Machine learning-enabled multi-omics discovery of prognostic biomarkers and signaling targets in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains difficult to subtype using single omics layers. We conducted an exploratory investigation integrating reverse-phase protein array (RPPA) and DNA methylation data from the cancer genome atlas (TCGA)- pancreatic adenocarcinoma (PAAD) to assess the feasibility of multi-omics subtyping, alongside a supervised machine learning analysis of a small gene expression omnibus (GEO) transcriptomic cohort (n = 26) to identify candidate diagnostic genes. RPPA-based K-means clustering suggested a weak, possible two-subtype structure (silhouette ≈ 0.16) that remained unassociated with overall survival (log-rank p = 0.113) and lacked independent prognostic value. An independently performed similarity network fusion (SNF) analysis integrating RPPA and methylation data showed low concordance with RPPA-derived subtypes (Adjusted Rand Index (ARI) = 0.014), indicating limited convergence between molecular modalities. Supervised machine learning analysis of the GEO cohort using a fully nested leave-one-out cross-validation pipeline achieved a mean (area under the curve) AUC of 0.896 across four classifiers and identified four-fold-stable candidate genes (ESCO2, COL17A1, BCL2L14, and SOWAHB). However, this gene panel demonstrated limited external validity across two independent PDAC cohorts (log-rank p = 0.438 for both GSE62452 and GSE28735), indicating limited generalizability despite robust internal performance. Collectively, these findings provide limited evidence for a robust, prognostically significant multi-omics subtype or a validated diagnostic gene signature; instead, this study serves as a hypothesis-generating resource and highlights the importance of rigorous cross-validation and independent external validation in small-sample transcriptomic biomarker discovery.