Abstract
Predicting neoadjuvant chemotherapy response in breast cancer remains critical for optimizing treatment strategies, yet robust predictive biomarkers are lacking. This study implemented an ensemble machine learning approach to identify a gene expression signature predicting pathological complete response (pCR) versus residual disease (RD) using bulk RNA-sequencing data from GSE163882 (138 RD, 80 pCR). We employed TMM normalization with differential expression analysis (250 genes, FDR < 0.05, |log2FC| ≥ 1), ensemble feature selection across five classifiers (Random Forest, Gradient Boosting, SVM, k-NN, and Neural Network) with 10-fold repeated cross-validation, and stacked ensemble development. Consensus selection identified a 17-gene signature consistently ranked across algorithms. The stacked ensemble achieved 0.97 AUC post-testing on hold-out test data. External validation on the independent GSE240671 cohort (37 pCR, 25 RD) following ComBat batch correction achieved ROC AUC of 0.78 and PR AUC of 0.85 with isotonic calibration, demonstrating balanced accuracy of 0.71 and 0.86 sensitivity for pCR detection. Pathway enrichment revealed associations with cell cycle regulation (E2F3, MKI67), DNA repair (BRCA2), and transcriptional control (MED1), with six priority genes (MED1, BRCA2, E2F3, PITPNB, H1-1, and FARP2) showing established breast cancer relevance. This externally validated 17-gene signature provides a biologically grounded tool for NAC response prediction in precision oncology.