Analysis Of Oral Salivary Microbiota In Patients With Esophageal Squamous Cell Carcinoma And Its Clinical Significance

Background&PurposeEsophageal cancer is the ninth most common cancer in the world and the sixth leading cause of cancer deaths in the world.Esophageal squamous cell carcinoma accounts for about 90%of the total incidence of esophageal cancer,and China accounts for about 50%of the global incidence of esophageal cancer.Studies have shown that smoking and drinking are positively correlated with the risk of esophageal squamous cell carcinoma.However,recent studies from China,India,Iran and other regions and other regions including Latin America and Japan with high incidence of esophageal cancer show that there is a correlation between human poor oral health indicators and esophageal squamous cell carcinoma.With the rise and development of microecological research,the relationship between oral bacteria and microecological environment imbalance and esophageal squamous cell carcinoma is worthy of research.Oral bacteria may play a pathogenic role in cancer and other chronic diseases by directly metabolizing chemical carcinogens and inflammatory factors.Studies have reported that tooth loss and poor oral hygiene are closely related to upper gastrointestinal cancer,indicating the possible role of salivary microbiota in the development of upper gastrointestinal cancer.And saliva is the main source of colonization of oral flora.Therefore,in this study,we used high-throughput sequencing to study the diversity of salivary flora in patients with esophageal squamous cell carcinoma,explore the relationship between esophageal squamous cell carcinoma and salivary flora,and screen out bacteria specifically expressed in patients with esophageal squamous cell carcinoma,Aiming to provide a simple and non-invasive method for the early screening of esophageal squamous cell carcinoma.MethodsA total of 50 patients with saliva from esophageal squamous cell carcinoma(ESCC group)and 40 patients from healthy control group(HC group)were selected for treatment at the Weilun Digestive Endoscopy Center of Guangdong People’s Hospital(Guangdong Academy of Medical Sciences).All patients were confirmed by pathological biopsy.All patients with esophageal cancer were esophageal squamous cell carcinoma,and were not accompanied by other systemic diseases that affect the saliva flora,such as diabetes,hepatitis,fatty liver,and so on.Saliva DNA was extracted using a kit purchased from the US MOBIO company,16S rDNA V4 region was amplified by PCR,high-throughput sequencing was performed,and then a library was constructed according to standard procedures,and then the established amplicon library was sequenced on Illumina Hiseq2500 PE250 Platform for sequencing.Finally,the sequencing data were analyzed by OUT clustering,including species annotation,Alpha diversity analysis,Beta diversity analysis,and LEfSe analysis.The target bacteria were screened out.Finally,58 patients with esophageal squamous carcinoma and 46 healthy controls were tested for target bacteria.ResultDiversity analysis showed that the differences in overall salivary flora between the ESCC group and the HC group were small(P>0.05).Species analysis showed that compared with the HC group,the proportion of Fusobacterium,Porphyromonas,and Streptococcus in the ESCC group was higher(the proportions were:11.1%,6.9%,and 5.8%).The proportion of Prevotella was significantly reduced(the proportion is 1.3%).The proportion of Fusobacterium,Porphyromonas,and Streptococcus in the HC group was lower(the proportions were:6.1%,4.8%,and 2.9%),while the proportion of Prevotella was significantly higher(the proportion is 13.6%).LEfSe analysis showed that The ESCC group showed increased expression of Porphyromonas,Fusobacterium and Streptococcus,and there were statistical differences(P>0.05).Then We used absolute quantitative PCR to validate P.gingivalis,F.nucleatum,and S.salivarius in saliva DNA.After statistical examination,detection of P.gingivalis,F.nucleatum,and S.salivarius was significantly higher in the ESCC group than in the healthy control group.(P<0.05).We draw ROC curve based on the copy amount of Porphyromonas gingivalis,Fusobacterium nucleatum and Streptococcus salivarius in the saliva sample and the combination of these three bacteria,AUC are:60%,60.1%,78.9%,71.1%respectively,the sensitivity are:32.7%,56.9%,81%,69%respectively,the specificity are:93.4%,63%,76.1%,65.2%respectively.Combined Logistic regression of meaningful indicators,like as smoking,drinking,hot food,place of residence(Chaoshan and non-Chaoshan),gender,age and other indicators with the copy amount of P.gingivalis，F.nucleatum,S.salivarius and three bacteria to draw ROC curves,AUC is 94.5%、93%、93.4%、94.5%,sensitivity is:86.2%、96.6%、86.2%、86.2%,and specificity is:91.3%,78.3%,84.8%,93.5%respectively.Conclusion1.Compared with healthy people,saliva in patients with esophageal squamous cell carcinoma has characteristic flora,including Fusobacterium,Porphyromonas,and Streptococcus.2.Compared with healthy people,the expression of P.gingivalis,F.nucleatum,and S.salivarius in saliva of patients with esophageal squamous cells was significantly higher.3.Smoking,drinking,hot food,place of residence(Chaoshan and non-Chaoshan),gender,age combined with the copy number of Porphyromonas gingivalis,Fusobacterium nucleatum and Streptococcus salivarius can improve the predictive diagnosis value of esophageal squamous cell carcinoma.