The Clinical Features、Prognosis、Bioinformatics Analysis Of NPM1/FLT3-ITD/DNMT3A Triple-mutated Acute Myeloid Leukemia

Purpose:To explore the clinical features,overall survival and prognostic factors of acute myeloid leukemia（AML）patients with NPM1/FLT3-ITD/DNMT3A triple mutations.Methods:1.Our study included a total of 165 patients with newly diagnosed non-acute promyelocytic leukemia（non-APL）AML at The First Hospital of Lanzhou University between January 2018 and June 2021.A retrospective analysis was performed using the clinical records to study clinical features and overall survival of AML patients with NPM1/FLT3-ITD/DNMT3A triple mutations.2.We selected a larger sample size dataset in the GEO database:GSE146173,which contain clinical and survival data,especially gene mutations.We divided AML patients with NPM1/FLT3-ITD co-mutations into two groups by DNMT3A wild-type or mutant.The quest for hub gene,firstly we identified the differentially expressed genes（DEGs）between two groups,then enrichment analysis which included Gene Ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis was performed,lastly was the protein–protein interaction（PPI）network.Finally,the hub gene was selected.3.Pan-cancer data from the The Cancer Genome Atlas（TCGA）database and healthy population data from the Genotype-Tissue Expression Project（GTEx）database were selected.Differences in the hub gene expression between patients with multiple tumors and healthy population were compared,and to analyze the correlation between the hub gene expression and immune cells infiltration.AML patients were divided into2 groups with high and low of the hub gene expression,and the significance of the hub gene expression in AML was further understood by identifing DEGs,GO,KEGG and Gene Set Enrichment Analysis（GSEA）.In a final step,we analysed the correlation between the hub gene expression and clinical features,and constructed a prognostic model for AML.Further understand the clinical significance of the hub gene.Results:1.We identified 6.7%AML patients with NPM1/FLT3-ITD/DNMT3A triple mutations.Compared to non-triple-mutated AML,the patients with triple-mutated AML had clinical features of white blood cell（WBC）count≥20×10~9/L,platelet count≥20×10~9/L,higher bone marrow blasts,and predominantly M4/M5 French-American-British（FAB）categories.Notably,some patients bone marrow morphologies had mature monocyte characteristic and they were difficult to distinguish from chronic myelomonocytic leukemia（CMML）.The patients with triple-mutated AML had a poor prognosis,with the median overall survival of only 4 months.2.The comparison of survival analysis between the two groups from GSE146173showed the poor prognosis of patients with NPM1/FLT3-ITD/DNMT3A triple-mutated AML（P<0.05）.A total of 480 DEGs were identified,including 196 upregulated genes and 284 downregulated genes.We found that the DEGs played important roles in positive regulation of cell adhesion,extrinsic component of membrane,receptor ligand activity,and cytokine-cytokine receptor interaction.Guanine nucleotide-binding proteinγsubunit 4（GNG4）gene was a critical node in PPI and selected as the hub gene.3.TCGA data showed high expression of GNG4 gene in 17 tumors and low expression in 7 tumors including AML compared to the healthy population.Tumor-infiltrating immune cells analysis showed that GNG4 gene expression was positively correlated with plasmacytoid dendritic cell（p DC）and negatively correlated with regulatory T cell（Treg）in AML.Multivariate Cox regression analysis indicated that 1-year survival rate was less than 30%in the NPM1/FLT3-ITD/DNMT3A triple-mutated AML,and GNG4 gene low expression group that was much less than 20%among triple-mutated AML.Conclusion:1.Our study confirmed that the high frequency of NPM1/FLT3-ITD/DNMT3A triple mutations（approximately 6.7%）and patients had specific clinical features.Some of triple-mutated AML had mature monocyte characteristic.2.Clinical and bioinformatics data analysis demonstrated that NPM1/FLT3-ITD/DNMT3A triple-mutated AML with specific clinical features have extremely poor survival,with 1-year survival rate below 30%.3.By bioinformatics analysis,we found GNG4 gene expression was low in AML.Tumor-infiltrating immune cells analysis showed that GNG4 gene expression was positively correlated with p DC and negatively correlated with Treg in AML.