Detection of NPM1 mutation in Acute Myeloid Leukemia by multiple droplet digital PCR and its clinical application value

Abstract Multiple droplet digital PCR detection of measurable residual disease in NPM1 mutation in acute myeloid leukemia Objective The NPM1 gene serves as a crucial prognostic marker for acute myeloid leukemia (AML) and an ideal target for monitoring minimal residual disease (MRD). Current clinical approaches for detecting NPM1 mutations include fluorescence PCR, Sanger sequencing, and next-generation sequencing (NGS). However, the limited sensitivity of these traditional methods restricts early assessment of treatment responses and recurrence risks in patients. Methods This study aims to systematically verify the application value of multiple droplet digital PCR (Multiple ddPCR) technology in the detection of common NPM1 mutation MRD.The experiment utilized a self-developed Multiple ddPCR kit, which designed specific primers and probes targeting the seven most common NPM1 gene mutation types (NPM1-I, NPM1-II, NPM1-III, NPM1-IV, NPM1-V, NPM1-VI, NPM1-VII). The test samples included: 28 confirmed NPM1-positive patient samples and 10 wild-type control samples. All samples underwent simultaneous next-generation sequencing (NGS) testing, with NGS results serving as the verification standard.In addition, continuous bone marrow samples were collected from 7 AML patients with NPM1 positive before and after chemotherapy, and the above Multiple ddPCR kit was used for dynamic monitoring. Results In 28 NPM1-positive diagnostic samples, Multiple ddPCR accurately detected all NGS-confirmed NPM1 mutations, with results showing complete consistency with NGS findings. None of the 10 wild-type control samples exhibited false-positive signals. Through serial dilution validation of clinical samples, the detection limit reached 0.1% at 10 ng/μL. Continuous detection of 7 mutation types across allele frequency ranges from 1% to 0.05% yielded R² values>0.97 for all variants. At allele frequencies of 20% and 4%, the coefficient of variation (CV) for these 7 mutation types remained below 5%.In the dynamic monitoring of 7 patients, 4 cases were persistently negative for NPM1 and sustained complete hematological remission after treatment; 3 cases showed no abnormal cells detected by flow cytometry after treatment, but multiple droplet digital PCR (Multiple ddPCR) and fluorescence PCR could still detect NPM1 mutation, and all 3 patients relapsed in a short period. Conclusion This study confirmed that multiple droplet digital PCR (Multiple ddPCR) has high specificity and sensitivity, linearity and precision, and can accurately detect NPM1 mutation, which is common in AML patients. Its detection limit of 0.1% meets the needs of MRD monitoring, and it can be used as a reliable method for MRD detection in NPM1 mutation AML patients.However, due to the limited number of clinical samples, our multiplex digital PCR system still needs further multi-center clinical studies to further confirm the clinical utility of the multiplex digital PCR system. Keyword Acute myeloid leukemia; Multiplex digital PCR; NPM1 mutation; measurable residual disease Disclosures No declaration of conflicts of interest is required.