| BackgroundAll over the world,the morbidity and mortality of lung cancer are extremely high and have an increasing trend year by year.The mortality rate ranks first among all malignant tumors,resulting in a serious disease burden.In 2020 alone,228,820 cases of lung cancer were diagnosed in the United States,resulting in 135,720 deaths.Patients with lung cancer by histopathologic type can be segmented into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),accounting for about 80%of non-small cell lung cancer,according to the pathological type mainly includes pulmonary adenocarcinoma,adenosquamous carcinoma and pulmonary large cell carcinoma,etc.Most patients with lung cancer have advanced stage when first diagnosed,and some of them have developed distant metastasis.Although targeted drugs and immunotherapy have achieved good results at present,most patients still can only accept traditional radiotherapy and chemotherapy,with poor prognosis.Therefore,it is crucial to find new technologies to improve the diagnosis,treatment and prognosis of lung cancer.With the development of microecology research,it has been proved that the development of many malignant tumors is closely related to microecology.Helicobacter pylori has been confirmed to be involved in the occurrence of gastric cancer,and intestinal flora has been confirmed to be involved in the occurrence of primary hepatocellular carcinoma,colorectal cancer,pancreatic cancer and other diseases,with a unique microbial profile,which provides microbial markers for diagnosis and treatment.It was previously believed that lung was a sterile space,but with the development of second-generation sequencing technology,it was found that healthy lungs also had abundant bacteria.Studies have confirmed that compared with the lung microecology of healthy people,the microbial diversity of lung cancer patients is reduced,and there are different structural changes and differences in the abundance of bacteria.In this study,sequencing analysis of the lower respiratory tract microecology of patients with lung cancer was conducted to explore the possible mechanism of microecology in the occurrence and development of lung cancer,which will bring clinical benefits.ObjectiveThe alveolar lavage fluid of lung cancer patients was sequenced by 16SrDNA technology to analyze the differences of lower respiratory tract microecology among different clinical characteristics of lung cancer patients,including pathological type,gender,smoking history,stage,gene mutation status.MethodsThe data of 119 patients who were admitted to the First Affiliated Hospital of.Zhengzhou University and first diagnosed with lung cancer by the Department of Pathology of our hospital from January 2019 to December 2019 were collected.Including name,gender,age,smoking history,comorbidity,imaging examination,immunohistochemical Ki67 expression level,TNM staging,gene testing,PDL-1 expression,treatment plan and efficacy.All cases were examined by fiberoptic bronchoscopy,bronchial lavage fluid was collected from patients at the side of the lesion,and pulmonary microbiome sequencing analysis was performed to analyze the microecological differences of lower respiratory tract in patients with different TNM stages,gene mutation status.The non-parametric Chao1 diversity index and Shannon-Wiener(SW)diversity index were used to evaluate the alpha diversity of the samples.Beta diversity was studied by Principal component analysis(PCA).Linear discriminant analysis(LDA)effect size(LEFSE)was used to detect the abundance of differences at the taxon level.The LDA value>was 2:0,and P<0.05 indicated a statistically significant difference.The test level α=0.05.Results1.Research objects:119 patients were included in this study.76 were males and 43 were females.With a median age of 63 years,64 patients had a history of smoking and 55 patients had no history of smoking.Pathological.classification:22 cases of small cell lung cancer,97 cases of non-small cell lung cancer(63 cases of lung adenocarcinoma,34 cases of lung squamous carcinoma).All lung cancer patients were staged according to the eighth edition of TNM,including 62 patients in theⅠ-ⅢA group and 57 patients in the ⅢB-Ⅳ stage.Forty-three patients with lung adenocarcinoma completed EGFR gene testing,and were divided into gene mutation group(n=32)and non-mutation group(n-11)according to the presence or absence of EGFR gene mutation.Eighteen patients with lung squamous cell carcinoma had completed EGFR gene testing,including 1 in the mutant group and 17 in the non-mutant group.2.Pathology:The alpha diversity of respiratory microflora of patients with small cell lung cancer was higher than that of lung adenocarcinoma and lung squamous cell carcinoma.Beta diversity showed that there was no statistical significance between different pathological types of flora.At the level of phylum,actinomycetes had a high abundance in patients with lung adenocarcinoma.Bacteroidetes have a high abundance in patients with small cell lung cancer.At the genus level,Paenarthrobacter and Neisseria have higher abundance in patients with lung adenocarcinoma,while Veronella and Actinobacteria have lower abundance in patients with lung adenocarcinoma than the other two.Haemophilus and Fusobacterium were significantly reduced in SCLC patients,while Prevotella-7,Alloprevotella and were higher in abundance than lung adenocarcinoma and lung squamous cell carcinoma patients.3.Year:The microflora richness of lower respiratory tract in SCLC patients aged ≥60 years was higher than that in SCLC patients aged<60 years,while the opposite result was obtained in non-SCLC patients(lung adenocarcinoma,lung squamous cell carcinoma).In terms of Beta diversity analysis,there was no significant difference in the microflora of lower respiratory tract among lung cancer patients of different ages(P>0.05).On the whole,phylum and genus levels,the lower respiratory tract of lung cancer patients aged ≥60 years and the group aged<60 years had similar microflora composition.The abundance of Actinobacteria was high in each ’subgroup,while the abundance of Proteobacteria was decreased in the group of patients with small cell lung cancer and lung squamous cell carcinoma aged≥60 years.4.gender:In terms of alpha diversity,female microflora diversity of small cell lung cancer patients and lung adenocarcinoma patients was higher than that of male patients,while the opposite result was obtained in the analysis of microflora of lower respiratory tract of lung squamous cell carcinoma patients.By PCA comparison,there was no significant difference in BETA diversity among patients with different pathological types of lung cancer(P>0.05).The abundance of Actinomycetes in women was higher than that in men,while the abundance of Firmicutes was lower than that in men,both in the overall lung cancer patients and in patients with different pathological types of lung cancer.At the genus level,Panellosis was found to be higher in women with lung cancer than in men,while Streptococcus was lower in women than in men.5.Smoke:Alpha diversity.The microbial population richness of smoking patients with small cell lung cancer,lung adenocarcinoma and lung squamous cell carcinoma was lower than that of non-smoking patients.In terms of Beta diversity,smoking or not had statistically significant difference in the microflora composition of lung adenocarcinoma patients(P=0.035),while there was no statistically significant difference in the microflora composition between small cell lung cancer patients and lung squamous cell carcinoma patients.Based on phylum and genus microflora analysis,smoking decreased the abundance of Actinobacteria and Panellosis in lung cancer patients,and the same changes could be seen in lung adenocarcinoma and lung squamous cell carcinoma patients.It was not seen in patients with small cell lung cancer.6.TNM:The alpha diversity of the lower respiratory microflora in patients with small cell lung cancer and squamous cell carcinoma was higher than that in patients with Ⅰ-ⅢA,while the opposite result was found in patients with lung adenocarcinoma.In terms of Beta diversity,there was no statistically significant difference in the microflora of small-cell lung cancer,lung adenocarcinoma and lung squamous cell carcinoma patients in the ⅠⅢA group and the ⅢB-Ⅳ stage.There was a decrease in the abundance of proteobacteria in the stage ⅢB-Ⅳ lung cancer group among different pathological types of lung cancer at the phylum level.At the genus level,it was found that the abundance of Haemophilus and Neisseria in stage ⅢB-Ⅳ was lower than that in stage Ⅰ-ⅢA in all pathological types.The abundance of Prevotella-7,Veronella and Prevotella in stage ⅢB-Ⅳ group was higher than that in stageⅠ-ⅢA group.7.EGFR:In patients with lung adenocarcinoma,the Alpha diversity of EGFR mutant was lower than that of wild-type group,and the Beta diversity analysis showed no statistically significant difference(P>0.05).At the level of phyla,the abundance of actomyces in the EGFR mutant group was lower than that in the wild-type group,while the abundance of Firmicutes and Proteobacteria was lower than that in the wild-type group.At the genus level,the abundance of Paenarthrobacter and Neisseria in the EGFR mutant group was lower than that in the wild-type group,while the abundance of Streptococcus and Haemophilus in the EGFR mutant group was higher than that in the wild-type group.At the genus level,the EGFR wild-type group had different bacteria flora of Peptostreptococcus.8.Through the TNM subgroup,it was found that veronella was increased in NSCLC patients at stage IIIB-IV.NSCLC patients at stage ⅢB-Ⅳ were divided into high abundance group(n=13)and low abundance group(n=18)according to the median abundance of veronella.The median OS and mean OS in the high abundance group were both lower than those in the low abundance group.Conclusions1.The microflora distribution of lung cancer patients with different clinical characteristics is different,and the microflora of the lower respiratory tract of lung cancer patients is different from the common pathogens of CAP,which provides the etiological basis for lung cancer patients complicated with pulmonary infection.2.The diversity of microflora in lower respiratory tract of patients with small cell lung cancer was higher than that of patients with non-small cell lung cancer.Smoking can reduce the diversity of respiratory microflora in lung cancer patients,and reduce the abundance of Actinobacteria and Panellosis in lung cancer patients.3.Veillonella was increased in the ⅢB-Ⅳ group of NSCLC patients,and the median OS of patients in the high abundance group was lower than that in the low expression group,which suggested that Veillonella might have the value of predicting the prognosis of the disease.4.In lung adenocarcinoma,the bacteria richness of EGFR mutant group was lower than that of EGFR wild-type group,and Streptococcus digestidis was the different bacteria genus between the two groups. |
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