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.

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

Revista Romana de Medicina de Laborator

Romanian Journal of Laboratory Medicine

Journal Information

IMPACT FACTOR 2017: 0.400
5-year IMPACT FACTOR: 0.320

CiteScore 2017: 0.31

SCImago Journal Rank (SJR) 2017: 0.144
Source Normalized Impact per Paper (SNIP) 2017: 0.195


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