34 NPM1 Mutation Spectrum as a Quality Assurance Metric for Next-Generation Sequencing in Myeloid Neoplasm Mutation Profiling

NPM1, coding nucleophosmin 1, is the most frequently mutated gene in adult acute myeloid leukemia (AML) and is typically associated with a favorable prognosis. NPM1 mutations are usually four base pair frameshift insertions in exon 12, with type A (TCTG insertion) being the most common (75-80%), followed by type B (CATG, 5-10%) and D (CCTG, 5-10%). Periodic review of molecular genetic statistics, as outlined by the College of American Pathologists (CAP) checklist item MOL.20550, is essential for identifying changes in test performance and preventing potential errors. A study conducted by the CAP Molecular Oncology Committee, assessing 422 molecular oncology laboratories from 2018 to 2020, identified MOL.20550 as the second most common deficiency, with 4% of laboratories cited for non-compliance. Our study aimed to analyze the NPM1 mutation spectrum to establish a statistical baseline for our patient population, ensure compliance with CAP quality assurance standards, and support the triaging of minimal residual disease (MRD) testing. NPM1 mutation-positive samples from January 2023 to January 2025 were retrieved from our internal database within the variant interpretation pipeline (GenomOncology Pathology Workbench, Version R31), documenting date, diagnosis, variant details, and variant allele frequency (VAF). A total of 79 cases with pathogenic NPM1 mutations were identified (35 cases in 2023 and 44 cases in 2024). Of these, 94% were diagnosed with AML, and 6% had other diagnoses (MDS, MPN, and others). The mutation subtypes were as follows: typical mutations in 86% of cases, comprising Type A (73%), Type B (5%), and Type D (8%). Non-typical (non-ABD) mutations represented 14% of cases. In 2023, 88.6% of cases (31/35) exhibited typical mutations, while 11.4% (4/35) had non-ABD mutations. In 2024, typical mutations were observed in 84% of cases (37/44), with non-ABD mutations present in 16% (7/44). The VAF percentage ranged from 1% to 45%, with an average of 28%. The average VAF was 28.6% in 2023 and 27.4% in 2024. Notably, most of the non-ABD subtype mutations involve a four base pair insertion, which could contribute to future test development for MRD. In conclusion, our internal variant database facilitated an efficient workflow for reviewing mutation statistics. The findings revealed a mutation spectrum consistent with that published on AML and demonstrated a stable VAF range over the past two years. Moving forward, we plan to conduct regular statistical analyses to monitor test performance and mutation spectrum, particularly focusing on non-ABD mutations as part of quality assurance effort to minimize errors. Furthermore, a future study is planned to investigate the clinical relevance of uncommon NPM1 mutations.