Human Breast Microbiome Correlates with Prognostic Features and Immunological Signatures in Breast Cancer

Background: Currently, over half of breast cancer cases are unrelated to known risk factors, highlighting the importance of discovering other cancer-promoting factors. Since crosstalk between gut microbes and host immunity contributes to many diseases, we hypothesized that similar interactions could occur between the recently described breast microbiome and local immune responses to influence breast cancer pathogenesis.Methods: Using 16S rRNA gene sequencing, we characterized the microbiome of human breast tissue in a total of 221 patients with breast cancer, 18 individuals predisposed to breast cancer, and 69 controls. We performed bioinformatic analyses using a DADA2-based pipeline and applied linear models with White's t or KruskalWallis H -tests with Benjamini-Hochberg multiple testing correction to identify taxonomic groups associated with prognostic clinicopathologic features. We then used network analysis based on Spearman coefficients to correlate specific bacterial taxa with immunological data from NanoString gene expression and 65-plex cytokine assays.Results: Multiple bacterial genera exhibited significant differences in relative abundance when stratifying by breast tissue type (tumor, tumor adjacent normal, high-risk, healthy control), cancer stage, grade, histologic subtype, receptor status, lymphovascular invasion, or node-positive status, even after adjusting for confounding variables. Microbiome–immune networks within the breast tended to be bacteriacentric, with sparse structure in tumors and more interconnected structure in benign tissues. Notably, Anaerococcus , Caulobacter , and Streptococcus , which were major bacterial hubs in benign tissue networks, were absent from cancer-associated tissue networks. In addition, Propionibacterium and Staphylococcus , which were depleted in tumors, showed negative associations with oncogenic immune features; Streptococcus and Propionibacterium also correlated positively with T-cell activationrelated genes.Conclusions: This study, the largest to date comparing healthy versus cancerassociated breast microbiomes using fresh-frozen surgical specimens and immune correlates, provides insight into microbial profiles that correspond with prognostic clinicopathologic features in breast cancer. It additionally presents evidence for local microbial–immune interplay in breast cancer that merits further investigation and has preventative, diagnostic, and therapeutic potential.