZA was associated to recurrence of MPD (2 TE and 1 PV) requiring restart of cytoreductive therapy in 2 of them.Conclusion: 5 AZA gives encouraging results in MDS or AML occurring in the course of MPD with an overall response rate of 59%. An update, with cases recently included in the program, will be presented.

Blast cells from patients treated for secondary AML (sAML) show less P-glycoprotein (Pgp)-mediated efflux of amonafide compared to daunorubicin: Implications for therapy

13536 Background: Multidrug resistance (MDR) associated with Pgp overexpression in blasts is common in high-risk AML and is associated with poor outcomes. Erba et al (JCO 2007) reported a 42% complete remission (CR) rate in patients (pts) with sAML [prior MDS or treatment-related AML, (tAML)] (N = 88) treated with amonafide and std dose cytarabine. In laboratory studies of Pgp+ human leukemia cells, amonafide was neither a substrate nor an inhibitor of Pgp-mediated efflux, in contrast to daunorubicin (DNR). We sought to correlate the lack of Pgp effect with treatment outcome. Methods: AML blasts from 15 pts from the Phase 2 trial cited above were retrospectively assessed for Pgp expression and function as well as amonafide and DNR uptake and retention in the presence and absence of the Pgp inhibitor cyclosporin A. Pts: median age, 62 yrs (range 51–87); 7 with prior MDS; 8 had tAML; 10 with unfavorable cytogenetics. Pgp-mediated efflux was assessed by comparing uptake of the Pgp substrate DiOC2(3) in the presence and absence of the Pgp inhibitor PSC-833. Pgp- mediated transport (effluxapp) was calculated as differential uptake and retention of amonafide and DNR with (a) and without (b) PSC-833, normalized to apparent influx, using the formula [(a-b)/a]×100, reported as the mean±s.e.m. Results: The 15 sAML samples showed significantly less effluxapp of amonafide (5.2%±3.2) than of DNR (16%±2.1; p=0.0083). The unfavorable cytogenetic subset showed much less effluxapp of amonafide (0.13%±3.7) compared to DNR (16%±2.1; p=0.0015). CR pts also showed less effluxapp of amonafide (4.0%±6.7) than of DNR (21%±2.9; p=0.035). Conclusions: The relative lack of Pgp-mediated efflux of amonafide, compared to DNR, from sAML blasts provides a rationale for its observed clinical efficacy in the Phase 2 trial. Prospective assessment of these MDR parameters is underway in a randomized Phase 3 clinical trial in sAML comparing amonafide to DNR in combination with std dose cytarabine for remission induction. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Xanthus Pharmaceuticals Xanthus Pharmaceuticals Xanthus Pharmaceuticals

Phase I Study of Clofarabine with Standard-Dose Infusional Cytarabine (Ara-C) as Intensive Induction Therapy for Newly-Diagnosed De Novo Acute Myeloid Leukemia in Older Adults Age ≥60 Years.

Background The median age of AML is approximately 70 years, and the survival of older adults age ≥60 years remains poor with standard therapy. Clofarabine (CLO) has significant single agent activity in AML, and in vitro studies demonstrate synergy with Ara-C. We therefore performed a phase I study to determine the maximum tolerated dose (MTD) of CLO with standard dose Ara-C (100mg/m2/day D1-7 by 24hr continuous infusion) as AML induction therapy. CLO was administered daily × 5 days beginning D2 to allow for pharmacokinetic (PK) & pharmacodynamic (PD) studies.Methods Enrollment was restricted to newly-diagnosed de novo AML patients age ≥60 years considered candidates for intensive therapy; those with prior MDS or hypoplastic AML (<20% bone marrow cellularity) were excluded. Pts were treated in cohorts of 3–6 to determine MTD of CLO with standard dose infusional Ara-C (100mg/m2 × 7). DLT was defined as grade III/IV non-hematologic toxicity occurring in >1 pt per cohort. The CLO starting dose was 30mg/m2/day ×5 (dose level 1), with the intention to increase to CLO 40mg/m2 if tolerated. However, CLO dose reductions of 25% (CLO 22.5 mg/m2/day × 5, dose level -1) & 50% (CLO 15mg/m2/day ×5, dose level -2) were allowed in the event of dose-limiting toxicity (DLT). Pts achieving complete remission (CR) received 2 cycles consolidation with CLO ×5 & Ara-C 100mg/m2 × 5.Results A total of 13 pts (median age 69 yrs, range 61–77; 10 male) have been treated. DLT was observed at dose level 1 (Table 1), and the protocol was amended to mandate additional hydration with CLO, and antibiotic/antifungal prophylaxis. At dose level -1, 1 pt died from PE on D27 after hematologic recovery, considered treatment-related per protocol, mandating dose de-escalation to dose level -2. Pt 13 is in active induction therapy, and not yet evaluable for response, although DLT has not been encountered. Febrile neutropenia occurred in 11/13 pts. The MTD in this study is CLO 15mg/m2/day × 5 with standard infusional Ara-C (dose level -2). Significant clinical activity was observed, particularly at higher CLO doses, including CR in 2 pts with complex karyotype. In contrast, only 1/5 evaluable pts at dose level-2 achieved CR. PK & PD studies are in progress.Conclusions In contrast to prior reports using intermediate dose bolus Ara-C (1g/m2), the MTD of CLO combined with standard dose infusional Ara-C in older adults with AML is lower, and the toxicity profile appears different. Significant clinical activity was noted at higher CLO dose levels & with complex karyotype. Based on this observation an escalation to CLO 20mg/m2 is now planned.CLO & Standard Dose Infusional Ara-C: Toxicity & EfficacyPatientCLO Dose LevelAgeCytogeneticsResponse (CR Duration)DLT11 (30mg)66−7DeathInfection/Renal2161−7, complexCR (15 mths)none3169intermediateCR (2 mths)none41775q-, complexDeathInfection/Renal/Vasc Leak5−1 (22.5mg)75+8DeathPneumonia6−167diploidCR (5 mths)none7−1719q-DeathPulm Embolism Day 278−2 (15mg)745q-, complexFailnone9−264diploidFailnone10−263diploidFailnone11−273complexCR (5 mths)none12−272diploidFailnone13−263diploid--Fail - failure to achieve CR

Risk Factors for Disease Progression in del(5q) MDS Patients Treated with Lenalidomide.

Introduction: Lenalidomide is a novel immunomodulatory drug that is highly effective in patients with transfusion-dependent MDS with del(5q.31) chromosomal abnormality. In the recent MDS-003 study of lenalidomide, there was a 76% erythroid response rate, including 67% transfusion independence. Cytogenetic complexity or bone marrow blast percentage did not affect this response rate. A number of patients experienced disease progression to higher FAB subtypes or AML. We questioned whether lenalidomide might promote disease progression in del(5q) MDS and performed a retrospective analysis to identify risk profiles.Methods: Fifty patients from three institutions were included in this analysis. They were partly treated within the Lenalidomide-MDS003-study. Patients were treated with an initial lenalidomide dose of 10 mg po daily. In case of grade >2 neutropenia, G-CSF and antibiotics were administered.Results: The median age was 71 years; 31 patients were female and 20 were male. Disease progression to a higher FAB subtype or to AML occurred in 13 patients (29.5%). 7 of the 13 patients had RAEB at the first lenalidomide dose. In addition, 3 of these had additional chromosomal aberrations (2, trisomy 21; 1, complex karyotype). Of the remaining 6 patients, 2 had a complex karyotype at the first lenalidomide dose, 1 had an additional inv(9)(p11q12), and 1 had hypocellular bone marrow so no FAB subtype could be assigned. Only 2 of 50 patients (4.3%) with 5q-syndrome progressed to AML; both patients developed acute erythroid leukemia (FAB M6).Conclusion: Within the del(5q) MDS subgroup, patients with an isolated del(5q) chromosomal aberration and a bone marrow blast count of <5% have the longest overall survival. Patients with additional chromosomal abnormalities or a higher blast percentage have a much shorter overall survival and a higher risk for progressing to AML. Our data show that progression to higher MDS subtypes or AML occurs almost exclusively in patients with additional risk factors such as >5% bone marrow blasts or additional chromosomal anomalies. Lenalidomide does not seem to increase the risk of transition of del(5q) MDS to higher stages of disease.

Multivariate Analysis Suggests That the Prognostic Impact of Poor Cytogenetics Is Potentially Underestimated in the IPSS.

Introduction: The IPSS-Score, published by Greenberg et al., defines the gold standard in risk stratification of patients suffering from MDS. Nevertheless, the assessment of cytogenetic findings within the IPSS was discussed intensively since its implementation in 1997. Within the IPSS, patients with a high number of bone marrow blasts are rated with a higher risk as compared to those with unfavourable cytogenetics. In a further univariate anylysis, we were able to demonstrate that poor cytogenetics are associated with the same prognostic risk as blast counts >20%. The aim of our present study was to investigate the prognostic impact of poor cytogenetics and bone marrow blast count in a multivariate analysis of a large patient cohort.Patients and Methods: 3169 patients were extracted from our large database, including 3860 patients with MDS and secondary AML following MDS. This data pool was collected in the framework of a cooperative project merging data from the German-Austrian Cooperative MDS Study Group (GACMSG; 55% of pts.) and the MD Anderson Cancer Centre, Houston, USA (45% of pts.). Median age of all study patients was 65.9 years, the female-male ratio 1:1.53. Complex abnormalities occurred in 21.2% of all patients. Multivariate analysis was done using a Cox-regression hazard model with overall survival as primary endpoint. The following groups were established for analysis: Blasts <5%, 5–10%, 11–20% and >20%, normal karyotype, complex abnormalities including chromosomes 5 or 7, complex abnormalities not including abnormalities of chromosomes 5 or 7 and −7/7q- (as single abnormality).Results: Former Kaplan-Meier analysis (data presented last year) showed that median survival (ms) in patients with unfavourable cytogenetics (n=516, ms=7.9 months) is similar to those showing >20% bone marrow blasts (n=197; ms=8.9 months). In multivariate analysis, relative Hazard ratio (HR) was 1.50 in pts. with 5–10% blasts, 1.64 in 11–20% blasts and 1.81 in >20% blasts (baseline for relative HR: <5% blasts, all p-values <0.0001). On the other hand, pts. with unfavourable cytogenetics show an considerable increased risk (normal karyotype was defined as baseline, all p-values <0.001): HR was 3.88 in pts. showing complex abnormalities including chromosomes 5/7 and 2.09 when −7/7q- occurred. Complex abnormalities not including chromosomes 5/7 were associated with a HR of 1.76.Conclusions: Our data suggests that complex abnormalities constitute a prognostic risk factor that is as unfavourable as high bone marrow blast counts. As demonstrated by calculated HR, the occurrence of unfavourable cytogenetics raises the prognostically risk disproportionately. Nevertheless, the IPSS assesses poor cytogenetics (rated with 1.0 points) with a lower risk as compared to increased bone marrow blast counts (rated with 1.5 points for 11–20% blasts; 2.0 points for 21–30% blasts). We conclude that the prognosic impact of unfavourable, especially complex abnormalities, is potentially underestimated in the IPSS. Further data will be shown and presented in detail.

Multivariate Evaluation of the Prognostic and Therapeutic Relevance of Cytogenetics in a Merged European-American Cohort of 3860 Patients with MDS.

This study investigated the influence of cytogenetic findings on survival in MDS, after accounting for other known prognostic parameters and type of treatment. We identified 3210 patients with a known therapy regimen from our large database, including 3860 patients with MDS and secondary AML following MDS. This data pool was collected in the framework of a cooperative project merging data from the German-Austrian MDS study group (55% of pts.) and the MD Anderson Cancer Centre (MDA), Houston, USA (45% of pts.). Median age of all study patients was 65.9 years, the female-male ratio 1:1.53. After accounting for age, sex, % marrow blasts, de novo vs. primary MDS, treatment (supportive care, AML-type, and epigenetic therapy) and site of treatment(Germany/Austria vs.MDA) we looked at the effect of cytogenetic subgroups on survival. Survival data was available for 95.8% of all pts. The distribution of main cytogenetic categories was as follows: normal (1.727, 45%), 5q- (208, 5%), 5q- +1 abnormality (73, 2%), −7/7q- not complex (222, 6%), +8 not complex (203, 5%), complex with −5/−7 (627, 17%), other complex (195, 5%), 20q- not complex (69, 2%), and other not complex (475, 13%). Chemotherapy (c) was applied in 32.9% while 67.1% were treated with supportive care (sc) or epigenetic therapy. The cytogenetic prognosis according to IPSS-criteria was favourable in 1704 (53.1%) and unfavourable in 839 (26.13%) of the 3210 study patients. Multivariate analysis revealed the following non-cytogenetic parameters as unfavourable: age >60, male sex, blasts >5%, secondary MDS, AML-like chemotherapy, MDA as site (due to overrepresentation of high risk MDS). After accounting for these variables the following relative Hazard ratios (HR of 1.0 for normal karyotype) were calculated allowing a relative ranking of cytogentic findings: 5q-: 0.93, 5q- +1: 1.06, other not complex: 1.11, 20q-: 1.31, +8: 1.65, complex without −5/−7: 1.76 −7/7q-: 2.09, complex with −5/−7: 3.88. The effect of cytogenetics in pts. with either supportive care (sc) or chemotherapy (c) measured as relative HR (see above) were: 20q- (sc: 1.25, c: 1.51), 5q- (sc: 0.93, c: 0.73), other not complex (sc: 1.07, c: 1.04), −7/7q- (sc: 2.10, c: 2.09), +8 (sc: 2.05, c:1.54), complex without −5/−7 (sc: 2.23, c: 1.75), complex with −5/−7 (sc: 4.78, c: 3.77) showing that cytogenetics remains it's prognostic relevance independent from the therapy applied. In Kaplan-Meier-analyses median survival in pts. showing favourable karyotypes was 37.9 months with supportive treatment as compared to 26.4 months with chemotherapy (p<0.01). We observed no therapy-related survival differences with regard to patients of the unfavourable cytogenetic subgroup. The occurrence of complex abnormalities (n=670) was associated with an identical median survival comparing the therapy groups (c: 7.0, sc: 7.1 months). We also investigated groups with distinct abnormalities (5q-, −7/7q-, +8, 20q-) and found significant differences in survival between the therapy groups: Patients with 5q- and 20q- benefited from supportive care while −7/7q- was related with a better survival when chemotherapy was applied. Patients with trisomy 8 showed no differences in survival. Regarding these data, chemotherapy response, expressed by survival, is closely associated with cytogenetics. Complex abnormalities always show a dramatically reduced outcome, independent from the therapeutic strategy.

362: Superior EFS and reduced TRM in patients with Int-2 and High risk MDS and secondary AML after initial cytoreduction with 5-azacytidine

362: Superior EFS and reduced TRM in patients with Int-2 and High risk MDS and secondary AML after initial cytoreduction with 5-azacytidine

Phase II Study of Clofarabine and Cytosine Arabinoside in Adult Patients with Relapsed AML and in Elderly Patients with Untreated AML Who Are at High Risk of Anthracycline Toxicity.

PURPOSE: To evaluate the efficacy and safety of clofarabine, a novel deoxyadenosine analog, in adult patients with refractory or relapsed acute myeloid leukemia and in selected elderly patients with untreated AML and heart disease.PATIENTS AND METHODS: In a phase II, open-label trial, 23 patients with de-novo AML, AML evolved from MDS or relapsed AML received a 5 day regimen consisting of clofarabine 40 mg/m2 intravenously over 1 hour followed within 4 hours by Ara-C 1000 mg/m2. The median age was 68 years (range 39–79 years). Fifteen (65%) had received at least one prior cytotoxic regimen (excluding 5-AZA). Significant cardiovascular disease (history of myocardial infarction, bypass grafting, cardiomyopathy) was present in 52% (12/23) prior to therapy.RESULTS: 22/23 received at least one cycle of therapy and 5 received 2 cycles. One early death was due to disease progression. Grade 4 neutropenia developed in all patients. There were no cases of regimen-related cardiac toxicity. Most patients had some degree of edema and third-spacing syndrome. Several developed a significant but reversible acral rash. 20 patients are evaluable for response. The histologic response rate (defined as marrow blasts <5%) is 60% (12/20) consisting of 10 (50%) complete remissions and 2 (10%) partial responses. Complete cytogenetic remissions occurred in 9 patients. Durable remissions and low toxicity allowed some patients to proceed to nonablative allogeneic stem cell transplantation.CONCLUSION: Clofarabine (40mg/m2) and Ara-C (1000mg/m2) x 5 days is an active and well tolerated regimen in myeloid malignancies including “elderly AML”—a distinct entity usually associated with poor response rate and high treatment-related toxicities. Other drug combinations with clofarabine are being explored for use in allogeneic transplant regimens and with other high-risk patient groups.

Impact of Cytogenetics on Outcome of Patients with MDS or Secondary AML Undergoing Allogeneic HSCT from HLA-Identical Siblings: A Retrospective Analysis of the EBMT-CLWP.

Cytogenetics (CG) represent one of the most important factors in determining survival for pts with MDS, being therefore required by the IPSS to predict life-expectancy. However, the impact of CG on outcome of pts undergoing allo-HSCT for MDS is unknown. The aim of this EBMT-CLWP study was to carry out a retrospective analysis of the impact of CG on overall survival (OS), relapse-free survival (RFS), relapse probability (REL), and transplant related mortality (TRM) in pts with MDS/sAML undergoing allo-HSCT from HLA-identical siblings.Data from 1506 pts with MDS/sAML who underwent a first allograft from HLA-identical siblings from 1984 to 2004 reported to EBMT were assessed. The following covariates were included: CG (good- vs standard- vs high-risk according to the IPSS); stage at HSCT (untreated vs treated in CR vs treated not in CR); FAB classification (RA/RARS vs RAEB/CMML vs RAEB-t/sAML); age (as a continuous covariate in the COX models); time from dx to HSCT (<5 vs 5 to 8 vs > 8 months); calendar year in which HSCT was performed; type of conditioning (standard vs RIC); source of stem cells (BM vs PB). A complete information dataset was available in 692 pts, who are the subject of this analysis.20% of pts had RA or RARS, 28% had RAEB or CMML, and 52% had RAEB-t or sAML. Age was ≥50 years in 30%. CG classified 55% of pts as good-, 24% as intermediate-, and 21% as high-risk. At the time of HSCT, 38% were untreated; among treated pts, 222 (52%) were in CR. A RIC regimen was administered to 93 pts (13%). Source of stem cell was PB in 38%. By univariate analysis, subdivision of pts in the IPSS risk-categories for CG associated with significantly different OS (at 60 mos, alive pts in good-, interm.-, and high-risk groups were 47%, 40% and 31%, respectively), RFS (alive pts at 60 mos 40%, 35% and 21%, respectively), relapse probability (34%, 35% and 57% at 60 mos, respectively) and TRM (33%, 42% and 46% at 60 mos, respectively).By multivariate COX analysis, age and CG associated with all the outcome variables (for OS, high- vs good-risk HR 1.4; interm.- vs good-risk HR 1.2; p<0.02). OS, RFS and REL were also determined by FAB category (for OS, RAEB-t/sAML vs RA/RARS HR 1.5, p<0.01) as well as by stage at HSCT (for OS, CR vs untreated HR 0.65, p<0.005). Conditioning intensity associated both with TRM (reduced vs standard HR 0.5, p<0.01) and REL (reduced vs standard HR 1.63, p<0.02). Concerning REL, the interval between dx and HSCT was also significant, whereas the source of stem cells associated only with OS (PB vs BM HR 0.78, p<0.05). In summary, this study provides evidence that CG have strong prognostic impact on outcome of pts undergoing allo-HSCT from HLA-identical siblings for MDS/sAML and should be taken into consideration when selecting candidates for this treatment strategy. Detailed analyses will be presented.

Impact of Hepatic Toxicities on Survival after Allogeneic Hematopoietic Cell Transplantation (HCT): Comparison of Reduced-Intensity Versus Conventional Conditioning Regimens at a Single Center.

[Purpose] To compare the incidence, onset and severity of hepatic toxicities and their ultimate impact on survival after reduced-intensity HCT (RIST) and conventional HCT (CST).[Patients and methods] We retrospectively reviewed the data of 151 patients with AML (n=86), MDS (32) and overt AML (33) who underwent allogeneic HCT after either RIST (n=78) or CST (73) between Jan, 2000, and Oct, 2004. Conditioning regimens for RIST included busulfan plus fludarabine or cladribine with or without 2–4 Gy TBI, and those for CST included cyclophosphamide with 12 Gy TBI or busulfan. The median age of the patients was 55 years (range, 15–67) for the RIST group and 37 years (1–57) for the CST group. Patients received marrow (n=64), G-CSF-mobilized PBSC (77) or cord blood (10) from either an HLA-matched relative (59), a mismatched relative (19) or an unrelated volunteer (73). The severity of liver toxicities was assessed by maximal measurements of total bilirubin, ALP, AST and ALT according to CTCAE v.3. Causes of hepatic toxicities were assessed basically by the clinical presentation and, in selected cases, by liver biopsy and autopsy results. The median follow-up of surviving patients was 1,401 days (598–2,317).[Results] The incidences of grade 3–4 hepatic toxicities in the RIST and CST groups were, respectively, 38% vs 38% at day 30, and 60% vs 56% at day 100. The timing of maximum hepatic toxicity was also similar between the two groups. However, the incidence of grade 3–4 hyperbilirubinemia was higher in the RIST group after day 60, probably due to the late onset of acute GVHD after the early tapering of immunosuppression in this group. In contrast, there was a trend for more grade 3–4 abnormal transaminase levels in the CST group than in the RIST group, probably due to the toxicities of drugs including myeloablative conditioning. The causes of grade 3–4 hepatic toxicities were GVHD (n=27), drugs (27), conditioning regimen (7), VOD (3), and others (13). Among the 68 RIST and 66 CST patients who survived more than 100 days after HCT, overall survival (OS) was significantly worse in those who developed grade 3–4 hyperbilirubinemia during the first 100 days after HCT than in those who did not (Figure; 1-year OS, 38% vs 76% after RIST and 33% vs 79% after CST). In a multivariate ananysis, grade 3–4 hyperbilirubinemia (HR 1.8 [95% CI, 1.2–2.7], p=0.02) and non-remission status before HCT (HR 2.5 [95% CI, 1.5–4.4], p=0.0001) were associated with an increased risk of mortality. However, grade 3–4 abnormality in ALP and AST/ALT during the first 100 days after HCT was not a significant risk factor for mortality.[Conclusions] Contrary to our expectations, our data showed a similar incidence of hepatic toxicities between the RIST and CST groups. This may be due to an older age population, the use of busulfan in the conditioning regimen, and the rapid tapering of immunosuppression in the RIST group. The outcome of patients who developed severe grade 3–4 hyperbilirubinemia was poor regardless of the conditioning regimen. [Display omitted]

Successful treatment of relapsed AML after allogeneic stem cell transplantation with azacitidine

Therapeutic options for patients with relapse of MDS or high risk AML after allogeneic stem cell transplantation are limited. We here present the case of a 64-year-old female patient with MDS, who received peripheral blood stem cells from her HLA-identical brother after a non-myeloablative conditioning regimen. Two months after allogeneic transplantation she suffered from a relapse, now fulfilling WHO criteria for AML with a bone marrow blast count of 91%. We then decided to treat her with azacitidine, a DNA methyltransferase inhibitor with proven antileukemic activity. The patient achieved a complete haematological response after two cycles and full donor chimerism after a single dose of donor lymphocytes. We postulate that azacitidine acts through a direct reduction of malignant cells and may in addition augment the immunologic effects of donor lymphocyte infusions.

Epigenetic therapy in MDS and acute AML: focus on decitabine

Epigenetic therapy in MDS and acute AML: focus on decitabine

Improved Remission Induction Rate of Childhood AML: Preliminary Results of the AML02 Trial.

The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine (A), daunorubicin (D), and etoposide (E) as Induction I; all subsequent therapy is risk-adapted. Pharmacokinetic, pharmacodynamic, and gene expression studies are performed after exposure to high-dose or low-dose cytarabine. Patients with no response (NR, ≥ 25% bone marrow blasts) to Induction I receive low-dose ADE + gemtuzumab ozogamicin (GO) as Induction II, whereas all others receive ADE. Minimal residual disease (MRD) is monitored by flow cytometry and is defined as ≥ 0.1% cells with leukemia-associated immunophenotypes among bone marrow mononuclear cells. Patients who are MRD+ after induction therapy receive GO as a single agent before consolidation chemotherapy or stem cell transplantation. Among the 112 patients enrolled since October 2002, 37 had -7, FAB M6 or M7, FLT3 internal tandem duplication (ITD), or MDS or treatment-related AML and were classified as high-risk (HR); 32 had AML1-ETO, CBFβ-MYH11, or MLL-AF9 (low-risk; LR); the remaining 43 were considered as standard-risk (SR). The rate of complete response (CR, <5% bone marrow blasts) was 84% after Induction I and 97% after Induction II. Of the 3 patients who did not attain CR, 2 had partial responses and 1 died of streptococcal sepsis during Induction II. CR rates differed according to risk group after Induction I (LR, 100%; SR, 91%, HR 61%) but not after Induction II (LR, 100%; SR, 98%, HR 94%). The association between risk groups and MRD (determined successfully in 95% of cases) was more striking: the rate of MRD negativity after Induction I was 88% in LR, 59% in SR, and 26% in HR patients; after Induction II, rates were 97%, 82%, and 53%, respectively. Response to initial therapy was particularly poor among patients with a FLT3-ITD: only 1 of 11 patients was MRD-negative after Induction I and only 3 were MRD-negative after Induction II. 5 of the 6 patients who had NR to Induction I attained CR after subsequent treatment with ADE + GO and all 6 had dramatic reductions in MRD levels (from a median of 26% to a median of 0.24%). An additional 14 patients with MRD levels 0.1% to 5.6% received GO at the beginning of consolidation: 9 became MRD-negative. Overall, there were 4 deaths from infection (2 bacterial, 1 fungal, 1 viral), which occurred during induction (1), consolidation (1), and after completion of chemotherapy (2). Because CNS relapse occurred in 3 of the first 33 patients treated, intrathecal therapy was changed from cytarabine alone to triple combination therapy with methotrexate, hydrocortisone, and cytarabine; none of the 79 subsequent patients has had a CNS relapse. The 1-year event-free and overall survival estimates are 76.8% (SE, 5.1%) and 89.1% (SE, 3.8%). In conclusion, AML02 therapy has produced a high overall remission rate and low treatment-related mortality, which we attribute to excellent supportive care and to the use of risk- and MRD-adapted therapy.

Relapse Free Survival of Elderly AML Patients Is Comparable to That of Younger Adults When Treated with a Modified Dose Intensive Cytarabine Regimen.

Introduction and Methods: Despite the development of new targeted therapies for treating AML, cytarabine (Ara-C) remains the most reliably effective agent, particularly when given in high dosage (HIDAC). The outcome of AML therapy in pts > 60 years of age remains dismal with slightly more than half of pts achieving remission, and < 15% becoming long term survivors. Elderly pts do not tolerate typical HIDAC regimens because of cerebellar and other toxicities, so we have employed a modified HIDAC regimen: Ara-C 2 gm/m2/day over 4 hours once daily on days 1–6, combined with daunorubicin 45 mg/m2 days 2,3,4 for induction. Consolidation consisted of one cycle of the same chemotherapy followed by two additional cycles of mHIDAC alone, unless the pt went on to autologous or allogeneic HPCT. We report a single institution experience with 59 consecutive pts ≥ 60 y/o (including 20 pts ≥ 70 y/o), and compare outcomes with 139 similarly treated pts < 60 y/o. Excluded were pts with antecedent MDS or chemotherapy-related AML, APL, and pts with major organ dysfunction.Results and Discussion: The CR rate for pts age ≥ 60 was 66% vs 81% for those < 60 (p=0.02). The distribution of pts into cytogenetic risk categories was similar, except for an under-representation of good risk cytogenetics among older pts (3% vs 14%, p=0.025). Overall survival for older pts was worse than that for younger individuals primarily due to an excess of early deaths, however, disease free survival among complete responders was similar for the 2 age groups (see below). There were 11 (16%) early deaths within 60 days among older pts, 4 with residual leukemia and 7 during aplasia. Early death occurred in 7 (5%) of the younger pts, 2 with residual leukemia and 5 during aplasia. There were 4 deaths (3 infectious and 1 stroke) during consolidation, 3 in the older and 1 in the younger group. There were also 2 late deaths in remission (both older pts) which were unrelated to AML or therapy. Clinically significant cerebellar toxicity was observed in 4 (6.8%) of the older pts and 3 (2%) of the younger pts, for a total of 7 episodes associated with 535 courses of Ara-C delivered (1.3%). 60% of older pts were able to complete 2 or 3 courses of consolidation therapy. Our data confirm the challenges of treating older pts with AML, but suggest that a significant subgroup can tolerate and benefit from aggressive therapy which includes dose intensification of Ara-C. This mHIDAC regimen is effective and well tolerated, and repeated administration to older pts is feasible. [Display omitted] [Display omitted]

Nuclear phospholipase C β1, regulation of the cell cycle and progression of acute myeloid leukemia

A large number of observations have hinted at the fact that location impinges on function of some of the main players of nuclear inositol lipid cycle. PLC beta1 is a well-known example, given that it has been shown that only the enzyme located in the nucleus targets the cyclin D3/cdk4 complex, playing, in turn, a key role in the control of normal progression through the G1 phase of the cell cycle. The PLC beta1 gene, which is constituted of 36 small exons and large introns, maps on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene itself. The chromosome band 20pl2 has been shown to be rearranged in human diseases such as solid tumors without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes. Moreover, non-specific alterations in chromosome 20 have been found in patients affected by MDS and acute myeloid leukemia AML. MDS is an adult hematological disease that evolves into AML in about 30% of the cases. The availability of a highly specific probe gave an opportunity to perform in patients affected with MDS/AML, associated with normal karyotype, painting and FISH analysis aimed to check the PLC beta1 gene, given that this signaling molecule is a key player in the control of some checkpoints of the normal progression through the cell cycle. FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PLC beta1 gene. In contrast, PLC beta4, another gene coding for a signaling molecule, located on 20pl2.3 at a distance as far as less than 1 Mb from PLC beta1, is unaffected in MDS patients with the deletion of PLC beta1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML, whilst the normal karyotype MDS patients, showing non-deletion of PLC beta1 gene, are still alive at least 24 months after the diagnosis. The immunocytochemical analysis using an anti PLC beta1 monoclonal antibody showed that all the AML/MDS patients who were normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC beta1. In contrast, the monoallelic deletion gave rise to a dramatic decrease of the nuclear staining suggesting a decreased expression of the nuclear PLC beta1. The reported data strengthen the contention of a key role played by PLC beta1 in the nucleus, suggest a possible involvement of PLC beta1 in the progression of MDS to AML and pave the way for a larger investigation aimed at identifying a possible high risk group among MDS patients with a normal karyotype.