Christiane Walter1, Christian Pozzorini2, Katarina Reinhardt1, Robert Geffers3, Zhenyu Xu2, Dirk Reinhardt1, Nils von Neuhoff1, Helmut Hanenberg1,4. 1. Department of Pediatrics III, University Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany. 2. Sophia Genetics Inc., Saint-Sulpice, Switzerland. 3. Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany. 4. Department of Otorhinolaryngology and Head/Neck Surgery (ENT), Heinrich Heine University, Düsseldorf, Germany.
Abstract
BACKGROUND: The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC-enriched blasts from pediatric patients. PROCEDURE: For three AMLs with mutations in nucleophosmin 1 and/or fms-like tyrosine kinase 3, we performed whole genome amplification on sorted single-cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies. RESULTS: Analysis revealed that read coverage statistics were comparable between single-cell and bulk WES data, indicating high-quality whole genome amplification. From 102 single-cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50-72× failed to detect rare AML subclones in the bulk DNAs. CONCLUSIONS: In summary, our study demonstrated that single-cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs.
BACKGROUND: The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC-enriched blasts from pediatric patients. PROCEDURE: For three AMLs with mutations in nucleophosmin 1 and/or fms-like tyrosine kinase 3, we performed whole genome amplification on sorted single-cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies. RESULTS: Analysis revealed that read coverage statistics were comparable between single-cell and bulk WES data, indicating high-quality whole genome amplification. From 102 single-cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50-72× failed to detect rare AML subclones in the bulk DNAs. CONCLUSIONS: In summary, our study demonstrated that single-cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs.
Authors: Wendy J Huss; Qiang Hu; Sean T Glenn; Kalyan J Gangavarapu; Jianmin Wang; Jesse D Luce; Paul K Quinn; Elizabeth A Brese; Fenglin Zhan; Jeffrey M Conroy; Gyorgy Paragh; Barbara A Foster; Carl D Morrison; Song Liu; Lei Wei Journal: Hum Hered Date: 2019-01-22 Impact factor: 1.455