Network-Based Stratification Refines Stratification of Intermediate-Risk Acute Myeloid Leukemia Samples

Abstract The European LeukemiaNet (ELN) risk stratification of acute myeloid leukemia (AML) uses genetic and molecular markers to categorize patients. However, disease heterogeneity, particularly in the intermediate-risk group, complicates stratification. Ideker et al. developed the Network-Based Stratification (NBS) method, combining protein network analysis and mutation profiling via machine learning. We applied NBS to intermediate- risk AML patients to refine prognosis and identify distinct molecular subtypes compared to the 2022 ELN scheme. We selected 170 intermediate-risk AML patients based on the 2022 ELN classification from TCGA (n=58), BEAT AML (n=87), and FIMM (n=25) datasets. Using NBS, we analyzed 3,108 genes from WGS or WES data, mapping them onto a cancer-specific protein network for clustering based on network-propagated mutation profiles. We conducted 200 iterations of sub-sampling, considering patients with at least 3 mutated genes and using consensus clustering for robust stratification, assessing associations with clinical and transcriptomic features. NBS identified five distinct molecular subgroups characterized by unique mutation patterns: IDH1-dominant (Cluster 1), DNMT3A-dominant (Cluster 2), low-frequency multi-mutated (Cluster 3), FLT3/NPM1/DNMT3A co- mutated (Cluster 4), and FLT3-dominant (Cluster 5). Cluster 4 showed significantly worse overall survival (HR = 1.81; p = 0.05). In addition, ex vivo drug sensitivity and transcriptomic analyses revealed significant variation in therapeutic response and pathway activation across clusters. These findings underscore the power of machine learning–driven approaches like NBS to uncover hidden molecular structure within intermediate-risk AML groups, enabling more precise prognostication and potentially informing personalized therapeutic strategies. Key points ML-based NBS stratification reveals distinct subgroups within intermediate-risk AML with unique molecular and clinical profiles. AML with NPM1/FLT3-ITD/DNMT3A mutations define a high-risk group with distinct drug sensitivities, including FLT3 inhibitors. Competing Interest Statement All authors met the criteria set forth by the International Committee of Medical Journal Editors (ICMJE) and hence adequately contributed to manuscript development. C.A.H received funding from the Research Council of Finland (grant no. 334781, 352265, 357686, and 320185), the Sigrid Juselius Foundation, and the Cancer Foundation Finland to support this study. CAH has received funding from Kronos Bio, Novartis, Celgene, Orion Pharma and the IMI2 consortium project HARMONY unrelated to this work. JW received funding from the American Cancer Society to support this study and received funding from Sobi and Servier unrelated to this work. PS received funding from the University of Helsinki and Instrumentarium Foundation. Funding Statement This work has been supported by funding the Research Council of Finland (grant no. 334781, 352265, 357686, and 320185), the Sigrid Juselius Foundation, the Cancer Foundation Finland and the American Cancer Society to support this study. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: An ethical committee of Helsinki University Hospital Comprehensive Cancer Center and Helsinki University Hospital approved the study (permit numbers 239/13/03/00/2010, 303/13/03/01/2011). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors