Pb1894: npm1 mutated acute myeloid leukemia: the co-mutation patterns may be associated with prognosis

Topic: 4. Acute myeloid leukemia - Clinical Background: Acute myeloid leukemia (AML) with mutated NPM1 (NPM1 +) – a distinct clinical entity – is characterized by heterogeneity in terms of prognosis. The concurrence of FLT3-ITD mutations categorizes the disease as intermediate risk (ELN 2022). Recent evidence suggests that detecting other myeloid lineage-related gene mutations by Next Generation Sequencing (NGS) may help identify patients at increased risk of relapse. Aims: To describe the gene profile of AML NPM1+ patients using NGS techniques and determine their possible prognostic role during the disease. Methods: DNA was isolated from bone marrow using the QIAamp DNA MiniKit (QIAGEN). Mutations of exon 12 of the NPM1 gene were detected using fragment analysis. Exon 12 of the NPM1 gene was amplified and the products were analyzed by capillary electrophoresis technique. A difference of 4 bases between wild type DNA versus mutated DNA is obtained. The LeukoStratFLT3 Mutation Assay (Invivoscribe) was used to detect FLT3-ITD and FLT3-TKD. The NGS panel involved variants in 30 genes associated with myeloid malignancies, and analysis was performed in the Illumina platform. We reported genetic variants classified as pathogenic or potentially pathogenic in the COSMIC database, met the criterion of x500 reads and were found at a frequency (VAF) greater than 5%. Results: We studied 21 AML NPM1+ patients with a median age of 50 years (26-71) that were diagnosed between 2018-2022. Nineteen patients received intensive chemotherapy and two hypomethylating agents +/- venetoclax (cycles 8-9). The median follow-up was 14 months (1-49). All patients, but 1 with del9(q22q34), had normal karyotypes. FLT3-ITD mutations were detected in 5 patients classified as intermediate risk, and 3/5 underwent allogeneic transplantation in CR1. The most frequent co-mutations revealed by NGS were DNMT3A (14/21), NRAS (8/21), and IDH1/2 (6/21). The coexistence of 2 or more variants (other than NPM1 or FLT3) was found in 13/21 patients. Eleven out of 25 patients relapsed, 4 of which ultimately underwent allogeneic HCT. Kaplan-Meier survival analysis estimated RFS at 18.3 months and OS at 35.4 months. FLT3-ITD and DNMT3A co-mutations adversely impacted RFS and OS (HR 1.9, 95%CI: 0.48-7.4, HR 1.31, 95%CI: 0.3-5.69 for NPM1+/FLT3-ITD+ respectively and HR 1.12, 95%CI: 0.11-11.38, HR 2.52, 95%CI: 0.29-21.2 for NPM1+/DNMT3A+ respectively, p=ns). On the contrary, NRAS mutations were associated with better prognosis (RFS: HR 0.27, 95%CI: 0.034-2.29, OS: HR 0.27, 95%CI: 0.34-1.69 for NPM1+/NRAS+, respectively). The triple mutation pattern of NPM1+/DNMT3A+/ FLT3-ITD+ identified as an adverse risk subgroup previously could not be independently confirmed as all 5/5 NPM1+/ FLT3-ITD+ patients carried DNMT3A mutations. Lower RFS and OS in this subgroup (HR 1.9, 95%CI: 0.48-7.4, HR 1.31, 95%CI: 0.3-5.69, respectively, p=ns) show a trend for worse outcomes. Summary/Conclusion: Using NGS techniques in AML NPM1+ patients detects concurrent mutations with potential prognostic significance and can contribute to daily clinical practice. These techniques must be carried out in patients diagnosed with AML who will receive treatment and only in specialized and accredited centers. Keywords: Molecular markers, Acute myeloid leukemia, Prognostic groups