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.
Highlights
Muations in the fms-related tyrosine kinase 3 (FLT3) gene are prevalent in newly diagnosed acute myeloid leukemia (AML) cases, affecting up to 39% patients
For our comparative analysis we selected 250 AML patient samples obtained at initial diagnosis, all FLT3ITD positive according to routine diagnostics (Figure 1), as well as 17 FLT3-internal tandem duplications (ITDs) negative AML samples
high-throughput amplicon sequencing (HTAS) detected a total of 312 FLT3-ITDs in 242 of 250 FLT3-ITD positive patients (97%), compared to 274 ITDs detected by fragment analysis (Supplementary Table 1)
Summary
Muations in the fms-related tyrosine kinase 3 (FLT3) gene are prevalent in newly diagnosed acute myeloid leukemia (AML) cases, affecting up to 39% patients. Patients carrying FLT3-ITD mutations with high allelic ratio benefit from a more intensive consolidation treatment such as allogeneic stem cell transplantation [7, 10,11,12]. A prognostic relevance was observed for the mutant to wild-type (WT) allelic ratio which corresponds to the size of the clone(s) with the FLT3-ITD. Up to five distinct clones with different FLT3-ITDs were observed per patient [16]. The FLT3-ITD status is assessed by capillary electrophoresis of PCR-amplified cDNA (hereafter referred to as ‘fragment analysis’) [26]. This assay only provides the length but not the position and the sequence of the insertion. To evaluate the applicability and accuracy of NGS-based FLT3-ITD detection for routine diagnostics, we compared FLT3-ITD detection by HTAS and fragment analysis in 250 adult FLT3-ITD positive AML patients
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