Comprehensive profiles and diagnostic value of menopausal-specific gut microbiota in premenopausal breast cancer

Breast cancer is a leading cause of cancer mortality worldwide, with a younger median onset age in Asian countries compared to Western countries. In Taiwan, over 30% of breast cancer cases occur in premenopausal women. While genetic factors like *BRCA1/2*, *TP53*, and *RAD50* mutations contribute to some cases, they only account for a small percentage. Westernization, encompassing environmental factors (endocrine-disrupting chemicals), dietary habits (high fat and alcohol intake), and reproductive factors (delayed childbearing), may play a significant role. The gut microbiota, a complex community of microorganisms in the gut, has been increasingly implicated in various diseases including cancer, known as the "oncobiome." In breast cancer, the "estrobolome," gut bacterial genes metabolizing estrogens, is a key mechanism. However, most research has focused on postmenopausal breast cancer, leaving a gap in understanding the role of gut microbiota in premenopausal breast cancer. This study aimed to comprehensively characterize gut microbiota profiles in premenopausal breast cancer patients, identify key microbial markers, assess their diagnostic value, and explore related functional pathways, ultimately aiming for a non-invasive approach for breast cancer detection and prevention.

Existing research demonstrates a strong link between the gut microbiota and breast cancer, primarily focusing on the "estrobolome"—the impact of gut bacteria on estrogen metabolism. Studies show that gut bacteria can deconjugate estrogens, leading to increased reabsorption and potentially higher risk of breast cancer, particularly in postmenopausal women. The role of bacterial metabolites transported to distant sites and influencing cancer development is also highlighted. However, the estrobolome concept largely relates to postmenopausal cancer, and many studies lack a focus on the menopausal status of breast cancer patients. There's limited research specifically addressing premenopausal breast cancer and the gut microbiome.

This study recruited 267 participants (October 2018-December 2020) from Kaohsiung Medical University Chung-Ho Memorial Hospital: 67 age-matched female controls (50 premenopausal, 17 postmenopausal) and 200 breast cancer patients (100 premenopausal, 100 postmenopausal). Patients with other malignancies or recent antibiotic/probiotic use were excluded. Fecal samples were collected before any treatment. Clinical data (age, BMI, grade, stage, tumor size, receptor status) were recorded. DNA was extracted from fecal samples using QIAamp Fast DNA Stool Mini Kit. The V3-V4 hypervariable region of 16S rDNA was amplified and sequenced using Illumina MiSeq platform. Bioinformatic analysis included quality filtering, OTU assignment (Greengene 13.8 database), α-diversity (Shannon entropy), β-diversity (PCoA-D_0.5 UniFrac), LEfSe analysis (LDA effect size), heatmap analysis, correlation matrix analysis (Spearman's rank correlation), and functional profile inference (PICRUSt2). Statistical analysis involved t-tests, ANOVA, ROC curves, and AUC calculations. The study was approved by the Internal Review Board, and informed consent was obtained.

Premenopausal breast cancer patients had significantly reduced α-diversity (Shannon entropy) compared to premenopausal controls, but no difference was seen between postmenopausal groups. β-diversity analysis showed significant differences in microbial composition among all groups. At the phylum level, Actinobacteria was enriched in premenopausal controls, Verrucomicrobia in postmenopausal controls, and Proteobacteria in postmenopausal breast cancer patients. LEfSe, Venn diagrams, and heatmap analyses identified 14 bacterial taxa as potential markers. Five were unique to premenopausal breast cancer (*Bifidobacterium* spp., *Anaerostipes*, *Bacteroides fragilis*), four to postmenopausal cancer (*Akkermansia muciniphila*, *Phascolarctobacterium*, *Proteobacteria*, *Klebsiella pneumoniae*), and five were universal markers across both menopausal statuses (*Faecalibacterium prausnitzii*, *Ruminococcus gnavus*, *Rothia mucilaginosa*, *Sutterella*, *Haemophilus parainfluenzae*). *Bacteroides fragilis* was negatively correlated with age in the breast cancer group, while *Klebsiella pneumoniae* showed a positive correlation. Analysis of diagnostic value showed excellent discrimination (AUC > 0.8) in distinguishing premenopausal and postmenopausal breast cancer patients from controls using specific combinations of microbial markers. Functional pathway analysis revealed enrichment of steroid-related and oncogenic pathways in premenopausal cancer and steroid-related and chemical carcinogenesis pathways in postmenopausal cancer.

This study provides novel insights into the menopausal-specific gut microbiota profiles associated with breast cancer. The findings of reduced α-diversity in premenopausal breast cancer patients highlight dysbiosis and a potential loss of beneficial bacteria. The identified microbial markers, particularly the menopausal-specific ones, offer promising diagnostic potential. The correlation between specific microbes and age within the breast cancer groups underscores the influence of age-related factors on the gut microbiome in the context of breast cancer. Functional pathway analyses provide further mechanistic clues, highlighting steroid metabolism and oncogenic pathways. This research expands the understanding of the complex interplay between the gut microbiota and breast cancer across different menopausal stages, suggesting potential for personalized diagnostic and preventive strategies.

This study comprehensively characterized the gut microbiota in premenopausal and postmenopausal breast cancer patients, identifying distinct menopausal-specific microbial markers with high diagnostic value. The findings highlight the importance of considering menopausal status when investigating the role of gut microbiota in breast cancer. Future research should focus on validating these findings in larger, independent cohorts and investigating the mechanistic pathways linking specific bacteria to breast cancer development. The potential for non-invasive diagnostic tools and preventive interventions based on these findings offers exciting possibilities.

The cross-sectional nature of the study limits the ability to establish causality. The sample size, while substantial, might benefit from further expansion to improve statistical power. The study focused on stage I-II breast cancer, potentially limiting generalizability to other stages. Further research is needed to investigate the impact of specific dietary and lifestyle factors on the observed microbial profiles.