The Study Of Performance Of Serum Autoantibodies In The Early Diagnosis Of Esophageal Squamous Cell Carcinoma

Background Esophageal cancer is the sixth leading cause of cancer deaths worldwide.In China,the incidence of esophageal squamous cell carcinoma(ESCC)is the highest in the world.The current five-year survival rate of ESCC is low,mainly because ESCC patients have no specific clinical symptoms in the early stage.The diagnostic rate of early-stage ESCC by esophageal endoscopy and pathological biopsy and other diagnostic methods is low and has some limitations.The traditional tumor-associated antigens(TAAs)serological indicators have different degrees of low sensitivity or low specificity.Autoantibodies related to ESCC are a hot topic in recent years.The study found that tumor-associated autoantibodies were detected in serum from 3-5 years before the development of ESCC to clinical symptoms.Therefore,the detection of tumor-associated autoantibodies is expected to bring new breakthroughs in the early diagnosis of ESCC.Objectives In our previous study,Based on a screening and subsequent-validation procedures by high-throughput protein chips(Hu Prot v3.0,containing 20,240 proteins provided by the High-throughput Biology Center of Johns Hopkins School of Medicine,USA),eight Ig G serum autoantibodies such as anti-TP53,ETHE1,CTAG1 A,C1QTNF1,TEX264,CLDN2,NSG1,HRAS were demonstrated the good diagnostic values for early-stage lung cancer(LC).Interestingly,in a small cohort study,the positive rates and levels of anti-TP53,HRAS,CTAG1 A and NSG1 in eight known autoantibodies were significantly higher in the early-stage ESCC group and the advanced-stage ESCC group than those in the esophageal benign lesion(EBL)group or the healthy control(HC)group(P <0.01).In view of the obvious advantages of autoantibodies in the early diagnosis of tumors,whether the above four autoantibodies combined with antigenic tumor markers have a good early diagnostic value for ESCC is worth exploring.Methods1.Enzyme-linked immunosorbent assay(ELISA)method was established to detect the value of eight Ig G-type autoantibody markers against TP53,ETHE1,CTAG1 A,C1QTNF1,TEX264,CLDN2,NSG1 and HRAS antibodies in the early diagnosis of ESCC.The small sample cohort included 50 patients with early-stage ESCC,50 patients with advanced-stage ESCC,50 patients with EBL,and 50 patients with HC.2.Western blotting was used to verify the ELISA results of anti-TP53,HRAS,CTAG1 A and NSG1 antibodies in early-stage ESCC serum.3.We evaluated the performance of the four-autoantibody panel for early diagnosis of ESCC using ELISA in a large cohort of 569 retrospective sera samples,including the following: a training set(n = 409)consisting of 129 patients with early-stage ESCC;130 patients with EBL;150 HC;and an independent matched validation set(n =160)consisting of 50 patients with early-stage ESCC;50 patients with EBL;60 HC.4.The anti-TP53,HRAS,CTAG1 A and NSG1 antibodies in the independent matched validation set(n =160)were detected by the constructed Luminex x MAP multiplex assay.5.The expression of TP53,HRAS,CTAG1 A and NSG1 protein in early-stage ESCC cancer tissues and matched paracancerous tissues was detected by immunohistochemistry.6.The levels of four ESCC antigenic tumor markers such as CEA,CYFRA21-1,CA19-9 and SCCAg in the training set were detected by chemiluminescence method.The value of separate detection of four antigenic tumor markers for early diagnosis of ESCC was analyzed.7.Comparison of combination model of four autoantibodies and combination model of four antigenic tumor markers in the training set of the early diagnosis of ESCC.The combined detection model of four autoantibodies and antigenic tumor markers were analyzed and established to further improve the performance of early diagnosis of ESCC.Results1.The results of ELISA in the small sample cohort showed that among the eight serum autoantibodies,the positive rates and levels of anti-TP53,HRAS,CTAG1 A and NSG1 in the early-stage ESCC group and the advanced-stage ESCC group were significantly higher than those in the EBL group or the HC group(P <0.01).Among the eight serum autoantibodies,the positive rates and levels of anti-ETHE1,C1QTNF1,TEX264 and CLDN2 were not statistically significant between the early-stage ESCC group,advanced-stage ESCC group and the EBL group or the HC group(P >0.05).2.The results of western blotting of anti-TP53,HRAS,CTAG1 A and NSG1 antibodies were consistent with the results of ELISA.3.In the training set,anti-TP53,HRAS,CTAG1 A and NSG1 antibodies showed higher positive rates and levels in the early-stage ESCC than the control group(P <0.01).The AUC,sensitivity and specificity of the combination consisted of all four autoantibodies were 0.823,62.8% and 88.9% for the early diagnosis of ESCC,respectively.In the validation set,the four autoantibodies showed higher positive rates and levels in the early-stage ESCC than the control group(P <0.01).The sensitivity and specificity of the combination consisted of all four autoantibodies were 58.0% and 90.0% for the early diagnosis of ESCC,respectively.4.The results of the validation set of the four autoantibodies were found to have a good correlation between the ELISA and Luminex methods.The sensitivity and specificity of the the combination consisted of all four autoantibodies in the serum samples of the validation set were 66.0% and 90.9%,respectively,by using the Luminex x MAP multiplex assay.At the same time,four kinds of autoantibodies were detected by ELISA and Luminex,and the agreement of positive hits for the combination was 73.0%.5.The expression of TP53,HRAS,CTAG1 A and NSG1 protein were detected by immunohistochemistry.The strong positive expression rates of four proteins in the cancer tissues of early-stage ESCC patients with positive(+)ELISA results were significantly higher than those of matched paracancerous tissues(P <0.01).The four proteins in the early-stage ESCC patients and the matched paracancerous tissues with negative(-)ELISA results showed a higher weak positive expression rate.6.In the training set,the positive rates and levels of CEA(P <0.05)and CYFRA21-1,CA19-9,SCCAg(P <0.01)in the early-stage ESCC group were higher than those in the EBL group or HC group.There were no significant differences in the positive rates and levels of four antigenic tumor markers between EBL group and HC group(P >0.05).The sensitivity of single antigenic tumor markers to the early diagnosis of ESCC was low,and the value of early diagnosis was limited(AUC ? 0.646).7.In the training set,the specificity of the combination of four autoantibodies and the combination of four antigenic tumor markers in the early diagnosis of ESCC were 88.9% and 90.4%,respectively.The AUC and sensitivity of the combination consisted of four autoantibodies were 0.823 and 62.8% for the early diagnosis of ESCC,respectively,which was significantly higher than the AUC(0.687)and sensitivity(39.5%)detected by the combination of the four antigenic tumor markers(P <0.01).The sensitivity of the combination of the four autoantibodies was not significantly different between the EBL group and the HC group(P >0.05).The sensitivity of the combination of the four antigenic tumor markers was not significantly different between the EBL group and the HC group(P >0.05).8.In the training set,the combined detection of four autoantibodies with CYFRA21-1,CA19-9 and SCCAg had the highest diagnostic performance for early ESCC patients,and the AUC could be increased to 0.842.Conclusions1.Anti-TP53,HRAS,CTAG1 A and NSG1 antibodies have good early diagnostic performance for ESCC,and the anti-CTAG1 A antibody has the highest early diagnostic performance for ESCC.The combined detection of four autoantibodies helps to improve the early diagnostic performance of ESCC.2.CEA,CYFRA21-1,CA19-9 and SCCAg have certain value for the early diagnosis of ESCC.Among them,CYFRA21-1 has the highest positive rate for early diagnosis of ESCC and SCCAg has the highest AUC for early-stage ESCC patients.However,the single indicator has a low positive rate for early diagnosis of ESCC,and the value of early diagnosis is limited.The combination of four antigenic tumor markers can improve the diagnostic performance of early-stage ESCC patients while ensuring specificity.3.The diagnostic performance of combination model of four autoantibodies in the early-stage of ESCC patients is significantly higher than the combination model of four antigenic tumor markers.The combined detection model of four autoantibodies with CYFRA21-1,CA19-9 and SCCAg can be significant improve diagnostic performance in early-stage ESCC patients.