This is a prospective, single-center diagnostic study testing whether a new, minimally invasive analysis of nipple fluid can distinguish benign from malignant causes of pathologic nipple discharge. Many patients with bloody or blood-tinged nipple discharge undergo surgery to make a diagnosis, yet most are ultimately found to have benign disease. The investigators aim to develop a laboratory test that analyzes DNA in nipple fluid to help avoid unnecessary operations while still identifying cancers.

Approximately 30 adults with spontaneous, single-duct, unilateral bloody or serosanguinous nipple discharge who are already scheduled for standard diagnostic surgery will be enrolled at Hubei Cancer Hospital. Before surgery, the investigators will collect a small sample of nipple fluid (or gently obtain nipple aspirate fluid using a soft suction cup if needed) and one tube of blood. The investigators will analyze the fluid's DNA using two approaches: low-pass whole-genome analysis to look for copy number changes and fragmentation patterns, and genome-wide DNA methylation profiling. Surgical pathology will serve as the reference standard. Using these data, the investigators will build and validate a model to classify lesions as benign or malignant.

The primary outcome is diagnostic accuracy (area under the ROC curve, sensitivity, and specificity). Secondary outcomes include positive and negative predictive values, model calibration, subgroup performance (e.g., ductal carcinoma in situ vs invasive cancer), and an estimate of potential clinical impact (for example, how many benign cases might safely avoid surgery at a high-sensitivity threshold). Study test results will not affect current clinical care; all participants will receive usual evaluation and surgery. Risks are minimal and may include brief nipple discomfort or skin irritation from gentle suction and routine blood-draw risks (bruising, lightheadedness). There is no direct benefit to participants, but the findings may support a future noninvasive test to guide care and reduce unnecessary surgery. Data will be de-identified and stored securely. Expected enrollment is from September 2025 to May 2026.

Inclusion Criteria:

1. Age ≥18 years.
2. Spontaneous, unilateral, single-duct pathologic nipple discharge, predominantly bloody or serosanguinous, raising clinical suspicion of intraductal disease.
3. Planned diagnostic breast surgery/biopsy after specialist assessment (e.g., duct excision/microdochectomy, lumpectomy); patients who had ductoscopy but are still scheduled for surgery remain eligible.
4. Study sampling (nipple discharge and one peripheral blood tube) feasible before surgery/invasive diagnostics without delaying standard care.
5. Able and willing to provide written informed consent and allow access to surgical pathology and relevant clinical data.

Exclusion Criteria:

1. Physiologic or non-pathologic discharge (typically bilateral, multiduct, expressible only with manipulation; milky/clear/green) or galactorrhea due to endocrine/drug causes.
2. Active breast infection/inflammatory disease (e.g., abscess) as the source of discharge.
3. Prior diagnosis and treatment of breast cancer (surgery/radiation/systemic therapy).
4. Recent invasive ductal manipulation likely to confound analysis (e.g., ductoscopy, duct cannulation/irrigation), per investigator judgment.
5. Pregnant or lactating patients.
6. Significant hematologic disease/coagulopathy precluding safe sampling.
7. Inability to complete preoperative study sampling, refusal/withdrawal of consent, or poor compliance.
8. Any condition judged by investigators to compromise sample quality, data interpretation, or participant safety.

Prospective, single-center consecutive series at Hubei Cancer Hospital. Adults presenting with spontaneous, unilateral, single-duct bloody/serosanguinous nipple discharge and scheduled for diagnostic surgery/biopsy will undergo preoperative nipple-discharge collection for low-coverage whole-genome sequencing and methylome profiling. Postoperative pathology serves as the gold standard to classify benign vs malignant lesions. Enrollment is not restricted by gender identity; individuals with relevant breast anatomy who meet criteria may be included. Excludes physiologic discharge, infection, prior treated breast cancer, pregnancy/lactation, recent duct manipulation, and unsafe sampling conditions. Target sample size ≈30; no healthy volunteers.

We do not plan to share individual participant data (IPD). The informed consent for this study and institutional/regulatory constraints do not permit external sharing of participant-level datasets. Aggregate results will be posted on ClinicalTrials.gov and reported in peer-reviewed publications. The study protocol, statistical analysis plan, and a CSR synopsis will be made available upon publication.