A new assay to identify recurrent mutations in acute myeloid leukemia using next-generation sequencing

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Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a late onset (it is rare in children), aggressive phenotype and dismal prognosis especially in patients in the older group (>65 years). For risk stratification of patients standard cytogenetic is used along with molecular techniques for point mutation identification. Here we describe a new method using next generation sequencing for identification of mutation in 5 AML recurrently mutated genes - RUNX1, FLT3, DNMT3A, IDH1 and IDH2. Materials and methods: Samples from 40 patients with normal karyotype AML referred to Fundeni Clinical Institute were sequenced. Primer design was performed using LaserGene Genomics suit. Next generation sequencing was performed on MiSeq (Illumina) and results were analyzed using LaserGene Genomics suit. Results of next generation sequencing were compared to Sanger sequencing. Results: No additional mutations were identified in samples from nine patients presenting FLT3-ITD and/or NPM1 mutations. In 25 out of 31 patients with normal karyotype and no FLT3-ITD and NPM1 mutations, we identified mutations in one of the 5 aforementioned genes. All these mutations identified by next generation sequencing were confirmed using the classical Sanger sequencing. Conclusions: We validated a very useful method for mutation identification in AML patients using next generation sequencing. There are many advantages exhibited by this method: it is more cost efficient and it has a higher sensitivity of mutation detection than Sanger sequencing, it has been described as being quantitative and in our case it allowed risk stratification for most of the normal karyotype AML samples which were FLT3-ITD and NPM1 negative.

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  • 1. Vardiman JW Thiele J Arber DA Brunning RD Borowitz RD Porwit A et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009 July 30;114(5):937-951. DOI: 10.1182/blood-2009-03-209262

  • 2. Ley TJ Mardis ER Ding L Fulton B McLellan MD Chen K et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature. 2008 Nov 6;456(7218):66-72. DOI: 10.1038/nature07485

  • 3. Ding L Ley TJ Larson DE Miller CA Koboldt DC Welch JS et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature. 2012 Jan 11;481(7382):506-10. DOI: 10.1038/nature10738

  • 4. Damm F Heuser M Morgan M Wagner K Görlich K Großhennig A et al. Integrative prognostic risk score in acute myeloid leukemia with normal karyotype. Blood. 2011 April 28;117(17):4561-4568. DOI: 10.1182/ blood-2010-08-303479

  • 5. Patel JP Gönen P Figueroa ME Fernandez H Sun Z Racevskis J et al. Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia. N Engl J Med. 2012;366:1079-1089. DOI: 10.1056/NEJMoa1112304

  • 6. Patel KP Ravandi F Ma D Paladugu A Barkoh BA Medeiros LJ et al. Acute Myeloid Leukemia With IDH1 or IDH2 Mutation Frequency and Clinicopathologic Features. Am J Clin Pathol. 2011 Jan;135(1):35-45. DOI: 10.1309/AJCPD7NR2RMNQDVF

  • 7. Ley TJ Ding L Walter MJ McLellan MD Lamprecht T Larson DE et al. DNMT3A Mutations in Acute Myeloid Leukemia. N Engl J Med. 2010;363:2424-2433. DOI: 10.1056/NEJMoa1005143

  • 8. Estey EH. Acute myeloid leukemia: 2012 update on diagnosis risk stratification and management. Am J Hematol. 2012 Jan;87(1):89-99. DOI: 10.1002/ajh.22246

  • 9. van Dongen JJ Macintyre EA Gabert JA Delabesse E Rossi V Saglio G et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia. 1999 Dec;13(12):1901-28. DOI: 10.1038/sj.leu.2401592

  • 10. Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CR et al. Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study. Blood 2006. 107(9):3463-8. DOI: 10.1182/blood-2005-09-3640

  • 11. Care RS Valk PM Goodeve AC Abu-Duhier FM Geertsma-Kleinekoort W Wilson GA et al. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol. 2003 Jun;121(5):775-7. DOI: 10.1046/j.1365-2141.2003.04362.x

  • 12. Ladetto M Bruggemann M Ferrero S Pepin F Drandi D Monitillo L et al. Next-Generation Sequencing and Real-Time Quantitative PCR for Minimal Residual Disease (MRD) Detection Using the Immunoglobulin Heavy Chain Variable Region: A Methodical Comparison in Acute Lymphoblastic Leukemia (ALL) Mantle Cell Lymphoma (MCL) and Multiple Myeloma (MM). ASH 2012 abstract 788.

  • 13. Schmitt MW Kennedy S R Salk JJ Fox E J Hiatt JB Loeb LA. Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci USA. 2012 Sep 4;109(36):14508-13. DOI: 10.1073/pnas.1208715109

  • 14. Gaidzik VI Bullinger L Schlenk RF Zimmermann AS Röck J Paschka P et al. RUNX1 mutations in acute myeloid leukemia: results from a comprehensive genetic and clinical analysis from the AML study group. J Clin Oncol. 2011 Apr 1;29(10):1364-72. DOI: 10.1200/ JCO.2010.30.7926

  • 15. Wang F Travins J DeLaBarre B Penard-Lacronique V Schalm S Hansen E et al. Targeted Inhibition of Mutant IDH2 in Leukemia Cells Induces Cellular Differentiation. Science. 2013 May 3;340(6132):622-6. DOI: 10.1126/science.1234769

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