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Schranz, Katrin; Hubmann, Max; Harin, Egor; Vosberg, Sebastian; Herold, Tobias; Metzeler, Klaus H.; Rothenberg-Thurley, Maja; Janke, Hanna; Bräundl, Kathrin; Ksienzyk, Bianka; Batcha, Aarif M. N.; Schaaf, Sebastian; Schneider, Stephanie; Bohlander, Stefan K.; Görlich, Dennis; Berdel, Wolfgang E.; Wörmann, Bernhard J.; Braess, Jan; Krebs, Stefan; Hiddemann, Wolfgang; Mansmann, Ulrich; Spiekermann, Karsten; Greif, Philipp A. (2018): Clonal heterogeneity of FLT3-ITD detected by high-throughput amplicon sequencing correlates with adverse prognosis in acute myeloid leukemia. In: Oncotarget, Vol. 9, No. 53: pp. 30128-30145
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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.