Matched germline and tumor profiling in male breast cancer for the discovery of molecular subtypes with clinical relevance

Background: Breast cancer (BC) in men is a rare disease, but morbidity and mortality in male BC (MBC) patients is a serious concern. Because of its rarity, to date, clinical management of men with BC has been informed almost entirely by female BC research. However, BC in men and women may behave differently. Inherited mutations in homologous recombination (HR) genes, mainly BRCA1,BRCA2 and PALB2, play a major role in MBC predisposition. MBCs associated with mutations in HR gene are likely to represent a subgroup of tumors with a peculiar tumor phenotype. Here, we performed a transcriptome analysis of MBCs characterized for germline mutations in HR genes in order to provide insight into underlying HR pathways and characterize distinct molecular subtypes of MBC with possible clinical relevance. Materials and methods: MBC cases were selected to include cases with and without germline mutations in HR genes. Tumor samples with pathology and outcome data were selected within the Italian multicenter study on MBC that comprises 750 MBCs with information on germline mutations in 50 cancer-related genes. To enrich the series of MBC cases with germline mutations in HR genes, tumor samples from kConFab were also included. Overall, 63 tumor samples, including 26 cases with germline mutations in HR genes (BRCA2, BRCA1, PALB2, RAD50 and RAD51D) and 37 without germline mutations, were analyzed. Whole transcriptome data were obtained by RNA-sequencing using Illumina technology. A bioinformatic pipeline including FastQC tool for quality control, STAR for alignment, RSeQC-FPKM for counting reads, DESeq2 package for differential expression and DAVID for enrichment, was used. Tumor immunophenotype was evaluated using CIBERSORT, which accurately determines the fraction of 22 immune cell types. Statistical analyses were performed using Fisher exact test, t-test and log-rank test as appropriate. Results: A total of 724 differentially expressed genes, of which 355 up-regulated and 369 down-regulated, emerged between MBC cases with and without germline mutations in HR genes. Pathway-based analyses revealed different transcriptome profiles for genes involved in key biological processes, including translational initiation, cell cycle, DNA damage and repair. A higher fraction of activated CD4 memory T cells was observed in tumors with germline mutations in HR genes compared to those without mutations (p=0.04). Unsupervised clustering approach, based on the 2000 most variable transcripts among tumors, revealed two distinct subgroups with different biologic features and clinical outcome. In particular, tumors in subgroup 1 showed higher expression of genes involved in cell proliferation, were enriched for cases with germline mutations in HR genes (p=0.03), had lymph node involvement (p=0.04) and displayed a trend toward worse overall survival (p=0.09), when compared to tumors in subgroup 2. Conclusions: Integration of matched germline and tumor profiling seem to be a valuable approach for the identification of biologically and clinically relevant subtypes of MBC. Germline mutations in HR genes could impact on MBC transcriptome, defining molecular subgroups with peculiar biological features. Overall, these results may help to improve the clinical managements of MBC patients. Study supported by AIRC (IG 21389) to L.O.