Monitoring MRD and clinical utility of DNMT3A R882 in AML by multiplex droplet digital PCR

Abstract ObjectiveCurrent clinical detection of the DNMT3A R882 variant is usually by NGS and sanger sequencing, but the traditional methods have limited sensitivity and do not allow for timely assessment of the disease remission process in patients.The aim of this study is to validate the application of multiplex droplet digital PCR (Multiple PCR) in detecting measurable residual disease (MRD) in patients with DNMT3A R882 (R882C/H/S/P) mutation. MethodsHighly sensitive and precise MdPCR was used in this study. The DNMT3A R882-related samples(10 DNMT3A R882-positive patients as a case group and 53 wild-type samples as a control group) were evaluated for MRD by MdPCR using a self-made reagent kit, and the accuracy was verified by BDA + Sanger sequencing and NGS.In addition, five AML patients with DNMT3A R882 mutation were followed up by MdPCR. ResultsEvaluation of MRD performance of MdPCR. In terms of accuracy, all known mutations were identified, and no false positives were detected. In terms of detection limit, it is determined that samples with a mutation rate of 0.1% at 5 ng/μl can be detected. In terms of linearity, except for R882S, the correlation coefficients of all assays were greater than 0.98 between 1%-0.05% concentrations. In terms of precision, the coefficient of variation was less than 5% at 10% and 1% allele frequencies.Assessment of clinical utility of MdPCR. We continuously monitored 5 patients with DNMT3A R882 mutation, and the results showed that 4 patients had the same changes in MdPCR monitoring and hematological detection, and the DNMT3A R882 mutation gradually decreased after treatment and reached remission. One case showed a decrease in flow cytometry after treatment, but the expression level of DNMT3A R882 continued to rise, and relapsed in a short period of time. ConclusionsMdPCR is suitable for monitoring the minimal residual disease (MRD) of DNMT3A R882 with a sensitivity of 0.1%, featuring high accuracy, sensitivity, precision, and linearity.So it can be used as a reliable method for MRD detection in DNMT3A R882 mutation AML patients.However, our study was a single-center study limited by the small patient base, and with the expansion of sample size, more meaningful results will be found in the future. KeywordsAcute myeloid leukemia; Multiplex droplet digital PCR; DNA methyltransferase 3A; Measurable residual disease DisclosuresNo relevant conflicts of interest need to be declared.