Body Microbiota and Its Relationship With Benign and Malignant Breast Tumors: A Systematic Review

Breast cancer is the most frequent type of cancer as well as one of the main causes of cancer-related mortality in women. Human microbial dysbiosis, which has been related to a range of malignancies, is one of the variables that may impact the chance of developing breast disorders. In this review, we aimed to investigate the relationship between breast cancer and benign breast tumors with dysbiosis of the microbiome at different body sites. We performed a systematic review of MEDLINE, Scopus, Ovid, and Cochrane Library to identify original articles published until July 2020 that reported studies of breast disease and microbiota. Twenty-four original articles were included in the study, which looked at the features and changes in breast, gut, urine, lymph node, and sputum microbial diversity in patients with benign and malignant breast tumors. In breast cancer, the breast tissue microbiome demonstrated changes in terms of bacterial load and diversity; in benign breast tumors, the microbiome was more similar to a malignant tumor than to normal breast tissue. Triple-negative (TNBC) and triple-positive (TPBC) types of breast cancer have a distinct microbial pattern. Moreover, in breast cancer, gut microbiota displayed changes in the compositional abundance of some bacterial families and microbial metabolites synthesis. Our review concludes that breast carcinogenesis seems to be associated with microbial dysbiosis. This information can be further explored in larger-scale studies to guide new prophylactic, diagnostic, and therapeutic measures for breast cancer.


Introduction And Background
Breast cancer is the most frequent type of cancer in women and is one of the main causes of cancer-related mortality in women [1,2]. Older age, prolonged exposure to female hormones, BRCA1 and BRCA2 genes mutations, and the presence of a personal or family history of breast and other cancers are all well-known risk factors for developing breast illnesses [3,4].
One of the factors that might influence the risk of the development of breast diseases is human microbial dysbiosis [5,6]. The microbiome, as defined by Lederberg and McCray, is the ecological community of commensal, symbiotic, and pathogenic microorganisms that share our body spaces [7]. In 1960, it was difficult to understand the role of microbiota. Our understanding of microbiota has improved as genomeanalyzing tools for complex microorganisms have advanced, but we still do not know much about its clinical significance [8].
Microbial dysbiosis results when maladaptation or abnormal composition occurs within the microbial community of a given organ or tissue [9]. The literature has reported a link between microbial dysbiosis and the development of a variety of cancers [10][11][12]. The microbiota have also been shown to help increase drug efficacy, decrease drug toxicity, and prevent cancer [13]. Other studies have concluded that microbiota could be used in the diagnosis, prediction of risk and course, and prevention of disease [14].
The association between different types of microbiota and gastrointestinal pathological conditions has been well studied. Some investigators propose an association between colorectal cancer and certain microbiota detected by fecal and oral swabs [15]. In a new field of research, recent studies have suggested an association between inflammatory bowel diseases and microbiota [16,17]. Research on different breast pathological conditions and their links to microbiota that inhabit breast tissue and other organs is limited. In this comprehensive review, we, therefore, aimed to study the characteristics and changes (dysbiosis) in breast,

Study selection
The primary screening of the studies was done by two authors based on the title and abstract by using the search terms described above independently, and duplicate studies were removed. A full-text review was undertaken independently. When the inclusion and exclusion criteria were not met during the initial review, the articles were discarded. When uncertainty existed, a third senior author resolved the disagreement. We included all studies that matched all of the following criteria.

Study Design
We included retrospective cohort studies and secondary analyses in which the main purpose was to evaluate the microbial diversity and characteristics of breast or gastrointestinal tissue or any other body site in patients with breast cancer or benign breast tumors.

Study Subjects
Studies included adult females, 18 years and over, with breast malignancy or benign breast tumors.

Outcomes
The outcomes were the characteristics and changes (dysbiosis) in breast, gut, and other body microbiomes in relation to benign and malignant breast tumors.
Studies considered by the authors to be unrelated to the subject, non-human studies, and non-English studies were excluded.

Breast microbiota
A total of 5616 articles were screened after the initial search, cross-referencing, and removal of duplicate studies. Inclusion and exclusion criteria were applied by reading the title and abstract. Following this step, 58 studies were included in the full-text reading. After a full-text review, 34 more studies were excluded, and a final 24 studies were selected and included in this review ( Figure 1). All included studies and their main characteristics and findings are summarized in Table 1.

Breast Tissue Microbiota
The existence of microbes in breast tissue has recently been explored. Different types of microbiota have been found that are distinct and clearly distinguishable from those found in the overlying skin tissue [18,19]. Proteobacteria were the most prevalent bacterial phyla identified in the included articles among breast tissue samples taken from benign, malignant, and healthy breasts, followed by Actinobacteria and Firmicutes [5,18,19,32].

Benign vs. Malignant vs. Healthy
The microbiota appears to have a role in both benign and malignant disorders, as the profiles of benign tumors were more similar to those of normal neighboring tissue from women with malignant tumors than to those of tissue from healthy patients [6]. Nevertheless, studies have shown some differences in microbial profiles between the two-disease status [5,19].
Normal adjacent tissues from patients with breast cancer exhibited a considerably greater number of certain bacteria, including Bacillus, Staphylococcus, and members of the Enterobacteriaceae family, as compared to patients with benign breast tumors and healthy women [6]. Moreover, proteobacteria relative abundance was found to be significantly higher in malignant disease than in benign disease [5].
Furthermore, the fluid aspirated from the nipple in patients with breast cancer (ductal carcinoma) and from healthy controls differed significantly in beta diversity [23], suggesting a possible role of bacterial dysbiosis in cancer formation.

Malignant vs. Normal Adjacent
Several studies showed that tissue from malignant tumors and normal adjacent tissue of the same breast shared similar bacterial diversity [6,26,32]. However, other studies found differences in microbial profiles in terms of the relative abundance of some bacteria. At the genus level, Xuan et al. found the bacterium Sphingomonas yanoikuyae was most prevalent in adjacent normal tissue, and the bacterium Methylobacterium radiotolerans was most prevalent in tumor tissue [12,18]. In contrast, Wang et al. [26] found that the genus Methylobacterium was considerably lower in cancer patients compared to non-cancer individuals. This difference between studies might be due to different extraction techniques or types of sample preparation.
When compared to non-cancerous surrounding samples, Mycobacterium phlei and Mycobacterium fortuitum were shown to be the most abundant species in tumor tissues [5].
On quantitative analysis, Xuan et al. found that the tissue of estrogen receptor-positive breast cancer has 10-fold more bacteria when compared to paired normal tissue from the same patients [18]. Nevertheless, despite this increase in microbial load in breast cancer tissue, antibacterial responses within cancer tissues were found to be significantly downregulated compared to normal breast tissue [12]. These data suggest a possibility that microbiota has the ability to influence the local immune microenvironment of the breast.

Role in carcinogenesis
The bacterial load in breast tissue seems to be inversely correlated with the stage of breast cancer, as the highest copy numbers of bacterial DNA were found in tumor tissues from patients with early-stage cancer, and it decreased with the more advanced stage. However, this association was not found with adjacent normal tissue from the same patients as no difference in bacterial load was found across stages [18]. These observations have significance in guiding new diagnostic implications for breast cancer.
The histological grade of breast cancer has also shown some effects on the tumor microbial profile [31]. Meng et al. discovered that when tumor grade increased, the compositional abundance of the Bacteriodaceae family was reduced. Furthermore, as malignancy grows, so does the prevalence of the genus Agrococcus [31].
Luminar type of breast cancer may have an influence on the microbial community of tumor tissue. Banerjee et al. discovered unique patterns of bacterial, viral, fungal, and parasitic profiles in triple-negative (TNBC) and triple-positive (TPBC) breast cancer samples. The microbial profiles of human epidermal growth factor receptor 2 (HER2/neu)-positive and estrogen receptor (ER)-positive samples, on the other hand, were similar [21,29]. When hormone receptor-positive breast cancer samples were compared to hormone receptornegative samples, Wang et al. discovered a substantial drop in Methylobacterium [26]. Furthermore, samples with histopathologic evidence of lymphovascular invasion contained fewer methylobacterium than those without lymphovascular invasion.
It has not yet been fully elucidated whether there is a specific microbial species that plays a role in breast cancer development. However, Escherichia coli isolated from adjacent normal tissue of patients with breast cancer has been shown to generate DNA double-stranded breaks, the most harmful sort of DNA damage [6].
16S rRNA sequencing data of breast tumors from untreated patients and from those treated with neoadjuvant chemotherapy indicates that chemotherapy increases the tumor proportional abundance of Pseudomonas by 85%, suggesting that chemotherapy induces preferential growth or survival of some types of bacteria [38].

Gut microbiota
The gut microbiota also seems to undergo changes in the presence of breast cancer. The number of these species in postmenopausal patients with breast cancer is higher than that in healthy controls, with a higher abundance of specific species, including E. coli, Prevotella amnii, and Enterococcus gallinarum [30].
The prevalence of Blautia species in the gut microbiota increased substantially with breast cancer grade. The number of Clostridium coccoides, Clostridium leptum, and Bacteroidetes clusters was significantly higher in clinical stage II/III breast cancer than in clinical stage 0/I breast cancer [25].
In early-stage breast cancer, bacterial cadaverine biosynthesis is decreased in the gut, resulting in less production of anti-cancer bacterial metabolites [33]. Lithocholic acid (LCA), another bacterial metabolite that can slow the growth of breast cancer, was also shown to be decreased in the guts of individuals with early-stage breast cancer [28]. The level of expression of tumor-infiltrating lymphocytes (TIL), an indicator of tumor immunity in solid cancers with prognostic value, was associated with gut microbial diversity in patients with breast cancer [34].
Obesity was linked to a considerably decreased total number of Eggerthella lenta bacteria, Blautia, Faecalibacterium prausnitzii, and Firmicutes species in early-stage breast cancer as a probable cofounder [25]. Furthermore, the proportions of Akkermansia muciniphila (AM) in the gut microbiota were lower in individuals with breast cancer who had increased body fat [39].

Microbiota of other body sites
The difference in diversity of the urinary microbiome between patients with breast cancer and healthy women was largely related to menopausal status, with peri/postmenopausal samples being significantly more diverse than premenopausal samples [26].
Oral rinse microbiomes have also been investigated. There was no significant difference between breast cancer patients and healthy women [26].
The presence of Methylobacterium radiotolerans, a species that was more abundant in tumor tissue than in normal paired tissue [18], was investigated in pathologically negative sentinel lymph nodes and found to be significantly associated with a higher stage of breast cancer [22].
No significant difference was observed in alpha and beta diversity in the microbiome of sputum samples in a comparison of samples from patients with nontuberculous mycobacterial lung disease with and without breast cancer [35].

Discussion
In benign and malignant breast cancer, the role of microbiota is poorly understood in the literature. Several hypotheses have been proposed, including its ability to induce chronic inflammation and regulate immunity, induce DNA damage, and modulate estrogen metabolism [40,41]. This review summarized the currently available data on the role that human microbiota plays in benign and malignant breast tumors.
A number of characteristics of breast microbiota have been discovered in the last few years. A wide diversity of microorganisms is specific to breast tissue and distinct from the microbiota of other body sites [29,24]. The most numerous microbial phyla detected in breast tissue, according to the evaluation of these microbiomes, are Firmicutes and Proteobacteria [5,18,19,32]. However, in one study, bacteria from the phylum Bacteroidetes were the most prevalent, with very few Proteobacteria [24]. Possible explanations for the presence of a microbiome within breast tissue include the passage of skin microbiome through the nipple, during lactation or sexual contact through nipple-oral contact, and finally, translocation of the gut bacteria [36], with the data suggesting more support for the latter hypothesis [42][43][44].
Microbial dysbiosis, which occurs when the abundance of some species within the microbial community changes relative to others, can result in the microbial community's typical function being lost [9]. Some authors reported dysbiosis of the breast tissue microbiome in patients with breast cancer [5,6,18,21,23,29,31,32,36]. Moreover, the bacterial load also differed according to the clinical stage of breast cancer [18]. These findings demonstrate an association between dysbiosis and breast cancer. However, it is unclear whether microbial dysbiosis is merely secondary to cancer development or a causative agent in breast carcinogenesis.
We have described the potential role of some types of bacteria in carcinogenesis, including E. coli and Staphylococcus species. Both were shown to be more common in breast tissue from breast cancer patients and had the ability to produce DNA double-stranded breaks, a condition known to possibly cause cancer [6,45]. Benign breast tumors, unexpectedly, have a microbial profile similar to that of malignant tumors [6]. Urbaniak et al. [6] hypothesized that a lower level of DNA-damaging bacteria in benign tumors could be a possible factor in preventing malignant transformation.
The gut microbiota and its relationship with estrogens have been well investigated in the literature. βglucuronidase secreted by gut bacteria allows gut microbiota to bind to estrogen receptors (ER) [10]. ER activation promotes cell proliferation, which is a well-defined process in breast cancer [46].
The studies in our review showed that the gut microbiomes display changes among breast cancer patients. Postmenopausal women with breast cancer had an altered gut microbial composition in both alpha and beta diversity metrics [20,30]. Moreover, although not statistically significant, this change was associated with a higher level of urinary estrogens in patients with breast cancer [35]. Furthermore, a significant correlation between estrogen-independency and immunoglobulin A IgA+/IgA− gut microbiota was found in postmenopausal patients with breast cancer [27]. These data suggest a relationship between gut microbiota, estrogen levels, and breast cancer.
Gut bacterial metabolites play a major role in microbiome-to-host signaling [47][48][49][50]. According to one study, the levels of LCA, one of the gut microbial metabolites, are lower in patients with breast cancer, especially in the early stages 0/I [28]. According to the same study, LCA performs an antineoplastic function in breast cancer cells. It specifically inhibits vascular endothelial growth factor production, epithelial-tomesenchymal transition, and metastasis development. Furthermore, in animal models, it stimulates antitumor immunity and alterations in metabolism [28]. These findings are concordant with previous reports in which the same metabolite was found to be inversely correlated with Ki-67, a proliferation index in breast cancer [51].
Cadaverine is another microbial metabolite generated by the LdcC and CadA enzymes during the decarboxylation of lysine [52,53]. It exerts its effects through the trace amine-associated receptors (TAARs) TAAR1, TAAR8, and TAAR9. Previously, TAAR1 was also found to be related to inhibition of breast cancer growth and with a favorable effect on the overall survival of patients with primary breast cancer [54]. In our review, we described the results of a study by Kovács et al. [33], who showed that DNA coding for bacterial enzymes responsible for cadaverine production is decreased in the fecal microbiome of patients with early breast cancer. Moreover, prolonged survival among patients with early-stage breast cancer was associated with higher expression of lysine decarboxylase [33]. These findings suggest that gut microbial dysbiosis in patients with breast cancer leads to a a decreased production of antineoplastic bacterial metabolites.
In breast cancer, immune cell infiltration, specifically cytotoxic CD8+ T cells, has predicted an improved prognosis [55][56][57]. Additionally, tumor-infiltrating lymphocytes (TILs) found in breast cancer prior to chemotherapy can predict pathological complete response and improve prognosis [58]. Shi et al. [34] investigated the TILs expression in breast cancer in relation to the gut microbiome and found that microbial diversity is different among different expression levels of TILs. Higher TILs expression was associated with a higher diversity of gut microbes. Moreover, the compositional abundance of some microbiota species was different according to TILs expression levels. These findings suggest a potential role for gut microbiota in the prognosis of patients with breast cancer.
To the best of our knowledge, this is the first systematic review to examine the relationship between human microbiota and benign and malignant breast tumors. The limitations of this review are the inclusion of small retrospective studies and the heterogenicity of the results due to the use of different sampling techniques; the description of different body site microbiomes, and the study of different pathological characteristics.
Further large-scale studies, including clinical trials, are needed to confirm this association and to discover the possible clinical applications of microbiota in the prevention and diagnosis of breast cancer, as well as therapeutic interventions. Furthermore, studies are required that focus more on specific members of the microbiome, such as E. coli and Staphylococcus, which could contribute to a better understanding of the pathophysiological characteristics of breast cancer.

Conclusions
Current data have linked microbial dysbiosis with breast cancer. Across different histological types, stages, and grades of breast cancer, both the breast and the gut microbiome display changes. However, the exact mechanism underlying these clinical observations is poorly understood. Moreover, yet to be fully identified is whether this microbial alteration causes cancer or is one of the consequences of carcinogenesis, and whether there are specific microbial agents that contribute to the pathophysiological characteristics of this disease.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.