Clonal heterogeneity of FLT3-ITD detected by high-throughput amplicon sequencing correlates with adverse prognosis in acute myeloid leukemia
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Katrin Schranz1,2,3,4,*, Max Hubmann2,*, Egor Harin2, Sebastian Vosberg1,2,3,4, Tobias Herold2,3,4, Klaus H. Metzeler1,2,3,4, Maja Rothenberg-Thurley2,3,4, Hanna Janke2, Kathrin Bräundl2,3,4, Bianka Ksienzyk2, Aarif M.N. Batcha5, Sebastian Schaaf5, Stephanie Schneider2,6, Stefan K. Bohlander7, Dennis Görlich8, Wolfgang E. Berdel9, Bernhard J. Wörmann10, Jan Braess11, Stefan Krebs12, Wolfgang Hiddemann1,2,3,4, Ulrich Mansmann5, Karsten Spiekermann1,2,3,4 and Philipp A. Greif1,2,3,4
1Experimental Leukemia and Lymphoma Research, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany
2Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany
3German Cancer Consortium, partner site Munich, Germany
4German Cancer Research Center, Heidelberg, Germany
5Department of Medical Data Processing, Biometrie and Epidemiology, LMU Munich, Munich, Germany
6Institute of Human Genetics, University Hospital, LMU Munich, Munich, Germany
7Leukaemia and Blood Cancer Research Unit, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
8Institute of Biostatistics and Clinical Research, University of Münster, Münster, Germany
9Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany
10Division of Hematology and Oncology, Charité Universitätsmedizin Berlin, Campus Virchow, Berlin, Germany
11Department of Hematology and Oncology, Barmherzige Brüder Hospital, Regensburg, Germany
12Laboratory for Functional Genome Analysis, Gene Center, LMU Munich, Munich, Germany
*These authors contributed equally to this work
Keywords: acute myeloid leukemia (AML); fms-related tyrosine kinase 3 (FLT3); iternal tandem duplication (ITD); next generation sequencing (NGS); fragment analysis
Received: February 18, 2018 Accepted: June 19, 2018 Published: July 10, 2018
In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of FLT3 are frequent mutations associated with unfavorable prognosis. At diagnosis, the FLT3-ITD status is routinely assessed by fragment analysis, providing information about the length but not the position and sequence of the ITD. To overcome this limitation, we performed cDNA-based high-throughput amplicon sequencing (HTAS) in 250 FLT3-ITD positive AML patients, treated on German AML Cooperative Group (AMLCG) trials. FLT3-ITD status determined by routine diagnostics was confirmed by HTAS in 242 out of 250 patients (97%). The total number of ITDs detected by HTAS was higher than in routine diagnostics (n = 312 vs. n = 274). In particular, HTAS detected a higher number of ITDs per patient compared to fragment analysis, indicating higher sensitivity for subclonal ITDs. Patients with more than one ITD according to HTAS had a significantly shorter overall and relapse free survival. There was a close correlation between FLT3-ITD mRNA levels in fragment analysis and variant allele frequency in HTAS. However, the abundance of long ITDs (≥75nt) was underestimated by HTAS, as the size of the ITD affected the mappability of the corresponding sequence reads. In summary, this study demonstrates that HTAS is a feasible approach for FLT3-ITD detection in AML patients, delivering length, position, sequence and mutational burden of this alteration in a single assay with high sensitivity. Our findings provide insights into the clonal architecture of FLT3-ITD positive AML and have clinical implications.
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