Username:
Password:
Log me on automatically each visit
 
 
It is currently Thu Mar 28, 2024 5:12 am
Post a new topic Post a reply  [ 1 post ]   
Author Message
PostPosted: Thu Aug 04, 2016 1:10 pm 
Site Admin
User avatar

Joined: Wed May 27, 2015 10:20 am
Posts: 515
The Microbiome of Aseptically Collected Human Breast Tissue in Benign and Malignant Disease

Quote:
Introduction
Globally, breast cancer accounts for nearly one quarter of all cancers and is the leading cause of cancer death among women1. While there are established risk factors for breast cancer, at least 70% of breast cancer cases occur in women of average risk, and current prediction models provide poor risk discrimination for individuals2,3. Mounting evidence suggests that shifts in microbial assemblages are linked to cancer development and aggressiveness while eradication of the causative organism or restoration of the normal microbiota may reverse this process4,5,6,7,8,9. Our group has recently identified immune effectors in breast lobules, with cytotoxic T lymphocytes and dendritic cells intimately associated with normal breast epithelium10. Since mucosal immune systems develop as a direct result of microbial exposure, the presence of immune effectors within the complex microenvironment of the breast is suggestive of a breast microbiome.

In cancer, the microbiome has been implicated at a variety of body sites including stomach, colon, liver, lung, and skin with the strongest linkage thus far with gastrointestinal tract carcinogenesis, notably gastric cancer and lymphoma (Helicobacter pylori) and colon cancer (Fusobacterium)5,8,11,12,13,14. The distinct nature of the microbiome of each body niche suggests a similar organ specificity to microbial effects on inflammation and carcinogenesis14. Work with 3,2′-dimethyl- 4-aminobiphenyl hydrochloride (DMAB)-treated mice suggests a microbial role in breast tumorigenesis, but this has not been examined in humans15. More recently, 16S rDNA analysis has shown that milk samples from lactating women have diverse bacterial communities, but it is unknown whether these also are present in breast tissues16. Interestingly, the milk microbiome seems to recapitulate some risk features that are associated with breast cancer. For instance, the breast milk microbiome appears to differ from other human niches and a different, less diverse bacterial community has been observed in obese versus lean mothers17. Additionally, the gut microbiome influences estrogen metabolism via the enterohepatic circulation18,19. These data and the association of obesity, estrogen levels and inflammation with breast cancer risk provide a rationale for exploring a cancer-associated microbiome in breast tissue. Past studies have cultured bacteria from intraoperatively-obtained breast tissue, consistent with the presence of endogenous bacteria20,21. However, potential confounding factors, such as contamination from the skin, were not explored and genomic technology for testing was not available.

The lack of data on the microbiome of sterilely obtained human breast tissue in benign and malignant disease states analyzed with paired skin tissue samples by a culture-independent molecular genetic approach prompted this investigation. We hypothesized a resident microbiome in human breast tissue that is distinct from that of the overlying skin. We further explored differences in the microbiome of human breast tissue from women with benign and malignant disease in order to evaluate a potential role for a breast microbiome in carcinogenesis.

Discussion
We investigated the microbiome of sterilely obtained human breast tissue in women with benign and malignant breast disease. Two major findings from our study are that breast tissue obtained under surgically sterile conditions does indeed have its own distinct microbiome and that it is distinct from that of the overlying breast skin. The unique features of our study include (1) simultaneous collection of breast tissue, skin tissue and skin swab samples in the operating room under aseptic conditions and (2) comparison of the breast tissue microbiome in women with benign versus malignant disease. Our other key finding is that the background breast microbiome in women with malignant disease is notably different from the breast microbiome in women with benign disease. These data form the foundation for exploration of the core microbial community in breast tissue and microbial dysbiosis in association with health and disease including both cancer and infection. Dysbiosis of this intrinsic microbial community may contribute to cancer development and clinically apparent infection.

Previous work investigating the breast tissue microbiome using next-generation sequencing includes two studies. Xuan et al. compared breast tumor and unspecified negative control breast tissue from 20 patients with estrogen receptor-positive breast cancer24. One of the main confounding factors they discussed was the presence of potential contamination, which they were unable to address using retrospective collection of non-sterile formalin-fixed tissue. However, their results showed 1614 OTUs with 11 exhibiting differential abundance between tumor and normal tissue samples, with the predominant finding enrichment of Sphingomonas in normal tissue. The high number of OTUs they report possibly may be due to sample contamination. While this study assumes that contamination would be similar in the case and control samples, this study design makes it difficult to assess the true role of individual microbes in breast cancer. More recently Urbaniak et al. reported on 16S sequencing in unaffected breast tissue 5 cm from tumors in patients with malignant and benign tumors and in women undergoing breast reduction25. Also sterilely collected, they identify 121 OTUs in the breast microbiome and compared the relative abundance of taxa between women from Canada versus Ireland. The most abundant phyla in that report were Proteobacteria followed by Firmicutes, Actinobacteria and Bacteroidetes in descending order, consistent with our own findings. The investigators were unable to culture some of the most abundant taxa they identified, raising the question of whether some of these microbes were introduced from contaminated DNA rather than being intrinsic to the tissue samples. Further, the most prominent finding was a geographical difference between Canadian and Irish breast tissue microbiomes (although this may be artifactual as the samples were processed in different laboratories using different protocols and reagents), and there was no subsequent examination of benign versus malignant disease.

A second major finding from our study is that the breast tissue microbiome is distinct from that of the overlying skin tissue and has greater species richness. This would suggest that although the breast tissue microbiome may be derived from or contributed to by the skin microbiome, breast tissue has its own distinct environment and ecosystem. These differences may be attributable to differences in tissue microenvironments such as pH and oxygen levels which may facilitate relative dominance of certain taxa. Our data are supported by the findings from the Human Microbiome Project analyses that demonstrate the diversity and abundance of the signature microbes in each habitat varied among healthy subjects, with strong niche specialization both within and among individuals26.

Interestingly, while we found evidence using deep sequencing techniques that microorganisms are present in breast tissue samples obtained under aseptic conditions in the absence of clinical infection, this finding was suggested approximately 30 years ago in two studies after cultures of breast tissue obtained sterilely from the operating room resulted in positive cultures in up to 90% of samples20,21. In both studies, the most commonly cultured organism was coagulase-negative Staphylococcus and the most common anaerobe was Propionibacterium acnes, both predominantly skin bacteria that had potentially contaminated the surgical site. Indeed, this was the major weakness of these findings—one which we now have addressed using skin tissue and swabs. In the current study, samples of skin tissue from the incision edge were also evaluated in order to determine if the findings in breast tissue might be attributable to the skin organisms. With much more sensitive microbial detection techniques using genomic sequencing technology, we have confirmed that breast tissue indeed harbors a microbiome which is distinct from the microbiome of the overlying breast skin.

Perhaps our most intriguing finding in the present study was the observation of demonstrable differences in the breast tissue microbiome between women with benign versus malignant disease where we identified notable differences in beta diversity. Specific genus-level taxa that were significantly enriched in breast tissue from women with malignant disease include Fusobacterium, Atopobium, Gluconacterobacter, Hydrogenophaga and Lactobacillus. Fusobacterium has been reported in association with other epithelial malignancies including colon cancer and may act by secreting virulence factors as well as creating a pro-inflammatory environment that promotes carcinogenesis4,5,6,7,27. Therefore, we further investigated the functional role of these bacteria within these microenvironments. Using KEGG pathways as the basis for our analysis, we identified 6 differentially abundant pathways between benign and malignant disease states. In patients with malignant disease, pathways involving cysteine and methionine metabolism, glycosyltransferases, fatty acid biosynthesis and C5-branched dibasic acid metabolism were depleted. Interestingly, methionine dependence is a general metabolic derangement across multiple cancers and it is postulated that depleting methionine might reverse cancer progression, either using methioninase or a methionine-restricted diet28,29.

Our findings invite further study to define the origin of the breast tissue microbiome. Possible routes of bacterial access to breast tissue include passage from the skin via the nipple-areolar orifices, nipple-oral contact via lactation or sexual contact and bacterial translocation from the gut. Data supporting the first two hypotheses stems mainly from observations showing that the microbial composition of human breast milk in healthy lactating women contains many of the same bacteria found commonly in skin16 and shifts from a skin flora-dominant pattern in colostrum to an oral cavity-dominant pattern after 6 months of lactation17. In support of bacterial translocation from the gut, studies have shown that orally administered probiotics are recovered in the milk and treat the clinical infection lactational mastitis with better efficacy than orally administered antibiotics30. Further, in a clinical trial of orally administered Lactobacillus salivarius in late pregnancy, the rate and severity of lactational mastitis was significantly decreased31. Taken together, these studies demonstrate potential translocation of beneficial microbes from the gastrointestinal tract to the breast, although an alternative explanation of the protection from mastitis might be homing to the breast of anti-microbe B cells made in the gut. Additional indirect evidence of the possibility of gut to breast translocation comes from mouse studies showing increased rates of mammary gland carcinogenesis with enteral administration of certain pathogenic bacteria that appear to act in an immune cell-dependent fashion, although no data yet exists on the breast tissue microbiome in those experiments32. Future work to examine the relationship of multiple microbiome sites including gut, oral, skin and breast may help resolve some of these questions.

A limitation of our study is small sample size. Although we were able to detect an overall microbiota difference between tissue types and between disease states based on the MiRKAT test, the study was underpowered to identify specific differential taxa/KEGG pathways if a multiple testing correction procedure such as false discovery rate control is applied. Thus, in differential abundance analysis for both taxa and function data, we did not perform multiple testing correction in order to increase the power to identify true positives at the cost of a greater likelihood of false positives. A future study with a larger sample size is needed to identify with confidence a microbial signature for malignant breast tissue. The patients in this study with malignant disease all had estrogen receptor-positive tumors, as do 85% of breast cancer patients, but a different microbial signature may be present in the breast tissues of women with other biologic subtypes of breast cancer. To address the potential for contamination we did run negative controls as detailed in the methods. The significant differences that we identified in the microbial communities among breast tissue, skin tissue, skin swab and buccal swab samples suggest that contamination in collection, storage or processing does not account for the breast microbiome.

Conclusions
Here we confirm the presence of a distinct breast tissue microbiome using culture-independent methods to analyze samples obtained and processed under aseptic conditions. Further, we show for the first time that this breast tissue microbiome is distinct from the overlying breast skin tissue, as well as from skin and buccal swab samples. In addition, we identified significant differences in the microbial composition of the breast tissue microenvironment in patients with benign versus malignant disease. While it is unclear whether small shifts in microbial communities or the presence of a virulent pathogenic strain or absence of a beneficial one might be responsible for promoting carcinogenesis, these findings are hypothesis-generating and support further investigation to identify a microbial risk signature for breast cancer and potential microbial-based prevention therapies.

Top
OfflineProfileReply with quote
 
Display posts from previous:  Sort by  
Post a new topic Post a reply  [ 1 post ] 
 

Who is online

Users browsing this forum: No registered users and 5 guests

You cannot post new topics in this forum
You cannot reply to topics in this forum
You cannot edit your posts in this forum
You cannot delete your posts in this forum
You cannot post attachments in this forum
 
Search for:
Jump to:  
cron