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Miseq based next generation sequencing to perform molecular profiling in acute myeloid leukemia and its prognostic significance, a retrospective review at a large tertiary care community hospital
Muhammad Zubair Afzal, Cong Liu, Sheryl Alberta, Anne Wilds and Brett Brinker

Introduction: Acute myeloid leukemia is routinely stratified by cytogenetic analysis and common molecular alterations of FLT3, NPM1, c-KIT, IDH1/IDH2, and CEBPA genes. Although less known genetic aberrations can impact the prognosis significantly; comprehensive DNA analysis to identify these mutations is not routinely done due to time constrains and various technical challenges. These days next generation sequencing (NGS) is being used to identify less known genetic aberrations. We evaluated the feasibility and patient outcomes of an NGS panel based on MiSeq for patients with AML at a community-based, tertiary hospital system.

Methods: A retrospective cohort study was performed on specimens collected between June 2013 and December 2014 from patients with a pathological diagnosis of AML and of 18 years of age or older. Stored bone marrow DNA samples were run on the Illumina MiSeq instrument using two custom panels: the Qiagen GeneRead panel and the Agilent Haloplex panel.

Results: 31 patients were included. Other than the four common mutations, sixteen less common gene mutations were identified including DNMT3A, IDH2, WT1, TET2, NRAS, SRSF2, TP53. TP53 had significant impact on overall survival on univariate and multivariate analyses (P= 0.016 and P=0.021 respectively) and TET2 had a significant impact on event free survival (EFS) on univariate and multivariate analyses (P = 0.042 and P = 0.026 respectively). On stratifying patients by mutations within cytogenetically intermediate risk category, there was a significant difference (P= 0.0054) in EFS between intermediate and unfavorable groups. Mutation load also increased significantly from favorable to unfavorable categories based on cytogenetics and mutations.

Conclusion: Comprehensive mutational analysis beyond routinely-tested mutations can detect rare mutations that may have significant prognostic value. Cytogenetically intermediate category benefits the most of such analysis.

Keywords: Next generation sequencing, Acute Myeloid Leukemia, Cytogenetics based risk stratification, Mutation based risk stratification

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