This prospective interventional diagnostic study evaluates the clinical utility of methylation analysis of BRCA1, RASSF1A and PTEN genes in breast lesions of uncertain malignant potential (B3 lesions).

Women with suspicious non-palpable breast lesions undergo standard diagnostic procedures including clinical examination, imaging assessment, and image-guided core needle biopsy when indicated.

As part of the study protocol, breast tissue samples are prospectively assigned to additional molecular diagnostic testing using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA).

The study evaluates whether gene promoter methylation status improves diagnostic assessment by comparing methylation results with final histopathological diagnosis.

Breast cancer is the most common malignancy in women. Epigenetic alterations, particularly DNA promoter methylation of tumor suppressor genes, play an important role in breast carcinogenesis and may occur early in disease development.

This single-center prospective interventional diagnostic study is conducted at the Institute of Oncology Ljubljana. Participants are women with suspicious non-palpable breast lesions referred through the Slovenian national screening program DORA or other diagnostic pathways.

All participants undergo standard diagnostic work-up, including:

• clinical breast examination,
• breast imaging (mammography and/or ultrasound),
• image-guided core needle biopsy when indicated.

Breast tissue samples obtained during biopsy or surgical excision are prospectively assigned to additional molecular testing using MS-MLPA to determine methylation status of BRCA1, RASSF1A and PTEN.

Histopathological classification includes:

• benign lesions,
• lesions of uncertain malignant potential (B3 lesions),
• malignant lesions.

The primary objective is to evaluate the diagnostic performance of methylation analysis compared with histopathological diagnosis.

Statistical analysis will be performed using SPSS and GraphPad Prism. Sensitivity, specificity, positive predictive value, and negative predictive value will be calculated. Group comparisons will be performed using chi-square test or Fisher's exact test. Statistical significance is defined as p < 0.05.

Inclusion Criteria:

• Female participants aged 40 years or older
• Suspicious non-palpable breast lesion referred for diagnostic evaluation at the Institute of Oncology Ljubljana
• Undergoing image-guided core needle biopsy or surgical excision as part of standard diagnostic work-up
• Availability of sufficient breast tissue sample for methylation analysis Signed written informed consent

Exclusion Criteria:

• Contraindications to standard diagnostic procedures
• Insufficient tissue sample for molecular analysis
• Withdrawal of informed consent