Salivary Microbiota Composition in Patients with Oral Squamous Cell Carcinoma: A Systematic Review

Simple Summary This review aimed to analyse the current knowledge regarding the composition of salivary microbiota of patients with oral squamous cell carcinoma (OSCC). The protocol for this study was designed following the PRISMA guidelines. Observational studies, in human subjects with histological diagnosis of OSCC, concerning the analysis of salivary microbiota, were selected. Eleven papers were included. The salivary microbiomes of 1335 patients were analysed. Periodontal pathogens were the most frequent bacteria detected in patients with OSCC. We have found that although there are evident alterations in the composition of the salivary microbiota in OSCC patients, due to the great heterogeneity of the studies, it is still a challenge to identify a specific microbiota pattern. If the associations between alterations in the salivary microbiome and OSCC are confirmed, microbiome analysis could represent a useful tool for the screening and follow-up of patients affected by OSCC. Abstract Background: Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide. Despite recent advances in diagnosis and treatment, in recent years, an increase in the incidence of OSCC has been registered, and the mortality rate is still high. This systematic review aims to identify a potential association between the composition of salivary microbiota and OSCC. Materials and Methods: The protocol for this study was designed following the PRISMA guidelines. Records were identified using different search engines (e.g., Medline/PubMed). Observational studies, in human subjects with histological diagnosis of OSCC, concerning the analysis of salivary microbiota, were selected. Results: Eleven papers were included. The salivary microbiomes of 1335 patients were analysed (n.687 OSCC and n.648 controls). Due to the great heterogeneity of the studies, it was not possible to profile a specific microbiota associated with OSCC. However, periodontal pathogens were the most common bacteria detected in patients with OSCC (i.e., Fusobacterium, Prevotella). Conclusions: Although there are evident alterations in the salivary microbiota composition in OSCC patients, it is still a challenge to identify a specific microbiota pattern in OSCC patients. If the associations between specific salivary microorganisms and OSCC are confirmed, microbiome analysis could be a useful tool for the screening and follow-up of patients affected by OSCC.


Introduction
In recent years, an increase in the incidence of oral cancer has been reported, and in 2020, approximately 377,713 new cases were diagnosed worldwide [1]. More than 90% of oral cancer is oral squamous cell carcinoma (OSCC) [2,3]. Despite recent advances in

Data Sources and Search strategy
A selection of studies concerning the analysis of salivary microbiota of patients affected by oral cancer was performed. Records were identified using different search engines (e.g., Medline/PubMed) and by scanning references lists of articles. For the search strategy, MeSH terms and free text words were combined through Boolean operators as follows: Microbiota AND ((oral cancer) OR (Squamous Cell Carcinoma of Head and Neck) OR (oral carcinoma)). Research was completed in January 2022.

Eligibility Criteria
The inclusion criteria for the studies were as follows: Human studies; -English language; -At least 30 patients for study; -Only diagnosis of OSCC histologically confirmed with a well-defined site classification; -Only saliva analysis; -Analysis of salivary microbiota in patients affected by OSCC, before therapy.
Exclusion criteria were: studies focused on tumours different from OSCC; narrative and systematic reviews and meta-analyses; case reports and studies with less than 30 patients; in vitro studies; in silico studies; animal studies; analysis of oral microbiome in patients affected by OSCC, during or after cancer therapy; studies with H&N or OP-SCC, no defined information for subgroup of OSCC; supragingival plaque analysis and tongue swab analysis; oral scraping.

Study Selection and Data Collection Process
The initial search strategy identified 1226 records, of which based on the inclusion criteria 668 studies were excluded. The screened process of 558 studies was performed based on the title and abstract, and 510 records were excluded. Subsequently, a full-text evaluation of 92 studies was carried out. Finally, 81 records were excluded, and 11 papers were included in the current review; a detailed flow chart of the selection process is provided in Figure 1.

Statistical Analysis
Selected studies were reviewed to detect outcomes of interest. For each study, the following data were extracted using a pre-designed data extraction Excel sheet. The following parameters were collected: i.
Study characteristics: name of the first author, year of publication, name of the country where the study was performed, study design. ii. Case-control groups characteristics: case population, age of case population, control population, age of control population, risk factors. iii. Methodology: sample collection, methods of sample collection, methods of DNA extraction, DNA amplification, sequencing platforms, reference database. iv. Outcomes: raw reds detected, microbial abundance, genera detected, species detected, phyla detected, and OTUs detected.
Some results were not present in all the studies included in the review. Continuous variables were summarized with mean values and standard deviations, while categorical variables were expressed as counts and percentages.

Study Characteristics
Eleven articles were included for this review. The main characteristics of the selected studies are described in Tables 1 and 2. In detail, the characteristics of the included studies and the methodology employed for the analysis of the salivary microbiota are reported in Table 1; the exclusively statistically significant abundance of phyla, genera, and species in the study group are summarized in Table 2 (all results subdivided into phyla, genera, and species are described in Supplementary Materials).  Streptococcus pneumoniae * Statistically significant differences only in OSCC diagnosed at the late stage. ↑ phyla, genera, and species increased in the OSCC group. ↓ phyla, genera, and species reduced in the OSCC group All the articles included were observational studies published between 2017 and 2021. However, a wide heterogeneity was observed which made it impossible to perform an advanced statistical analysis. Eight studies were case-control [27][28][29][30][31][32][33][34], two were cohort studies [35,36], and one was a cross-sectional cohort study [37]. Of the eleven studies, five were from Taiwan [30,[34][35][36][37], three were from China [28,29,32], one was from the USA [27], one was from Sudan [31], and one was from Japan [33].
In total, the oral microbiomes of 1335 patients were analysed, of which 687 were affected by OSCC and 648 were controls. Among the control groups, 480 were healthy patients, 153 were affected by oral potentially malignant disorders, and 15 suffered from periodontitis.
Four studies compared salivary samples of patients affected by OSCC with healthy controls [28,31,33,34]. One study analysed salivary sample of patients affected by OSCC without a control group [35]. Three studies compared the oral microbiome of patients affected by OSCC, healthy controls, and oral potentially malignant disorders (OPMD) [27,30,36]. One study compared the oral microbiomes of patients affected by OSCC, healthy controls, and patients affected by gingivitis or periodontitis [29]. Two studies analysed the microbiome of cancer lesion and of anatomically matched normal sites of the same patients [32,37].

Sample Collection
The main information about the salivary collection and analysis technique is described in detail in Table 1. Generally, participants were instructed to refrain from eating, drinking, smoking, or oral hygiene prophylaxis for 30 min to 2 h before the saliva collection. In one study, the participants were asked not to take in any food and not brush or floss for at least 12 h before the sample collection [28]. Saliva samples were collected after mouth rinsing with 10 mL of sterile saline for 30 s. Salivary samples were collected by different techniques, such as sputum [30,31,[33][34][35], oral rinse [27], and mucosa swab [32,37].

DNA Sequencing and Analysis of Data
After DNA amplification was completed, DNA sequencing was implemented. Eleven studies carried out sequencing by using the Illumina MiSeq System ® . Just one study analysed different fungal genera using 454 GS FLX ® [31]. The processed sequencing reads were clustered into operational taxonomic units (OTU), and taxonomy classification was assigned according to the information retrieved from different databases (e.g., Greengenes database, SILVA database).

Microbial Abundance
The main data regarding microbial abundance are described in detail in Table 2. At the phylum level, only two studies defined the number of phyla detected, 11 and 12 phyla, respectively [27,32]. The most abundant phyla identified in the OSCC group were Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria compared to healthy controls [30,35,37].
In the salivary samples of patients affected by OSCC, a significant abundance of Spirochaetes, Fusobacteria, and Bacteroidetes and a decrease in Firmicutes and Actinobacteria was observed by Zhao et al. [32].
Additionally, in the OSCC patients when compared to healthy controls, a decrease in Actinobacteria and Fusobacteria was reported by Su et al. and Yang et al., respectively [35,37].
With respect to genera, four studies defined the number of genera; the average number ranged from 36 to 130, with a mean number of 91.7 ± 36 genera detected [27,[31][32][33].
A decrease in Streptococcus in the OSCC group compared to healthy controls was reported in several studies [27,37].
Zhao et al. observed a significant abundance of 17 different taxa in the OSCC samples compared to the control group (see Supplementary Materials) [32], while the taxa Megasphaera, Stomatobaculum, Granulicatella, Lautropia, Veillonella, Streptococcus, Scardovia, Rothia, and Actinomyces were observed to be remarkably prevalent in the control group [32].
Lee at al. reported the prevalence of Prevotella, Veillonella, Streptococcus, Neisseria, Rothia, Fusobacterium, Haemophilus, Actynomices, and Porphyromonas in all groups and the exclusive presence of Megasphaera in healthy controls, Escherichia in the PMD group, and Atopobium in the OSCC group [30].
Notably, the prevalence of Capnocytophaga was observed in relation to the advanced stage of OSCC by Yang et al. [35]. Capnocytophaga was observed to be significantly more abundant in the saliva of patients with a recurrence of OSCC also by Ganly et al. [27]. Moreover, Ganly et al. observed a high level of Veillonella and Fusobacterium in patients suffering from PMD lesions [27].
Takahashi et al. detected a significant abundance of Streptococcus and a low level of Haemophilus in female subjects compared to male subjects in the OSCC group [33].
A low level of Rothia, Haemophilus, and Porhyromonas was described in OSCC patients by Takahashi et al. and Yang et al. [33,35].
In healthy patients, Zhou et al. observed the exclusive presence of Rothia and high level of Streptococcus, Neisseria, Prevotella, Porphyromonas, and Haemophilus compared to OSCC groups [28].
Lastly, the fungal composition of oral microbiome was analysed only by Mohamed et al., who observed that Candida and Saccharomyces were more abundant in the saliva of OSCC patients, while Cyberlindnera was more abundant in healthy controls. Moreover, Candida was observed to be more abundant in female patients than in males [31].
In relation to species, only three studies reported specific data; the average number ranged from 172 to 389, with a mean number age of 253. 7 [36], while in the control group, an abundance of Streptococcus pneumoniae was observed [37].
Mohamed et al. observed the presence of several species of Candida, Malassezia, and Saccharomyces cerevisiae in all salivary samples, while C. orthopsilosis and C. sake were identified exclusively in the saliva of OSCC patients [31].
Regarding the association between salivary microbial composition and other clinical factors, only three authors investigated it. Hsiao et al. detected several important associations: between higher levels of P. intermedia and alcohol drinking, and betel quid chewing, and between higher percentage of F. nucleatum and cigarette smoking. Moreover, it was observed that tooth brushing less than two times per day and a lack of regular dental visits were both associated with a significantly higher percentage of P. tannerae, while no use of dental floss was associated with a significantly higher percentage of F. nucleatum [34]. Additionally, a high prevalence of Haemophilus in patients who drink was observed by Takahashi et al. [33].
Chen et al. reported the association between HPV infection and Haemophilus and Gemella [36].
Mohamed et al. detected the presence of Lodderomyces only in the saliva of smokers, while Phlebiopsis and Filobasidium were detected only in the saliva of non-tobacco users. Furthermore, in the same study, it was reported that OSCC patients with locoregional lymph node involvement showed a higher abundance of Candida and Aspergillus and a lower abundance of Malassezia compared to the group with no lymph node involvement [31].
In the salivary samples of healthy patients, an abundance of Streptococcus, Neisseria, Prevotella, Porphyromonas, Haemophilus, and Rothia was reported by Zhou et al. [28] Lee et al. observed the presence of the genus Megasphaera exclusively in the healthy group [30].
Regarding the fungi composition, Mohamed et al. reported the abundance of M. arunolokei in the salivary microbiota of healthy patients [31].
With regard to the main phyla, genera, and species reported as statistically significant associated with OSCC presence, it was not possible to profile a specific microbiota associated with OSCC. However, based on the present findings, periodontal pathogens (e.g., Fusobacterium, Prevotella) were the most common bacteria detected in patients with OSCC.

Discussion
OSCC is one of the most prevalent cancers worldwide [40]. Despite recent advances in diagnosis, surgical techniques, and adjuvant therapy, in recent years, an increase in the incidence of OSCC has been registered, and the mortality rate is still high [41]. OSCC is of multifactorial origin; several factors act individually or in combinations in the pathogenesis. Most of the risk factors that have been identified are lifestyle related; however, about 15% of OSCC is not associated with any known risk factors [42].
Regarding the potential role of oral microbiota in the carcinogenesis process, very few studies have investigated the salivary microbiota composition in patients affected by OSCC compared to healthy control.
Unlike the study of the microbiome of different regions of the body, the oral one possesses some criticalities. Indeed, oral microbiota is a complex dynamic ecological community conditioned by continuous changes in the availability of oxygen, nutrients, and the pH of saliva, since it contains very distinct niches adhering to various surfaces [18]. According to the Human Oral Microbiome Database (HOMD), only approximately 57% of the oral bacterial species were identified, 13% were cultivated but they remain nameless, and 30% are neither isolable nor replicable in cultures microbiological [43].
This review aimed to analyse the current knowledge regarding the composition of salivary microbiota of patients with OSCC and the possible association between salivary changes and OSCC.
Eleven studies were included from different parts of the world, most of these from Asia. Although all studies analysed oral microbiota composition of patients affected by OSCC, different techniques of saliva collection, DNA extraction, and data analysis were employed.
The most common technique for saliva collection is sputum, followed by mucosa swab, and oral rinse.
There are more selective methods to analyse the microorganisms that colonize specific oral surfaces separately (e.g., tongue, buccal mucosa) [18]. However, the procedures required are often complicated, since it is difficult to collect saliva from a determined surface with precision and selectively [44].
To evaluate microbiota composition from the whole saliva, different methods have been developed. Currently, the study of the oral microbiota is based on the sequencing of the genome (next-generation sequencing, NGS). This technology is mainly employed to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA. This methodology to characterize the microbiome structure is probably the most utilized because it is relatively quick and easy [45].
The two most used approaches of NGS technique are the shotgun metagenome sequencing, which analyses the entire metagenome, and sequencing of the 16S ribosomal RNA amplicon, generally 16S rRNA for bacteria and 18S rRNA/ITS for fungi. Both techniques read the DNA sequences of the microbes present in a sample comparing them with a sequence database to establish relative quantities of different organisms present in that sample [18,46]. In almost all the included studies, the sequencing of 16S ribosomal RNA amplicon was the method applied. All these studies used the Illumina MiSeq System ® , except one study that analysed fungal genera composition, using the 454 GS FLX ® [31].
Despite the great heterogeneity of the included articles, the most common genera detected in the OSCC patients were Fusobacterium and Prevotella, and in some studies, Streptococcus. Interestingly, these three bacteria are periodontal pathogens: Fusobacterium and Prevotella are Gram-negative anaerobic pathogens, and Streptococcus is a Gram-positive facultative anaerobic pathogen.
We believe it is possible that these periodontopathic bacteria are directly and indirectly involved in oral carcinogenesis as they participate in three mechanisms: the development of chronic pro-inflammatory processes, direct anti-apoptotic action, and the production of carcinogenic metabolites [17,18,47].
A direct anti-apoptotic action is stimulated by P. gingivalis and F. nucleatum infection. P. gingivalis stimulates the secretion of antiapoptotic factors (e.g., Bcl-2) and reduces the expression of the tumour suppressor gene p53, promoting the migration of cancer cells [53,54]. Additionally, F. nucleatum participates in tissue invasion and metastasis development, activating p38 with the consequent secretion of MMP-9 and MMP-13 [19,55].
Lastly, the production of bacteria carcinogenic metabolites can also promote carcinogenesis through the release of free radicals in chronic inflammatory processes. Several Streptococcus species have been observed to be associated with the production of H 2 O 2 , NO 2 , and acids (e.g., lactic acid), which lower the tissue pH, contributing to the development of hypoxia in the tumour microenvironment and increasing the risk of metastasis [56,57]. Furthermore, thanks to the enzyme alcohol dehydrogenase, these bacteria metabolite alcohol into acetaldehyde, a carcinogenic substance [58]. Finally, F. nucleatum, P. gingivalis, and P. intermedia have the ability to produce volatile sulphur compounds (e.g., sulphuric acid), which promote the development and accumulation of genetic mutations [59].
Regarding the fungi potential involvement in oral carcinogenesis, only one included study investigated the fungi microbiota composition in patients with OSCC [31]. Fungi represent about 1% of the microbiota, and the genus Candida is the most easily isolated and, therefore, the most studied. Candida genus may be involved in OSCC onset through the production of potentially carcinogenic substances, such as endogenous nitrosamines starting from the nitrites present in the saliva [56]. Additionally, it has been observed that C. albicans promotes the adhesion of bacteria on the oral mucosa (e.g., S. Mutans), making bacteria more resistant to antibiotic treatments and consequently stimulating the creation of microbial infections, promoting chronic inflammation [60].
To be best of our knowledge, this is the first study that has attempted to compare the results from different studies to identify a potential association between a specific salivary microbiota and OSCC.
This review possesses some limitations derived from the great heterogeneity of the few studies present in the literature to date. Of these, some studies compared salivary samples of patients affected by OSCC with healthy controls, others analysed cancer lesions and anatomically matched normal sites of the same patients, and only one study compared the salivary samples of patients with OSCC with patients affected by periodontitis and healthy controls.
The wide variation of the results observed could be explained by two variables: the absence of a standardized protocol of oral microbiome analysis and the extreme heterogeneity of the oral microbiome composition associated with healthy and OSCC patients. Therefore, further investigations are required to identify and validate standardized protocols of microbiota analysis and to investigate its implication in oral carcinogenesis.
Even if saliva represents a very attractive sample because it is easily sampled, noninvasive, and reflects systemic conditions, to date the gold standard for OSCC diagnosis is still the incisional biopsy for histological confirmation [22,61]. However, if the associations between specific salivary microorganisms and OSCC are confirmed, microbiome analysis could be a useful screening test to identify "high-risk" patients. Indeed, we believe that in the near future, salivary microbiota analysis may be the litmus test for clinicians to intercept rapidly and easily the possible changes in saliva composition. Additionally, in the case of dysbiosis, saliva analysis could promote personalized approaches to restore the "good" oral microbiome and prevent oral diseases, as well as carcinogenesis, if its connection is proven. Consequently, these patients could undergo regular oral examinations and increase the frequency of check-ups to improve early diagnosis and focused therapy.

Conclusions
There is an increasing interest in the analysis of the saliva and oral microbiome composition to identify a potential association between oral dysbiosis and OSCC. However, although there are evident alterations in the composition of the salivary microbiota in OSCC patients, it is still a challenge to identify a specific microbiota pattern in OSCC patients. We encourage future standardized studies to increase the knowledge about salivary microbiota and its potential applications.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers14215441/s1, Table S1: Description of all the results about phyla, genera and species of the studies included.