Oral Bacterial Microbiota in Digestive Cancer Patients: A Systematic Review

The relation between the gut microbiota and human health is increasingly recognized. Recently, some evidence suggested that dysbiosis of the oral microbiota may be involved in the development of digestive cancers. A systematic review was conducted according to the PRISMA guidelines to investigate the association between the oral microbiota and digestive cancers. Several databases including Medline, Scopus, and Embase were searched by three independent reviewers, without date restriction. Over a total of 1654 records initially identified, 28 studies (2 prospective cohort studies and 26 case-controls) were selected. They investigated oral microbiota composition in patients with esophageal squamous cell carcinoma (n = 5), gastric cancer (n = 5), colorectal cancer (n = 9), liver carcinoma (n = 2), and pancreatic cancer (n = 7). In most of the studies, oral microbiota composition was found to be different between digestive cancer patients and controls. Particularly, oral microbiota dysbiosis and specific bacteria, such as Fusobacterium nucleatum and Porphyromonas gingivalis, appeared to be associated with colorectal cancers. Current evidence suggests that differences exist in oral microbiota composition between patients with and without digestive cancers. Further studies are required to investigate and validate oral–gut microbial transmission patterns and their role in digestive cancer carcinogenesis.


Introduction
Digestive cancers include cancers located in the esophagus, stomach, liver, pancreas, colon, and rectum. Their incidence and related mortality are increasing worldwide, but with some characteristic geographical differences [1]. According to the GLOBOCAN, i.e., Cancer Incidence in Five Continents, and the World Health Organization (WHO) mortality databases in 2018, the majority of new cases of digestive cancers (63%) and related deaths (65%) occurred in Asia, followed by Europe and North America. Moreover, esophageal, gastric, and liver cancers appear to be more prevalent in Asia, whereas colorectal and pancreatic cancers are more common in Europe and North America [2]. Most of these cancers are considered sporadic and are influenced by several potentially modifiable environmental factors, such as tobacco smoking, diet, alcohol consumption, physical inactivity, obesity, and immunosuppressive drugs [1]. Some recent evidence suggested a role of the human microbiota in the development of digestive cancers, not only related to the composition and changes in relative abundance of microbes of the gut microbiota [3][4][5] but also linked to a state of dysbiosis of the oral microbiota [6,7]. In this review, we define "dysbiosis" as any change to the composition of resident bacterial communities relative to the community found in healthy individuals [8].
The oral cavity is a reservoir of more 700 species or phylotypes of bacteria, of which approximately 35% have not been cultured yet [9]. The equilibrium of this complex ecosystem is essential for oral health and influences host responses to disease [10]. The disruption in the homeostasis, i.e., dysbiosis, can result in significant metabolic and immunologic effects on the host, ultimately leading to local and systemic diseases [11,12]. These mechanisms are well documented in the pathogenesis of periodontitis, a chronic multifactorial inflammatory disease of the tooth-supporting tissues including the gums, the periodontal ligament, and the alveolar bone [13], but it has also been observed for cardiovascular, neurological, and metabolic disorders as well as digestive cancers and inflammatory bowel diseases [7,[13][14][15].
In particular, specific oral bacteria that are typically found in the oral cavity of individuals suffering from periodontal diseases, such as Fusobacterium nucleatum [16] and Porphyromonas gingivalis [17], have been found in significantly high abundance in tumoral tissues and fecal samples of patients affected by colorectal cancer (CRC) [18,19], supporting the ability of these bacteria to migrate through the gastrointestinal tract where they can induce inflammation, alter the host immune response, and create an environment that may eventually favor tumor growth [20][21][22][23]. Specifically, data from a mouse model of intestinal tumorigenesis suggest that fusobacteria generate a proinflammatory microenvironment that is conducive to CRC progression through recruitment of tumor-infiltrating immune cells [24][25][26]. This was confirmed in clinical studies analyzing Fusobacterium nucleatum abundance by quantitative real-time polymerase chain reaction of DNA extracted from colorectal tissue biopsies and surgical resection specimens and observing that Fusobacterium nucleatum was more abundant in stool samples from CRC patients compared with adenomas or controls [27][28][29]. Some authors concluded that Fusobacterium nucleatum could be a novel risk factor for disease progression from adenoma to cancer, possibly affecting patient survival outcomes [27].
In this perspective, recent evidence suggested that certain oral bacteria and in general the characterization of the oral microbiota composition may be used as biomarkers for the detection of certain digestive cancers, with a potential role as a non-invasive screening tool [18,30,31].
The present systemic review aims to provide an updated appraisal of the current literature investigating the association between the oral microbiota and different types of digestive cancers, by (i) describing the observed differences in oral bacterial composition and abundance in cancer patients vs. controls and (ii) suggesting the possible impact of oral microbiota on digestive cancer risk.

Protocol Development and Literature Search
The present systematic review was performed according to the Cochrane collaborationspecific protocol [32] and was reported following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist (Table S1) [33].
Studies that investigated the association between oral microbiota and digestive cancers were searched in the following databases without date restrictions: Medline (through PubMed), Scopus, Embase, Cochrane Library and Google Scholar.
A specific research equation was used for each database, using the following keywords and MeSH terms: oral microbiome, oral microbiota, mouth microbiome, gastrointestinal neoplasms, gastrointestinal cancers, gastrointestinal carcinoma, digestive cancer, digestive neoplasms.
According to the PICOS schema, the following criteria were used to construct the literature search and to select the pertinent articles: Population: adult patients with a diagnosis of digestive cancer (all types of cancers located in the digestive apparatus). Intervention: analysis of oral microbiota composition and/or oral microbiome with or without a concomitant oral/periodontal examination. No restriction was applied for the type of microbiological technique used to sample and analyze the oral microbiota/microbiome, which include culture-dependent and genome sequencing methods. Comparison(s): adult patients without cancer. Outcome(s): oral microbiota composition (quality and quantity of bacterial species and pathogens related to oral diseases, particularly periodontitis). Study designs: All types of descriptive (case series, cross-sectional) and analytic studies (cohort, case-control, clinical trial) estimating the magnitude of the association between oral microbiota dysbiosis and digestive cancers.
Results were limited to human clinical studies with review articles, with case reports being excluded. The literature review was completed by an extensive search using the "related articles" function in PubMed. The reference lists of the eligible records and of review articles were double-checked to identify potential additional pertinent articles. Articles were selected and reviewed if written in English only.
The literature search and selection were performed independently and blindly by three reviewers (E.R., G.B. and M.C.C.). Records were removed from the selection if all reviewers excluded the articles at the title/abstract screening levels. Disagreements were resolved with discussion with a third reviewer (N.d.A.).

Data Extraction
The reviewers performed an independent full-text analysis and data extraction by filling an electronic database. Extracted data included first author name, country, journal, year of publication, number of patients, age of the patients, type of cancer, oral diagnosis, type of microbiota analysis, sample extraction, detection method, and main results.

Study Quality Assessment and Risk of Bias
The reviewers also carried out a study quality assessment and risk of bias evaluation of the selected articles. According to the type of study design, the Newcastle-Ottawa scale [34] (NOS) was used.

Literature Search and Selection
The merged search yielded 1805 results. After removing duplicates, 1654 articles were screened for eligibility based on title and abstract. Twenty-eight articles fulfilled the inclusion criteria and were selected for the present systematic review, as shown in Figure 1.
All studies described microbiota composition differences between cases (cancer patients) and controls, and several studies [6,[39][40][41]47,49,50,56,57] also analyzed the oral microbiota as being possible biomarkers for cancer screening and early diagnosis. The overall total number of digestive cancer patients considered was 2708, who were compared with 2593 controls. Twenty-six studies used DNA analysis, with 16S rRNA V4 sequencing and PCR as the principal method for microbiota investigation, whereas indirect immunofluorescence was used in one study [36] and shotgun sequence in another one [38]. Study outcomes are displayed in Table 2.

Oral Microbiota and Esophageal Cancer (EC)
Five articles investigated the association between oral microbiota and EC [6,[53][54][55][56]. Chen et al. [53] demonstrated that patients with esophageal squamous cell carcinoma (ESCC) have a decreased oral microbiota diversity when compared with non-ESCC controls. The authors described a decreased salivary carriage of genera Lautropia, Bulleidia, Catonella, Corynebacterium, Moryella, Peptococcus, and Cardiobacterium and higher relative abundance of Prevotella, Streptococcus, and Porphyromonas in the ESCC group [53]. Wang et al. [54] showed that Actinomyces and Antopobium were related to a higher risk of ESCC, whereas the healthy group of the study was closely related to Fusobacterium and Porphyromonas. Zhao et al. [55] showed that ESCC is associated with an increased salivary carriage of Firmicutes, Negativicutes, Selenomonadales, Prevotellaceae, Prevotella, and Veillonellaceae and with a decreased taxa of Proteobacteria, Betaproteobacteria, Neisseriales, Neisseriaceae, and Neisseria.
Kawasaky et al. and Peters et al. concluded that differences in microbiota compositions between cases and controls could be used as a possible biomarker for cancer screening [6,56]. Only one study investigated the microbiota in relation to an esophageal adenocarcinoma, showing an association with an increased oral rinse carriage of Tannarella forsythia and a depletion of the commensal genus Neisseria and Streptococcus Pneumoniae [6].

Oral Microbiota and Liver Cancer (LC)
Only two articles investigated the association between oral microbiota dysbiosis, and LC. Lu et al. [57] identified Oribacterium and Fusobacterium as possible biomarkers to identify LC patients, showing a significant difference in their relative abundance between healthy controls and LC patients [57]. Li et al. compared the oral microbiota of patients with LC with healthy controls and patients with cirrhosis in different stages, finding an association between cancer and Haemophilus, Porphyromonas, and Filifactor.

Oral Microbiota and Pancreatic Cancer (PC)
In the seven studies dealing with PC, the microbiota analysis was conducted on salivary samples in five studies [30,[42][43][44]47], on tongue coating in one study [46], and on oral rinse [45] in the remaining study. Results were heterogeneous. Lu et al. as well as Fan et al. observed an increased risk of cancer linked to Porphyromonas gingivalis, but contrasting findings were reported in relation to Fusobacterium, which was respectively linked to an increased and decreased risk of cancer [45,46]. The remaining studies found different bacteria linked to PC risk, without highlighting the predominance of a specific microbiota.

Oral Microbiota and Gastric Cancer (GC)
The microbiota analysis was conducted on tongue coating in four studies [39,48,51,52] and on salivary sample in two studies [49,50]. Hu et al. [48], Xu et al. [52], and Han et al. [39] reported that tongue coating characteristics significantly differ between cases and controls, with thicker tongue coating associated with an increased risk of GC. Xu et al. [52] divided the sample of GC patients into five subgroups based on the tongue coating characteristics, reporting different microbial associations in the different groups. However, also for this type of cancer there was no evidence of a specific microbiota being related to the development of GC.

Oral Microbiota and Colorectal Cancer (CRC)
Microbial samples were collected by oral swab in three studies [3,16,40], oral rinse in one study [45], saliva in three studies [36,38,41], subgingival plaque in one study [35], and tongue coating in one study [39]. Overall, these studies showed the most consistent results. Fusobacterium nucleatum was found to be linked to an increased risk of CRC in five studies [35,36,[39][40][41]. Four studies agreed on the association of different species of Prevotella and CRC risk [3,35,37,40], while a borderline association was found in one study [39].

Selection
Comparability Outcome Total Score

Discussion
The present systematic review provides a synthesis of studies investigating the association between oral microbiota composition and digestive cancers with a systematic approach. The available evidence suggests that digestive cancer patients present an oral microbiota composition that differs from non-cancer controls and specific oral bacteria may be linked to increased odds for digestive cancers. The predominance and/or relative abundance of these bacteria could have a synergistic effect on digestive cancers' etiology [3,6,36,37,40,44,45,49,51,55], suggesting a possible role of oral microbiota characterization for screening and risk assessment of some types of cancer [40,47,49,56,59]. However, it is important to highlight that the current body of evidence is still limited, and some digestive cancers are under-investigated. For instance, only two studies were found concerning LC [57,58]. More studies are available concerning the role of oral microbiota in PC, GC, EC, and CRC patients, but further research is needed before advancing any solid conclusion. Notwithstanding this, the overall body of evidence consistently suggests that the oral microbiota is linked to the risk of digestive cancers.
In the digestive tract, the microbiota synthetizes essential amino acids and vitamins taking part in the digestion process. A symbiotic microbiota is normally characterized by a high diversity, and it is able to resist changes that occur during physiological stress [60], and to stimulate an immune response [20]. In recent years, different studies focused on the possible role of intestinal and oral microbiota dysbiosis in disease development, especially in carcinogenesis [7,20,23,53]. The digestive tract microbiota is involved in inflammation, metabolism, and genotoxicity mechanisms taking part in the oncogenic process [60]. Meanwhile, a moderate inflammatory reaction is protective against cancer development, and excessive inflammatory response is correlated with a higher risk of carcinogenesis [59]. Different oral bacteria associated with carcinogenesis, such as Porphyromonas, Prevotella, and Fusobacterium, can contribute to a chronic inflammatory reaction by stimulating the production of inflammatory mediators such as interleukin-1β, interleukin-6, and matrix metalloproteinases [61,62]. Prevotella and Fusobacterium have also been linked to carcinogenesis by the mechanisms of suppressing host immunological functions and promoting the malignant transformation of epithelial cells [28,29,59].
The potential translocation ability of the oral microbiota through the circulation system (hematogenous route) or following the flow of food and fluids into the digestive system (enteral route) could explain its presence in the gut and its role in carcinogenesis [59]. Three possible mechanisms of bacterial translocation in animal models were suggested: (1) disruption of the digestive equilibrium with intestinal bacterial overgrowth, (2) increased permeability of the intestinal mucosal barrier, and (3) deficiencies in host immune defenses. However, the transmission pathway remains questionable, and no definitive reports are currently available on the topic [63]. Zhang et al. [40] conducted an additional functional analysis using PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) and showed that the membrane transport pathway was decreased in CRA and CRC patients compared with healthy controls, with a potential impact on the anti-tumor immune response (such as the response mediated by bacterial outer membrane vesicles). Moreover, the cell motility pathway was found to be overrepresented in cancer patients, promoting tumor invasion and migration [40].
Finding the exact transmission pathways between the oral and the gut microbiota might provide evidence to support new methods for non-invasive cancer screening and could lead to control specific bacteria in their source, i.e., the oral cavity ( Figure 2). Schmidt el al. [38] suggested an oral bacteria translocation in patients with CRC cancers. Flemer et al. [3] reported that oral swabs are associated with a sensitivity of 76% and a specificity of 90% in predicting colon adenomas; and a sensitivity of 98% and a specificity of 70% in predicting CRC. Sun et al. [50], found that salivary samples have a sensitivity of 97% and a specificity of 92% in predicting a GC, whereas Lu et al. [46] showed a sensitivity of 77% and a specificity of 78% for tongue coating to predict PC. Overall, the available studies suggest that a single oral bacterium has a limited ability to detect digestive cancer; conversely, multi-bacteria models have better performance and screening accuracy to differentiate patients with PC, EA, ESCC, GC, and CRC and to support the evaluation of the oral microbiota as a potential tool for prediction and prevention of digestive cancers [31]. Some evidence also supports a significant association between specific oral-gut bacteria and tumor stage, cancer-specific survival, and response to treatment [64][65][66][67][68], which, if confirmed and validated, would represent a novel and relevant strategy for reducing digestive cancer risk and improving cancer patients' outcomes. Indeed, once the putative mechanisms of bacterial carcinogenesis can be elucidated and the role of oral microbiota precisely defined, its detection and characterization could be potentially used as a cancer biomarker and in the treatment of oral dysbiosis (e.g., periodontitis) as a preventive measure. Due to their clinical relevance, these findings need to be validated in future studies with a large population of patients and reproducible protocols for oral microbiota sampling and analysis. The available literature is characterized by a high degree of heterogeneity in the methodology used. Moreover, the role of potential confounders, such as alcohol intake and smoking, which are known to drastically influence oral health and oral microbiota homeostasis [69,70], has been rarely considered. Alcohol abuse and smoking are also related to different cancers [70], but only a few of the studies selected in the present systematic review considered these factors in their analyses of the association between oral microbiota composition and digestive cancers [6,10,40,42,44,45,[54][55][56]. Moreover, other confounders, such as oral hygiene habits [71], periodontal health status and severity of periodontitis, presence of other oral diseases, and dietary patterns [72], were disregarded. Tooth loss has been linked to an increased risk of GC development [73] and to an overall increased mortality rate [40]. Poor oral hygiene and tooth loss have also been linked to an increased risk of CRA occurrence [74,75]. Overall, these variables, which may influence oral dysbiosis and cancer development, need to be systematically considered in future research.
Another relevant issue to consider is the sampling method used in the different studies (e.g., saliva, tongue coating, oral rinse, subgingival plaque). The oral cavity consists of different sites (i.e., teeth, tongue, cheeks, hard and soft palates) that create specific niches for microbial colonization characterized by different oxygen levels, nutrient availability, and mechanical stress conditions. Consequently, distinct microbial communities can be found in the oral cavity according to the explored sites and the presence of diseases (e.g., periodontitis) [76]. The type of sample used could therefore affect the results while assessing the link between oral microbiota and cancer. Saliva represents the preferred sampling site to obtain oral microbiota DNA to process since it tends to reflect the microbiota from all oral sites and the associated disease [77]. Ryutaro et al. [78] showed no differences in microbiota diversity in samples of unstimulated saliva, stimulated saliva (through chewing a paraffin gum), and mouth rise water collected in specimen tubes. The authors concluded that mouth rise is a reliable sample for microbiota analysis and that it could be particularly useful in specific subsets of patients, e.g., in elderly patients or in patients with low salivary flow, often providing results comparable with pure saliva samples. Conversely, the study of Gomar-Vecher et al. [79] comparing unstimulated saliva collected on paper points and stimulated saliva collected after chewing paraffin gum showed significant differences in microbiota composition between the two sampling methods. Again, Ryutaro, J. et al. [78] showed a different microbiota composition in tongue coating samples collected by scraping the dorsum of the tongue with a specific specimen compared with saliva and mouth rise water samples. Xu, J. et al. [52] focused on the landmark flora of the four common tongue coatings in GC patients. The samples were collected from the middle section of tongue dorsum using a toothbrush and put into the test tube with saline. By describing the oral microbiota composition, the authors concluded that these data could be useful for future standardization of GC diagnosis based on tongue microbiota. Tongue coating is a common sampling method in Chinese medicine. However, its biological and molecular bases are not completely validated, and thus it is not a widespread sampling method in the Western world [80]. Han, S. et al. [39] showed a relationship between the risk of PC and the tongue coating microbiota collected by scraping the front and middle section of the tongue with a sterile spoon and put into a test tube with saline (repeated three times and centrifuged (2000 r/min) for 5 min). Finally, subgingival plaque can also be used. Theoretically, it reflects the local microbiome composition much more specifically than a salivary sample and should be preferred when simultaneously evaluating periodontal diseases. Nevertheless, this sample collection method is more invasive and requires qualified trained staff to harvest subgingival plaque [56,81]. Moreover, in the presence of periodontitis, great heterogeneity can be found in the microbiota composition of the subgingival plaque sampled in deep vs. shallow periodontal pockets or following periodontal treatments [82]. Kawasaki et al. [56] examined six common periodontal pathogens in the subgingival microbiota of patients with EC and compared them with the salivary microbiota using real-time PCR analysis. A stronger association was found for the microbiota sampled in subgingival plaque. Thus, methodological issues should be considered in the interpretation and generalization of the results [83].
The present systematic review has some limitations related to the type and quality of the pertinent literature considered. Overall, the body of evidence is of low-to-moderate quality, but the results support the need of further studies investigating the role of oral microbiota composition in digestive cancer patients. The qualitative synthesis of the literature highlights the need for standardization in study design and methods to obtain comparable results contributing to the emerging evidence and elucidating the current knowledge. Indeed, the literature is characterized by high heterogeneity related not only to methodological aspects (e.g., sample selection, microbiome preservation, collection methods, taxonomical assignments, DNA extraction methods, and statistical analyses) but also to population characteristics, lifestyle habits, and culture, which may influence oral microbiota composition. Indeed, geography, ethnicity, subsistence-specific variations in human microbiome composition [84], and the genetic risk of cancers [85] could influence the results. Moreover, while most of the studies included healthy controls from comparable populations of those of cancer patients, only two studies reported to have performed endoscopies in controls to rule out the presence of cancer or precancerous lesions. Globally, there is no evidence of external validity so far.

Conclusions
Despite the limited evidence available, the literature is consistent in suggesting a significant association between oral microbiota composition and digestive cancers. To date, it is not possible to identify specific bacteria species involved in digestive cancer development, but these findings support further research to characterize the oral microbiota of these patients. Indeed, future studies should analyze the possible microbiota dissemination patterns from the oral cavity and the rest of the digestive tract and the possible role of oral microbiota characterization as a screening tool for digestive cancers.
contributed to data interpretation, article drafting, and manuscript critical revision. All authors read and approved the final version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.