| Esophageal cancer is one of the most common malignant tumors.China is of the highest incidence and mortality rate of esophageal cancer.The most prevalent type is esophageal squamous cell carcinoma(ESCC),the annual incidence of new cases account for 50%.Recent studies have shown that tumor heterogeneity(ITH)is prevalent in ESCC based on DNA sequencing and chromosome analysis in multiple regions from the same tumor.In light of the evidence that ITH is the major cause of drug resistance and treatment failure,the 5-year survival rate after ESCC surgery is less than 30%.Deciphering the genomic diversity of ESCC tumors,identifying new targets,screening early diagnostic markers,and design individualized treatment strategies are hot and difficult to study today.Objective: To investigate intra-tumoral heterogeneity and the expression of ZNF750,EP300,MTOR and KMT2 D on the prognosis of patients with esophageal squamous cell carcinoma.Methods:1 Retrospective analysis of 1782 patients with ESCC and then having the operation of esophageal cancer resection who were admitted to the Fourth Hospital of Hebei Medical University from January 2008 to December 2010.1550 cases were followed-up successfully,while 232 cases lost.The follow-up rate was 86.9%.106 cases with heterogeneity within the tumor were screened from the 1550 patients according to HE staining.There were histological or morphological differences in the two lesion regions in each case.All the 106 patients had no previous treatment before surgery,no other tumor history,and all of them had complete clinical data.2 The expression levels of ZNF750,KMT2 D,EP300 and MTOR in the two lesion regions were detected by immunohistochemistry(IHC)HRP method,and the difference was observed between different lesions.Specimens were fixed with 10% formalin,conventional materials,paraffin embedded tablets,HE staining and immunohistochemical staining,light microscopy.The method of immunohistochemistry was as follows: the degree of staining was scored according to the percentage of the positive stained tumor cells,and the high expression group was 4 ~ 9.The low expression group included negative expression or low expression with a total score of 0 to 3.3 Using a novel method based on RNA in situ hybridization(RNAScope),24 lesions of 12 patients with formalin-fixed paraffin-embedded tissue were randomly selected from 106 patients with ESCC,using specific RNA probes.The m RNA transcription levels of ZNF750,KMT2 D,EP300 and MTOR were detected and the differences in expression between different lesions were detected.The number of signals in m RNA cells with high expression of m RNA is more(3 or 4),and the number of signals in low m RNA expression of m RNA is less(0 ~2).4 Statistical analysis was performed by SPSS21.00.Two independent samples were analyzed by Kaplan test,Kaplan-Meier single factor analysis for survival analysis,and COX regression for multivariate analysis.With α = 0.05 as the test standard,P <0.05 was statistically significant.Result:1 Clinicopathological featuresAmong the 1550 ESCC patients,there were 1038 males,512 females,among 106 patients with heterogeneous ESCC,80 males and 26 females.The difference was not statistically significant(P = 0.071).1550 cases of ESCC patients aged 18 to 81 years,the median age of60 years,mean age 59.93 years,tumor location in the upper segment 15 cases,62 cases of the middle,the lower 29 cases,lymph node metastasis rate was 38.58%(598/1550),the positive rate of vascular tumor thrombus was 25.61%(397/1550),histological grade G1 was 121,G2 was 1161,G3 was 268,clinical stageⅠwas 236,stageⅡwas 747,stageⅢ was 549,stageⅣ was 18.The depth of tumor T1 was 281,T2 was 259,T3 was 1010,the survival period was from 1 to 108 months,5-year survival rate was48.30%,the median survival time were 57 months;106 cases with heterogeneous aged 39-76 years,median age was 59 years,mean age was59.28 years,15 cases were of upper part of the disease,the middle 62 cases,the lower 29 cases,lymph node metastasis rate was 55.67%(59/106),the positive rate of vascular tumor thrombus was 36.79%(39/106),histological grade G1 was 5,G2 was 73,G3 was 28,clinical stage I was 7,stageⅡwas 39,stageⅢ was 60,depth of T1 was in 9 cases,T2 in 27 cases,T3 in 70 cases,the survival period was from 4 to 105 months,5-year survival rate was 34.00%,the median survival time was30 months.The difference between the two groups was statistically significant(P <0.005).2 Immunohistochemical results2.1 Expression of ZNF750 protein and its correlation with clinical index and tumor heterogeneityAmong the106 cases of esophageal squamous cell carcinoma,58cases(54.72%)were of high expression,48 cases(45.28%)of low expression.The expression of ZNF750 protein was closely related to histological grade,clinical stage,depth of invasion,vascular thrombosis and lymph node metastasis(P<0.05).There was no significant correlation between ZNF750 expression and tumor location,sex,and age.The high expression group of ZNF750 low expression group had a poor prognosis(χ2 = 25.821,P = 0.000).ZNF750 showed heterogeneity in 44 patients(41.51%)in 106 patients with ESCC screening,and the high expression rate of ZNF750 in212 lesion areas was 33.96%(72/212),because both lesions were not highly expressed,lower than 106 patients with ESCC(54.72%).2.2 Expression of EP300 protein and its correlation with clinical index and tumor heterogeneityAmong the 106 cases of esophageal squamous cell carcinoma,66cases(62.26%)were of high expression and 40(37.72%)of low.The expression of EP300 protein was closely related to histological grade,clinical stage,portal vein thrombosis and lymph node metastasis(P<0.05).There was no significant correlation between EP300 expression and tumor site,depth of invasion,gender and age.The low expression group of EP300 had a poor prognosis(χ2 = 14.037,P = 0.000).EP300 showed heterogeneous expression in 37(34.90%)patients,and the high expression rate of EP300 in 212 lesion areas was 44.81%(95/212),because both lesions were not highly expressed,lower than 106 patients with ESCC(62.26%).2.3 Expression of MTOR protein and its correlation with clinical index and tumor heterogeneityAmong the 106 cases of ESCC,75(70.75%)cases showed high expression of MTOR,31 cases(29.25%)of low expression.The expression of MTOR protein was closely related to histological grade,clinical stage,portal vein thrombosis and lymph node metastasis(P<0.05).There was no significant correlation between high expression of MTOR and tumor site,depth of invasion,sex and age.The lower expression group of MTOR highexpression group had a poor prognosis(χ2 = 29.856,P = 0.000).MTOR was an independent prognostic factor of multivariate analysis(χ2 = 12.166,P = 0.000).The expression of MTOR in 39 cases(36.79%)showed heterogeneity in both lesion areas,and 52.36%(111/212)was of high expression in 212 lesions,lower than 106 patients with ESCC(70.75%).2.4 KMT2 D protein expressionKMT2D was not detected by immunohistochemical of the 106 cases with esophageal squamous cell carcinoma.3 RNA scope results3.1 Expression of ZNF750 m RNA and its correlation with clinical index and tumor heterogeneityAccording to the RNA scope staining score,of the 12 cases of esophageal squamous cell carcinoma,66.67% of ZNF750 m RNA was high expressed: 6 cases scored 4 points,2 cases score 3 points,33.33%was low: 4 cases scored 1 point.The expression of ZNF750 was closely related to histological grade,clinical stage,portal vein thrombosis,lymph node metastasis and age(P <0.05).There was no significant correlation between ZNF750 m RNA expression and tumor location,sex.The heterozygous expression of ZNF750 mRNA in 8 patients was consistent with that of immunohistochemistry.3.2 Expression of EP300 mRNA and its correlation with clinical index and tumor heterogeneityOf the 12 cases of esophageal squamous cell carcinoma,83.33% of the EP300 was highexpressed: 2 had a score of 4,8 had a score of 3;33.33% had low expression of EP300: 2 had a score of 2.The expression of EP300 was closely related to histological grade,clinical stage,lymph node metastasis,sex and tumor location(P <0.05).There was no significant correlation between EP300 expression and tumor age,depth of invasion,portal vein thrombosis.The heterogeneity of EP300 m RNA was consistent with that of immunohistochemistry.3.3 Expression of MTOR mRNA and its correlation with clinical index and tumor heterogeneityAmong the 12 cases of esophageal squamous cell carcinoma,33.33% of the MTOR were highexpressed: 2 cases were scored 4 points,2 cases scored 3 points,66.67% MTOR low expression: 6 cases score 2points.The expression of MTOR was closely related to histological grade,clinical stage,portal vein thrombosis,depth of invasion,lymph node metastasis and age(P <0.05).There was no significant correlation between MTOR expression and tumor location,sex.4 cases of MTOR m RNA showed heterogeneous expression,4cases of heterogeneous expression consistent with immunohistochemistry.3.4 Eexpression of KMT2 D m RNA and its correlation with clinical index and tumor heterogeneityIn 12 cases of esophageal squamous cell carcinoma,50% of KMT2 D was highexpressed:6 cases scored 3 points,50% was low: 4scored 2 and 2 scored 1 point.There was no significant correlation between KMT2 D overexpression and histological grade,clinical stage,portal vein thrombosis,depth of invasion,lymph node metastasis,age,tumor location and sex.Four patients with KMT2 D mRNA showed heterogeneous expression.Conclusion:1 Patients with heterogeneous esophageal squamous cell carcinoma have a worse survival and prognosis.2 In esophageal squamous cell carcinoma,the expression of ZNF750,EP300 and MTOR protein expression by immunohistochemistry are colorated with tumor heterogeneity.3 In esophageal squamous cell carcinoma,the expression of ZNF750 protein was positively correlated with the prognosis of ESCC patients.The expression of EP300 and MTOR protein was negatively correlated with the prognosis of ESCC patients.MTOR was the independent prognostic factor of ESCC patients.4 RNAscope detection method has good stability and higher sensitivity,it is a more reliable exploration of new tumor markers and detection of tumor heterogeneity.ZNF750,EP300,KMT2 D and MTOR in situ m RNA detection can be used as genes of detection of esophageal cancer gene mutation detection,with potential clinical application value. |
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