Diagnosis Of Mediastinal And Hilar Diseases By Transesophageal And/or Transtracheal Ultrasound Endoscopic Puncture Combined With Genetic Testing

Mediastinal and hilar lesions are common diseases in clinical work,but the diagnosis is often difficult due to the complex anatomical structure.Transesophageal/tracheal endoscopic ultrasound-guided puncture mainly include Endoscopic ultrasonography guided fine-needle aspiration(EUS-FNA)and Endobronchial ultrasound-guided transbronchial needle aspiration(EBUS-TBNA),both of which are less traumatic,and can be performed under local anesthesia.The cost is low and the operation can be repeated.The combined use can form a complement,almost achieve the purpose of full mediastinal biopsy and staging,and has a tendency to replace the "gold standard" mediastinoscope.Genetic diagnosis plays a very important role in the diagnosis and treatment of cancer patients.More and more new gene targets have been discovered and applied to the clinic,but drug resistance or poor response often occurs.Considering that there may be other mutated genes.Therefore,Discovering new target gene is the primary task of genetic diagnosis and treatment.At present,there are many research reports on the diagnosis and staging of mediastinal and hilar lesions by EUS-FNA and EBUS-TBNA,but there are few clinical studies on large samples and few studies on the exploration of new target genes and verification in puncture specimens.This study will review a large sample of cases,analyze the diagnosis of mediastinum and hilar space-occupying lesions by EUS-FNA and EBUS-TBNA,and use bioinformatics methods to discover differential genes for poor prognosis of non-small cell lung cancer(NSCLC).It was verified in tissue samples to evaluate the diagnostic value of transesophageal/transtracheal endoscopic ultrasound guided puncture combined with genetic testing for mediastinal and hilar space-occupying lesions.This research will be conducted from the following three aspects:Part one:Clinical application of transesophageal and/or transtracheal ultrasound endoscopic puncture in the diagnosis of mediastinal and hilar lesionsObjective: To study the large sample size of EUS-FNA and EBUS-TBNA in the diagnosis of mediastinal and hilar lesions,and to explore the clinical application value of EUS-FNA and/or EBUS-TBNA in mediastinal and hilar lesions.Methods: A retrospective study of patients who underwent EBUS-TBNA and/or EUS-FNA after CT/PET-CT suggested mediastinal and hilar lesions in the Cancer Hospital of Guangxi Medical University from June 2013 to December2019.According to the puncture results and the final results,the sensitivity,specificity,positive predictive value,negative predictive value,accuracy rate of the patient’s diagnosis were counted,and the differences in diagnosis between EBUS-TBNA and EUS-FNA were compared.The learning curve of doctors A and B were drawed and cumulative accuracy of their diagnosis was compared.Results: A total of 710 sites were punctured in the 424 patients enrolled,covering almost the entire mediastinum and hilar zones(except for zone 6).The most common disease is non-small cell lung cancer.There was no difference in gender and age between EBUS-TBNA group and EUS-FNA group(p>0.05).The overall sensitivity,specificity,positive predictive value,negative predictive value and accuracy rate respectively were 86.43%,87.30%,97.50%,52.88%and 86.56%;There was no difference in accuracy(p=0.133)between EBUS-TBNA and EUS-FNA group,but the sensitivity of the EBUS-TBNA group was higher than that of EUS-FNA(p=0.046).No serious complications occurred in all patients.Among the two groups of patients with confirmed diagnosis,more than 60% need to relied on routine pathology plus immunohistochemistry and genetic testing,and there was no statistical difference between the two groups(p=0.541).From the perspective of the learning curve,the two doctors A and B can achieve the final diagnostic accuracy when operating 125 cases and 30 cases respectively,but there is no statistical difference in the cumulative diagnostic accuracy of the two when operating 30 patients(p =0.374)).Conclusion: EBUS-TBNA and EUS-FNA are efficient and safe for the diagnosis of mediastinal and hilar lesions;EBUS-TBNA is more sensitive than EUS-TNA and can be selected first;the diagnosis of the two groups of lesions often depends on immunohistochemistry and Genetic testing;EBUS-TBNA is easy to master under the guidance of an experienced doctor.Part two:Analysis and identification of important genes with poor prognosis for non-small cell lung cancer through bioinformatics methodsObjective: This study uses bioinformatics methods to analyze important genes with poor prognosis of NSCLC and their underlying mechanisms,explore and identify potential biomarkers of NSCLC,and provide evidence for clinical genetic diagnosis and treatment of NSCLC.Methods: Download the relevant microarray data(GSE5816,GSE6695 and GSE74572)from the GEO database of the National Center for Bioinformatics Information,and select the differentially expressed genes(DEG)between NSCLC tissues and normal tissues through GEO2 R tools and Venn software;use the bioinformatics annotation database(DAVID)Perform gene ontology(GO)analysis on DEG,and use Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis;then use Cytoscape software to construct protein-protein interaction(PPI)network analysis to screen out core genes;The Kaplan Meier plotter online tool was used to analyze the prognosis of the core genes and use the gene expression profile interactive analysis(GEPIA)to verify,and finally through the clinically confirmed NSCLC tissue section for immunohistochemical verification.Results: The three chip data contains 41 NSCLC tissues and 25 normal tissues,a total of 124 DEGs,including 54 up-regulated genes and 70 down-regulated genes,mainly enriched in the cytosol,endoplasmic reticulum,adhesion plaques,and extracellular matrix,regulate transcription factor activity,sequence-specific DNA binding,RNA polymerase II core promoter proximal region sequence specific DNA binding,actin binding,etc.Pathway analysis showed that the four pathways mainly involved extracellular matrix receptor interaction,focal adhesion,arrhythmogenic right ventricular cardiomyopathy,alanine,aspartic acid and glutamate metabolism.In the PPI network,six core genes were found.The results of prognostic analysis showed that 4 out of 6 genes had a significantly poorer prognosis.Further GEPIA verification found that compared with normal non-tissue,2 out of 4 genes are different in NSCLC tissue.Finally,the results of immunohistochemical verification on clinically diagnosed NSCLC tissue sections showed that in adenocarcinoma and squamous cell carcinoma,FSLT1 and SPP1 were significantly different from normal tissues(p<0.05).Conclusion: FSTL1 and SPP1 can be used as potential biomarkers for the poor prognosis of NSCLC,and can provide a new theoretical basis for the diagnosis and treatment of NSCLC.Part three : A prospective study to analyze the significance of transesophageal /transtracheal endoscopic ultrasound puncture combined with genetic testing in the diagnosis of mediastinal and hilar lesionsObjective: To study and analyze the clinical application of EUS-FNA and EBUS-TBNA in the diagnosis of mediastinal and hilar disease,and to explore the significance of EUS-FNA and/or EBUS-TBNA in the diagnosis of mediastinal and hilar disease.Methods: Prospective study of patients who underwent EBUSTBNA/EUS-FNA in the Endoscopy Center of the Cancer Hospital of Guangxi Medical University.The tissues were punctured for pathology,cytology,liquid-based cytology and genetic testing,based on the final diagnosis.Judgment to evaluate the significance of genetic testing,ultrasound endoscopic puncture combined with genetic testing in the diagnosis of mediastinal and hilar lesions.Results: Among 38 patients,the FSTL1 test,EBUS-TBNA/EUS-FNA,and EBUS-TBNA/EUS-FNA combined with FSTL1 test results were compared with the final diagnosis results,sensitivity,specificity,positive predictive value,negative predictive value,and diagnostic accuracy respectively are:86.36%vs90.63%vs96.88%,31.25%vs100.00%vs100.00%,63.33%vs100.00%vs100.00%,62.50%vs66.67%vs85.71%,63.16%vs92.11%vs91.37%,EBUSTBNA/ EUS-FNA and EBUS-TBNA/EUS-FNA combined with FSTL1 test results were compared,although there was an upward trend,the difference was not statistically significant(p>0.05).Conclusion: EBUS-TBNA/EUS-FNA combined with FSTL1 gene detection has a high efficiency for the diagnosis of non-small cell lung cancer,and it is expected to become a new combination method for the diagnosis of non-small cell lung cancer.