Multiparametric Flow Cytometry Combined with Multi-Mutated Genes Measurable Residual Disease Assessed By Droplet Digital PCR to Predict Relapse in Acute Myeloid Leukemia Patients: A Single Center Experience

Background:Monitoring the dynamic changes of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) to predict prognosis and guide treatment has become a consensus among hematologists.The NPM1 mutation has emerged as a crucial method for MRD monitoring. Digital droplet PCR (ddPCR), as a third-generation PCR technique, offers greater objectivity through absolute quantification and is more cost-effective than NGS, thus assuming a more prominent role in MRD monitoring. Therefore, it is necessary to investigate the utility of detecting mutated gene MRD by ddPCR for non-allo-HSCT AML patients with regard to their prognosis and whether it can serve as a complementary method for MFC-based MRD monitoring.

Methods:Collect the data of 103 newly diagnosed AML (non-acute promyelocytic leukemia) patients was who received at least one MRD detection method and were treated at the Hematology Department of the Affiliated Hospital of Hebei University from January 1, 2018 to March 15, 2024. All patients had an AML diagnosis that met the 2022 European LeukemiaNet (ELN) criteria and achieved CR/CRi/CRp efficacy. At least one MRD monitoring method, including MFC, RT-qPCR, and ddPCR, was applied.According to the 2021 ELN-MRD guidelines , MRD should be monitored once before each chemotherapy cycle using MFC, RT-qPCR, and ddPCR. 1) MFC: detection of leukemia-related immunophenotype (LAIP) with <0.1% as negative. 2) RT-qPCR: negative with at least 2 of 3 repeat reactions having a cycle threshold ≤40. 3) ddPCR: negative with all monitored gene VAF <0.001%.

Results:The median follow-up duration was 13.7 (1.7-77.1) months, with 98 patients utilizing MFC for monitoring MRD. 57 patients with MRD were monitored mutant genes using ddPCR, including 22 with NPM1, 14 with CEBPA, 5 with FLT3-ITD, 5 with IDH1, 4 with IDH2, 2 with DNMT3A, 2 with TP53, 2 with CSF3R, 2 with RUNX1, and one each for TET2 and JAK2. Additionally, 32 MRD-positive patients were monitored using reverse transcription quantitative polymerase chain reaction (RT-qPCR), all of whom had fusion genes: AML1::ETO in eighteen cases and CBFβ::MYH11 in nine cases.The RFS of the MRD-negative group in the MFC-MRD cohort exhibited significantly longer duration compared to the positive group. No significant difference in RFS was observed between the positive and negative groups in the ddPCR-MRD cohort at a threshold of 0.001%. However, upon lowering the threshold to 0.1%, the RFS of the <0.1% group demonstrated a significantly longer duration than that of the ≥0.1% group in this cohort. Amongst the 56 patients monitored by both MFC-MRD and ddPCR-MRD, those belonging to the two-negative group displayed a significantly longer median RFS compared to those categorized as one-positive or two-positive groups.Although MRD monitoring by RT-qPCR was conducted, no significant difference in RFS was observed between the positive and negative groups. This may be attributed to the limited sample size and the predominance of core binding factor (CBF) fusion gene among the included patients. The prognosis of CBF is primarily associated with achieving a 3-log reduction compared to baseline after consolidation treatment.MRD proved capable of predicting relapse risk across both treatment cohorts; notably, MFC monitoring MRD exhibited stronger predictive efficacy within non-intensive therapy while ddPCR monitoring MRD conferred benefits within intensive therapy.

Conclusions:The ddPCR method holds significant value in monitoring the MRD of multi-mutated genes for predicting the risk of relapse and prognosis of AML patients. The combination of MFC with ddPCR for MRD detection can offer a more precise prediction of relapse risk in patients.

No relevant conflicts of interest to declare.