The Research Of Oral Microbiome In Non-small Cell Lung Cancer And Its Clinical Significance

BackgroundLung cancer is one of the major causes of cancer-related deaths worldwide,placing a heavy economic burden on society and families.Lung cancer mainly includes non‐small cell lung cancer(NSCLC)which most common pathological subtypes are adenocarcinoma and squamous cell carcinoma.It is characterized by nonspecific clinical symptoms,difficult diagnosis,late diagnosis and poor prognosis.Currently,the available diagnostic and prognosis evaluation indicators have limited sensitivity and specificity include carcinoembryonic antigen(CEA),carbohydrate antigen 125(CA125),neuron-specific enolase(NSE),etc.so we need to explore more ideal diagnostic markers.Human microorganisms(including bacteria and fungi)coexist with the host.Microorganisms play an important role in symbiotic balance that is significant factor in growth,nutrition,immunity,metabolism,tumorigenesis,etc.At present,with the development of Highthroughput sequencing technology,We have found that in many systemic diseases such as Depression,Pancreatic Cancer,Colon Cancer,Stomach Cancer,the ecological balance between human and microorganisms(including bacteria and fungi)is broken.Oral microflora,the second largest microbiota after the gut,play an important role in the human immune and inflammatory system.Mouthwash detection is a non-invasive detection method for detecting oral microorganisms.It has unique advantages in convenient sampling,high acceptance,high repeatability,and suitable for large-scale popularization.However,There are many studies on the relationship between oral bacterial changes and non-small cell lung cancer,but few studies on fungi.Therefore,we have designed this research.ObjectiveTo explore differences in oral bacteria,fungi and metabolomic changes between healthy controls and patients with NSCLC(adenocarcinoma and squamous cell carcinoma)and to screen out potential markers associated with NSCLC.MethodOur research is based on Mouthwash samples from the healthy control and patients with NSCLC diagnosed by intraoperative freezing and postoperative pathology.Patients were recruited from Second Affiliated Hospital of Naval Medical University between March2021 to January 2022.We used Novaseq 6000 sequencing technology platform to analyze the v3-v4 region of 16 S r RNA gene and ITS2 region of fungal that measured by 250 bp double ended sequencing.At the same time,we have studied samples by bioinformatics analysis methods to analyze the structural composition,relative abundance,diversity changes and differences of microbial colonies.The microbial species with significant differences in relative abundance between two groups were analyzed to find potential biomarkers.Then,the bacterial groups with diagnostic efficacy were screened by drawing the ROC curve.At last,using liquid chromatography mass spectrometry(LC-MS)technology,a non-targeted metabolomics research method was used to study the mouthwash of patients with NSCLC to screen different metabolites associated with NSCLC.Then,the metabolites with diagnostic efficacy were selected from two groups by drawing the ROC curve.Result一、Characteristics of Oral bacterial microbiome and comparative study results in healthy people and NSCLC patients1.Comparing the two groups,the α diversity of NSCLC patients was reduced.Considering the oral bacterial flora abundances of two groups,the abundance of the Proteobacteria and Actinobacteria in NSCLC were significantly increased and Firmicutes was significantly reduced in NSCLC patients.Burkholderia-Caballeronia-Paraburkholderia is significantly elevated in lung cancer patients.Neisseria,Streptococcus,Haemophilus and Rothia are reduced in patients with NSCLC.2.The Oral bacterial microbiome mainly includes four dominant phylums:Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria.six dominant genera:Neisseria,Streptococcus,Haemophilus,Rothia,Prevotella and Veillonella.3.Combined with LEf Se analysis,the increased levels of Proteobacteria,Paraburkholderia,and Sphingopyxis in NSCLC showed significant differencebetween the two groups.ROC diagnostic analysis of NSCLC patients and the healthy control group by 3 genera with significant differences,found that two genera: Burkholderia-CaballeroniaParaburkholderia and Sphingopyxis has a high diagnostic value for the identification of NSCLC.二、Characteristics of oral fungal microbiome and comparative study results in healthy people and NSCLC patients1.Comparing the two groups,there was no significant difference in α diversity of NSCLC patients and there were significant differences in β diversity.Comparison of microbiome abundance between the two groups,Ascomycota(Cancer : 60.55%;Control:35.34%)and Basidiomycota(Cancer : 32.17%;Control:13.63%)were was significantly higher in NSCLC patients.The abundance of zygomycota was significantly increased in the control group(Cancer:0.27 %;Control:32.98%).There were significant differences in fungal species and abundance between the two groups at the genus level:Actinomucor and Monascus specifically existed in the control group,while Candida specifically existed in the NSCLC group.2.The Oral fungal microbiome mainly includes two dominant phylums:Ascomycota(Cancer:60.55%;Control:35.34%)and Basidiomycota(Cancer:32.17%;Control:13.63%).3.Combined with LEf Se analysis,Malassezia、Candida、Cryptococcus、Sarocladium、Setosphaeria 、 Setophoma showed significant difference between the two groups.ROC diagnostic analysis of lung cancer patients and the healthy control group by 2 genera with significant differences,found that two genera: Candida and Cryptococcus has a high diagnostic value for the identification of lung cancer.三、Characteristics of metabolomics and comparative study results in healthy people and NSCLC patients1.A sample of 53 mouthwash samples was collected for analysis of metabolites.There were 15 cases of healthy control,14 cases of NSCLC.2.The number of significant differential Oral metabolites screened for NSCLC patients was 50(45 decreased and 5 increased among them).Among them,the main metabolites decreased in the NSCLC group were mainly lipid metabolites(estrone glucuronide),oxidative metabolites(Octanoylglucuronide),organic acids(Kinetensin 4-7).the main metabolites increased in the NSCLC group were mainly organosulfur compounds(Methyl2-propenyl tetrasulfide),Organoheterocyclic compounds(Xanthine).Major disorders of metabolic pathways include Lipid metabolism,t RNA aminoacylation,small molecule transport,etc.3.4 differential Oral metabolites with AUC greater than 0.8 were screened.At the same time,4 Oral metabolites with potential predictive value were found(normetanephrine,biliverdin,pantetheine and octanoylglucuronide).Conclusion:Using the Nova Seq6000 high-throughput second-generation sequencing technology platform and bioinformatics analysis platform,we analyzed mouthwash samples from the healthy control group and non-small cell lung cancer.The oral bacterial microbiome mainly includes 4 major phyla and 6 major genera.The alpha diversity of the Oral microbiomes in NSCLC patients decreased.It means that The relative abundance of some phyla and genera may increase,while others may decrease.As a result,Burkholderia-CaballeroniaParaburkholderia and Sphingopyxis may be of great significance for the identification of NSCLC.The oral fungal flora mainly includes Ascomycota and Basidiomycota.there was no significant difference in α diversity of lung cancer patients and there were significant differences in βdiversity.Meanwhile,at the level of fungi,there were significant differences between two groups.Candida was of great significance for the identification of NSCLC.We used metabolomics research method(Liquid chromatography-mass spectrometry,LC-MS)to analyze mouthwash samples from healthy people and lung cancer patients.Multiple metabolic pathways which are dysfunctional in NSCLC patients have been found and multiple potentially predictive Oral metabolites have been screened.