Male breast cancer (MBC) is a rare disease with incompletely defined clinical management. Pathogenic variants (PVs) in BRCA1/2 are established risk factors and inform personalized treatment strategies. Liquid biopsy represents a minimally invasive approach to detect molecular biomarkers, with circulating microRNAs (cmiRNAs) being particularly attractive due to their stability and tissue specificity. This study aimed to perform a comprehensive miRNome analysis in both plasma and tissue samples to identify novel biomarkers associated with MBC risk and to compare cmiRNA profiles between MBC patients with and without BRCA PVs, with the goal of improving early detection and informing clinical management.

Plasma samples from 95 MBCs (30 BRCA+ and 65 BRCA-) and 64 male healthy controls (3 BRCA+ and 61 BRCA-) were analyzed. Clinicopathological data were available for all subjects. FFPE samples from 24 (18 BRCA+ and 6 BRCA-) of 95 the MBCs were included in the study. miRNAs were isolated from plasma and FFPE samples using the miRNeasy Serum/Plasma Advanced Kit and miRNeasy FFPE kit, respectively. miRNA quality and quantity were assessed using Qubit flex 4.0 and 2100 Bioanalyzer. miRNome profiling was carried out using the QIAseq miRNA Library Kit, and libraries were sequenced on the Illumina MiSeq i100+ platform. All sequencing runs underwent quality control to ensure data reliability. Differential expression analysis was performed with RNA-seq Analysis portal.

A total of 199 cmiRNAs were significantly deregulated in plasma samples from 95 MBCs compared with 64 healthy male controls. Among these, miR-885-5p and miR-3613-5p showed the most significant fold changes (FDR-adjusted p-value < 0.05). We then restricted the analysis to MBCs to evaluate the impact of BRCA status on cmiRNA profiles. In this subgroup analysis, 17 cmiRNAs were identified as significantly deregulated in plasma samples from 30 BRCA+ compare with 65 BRCA- MBCs. miR-31-5p and miR-184 exhibited the most significant fold changes (FDR p-value < 0.05). Preliminary analysis of the 24 FFPE tumors samples identified 7 miRNAs differentially expressed between BRCA+ and BRCA- MBCs, one of these, hsa-miR-187-3p, was also found to be deregulated in the plasma analysis. All differentially expressed miRNAs identified to date are known to be involved in biology pathways related to breast cancer.

This study provides the first evidence that cmiRNAs may serve as potential biomarkers to assess MBC risk and to distinguish between MBCs with and without BRCA PVs, thus supporting early diagnosis and precision clinical management. Ongoing studies are evaluating these findings in a larger cohort. In addition, FFPE analyses are being extended to a larger series of MBCs to validate liquid biopsy results and to compare tissue-derived and plasma-derived miRNA profiles.

Study supported by AIRC (IG28775) and PNRR M4C2-I1.5 ECS 0000024 Rome Technopole to LO.