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 Kruskal–Wallis 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 bacteria-centric, 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 activation-related genes. Conclusions This study, the largest to date comparing healthy versus cancer-associated 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.

PURPOSE We evaluate potential contributors to the development of autoimmunity and other phenotypes consistent with immune dysregulation in individuals with germline mutations in the tumor suppressor gene PTEN in this observational report. MATERIALS AND METHODS Illumina sequencing of bacterial and fungal microbes was carried out on patient-donated fecal samples in a cohort of 67 patients with pathogenic germline PTEN mutations, including 41 individuals with autoimmunity and/or phenotypes consistent with immune dysregulation (cases) and 26 individuals without (controls). From these data, we measured differences in alpha and beta diversity between cases and controls and identified differentially abundant bacterial and fungal taxa using phyloseq and MicrobiomeSeq packages in R. We analyzed correlations between these taxa and specific HLA genotypes, along with correlations between HLA diversity and microbial diversity, by conducting high-resolution HLA genotyping at four class II loci (DRB1, DRB345, DQA1, and DQB1). RESULTS We found that alpha diversity distributions for both bacterial and fungal genera were statistically different between cases and controls. We identified differentially abundant bacterial and fungal taxa between cases and controls. Network analysis of differentially abundant bacterial taxa revealed some co-varying bacterial genera. We additionally found significant correlations between certain HLA genotypes and certain taxa and significant correlations between HLA diversity and alpha diversity. CONCLUSION PTEN-associated immune phenotypes might be influenced by the gut microbiome, and class II HLA molecules, in part, crosstalk with the gut microbiome. These preliminary observations should lay the groundwork for future studies to ultimately derive clinical measures, which could use gut microbiome and HLA molecule biomarkers to predict, and perhaps prevent, immunity and inflammation in patients predisposed to cancer because of germline PTEN mutations.

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This a preprint and has not been peer reviewed. Data may be preliminary.