Validation of a DNA Methylation Signature of Favorable Prognosis in Newly Diagnosed Acute Myeloid Leukemia.

Abstract Abstract 1615 Poster Board I-641 Epigenetic modifications are defined as heritable alterations in gene expression with no accompanying change in DNA sequence. Disruption of the epigenetic balance has major impact on chromatin structure and transcriptional activity. Hypermethylation of CpG islands within or near gene promoter regions is associated with gene silencing via transcriptional inactivation in human carcinogenesis. This gene silencing has the potential not only to affect disease progression, but also drug resistance and clinical outcome. We have previously reported that patients with Acute Myeloid Leukemia (AML) who are cured by conventional chemotherapy often display intense and simultaneous hypermethylation of multiple genes. To confirm these data, we evaluated the aberrant promoter methylation of these 9 genes in 68 patients with AML [excluding Acute Promyelocytic Leukemia, patients with inv-16 or t(8:21) and patients over the age of 65] enrolled on a clinical trial of conventional chemotherapy (idarubicin + cytarabine). Methylation at diagnosis was studied using bisulfite pyrosequencing. The patients had a median age of 52.5 years, median WBC count of 8,450/mm3, and the cytogenetic distribution was 3% favorable, 48% diploid and 49% poor-risk. Complete response rate was 71%. After a median follow-up of 24 months, median survival was 17 months, and median relapse-free survival 14 months. Dense methylation (>25% of CpG sites) was present in 7% of cases for HIN1; 12% for NOR1 and OLIG2; 15% for SLC26A4; 19% for NPM2 and P15INK4b; 24% for PGRA; 25% for CDH13 and 26% for PGRB genes. In univariate analyses, traditional factors like age, achievement of a complete remission, cytogenetics, history of myelodysplastic syndrome or myeloproliferative neoplasm, a sustained hematological response, and platelet count at diagnosis had their usual prognostic impact suggesting that this cohort is typical of the general AML population. Individually, dense methylation of each of NOR1, NPM2, HIN1, P15INK4b, SLC26A4, PGRA and PGRB genes was associated with a trend for improved overall survival, which was significant for PGRB (p=0.03) and near-significant for HIN1 (p=0.08) and NPM2 (p=0.1). A subgroup of 8 patients out of the 68 (11.8%) was strikingly distinct with 5 genes or more methylated in each case, and corresponded to a previously described CpG Island Methylator Phenotype (CIMP) in AML. These cases had similar age and cytogenetics as CIMP-negative cases. Median OS in CIMP-positive cases had not yet been reached at the time of analysis, compared to 15 months for CIMP-negative cases (p=0.03). The estimated 2-year survival rate was of 88% for CIMP-positive cases, compared to 40% for CIMP-negative cases (p=0.01). These results validate our previous findings of an association between increased DNA methylation and good prognosis in relatively young patients with AML who receive standard induction chemotherapy. The mechanism of this association between CIMP and survival is unknown, and we speculate that it relates to the inactivation of a gene that protects cells from the effects of chemotherapy. We conclude that epigenetic profiling using DNA methylation can help identify an AML patient subpopulation that may particularly benefit from conventional chemotherapy. Disclosures No relevant conflicts of interest to declare.

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Tritsomy 8 Acute Myeloid Leukemia Analysis Reveals New Insights of DNA Methylome with Identification of HHEX as Potential Diagnostic Marker

Biomarkers in Cancer ◽

10.4137/bic.s19614 ◽

2015 ◽

Vol 7 ◽

pp. BIC.S19614 ◽

Cited By ~ 4

Author(s):

Marwa H. Saied ◽

Jacek Marzec ◽

Sabah Khalid ◽

Paul Smith ◽

Gael Molloy ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Diagnostic Marker ◽

Cpg Island ◽

Global Analysis ◽

Extra Chromosome ◽

Chromosome 8 ◽

Trisomy 8 ◽

Acute Myeloid

Trisomy 8 acute myeloid leukemia (AML) is the commonest numerical aberration in AML. Here we present a global analysis of trisomy 8 AML using methylated DNA immunoprecipitation-sequencing (MeDIP-seq). The study is based on three diagnostic trisomy 8 AML and their parallel relapse status in addition to nine non-trisomic AML and four normal bone marrows (NBMs). In contrast to non-trisomic DNA samples, trisomy 8 AML showed a characteristic DNA methylation distribution pattern because an increase in the frequency of the hypermethylation signals in chromosome 8 was associated with an increase in the hypomethylation signals in the rest of the chromosomes. Chromosome 8 hypermethylation signals were found mainly in the CpG island (CGI) shores and interspersed repeats. Validating the most significant differentially methylated CGI ( P = 7.88 · 10–11identified in trisomy 8 AML demonstrated a specific core region within the gene body of HHEX, which was significantly correlated with HHEX expression in both diagnostic and relapse trisomy 8 AMLs. Overall, the existence of extra chromosome 8 was associated with a global impact on the DNA methylation distribution with identification of HHEX gene methylation as a potential diagnostic marker for trisomy 8 AML.

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Trisomy 8 in the Light of Recent Cytogenetic Classification Advances. A Study From the French Acute Myeloid Leukemia Intergroup.

Blood ◽

10.1182/blood.v114.22.2594.2594 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2594-2594 ◽

Cited By ~ 1

Author(s):

Nicolas Boissel ◽

Christine Terré ◽

Pascale Cornillet-Lefebvre ◽

Odile Maarek ◽

Eric Lippert ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Complete Remission ◽

Gene Mutation ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Prognostic Impact ◽

Trisomy 8 ◽

Factors Associated ◽

Acute Myeloid ◽

Similar Incidence

Abstract Abstract 2594 Poster Board II-570 Background: Trisomy 8 (+8) is one of the most common cytogenetical abnormality observed in acute myeloid leukemia (AML). The prognostic impact of +8 as sole aberration remains unclear and +8 may be classified either within intermediate- or high-risk subgroups. Recently, the prognostic impact of cytogenetic in AML has been refined by the identification of: 1) favorable genotypes in cytogenetically normal (CN) AML defined by the presence of either NPM1 gene mutation (NPM1m) or CEBPA gene mutation (CEBPAm) and the absence of FLT3 duplication (FLT3/ITD); 2) highly unfavorable AML with monosomal karyotype (MK). The aim of this study was to precise the prognostic impact of: 1) additional +8 in various cytogenetic risk subgroups; and 2) +8 as sole aberration when compared to different CN-AML genotypes. Patients: A total of 2087 patients with AML (AML-M3 excluded) were treated in the LAM-2001, LAM-SA-2002, ALFA-9802 and ALFA-9801 studies from the French AML Intergroup. After central review, cytogenetic analysis was considered successful in 1796 patients. Abnormalities were categorized according to the French AML Intergroup classification. All analysis (complete remission, CR; overall survival, OS; probability of continuous complete remission, %CCR) were stratified on studies. Results: +8 was present in 171/1796 (9.5%) with a similar incidence among the different cytogenetic subgroups: 22/243 fav-risk (9.1%), 99/1121 int-risk (8.8%), and 50/432 unfav-risk (11.6%). The incidence of +8 was significantly higher in MK-AML versus non MK-AML (30/223, 13.5%, p=.04). In none of these subgroups (fav, int, unfav, and MK), the presence of +8 was associated with a significantly different outcome (CR, OS, %CCR). When compared to patients with CN-AML, the 78 patients with +8 as sole anomaly had a similar age, a lower WBC (median WBC: 5 G/L vs 11.5 G/L, p=.004), a similar incidence of FLT3/ITD (22.2% vs 23.7%, 6/27 vs 101/426, p=.99), and a lower incidence of NPM1m (23.8% vs 46.5%, 5/21 vs 187/402, p=.05). In patients with +8 as sole anomaly, prognostic factors associated with a shorter OS were age (p=.01), high WBC (p=.01), and presence of +8 in all analyzed metaphases which was found in 1/3 of patients (p=.05). In those patients, when compared to CN-AML in general, CR rate was similar (88% vs 87%, p=.99), but %CCR and OS were shorter without, however, reaching significance (5y-%CCR: 31.8% vs 45.7%, p=.18). When compared to CN-AML patients with favorable genotypes (NPM1m or CEBPAm w/o FLT3/ITD), patients with +8 as sole anomaly had now a lower CR rate (87% vs 93%, p=.13) and significantly shorter %CCR and OS (5y-%CCR: 37.4% vs 57.8%, p=.05; 5y-OS 35.6% vs 59.0%, p=.05). Conversely, the prognosis of patients with +8 as sole anomaly appeared similar to that of patients with CN-AML w/o favorable genotypes (5y-OS: 32.6%). Conclusion: We report here the largest cohort of patients with +8. Additional +8 is equally distributed among cytogenetic risk subgroups and does not impact prognosis in each of these subgroups. Patients with AML with +8 as sole anomaly have an outcome comparable to that of CN-AML without favorable genotypes, suggesting that these patients should be managed similarly. Disclosures: No relevant conflicts of interest to declare.

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DNMT3A mutations Predict for Inferior Outcome in NPM1-Wildtype and Molecular Unfavorable Cytogenetically-Normal Acute Myeloid Leukemia: A Study of the German-Austrian AMLSG

Blood ◽

10.1182/blood.v118.21.415.415 ◽

2011 ◽

Vol 118 (21) ◽

pp. 415-415 ◽

Cited By ~ 1

Author(s):

Verena I. Gaidzik ◽

Richard F. Schlenk ◽

Peter Paschka ◽

Anja Stölzle ◽

Andrea Corbacioglu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Negative Impact ◽

Epigenetic Modification ◽

Methylation Status ◽

Prognostic Impact ◽

Sequencing Analysis ◽

Mutational Status ◽

Acute Myeloid

Abstract Abstract 415 Background: Alteration of DNA methylation, a hallmark of epigenetic modification, is currently discussed as one important pathomechanism in leukemogenesis. Using a next-generation sequencing approach, a frameshift mutation of the gene encoding the DNA methyltransferase (DNMT3A) in an acute myeloid leukemia (AML) case was identified. DNMT3A catalyses the addition of a methyl group to the cytosine residue of CpG dinucleotides, thereby affecting promoter methylation status and gene expression. Subsequent sequencing analysis in an independent cohort of 288 AML patients (pts) revealed DNMT3A mutations (DNMT3Amut) in 22% of the pts; mutations were associated with intermediate-risk cytogenetics and poor outcome. Aims: To evaluate frequency and clinical impact of DNMT3Amut in pts with AML aged 18 to 61 years who were treated within AMLSG treatment trials AML HD98A (Schlenk et al., J Clin Oncol 2010;28:4642–8) and AMLSG 07–04 (NCT00151242). Methods: DNMT3A mutation analysis was performed in 1218 AML (HD98A, n=685; AMLSG 07–04, n=533; de novo AML, n=1102; s-AML, n=45; t-AML, n=69) using a DNA-based PCR assay for all coding exons (1 to 23) followed by direct sequencing. The median follow-up was 5.06 years. Results: DNMT3A mut were found with an overall frequency of 19.6% (239/1218); 189 mutations were located in the MTase domain clustering at amino acid R882 (79%). All but one mutation were heterozygous; only 4 cases had two mutations. DNMT3A sequence alterations included 17 frameshift, 4 nonsense, and 222 missense mutations. DNMT3A mut pts were significantly older (P=.01), more frequently females (P=.001), had higher white blood cell and platelet counts (both P<.0001), and higher bone marrow blasts percentage (P=.001). DNMT3Amut were associated with cytogenetically-normal AML (CN-AML, P<.0001), while DNMT3Amut were rare in favorable and adverse-risk karyotypes (P<.0001). Correlations with other molecular markers (NPM1, CEBPA, FLT3, IDH1/2, TET2, ASXL1) revealed a significant association with NPM1 (P<.0001), FLT3-ITD (P<.0001), and IDH1/2 (IDH1R132, P<.0001; IDH2R140, P=.0003; IDH2R172, P=.03) mutations, while co-occurrence of CEBPA (P=.02) and ASXL1 (P=.02) mutations was less frequent. DNMT3A mutational status did not impact complete remission (CR) rate, event-free (EFS) and relapse-free survival (RFS), neither in the whole cohort (P=.09, P=.98, P=.11; respectively) nor in the subgroup of CN-AML (P=.39, P=.79, P=.19, respectively). DNMT3Amut had a negative impact on overall survival (OS) in trend in the whole cohort (P=.07) and significantly in CN-AML (P=.02). In multivariable analyses, DNMT3Amut were in trend associated with a negative prognostic impact on OS (hazard ratio, 1.24; P=.06). In addition, we performed subgroup analyses according to (1) the NPM1 mutational status, and (2) the molecular risk groups of CN-AML (as defined by the European LeukemiaNet classification). DNMT3Amut did not impact OS in NPM1-mutated patients in the whole cohort as well as in CN-AML (P=.34; P=.22; respectively), while in NPM1-wildtype patients DNMT3Amut were associated with inferior OS in both, the whole cohort and in CN-AML (P=.001; P=.005; respectively). In molecular unfavorable CN-AML (NPM1-wildtype with or without FLT3-ITD, NPM1-mutated with FLT3-ITD, CEBPA-wildtype), DNMT3Amut were significantly associated with worse OS (P=.002) compared with DNMT3A-wildtype pts, even outweighing FLT3-ITD as an unfavorable prognostic marker. There was no effect of DNMT3Amut in molecular favorable-risk CN-AML. Conclusions: DNMT3A mutations are confirmed as frequent genetic aberrations in AML, associated with normal karyotype, NPM1, FLT3-ITD, and IDH1/2 mutations. DNMT3Amut predicts for inferior outcome in molecularly-defined subsets of AML, that is, NPM1-wildtype AML and molecular unfavorable CN-AML. As a single marker, DNMT3Amut only had a moderate effect on outcome. Disclosures: No relevant conflicts of interest to declare.

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EZH2 Mutations Are Associated with Low Blast Percentage in Bone Marrow and −7/Del(7q) Karyotype in De Novo Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2544.2544 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2544-2544

Author(s):

Xiuli Wang ◽

Haiping Dai ◽

Qian WANG ◽

Qinrong Wang ◽

Yang Xu ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Myelogenous Leukemia ◽

Prognostic Impact ◽

Coding Region ◽

Acute Myeloid

Abstract Abstract 2544 Somatic mutation of the EZH2 gene is seen in myelodisplastic syndrome, myelofibrosis, and chronic myelomonocytic leukemia patients. The prevalence and prognostic impact of somatic mutations of EZH2 in patients with acute myelogenous leukemia (AML) remains unknown. In this study, we sought to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo AML by PCR amplification of the entire coding region followed by direct bidirectional DNA sequencing. EZH2 mutations were identified in 13/714 (1.8%) of AML patients and occurred almost exclusively in males (11/13, P=0.033). In univariate analysis, the presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P=0.0001) and −7/del(7q) (P=0.025). There was no difference in the incidence of mutations in 13 genes, including ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. Complete remission, event-free survival or overall survival was similar between AML patients with and without EZH2 mutation (p>0.05). These results demonstrated EZH2 mutation as a recurrent genetic abnormality associated with lower blast percentage in BM and −7/del(7q) in de novo acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

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Loss of H3K27 Methylation Identifies Poor Outcome in Adult-Onset Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-139387 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 24-24

Author(s):

Anneke D. van Dijk ◽

Fieke W Hoff ◽

Yihua Qiu ◽

Eveline S. de Bont ◽

Sophia W.M. Bruggeman ◽

...

Keyword(s):

Dna Methylation ◽

Overall Survival ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Molecular Genetic ◽

Functional Enrichment ◽

Prognostic Impact ◽

Cox Regression Analysis ◽

H3k27 Methylation ◽

Acute Myeloid

Background: Acute myeloid leukemia (AML) is an epigenetically heterogeneous disease. The intensity of treatment is currently guided by cytogenetic and molecular genetic risk classifications; however these incompletely predict outcomes, requiring additional information for more accurate predictions. We aimed to identify potential prognostic implications of epigenetic modification of histone proteins, with a focus of H3K27 methylation in relation to mutations in chromatin, splicing and transcriptional regulators. Material and methods: Histone methylation mark expressions were evaluated in a cohort of 241 AML bone marrow (BM) and peripheral blood (PB) samples from patients admitted at the MD Anderson Cancer Center relative to their expression in CD34+ BM derived samples from healthy donors. Simultaneous analysis of 230 proteins was performed using the reverse phase protein array - a high-throughput, quantitative proteomic platform that enables identification of aberrant expressed proteins and the pathways they act in. Additional mutational analysis was performed on 65 BM samples. Results:H3K27Me3 was significantly lower in both BM and PB leukemic-derived samples compared to their expression in normal BM (figure 1A). A greater loss of H3K27Me3 associated with increased proliferative potential and shorter overall survival (OS) in the whole patient population (n=241, HR=0.64, 95% CI=0.47-0.87, p<0.01), as well as in subsets, e.g. cytogenetically normal AML (n=110, HR=0.62, 95% CI=0.40-0.97, p=0.03). To study the prognostic impact of H3K27Me3 in the context of cytogenetic aberrations and mutations, multivariate cox regression analysis was performed which identified H3K27Me3 level as an independent favorable prognostic factor in all (HR=0.74, 95%CI=0.57-0.95, p=0.02) as well as in P53 mutated AML (n=54, HR=0.48, 95%CI=0.26-0.87, p=0.02). A total of 78 AML patients had molecular data available for the major methylation affecting genes, i.e. IDH1, IDH2, DNMT3A and TET2. The level of H3K27Me3 was not prognostic in patients without any DNA methylation affecting mutation present, but patients with at least one mutation in any of these had better outcome when H3K27Me3 levels were high (highest tertile, figure 1A) compared to those with lower levels (median OS 7.1 vs. 24.1 months, HR=0.42, 95% CI=0.21-0.83, p=0.01, figure 1B). Mutations in U2AF1 and SRSF2 affect the spliceosome and are frequently found in antecedent hematological disorders (AHD), as well as are mutations in chromatin regulating genes ASXL1 and BCOR. We observed significant decreased H3K27Me3 in patients with these mutations corresponding with observed lower levels of H3K27Me3 in patients with AHD than those without (p=0.035). BCOR, SRSF2, U2AF1 and ASXL1 mutations confer poor prognosis in myeloid malignancies, however, in our cohort of 65 sequenced AML patients; not individual or a combination of these mutations were independent prognostic factors, but the degree of H3K27Me3 in these patients (HR= 0.49, 95% CI=0.25-0.95, p=0.03). To recognize dysregulated pathways in AML patients with the identified loss of H3K27Me3, we examined correlations of H3K27Me3 with the other 229 proteins on the array. H3K27Me3 is catalyzed by the polycomb group protein EZH2 and is linked to transcriptional repression via the formation of heterochromatin regions. To identify upregulated proteins and pathways upon the loss of H3K27Me3, we focused on significant negatively correlated proteins with H3K27Me3 leading us to the identification of 20 total and 6 phospho-proteins that showed increased expression upon decreased H3K27Me3. Functional enrichment analysis of this protein set revealed an upregulated anti-apoptotic phenotype. Conclusion:This study shows that proteomic profiling of epigenetic modifications on the histone level have clinical implications in AML and support the idea that epigenetic patterns contribute to a more accurate picture of the leukemic state complementing cytogenetic and molecular genetic subgrouping. Figure 1. A) Lower H3K27Me3 in BM and PB derived AML samples compared to normal CD34+. **** represents p<0.0001, ns = not significant. B) Overall survival probability in AML patients with any DNA methylation affecting mutation (i.e. IDH1/2, DNMT3A, TET2, n=53) according to H3K27Me3 low (blue) and high (orange) status. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Prognostic Value of Monosomal Karyotype in Patients with Primary Acute Myeloid Leukemia On Behalf of Spanish CETLAM Group.

Blood ◽

10.1182/blood.v114.22.1003.1003 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1003-1003 ◽

Cited By ~ 3

Author(s):

Isabel Granada ◽

Salut Brunet ◽

Montserrat Hoyos ◽

Dolors Costa ◽

Anna Aventín ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Conflicts Of Interest ◽

Treatment Strategies ◽

Disease Free Survival ◽

Prognostic Impact ◽

Complex Karyotype ◽

Monosomal Karyotype ◽

Acute Myeloid

Abstract Abstract 1003 Poster Board I-25 Introduction: Recently, the cooperative group HOVON-SAKK has refined the prognostic impact of cytogenetic abnormalities in acute myeloid leukemia (AML) by introducing the concept of monosomal karyotype (MK). This consists of ≥ 2 autosomal monosomies or one autosomal monosomy in addition to a structural alteration. In their experience, MK would explain the poor prognosis of AML with a complex karyotype. Objective: To investigate the prognostic impact of MK in patients with primary (de novo) AML enrolled in the Spanish CETLAM group protocols (AML 94/99/03). Also, to determine whether considering MK added predictive value to the cytogenetic classification of the Medical Research Council (MRC). Methods: Retrospective analysis of data from 1149 AML patients. Chromosomal formula was centrally reviewed with karyotypes being classified by the presence of MK and allocated into the MRC risk categories. Complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were calculated. Results: The karyotype was assessable in 904 (79%) of the 1149 cases. In 145 of the 904 cases (16%), abnormalities involving CBF gene were detected and in 437 (48%) the karyotype was normal (NK). In 253 (28%) additional patients the karyotype was not monosomal; of them, 61 (24%) belonged to the unfavorable MRC with 17 cases harboring a complex karyotype ≥ 5 abnormalities, 7 cases with rearrangements 3q, 13 cases with -7, 9 cases with 5q abnormalities and 16 cases with t(6;9)). The remaining 69 (7.7%) patients had a MK; of them, 59 (85.5%) were from the unfavorable MRC category and included 43 cases with complex karyotype ≥ 5 abnormalities, 6 cases with rearrangements 3q, 5 cases with -7, 5 cases with alterations of 5q). The following table summarizes the results in terms of CR rate, DFS and OS: Conclusions: The addition of MK to the MRC cytogenetic classification refines the prognostic prediction. In our series, the dismal outcome of patients with MK is confirmed; these patients had worse prognosis than those with adverse cytogenetics without MK. Alternative treatment strategies are mandatory for MK+ patients. Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures: No relevant conflicts of interest to declare.

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Detection, Phenotype and Prognostic Significance of the Leukemic Stem Cell Side PopulationHo342low in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v120.21.2586.2586 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2586-2586

Author(s):

Juana Serrano-Lopez ◽

Josefina Serrano ◽

Joaquín Sanchez-Garcia ◽

Noemi Fernandez-Escalada ◽

Maria del Carmen Martinez-Losada ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Complete Remission ◽

Myeloid Leukemia ◽

Common Finding ◽

Prognostic Impact ◽

Leukemic Stem Cell ◽

Conventional Chemotherapy ◽

Healthy Donors ◽

Acute Myeloid

Abstract Abstract 2586 Introduction: Acute Myeloid Leukemia (AML) is a heterogeneous disorder arising from a clonal expansion of Leukemic Stem Cell (LSC). The characterization of LSC is crucial because it is resistant to conventional chemotherapy and is ultimately responsible for leukemic relapses. The LSC in AML is a phenotypically heterogeneous population (CD34+CD38-, CLL1 +, CD96 +…). In this sense, “Side Population” cells (SPHo342Low) are considered to be a type of stem cells that can self-renew and differentiate into tissues. SP are characterized by their ability to efflux the vital dye Hoechst 33342 through the drug ABCG2 pump. SPHo342Low cells have been described in many types of solid tumors and AML as potential LSC. The objective in this study is to analyze the frequency of SPHo342Low in AML, their phenotype and the possible prognostic impact on outcomes. Patients and Methods: Bone marrow samples (BM) obtained from 57 patients (median age 58 years, range: 4–82), diagnosed with AML between Mar-07 to Mar-12, were included. Distribution of cytogenetic risk groups was: Favorable (12.5%), Intermediate (60.7%) and Unfavorable (26.8%). NPM1mut was present in 11 cases and FLT3-ITD in 6 cases. Prior MDS was present in 10 cases. After achieving complete remission (CR) with conventional chemotherapy, allogeneic or autologous stem cell transplantation was performed in 17 and 12 patients respectively, according to individual risk and availability of donor. Eleven frail patients received as front-line, low intensity therapy with Azacytidine. We detected LSC, SPHo342Low in marrow MNCs obtained at diagnosis (N=40), at morphologic complete remission (CR) (N=21) or at relapsed / resistant (N=16) disease. For detection, 2×10(6) MNC/ml were resuspended in HBSS medium with 5 ug/ml of Ho342 dye and CD45-FITC, CD34-PE Mn-Abs, analyzing at least 1×105 viable cells in UV laser FACSVantage cytometer with the combination of filters BP 670/40 for emission in red and BP 450/30 for the blue emission. We verified SP region by inhibiting ABCG2 pump with Verapamil (50μM/mL). As controls we analyzed MNCs from BM aspirates from healthy donors (N=5). Results: In all BM samples from healthy donors, SPHo342Low population was detected accounting for 0.5% (range: 0.1 to 0.9%) and it was CD34negCD45neg phenotype in 80% of cases. SPHo342Low cells were detected in 23/40 cases (57.5%) of samples from AML diagnosis with a median of 0.08% (range 0.01–2.3%). Phenotype of SPHo342Low cells at diagnosis was CD34+CD45+/− in 36% of cases. The presence of SPHo342Low cells presented in AML at diagnosis did not statistically correlate with any prognostic clinical variables such as age, cytogenetic-molecular risk or prior MDS. Interestingly, the detection of LSC SPHo342Low at diagnosis was statistically associated to the presence of >0.1% of CD34+CD38- AML cells (P=0,03). In BM samples obtained from AML patients in CR, SPHo342Low cells were detected in 17/21 (81.0%) with a median of 0.17% (range: 0.1 to 0.76%), with a phenotype mostly CD34 negative. In BM samples obtained from AML patients in relapsed/refractory situation, SPHo342Low cells were detected in 14/16 (87.5%) with a median of 0.22% (range: 0.2 to 0.91%) with a phenotype of CD34+ CD45+/− in 33% of cases. Interestingly, patients who did not achieve CR, have a significantly higher percentage of SPHo342Low at diagnosis (0.42% vs. 0.06%, P = 0.044) as well as those who need more than one cycle to achieve CR (0.52% vs. 0.07%, P = 0.04). Moreover, for those patients achieving CR, persistence of Minimal Residual Disease (MRD+) was associated to a higher percentage of SPHo342Low at diagnosis (0.28% vs. 0.05%, P = 0.021). Likewise, Relapse-free survival (RFS) was significantly higher in AML patients lacking SPHo342Low at diagnosis (70 ± 18.2% vs. 43.3 ± 17.6%, P = 0.0324, Log rank test). Conclusions: Detection of LSC SPHo342Low+CD34+CD45+/− phenotype in AML at diagnosis is a common finding that is associated with increased resistance to achieve CR, clearance of MRD and lower RFS. During progression of disease this SPHo342Low+ population increases and maintains CD34+CD45neg phenotype. BM samples obtained from AML patients at CR were SPHo342Low+ CD34negCD45+/− phenotype which can be considered responsible for normal hematopoietic regeneration. Disclosures: No relevant conflicts of interest to declare.

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RUNX1 and GATA2 Regulate the Expression of the SET Oncogene in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.879.879 ◽

2014 ◽

Vol 124 (21) ◽

pp. 879-879

Author(s):

Raffaella Pippa ◽

Ana Dominguez ◽

Nerea Marcotegui ◽

Raquel Malumbres ◽

Elizabeth Guruceaga ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Promoter Region ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Minimal Promoter ◽

Research Network ◽

Luciferase Reporter ◽

Prognostic Impact ◽

Minimal Promoter Region ◽

Acute Myeloid

Abstract INTRODUCTION. The protein SET (I2PP2A), a potent protein phosphatase 2A (PP2A) inhibitor, has been implicated in many cell processes such as DNA replication, chromatin remodeling and gene transcription, differentiation, migration, and cell-cycle regulation. In fact, SET has been described as an oncogene that regulates important signaling pathways. Our group reported that PP2A inhibition is a common event in AML, and that SET is overexpressed in 28% of acute myeloid leukemia (AML) cases, where it is associated with short overall survival. Moreover, the anti-leukemic effects of the FTY720 and OP449 PP2A-activating drugs in AML cells depend on interaction/sequestration of SET. However, despite the importance of SET overexpression and its prognostic impact in both hematological and solid tumors, there are few data about the mechanisms involved in its regulation. AIM. To characterize the functional promoter region of the SET gene, and to identify transcription factors (TFs) involved in its regulation. RESULTS. Luciferase reporter assays with five truncatedconstructs allowed us to determine a 163bp-region as the minimal promoter region of SET that contains consensus sites for several TFs. Chromatin immunoprecipitation (ChIP) assays confirmed the binding of RUNX1, GATA2, MYC, and SP1. RUNX1 and GATA2 are two essential TFs in hematopoiesis, and localized on the SET promoter when the acetylation state of both histone H3 and H4 and the tri-methylation on H3K4 is high, confirming that they both could act as positive regulators of SET transcription. In silico analysis in large series of adult patient samples with de novo AML recently published by The Cancer Genome Atlas Research Network showed a significant positive correlation between SET and RUNX1 and GATA2 at mRNA level. Furthermore, knockdown of RUNX1 and/or GATA2 triggered SET downregulation, whereas only a simultaneous overexpression of these two TFs caused a significant up-regulation of SET. Interestingly, RUNX1 interacts with GATA2 in both HL-60 and HEL cell lines. Moreover, we found that SP1 is also part of this transcription complex. Altogether, these results show that RUNX1 and GATA2, together with SP1, regulate the transcription of the SET gene. CONCLUSIONS We have defined the minimal promoter region of the SET gene, and have demonstrated that RUNX1 and GATA2 regulate its expression in AML. Moreover, our functional studies demonstrate that RUNX1 and GATA2 form a complex with SP1 that activates the transcription of SET in AML cells. This study opens new directions to further understand the mechanisms of SET overexpressing leukemias. Disclosures No relevant conflicts of interest to declare.

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Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse

Blood ◽

10.1182/blood-2007-11-126227 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1366-1373 ◽

Cited By ~ 103

Author(s):

Heike Kroeger ◽

Jaroslav Jelinek ◽

Marcos R. H. Estécio ◽

Rong He ◽

Kimie Kondo ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Leukemia Cell ◽

Transcription Start Sites ◽

Remission Maintenance ◽

Acute Myeloid

AbstractDNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.

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Methscore As a New Prognostic Tool for Complex DNA Methylation Changes Assessment in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-137380 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-33

Author(s):

Sarka Sestakova ◽

Cyril Šálek ◽

Dávid Kundrát ◽

Ivana Ježíšková ◽

Jiří Mayer ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Methylation Level ◽

Myeloid Leukemia ◽

Research Funding ◽

Cox Regression ◽

Validation Cohort ◽

Prognostic Impact ◽

Logrank Test ◽

Acute Myeloid

Changes in DNA methylation are characteristic for patients with acute myeloid leukemia (AML) and many studies have reported the prognostic significance of these epigenetic aberrations. We aimed for a complex evaluation of DNA methylation changes in AML patients at diagnosis. Therefore, we designed a sequencing panel targeting 239 regions annotated to 186 genes previously described in literature as having a prognostic impact or being commonly associated with AML pathogenesis (e.g. WT1, HOX genes). We used diagnostic whole-blood DNA samples of adult AML patients who were treated with curative intent starting with 3+7 induction regimen. In the testing cohort, we sequenced 128 AML patients and 11 samples from healthy donors. We analyzed another 50 AML patients from partner institution University Hospital Brno as an independent validation cohort. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Data were processed in Linux opensource software and further analyzed in R. For each sample, we measured the methylation level of nearly 50 000 CpGs. In the testing cohort, we used the Cox regression to evaluate the effect of each CpG's methylation level on overall survival (OS). As a result, we found 1961 CpGs significantly effecting the OS (p<0.05) annotated to 141 genes. In gene ontology analysis, these loci were mainly connected to transcription and RNA regulation, DNA binding, and embryonic development. In 1097 CpGs, higher methylation indicated better outcome and, on the contrary, a poorer prognosis in the remaining 864 CpGs. Next, we used linear combination of the methylation levels and Cox's beta regression coefficients for each CpG to count a summarizing value that we called MethScore. Patients with lower MethScore (n=64) had markedly longer OS and event-free survival (EFS) than patients with higher MethScore (n=64, Logrank test for OS: p<2e-16, for EFS: p<2e-12). To further specify the prognostically most influential CpGs out of the selected 1961 loci, we subjected them individually to Cox multivariate regression analysis together with age, leukocytes count, cytogenetics, FLT3-ITD mutation, and transplantation in the 1st complete remission. Only 625 CpGs remained significant (p<0.05) and from these, we calculated MethScore-MVA via the same procedure as MethScore. MethScore-MVA was also very well predictive of patients' survival (Logrank test comparing patients with higher and lower MethScore-MVA for OS: p<7e-14, for EFS: p<2e-10). MethScore and MethScore-MVA proved to be the most significant variables in multivariate Cox regression for both OS (p=2e-12 and p=2e-13) and EFS (p=2e-12 and p=3e-12, respectively). To validate these results, we counted MethScore and MethScore-MVA for 50 patients from the validation cohort and performed the same Cox multivariate analysis. MethScore remained highly significant for both OS and EFS (p=0.002 and p=0.004, respectively). MethScore-MVA was significant for OS (p=0.02) but not for EFS (p=0.09). Here, we present the MethScore as a novel complex characterization of predictive DNA methylation changes in patients with AML. MethScore might be used as a new surrogate marker that could enrich the currently used cytogenetic and genetic markers to refine the prognostication of newly diagnosed AML patients. This study was supported by the Ministry of Health of the Czech Republic, project for conceptual development of research organizations (00023736, IHBT). Disclosures Šálek: Amgen: Consultancy, Honoraria, Research Funding. Mayer:Celgene: Research Funding.

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