THR-6E: A Six-Gene Cell-of-Origin Signature Stratifies Risk and Predicts Systemic Therapy Response in ER+/HER2- Breast Cancer

BackgroundGenomic assays such as Oncotype DX, MammaPrint, and Prosigna have transformed risk stratification and treatment selection in early-stage, estrogen receptor-positive (ER+), HER2-negative breast cancers by enabling more precise prognostication and chemotherapy de-escalation in selected patients. However, their clinical utility is limited in lymph nodes positive disease. A major unmet need is the development of compact, mechanistically grounded biomarkers that extend risk and treatment-response prediction to clinically challenging ER+/HER2- subgroups, including lymph node-positive patients. MethodsBuilding on a cell-of-origin framework, we previously established a 70-gene triple hormone receptor (THR; ER, AR, VDR) signature (THR-70) that reflects luminal epithelial differentiation programs and is prognostic across breast cancer subtypes. Here, we refined this framework using interactome-guided clustering to derive a six-gene cell-of-origin signature (THR-6E: KIF4A, KIF2C, CDC20, FAM64A, TPX2, and LMNB2). We evaluated the prognostic and predictive performance of THR-6E across >7,000 breast cancer cases from multiple independent cohorts, assessed treatment-response prediction using endocrine- and chemotherapy-annotated datasets, and performed independent validation in the I-SPY2 adaptive clinical trial. FindingsTHR-6E robustly stratifies relapse-free survival (RFS) in ER+/HER2- breast cancer independent of tumor grade, proliferation status, and subtype. Hazard ratios for RFS were 2.41 (p<1x10-{superscript 1}), 1.61 (p=4.9x10-), and 1.50 (p=6.2x10-3) for grades 1, 2, and 3, respectively, and 2.16 and 1.33 for Luminal A and Luminal B subtypes. THR-6E maintained predictive value across endocrine- and chemotherapy-treated, untreated, lymph node-positive, and lymph node-negative subgroups. Beyond prognosis, THR-6E predicted endocrine therapy response in ER+/HER2-, node-negative disease and chemotherapy response in ER+/HER2-, node-positive disease, achieving approximately 70% sensitivity and specificity (AUC=0.714, p=3.6x10-), with consistent performance across taxane-, anthracycline-, and FEC-based regimens (AUCs 0.71-0.72). Single-cell transcriptomic and proteomic analyses demonstrated that THR-6E expression is specific to normal and malignant breast glandular epithelium, preserved during transformation, and further enriched in metastatic disease. Consistent with a cell-of-origin program, THR-6E genes were rarely mutated in breast cancer and retained normal tissue-like co-expression patterns. In the I-SPY2 trial, THR-6E achieved robust prediction of pathologic complete response across multiple treatment arms (AUCs 0.72-0.94), with an overall AUC of 0.741. InterpretationThese results support a cell-of-origin-anchored approach to biomarker development and challenge purely tissue-agnostic models of therapeutic response. THR-6E represents a compact, biologically interpretable signature that extends prognostic and predictive assessment to clinically relevant ER+/HER2- subgroups, including lymph node-positive disease. Its mechanistic grounding and stable performance across cohorts position THR-6E as a complementary tool to existing multigene assays, warranting prospective diagnostic accuracy studies to define its clinical utility and impact on treatment decision-making.