Integrated Circulating miRNA–EPCAM–Protein Liquid Biopsy Signature for Early Breast Cancer Detection in Iraqi Women: A Multi-Layer Molecular Framework Early Breast Cancer Detection in Iraqi Women: A Multi-Layer Molecular Framework
Abstract
Background: Early breast cancer is characterized by subtle molecular alterations that may escape detection by conventional single-marker assays. Liquid biopsy offers a non-invasive approach to capturing this complexity, yet most circulating biomarker studies remain limited to a single molecular layer. This study aimed to develop and evaluate an integrated circulating signature combining microRNAs, EPCAM-associated epithelial dissemination signals, and tumor-associated serum proteins for early breast cancer detection in Iraqi women.
Methods: This case–control study included 75 women (25 patients with early breast cancer and 50 healthy controls) recruited at the National Hospital for Oncology and Hematology, Najaf, Iraq, between February and August 2025. Three computationally prioritized circulating miRNAs (hsa-miR-4734, hsa-miR-5787, hsa-miR-4532) identified from the public serum-miRNA dataset GSE73002 were quantified by SYBR-Green qRT-PCR using hsa-miR-16 as reference. EPCAM expression was assessed by qPCR with GAPDH normalization in a subset of 13 subjects. Serum CA15-3, CEA, and mammaglobin-1 (MGB1) were measured by sandwich ELISA. An integrated multi-marker classifier was constructed using leave-one-out cross-validated logistic regression.
Results: hsa-miR-4734 (4.2-fold; p=0.0005, AUC 0.75), hsa-miR-5787 (4.0-fold; p=0.013, AUC 0.68), and hsa-miR-4532 (5.0-fold; p=0.087, AUC 0.62) were upregulated in patients. EPCAM expression was also elevated in a 13-subject subset (7 patients, 6 controls; p=0.043, AUC 0.86, 95% CI 0.50–0.97). The integrated five-marker classifier achieved a leave-one-out cross-validated AUC of 0.91 (95% CI 0.77–0.96), sensitivity of 84%, and specificity of 90%.
Conclusion: An integrated circulating signature encompassing post-transcriptional regulation, epithelial shedding, and tumor-associated protein release may support noninvasive early detection of breast cancer. Diagnostic performance estimates should be considered exploratory pending external prospective validation in age-matched cohorts.
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Copyright (c) 2026 Adam Mohammed, Rasha Abd Ali Al-khalidi
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