Whole exome sequencing of sorted leukemic cells as a complementing method in genetic characterization of acute myeloid leukemia

Erik Malmberg1,*, Sara Ståhlman1, Sofie Johansson Alm1, Tore Samuelsson2, Lars Palmqvist1,3, Linda Fogelstrand1,3

1Department of Clinical Chemistry and Transfusion Medicine, 2Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, 3Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden

Introduction: Comprehensive genetic characterization of acute myeloid leukemia (AML) is necessary for accurate diagnosis, treatment stratification and follow-up of patients. Today, this is achieved using a combination of techniques; karyotyping, fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR) assays for analysis of genes like FLT3 and NPM1.

Objectives: The aim of this study was to test if exome sequencing of sorted leukemic cells could be an alternative method for identification of genetic aberrations in AML.

Methods: Leukemic cells and normal lymphocytes from newly diagnosed cases of AML (n=10) were sorted using fluorescence activated cell sorter (FACS). Exome sequencing was performed on the Illumina platform with HiScanSQ. Variant calling was performed with the Genome Analysis Toolkit (GATK) package. Leukemia-specific single nucleotide variants (SNVs) and short insertions and deletions were verified with Sanger sequencing. Analysis if copy number variation was performed using Nexus software. Results from exome sequencing were compared with results from karyotyping, FISH, and PCR.

Results: Leukemia-specific SNVs (with coverage spanning between 10 and 250) were detected in all patient samples using our exome sequencing and data analysis approach. Mutations in NPM1 were present in three cases and could all be identified using our approach. Internal tandem duplication (ITD) in FLT3 was present in two cases, but could not be identified. Copy number analysis of exome sequencing data revealed similar, but not identical, results as conventional karyotyping. Balanced translocations could not be identified.

Conclusion: Using GATK-based analysis of exome sequencing data alone can currently not replace conventional genetic methods, apart from some PCR assays (e.g. for NPM1). Leukemia-specific mutations in the diagnostic sample provide additional information that could serve as a basis for analysis of minimal residual disease in follow-up samples.

Keywords: Blood, Genomics, Method