| Objective:With the development of endoscopic technology,endoscopic resection(ER)has become one of the standard treatments for superficial esophageal squamous cell carcinoma and precancerous lesion(SESCC-PL),while endoscopic mucosal dissection(ESD)and endoscopic submucosal tunnel dissection(ESTD)have gradually replaced endoscopic mucosal resection(EMR)as the first choice for endoscopic treatment of SESCC-PL.However,due to the different pathological changes and basic conditions of patients,the outcome of ESD(including ESTD)may be different in SESCC-PL patients.The treatment outcome of ESD in some special SESCC-PL patients is still unclear.In addition,superficial esophageal squamous cell carcinoma lacks typical clinical symptoms.Most patients are in advanced stage when they are confirmed diagnosis because of progressive dysphagia or other symptoms,and the survival of these patients is severely limited.Therefore,early diagnosis and early treatment of esophageal cancer is important to improve the short-term treatment effect and long-term prognosis.At present,the early screening of esophageal squamous cell carcinoma in China mainly depends on endoscopy,which acceptance of patients is low and the medical cost is high.However,the diagnostic efficacy of the existing serological markers of esophageal squamous cell carcinoma is low,and there is a lack of specific serological markers for early esophageal cancer.Exosome,as a kind of microvesicles,can be secreted from tumor cells and distributed to the whole body through circulatory blood flow.Many studies have proved that mi RNA(micro RNA)in exosome has a specific expression profile,which has potential clinical value.The purpose of this study was to analyze the therapeutic evaluation of ESD in SESCC-PL patients,especially for the patients with the lesion depth reaching or over the muscularis mucosae(M3),elderly(≥65 years old)and with liver cirrhosis,and to explore the safety and efficacy of ESD for these patients.In addition,based on the peripheral blood samples of patients with superficial esophageal squamous cell carcinoma treated with ESD and patients with advanced esophageal squamous cell carcinoma treated by surgery,the expression profiles of circulating exosomal mi RNA in patients with esophageal squamous cell carcinoma of different clinicopathological stages were established to find the early diagnostic markers of exosomal mi RNA in superficial esophageal squamous cell carcinoma,which aim to explore its diagnostic value and provide scientific evidence for early diagnosis and early treatment for superficial esophageal squamous cell carcinoma.Methods:1.In chapter 1 of this study,we retrospectively analyzed the clinical data,surgical data and follow-up data of SESCC-PL patients who received ESD(including ESTD)in the endoscope center of West China hospital of Sichuan University from January 1,2014 to May 1,2020.First of all,according to the depth of lesions,all patients were divided into three groups:the epithelium and lamina propria group(M1-M2),the muscularis mucosae and submucosal superficial layer group(M3-SM1)and the submucosal deep layer group(≥SM2).The efficacy and safety of ESD in three groups of patients with different depth of lesions were compared and analyzed.The risk factors of non-R0 resection and postoperative esophageal stenosis in SESCC-PL patients after ESD were further analyzed.Secondly,according to the age of patients,all SESCC-PL patients were divided into two groups:elderly group(≥65 years old)and non-elderly group(<65 years old).All SESCC-PL patients were divided into two groups again:patients with liver cirrhosis and patients without liver cirrhosis.The efficacy and safety of ESD in above patients were compared and analyzed.Finally,according to the follow-up data of the patients,the five-year overall survival rate,five-year progress free survival rate and five-year disease-specific survival rate of SESCC-PL patients in three groups with different lesion depth,elderly group and non-elderly group were analyzed by Kaplan-Meier analysis.COX analysis was used to analyze the risk factors that may affect the long-term survival of SESCC-PL patients after ESD.2.In chapter 2 of this study,we collected 7 peripheral blood samples from patients with superficial esophageal squamous cell carcinoma who received esophageal ESD in West China hospital of Sichuan University from May 2020 to February 2021,which were selected as superficial esophageal squamous cell carcinoma group(superficial ESCC group).4 peripheral blood samples from patients with advanced esophageal squamous cell carcinoma who underwent esophagectomy at the same time were selected as advanced esophageal squamous cell carcinoma group(advanced ESCC group),and 4 peripheral blood samples from healthy subjects who received endoscopy without obvious diseases were selected as normal control group.In this study,exosome was isolated and purified by ultra-high speed centrifugation,identified by transmission electron microscope(TEM)and Western blot.mi RNA microarray was used to detect the expression level of circulating exosomal mi RNA in three groups.The expression profiles of circulating exosomal mi RNA in patients with superficial esophageal squamous cell carcinoma and advanced esophageal squamous cell carcinoma were established,and the differently expressed circulating exosomal mi RNA was screened.The circulating exosomal mi RNA which can be used as a potential diagnostic marker for superficial esophageal squamous cell carcinoma was found in this study.The target genes were predicted by DIANA-micro T,Target Scan,mi RDB and mi Randa database,and the possible biological functions and pathways of mi RNA differentially expressed in different stages of esophageal squamous cell carcinoma were explored by GO and KEGG analysis,and its role in the occurrence and development of esophageal squamous cell carcinoma was speculated.Results:1.A total of 641 patients with 744 lesions were included in the study,including 436 males and 205 females.The average age of the patients was 62.0±8.2 years old.According to the depth of lesions,all patients with esophageal squamous cell carcinoma treated with ESD were divided into 3 groups.The lesion depth was mucosal epithelium and lamina propria(M1-M2)in 460 patients(71.8%),muscularis mucosae and submucosa superficial layer(M3-SM1)in 133patients(20.7%),and the lesion depth reached or exceeded the depth of submucosa(≥SM2)in 48 patients(7.5%).The lesion area of the three groups of esophageal squamous cell carcinoma patients with different lesion depth was1222.3±1282.3 mm~2and 2137.5±2460.6 mm~2respectively.The lesion area was positively correlated with the lesion depth(P<0.001).The lesion length of three groups of esophageal squamous cell carcinoma patients with different lesion depth was 6.5±2.4 cm and 8.6±3.6 cm,respectively.The lesion length was positively correlated with the lesion depth(P<0.001).The circumferential ratio of lesions in M1-M2 group(0.6±0.2)was lower than that in M3-SM1 and≥SM2 groups(0.7±0.2)(P<0.001).The operation time of the three groups was 66.6±39.7,81.1±45.7 and 93.6±50.6 minutes,respectively.With the depth of the lesion,the operation time increased with the depth of the lesion(P<0.001).The lesion resection speed of the three groups was 20.2±16.3,22.9±14.6 and 23.5±18.0mm~2/min,respectively,and there was no significant difference(P=0.155).The en bloc resection rates of the three groups were 97.6%,97.7%and 100.0%,respectively,and there was no statistical difference(P=0.558).The R0 resection rates of the three groups were 91.1%,79.7%and 62.5%,respectively.The R0resection rate among the three groups decreased with the increase of lesion depth(P<0.001).Among the three groups,38.5%,39.1%,29.2%and 0.9%,1.5%,0%of the patients had intraoperative and postoperative bleeding respectively,and there was no statistical difference(P=0.427,P=0.626).27.8%,31.6%and 18.8%of the patients in the three groups had muscularis propria injury(P=0.25),and0.7%,1.5%and 2.1%of the patients had intraoperative perforation,respectively,with no statistical difference(P=0.462).11.3%,18.8%and 14.6%of the patients in the three groups developed postoperative esophageal stenosis,respectively,and there was no statistical difference either(P=0.075).2.The risk factors of non-R0 resection in patients with SESCC-PL after ESD were analyzed.Univariate and multivariate analysis showed that the depth of lesion invasion≥M3(OR 2.0,95%CI 1.1-3.5),lesion length>9.5 cm(OR 3.6,95%CI 2.1-6.2),lesion circumference ratio≥3/4(OR 2.1,95%CI 1.1-4.1)and whole esophageal lesions(OR 13.7,95%CI 11.1-173.1)were risk factors for non-R0 resection(all P<0.05).The risk factors of esophageal stenosis in patients with SESCC-PL treated with ESD were analyzed.It was found that the lesion was located in the upper segment of esophagus(OR 3.4,95%CI 1.8-6.2),muscularis propria injury(OR 1.7,95%CI 1.0-2.7),circumferential ratio of lesion≥3/4(OR15.8,95%CI 8.7-28.8),operation time≥90.5 min(OR 7.2,95%CI 4.3-11.9),lesion area≥1470 mm~2(OR 11.6,95%CI 6.4-20.7),lesion length>4 cm(OR 6.1,95%CI 1.9-19.8),lesion depth≥M3(OR 1.8,95%CI 1.1-3.1)were risk factors for postoperative stenosis in patients with SESCC-PL treated with ESD(all P<0.05).However,all patients with dysphagia were relieved by multiple esophageal dilatation(Stooler score decreased 2.1±1.0).3.All patients were divided into elderly group(≥65 years old)(N=243)and non-elderly group(<65 years old)(N=398).There was no significant difference in the ratio of male to female,lesion location,lesion area,lesion ring ratio,lesion length and lesion depth between the elderly group and the non-elderly group.In terms of efficacy,there was no significant difference in operation time,lesion resection speed and en bloc resection rate between the non-elderly group and the elderly group(all P>0.05).The R0 resection rate in the elderly group was 83.5%,which was slightly lower than that in the non-elderly group(88.7%)(P=0.045).The positive rate of horizontal margin in the elderly group was higher than that in the non-elderly group(15.6%vs.9.8%,P=0.027).There was no significant difference in the positive rate of vertical margin(P>0.05).In terms of safety,except that the postoperative stenosis rate in the elderly group was slightly higher than that in the non-elderly group(16.5%vs.11.1%,P=0.049),there was no significant difference in the rate of muscularis propria injury,intraoperative and postoperative bleeding rate,perforation rate and lymph-vascular invasion rate(all P>0.05).All patients were divided into cirrhosis group(N=17)and non-cirrhosis group(N=624)according to whether they were complicated with liver cirrhosis or not.There was no significant difference in age,sex ratio,lesion area,lesion length and lesion depth between the two groups.The circumferential ratio of lesions in cirrhosis group was higher than that in non-cirrhosis group(0.7±0.2 vs.0.6±0.2,P=0.012).In terms of efficacy and safety,there was no significant difference in operation time,resection speed,en bloc resection rate,R0 resection rate,muscularis propria injury,postoperative bleeding rate,intraoperative perforation rate and postoperative stenosis rate between the two groups.The intraoperative bleeding rate in patients with liver cirrhosis was higher than that in patients without liver cirrhosis.However,after matching by PSM,there was no significant difference in operation time,lesion resection speed,en bloc resection rate,R0 resection rate,postoperative bleeding rate,intrinsic muscle injury rate,perforation rate and the incidence of postoperative stenosis between the two groups(all P>0.05).4.A total of 545 patients were followed up after ESD,with an average follow-up time of 38.0±20.4 months.Through Kaplan-Meier analysis,it was found that the five-year overall survival rates of SESCC-PL patients with different lesion depths in M1-M2,M3-SM1 and≥SM2 groups were 93.2%,94.7%and84.6%,respectively,and the five-year progress-free survival rates were 96.7%,90.1%and 95.2%,respectively.The disease-specific survival rates were 99.7%,94.7%and 92.3%,respectively.The Kaplan-Meier results showed that the five-year overall survival rate was 94.7%in the non-elderly group,90.4%in the elderly group.The five-year progress-free survival rate was 96.3%in the non-elderly group and 98.7%in the elderly group.The five-year disease-specific survival rate of non-elderly patients was 98.6%,and the five-year disease-specific survival rate of elderly patients was 97.6%.There was no significant difference in the five-year survival rate between the two groups(P>0.05).COX analysis showed that positive lymph-vascular invasion(OR 6.9,95%CI 1.5-32.2,P=0.014)and liver cirrhosis(OR 7.8,95%CI 1.7-36.6,P=0.009)were risk factors for overall survival,while positive lymph-vascular invasion(OR 52.3,95%CI5.2-521.8,P<0.001)and positive vertical margin(OR 13.6,95%CI 5.2-521.8,P=0.027)were risk factors for disease-specific survival.5.The mi RNA microarray scans a total of 2549 human target circulating exosomal mi RNA for 15 subjects in three groups.By analyzing the scanning results,it was found that 646 mi RNA expression levels were up-regulated and 399mi RNA expression levels were down-regulated between the superficial ESCC group and the normal control group(Fold Change>1.00,P<0.001).Among the differentially expressed mi RNA,with Fold Change value≥1.5 and P<0.05,43mi RNA were up-regulated and 5 mi RNA were down-regulated.It was found that525 mi RNA expression levels were up-regulated and 520 mi RNA expression levels were down-regulated between the advanced ESCC group and the normal control group(Fold Change>1.00,P<0.001).Among the differentially expressed mi RNA,with Fold Change value≥1.5 and P<0.05,43 mi RNA were up-regulated and 13 mi RNA were down-regulated.Compared with the superficial ESCC group and the advanced ESCC group,531 mi RNA expression levels were up-regulated and 514 mi RNA expression levels were down-regulated(Fold Change>1.00,P<0.001).Among the differentially expressed mi RNA,with Fold Change value≥1.5and P<0.05,39 mi RNA were up-regulated and 41 mi RNA were down-regulated.6.After screening the differentially expressed mi RNA among the three groups,it was found that the 11 circulating exosomal mi RNA in superficial ESCC group and advanced ESCC group were up-regulated compared with the normal control group,which were hsa-mi R-1825,hsa-mi R-200a-5p,hsa-mi R-2116-3p,hsa-mi R-3944-5p,hsa-mi R-4537,hsa-mi R-4649-3p,hsa-mi R-5699-3p,hsa-mi R-3122,hsa-mi R-379-5p,hsa-mi R-4654 and hsa-mi R-8087,respectively.None mi RNA was down-regulated in the early and advanced ESCC group compared with the normal control group.hsa-mi R-4654 is the only mi RNA,with progressive high expression in normal control group,superficial ESCC group and advanced ESCC group.7.A total of 1538 target genes were predicted from 11 differentially expressed circulating exosomal mi RNA.The target genes related to GO analysis was mainly involved in biological processes such as synapse and axon formation,neuron differentiation,tubular epithelial fineness,polarization,specific DNA binding,GTPase binding,DNA binding transcriptional activation,GTPase activity regulation and so on.KEGG analysis showed that related target genes were mainly involved in tumor-related signal pathways such as MAPK,Fox O,Hippo,Wnt,c AMP,PI3K-Akt,m TOR and so on.The target genes of hsa-mi R-4654 are mainly involved in protein synthesis and modification,signal pathway regulation,transcriptional regulation,cell differentiation,cell proliferation and migration and other biological functions.Conclusions:This study confirmed that ESD is safe for all SESCC-PL patients.In terms of efficacy,for elderly patients and patients with lesion depth≥M3,although R0resection rate of ESD was lower than that of M1-M2 and non-elderly patients,there was no significant difference in five-year overall survival rate and five-year disease-specific survival rate.We believe that these patients can still benefit from ESD.As for the patients with liver cirrhosis,we found that there was no significant difference in efficacy and safety of ESD between patients with or without liver cirrhosis,indicating that ESD can be used as the first choice for the patients with early esophageal lesions who has severe liver diseases.In this study,it was also found that the risk factors of non-R0 resection after ESD were mainly concentrated in lesion depth,lesion length,lesion circumferential ratio and lesion location,while the risk factors leading to esophageal stenosis after ESD were lesion location,lesion area,lesion length,lesion circumferential ratio and muscularis propria injury.We should pay great attention to these risk factors in clinical work and give corresponding precautions.For patients with non-R0resection after ESD,the positive margin of non-R0 resection should be evaluated.Patients with positive horizontal margin should be closely followed up,and patients with positive vertical margin should be actively treated with surgery or chemoradiotherapy.For patients with postoperative esophageal stenosis,multiple endoscopic esophageal dilatations can significantly relieve dysphagia and improve the life quality of patients.In the exploration of diagnostic markers,this study also found that the expression profiles of circulating exosomal mi RNA in patients with superficial esophageal squamous cell carcinoma and advanced esophageal squamous cell carcinoma were significantly different from those in normal subjects.The expression level of circulating exosomal hsa-mi R-4654 was positively correlated with the progression of esophageal squamous cell carcinoma.Related bioinformatics analysis suggests that hsa-mi R-4654 may participate in the differentiation,proliferation and migration of esophageal squamous cell carcinoma and play an important role in the progression of the disease.However,the relationship between the differential expression of hsa-mi R-4654,the occurrence and development of esophageal squamous cell carcinoma and its specific pathway still need to be further studied,in order to determine whether hsa-mi R-4654 can be used as a potential diagnostic marker for screening superficial esophageal squamous cell carcinoma and predicting the development of the disease. |
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