Non-blood liquid biopsy in nipple discharge for decoding tumor-derived CNV signatures for breast cancer detection and prognostication.

Abstract

e12578 Background: Nipple discharge is a prevalent symptom affecting about 80% of women during reproductive years, yet only less than 5% of such cases are malignant. However, accurate non-invasive diagnosis in these women remains a longstanding clinical challenge. Here, we evaluated somatic copy number variations (CNVs) in nipple discharge DNA (ndDNA) as diagnostic biomarkers and explored their cellular origins. Methods: Breast cancer-specific CNV features were first derived from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA; 1,086 tumors and 942 controls). A diagnostic model constructed from these features was then tested in two in-house cohorts: nipple discharge (39 malignant, 18 benign) and plasma cell-free DNA (cfDNA; 149 malignant, 34 benign). To trace the cellular origin of nipple discharge CNVs, we performed single-cell RNA-sequencing on matched tumor tissues (n=5) and integrated the resulting inferCNV profiles with nipple discharge CNV profiles at single-cell level. Results: We identified 20 recurrent somatic CNVs (12 amplifications and 8 deletions). The diagnostic model demonstrated robust diagnostic performance in four external validation datasets. In the nipple discharge cohort, it achieved 74.4% sensitivity and 94.4% specificity for malignancy. In contrast, no significant CNV feature was detected in the plasma cfDNA cohort. Paired analysis revealed high concordance between nipple discharge CNVs and inferCNV. CNV release fidelity varied among epithelial subclusters. High-fidelity subsets displayed greater CNV burden, active estrogen signaling, and stronger immune interactions, and were associated with better prognosis. Conclusions: Our findings provide the first definitive evidence that nipple discharge is a tumor-proximal, highly specific non-blood liquid biopsy for breast cancer—surpassing plasma cfDNA by preserving tumor genomic signatures with minimal dilution and background noise. Beyond diagnostics, single-cell tracing deciphers luminal epithelial subcluster-selective ndDNA shedding, illuminating biomarker release mechanisms and advancing liquid biopsy paradigms across cancers.