Clinical Relevance of NPM1 Quantitative PCR As Standard of Care with Different Cut-Off Values on Treatment Outcomes in NPM1-Mutated Acute Myeloid Leukemia Patients

Introduction: The European LeukemiaNet (ELN) 2022 guidelines recommend NPM1 real-time quantitative PCR (qPCR) for measurable residual disease (MRD) monitoring post-two cycles of intensive chemotherapy, end of treatment, and every 3 monthly thereafter on bone marrow, and every 4-6 weeks on peripheral blood. It was reported that low level NPM1 positivity is not prognostic for relapse while previous studies reported persistence of NPM1 transcripts >10-20 copies/104 ABL is associated with higher relapse risk. Thus, this threshold is recommended by the ELN 2022 guidelines. However, this threshold is not standardized and its clinical relevance is not fully evaluated. Accordingly, the present study aimed to investigate if our approach assessing NPM1 MRD status after one cycle of induction chemotherapy, which is standard practice in North America, and every 3 months following initial assessment, can stratify prognosis based on different NPM1 absolute transcript cutoff levels. Methods and Patients: 78patients with newly diagnosed NPM1-mutated acute myeloid leukemia (AML) between February 2021 and January 2024 at Princess Margaret Cancer Centre who underwent chemotherapy and had NPM1 MRD testing with a lower limit of detection of 10-5 performed within the first 12 months after initial diagnosis, were included. To define the optimal NPM1 cut-off level, qPCR values as copy numbers/10,000 ABL were evaluated for recursive partitioning (rpart) analysis using relapse-free survival (RFS) as a statistical endpoint. Relapse-free survival (RFS) was a primary endpoint, defined as the time from the date of MRD assessment to the date of relapse or death from any cause, while overall survival (OS) was calculated to the date of death of any cause and censored at the date of last follow-up. Cumulative incidence of relapse (CIR) was calculated from the date of MRD assessment to the date of relapse considering death from other causes as a competing risk. Cases that received allogeneic stem cell transplantation were not censored in the present analysis. The Kaplan-Meier method using a log-rank test, and a Cox proportional hazard model was used for univariate and multivariate analyses. Results: The median age was 60.8 years (range, 56.7-63.9), FLT3-ITD positive in 30 (39%) patients, FLT3-TKD positive in 7 (9%) patients, and normal karyotype in 50 (64%) patients. 69 (89%)patients received intensive chemotherapy with majority of them receiving 7+3 based induction chemotherapy. 70 (90%) patients achieved first complete remission. In the recursive partitioning analysis, cut-off values of 5, 50, 500 copies/104 ABL were suggested to be of prognostic value, therefore NPM1 results were divided into 4 groups, denoted by undetectable transcripts (group 1; n=139), >0 to <50 copies/104 ABL (group 2, n=40), >50 to <500 copies/104 ABL (group 3; n=17), and >500 copies/104 ABL(group 4, n=22). Median follow-up was 17.7 months (range, 14.1-19.7) for the entire cohort, while median follow-up for survivors (60 patients) was 18.2 months (range, 15.8-21.3). RFS, OS and CIR rate at 2 years was 65.6% (95% CI, 57.8-72.3%), 75.4% (65.5-82.9%), and 26.3% (20.4-32.5%), respectively. RFS rate at 2 years was 77.1%, 68.8%, 38.9% and 17.4% in groups 1, 2, 3 and 4 (p<0.0001), while the OS rate at 2 years was 84.8%, 61.5%, 50.0% and 57.0%, respectively (p=0.0002). Relapse risk was well stratified with the cutoff values: CIR rate at 2 years 13.7%, 23.2%, 61.1% and 73.9% in groups 1, 2, 3 and 4 (p<0.0001), respectively. NPM1 MRD was conducted not just at the time of CR1 and end of consolidation, but also within the first 12 months of initial diagnosis, of which its prognostic relevance is not completely clear although we frequently use it in our clinical practice. To evaluate its clinical relevance, we divided MRD testing into 3 different time periods after initial diagnosis (i.e., 0-120 days, 121-240 days, 240-365 days), and evaluate the implication of NPM1 copy numbers by the 4 groups, on RFS. Regardless of time periods for MRD assessment, NPM1 qPCR provided excellent prognostic risk stratification: p=0.05 on day 0-120, p<0.0001 on day 121-240, and P<0.0001 on day 241-365. Conclusion: NPM1 qPCR MRD monitoring has become standard of care in AML management. The present study demonstrated that it provides excellent prognostic risk stratification for RFS, OS and CIR. Also, the cutoff values of 5, 50 and 500 copies/104 ABL performed well in clinical practice at our centre.

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