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

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.

Association between increased mutation rates in DNMT3Α and FLT3‑ITD and poor prognosis of patients with acute myeloid leukemia

“…It has been previously reported that, compared with patients harboring no DNMT3A mutations, patients carrying DNMT3A mutation were found to have higher CD34 + , CD15 + and HLA-D + rates (29). Furthermore, another previous study revealed that patients carrying FLT3-ITD mutations exhibited higher rates of CD34 + and CD38 + compared with patients without this mutation (30). Therefore, chemotherapy efficacy was compared between the four groups in the current study.…”

Assessment of the clinical utility of four NGS panels in myeloid malignancies. Suggestions for NGS panel choice or design

“…In our cohort, two FLT3-ITD mutations of 36bp in length (detected by classical molecular techniques in our laboratory) were not called by any of the NGS gene panels tested in this study, which means that conventional diagnostics techniques are still essential for hematological malignancies diagnosis [81]. NGS difficulty for long FLT3-ITD detection has been reported before [62] [82]; this is because current NGS chemistries employ short reading sequencing (read length 50-300bp) and this makes it prone to lose structural variants such as long indels [83] [84]. In support of this observation, in our cohort, the three variants missed by SureSeq panels (sequenced at shorter read length than the other panels, 150bp vs >200bp), were indels.…”

Clinical implications of recurrent gene mutations in acute myeloid leukemia

“…A high mutant/wild-type ratio appears to have a major impact on the prognostic relevance [29]. Patients with more than one ITD had a significantly shorter OS and RFS [30]. Retrospective validation study of the ELN-2017 guidelines on the classification for AML with NPM1 and FLT3-ITD genotypes demonstrated that the ELN-2017 was more accurate to distinguish prognosis in patients with newly diagnosed AML [25].…”