Clonal heterogeneity of FLT3 -ITD detected by high-throughput amplicon sequencing correlates with adverse prognosis in acute myeloid leukemia

// Katrin Schranz 1, 2, 3, 4, * , Max Hubmann 2, * , Egor Harin 2 , Sebastian Vosberg 1, 2, 3, 4 , Tobias Herold 2, 3, 4 , Klaus H. Metzeler 1, 2, 3, 4 , Maja Rothenberg-Thurley 2, 3, 4 , Hanna Janke 2 , Kathrin Braundl 2, 3, 4 , Bianka Ksienzyk 2 , Aarif M.N. Batcha 5 , Sebastian Schaaf 5 , Stephanie Schneider 2, 6 , Stefan K. Bohlander 7 , Dennis Gorlich 8 , Wolfgang E. Berdel 9 , Bernhard J. Wormann 10 , Jan Braess 11 , Stefan Krebs 12 , Wolfgang Hiddemann 1, 2, 3, 4 , Ulrich Mansmann 5 , Karsten Spiekermann 1, 2, 3, 4 and Philipp A. Greif 1, 2, 3, 4 1 Experimental Leukemia and Lymphoma Research, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany 2 Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany 3 German Cancer Consortium, partner site Munich, Germany 4 German Cancer Research Center, Heidelberg, Germany 5 Department of Medical Data Processing, Biometrie and Epidemiology, LMU Munich, Munich, Germany 6 Institute of Human Genetics, University Hospital, LMU Munich, Munich, Germany 7 Leukaemia and Blood Cancer Research Unit, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand 8 Institute of Biostatistics and Clinical Research, University of Munster, Munster, Germany 9 Department of Medicine A, Hematology and Oncology, University of Munster, Munster, Germany 10 Division of Hematology and Oncology, Charite Universitatsmedizin Berlin, Campus Virchow, Berlin, Germany 11 Department of Hematology and Oncology, Barmherzige Bruder Hospital, Regensburg, Germany 12 Laboratory for Functional Genome Analysis, Gene Center, LMU Munich, Munich, Germany * These authors contributed equally to this work Correspondence to: Philipp A. Greif, email: Philipp.Greif@med.uni-muenchen.de , p.greif@dkfz-heidelberg.de 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 ABSTRACT 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.