Introduction
Oral cancer represents a major public health problem worldwide, and oral squamous cell carcinomas (OSCC) account for more than 90% of oral cancers [
1]. Of these 90%, OSCCs of the tongue (TSCC)are reported to occur with rates of up to 40–50% [
2]. TSCC is significantly more aggressive than other types of OSCC [
3]. The etiology of cancer is complicated by the fact that a high proportion is attributable to environmental and heritable risk factors. An emerging concept in cancer biology implicates the microbiome as an influential environmental factor modulating the carcinogenic process [
4]. Changes in the human microbiome are hypothesized to increase tumor formation and contribute to carcinogenesis through a few potential mechanisms: triggering cellular antiapoptotic signals; releasing carcinogenic factors; unleashing chronic inflammatory responses, and modifying anticancer immunity [
5,
6].
The microbiota is a collection of microbial taxa associated with humans [
7]. This microorganism is a double-edged sword that may have beneficial or deleterious effects on its host [
8]. The benefit side: active communication (‘cross-talk’) between some resident bacteria and host cells has been proven, and micro-organisms are maintained in an environment that is supplied with a diverse array of host molecules that serve as nutrients [
9]. The resident oral microbiota can also contribute to the host defenses by preventing the establishment of many exogenous micro-organisms [
9]. More importantly, the normal oral microbiome is also vital in maintaining oral as well as systemic health [
10]. The bad side: microorganisms and their products, including endotoxins (lipopolysaccharides), enzymes, and metabolic byproducts are toxic to host cells and may directly induce mutations or alter signaling pathways that affect cell proliferation and/or survival of epithelial cells, and eventually lead to cancer [
11].
The occurrence and development of OSCC is a complicated process, and cell cycle regulation is crucial for tumorigenesis. Protein p16 is a cyclin-dependent kinase inhibitor that inhibits retinoblastoma protein (pRb) phosphorylation and blocks cell cycle progression at the G1 to S checkpoint [
12]. The loss of p16 expression results in a worse prognosis for head and neck squamous cell carcinomas (HNSCC). Bova et al. [
13] considered that the loss of p16 expression is associated with a reduction in the 5-year overall survival rate, and they also observed that overexpression of cyclin D1 and loss of expression of p16 are independent death predictors in TSCC [
13]. In patients with TSCC, p16 positivity correlated with improved relapse-free survival [
14]. Researchers also suggested that p16-positive HNSCC had better treatment outcomes than p16-negative HNSCC [
15]. In addition, the expression of p16 occurs as a result of the functional inactivation of the pRb by the HPV E7 protein [
16]. Some studies found that the p16 expression has high concordance with other methods of HPV-DNA detection, suggesting that p16 is a surrogate marker for HPV [
17]. In another aspect, the role of oral microbiota on OSCC/TSCC has been raised. Researchers have proposed the hypothesis that oral bacterial infections can promote OSCC/TSCC carcinogenic processes [
18,
19]. However, very little is currently known about the relationship between p16 status and the microbial taxa in TSCC. In previous studies on the microorganism of OSCC, including TSCC, the vast majority of survey data were detected from oral saliva [
20‐
22] or fresh tissue samples [
19]. However, compared with these traditional clinical microbial collection methods, pathological formalin-fixed paraffin-embedded (FFPE) tissue samples have the advantages of long-term storage at room temperature and retrospective analysis at any time. Moreover, FFPE tissues are widely available from biobanks in pathology departments. Paraffin samples can also be well used to conveniently detect the expression of specific proteins in disease states, such as p16 expression analysis by immunohistochemistry. Some researchers have studied the changes of microorganisms in tumors [
11,
19] or other diseases (e.g., IgA nephropathy [
23]) by paraffin tissue.
In this study, to investigate the difference in bacterial communities between p16-positive and p16-negative TSCC, we detected the microbiota changes by using the tongue paraffin tissue.
Discussion
Generally, OSCC is caused by the long-term impact of known risk factors: tobacco and alcohol, along with chronic traumatization. In recent years, researchers have also paid more attention to the role of bacteria in many cancers, from gastric cancer (
Helicobacter pylori [
29]), which was first discovered, to gallbladder cancer (
Salmonella typhi [
30]). Oral carcinogenesis is also associated with bacteria [
31‐
33]. P16 is a cellular protein involved in cell cycle regulation and is expressed at a very low level in normal cells. The expression of p16 is associated with oral tumor stage and progression [
34], making it helpful for determining treatment prognosis in OSCC [
15]. Recently, meta-analysis suggests that p16 overexpression in oral potentially malignant disorders is significantly associated with a greater risk of malignant transformation to OSCC [
35]. However, in the case of OSCC, the relationship between p16 status and oral bacteria has not been thoroughly characterized. Therefore, a detailed analysis of the microbial status in p16-positive or p16-negative OSCC is necessary.
In the human mouth, most microbes are site specialists, the most important cause of this is that the mouth is not a unitary environment and sites within the mouth, although connected by salivary flow, constitute distinct habitats [
36]. Hence, to avoid the influence of different sites on the results, only tongue squamous cell carcinoma was selected in this study.
With the emergence of next-generation sequencing (NGS), 16 S rRNA sequencing promoted the study of associations between microbiota and OSCC. Several studies [
11,
33,
37‐
39] on oral microorganisms and OSCC have been carried out using this method. Among these studies, different samples were collected, including oral rinse [
38], saliva [
11] and fresh tumor samples [
33,
37,
39]. Compared with these traditional clinical microbial collection methods, FFPE tissue samples have several advantages, such as long-term storage at room temperature, and retrospective analysis at any time, representing a vast repertoire of genetic information and are widely available from biobanks in pathology departments. However, some challenges associated with FFPE tissues may also exist. For instance, for formalin-treated samples, retrieving nucleic acids from these tissues for downstream molecular analysis has proven challenging for researchers [
40]. Formalin fixation mainly induces damage in DNA by creating cross-links (DNA-DNA, protein-DNA), depurination, DNA fragmentation and sequence alterations.
In the present study, we encountered similar challenges. DNA extraction is one of the first challenges. Our team’s previous study detected the tonsillar microbiota changes in formalin-fixed paraffin-embedded tissue by the QIAamp DNA FFPE tissue kit [
23], however, the DNA extraction was not particularly satisfying. Hence, according to the previous experience of our teams, the advice from the sequencing companies and the literature report (DNA yields were significantly higher with either method of the FastDNA kit for soil than with the other kit, such as MoBio kit, Mobio Ultra Clean® Fecal DNA Isolation Kit; QIAamp® DNA Stool Mini Kit and FastDNA® SPIN Kit [
41,
42], we ultimately used the Fast DNA Spin Kit for soil to extract the DNA. It is crucial to note that we do not necessarily mean that this kit is the best, rather, we indicate that the DNA extraction procedure still needs to be improved. Another challenge is choosing the appropriate primers. We found that the replacement of some primers could not effectively improve the success rate of amplification. Some primers even reduce the success rate of amplification. This suggests that it is very important for us to choose appropriate primers in future studies. Moreover, storage time has also been considered a limiting factor for retrieving intact nucleic acids from FFPE samples [
43]. However, in our study, although there was no significant difference in the amplification efficiency of paraffin samples for different storage times, we noticed that the success rate of DNA amplification of paraffin samples with a short storage time had a higher trend than that of paraffin samples with a long storage time. Therefore, when selecting paraffin samples for analysis, the storage time of paraffin samples should be considered as much as possible to ensure the repeatability and reliability of the results. At the technical level, previous researchers [
44] detected the bacteria in blood samples from healthy and diseased humans, in which a nested-PCR was applied for DNA amplification and 16 S library preparation. Normally, bacterial DNA is present at very low abundance in the blood of healthy samples. In these situations, nested-PCR is necessary to increase the sensitivity and/or specificity of PCR [
45]. Hence, in future studies, the nested-PCR method may be an alternative method for DNA amplification in paraffin samples with low DNA content.
Overall, the results of 515 F/806R and 27 F/338R are quite similar, for example, the four most dominant phyla in the p16-positive group and p16-negative group are identical, including
Proteobacteria,
Firmicutes,
Bacteroidota, and
Actinobacteriota. These most dominant phyla in the oral cavity match those observed in earlier studies [
39,
46]. However, the results of 515 F/806R and 27 F/338R had some subtle differences, such as the diversity estimator of p16-positive patients and p16-negative patients at the phylum level being not the same. The possible reasons are mainly due to the sequencing length and capturing species of bacteria by different primers not being entirely consistent.
The current study identified a few bacterial classes with higher abundance among participants with p16 positivity, including
Desulfobacteria, Limnochordia,
Phycisphaerae, Anaerolineae,
Saccharimonadia and
Kapabacteria. Desulfobacteria is one of the six classes of dissimilatory phosphite oxidation microorganisms and can be detected in the environment. The role of this microbiome in human disease has not been fully reported. A recent report [
47] using concatenated protein marker trees resolved the class
Thermodesulfobacteria(
Desulfobacteria) as a clade within the
Deltaproteobacteria, which have the potential to play a role in the etiology of periodontal disease [
48]. Periodontitis can be considered as an independent risk factor for oral cancer [
49]. It is hypothesized that hydrogen sulfide produced by
Desulfobacteria as a virulence factor may damage the intestinal epithelium [
50] or periodontal tissue [
51] leading to cell death and chronic inflammation. Therefore, the role of this genus in oral cancer requires further investigation.
Limnochordia is placed in the phylum
Firmicutes [
52]. In the present study, the proportion of the
Firmicutes in the p16-positive group was higher than that in the of p16-negative group. To date, no evidence has been found associating
Limnochordia with oral cancer. Regarding
Phycisphaerae, although its role in human cancer has not yet been reported, a bacterial community analysis showed that reduced enrichment of
Phycisphaerae in the intestinal genera was observed in decreasing tendency from nonsmokers without hypertension, smokers without hypertension, and nonsmokers with hypertension to smokers with hypertension [
53]. This highlights the impact of smoke on the intestinal genera, especially
Phycisphaerae. Smoke is one of the main risk factors for TSCC, and future studies regarding the role of
Phycisphaerae in TSCC are therefore recommended.
Saccharimonadia, formerly known as phylum TM7, are ubiquitous members of the human oral microbiome, accumulating evidence linking their association with periodontal disease [
54]. Moreover, TM7 could modulate the oral microbiome structure hierarchy and functionality by affecting their bacterial host physiology [
54]. However, there are still many unanswered questions about whether this microbiota could affect tumor development. For the
Anaerolineae, it was positively correlated with the levels of the cytokines, IL-2 and IL-10 and the chemokines, CCL7, CCL11, CXCL12, and CXCL16, which contribute to the inflammatory process [
55]. Furthermore, these cytokines are not only related to the establishment of a protumor microenvironment and organ-directed metastasis, but they also mediate disease progression by promoting tumor cell growth and proliferation, including HPV-induced malignancies [
56]. In the present study, we found that
norank_o__Peptostreptococcales-Tissierellales had a positive correlation with the p16 proportion. A previous report demonstrated that significantly different levels of
Peptostreptococcus exist between oral dysplasia and OSCC [
57]. Lissoni A et al. [
58] also suggested that the main bacterial species that correlate with OSCC include
Peptostreptococcus.
On a functional level, we predicted that viral carcinogenesis was more enriched in p16-positive TSCC, and correlation analysis showed that some signatures of bacterial families and species were associated with tumor lymphatic metastasis (
Prevotellaceae) and muscle infiltration (
Pseudomonadaceae). Therefore, for clinically p16-positive TSCC patients, paying attention to the changes of these two bacteria may have reference significance and be helpful for judging the malignancy grade, but this specificity and accuracy need further research. For
Prevotellaceae, previous reports demonstrated that
Prevotellaceae was significantly increased in OSCC [
39], esophageal [
59] and gastric cancer tissues [
60]. Zhang et al. [
61] showed that the abundance of
Prevotellaceae was positively correlated with the severity of oral mucositis in patients with squamous cell carcinoma of the head and neck. Recently, Zhang et.al [
62] performed a largest study to assess the association between the oral microbiome and oral HPV DNA, and found that oral HPV infection is associated with a higher abundance of
Prevotellaceae. Mechanically, HPV-associated carcinogenesis is mediated through the influence of oncoprotein E6 and E7 expression on cell cycle regulatory pathways, which leads to p16 protein accumulation [
63,
64]. Overexpression of p16 has been indicated as a marker for HPV-related cancer [
17,
65] and p16 may be a clear potential marker in the detection of dysplasia in head and neck squamous mucosa [
66]. Regarding
Pseudomonadaceae, researchers revealed that
Pseudomonadaceae was linked to a higher risk of developing OSCC [
25] and pancreatic cancer [
67]. In particular, women with high-risk HPV had significantly higher relative abundances of
Pseudomonadaceae than those with low-risk-HPV or no HPV infection [
68]. This indicates that these bacteria play important roles in HPV-associated cancer. Therefore, it is advised that additional research be done with a greater emphasis on these microorganisms in the p16-positive TSCC.
There are limitations to our study. First, although we started with an effort to collect 60 paraffin samples, the number of samples successfully sequenced for microbial sequencing was very small. This problem is also a thorny one that is currently faced with the use of paraffin samples for microbiome research. We used different optimization schemes, such as different primers and exploring suitable amplification conditions, etc., which can provide a reference for future research. Second, its retrospective design and consequent scope for missing information about risk factors such as smoking and alcohol, in addition to the absence of TNM stage, treatment outcome and survival state, prevented a more complete analysis. Previous studies have shown that smoking and alcohol consumption drive microbial changes particularly in the saliva [
69,
70]. However, it is uncertain whether the microbiota status in TSCC tissue is similarly affected. Future investigations using large sample sizes are necessary to examine these potential risk factors and microbial alterations in TSCC.
In summary, we performed a small pilot study of microbiological studies of TSCC by paraffin tissue, and some challenges associated with DNA damage in FFPE tissues indeed exist. These results can be used as a reference for future workers who also use paraffin samples for research. Nevertheless, we have tentatively found some meaningful results. The p16 status is associated with microbiota diversity, this diversity was significantly increased in p16-positive patients compared with p16-negative patients. Desulfobacteria, Limnochordia, Phycisphaerae, Anaerolineae, Saccharimonadia and Kapabacteria had higher abundances among 16-positive participants. Moreover, functional prediction revealed that the increase in these bacteria may enhance viral carcinogenesis in p16-positive TSCC. These findings may provide insights into the relationship between p16 status and the microbial taxa in TSCC, and developing new drugs that target these bacteria may be a promising strategy for intervening in p16-positive TSCC progression.