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Rapid Screening of ASXL1, IDH1, IDH2, and c-CBL Mutations in de Novo Acute Myeloid Leukemia by High-Resolution Melting

2012; Elsevier BV; Volume: 14; Issue: 6 Linguagem: Inglês

10.1016/j.jmoldx.2012.06.006

ISSN

1943-7811

Autores

Mariam Ibáñez, Esperanza Such, José Cervera, Irene Luna, Inés Gómez‐Seguí, Maria López‐Pavía, Sandra Dolz, Eva Barragán, Óscar Fuster, Marta Llop, Rebeca Rodríguez‐Veiga, Amparo Avaria, Silvestre Oltra, M. Leonor Senent, Federico Moscardó, Pau Montesinos, David Martínez‐Cuadrón, Guillermo Martı́n, Miguel Á. Sanz,

Tópico(s)

Myeloproliferative Neoplasms: Diagnosis and Treatment

Resumo

Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1R132 and 6% with IDH2R140), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, FLT3, or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome. Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1R132 and 6% with IDH2R140), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, FLT3, or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome. In addition to the well-established mutations in the FLT3, NPM1, and CEBPA genes as prognostic markers,1Grimwade D. Hills R.K. Moorman A.V. Walker H. Chatters S. Goldstone A.H. Wheatley K. Harrison C.J. Burnett A.K. National Cancer Res Institute Adult Leukaemia Working Group Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials.Blood. 2010; 116: 354-365Crossref PubMed Scopus (1454) Google Scholar, 2Döhner H. Estey E.H. Amadori S. Appelbaum F.R. Büchner T. Burnett A.K. Dombret H. Fenaux P. Grimwade D. Larson R.A. Lo-Coco F. Naoe T. Niederwieser D. Ossenkoppele G.J. Sanz M.A. Sierra J. Tallman M.S. Löwenberg B. Bloomfield CD; European LeukemiaNet Diagnosis and management of acute myeloid leukaemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet.Blood. 2010; 115: 453-474Crossref PubMed Scopus (2607) Google Scholar, 3Renneville A. Roumier C. Biggio V. Nibourel O. Boissel N. Fenaux P. Preudhomme C. Cooperating gene mutations in acute myeloid leukemia: a review of the literature.Leukemia. 2008; 22: 915-931Crossref PubMed Scopus (294) Google Scholar various novel molecular abnormalities have been found in acute myeloid leukemia (AML), especially in patients with the normal karyotype.4Grand F.H. Hidalgo-Curtis C.E. Ernst T. Zoi K. Zoi C. McGuire C. Kreil S. Jones A. Score J. Metzgeroth G. Oscier D. Hall A. Brandts C. Serve H. Reiter A. Chase A.J. Cross N.C. Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms.Blood. 2009; 113: 6182-6192Crossref PubMed Scopus (308) Google Scholar Specific examples are mutations in the ASXL1 gene (6% to 30%),5Chou W.C. Huang H.H. Hou H.A. Chen C.Y. Tang J.L. Yao M. Tsay W. Ko B.S. Wu S.J. Huang S.Y. Hsu S.C. Chen Y.C. Huang Y.N. Chang Y.C. Lee F.Y. Liu M.C. Liu C.W. Tseng M.H. Huang C.F. Tien H.F. Distinct clinical and biological features of de novo acute myeloid leukemia with additional sex comb-like 1 (ASXL1) mutations.Blood. 2010; 116: 4086-4094Crossref PubMed Scopus (166) Google Scholar, 6Carbuccia N. Trouplin V. Gelsi-Boyer V. Murati A. Rocquain J. Adélaïde J. Olschwang S. Xerri L. Vey N. Chaffanet M. Birnbaum D. Mozziconacci M.J. Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias.Leukemia. 2010; 24: 469-473Crossref PubMed Scopus (82) Google Scholar, 7Boultwood J. Perry J. Pellagatti A. Fernandez-Mercado M. Fernandez-Santamaria C. Calasanz M.J. Larrayoz M.J. Garcia-Delgado M. Giagounidis A. Malcovati L. Della Porta M.G. Jädersten M. Killick S. HellströM-Lindberg E. Cazzola M. Wainscoat J.S. Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukemia.Leukemia. 2010; 24: 1062-1065Crossref PubMed Scopus (214) Google Scholar IDH1 and IDH2 genes (8% to 33%),8Mardis E.R. Ding L. Dooling D.J. Larson D.E. McLellan M.D. Chen K. Koboldt D.C. Fulton R.S. Delehaunty K.D. McGrath S.D. Fulton L.A. Locke D.P. Magrini V.J. Abbott R.M. Vickery T.L. Reed J.S. Robinson J.S. Wylie T. Smith S.M. Carmichael L. Eldred J.M. Harris C.C. Walker J. Peck J.B. Du F. Dukes A.F. Sanderson G.E. Brummett A.M. Clark E. McMichael J.F. Meyer R.J. Schindler J.K. Pohl C.S. Wallis J.W. Shi X. Lin L. Schmidt H. Tang Y. Haipek C. Wiechert M.E. Ivy J.V. Kalicki J. Elliott G. Ries R.E. Payton J.E. Westervelt P. Tomasson M.H. Watson M.A. Baty J. Heath S. Shannon W.D. Nagarajan R. Link D.C. Walter M.J. Graubert T.A. DiPersio J.F. Wilson R.K. Ley T.J. Recurring mutations found by sequencing an acute myeloid leukemia genome.N Engl J Med. 2009; 361: 1058-1066Crossref PubMed Scopus (1800) Google Scholar, 9Ward P.S. Patel J. Wise D.R. Abdel-Wahab O. Bennett B.D. Coller H.A. Cross J.R. Fantin V.R. Hedvat C.V. Perl A.E. Rabinowitz J.D. Carroll M. Su S.M. Sharp K.A. Levine R.L. Thompson C.B. The common feature of leukemia-associated idh1 and idh2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.Cancer Cell. 2010; 17: 225-234Abstract Full Text Full Text PDF PubMed Scopus (1509) Google Scholar, 10Gross S. Cairns R.A. Minden M.D. Driggers E.M. Bittinger M.A. Jang H.G. Sasaki M. Jin S. Schenkein D.P. Su S.M. Dang L. Fantin V.R. Mak T.W. Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations.J Exp Med. 2010; 207: 339-344Crossref PubMed Scopus (598) Google Scholar, 11Paschka P. Schlenk R.F. Gaidzik V.I. Habdank M. Krönke J. Bullinger L. Späth D. Kayser S. Zucknick M. Götze K. Horst H.A. Germing U. Döhner H. Döhner K. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication.J Clin Oncol. 2010; 28: 3636-3643Crossref PubMed Scopus (650) Google Scholar and c-CBL proto-oncogene (∼1%).12Makishima H. Cazzolli H. Szpurka H. Dunbar A. Tiu R. Huh J. Muramatsu H. O'Keefe C. Hsi E. Paquette R.L. Kojima S. List A.F. Sekeres M.A. McDevitt M.A. Maciejewski J.P. Mutations of e3 ubiquitin ligase cbl family members constitute a novel common pathogenic lesion in myeloid malignancies.J Clin Oncol. 2009; 27: 6109-6116Crossref PubMed Scopus (173) Google Scholar, 13Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1.Leukemia. 2010; 24: 1128-1138Crossref PubMed Scopus (432) Google Scholar Although their clinical relevance and prognostic implications are not yet well established, these mutations appear to exert a characteristic phenotype-modifying effect. In this regard, ASXL1 mutations seem to lead to epigenetic dysregulation14Bejar R. Levine R. Ebert B.L. Unraveling the molecular pathophysiology of myelodysplastic syndromes.Clin Oncol. 2011; 29: 504-515Crossref Scopus (258) Google Scholar; IDH mutations result in the generation of the aberrant metabolite 2-hydroxyglutarate, which induces DNA hypermethylation and impairs differentiation in hematopoietic cells through inhibition of TET215Figueroa M.E. Abdel-Wahab O. Lu C. Ward P.S. Patel J. Shih A. Li Y. Bhagwat N. Vasanthakumar A. Fernandez H.F. Tallman M.S. Sun Z. Wolniak K. Peeters J.K. Liu W. Choe S.E. Fantin V.R. Paietta E. Löwenberg B. Licht J.D. Godley L.A. Delwel R. Valk P.J. Thompson C.B. Levine R.L. Melnick A. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation.Cancer Cell. 2010; 18: 553-567Abstract Full Text Full Text PDF PubMed Scopus (2014) Google Scholar; and IDH1 single-nucleotide polymorphism (SNP) rs11554137 may alter IDH1 activity by alterations in RNA.16Wagner K. Damm F. Göhring G. Görlich K. Heuser M. Schäfer I. Ottmann O. Lübbert M. Heit W. Kanz L. Schlimok G. Raghavachar A.A. Fiedler W. Kirchner H.H. Brugger W. Zucknick M. Schlegelberger B. Heil G. Ganser A. Krauter J. Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid leukemia: sNP rs11554137 is an adverse prognostic factor.J Clin Oncol. 2010; 28: 2356-2364Crossref PubMed Scopus (198) Google Scholar Finally, c-CBL alterations produce an abnormal ubiquitination of MPL,17Saur S.J. Sangkhae V. Geddis A.E. Kaushansky K. Hitchcock I.S. Ubiquitination and degradation of the thrombopoietin receptor c-Mpl.Blood. 2010; 115: 1254-1263Crossref PubMed Scopus (70) Google Scholar KIT,18Bandi S.R. Brandts C. Rensinghoff M. Grundler R. Tickenbrock L. Köhler G. Duyster J. Berdel W.E. Müller-Tidow C. Serve H. Sargin B; Study Alliance Leukemias E3 ligase-defective Cbl mutants lead to a generalized mastocytosis and myeloproliferative disease.Blood. 2009; 114: 4197-4208Crossref PubMed Scopus (42) Google Scholar and FLT3,19Sargin B. Choudhary C. Crosetto N. Schmidt M.H. Grundler R. Rensinghoff M. Thiessen C. Tickenbrock L. Schwäble J. Brandts C. August B. Koschmieder S. Bandi S.R. Duyster J. Berdel W.E. Müller-Tidow C. Dikic I. Serve H. Flt3-dependent transformation by inactivating c-Cbl mutations in AML.Blood. 2007; 110: 1004-1012Crossref PubMed Scopus (104) Google Scholar promoting growth factor independence.35Gelsi-Boyer V. Trouplin V. Roquain J. Adélaïde J. Carbuccia N. Esterni B. Finetti P. Murati A. Arnoulet C. Zerazhi H. Fezoui H. Tadrist Z. Nezri M. Chaffanet M. Mozziconacci M.J. Vey N. Birnbaum D. ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia.Br J Haematol. 2010; 151: 365-375Crossref PubMed Scopus (178) Google Scholar Until recently, mutation detection has relied heavily on the use of methods, such as Sanger sequencing, that are expensive and time-consuming. The availability of reliable methods for a more rapid and efficient genetic-based testing is therefore critical for clinical diagnosis. A reasonable approach for mutation detection is to use screening methods to identify specific regions of sequence variation, thus reducing the amount of direct sequencing required. High-resolution melting (HRM) is a novel method to analyze genetic variations based on their sequence, length, GC content, or strand complementarity. Therefore, mutations are detectable due to changes in the shape of DNA melting curves. In this report, we screened acquired mutations of the ASXL1, IDH1, IDH2, and c-CBL genes in a relatively large cohort of patients with de novo AML from a single institution to learn their incidence and prognostic effect. For this objective, we developed a new combination of HRM assays. The study includes 175 adult patients with de novo AML (excluding acute promyelocytic leukemia) recruited between 1998 and May 2009 at the Hospital Universitario y Politécnico La Fe of Valencia, Spain. For the selection of the patients, availability of DNA at diagnosis was the limiting criterion for inclusion. The diagnosis and classification of AML were made according to the French-American-British criteria.11Paschka P. Schlenk R.F. Gaidzik V.I. Habdank M. Krönke J. Bullinger L. Späth D. Kayser S. Zucknick M. Götze K. Horst H.A. Germing U. Döhner H. Döhner K. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication.J Clin Oncol. 2010; 28: 3636-3643Crossref PubMed Scopus (650) Google Scholar, 12Makishima H. Cazzolli H. Szpurka H. Dunbar A. Tiu R. Huh J. Muramatsu H. O'Keefe C. Hsi E. Paquette R.L. Kojima S. List A.F. Sekeres M.A. McDevitt M.A. Maciejewski J.P. Mutations of e3 ubiquitin ligase cbl family members constitute a novel common pathogenic lesion in myeloid malignancies.J Clin Oncol. 2009; 27: 6109-6116Crossref PubMed Scopus (173) Google Scholar Biphenotypic AML classification was based on multiparameter flow cytometry. One hundred thirty-eight patients were enrolled in three successive trials (AML92 and PETHEMA, Spanish Program for Treatments in Hematology, 1999 and 2007) in which induction chemotherapy consisted of idarubicin plus cytarabine, with or without etoposide. Postremission therapy included 1 to 2 courses of consolidation chemotherapy with intermediate- or high-dose cytarabine followed by autologous transplantation when feasible (n = 18) or allogeneic stem cell transplantation (n = 34). Patients not receiving chemotherapy (n = 37) were excluded for the survival analysis. The institutional ethics committee for clinical research approved this study, and a written informed consent in accordance with the recommendations of the Declaration of Human Rights, the Conference of Helsinki, and institutional regulations were obtained from all patients. The Hospital La Fe Biobank provided all study samples and controls. Bone marrow samples were obtained from all patients at the time of diagnosis. Genomic DNA was isolated using a MagNA Pure LC large volume DNA Isolation Kit (Roche, Mannheim, Germany) with the automatic MagNA Pure LC System (Roche Molecular Biochemicals, Indianapolis, IN). The quality and concentration of DNA extracted were assessed with a Nano-Drop-1000 (NanoDrop Technologies Inc, Wilmington, DE). The genomic DNA was amplified using the REPLI-g Midi Kit (Qiagen GmbH, Hilden, Germany). All samples were diluted with Tris-borate-EDTA buffer to a final concentration of 20 ng/μL. Karyotype was performed following standard cytogenetic methods and chromosomal abnormalities described according to the International System for Human Cytogenetic Nomenclature.20Shaffer L.G. Slovak M.L. Campbell L.J. eds An International System for Human Cytogenetic Nomenclature: Recommendations of the International Standing Committee.Basel, S. Karger. 2009; Google Scholar Whenever possible, at least 20 metaphases were analyzed. Cytogenetic analysis of 24 patients could not be performed because of a lack of an appropriate sample at diagnosis. Cytogenetic risk stratification was established according to the refined Medical Research Council criteria.1Grimwade D. Hills R.K. Moorman A.V. Walker H. Chatters S. Goldstone A.H. Wheatley K. Harrison C.J. Burnett A.K. National Cancer Res Institute Adult Leukaemia Working Group Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials.Blood. 2010; 116: 354-365Crossref PubMed Scopus (1454) Google Scholar FLT3-ITD and FLT3-D835,21Moreno I. Martín G. Bolufer P. Barragán E. Rueda E. Román J. Fernández P. León P. Mena A. Cervera J. Torres A. Sanz M.A. Incidence and prognostic value of FLT3 internal tandem duplication and D835 mutations in acute myeloid leukaemia.Haematologica. 2003; 88: 19-24PubMed Google Scholar, 22Thiede C. Steudel C. Mohr B. Schaich M. Schäkel U. Platzbecker U. Wermke M. Bornhäuser M. Ritter M. Neubauer A. Ehninger G. Illmer T. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.Blood. 2002; 99: 4326-4335Crossref PubMed Scopus (1415) Google Scholar NPM1,23Schnittger S. Schoch C. Kern W. Mecucci C. Tschulik C. Martelli M.F. Haferlach T. Hiddemann W. Falini B. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (592) Google Scholar and CEBPA24Fuster O. Barragán E. Bolufer P. Such E. Valencia A. IbÁñez M. Dolz S. de Juan I. Jiménez A. Gómez M.T. Buño I. Martínez J. Cervera J. Montesinos P. MoscardÓ F. Sanz MÁ Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.Ann Hematol. 2012; 91: 1-7Crossref PubMed Scopus (11) Google Scholar mutations were assessed as previously reported. We checked the mutational status of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) in all 175 enrolled patients. PCR amplification and HRM were performed with a LightCycler480 Instrument (Roche Diagnostics, Rotkreuz, Switzerland) with a 96-well thermal block using specific primers (Table 1). All samples were tested in duplicate. One positive mutated DNA sample for each exon and two wild-type DNA samples were included in each run.Table 1Selected Primers for PCR and HRM AnalysisGeneGenBank accession no.Forward primerReverse primerAmplicon (bp)Annealing conditions[Mg2+]ASXL1_1ANM_015338.45′-AGGTCAGATCACCCAGTCAGTT-3′5′-CAGCAGTGGTGATGGTGGTGA-3′34165°C–55°C touchdown 1°C/cycle2.5 mmol/LASXL1_1BNM_015338.45′-TGCCATAGAGAGGCGGCCACC-3′5′-ACAGTTGGACTCACAGATGGGCTA-3′33665°C–55°C touchdown 1°C/cycle2.5 mmol/LASXL1_2ANM_015338.45′-AGAGGACCTGCCTTCTCTGAGAAA-3′5′-GGAACTGGCCAAGCTCTTGAC-3′34365°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_2BNM_015338.45′-GCAAGGACCCACCGTTCCTGC-3′5′-GCATTGGATACCCATCCCATCGAA-3′30365°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_3ANM_015338.45′-ACTTGAAAACCAAGGCTCTCGT-3′5′-CACGGTGAGTCCACGGATACA-3′35365°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_3BNM_015338.45′-TCTGGACTGTGCCATCTCGAG-3′5′-TACAAGGACAGATGGGATGGTTGC-3′30765°C–55°C touchdown 1°C/cycle2.5 mmol/LASXL1_4ANM_015338.45′-GGTGGACAAGGATGAGAAACCCAA-3′5′-CCATGGCTCGCTACGCATGGG-3′35865°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_4BNM_015338.45′-CCCACGATGACAGCATGTCAG-3′5′-AAGTCCGTGCTATGTCACAGGACA-3′38465°C–55°C touchdown 1°C/cycle2.5 mmol/LASXL1_5ANM_015338.45′-TGGATTCCAAAGAGCAGTTCTCTTC-3′5′-CCATGTCTGGTGGGGTACAGA-3′36665°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_5BNM_015338.45′-CTTTTTGGCTCTGGGAATGTGG-3′5′-TTGGAAGGGATGCCCTTTGTCATG-3′30665°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_6ANM_015338.45′-ACAGGAAAGCTACTGGGCATAGTC-3′5′-CACTGACAGCAGCACGGTGGA-3′34965°C–55°C touchdown 1°C/cycle2.0 mmol/LASXL1_6BNM_015338.45′-GCCTTGCTGGAAGTGTGGTG-3′5′-ACTCTTTAGGCAGGAGCACTCTTG-3′34765°C–55°C touchdown 1°C/cycle2.5 mmol/LIDH111NM_0058965′-CCATTTGTCTGAAAAACTTTGCT-3′5′-GTAAGTCATGTTGGCAATATTGTGA-3′36059°C2.0 mmol/LIDH211NM_0021685′-GCTGCAGTGGGACCACTATT-3′5′-CTCTGCAGTACAAGGCCACA-3′29360°C2.5 mmol/Lc-cbl84NM_0051885′-CAGTTATTTATTCAACT-3′5′-CTGGCTTTTGGGGTTAGGTT-3′28660°C2.0 mmol/Lc-cbl94NM_0051885′-CTGTTACTATCTTTTGCTT-3′5′-AAAGCCGTAAAACACTTAACGA-3′30958°C2.0 mmol/L Open table in a new tab All assays were performed in 10 μL of final volume reaction with 20 ng of DNA. Optimal PCR annealing temperatures and MgCl2 concentrations were evaluated for each amplicon. Thermal cycling consisted of an initial 10-minute hold at 95°C, followed by a 10-second hold at 95°C, a 13-second hold at the indicated annealing temperature (Table 1), and a 14-second hold at 72°C for 45 cycles. Finally, heteroduplexes were generated by adding a step at 95°C for 1 minute and cooling the reaction to 40°C for 1 minute, with a 60°C to 95°C melting gradient and a ramp rate of 0.04°C/s a and continuous acquisition mode set at 25 acquisitions/°C, regardless of the amplicon analyzed. Melting curves from the samples were automatically normalized and analyzed by direct comparison with LightCycler 480 Software, version 1.5 (Roche Diagnostics). All of the patient samples with curves that differed in shape and/or melting temperature were considered as potential variant carriers. The results were confirmed by direct sequence. The PCR products, from each sample, were mixed with ExoSap-IT (USB Corporation, Cleveland, OH) to remove the remaining primers and bidirectionally sequenced with forward and reverse primers using the Big Dye Terminator v1.1 Cycle Sequencing Reaction kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol using an ABIPRISM 3130 DNA Analyzer (Applied Biosystems). Sequence traces were analyzed using Applied Biosystems software and reviewed manually. The analysis was checked by its corresponding GenBank accession number. Differences in the distribution of variables among subsets of patients were analyzed using χ2 and Fisher exact tests with the SPSS statistical software package, version 17.0 (SPSS Inc, Chicago, IL). All tests were two-sided, and P < 0.05 was considered statistically significant. Unadjusted time-to-event analysis was performed using the Kaplan-Meier method.25Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration.Cancer Chemother Rep. 1966; 50: 163-170PubMed Google Scholar Variables included in the regression analyses were age, white blood cell count, platelet count, percentage of bone marrow blasts, cytogenetic risk group, and mutational status of the NPM1 (exon 12), FLT3 (ITD and D835), CEBPA, ASXL1, IDH1, IDH2,and c-CBL genes. Complete remission and resistant disease were defined according to the recommendations of Cheson et al.26Cheson B.D. Bennett J.M. Kopecky K.J. Büchner T. Willman C.L. Estey E.H. Schiffer C.A. Doehner H. Tallman M.S. Lister T.A. Lo-Coco F. Willemze R. Biondi A. Hiddemann W. Larson R.A. Löwenberg B. Sanz M.A. Head D.R. Ohno R. Bloomfield C.D. International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia.J Clin Oncol. 2003; 21: 4642-4649Crossref PubMed Scopus (2212) Google Scholar Overall survival was measured from the time of diagnosis to the time of last follow-up or death from any cause. Disease-free survival and relapse-free survival were measured from the date of complete remission. In the analysis of disease-free survival, relapse or death, whichever occurred first, was considered an uncensored event. For relapse-free survival, relapse was considered an uncensored event. The follow-up of the patients was updated on September 2011, and all follow-up data were censored at that time point. The median follow-up of surviving patients was 59 months (range, 26 to 144 months). One hundred seventy-five patients were screened for the presence of ASXL1, IDH1, IDH2, and c-CBL mutations. Melting curves revealed a variation between the group with wild-type DNA and those with mutations. This discrepancy was further enhanced with the difference plot, which establishes clusters of samples. For the duplicates of each sample, the curves practically overlapped either in the same assay or between assays. Direct sequencing produced no false-positive or false-negative results. All samples were classified in the correct cluster. The wild-type sample had a normal DNA sequence, and the patients in different clusters had sequence variations and mutations. The analysis of melting curves well distinguished between the group of wild-type DNA and the variant carrier samples (Figure 1). We found variations in 44 patients (25%) for ASXL1, 26 patients (15%) for IDH1, and 11 patients (6%) for IDH2. None were found in c-CBL. The analysis of the variations detected by HRM using direct sequencing identified 17 different mutations of ASXL1 in 16 patients (9%) (one patient presented with two simultaneous mutations). The anomalies were p.C606Xs, c.2898_2900delAAG (n = 3 patients), p.L1108F (n = 4 patients), p.E1102D, p.S1169F, p.R1171R (n = 2 patients), p.G1287D, p.G1278D, p.K1302L (n = 2 patients), and p.A1313E. We detected known SNP variants in 28 patients (16%). We also detected canonical IDH mutations in 16 patients (9%). Twenty-six patients (15%) carried variations in IDH1, 5 patients (3%) had the IDH1R132 mutation, and 22 patients (13%) had the SNP rs11554137. Eleven patients (6%) had the IDH2R140 mutation (Figure 1). Only two patients presented with the SNP rs11554137 and the IDH2R140 mutation simultaneously. As a whole, 32 patients (18%) harbored mutations in the ASXL1 or IDH genes. In addition, FLT3, NPM1, and CEBPA mutations were detected in 37 (21%), 44 (25%), and 8 patients (5%), respectively. Main characteristics of the series according to the mutational status are listed in Table 2. ASXL1 and IDH variations were correlated with no clinical or biological features studied (Table 1). We found no statistically significant pattern of cooperating mutations in the studied group of genes. However, patients with ASXL1 mutations did not harbor IDH1, FLT3, or CEBPA mutations. A combination of ASXL1 and IDH2 mutations was found only in a single patient. We found no concurrent IDH1 and FLT3 or CEBPA mutations and no IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. The concurrence of NPM1 mutations and ASXL1, IDH1, and IDH2 was 50%, 50%, and 20%, respectively. FLT3-ITD mutations were found in 25% of ASXL1 mutated patients and in 80% of IDH2 mutated patients. FLT3-D835 mutations were carried simultaneously in 17% of ASXL1 and 11% of IDH2 mutated patients (Figure 2).Table 2Main Characteristics of the SeriesCharacteristicsAll patientsASXL1IDH1IDH2Wild typeMutatedWild typeMutatedSNPWild typeMutatedOverall, no. (%)175159 (91)16 (9)148 (84)5 (3)22 (13)164 (94)11 (6)Mean age, years (range)62 (16–88)62 (17–88)62 (16–83)62 (16–88)43 (30–69)63 (21–87)61 (17–81)62 (16–88)Age, no. (%) ≥60 years99 (57)90 (57)9 (56)85 (57)1 (20)13 (59)93 (57)6 (46) <60 years76 (43)69 (43)7 (44)63 (43)4 (80)9 (41)71 (43)5 (44)Sex, no. (%) Male99 (57)91 (60)8 (50)79 (53)4 (80)16 (73)94 (57)5 (54) Female76 (43)68 (40)8 (50)69 (47)1 (20)6 (27)70 (43)6 (56)Cytogenetic risk assessment, no. (%) Favorable12 (8)11 (8)1 (8)8 (6)2 (50)2 (11)12 (9)0 (0) Intermediate102 (68)94 (69)8 (62)90 (71)1 (25)11 (53)96 (68)7 (70) Adverse37 (24)33 (23)4 (31)29 (23)1 (25)7 (37)33 (48)3 (30)Karyotype, no. (%) Normal67 (44)62 (45)5 (38)57 (45)0 (0)10 (53)64 (45)3 (30)FAB, no. (%) M021 (12)19 (12)2 (13)19 (13)0 (0)2 (9)21 (13)0 (0) M142 (24)37 (23)5 (31)34 (23)1 (20)7 (32)39 (24)3 (27) M240 (23)39 (25)1 (6)34 (23)0 (0)6 (27)39 (24)1 (9) M430 (17)28 (18)2 (13)24 (16)2 (40)4 (19)28 (17)2 (18) M520 (11)15 (9)5 (31)16 (11)1 (20)3 (14)18 (11)2 (18) M614 (8)13 (8)1 (6)14 (8)0 (0)0 (0)12 (7)2 (18) M72 (1)2 (1)0 (0)1 (1)1 (20)0 (0)1 (1)1 (9)Biphenotypic, no. (%)5 (3)5 (3)0 (0)5 (3)0 (0)0 (0)5 (3)0 (0)Unclassified, no. (%)1 (1)1 (1)0 (0)1 (1)0 (0)0 (0)1 (1)0 (0)White blood cell count, ×109/L Median11.709.4309.2520.326.512.42.6 Range1–3961–3962–3851–3964–1472–2501–3961–81Platelet count, ×109/L Median58585164694857,558 Range5–4085–40818–1315–40836–22214–1695–40818–133Hemoglobin, g/dL Median8.908.90138.98.99.298.3 Range5–175–170–895–175–175–145–1718–133PMNs, ×109/L Median13138.5013.76101214.2 Range0–890–890–460–890–560–620–890–35Blood blasts, % Median38308029.554503926.5 Range0–1000–1001–980–1001–900–970–1000–98FLT3-ITD, no. (%) Positive32 (21)28 (18)4 (25)26 (20)0 (0)6 (33)30 (21)8 (80) Negative122 (79)112 (82)10 (75)105 (80)5 (100)12 (67)114 (79)2 (20)FLT3-D835, no. (%) Positive7 (5)5 (4)2 (17)6 (5)0 (0)1 (6)6 (4)1 (11) Negative147 (95)135 (96)12 (83)125 (95)5 (100)17 (94)138 (96)9 (89)NPM1, no. (%) Mutated44 (30)38 (24)6 (50)36 (28)2 (50)6 (35)42 (30)2 (20) Wild type104 (70)98 (76)6 (50)91 (72)2 (50)11 (65)96 (70)8 (80)FLT3-ITD/NPM1 status, no. (%) FLT3-ITD negative/NPM1 mutation28 (20)24 (19)4 (33)22 (18)2 (50)4 (25)26 (20)2 (22) FLT3-ITD positive/NPM1 wild type14 (10)12 (9)2 (17)12 (10)0 (0)2 (13)13 (10)1 (11) FLT3-ITD negative/NPM1 wild type85 (60)81 (51)4 (33)75 (62)2 (50)8 (50)79 (60)6 (67) FLT3-ITD positive/NPM1 mutation14 (10)12 (8)2 (17)12 (10)0 (0)2 (13)14 (11)0 (0)CEBPA, no. (%) Mutated8 (10)8 (11)0 (0)7 (10)0 (0)1 (14)8 (11)0 (0) Wild type72 (90)65 (89)7 (100)65 (90)1 (100)6 (86)68 (89)4 (100)ASXL1, no. (%) Mutated16 (9)14 (9)0 (0)2 (9)15 (9)1 (10) Wild type159 (91)134 (91)5 (100)20 (91)149 (91)10 (90)IDH1, no. (%) Mutated5 (3)0 (0)0 (0)5 (3)0 (0) Wild type148 (84)134 (87)14 (88)139 (97)9 (100) SNP22 (13)20 (13)2 (12)20 (12)0 (0)IDH2, no. (%) Muta

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