FLT3, NPM1 and MLL Mutations Help Risk Stratification in Pediatric Acute Myeloid Leukemia.

Abstract Abstract 1574 Poster Board I-600 Introduction Acute myeloid leukemia (AML) is a heterogeneous disease which harbors various genetic alterations. Among theses genetic events, Mutations of FLT3, NPM1, MLL and other genes often predict prognosis, particularly in cases cytogenetic normal (CN-AML). Could these be criteria for risk stratification in Pediatric AML ? Patients and Methods 155 cases of de novo AML were diagnosed routinely according to morphology, immunology, cytogenetics, and molecular biology examination on bone marrow (BM) aspirates between Jan. 2002 and Dec. 2008. All patients received chemotherapy according to the AML-XH-99 protocol, which consist of Daunorubicin, Cytosine arabinoside, Etoposide, Homoharringtonine. For acute promyelocytic leukemia, all-trans retinoic acid and Arsenic trioxide were also included. Meanwhile, total RNA of leukemic cells form all diagnostic BM samples were extracted, and then reverse transcribed. MLL partial tandem duplication (MLL/PTD) fusion transcripts were screened by real-time quantitative polymerase chain reaction. FLT3 internal tandem duplication (FLT3/ITD), FLT3 tyrosine kinase domain mutation (FLT3/TKD) and NPM1 mutation were examined by High resolution melting analysis. Results Of the 155 children with de novo AML, 121(78.1%) had received chemotherapy for more than one week with data available for analysis. Among them, 55(45.5%) was cytogenetically normal (CN-AML). In this total cohort of patients 49(27.09%) had FLT3/ITD (32.70% in CN-AML), 14 (9.03%) had FLT3/TKD (7.30% in CN-AML), 62 (40%) had NPM1 mutation (49% in CN-AML), and additional 8 (5.16%) had MLL/PTD (5.50% in CN-AML). In this cohort of patients 98 (63.22%) had at least one mutation. The clinical outcomes were listed in table 1. Generally, patients with FLT3 mutation (ITD or TKD mutation) usually have worse results after chemotherapy, as reported previously by other researchers. Meanwhile, NPM1 mutations usually predict better prognosis in our cohort of AML patients. MLL/PTD always predicts the worst outcome in AML as other MLL rearrangements in leukemia. Among CN-AML patients, 5-year EFS and OS were similar to whole cohort of patients according to those mutations. Cox regression analysis in a univariate model revealed that the presence of FLT3/ITD and NPM1 was significant prognostic factor of EFS, (P<0.05). We therefore proposed a molecular-risk classification of pediatric AML patients based on the data we got in this study. For the newly classified groups of low, medium and high risk groups, EFS rate was 62.03%±8.42%, 45.42%±4.52%, and 14.85%±2.99%, respectively, P=0.00. CRD for the 3 groups was 27.69±21.34 months, 22.62±19.64 months, 13.26±11.95 months, respectively, p=.022. Our results indicate that combinations of these couple of molecular events may be the useful tool for further classify AML in children. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Mutation of NPM1 and FLT3 Genes in Acute Myeloid Leukemia and Their Association with Clinical and Immunophenotypic Features

Disease Markers ◽

10.1155/2013/582569 ◽

2013 ◽

Vol 35 ◽

pp. 581-588 ◽

Cited By ~ 12

Author(s):

Pradeep Singh Chauhan ◽

Rakhshan Ihsan ◽

L. C. Singh ◽

Dipendra Kumar Gupta ◽

Vishakha Mittal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Fusion Gene ◽

Genetic Alterations ◽

Prognostic Indicators ◽

White Blood Count ◽

Npm1 Mutation ◽

De Novo Aml ◽

Acute Myeloid

Background.Mutations in NPM1 and FLT3 genes represent the most frequent genetic alterations and important diagnostic and prognostic indicators in patients with acute myeloid leukemia (AML).Objective.We investigated the prevalence and clinical characteristics of NPM1 and FLT3 mutations in 161 patients of de novo AML including adults and children.Results.NPM1 mutation was found in 21% and FLT3 mutation in 25% of the AML patients. Thirteen (8%) samples were positive for both NPM1 and FLT3/ITD mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (25.8% versus 8.8%;P=0.02). Further, NPM1 mutation was found to be more frequent in patients above 45 years of age (P=0.02). NPM1 mutation was significantly associated with higher platelet count (P=0.05) and absence of hepatosplenomegaly (P=0.01), while FLT3/ITD mutation was associated with higher white blood count (P=0.01). Immunophenotypically, NPM1 mutation was associated with the lack of CD34 (P<0.001) and HLD-DR expression (P<0.001), while FLT3/ITD mutation was positively associated with the expression of CD7 (P=0.04). No correlation was found between NPM1 mutation and fusion gene. Interestingly, FLT3/ITD mutation was found to be inversely associated with AML/ETO fusion gene (P=0.04).Conclusions.The results suggest that distinct clinical and immunophenotypic characteristics of NPM1 and FLT3/ITD mutations present further insight into the molecular mechanism of leukemogenesis.

Download Full-text

Genome-Wide Genotyping of Acute Myeloid Leukemia with t(9;11) Reveals New Recurrent Genomic Alterations,

Blood ◽

10.1182/blood.v118.21.3546.3546 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3546-3546

Author(s):

Michael W.M. Kühn ◽

Lars Bullinger ◽

Jennifer Edelmann ◽

Jan Krönke ◽

Gröschel Stefan ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Genetic Alterations ◽

Mixed Lineage Leukemia ◽

Genetic Event ◽

Genome Wide ◽

De Novo Aml ◽

Paired Analysis ◽

Acute Myeloid

Abstract Abstract 3546 Rearrangements of the mixed lineage leukemia (MLL) gene are associated with the development of acute leukemia, and a variety of translocation partners have been described to date. In acute myeloid leukemia (AML), the translocation t(9;11)(p22;q23), resulting in the MLLT3-MLL fusion gene, is the most common genetic event involving MLL. The translocation t(9;11) can occur de novo, or as a consequence of previous chemotherapy (t-AML). Both types exhibit significant biological and clinical heterogeneity, and cooperating genetic events have been implicated underlying these heterogeneous phenotypes. To identify additional genomic abnormalities in AML with t(9;11), we performed high-resolution, genome-wide analysis of DNA copy number alterations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 34 AMLs with t(9;11) [de novo AML, n=22; t-AML, n=12]. Samples were also analyzed for AML-associated mutations: FLT3 [internal tandem duplication (ITD; 2/33); tyrosine kinase domain (TKD; 2/26)], NPM1 (0/28), CEBPA (0/23), IDH1 (0/28), IDH 2 (0/28), DNMT3A (0/19), NRAS (0/6); and deregulated expression of EVI1 (8/16). Control DNA from remission bone marrow or peripheral blood was available for paired analysis in 12 (33%) cases. Data were processed using reference alignment, dChipSNP, and circular binary segmentation. Paired analysis revealed a mean of 1.9 somatic CNAs per case (range: 0–12); 45% of cases lacked any CNAs. Deletions were more common than gains (1.73 losses/case vs. 0.25 gains/case; p =0.04). There were no significant differences in the mean number of CNAs between de novo and therapy-related cases (de novo AML: 1.0, range: 0–2; t-AML: 2.7, range: 0–12; p =0.93). Recurrent deletions were detected at chromosomal bands 7q36.1–36.2 (n=2) and at the chromosomal translocation breakpoint at 11q23 (n=2). The del(7q36.1–36.2) overlapped with a minimally deleted region at 7q36.1 that we previously identified in 8% of core-binding factor AML containing only 4 genes (PRKAG2, GALNT11, GALNTL5 and MLL3). The only gene contained in both regions was MLL3, a member of the mixed-lineage leukemia gene family. The most recurrent CNA was trisomy 8 (n=5), also detected by conventional cytogenetics in all 5 cases. Novel recurrent focal gains were identified at 9p22.1 (n=2; size: 341 Kb) and at 13q21.33-q22.1 (n=2; size: 1021 Kb) with each region containing genes potentially involved in cancer pathogenesis (ACER2 in 9p; KLF5 in 13q). Analysis of CN-LOH revealed no such lesion in any of the cases. In summary, our data provide a comprehensive survey of CNAs in a well characterized cohort of AMLs with t(9;11). These data demonstrate a very low occurrence of CNAs, with no significant differences between de novo and therapy-related cases and complete absence of CN-LOH. Interestingly, a number of novel recurrent secondary genetic alterations were identified. Determining the functional role of these lesions in leukemogenesis and drug resistance should provide new insights into t(9;11)-bearing AMLs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Prognosis of Karyotypic Subgroups in Pediatric Acute Myeloid Leukemia Depends on the Alterations of KIT, MLL and FLT3 Genes: A Report from Japanese Childhood AML Cooperative Study Group.

Blood ◽

10.1182/blood.v108.11.2281.2281 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2281-2281

Author(s):

Akira Shimada ◽

Tomohiko Taki ◽

Ken Tabuchi ◽

Ryoji Hanada ◽

Akio Tawa ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Tandem Duplication ◽

Normal Karyotype ◽

Genetic Alterations ◽

Kras Mutations ◽

Childhood Aml ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Karyotypic abnormalities are associated with a prognosis of acute myeloid leukemia (AML). However, there are some patients who had poor prognosis among patients with the same karyotypic abnormalities. Recently, it is revealed that alterations of the genes including tyrosine kinases, lead to poor prognosis in AML. Genetic alterations associated with a poor prognosis remain to be clarified in karytotypic subgroups of AML. We performed the mutation analysis of FLT3, MLL, KIT, RAS and NPM in 158 pediatric AML patients who were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, including 33 with normal karyotype, 46 with t(8;21), 7 with inv(16), 20 with 11q23 translocations, 13 with t(15;17), 10 with Down syndrome (DS) FAB-M7 and 29 other karyotypic abnormalities. In the 33 patients with normal karyotype, 9 FLT3-internal tandem duplication (ITD), 2 FLT3-D835 mutation (D835Mt), 8 MLL-partial tandem duplication (PTD), 2 KIT, 2 NRAS and 3 KRAS mutations were identified. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Notably, NPM gene mutation was not identified in these patients. In the 46 patients with t(8;21), 2 FLT3-ITD, 1 D835Mt, 4 MLL-PTD, 8 KIT, 4 NRAS and 5 KRAS mutations were found. KIT mutations were associated with a poor prognosis. In the 7 patients with inv(16), 2 FLT3-D835Mt and 1 KIT mutations were identified. Only one patient with KIT mutation relapsed. In the 20 patients with 11q23 translocations, 1 D835Mt and 5 MLL-PTD were identified. MLL-PTD was associated with a poor prognosis in patients with 11q23 translocations. In the 13 patients with t(15;17), 3 FLT3-ITD and 3 D835Mt were found. FLT3-ITD was not associated with a poor prognosis. In the 10 patients with DS-M7, KIT and KRAS mutations were found in each one patient, showing no prognostic significance. In the 29 patients with other karyotypic abnormalities, 6 FLT3-ITD, 2 D835Mt, 4 MLL-PTD, 0 KIT, 2 NRAS and 2 KRAS mutations were found. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Fifteen patients (9.5%) who had alterations of 2 of these 4 genes showed a poor prognosis. RAS gene mutations were found in 26 (16.5%) of 158 patients, but were not associated with the prognosis. These results suggest that a new different therapeutic strategy for the patients with these gene alterations in each subgroup, for example KIT mutations in t(8;21)-patients and MLL-PTD in patients with 11q23 translocation, is needed to improve the prognosis of pediatric AML.

Download Full-text

Characteristics and Outcome Of Acute Myeloid Leukemia Following Breast Cancer: Analysis Of 408 Cases and Controls

Blood ◽

10.1182/blood.v122.21.1408.1408 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1408-1408

Author(s):

Marine Gilabert ◽

Jean-Baptiste Micol ◽

Austin G Kulasekararaj ◽

Stephane de Botton ◽

Claude Chahine ◽

...

Keyword(s):

Breast Cancer ◽

Acute Myeloid Leukemia ◽

Induction Chemotherapy ◽

Myeloid Leukemia ◽

De Novo ◽

Biological Data ◽

Matched Pair ◽

Npm1 Mutation ◽

De Novo Aml ◽

Acute Myeloid

Abstract Background With the improvement of cancer therapy, long term survivors are more exposed to the risk of secondary myeloid neoplasm including myelodysplasia and acute leukemias. The WHO 2008 classification individualize the therapy related acute myeloid leukemia (AML) as a specific entity and highlight the role of chemotherapy/radiotherapy in the pathogenesis of the disease. There is a demonstrated link between specific therapeutic agents, recurrent genetic lesions (such as t(15;17), CBF AML, monosomy 7, …), and outcome. However, most of the published series include multiple types of primary cancer and treatments. This heterogeneity may represent a problem as only limited data are available in patients with specific cancer subtypes, such as the most frequent one, breast cancer. This may be important at a public health level but also to homogenize age and types of prior treatment. Moreover, the complexity of t-AML could not be resumed to the association between treatment and karyotype, as some patients developed AML without chemotherapy, suggesting a potential predisposition to AML. In the present report, we focused on AML arising after breast cancer (BC) and describe the characteristics and outcome of this population. Patients and Methods This is a retrospective multicenter study. Patients were included if they had the diagnosis of breast cancer preceding diagnosis of acute myeloid leukemia whatever may be the interval between the 2 cancers and whatever treatment was administered for BC. All patients with AML were treated with induction chemotherapy. Clinical and biological data for both cancers were collected. Patient’s characteristics and results were compared with age, cytogenetic risk, and optionally sex matched (if possible) de novo AML with a 2/1 ratio. Results 408 patients were analyzed, including 136 AML associated with BC and 272 de novo AML. The median age at diagnosis of BC was 50 years. 47% of patients had invasive ductal carcinoma. Treatment of BC included chemotherapy in 81% of cases, radiotherapy in 91% of cases, and surgery in 99% of cases. The median time between BC and AML was 2.8 years. Median WBC was 3.4G/l and median platelets count was 49G/l. For AML-BC, Cytogenetics were abnormal in 82% of cases including 12% Complex Karyotype, 12% t(15;17), 17% CBF, and 20% MLL translocations. The recent introduction of taxanes in the treatment of BC did not seem to change the frequencies of these aberrations. With the exception of sex ratio, there was no significant difference of baseline characteristics as compared to control group. Regarding induction chemotherapy, CR rate was 81% and 8-week mortality 11.6%. In the control cohort, CR rate was 83% (p=NS) and 8 week mortality 7% (p=NS). Allogeneic transplantation was performed in 21% and 17% of patients respectively. In cytogenetic adjusted survival analysis, median overall survival and relapse free survival were similar between AML-BC and de novo AML for favorable risk (OS and RFS not reached for both groups; p=0.06 and p=0.3 respectively), and unfavorable risk (OS 12m vs. 13m p=NS, RFS 8m vs. 9m p=NS) groups. Interestingly, there was a difference for intermediate cytogenetics group, with median OS (21 months vs. 38 months p=0.01) and median RFS (14 vs. 25 months p=0.04). Difference was also confirmed for cumulative incidence of relapse (1 year probability 41% vs. 22%, p=0.04). Frequency of FLT3, NPM1, and CEBPA mutations were only available in a subset of patients. Only 1/12 pts in the intermediate AML-BC group was FLT3 mutated and 1/12 pt had isolated NPM1 mutation. In the de novo cohort, 11/28 pts had FLT3 mutations and 8/21 pts had isolated NPM1 mutation. Conclusion Our data showed that in AML arising after BC, the prior BC do not appear to impact the outcome in favorable and unfavorable cytogenetic risk groups. However, this is different for intermediate risk cytogenetics, our data suggesting a poorer outcome of AML-BC and potentially a different mutational profile. Regarding AML susceptibility, a matched pair analysis comparing the AML-BC and BC without AML will also be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Molecular and Clinical Aspects of Acute Myeloid Leukemia with Inv(3)(q21q26)/t(3;3)(q21;q26) Carrying Spliceosomal Mutations

Blood ◽

10.1182/blood-2020-139953 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-8

Author(s):

Hassan Awada ◽

Cassandra M Kerr ◽

Heesun J. Rogers ◽

Jaroslaw P. Maciejewski ◽

Valeria Visconte

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Dominant Gene ◽

Prognostic Significance ◽

Genetic Alterations ◽

Splicing Factor ◽

Clinical Aspects ◽

De Novo Aml ◽

Acute Myeloid

Inversion or translocation of the chromosome 3, specifically inv(3)(q21q26.2/ t(3;3)(q21;q26.2) are present in 1-2% of acute myeloid leukemia (AML) cases and are classified as a distinct entity in the 2016 WHO classification. Hallmark genetic alterations in this entity include mutations in GATA2 and MECOM. In fact, these rearrangements result in over activation of MECOM due to juxtaposition with a distal GATA2 enhancer. Cytomorphologic phenotypes include anemia, normal to elevated platelets and multilineage dysplasia in a hyperplastic bone marrow (BM). Studies have shown the frequent occurrence of NF1, NRAS and RUNX1 mutations. While proceeding towards our molecularly informed AML subtyping (Awada, Blood 2019;1406), we observed a high occurrence of somatic mutations in the splicing factor SF3B1 in inv(3)/t(3;3) AML. We were particularly intrigued by this observation considering several key aspects of SF3B1 mutations in the context of MDS. For instance, SF3B1 mutations are highly associated with clear phenotypic and morphologic features and carry favorable prognosis in MDS. These mutations are often found in patients carrying less deleterious abnormalities [e.g., del(5q)] and their founder clonal nature has been uncovered through experimental studies. Recent studies unveiled the occurrence of SF3B1 mutations in de novo AML and low complete remission rate when combined with other mutations (e.g., DNMT3A). To investigate whether SF3B1 mutations were unequivocally frequent in inv(3)/t(3;3) AML compared to other splicing factor mutations, we moved forward in dissecting the clinical, morphologic and molecular profiles of these cases. We analyzed results from whole exome sequencing and targeted deep sequencing from the Cleveland Clinic and publicly available data of AML with inv(3)/t(3;3) (de novo AML, n=32; secondary AML from antecedent myeloid neoplasms, n=11; t-AML, n=1). In our cohort, mutations in the most common components of the RNA splicing machinery (SF3B1, SRSF2, U2AF1, ZRSR2) were observed in 27% (n=12) of the patients. Among splicing factor mutations, SF3B1 was the most mutated gene (77%; 10/13 total mutations); 7 cases had inv(3) and 3 had t(3;3). Mutations were observed at canonical sites: K700E (70%) and K666N (30%) with no difference compared to the hotspots observed in MDS. Sixty% of patients were female. Median age was 61 years (range, 36-73). Anemia was present in 50%, leukopenia in 10% and thrombocytopenia in 50% of the patients. For 40% of the cases, BM smears for iron staining was available and showed absence of ringed sideroblasts. Complex karyotype (CK) was present in 20% of the patients; -7/del(7q) was present as the only cytogenetic abnormality in 30% or with CK in 10% pf the cases. Variant allele frequency (VAF) of SF3B1mutations in inv(3)/t(3;3) was not different than the those without inv(3)/t(3;3) (42% vs 40%). Survival analysis was performed in 3 subgroups: SF3B1MT AML (n=70), SF3B1MT AML + inv(3)/t(3;3) (n=10), AML + inv(3)/t(3;3) (n=34). SF3B1MT AML + inv(3)/t(3;3) and AML + inv(3)/t(3;3) had similar OS (11.7 vs 9.7 months) which was shorter than the that of SF3B1MT AML without any inv(3)/t(3;3) (19.4 months, P=0.002) suggesting that SF3B1MT in the context of inv(3)/t(3;3) might hold a different prognostic significance strongly due to the presence of inv(3)/t(3;3). Given this observation, we delved into the clonal diversity of SF3B1 mutations and its co-occurrence with other molecular mutations. Comparison of VAFs showed that SF3B1 mutations in relation to other mutations were dominant/founder in 30%, secondary/subclonal in 20% while co-dominant to another gene (VAF differences <5%) in 50% of the cases. The most common co-dominant gene mutation was GATA2 (60%, 3/5). Top mutations by frequency were in GATA2 (30%), ASXL1 (20%) and NRAS (20%). Hemizygous GATA2 mutations were detected in 15% of SF3B1MT AML + inv(3)/t(3;3). In our cohort, other RAS gene mutations were detected in 10% of the patients each, including CBL, NF1 and PTPN11). One SF3B1MT AML + inv(3)/t(3;3) case also harbored a SRSF2 mutation with a parallel median VAF of 40% and 41%, respectively. The results of our study are summarized in Fig. 1. In sum, we describe that SF3B1 mutations occur in combination with inv(3)/t(3;3) in AML and might represent a subclass of this entity in which lesions in SF3B1 gene could potentially hide a cryptic association between splicing abnormalities and disease phenotypes. Figure 1 Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Download Full-text

Genomic characterization of relapsed acute myeloid leukemia reveals novel putative therapeutic targets

Blood Advances ◽

10.1182/bloodadvances.2020003709 ◽

2021 ◽

Vol 5 (3) ◽

pp. 900-912

Author(s):

Svea Stratmann ◽

Sara A. Yones ◽

Markus Mayrhofer ◽

Nina Norgren ◽

Aron Skaftason ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Pediatric Patients ◽

De Novo ◽

Low Frequency ◽

Whole Genome ◽

Recurrent Mutations ◽

De Novo Aml ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Relapse is the leading cause of death of adult and pediatric patients with acute myeloid leukemia (AML). Numerous studies have helped to elucidate the complex mutational landscape at diagnosis of AML, leading to improved risk stratification and new therapeutic options. However, multi–whole-genome studies of adult and pediatric AML at relapse are necessary for further advances. To this end, we performed whole-genome and whole-exome sequencing analyses of longitudinal diagnosis, relapse, and/or primary resistant specimens from 48 adult and 25 pediatric patients with AML. We identified mutations recurrently gained at relapse in ARID1A and CSF1R, both of which represent potentially actionable therapeutic alternatives. Further, we report specific differences in the mutational spectrum between adult vs pediatric relapsed AML, with MGA and H3F3A p.Lys28Met mutations recurrently found at relapse in adults, whereas internal tandem duplications in UBTF were identified solely in children. Finally, our study revealed recurrent mutations in IKZF1, KANSL1, and NIPBL at relapse. All of the mentioned genes have either never been reported at diagnosis in de novo AML or have been reported at low frequency, suggesting important roles for these alterations predominantly in disease progression and/or resistance to therapy. Our findings shed further light on the complexity of relapsed AML and identified previously unappreciated alterations that may lead to improved outcomes through personalized medicine.

Download Full-text

DNMT3A Mutations in Acute Myeloid Leukemia-Stability During Disease Evolution and the Clinical Implication

Blood ◽

10.1182/blood.v118.21.409.409 ◽

2011 ◽

Vol 118 (21) ◽

pp. 409-409 ◽

Cited By ~ 3

Author(s):

Hsin-An Hou ◽

Yuan-Yeh Kuo ◽

Chieh-Yu Liu ◽

Wen-Chien Chou ◽

Ming Cheng Lee ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

De Novo ◽

Npm1 Mutation ◽

Poor Prognostic Factor ◽

Disease Evolution ◽

Molecular Abnormalities ◽

De Novo Aml ◽

Acute Myeloid

Abstract Abstract 409 Background: DNMT3A mutations are associated with poor prognosis in acute myeloid leukemia (AML), but the stability of this mutation during the clinical course remains unclear. Materials and Methods: Mutation analysis of DNMT3A exons 2–23 was performed by polymerase chain reaction and direct sequencing in 506 de novo AML patients. Their interaction with clinical parameters, chromosomal abnormalities and genetic mutations were analysed. Results: DNMT3A mutations were identified in 14% of total patients and 22.9% of patients with normal karyotype (CN-AML). 30 different kinds of DNMT3A mutations were identified in 70 patients. Twelve were missense mutations, eight were nonsense mutations, nine were frame-shift mutations and one, in-frame mutation. The most common mutation was R882H (26 patients), followed by R882C (15 patients), R882S (3 patients), R736H (3 patients) and R320X (2 patients). DNMT3A mutations were closely associated with older age, higher white blood cell (WBC) and platelet counts at diagnosis, FAB M4/M5 subtype, intermediate-risk and normal cytogenetics. Among the 70 patients with DNMT3A mutations, 68 (97.1%) showed additional molecular abnormalities at diagnosis. The most common associated molecular event was NPM1 mutation (38 cases), followed by FLT3-ITD (30 cases), IDH2 mutation (16 cases) and FLT3-TKD (9 cases). Patients with DNMT3A mutations had significantly higher incidences of NPM1 mutation, FLT3-ITD, IDH2 and PTPN11 mutations than those with DNMT3A-wild type (54.3% vs. 15.3%, P<0.0001; 42.9% vs. 19.3%, P<0.0001; 22.9% vs. 9.1%, P=0.0016; and 10% vs. 3.5%; P=0.007, respectively). On the contrary, CEBPA was rarely seen in patients with DNMT3A mutations (4.3% vs. 14.7%, P=0.0134). Totally, 40 patients (58.8%) had concurrent both Class I and Class II or NPM1 mutations at diagnosis. With a median follow-up of 55 months (ranges, 1.0 to 160), patients with DNMT3A mutation had significantly poorer overall survival (OS) and relapse-free survival (RFS) than those without DNMT3A mutation (median, 14.5 months vs. 38 months, P =0.013, and medium, 7.5 months vs. 15 months, P=0.012, respectively). In the subgroup of 130 younger patients (less than 60 years) with CN-AML, the differences between patients with and without DNMT3A mutation in OS (median, 15.5 months vs. not reached, P= 0.018) and RFS (median, 6 months vs. 21 months, P=0.004) were still significant. Multivariate analysis demonstrated that DNMT3A mutation was an independent poor prognostic factor for OS and RFS among total patients (HR 2.218, 95% CI 1.333–3.692, P=0.002 and HR 2.898, 95% CI 1.673–5.022, P<0.001, respectively) and CN-AML group (HR 2.303, 95% CI 1.088–4.876, P=0.029 and HR 3.496, 95% CI 1.773–6.896, P<0.001, respectively). Further, a scoring system incorporating DNMT3A mutation and eight other prognostic factors, including age, WBC count, cytogenetics, and gene mutations (NPM1/FLT3-ITD, CEBPA, AML1/RUNX1, WT1, and IDH2 mutations), into survival analysis was proved to be very useful to stratify AML patients into different prognostic groups (P<0.001). DNMT3A mutations were serially studied in 316 samples from 138 patients, including 35 patients with distinct DNMT3A mutations and 103 patients without mutation at diagnosis. Among the 34 patients with DNMT3A mutations who had ever obtained a CR and had available samples for study, 29 lost the original mutation at remission status, but five retained it; all these five patients relapsed finally within a median of 3.5 months and died of disease progression, suggesting presence of leukemic cells. In the 13 patients who had available samples for serial study at relapse, all patients regained the original mutations, including mutant clone was found by TA cloning in one patient. Among the 103 patients who had no DNMT3A mutation at diagnosis, none acquired DNMT3A mutation at relapse, while karyotypic evolution was noted at relapse in 39% of them. Conclusion: DNMT3A mutations are associated with distinct clinical and biological features and poor prognosis in de novo AML patients. Furthermore, the mutation may be a potential biomarker for monitoring of minimal residual disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Clonal Hematopoiesis Landscape in Patients with De Novo Acute Myeloid Leukemia By Deep Sequencing

Blood ◽

10.1182/blood.v128.22.598.598 ◽

2016 ◽

Vol 128 (22) ◽

pp. 598-598

Author(s):

Mariam Ibáñez ◽

Alexander Neef ◽

Carmen Martínez-Losada ◽

Esperanza Such ◽

Desiree Company ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Deep Sequencing ◽

Myeloid Leukemia ◽

De Novo ◽

Clonal Evolution ◽

Genetic Alterations ◽

Prognostic Impact ◽

Clonal Hematopoiesis ◽

De Novo Aml ◽

Acute Myeloid

Abstract Purpose: Acute myeloid leukemia (AML) is associated with progressive accumulation of genetic alterations in hematopoietic progenitors. Massive sequencing allows inference of the clonal architecture of hematologic malignancies, by determining the presence of subclones, their genetic composition and evolution. The objective of the present study was to determine the spectrum of mutations present at relapse and define the proportion of cellular clones and the genetic architecture of the evolution of patients with de novo AML. Methods: Paired samples (diagnostic/relapse) of 44 patients of the University Hospital La Fe with de novo AML and treated with consecutive PETHEMA schemes were studied. The samples were provided by the Biobanco La Fe. The median age was 59 years (range 17 - 89); 21M/13F; 17 patients with normal karyotype; 14 patients with FLT3-ITD positive and 9 with mutations in NPM1. Using an amplicon panel (Ampliseq, Life Technologies) for deep sequencing (10.000x) with an Ion Torrent Proton, the complete coding regions of the following genes were sequenced, BCOR, BRAF, CDKN2A, CEBPA, DNMT3A, ETV6, EZH2, GNAS, LUC7L2, NF1, PHF6, PTPN11, RAD21, RPS14, SF1, SF3A1, SMC3, SPARC, SRSF2, STAG2 and ZRSR2, as well as, the hotspot regions of ASXL1, MPL, NPM1, JAK2, KRAS, NRAS, TET2, U2AF1, KIT, IDH1, RUNX1, IDH2, SETBP1, TP53, WT1, CBL, SF3B1 and FLT3. Primary bioinformatic analysis was performed using an in-house protocol and variants were selected based on VAF ≥ 1%, its absence in the healthy population (UCSC Common SNPs; MAF < 0.01) and its putative effect on the protein. Results: At least one alteration was detected in 98% of patients, (n = 43). At a mean sequencing depth of 8967x, in total, 249 mutations were detected with an average of 3.3 mutations per patient and sample (range 0 - 8). Comparing the two time points, we noted that 45% of the mutations were present at both moments, with rather similar VAF values. However, 24% were acquired during progression while 31% went missing at the time of relapse. Regarding the mutated genes analyzed at diagnosis, in 8 of 44 patients one single gene clone was detected, in 26 two subclones and in 10 three or more subclones. In addition, two different patterns of clonal evolution were detected. In model 1 the dominant founder clone persisted at relapse (n = 32, 71%), occasionally acquiring new changes, either in the same clone (n = 5) or in a new subclone (n = 17). In model 2 the founder clone was displaced at relapse by new subclones (n = 12, 29%), probably due to selective pressure through competition between subclones or as a consequence of the chemotherapy. Furthermore, the clonal hematopoiesis models did not show an association with clinical variables or prognostic impact on OS or EFS (P = 0.317; P = 0.12, respectively). Conclusions: AML cells can acquire additional mutations at relapse. Some of those may contribute to the clonal selection responsible for disease progression. Two models of clonal evolution were observed: model 1, where the dominant founder clone persists during relapse, and, model 2, where the founder clone is displaced by new cell subclones, displaying, both models, a similar impact on outcome. Financed by the Spanish Foundation of Hematology (FEHH), PI12/01047, RD12/0036/0014, PIE13/00046, PI13/01640, PI13/02837, PT13/0010/0026, PI14/01649, ACOMP2015/0335 and PROMETEOII/2015/study/025. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Complete Molecular Risk Stratification of De Novo Acute Myeloid Leukemia with Intermediate Cytogenetics Using an Eight-Gene Panel

Blood ◽

10.1182/blood.v124.21.2333.2333 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2333-2333

Author(s):

Maya Thangavelu ◽

Ryan Olson ◽

Li Li ◽

Wanlong Ma ◽

Steve Brodie ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Risk Stratification ◽

Myeloid Leukemia ◽

De Novo ◽

Direct Sequencing ◽

Core Binding Factor ◽

Binding Factor ◽

Molecular Abnormality ◽

De Novo Aml ◽

Acute Myeloid

Abstract Background: Refining risk stratification of acute myeloid leukemia (AML) using molecular profiling, especially those with intermediate cytogentic risk, is becoming standard of care. However, current WHO and ELN classifications are focused on few markers, mainly FLT3, NPM1, and CEBPA. While these abnormalities are relatively common, not all patients with AML and intermediate or normal cytogenetics will have abnormalities in these genes leaving large percentage of patients without refined risk stratification. We demonstrate that using 8 different AML-related genes are adequate to provide one or more molecular markers to further risk stratify patients with de novo AML. Method: Using direct sequencing we analyzed 211 samples referred from community practice with the diagnosis AML for molecular analysis. All samples were evaluated prospectively for mutations in FLT3, NPM1, IDH1, IDH2, CEBPA, WT1, RUNX1, and TP53 using direct sequencing. Fragment length analysis was used in addition to sequencing for FLT3 and NPM1. Available morphology, cytogenetics, and clinical data along with history were reviewed. Results: Of the 211 samples tested 103 (49%) had at least one or more molecular abnormality adequate for refining the risk classification. The mutations detected in these 103 patients were as follows: 27 (26%) FLT-ITD, 10 (10%) FLT3-TKD, 30 (29%) NPM1, 7 (7%) CEBPA, 14 (14%) IDH1, 13 (13%) IDH2, 10 (10%) WT1, 38 (37%) RUNX1, and 2 (2%) TP53. There was significant overlap and most patients had more than one mutation as illustrated in the graph below. However, if the testing was restricted to FLT3, NPM1, CEBPA and DNMT3A, only 56 (54%) would have had refined risk classification and 46% of patients would have remained without subclassification. The most striking finding was that all the remaining patients, who had no molecular abnormality detected in any of these 8 genes, had either history of MDS evolved to AML, therapy-related AML, or cytogenetic abnormalities other than intermediate (multiplex cytogenetic abnormalities or core-binding factor abnormality). Conclusion: Using FLT3, NPM1, CEBPA, and DNMT3A is inadequate for the molecular characterization of patients with AML. Patients with de novo AML and intermediate risk cytogenetics can be adequately prognostically subclassified and molecularly studied by testing only 8 genes. More importantly, this data confirms that the molecular biology driving de novo AML is significantly different from that driving MDS, AML with myelodysplasia-related changes, therapy-related AML, or AML with core binding factor or multiplex cytogenetics. Unlike de novo AML, these entities should be molecularly studied using MDS-specific driver genes. Furthermore, this data suggests that different therapeutic approaches should be developed for MDS and MDS-related AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Association of TET2 Alterations with NPM1 Mutations and Prognostic Value in De Novo Acute Myeloid Leukemia (AML).

Blood ◽

10.1182/blood.v114.22.163.163 ◽

2009 ◽

Vol 114 (22) ◽

pp. 163-163 ◽

Cited By ~ 3

Author(s):

Olivier Nibourel ◽

Olivier Kosmider ◽

Meyling Cheok ◽

Nicolas Boissel ◽

Aline Renneville ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Prognostic Value ◽

De Novo ◽

Missense Mutations ◽

Npm1 Mutation ◽

Tet2 Mutation ◽

De Novo Aml ◽

Acute Myeloid

Abstract Abstract 163 In acute myeloid leukemia (AML), both cytogenetic and molecular abnormalities are strongly associated with prognosis. In particular, in cytogenetically normal AML (CN-AML), FLT3-ITD (internal tandem duplication) carries adverse prognostic factor whereas NPM1 or CEBPA mutations are associated with favorable outcome. Recently, mutations of the ten eleven translocation 2 gene (TET2) have been reported myeloid neoplasms. We evaluated the frequency and prognostic value of TET2 alterations, in a cohort of 111 de novo AML patients. We studied 111 patients aged between 15 years and 69 years with previously untreated de novo AML who had reached complete remission (CR) using intensive chemotherapy. 28 of them also received an allogenic bone marrow transplantation in first CR. Analysis of TET2 sequence variation was performed by direct sequencing of PCR products from 111 genomic DNA samples obtained at diagnosis. Frameshift and nonsense variations were all scored as mutation whereas missense mutations were retained when observed at diagnostic but absent in the CR paired sample obtained. Previously identified single nucleotide polymorphisms (SNP) were not considered. TET2 anomalies were numbered according to Genebank reference FM992369. Paired diagnosis and CR genomic DNAs were analyzed using Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA). Data were analyzed using Gene Chip Genotyping Console 3.0.2 and Partek Genomics Suite (www.partek.com/). Comparisons were made by Fisher's exact test for binary variables and the Mann-Whitney‘s test for continuous variables. Disease Free Survival (DFS) and overall survival (OS) were calculated according to the Kaplan-Meier method. Comparisons regarding DFS and OS were performed with the log-rank test. 24 acquired TET2 mutations were observed in 19 of the 111 (17%) de novo AML patients, suggesting the alteration of the two TET2 alleles in 5 patients. They included 21 different events: 6 frameshift, 7 non-sense and 11 missense mutations. Four of the missense mutations were located in conserved regions and 7 outside. All of them were detected in the diagnostic sample but were absent in the paired remission sample. Except for two missense mutations (S282F, T492S) both detected in two patients, no recurrent TET2 mutation was observed. Acquired mutations were spread over all exons. No case of uniparental disomy (UPD) was observed and only one patient presented a small deletion of 60Kb in the TET2 gene locus without TET2 mutation. No significant difference was observed between patients with or without TET2 alterations for gender, age, hemoglobin level, platelet count, FAB subtypes distribution and cytogenetics according to MRC classification, but there was a trend for higher WBC count in patients with TET2 alteration. No significant association was observed between TET2 mutations and FLT3 or CEBPA alterations. However, TET2 alterations were significantly associated with NPM1 mutations (p=0.032). In the entire patient cohort, no difference in DFS or OS was seen between patients with and without TET2 alteration. However, a significantly worse DFS was observed for patients presenting TET2 mutations within the subgroup of patients with NPM1 mutations (3y-DFS: 0% vs 66.4%, 95% CI [45.6–87.2], p=0.008) Considering both the favorable prognosis of NPM1 mutations without FLT3-ITD in CN-AML and the absence of clear association between FLT3-ITD and TET2 alterations in this study, prognostic value of the genotype characterized by NPM1 mutation without FLT3-ITD or TET2 alteration (NPM1+FLT3-ITD-TET2-) was compared to other patients within CN-AML group (N=54). NPM1+FLT3-ITD-TET2- patients showed a significantly better DFS and OS compared to other patients in CN-AML group (3y-DFS: 82.1%, 95% CI [59.1–100] vs 37.3%, 95% CI [20.2–54.3], p=0.01; 3y-OS: 80.8%, 95% CI [56.1–100] vs 42.3%, 95% CI [23.3–61.3], p=0.04). In conclusion, we observed point mutations of TET2 in 17% of patients, whereas TET2 deletion or UPD are very rare. In our study, TET2 mutations were clearly associated with NPM1 mutations and carried a negative prognostic impact in this subgroup. Screening for TET2 mutations may improve the characterization of CN-AML and help to identify within the low-risk subgroup with NPM1 mutation and without FLT3-ITD, patients at high risk of relapse. Disclosures: Fenaux: Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Ortho Biotech: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Download Full-text