post-MDS Acute Myeloid Leukemia Research Articles

Comparison of outcomes of HCT in blast phase of BCR-ABL1− MPN with de novo AML and with AML following MDS

AbstractComparative outcomes of allogeneic hematopoietic cell transplantation (HCT) for BCR-ABL1− myeloproliferative neoplasms (MPNs) in blast phase (MPN-BP) vs de novo acute myeloid leukemia (AML), and AML with prior myelodysplastic syndromes (MDSs; post-MDS AML), are unknown. Using the Center for International Blood and Marrow Transplant Research (CIBMTR) database, we compared HCT outcomes in 177 MPN-BP patients with 4749 patients with de novo AML, and 1104 patients with post-MDS AML, using multivariate regression analysis in 2 separate comparisons. In a multivariate Cox model, no difference in overall survival (OS) or relapse was observed in patients with MPN-BP vs de novo AML with active leukemia at HCT. Patients with MPN-BP in remission had inferior OS in comparison with de novo AML in remission (hazard ratio [HR], 1.40 [95% confidence interval [CI], 1.12-1.76]) due to higher relapse rate (HR, 2.18 [95% CI, 1.69-2.80]). MPN-BP patients had inferior OS (HR, 1.19 [95% CI, 1.00-1.43]) and increased relapse (HR, 1.60 [95% CI, 1.31-1.96]) compared with post-MDS AML. Poor-risk cytogenetics were associated with increased relapse in both comparisons. Peripheral blood grafts were associated with decreased relapse in MPN-BP and post-MDS AML (HR, 0.70 [95% CI, 0.57-0.86]). Nonrelapse mortality (NRM) was similar between MPN-BP vs de novo AML, and MPN-BP vs post-MDS AML. Total-body irradiation–based myeloablative conditioning was associated with higher NRM in both comparisons. Survival of MPN-BP after HCT is inferior to de novo AML in remission and post-MDS AML due to increased relapse. Relapse-prevention strategies are required to optimize HCT outcomes in MPN-BP.

Leukemic Stem Cell Phenotype Is Associated with Mutational Profile in Acute Myeloid Leukemia

Background: Understanding leukemic stem cell (LSC) is important for acute myeloid leukemia (AML) treatment. As such, understanding the relationship between LSC and genetically defined sub-clones can, in turn, help to understand the heterogeneity of AML. However, to date, there are only a few reports specifically focusing on this topic. To this end, we conducted this study to (1) examine the phenotypic diversity of AML-LSC, (2) explore the association between AML-LSC phenotypes and gene mutations, and (3) investigate the prognostic implications of AML-LSCs. Methods: Mononuclear cells (MNCs) were isolated from the patient's bone marrow aspirates by ficoll gradient centrifugation and cryopreserved in serum-free medium. Stored cells were thawed to Iscove's Modified Dulbecco's Medium (IMDM) and washed with fluorescence-activated cell sorting (FACS) buffer [1% FBS, Dulbecco's Phosphate-Buffered Saline (DPBS)]. Cells were stained with following anti-human monoclonal antibodies: CD45-APC/cy7, CD34-APC, CD38-BV421, CD90-PE, CD123-PE/Cy7, CD45RA-PerCP/Cy5.5. Analyses were performed on a FACSCanto II (HTS) (BD Bioscience) and FlowJo V 10.0 (BD Bioscience) program. For sequencing, the DNA capture probes for 76 target genes were designed using the Agilent SureDesign web-based application. The target regions included protein coding exons with 10 bp intron flanking regions and hot spot regions of the 20 genes involved in recurrent translocations. DNA was extracted on a Chemagic 360 instrument (Perkin Elmer, Baesweiler, Germany). The genomic DNA was sheared using Covaris S220 focused‐ultrasonicator (Covaris, Woburn, MA). We used 50ng of total input genomic DNA. A library preparation was performed according to Agilent's SureSelectQXT Target Enrichment protocol. Paired-end 150-bp sequencing was using NextSeq 550 Dx platform (Illumina, San Diego, CA). Targeted sequencing raw data was obtained in FASTQ format. Results: In secondary AML patients, MPP-like LSC was significantly higher than de-novo AML (p=0.0037), and was higher in MPN-AML than in MDS-AML (p=0.0485). There was no correlation between age and LSC phenotype, though CD34+CD38- subpopulation was enriched in younger patients (<65 yrs). Mutations of KRAS and NRAS were frequently observed in MPP-like LSC dominant patients (3/14 and 4/14), TP53 and ASXL1 mutations in LMPP-like LSC dominant patients (4/12 and 4/12) , and CEBPA, DNMT3A and IDH1 (6/12, 4/12, and 3/12) mutations in GMP-like LSC dominant patients. Furthermore, as shown in Figure, KRAS mutation was significantly associated with the percentage of MPP-like LSC phenotype (p=0.0540), and TP53 mutation with the percentage of LMPP-like LSC phenotype (p=0.0276). When the patients were separated according to the combined risk including next generation sequencing data, the poorer the prognosis, the higher the LMPP-like LSC expression (p=0.0052). The importance of our study lies in that we showed for a given AML patients there is a dominant LSC phenotype and LSCs are associated with clinical outcomes, supporting the significance of cancer stem cell model for human AML. First of all, based on detailed characterization of the surface immunophenotype of AML LSCs we found that AML show evidence of a hierarchical cellular organization. We also recognized that the composition of LSC phenotypes is associated with AML phenotypes. For example, secondary AML patients showed higher fraction of MPP-like LSCs compared to de novo AML patients. In this regard, the higher expression of MPP-like LSCs could explain the poor response to standard treatments traditionally associated with secondary AML. Furthermore, the higher expression of MPP-like LSCs in post-MPN AML compared to post-MDS AML could explain the dismal prognosis associated with post-MPN AML, despite the relative indolent clinical course in their chronic phase and the presence of druggable target. Conclusion: In conclusion, our findings provide better insights into the characteristics and clinical implications of LSC. Although in a small scale, we provide evidence that specific LSC phenotypes are associated with certain mutations thus should be in the AML risk stratification process. Figure Disclosures Yoon: Janssen: Consultancy; Kyowa Hako Kirin: Research Funding; Genentech, Inc.: Research Funding; Yuhan Pharma: Research Funding; MSD: Consultancy; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

A novel non-invasive monitoring assay of 5-azacitidine efficacy using global DNA methylation of peripheral blood in myelodysplastic syndrome.

Purpose: Monitoring response and resistance to 5-azacitidine (AZA) is essential when treating patients with myelodysplastic syndrome (MDS). To quantify methylated DNA not only in the promoter region but also in the gene body, we established a single-molecule methylation assay (SMMA).Patients and methods: We first investigated the methylation extent (expressed as methylation index [MI]) by SMMA among 28 MDS and 6 post-MDS acute myeloid leukemia patients. We then analyzed the MI in 13 AZA-treated patients.Results: Whole-blood DNA from all 34 patients had low MI values compared with healthy volunteers (P<0.0001). DNA hypomethylation in MDS patients was more evident in neutrophils (P=0.0008) than in peripheral mononuclear cells (P=0.0713). No consistent pattern of genome-wide DNA hypomethylation was found among MDS subtypes or revised International Prognostic Scoring System (IPSS-R) categories; however, we found that the MI was significantly increased for patients at very high risk who were separated by the new cytogenetic scoring system for IPSS-R (P=0.0398). There was no significant difference in MI before AZA, regardless of the response to AZA (P=0.8689); however, sequential measurement of MI in peripheral blood demonstrated that AZA non-responders did not have normalized MI at the time of next course of AZA (P=0.0352).Conclusion: Our results suggest that sequential SMMA of peripheral blood after AZA may represent a non-invasive monitoring marker for AZA efficacy in MDS patients.

Comparison of Outcomes of Allogeneic Hematopoietic Cell Transplantation in Patients with Acute Myeloid Leukemia (AML) with Antecedent History of Philadelphia-Negative Myeloproliferative Neoplasm with De Novo AML and with AML Arising from Myelodysplastic Syndrome: A Study from the Center for International Blood and Marrow Transplant Research (CIBMTR)

Allogeneic hematopoietic cell transplantation (HCT) is the only treatment modality associated with long-term disease control in patients with acute myeloid leukemia (AML) with antecedent history of Myeloproliferative neoplasms (MPN), however outcomes data are scanty. We compared the outcomes of 177 patients with AML with prior MPN (post MPN AML) with 4749 patients with de no AML, and 1104 patients with AML with prior myelodysplastic syndrome (Post MDS AML) with an aim to identify patient, disease and transplant related factors associated with outcomes. The study cohort included patients aged ≥ 40 years undergoing first HCT between 2001 and 2015. Patients with Philadelphia or BCR-ABL positive MPN, undergoing transplants from syngeneic donors, cord blood transplants, or ex vivo T-cell depletion were excluded. Comparisons of outcomes of HCT were done in post MPN AML versus de novo AML and post MPN AML versus post MDS AML using multivariate regression analysis. A higher proportion of patients with post MPN AML had monosomal or non-monosomal poor risk karyotype (39%) in comparison to de novo AML (23%) and post MDS AML (34%). Active leukemia at HCT was seen in 52%, 26% and 42% in patients with post MPN AML, de novo AML and post MDS AML, respectively. In multivariate cox model, patients with post MPN AML in morphological leukemia free state (blasts Patients with post MPN AML are at high risk of relapse compared to de novo AML and post MDS AML. Peripheral blood grafts result in lower relapse risk in patients with post MPN and post MDS AML. Relapse prevention strategies are required to optimize the outcomes of HCT in post MPN AML.

TheJAK2gene as a protagonist in chronic myeloproliferative neoplasms

The identification of the association of a JAK2 gene mutation with chronic myeloproliferative neoplasms (cMPN) negative for BCR-ABL(1,2) has allowed significant advances in the understanding of this group of hematologic diseases. The JAK2 gene, located on chromosome 9p24, encodes the JAK2 protein which is a cytoplasmic tyrosine kinase that plays an important role in the signal transduction of various hematopoietic growth factors. The JAK2V617F mutation results in the substitution of the amino acid valine for phenylalanine in the pseudokinase domain (JH2) causing constitutive activation of the kinase domain (JH1) and hypersensitivity to protein growth factors. Among the BCR-ABL negative cMPN cases, the JAK2617F mutation occurs at a frequency of 96% in polycythemia vera (PV), and 50% of essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (MF) patients(2). The association of this mutation with BCR-ABL negative cMPN has contributed to improve the diagnosis, classification and treatment of patients, in particular in respect to PV. Tefferi and Pardanani(3) suggested that an investigation of the JAK2V617F mutation in the peripheral blood should be integrated into the initial assessment of patients with suspected diagnosis of PV and of those of thrombocytosis of unknown cause, with thrombotic complications, including cerebral or abdominal thrombosis, and other clinical manifestations of myeloproliferative diseases. Other less frequent mutations were also found in the JAK2 gene in JAK2V617F-negative PV patients, as well as in other myeloproliferative neoplasms. Several studies report deletions, point mutations and duplications(4) mainly affecting the seven highly conserved amino acid residues (F537-F547) in the JAK2 protein. PV patients positive for these mutations are often heterozygous for the mutation and are characterized by the predominance of myelopoesis, serum erythropoietin levels below normal and lower age at diagnosis(3-5). The clinical evolution of these patients is similar to JAK2V617F-positive PV patients(5). The V617F mutation of the JAK2 gene triggers three clinical manifestations and there is evidence that genetic and additional epigenetic events contribute to the pathogenesis(6). In addition, other genes, such as the MPL, TET2 and ASXL1 genes, may also be mutated with the accumulation of mutations possibly explaining the different phenotypes observed in MPN. The MPL gene, located on chromosome 1p34, encodes the thrombopoietin receptor (cMPL). Its expression is important for growth and survival of megakaryocytes. Some mutations in this gene result in gain of function and have been associated with thrombocytosis, splenomegaly, myelofibrosis and an increased risk of thrombosis(7). Mutations in the transmembrane domain of cMPL were observed in nine patients negative for the JAK2V617F mutation (MPLW515L and MPLW515K); mutations were also detected in JAK2V617F-positive patients. The TET2 (4q24) gene has many mutations (frameshift, missense, nonsense) that are observed in JAK2V617F cMPN-positive (17%) and JAK2V617F-negative patients (7%), with mutational frequencies of approximately 16% in PV, 5% in ET, 17% in MF, 14% in post-PV MF, 14% in post-TE MF and 17% in blast-phase MPN(8). The main function of the TET2 protein is the conversion of 5-methyl-cytosine to 5-hydroxymethyl cytosine; it eventually affects the epigenetic regulation of transcription. The ASXL1 gene maps to chromosome 20q11.1 and belongs to the enhancer of trithorax and polycomb gene family. The function of this gene is believed to include dual activator/suppressor activity toward transcription and includes repression of retinoic acid receptor-mediated transcription. Mutations in this gene are associated with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CML)(9). In a recent study of 300 patients with a spectrum of non-MPN myeloid malignancies, ASXL1 gene mutations were found in 62 patients (~21%): ~7% in MDS without excess blasts, 11-17% in MDS with ring sideroblasts, 31% in MDS with excess blasts, 23% in post-MDS acute myeloid leukemia (AML), 33% in CML and 30% in primary AML. It was observed that mutations of the ASXL1 gene occur in MPNs in both chronic and blast phases. In a study of 64 patients with ET (n = 35), MF (n = 11), PV (n = 10), blast-phase MPN (n = 5) and unclassifiable MPNs (n = 3), mutations of this gene in heterozygosis were identified in five JAK2V617F-negative patients (~ 8%; 3 MF, 1 ET and 1 blast-phase ET)(10). There are other reports in the literature associating gene mutations to BCR-ABL-negative cMPN. The knowledge about genotype-phenotype interaction could elucidate the molecular mechanisms and contribute to improvements in diagnosis, in the status and in the treatment of patients with ET, MF and PV.

Hesk Ratio: A Complementary Tool for the Diagnosis of Myelodysplastic Syndromes and a Novel Method for Flow Cytometry Analysis

AbstractAbstract 4946Establishing the diagnosis of Myelodysplastic Syndromes (MDS) is a challenging task for hematologists due to the heterogeneity of this clinical entity. Several attempts have been made to include findings from advanced technologies to the diagnostic criteria of MDS, but still in the majority of cases, morphology of peripheral blood and bone marrow remains the cornerstone for the diagnosis. Flow cytometry(FC) can identify abnormal antigen expression on myeloid cells. FC has been proposed as a complementary method in the diagnosis of low and intermediate risk MDS, particularly for patients not exhibiting characteristic karyotype abnormalities. On the other hand, recent literature suggests that these findings are not MDS-related, questioning the specificity of immunophenotyping for the diagnosis of MDS.The aim of the present study is to maximize the utility of FC data and simplify their interpretation for the diagnosis of MDS, by developing new analytical approaches of digital data, other than the conventional sequential biparametric analysis. The applied methodology was based on a mathematical model of scale analysis.Bone marrow(BM) samples from 50 subjects were analysed for the expression of CD45PC7, CD11bPC5, CD16FITC and CD13PE (antigens by Beckman Coulter, FC500 flow cytometer Beckman Coulter). 36 patients were diagnosed with MDS (23 low risk, 13 high risk) and 14 patients had other than an MDS diagnosis (ITP, chronic idiopathic neutropenia, systemic lupus erythematosus, LGL leukemia, age-related cytopenias, aplastic anemia, myelofibrosis etc). Additionally, 3 BM samples of patients with post-MDS acute myeloid leukemia(AML) were analysed. 1.The data used for the development of the mathematical model were the following: two populations (neutro1, neutron2) were gated according to their CD45 and CD13/CD16 antigen expression (Figure 1i-1v).2.Seven subpopulations of Neutrophils were defined on CD11b/CD16 density plot N=g+h+i and O=k+j (Figure 1vi).In an attempt to identify correlations between data that cannot be routinely revealed by sequential biparametric analysis, we have developed the HeSK* ratio, which is given by: [Display omitted] where x is the median of CD11b in gate O, y is the median of CD16 in gate O, z is the median of CD45 in gate neutro, pO is the percentage of gate O in the total CD11b/CD16 diagram gated in neutro, pN is the percentage of gate N in the total CD11b/CD16 gated in neutro and 1000 is an empirical parameter.The HeSK ratio combines fluorescence levels of CD16, CD11b and CD45 with the percentage of two distinct neutrophil populations (N and O), which differ in their maturation and differentiation stage. The ratio can quantify the abnormal differentiation profile of mature myeloid cells and thus distinguish MDS from non-MDS samples with statistical significance P<0. 0001 (Kruskal Wallis test) as indicated in graph 1. Descriptive statistics are shown in table 1.· HeSK ratio is based upon a novel FC analysis method that could change the conventional biparametric routine FC analysis and quantify patterns that are not evaluated properly. Mathematical modeling of antigen expression patterns optimizes the interpretation of single immunophenotype findings.· The present study proposes HeSK as a complementary diagnostic tool for MDS and a strong indicator for the classification of the patients according to their prognosis as well.*the name HeSK comes from the initials of the 4 main authors (H=Hala, e=Evgenia, S=Smirni, K=Kolliopoulou).Table 1non MDSlow risk MDShigh risk MDSNumber of values142313Minimum50,764,7890,285025% Percentile304,826,1117,05Median213392,5247,6475% Percentile10650228,9144,3Maximum550403043671,7Mean10320316,1122,7Std. Deviation17860647,9185,1Std. Error4773135,151,33 [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Azacytidine and erlotinib exert synergistic effects against acute myeloid leukemia

The term myelodysplastic syndrome (MDS) identifies a heterogeneous group of clonal disorders originating from bone marrow stem cells that often progress to acute myeloid leukemia (AML). The reference treatments for MDS include the DNA methyltransferase inhibitors azacytidine and decitabine. Recently, the epidermal growth factor receptor (EGFR) inhibitor erlotinib has been shown to exert antileukemic activity in vitro and in vivo, independent of the EGFR. Thanks to this feature, erlotinib is currently being tested as an antileukemic drug in clinical trials. Here, we report that azacytidine and erlotinib mediate synergistic antineoplastic effects in several primary or secondary (post-MDS) AML cell lines. The combination of azacytidine and erlotinib blocked cell-cycle progression and induced caspase-dependent apoptosis more consistently than either of the two agents alone. These effects were not a consequence of cellular differentiation and could be discriminated from each other, as the former depended on caspases whereas the latter did not. The synergy between azacitidine and erlotinib, which involved the proteasomal degradation of the anti-apoptotic Bcl-2 family members MCL-1 and BCL2L10 and the upregulation of their pro-apoptotic counterpart PUMA, was abolished when azacytidine was replaced by decitabine but persisted when erlotinib was substituted with gefitinib, another EGFR inhibitor. Of note, the intracellular accumulation of azacytidine was exacerbated by both erlotinib and gefitinib, pointing to a pharmacokinetic mechanism of synergy. In approximately half of the cases studied, marrow and circulating blasts from MDS and AML patients, respectively, exhibited hyperadditive cytotoxic responses to the combination of azacytidine and erlotinib. These results strongly suggest that the combination of azacytidine and erlotinib may exert clinically relevant antileukemic effects.

IDH Mutations and Trisomy 8 In Myelodysplastic Syndromes and Acute Myeloid Leukemia

AbstractAbstract 4009Trisomy 8 is the most common among sole cytogenetic abnormalities in both acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In the very first paper published on isocitrate dehydrogenase (IDH) mutations in AML, 13 of the 16 IDH1 mutations detected were associated with normal karyotype, 2 with trisomy 8 and one with trisomy 13. Trisomy 8 was also recurrent in patients with IDH1/IDH2-mutated post-MDS AML. In the current study we examined the prevalence and disease distribution of IDH1 and IDH2 mutations in a large (n=157) group of patients with hematologic malignancies and isolated trisomy 8. The Mayo Clinic cytogenetic database allowed identification of 157 patients with isolated trisomy 8. Archived bone marrow cell pellets were used to extract DNA for IDH1 and IDH2 mutation analysis. Eighteen IDH mutations were identified: 15 IDH2 (14 R140Q and one R140W) and 3 IDH1 (2 R132C and one R132G). Seventeen of the 18 IDH mutations occurred in myeloid malignancies whereas one (IDH2R140W) occurred in a patient with angioimmunoblastic lymphoma who was not previously exposed to chemotherapy or radiotherapy, and in whom the IDH2 mutation disappeared after effective lymphoma chemotherapy. Among the 17 IDH-mutated myeloid malignancies, disease-specific IDH1/IDH2 mutational frequencies were as follows: 27% (3/11) for post-MDS AML, 25% (3/12) for therapy-related MDS/AML, 15% (8/54) for de novo MDS, 13% (2/15) for de novo AML and 3% (1/32) for myeloproliferative neoplasm (MPN). In contrast, IDH mutational frequencies were significantly lower among 64 additional patients with AML or MDS without isolated trisomy 8: 7% in de novo AML (n=28), 0% in de novo MDS (n=21), 0% in post-MDS AML (n=11) and 0% in therapy-related MDS/AML (n=4). In the 54 patients with trisomy 8-associated de novo MDS, prognosis was similar between IDH mutated (n=8; median survival 14 months) and unmutated (n=46; median survival 16 months) cases (p=0.7). The majority of IDH-mutated cases with de novo MDS belonged to high risk MDS disease category. However, 3 of the 8 IDH-mutated patients with de novo MDS and 2 of the 3 with therapy-related MDS did not display excess bone marrow blasts. The current study suggests a possible association between IDH mutations and trisomy 8 in AML and MDS but not in MPN or MDS/MPN. The fact that the mere presence of trisomy 8 did not result in a more than expected incidence of IDH mutations in MPN or MDS/MPN makes it unlikely that such an association would be secondary to trisomy 8-associated genetic or biologic changes. Instead, it is possible that IDH mutations, which have been shown to cluster with high-risk disease in both MPN and MDS associated with 5q-, promote a selective advantage for the survival of the clone that harbors trisomy 8. Consistent with this contention, we were able to demonstrate in one of our patients with relapsed AML, the presence of IDHR132C both at initial AML diagnosis and time of relapse, whereas the trisomy 8 abnormality was seen only at the time of relapse. Regardless, the presence of molecular heterogeneity among patients with trisomy 8-associated AML or MDS might explain the controversial prognostic influence of the specific cytogenetic abnormality. Disclosures:No relevant conflicts of interest to declare.

IDH mutations and trisomy 8 in myelodysplastic syndromes and acute myeloid leukemia

Abstract 4009 Trisomy 8 is the most common among sole cytogenetic abnormalities in both acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In the very first paper published on isocitrate dehydrogenase (IDH) mutations in AML, 13 of the 16 IDH1 mutations detected were associated with normal karyotype, 2 with trisomy 8 and one with trisomy 13. Trisomy 8 was also recurrent in patients with IDH1/IDH2-mutated post-MDS AML. In the current study we examined the prevalence and disease distribution of IDH1 and IDH2 mutations in a large (n=157) group of patients with hematologic malignancies and isolated trisomy 8. The Mayo Clinic cytogenetic database allowed identification of 157 patients with isolated trisomy 8. Archived bone marrow cell pellets were used to extract DNA for IDH1 and IDH2 mutation analysis. Eighteen IDH mutations were identified: 15 IDH2 (14 R140Q and one R140W) and 3 IDH1 (2 R132C and one R132G). Seventeen of the 18 IDH mutations occurred in myeloid malignancies whereas one (IDH2R140W) occurred in a patient with angioimmunoblastic lymphoma who was not previously exposed to chemotherapy or radiotherapy, and in whom the IDH2 mutation disappeared after effective lymphoma chemotherapy. Among the 17 IDH-mutated myeloid malignancies, disease-specific IDH1/IDH2 mutational frequencies were as follows: 27% (3/11) for post-MDS AML, 25% (3/12) for therapy-related MDS/AML, 15% (8/54) for de novo MDS, 13% (2/15) for de novo AML and 3% (1/32) for myeloproliferative neoplasm (MPN). In contrast, IDH mutational frequencies were significantly lower among 64 additional patients with AML or MDS without isolated trisomy 8: 7% in de novo AML (n=28), 0% in de novo MDS (n=21), 0% in post-MDS AML (n=11) and 0% in therapy-related MDS/AML (n=4). In the 54 patients with trisomy 8-associated de novo MDS, prognosis was similar between IDH mutated (n=8; median survival 14 months) and unmutated (n=46; median survival 16 months) cases (p=0.7). The majority of IDH-mutated cases with de novo MDS belonged to high risk MDS disease category. However, 3 of the 8 IDH-mutated patients with de novo MDS and 2 of the 3 with therapy-related MDS did not display excess bone marrow blasts. The current study suggests a possible association between IDH mutations and trisomy 8 in AML and MDS but not in MPN or MDS/MPN. The fact that the mere presence of trisomy 8 did not result in a more than expected incidence of IDH mutations in MPN or MDS/MPN makes it unlikely that such an association would be secondary to trisomy 8-associated genetic or biologic changes. Instead, it is possible that IDH mutations, which have been shown to cluster with high-risk disease in both MPN and MDS associated with 5q-, promote a selective advantage for the survival of the clone that harbors trisomy 8. Consistent with this contention, we were able to demonstrate in one of our patients with relapsed AML, the presence of IDHR132C both at initial AML diagnosis and time of relapse, whereas the trisomy 8 abnormality was seen only at the time of relapse. Regardless, the presence of molecular heterogeneity among patients with trisomy 8-associated AML or MDS might explain the controversial prognostic influence of the specific cytogenetic abnormality. Disclosures: No relevant conflicts of interest to declare.

Gentuzumab-Ozogamicin, Citosine Arabinoside, G-CSF Combination (G-AraMy) in the Treatment of Secondary Acute Myeloid Leukemia in Elderly Patients.

Backround: Gentuzumab Ozogamicin (GO) is effective as single agent in the treatment of poor risk acute myeloid leukemia (AML) patients (pts). The aim of this study was to evaluate the efficacy and safety of a chemotherapy including growth factors, cytabine and GO in the treatment of secondary AML (sAML) elderly pts.Patients and Treatments: From September 2003 to September 2006, a total of 23 pts, median age 69 years (range 58–77) with sAML were enrolled in G-AraMy protocol which was divided in two phases. In the first phase from September 2003 to December 2004 11/23 pts received G-AraMy-1 treatment: rhG-CSF (5 μg/kg, on days 1–8), Aracytin as continuous perfusion (100 mg/m2 on days 4–8), GO (6 mg/m2 iv on day 9). In the second phase from January 2005 to September 2006 12 pts was treated according G-AraMy-2 protocol: G-CSF (5 μg/kg, on days 1–8), Ara-C as continuous perfusion (100 mg/m2 on days 2–8), GO (6 mg/m2 iv on day 9). In pts reaching complete (CR) or partial remission (PR), consolidation therapy was performed. In G-AraMy-1 this consisted of: G-CSF(5 μg/kg, on days 1–6), Ara-C as continuous perfusion (100 mg/m2 on days 2–6), GO (6 mg/m2 iv on day 7). G-Ara-My-2 group was consolidated with: G-CSF(5 μg/kg, on days 1–5), Ara-C (1 g/m2 every 12 hours on days 2–5), GO (6 mg/m2 iv on day 6).Results: Among the 23 treated pts 11 (48%) presented a post-MDS AML while 12 pts (52%) had received chemotherapy for a prior malignancy (3 Hodgkin's lymphoma, 5 breast, 2 thyroid, 1 gut, 1 bladder). Ten out 23 pts (43.5%) had previously received chemotherapy for AML being relapsed (4) or primary resistant pts (6) while 13 (56.5%) were untreated pts. Cytogenetic study was performed in all pts; karyotype was at “intermediate prognosis” in 11 cases, at “worse prognosis” in 7 cases, at “good prognosis” in 2 cases. In 3 pts no metaphases were observed. After induction and consolidation therapy 14 pts (6 group 1; 8 group 2) (61%) achieved a CR and 2 pts obtained PR. Five pts (22%) resulted refractory to treatment and 2 died during the aplasia period post induction treatment (1 due to sepsis, 1 due to cerebral haemorrhage). The most common adverse event was myelosuppression, as expected. No VOD was recorded. Seven pts (30%) developed documented infection (including pulmonary aspergillosis in 2 cases). Two pts died while in CR, 1 due to bladder cancer relapse and 1 to ischemic stroke. Nine of CR pts (39%) relapsed; at March 2007 5 pts (22%) are alive, of whom 1 are still in CR (4%). Median time to treatment failure (TTF) and median overall survival (OS) of whole population were 7.1 months (range 1–40.6+) and 8.6 months (range 1.7–40.6+) respectively. Stratifying pts according the two treatment groups median TTF was 4.4 months (range 3–10.5) in the first group and 7.2 months (range 1–40.6+) in the second; median OS was 6 months (range 1–13.6) in the first group and 9.1 months (range 1.7–40.6+) in the second.Conclusions: G-AraMy protocol could be considered an useful approach for elderly sAML pts considering the low reported side effects with a CR rate similar to that reported in literature. Unfortunately CR duration is brief. The modification of protocol schedule in the G-AraMy 2 group with the addition of more aggressive consolidation therapy seems to improve the duration of CR and OS.

Sequential Treatment with 5-Azacytidine, Valproic Acid (VPA), and All-Trans Retinoic Acid (ATRA) in Patients with High-Risk Acute Myeloid Leukemia (AML).

Probably through inhibition of histone deacetylases, VPA ± ATRA has been associated with clinical responses in patients with myelodysplastic syndrome (MDS) or AML (Kuendgen, Blood 2004; Raffoux, Haematologica 2005). Interestingly, VPA/ATRA-induced differentiation of AML cells has been documented in vivo (Nervi, ASH 2004 #1805). On the other hand, the combination of VPA with a demethylating agent synergistically enhances its antileukemia activity in vitro (Yang, Leukemia Res 2005). In a pilot phase, we treated 5 high-risk AML patients with a sequential combination of 5-azacytidine, VPA, and ATRA. Each treatment cycle comprised 75 mg/m2/d 5-azacytidine SC from day 1 to 7, 20 mg/kg/d VPA (then adapted on serum concentration) from day 1 to 28, and 45 mg/m2/d ATRA from day 8 to 28. Six cycles were planned, each starting at day 29 of the previous one whatever the hematological status. High-risk AML (AML-M3 excluded) was defined as:previously untreated de novo or post-MDS AML in patients aged 65+ unfit for intensive chemotherapy; orpost-MDS AML in first relapse or AML in second or subsequent relapse.Eligibility criteria also comprised WBC<30 G/L, PS<2, no severe infection, liver enzyme levels ≤ 2xULN, and serum creatinine ≤ 1.5xULN. Main patient characteristics and response are indicated in the Table below:PatientAgeAMLPrior TxBM blastsCyclesResponseTime to response#171ypost-MDS441%6CRp4 cycles#273ypost-MDS028%6CR4 cycles#376yde novo262%2Progression–#468ypost-MDS026%2Stable–#555yde novo220%4+AML-free3 cyclesPatient #3 progressed after the 2nd cycle. Treatment was also discontinued after the 2nd cycle in patient #4, because of ovarian carcinoma diagnosis. Response was observed after at least 3 cycles in the 3 remaining patients (1 CR, 1 CRp, 1 AML-free state with platelet count normalization), all treated as out-patients. Safety profile was excellent (no significant treatment-induced cytopenia). Main side effects were pain at 5-azacytidine injection sites and constipation. This treatment approach appearing promising in terms of efficacy versus toxicity, a formal multicenter Phase 2 study has been initiated.

Elevated MLF1 expression correlates with malignant progression from myelodysplastic syndrome.

MLF1 is a novel protein identified as the NPM-MLF1 chimeric protein produced by a t(3;5)(q25.1;q34) chromosomal translocation, which is associated with myelodysplastic syndrome (MDS), often prior to acute myeloid leukemia (AML), except for M3. The clinical features of t(3;5)-positive myeloid disorders suggest that this chimeric protein is involved in dysregulation of progenitor cells with the capability to differentiate into multiple lineages. So far, involvement of wild-type MLF1 in hematopoiesis or in leukemogenesis has not been fully investigated. In the present study, 65 patients with AML and 44 patients with MDS were tested for the expression of MLF1 using the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. A significantly higher level of MLF1 expression (ratio of MLF1/beta-actin mRNA >0.4) was readily detected in seven of 65 patients with de novo AML, three of 12 with post-MDS AML and seven of 44 with MDS, but not in any patients with ALL (n = 18). According to the FAB classification, high levels of MLF1 were found in patients with relatively immature subtypes of AML (M1, M2, M6 and M7) and high risk MDS (RAEB and RAEB-T). These findings indicate that the pattern of MLF1 expression is identical to the clinical morphology appearing in the t(3;5)-positive myeloid disorders and is correlated to the MDS-associated AML and transformation phase of MDS in t(3;5)-negative myeloid disorders. A CD34+ population of normal bone marrow cells preferentially expressed MLF1 with obviously decreasing levels of expression during maturation. Therefore, MLF1 normally functions in multi-potent progenitor cells and its dysregulation may take part in leukemogenesis from MDS.

A sole del(15q) anomaly in post-myelodysplasia acute myeloid leukemia

We report the second case of post-myelodysplasia acute myeloid leukemia (post-MDS AML) with a sole chromosome change del(15q). This anomaly is rarely seen. To our knowledge, only seven cases so far have been reported in human neoplasias, including one case each of acute myeloid leukemia (AML), acute lymphoid leukemia, post myelodysplasia AML, myelodysplastic syndrome, myelofibrosis, macroglobulinemia, Hodgkin's lymphoma and uterine leiomyoma. This case suggests that del(15q) is related to lympho-myeloproliferative disorders. Moreover, we speculate that certain oncogene(s) located on 15q might have some role in the progression of the disease, since the del(15q) anomaly appeared only in the AML phase in this case.

Ecotropic Virus Integration Site-1 Gene Preferentially Expressed in Post-Myelodysplasia Acute Myeloid Leukemia: Possible Association With GATA-1, GATA-2, and Stem Cell Leukemia Gene Expression

We investigated expression of the human ecotropic virus integration site-1 (EVI1) gene in patients with leukemia and myelodysplastic syndrome (MDS) using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The EVI1 transcripts were detected in 5 (10.0%) of 50 patients with de novo acute myeloid leukemia (AML), including two AML patients with trilineage myelodysplasia, and in 8 (34.8%) of 23 patients with post-myelodysplastic syndrome AML (post-MDS AML). EVI1 expression was also detected in 6 (35.3%) of 17 MDS patients and three of six patients with chronic myeloid leukemia (CML) in myelomegakaryoblast crisis. No EVI1 transcripts were detected in patients with acute lymphoid leukemia (n = 15) or CML in lymphoid blast crisis (n = 4). Chromosomal abnormalities at the 3q26 region, where the EVI1 gene is located, were found in one patient with MDS and two patients with CML myelomegakaryoblast crisis who had EVI1 expression. Our results showed that EVI1 expression was frequent in patients with post-MDS AML and AML with trilineage myelodysplasia, regardless of the presence or absence of 3q26 abnormalities. EVI1 expression was accompanied by expression of GATA-1 and GATA-2, and often by stem cell leukemia (SCL) gene expression. In patients with post-MDS AML, EVI1 expression was not always associated with a 3q26 abnormality, whereas EVI1 expression in CML myelomegakaryoblast crisis was often linked to a 3q26 abnormality. Our results suggest that the leukemogenic role of EVI1 expression may differ between post-MDS AML and leukemia, with EVI1 expression associated with a 3q26 abnormality.