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Acute Myeloid Leukemia
Comprehensive genetic diagnosis of acute myeloid leukemia by next-generation sequencing
Ferrata Storti Foundation
Haematologica 2019 Volume 104(2):277-287
Elisabeth K.M. Mack,1 André Marquardt,1* Danny Langer,1* Petra Ross,1 Alfred Ultsch,2 Michael G. Kiehl,3 Hildegard I.D. Mack,4 Torsten Haferlach,5 Andreas Neubauer1 and Cornelia Brendel1
1Department of Hematology, Oncology and Immunology, Philipps-University Marburg, and University Hospital Gießen and Marburg, Marburg, Germany; 2Databionics, Department of Mathematics and Informatics, Philipps-University Marburg, Germany; 3Department of Internal Medicine, Frankfurt (Oder) General Hospital, Frankfurt/Oder, Germany; 4Institute for Biomedical Aging Research, Leopold-Franzens-University Innsbruck, Austria and 5MLL, Munich Leukemia Laboratory, Germany
*These authors contributed equally to this work.
ABSTRACT
Differential induction therapy of all subtypes of acute myeloid leukemia other than acute promyelocytic leukemia is impeded by the long time required to complete complex and diverse cyto- genetic and molecular genetic analyses for risk stratification or targeted treatment decisions. Here, we describe a reliable, rapid and sensitive diagnostic approach that combines karyotyping and mutational screen- ing in a single, integrated, next-generation sequencing assay. Numerical karyotyping was performed by low coverage whole genome sequencing followed by copy number variation analysis using a novel algorithm based on in silico-generated reference karyotypes. Translocations and DNA variants were examined by targeted resequencing of fusion tran- scripts and mutational hotspot regions using commercially available kits and analysis pipelines. For the identification of FLT3 internal tandem duplications and KMT2A partial tandem duplications, we adapted previ- ously described tools. In a validation cohort including 22 primary patients’ samples, 9/9 numerically normal karyotypes were classified correctly and 30/31 (97%) copy number variations reported by classical cytogenetics and fluorescence in situ hybridization analysis were uncov- ered by our next-generation sequencing karyotyping approach. Predesigned fusion and mutation panels were validated exemplarily on leukemia cell lines and a subset of patients’ samples and identified all expected genomic alterations. Finally, blinded analysis of eight additional patients’ samples using our comprehensive assay accurately reproduced reference results. Therefore, calculated karyotyping by low coverage whole genome sequencing enables fast and reliable detection of numer- ical chromosomal changes and, in combination with panel-based fusion- and mutation screening, will greatly facilitate implementation of sub- type-specific induction therapies in acute myeloid leukemia.
Introduction
Acute myeloid leukemia (AML) has recently been the object of thorough investi- gations at a molecular level, including whole genome next-generation sequencing (NGS) studies.1 According to current guidelines from the European Leukemia Network, recommended genetic testing in adult AML is predominantly directed towards risk stratification in order to identify an appropriate strategy for consolida- tion therapy.2 Genetic markers comprise t(8;21)(q22;q22.1)/RUNX1-RUNX1T1, inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/CBFB-MYH11, t(15;17)/PML-RARA, t(9;11)(p21.3;q23.3)/MLLT3-KMT2A, other translocations involving the KMT2A
Correspondence:
brendelc@staff.uni-marburg.de
Received: May 26, 2018. Accepted: September 5, 2018. Pre-published: September 6, 2018.
doi:10.3324/haematol.2018.194258
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/2/277
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