Development of a Next-generation Sequencing-based Gene Panel Test to Detect Measurable Residual Disease in Acute Myeloid Leukemia

Abstract

Background: AML is a heterogeneous disease, and despite intensive therapy, recurrence is still high in AML patients who achieve the criterion for cytomorphologic remission (re-sidual tumor burden [measurable residual disease, MRD]< 5%). This study aimed to de-velop a targeted next-generation sequencing (NGS) panel to detect MRD in AML patients and validate its performance. Methods: We designed an error-corrected, targeted MRD-NGS panel without using physi-cal molecular barcodes, including 24 genes. Fifty-four bone marrow and peripheral blood samples from 23 AML patients were sequenced using the panel. The panel design was validated using reference material, and accuracy was assessed using droplet digital PCR. Results: Dilution tests showed excellent linearity and a strong correlation between ex-pected and observed clonal frequencies (R> 0.99). The test reproducibly detected MRD in three dilution series samples, with a sensitivity of 0.25% for single-nucleotide variants. More than half of samples from patients with morphologic remission after one month of chemotherapy had detectable mutations. NGS-MRD positivity for samples collected after one month of chemotherapy tended to be associated with poor overall survival and pro-gression-free survival. Conclusions: Our highly sensitive and accurate NGS-MRD panel can be readily used to monitor most AML patients in clinical practice, including patients without gene rearrange-ment. In addition, this NGS-MRD panel may allow the detection of newly emerging clones during clinical relapse, leading to more reliable prognoses of AML.