| Objective:Stenosis,which is divided into benign lesions and malignant lesions,is a common disease of the pancreaticobiliary duct.The main cause of malignant stenosis is cholangiocarcinoma(CCA),whose unique biological characteristics often make it difficult to distinguish benign stenosis from malignant stenosis.Traditional diagnostic methods such as tissue sampling are not sensitive enough to accurately diagnosepatients with undetermined pancreaticobiliary strictures.Confocal laser endomicroscopy(CLE),which can provide real-time microscopic information of the lesions,have been used in the diagnosis of undetermined pancreaticobiliary strictures in recent years,but there are great differences in the diagnosis results between different studies.In the first part of this study,the diagnostic efficacy of CLE,tissue sampling and CLE combined tissue sampling for undetermined cholangiopancreatic stenosis was evaluated by means of meta-analysis,so as to provide a basis for the establishment of an accurate and effective diagnostic scheme.In addition,with the development of CLE research,the study of CLE molecular imaging has gradually become a hot spot.The selection of disease-specific targets is one of the key factors affecting CLE molecular imaging.In the second part of this study,weighted gene coexpression network analysis(WGCNA)was adopted to identify the modules and key genes related to CCA,so as to lay a foundation for CLE molecular imaging to be applied in the diagnosis of CCA and provide clues for a deeper understanding of the etiology and pathogenesis of CCA.Methods:1.A comprehensive computer search of the English literature on the use of confocal laser endomicroscopy for the diagnosis of undetermined pancreaticobiliary strictures was performed.Retrieval databases were three commonly used English databases,including Pubmed,the Cochrane Library as well as Embase.The studies which met the inclusion as well as exclusion criteria were collected.To conduct these literatures quality assessment,the QUADAS-2 was utilized.The number of true-positive,false-positive,false-negative and true-negative of the three methods for diagnosis of undetermined pancreaticobiliary strictures was extracted.We utilized the random-effects model to pool estimates of sensitivity as well as specificity.The summary receiveroperating characteristic curve was constructed to calculate the area under the curve.Meta-regression analysis was carried out to explore sources of heterogeneity.Therobustness assessment of results was performed in accordance with sensitivity analysis.And subgroup analysis was also performed to assess the diagnostic performance of confocal laser endomicroscopy.2.The raw gene expression dataset(GSE89747)was extracted from Gene Expression Omnibus(GEO)database.The weighted gene co-expression network analysis method was utilized to construct the co-expression modules of tumor and tumor-normal samples respectively.And enrichment of functions of the genes from important co-expression modules was conducted by bioinformatics methods.Two approaches were involved in order to screen modules which were significantly associated with tumorigenesis.Then,candidate hub genes were defined as genes with high module connectivity(module membership(MM)>0.8)as well as clinical trait relationship(GS > 0.2).Moreover,genes in the module most highly correlated with tumorigenesis were mapped to STRING database to establish the protein-protein interaction network,and genes with connectivity degree ?4 were defined as candidate hub genes.The common parts of two sets of candidate hub genes were ultimately defined as key genes.Results:1.A total of 12 studies in conformity to the criteria were obtained,involving a total of 591 patients with undetermined pancreaticobiliary strictures.All of these studies evaluated the diagnosis efficacy of confocal laser endomicroscopy in patients with undetermined pancreaticobiliary strictures.Of the 12 studies,nine evaluated the diagnosis efficacy of the new diagnostic modality,and four evaluated the diagnosis efficacy of joint application of confocal laser endomicroscopy and tissue sampling.The overall sensitivity as well as specificity of confocal laser endomicroscopy in thediagnosis of the cholangio-pancreatic disease were 87%(95%CI,83-91%)as well as76%(95%CI,70-81%),respectively.Besides,the area under the curve regarding the new endoscopic technology was 0.8705.The overall sensitivity as well as specificity of tissue sampling methods were 64%(95%CI,57-70%)as well as 94%(95%CI,90-97%),respectively.Furthermore,the area under the curve regarding tissue sampling methods was 0.8040.For joint application of confocal laser endomicroscopy and tissue sampling,the overall estimates of sensitivity as well as specificity were 93%(95%CI,88-96%)as well as 82%(95%CI,74-89%),respectively.The area under the curve regarding joint application of confocal laser endomicroscopy and tissue sampling was0.9377.Sensitivity analysis result suggest that the diagnostic efficiency of confocal laser endomicroscopy remained high,and the calculation result was stabilization.Results of subgroup analysis suggest that the pooled specificity in the studies using the Paris classification was higher than the studies using the Miami classification(79% versus70%).And the pooled specificity of confocal laser endomicroscopy when using a catheter was higher than the pooled specificity when using a cholangioscope(71%versus 68%).Meta-regression analysis results showed that none of the variables had any meaningful effect on heterogeneity.No publication bias was identified by Deeks' funnel plot with p =0.936.2.A total of 36 coexpression modules with biological significance for 7846 genes from 46 cholangiocarcinoma samples were obtained by using weighted gene coexpression network analysis method.Functional enrichment analysis of the first five largest network modules showed that genes in the modules significantly enriched in ubiquity mediated proteolysis,bile secretion,complement as well as coagulation cascades,the m TOR signaling pathway,PI3K-Akt signaling pathway and so on.Moreover,the co-expression modules were constructed between tumor and normaltissue samples and associated with sample types.Two modules,including black module and tan module significantly correlated with tumorigenesis were obtained,in which tan module had the highest correlation(cor=-0.74,p=2e-9).And weighted gene coexpression network analysis identified 49 genes with high module connectivity(MM >0.8)and clinical trait relationship(GS > 0.2)in the module as candidate hub genes.Then,the protein-protein interaction network of genes in tan module was constructed.We obtained a protein-protein interaction network with 133 nodes and 409 edges.Sixty-two hub nodes with connectivity degree ?4 were identified by utilizing Cyto NCA plugin.In the end,the common parts of two sets of candidate hub genes,including IL6,SELE,BDKRB1,CD79 A,IGJ,CTSG,FOSB,IL1R1,NAMPT,C5AR1,S100A8,CCL19,SOCS3,THBS1,ADAM and TS1,were regarded as key genes in association with tumorigenesis of cholangiocarcinoma.Conclusion:1.To our knowledge,this part of the study may be the first to evaluate the diagnostic efficacy of confocal laser endomicroscopy,tissue sampling,and joint application of the new endoscopic technique and tissue sampling for undetermined pancreaticobiliary strictures by meta-analysis.The results show that confocal laser endomicroscopy can be regarded as an effective diagnostic methods in undetermined pancreaticobiliary strictures.Compared with traditional tissue sampling methods,the new endoscopic technique can improve diagnostic sensitivity.Joint application of confocal laser endomicroscopy and tissue sampling may have good diagnostic efficacy in the patients with undetermined pancreaticobiliary strictures,and more studies should be needed to verify the result in the future.2.This part construct the gene co-expression module of cholangiocarcinomathrough weighted gene co-expression network analysis.A total of 36 coexpression modules with biological significance were obtained.We further identified 15 key genes that may negatively regulate the development of cholangiocarcinoma.The identified modules and key genes can lay a foundation for the CLE molecular imaging application in the diagnosis of CCA,and provide help for further understanding the molecular mechanism of CCA. |
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