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Updated: Jan 7, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
[Clinical application value of detecting the mutation load of the FLT3-ITD gene before allogeneic hematopoietic stem
Y C Shao1, Y X Zhang2, W L Sun3
1National Research Center for Translational Medicine, State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China; Department of Hematology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China.
Abstract:
Objective: This study aimed to develop a methodology for detecting FMS-like tyrosine kinase 3 (FLT3) gene internal tandem duplication (ITD) mutation burden using polymerase chain reaction-next-generation sequencing (PCR-NGS). The prognostic value of FLT3-ITD mutation burden detected with PCR-NGS in patients with acute leukemia (AL) before allogeneic hematopoietic cell transplantation (allo-HSCT) is investigated. Methods: This retrospective study developed a methodology for detecting FLT3-ITD mutational burden in bone marrow samples isolated from 65 patients with AL with FLT3-ITD mutations who received allo-HSCT from January 2021 to June 2024 at Ruijin Hospital. PCR-NGS was used for data analysis, and results were compared with conventional quantitative PCR (qPCR) . Results: The PCR-NGS assay demonstrated robust performance, with a sensitivity of 10(-6). Pretransplant complete remission with FLT3-ITD negativity via qPCR was achieved in 58 patients, comprising 25 with FLT3-ITD positivity in PCR-NGS [median VAF:0.629% (0.004% -26.350% ) ]. The 2-year probability of relapses and event-free survival (EFS) were 11.2% and 86.2% for patients with FLT3-ITD VAF of <0.1% and 30.6% and 64.5% for those having FLT3-ITD VAF of ≥0.1%, respectively (relapse: HR=3.159, 95% CI: 0.950-10.510, P=0.048; EFS: HR=2.846, 95% CI: 0.953-8.500, P=0.050). Two-year probability of relapses and EFS were 12.5% and 84.4% for patients with both negative PCR-NGS and qPCR, 26.2% and 73.8% for those with positive PCR-NGS and negative qPCR, and 28.6% and 57.1% for patients with both positive PCR-NGS and qPCR (relapse: HR=2.892, 95% CI: 1.122-7.451, P=0.0321; EFS: HR=1.784, 95% CI: 0.880-3.615, P=0.248), respectively, before allo-HSCT. Maintenance therapy with FLT3 inhibitors is a protective factor for reduced relapse rate and better overall survival rate after allo-HSCT. Conclusion: A highly sensitive PCR-NGS assay for FLT3-ITD mutational burden has been developed. Compared with qPCR, PCR-NGS demonstrates superior sensitivity and enables more accurate prediction of posttransplant outcomes in patients with AL undergoing allo-HSCT.
