The Intrapapillary Capillary Loop Changes And Expression Of Angiogenic Factors,Infiltrating Factors In Superficial Esophageal Lesions

BackgroudEsophageal cancer is one of the most serious gastrointestinal cancer worldwide and has poor prognosis,it ranks eighth in the incidence of malignant tumor and is the sixth largest cause of cancer related death.In China,esophageal cancer ranks fifth in the incidence of malignant tumor and is the fourth largest cause of cancer related death.Superficial esophageal cancer is defined as that with mucosal or submucosal invasion.Because of the low risk of lymph node metastasis in this type of cancer,endoscopic mucosal resection(EMR)and endoscopic submucosal dissection(ESD)has been established as a standard therapeutic approach for superficial esophageal squamous cell carcinoma(SESCC)within the superficial submucosa,which has the advantages of minimal invasive and high quality of life after treatment.So it is very important for us to detect esophageal cancer in its early stage.Previous reports have demonstrated the usefulness of endoscopic screening combined with iodine staining in the identification of esophageal cancer.In recent years,narrow band imaging(NBI)combined with magnifying endoscopy(ME)has created a new diagnostics method that involves observing capillaries and fine tissue structures.Clinical application of this combined technique(NBI-ME)facilitates diagnosis of esophageal neoplasia and diagnostic accuracy of invasion depth.It is reported that morphology of the intraepithelial papillary capillary loop(IPCL)in esophageal squamous epithelial is significantly different between benign and malignant lesions of esophagus.Visualizing these differences by NBI-ME has been reported to contribute to evaluate the depth of invasion.Intrapapillary capillaries arise from branching vessels in epithelial papillae and form single loops referred to as IPCLs.IPCLs inside cancer lesions show abnormal changes and different from that of normal tissue.There are several classifications of IPCL,Inoue’s classification and Arima’s classification were most commonly adopted in the past several years.However,both classifications are complicated to beginners.The Japan Esophageal Society put the two classifications together and developed a new classification in 2011.The new classification has been relatively popular in Japan recent years,because it is simple and useful for diagnosis of invasion depth of esophageal cancer.However,there was no report of research in China using the new classification.The classic morphological indicators of IPCL are dilatation,tortuosity,caliber change of individual IPCL,multiple IPCLs of various shapes,in our study,we found a new morphological indicator of IPCL,which was irregular arrangement.So we compared all the five morphological indicators of IPCL in SESCC.Calibers,areas and densities of IPCLs might be important factors to evaluate infiltration depth of ESCC.However,calculating areas and densities of vessels was very difficult undermicroscopes.With the development of the digital pathology technology,it became simple and feasible.In this study,we provided the measurement of calibers,areas and densities of IPCLs in different types classified by the Japan Esophageal Society classification to address this research gap.On the basis of above-mentioned study,immunohistochemistry(IHC)and quantitative real-time polymerase chain reaction(qRT-PCR)assays were used to detect the expression of VEGF,Ang-2,MMP-14,uPA,TGF-β in SESCC with different depth of invasion.The results of this study will help us to explore the features of SESCC and search for the ideal target for diagnosis and therapy of esophageal cancer.Part One:The Classification of the Intraepithelial Papillary Capillary Loop is Useful in Diagnosing Superficial Esophageal LesionsObjectiveTo investigate the diagnostic potential of different types of IPCLs observed by NBI-ME for superficial esophageal lesions.MethodPatients with abnormal esophageal mucosa found by white light gastroscopy indigestive endoscopy center of the Second Hospital of Shandong University during the period of May 2013 to October 2015 were enrolled in this study.IPCL was observed by NBI-ME and divided into different types according to Japan Esophageal Society Classification.Histopathology of biopsies or post operation(ESD or surgery)specimens were evaluated and used as the gold standard to evaluate the diagnostic value of Japan Esophageal Society Classification of IPCL.ResultsA total of 216 lesions from 195 patients with esophageal superficial lesions were collected.Among 216 lesions,102 were pathologically proven inflammation,16 were pathologically proven low-grade intraepithelial neoplasia(LGIN),26 were pathologically proven hign-grade intraepithelial neoplasia(HGIN),36 were pathologically proven lamina propria mucosae cancer(LPM),12 were pathologically proven muscularis mucosae cancers(MM),10 were pathologically proven superficial submucosal cancer(SM1),14 were pathologically proven deep submucosal cancer(SM2-3).According to the result of NBI-ME,123 were type A IPCL,32 were type B1 IPCL,25 were type B2 AVA-S IPCL,21 were type B2 AVA-M IPCL,5 were type B2 AVA-L IPCL,and 10 were type B3 IPCL.The sensitivity,specificity,accuracy of NBI-ME to differentiating non-cancerous lesion(inflammation and LGIN)from cancerous lesion(HGIN,LPM,MM,SMl-3)were 92.4%、85.7%、89.4%.The sensitivity,specificity of NBI-ME to diagnosing HGIN/LPM,MM/SM1,SM2-3 were 70.1%、91.6%,59.1%、95.9%,78.6%、98.0%,respectively.ConclusionNBI-ME can observe IPCL clearly.High diagnostic accuracy of IPCL classification indicates the feasibility of NBI-ME for the efficacious diagnosis of superficial esophageal lesions.Part Two:The Morphological Indicators of Intrapapillary Capillary Loop in Superficial Esophageal LesionsObjectiveThe aim of this study were to investigate morphological indicators of intrapapillary capillary loops,that is dilatation,tortuosity,caliber change of individual IPCL,multiple IPCLs of various shapes,irregular arrangement,in differnt superficial esophageal lesions.MethodsA total of 104 superficial esophageal squamous cell carcinoma tissue samples obtained from patients of the Second Hospital of Shandong University between March 2016 and September 2016 were selected for use in the present study.All of cases were divided into two groups according to histological diagnosis,that is,inflamation/LGIN group and HGIN/LPM group.All cases underwent NBI-ME and morphological indicators of IPCL including dilatation,tortuosity,caliber change of individual IPCL,multiple IPCLs of various shapes,irregular arrangement were recorded.ResultsIn univariate analysis,caliber change of individual IPCL,multiple IPCLs of various shapes,irregular arrangement were significantly associated with the diagnosis of HGIN/LPM(P<0.001).In multivariate analysis,multiple IPCLs of various shapes and irregular arrangement were confirmed to be independent factors.Odds ratios were 5.18(95%confidence interval:1.23-21.83)for multiple IPCLs of various shapes and 13.43(95%confidence interval:2.11-85.53)for irregular arrangement.ConclusionThe results of this observation showed multiple IPCLs of various shapes and irregular arrangement were independent and more important IPCL’s morphological indicators associated with the diagnosis of HGIN/LPM.Part Three:The Intrapapillary Capillary Loop Changes in Superficial Esophageal LesionsObjectiveThe aim of the present study were to investigate the calibers,areas and densities of IPCLs in different types of superficial esophageal lesions.Methods A total of 22 tissue samples obtained from patients underwent ESD/EMR orsurgery for superficial esophageal cancer at the Second Hospital of Shandong University between May 2013 and December 2014 were selected for use in the present study.All of cases were examined histologically by NBI-ME and divided into different IPCL groups according to the Japan Esophageal Society classification.The type B1 and type B2 were analyzed as one group.14 cases of Tla or T1bSM1 squamous carcinoma with IPCLs of type B1/B2 and 8 cases of T1bSM2 squamous carcinoma with IPCLs of type B3 were selected.In addition,12 cases of normal esophageal mucosa tissues from patients underwent surgery for esophageal or esophagogastric junction adenocarcinoma were added to the study,which were divided into the type A by NBI before treatments.The calibers,areas and densities of IPCLs were detected in all 34 cases using immunohistochemical analysis.ResultsStatistically significant differences in calibers,areas and densities of IPCLs were observed between type A,type B1/B2 and type B3 area(P<0.001).ConclusionThe results of this observation showed the calibers,areas and densities of IPCLs related to the quality of the lesion.The Japan Esophageal Society classification of IPCL would help endoscopists to diagnose the type and the invasion depth of superficial lesion in esophagus.Part Four:Expression of VEGF,PD-ECGF,ANG-2,MMP-14,uPA,and TGF-β1 in Superficial Esophageal Squamous Cell CarcinomaObjectiveThe aims of this study were to investigate the expression of VEGF,PD-ECGF,ANG-2,MMP-14,uPA,and TGF-β1 in SESCC with different depth of invasion.MethodsA total of 79 tissue samples of superficial esophageal cancer obtained from patients underwent ESD/EMR or surgery at the Second Hospital of Shandong University between October 2014 and October 2016 were selected for use in the present study.All cases were divided into three groups according to the depth of invasion,that is,HGIN/LPM group including 25 cases,MM/SM1 group including 23 cases and SM2/SM3 group including 31 cases.The expression of VEGF,Ang-2,MMP-14,VEGF,TGF-β1 were detected in all 79 cases using IHC analysis,then we detected the expression of VEGF gene and uPA gene in all the cases using qRT-PCR assays.ResultsThe level of VEGF expression was more and more high in HGIN/LPM group,MM/SM1 group and SM2/SM3 group,there was significantly different between the three groups using IHC analysis(P<0.001).The expression of uPA in SM2/SM3 group was significantly higher than HGIN/LPM group and MM/SM1 group using IHC analysis(P≤0.001),and there was no significant difference between HGIN/LPM group and MM/SM1 group.The expression of Ang-2,MMP-14,TGF-β1 had no significant differences between HGIN/LPM group,MM/SMlgroup and SM2/SM3 group using IHC analysis.The expression of VEGF gene and uPA gene between the three groups using qRT-PCR assays were same with the results of IHC.ConclusionThe results of this study showed the expression of VEGF and uPA were significantly different in SESCC with different depth of invasion,the expression of Ang-2,MMP-14 and TGF-β1 were not significantly different in SESCC with different depth of invasion.