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

In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of are frequent mutations associated with unfavorable prognosis. At diagnosis, the 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 ITD positive AML patients, treated on German AML Cooperative Group (AMLCG) trials. 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 ( = 312 vs. = 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 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 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 ITD positive AML and have clinical implications.