Study Of Circulating Autoantibodies To Immune Regulatory Factors And Tumor-associated Antigens In Lung Cancer And Esophageal Cancer

ObjectiveLung cancer and esophageal cancer are major malignant tumors in the chest;their morbidity and mortality are high in our country. Due to the lack of specificitymarkers available for clinical diagnosis, most patients have been diagnosed ashaving the malignant tumours at a late stage and a low survival rate in the first yearof diagnosis.. Therefore, early discovery and diagnosis with an early treatment foresophageal and lung cancers remains a top priority. In recent years, circulatingautoantibodies as tumor markers for early diagnosis have been widely recognizedand the first antibody-based diagnostic tool for lung cancer, called EarlyCD-Lung,has been commercially available in Europe and North America. In this study，wedeveloped a novel enzyme-linked immunosorbent assay (ELISA) for analysis of thelevels of circulating autoantibodies against some immuno-regulatory molecules(IRMs) and tumor-associated antigens (TAAs) in lung cancer and esophageal cancer,the correlations between circulating levels of these autoantibodies and their clinicalsignificance in early diagnosis and prognosis of these two cancers.MethodA total of7IRMs and TAAs were selected as antigens in this study, includingCD25，FOXP3，TGF-β1，p16，TP53，SOX2and CAGE. The bioinformatics databaseand software were used to analyse their structures and characters. Based on in silicomapping of MHC restricted epitopes derived from these proteins, linear antigenfragments were then designed; the linear peptide antigens were synthezied with achemical method and an ELISA test was then developed in-house to detectcirculating autoantibodies in patients with non-small cell lung cancer (NSCLC) and with esophageal cancer and healthy control subjects. All samples were testedin duplicate and quality control was performed with pooled plasma samples collectedfrom>200healthy blood donors. SPSS for Windows18.0was applied to analyzedata, Mann-Whiteney U test, chi-square (X2) test and analysis of the area under thereceiver operating characteristic (ROC) curve (AUC).Result1．The immune regulatory molecules：（1）The peptides：here are the sequenceof the three immune regulatory molecules：CD25：H-VQGYRAL HRGPA ESVFRRIKSGSLYML-OH；FOXP3：H-EIYHWFTR MFAFFRNHPAT WKNAI RHNLSLHKD-OH；TGF-β1：H-LYQKYSNNSWRYLSNRLLILSKLRLASPPSQ-OH.（2）The levels of autoantibodies：①The levels of circulating anti-CD25IgG weresignifacantly higher in patients with NSCLC than healthy controls (P <0.001). Thepositive rate of anti-CD25IgG was significantly higher in patients with NSCLC thanhealthy subjects; ROC analysis showed an AUC of0.70(95%CI:0.65-0.75) foranti-CD25IgG, with a sensitivity of35.0%against specificity of>90%. The levels ofanti-CD25IgG were signifacantly higher in patients with ESCC than healthycontrols (P <0.001); ROC analysis showed an AUC of0.68(95%CI0.57-0.73) foranti-CD25IgG, with a sensitivity of37.2%against specificity of90%. The levels ofanti-CD25IgA did not show a significant change in either patients with NSCLC orthose with ESCC. These results suggest that the anti-CD25IgG are a potentialmarker for diagnosis of NSCLC and early ESCC.②The levels of circulatinganti-FOXP3IgG were significantly higher in patients with NSCLC than healthysubjects (P <0.001), although there is a gender difference in patients with squamouscell cancer. Anti-FOXP3IgG had a certain diagnosis meaning in patient with lungadenocarcinoma,but the sensitivity and NPV were too low.The levels of circulatinganti-FOXP3IgG were also significantly higher in patients with ESCC than healthysubjects (P <0.001). ROC analysis showed an AUC of0.70(95%CI0.65-0.75) foranti-FOXP3IgG, with a sensitivity of20.3%against specificity of90.3%, in patientswith NSCLC, and0.72(95%CI:0.65-0.78), with a sensitivity of36.8%againstspecificity of90.4%in patients with ESCC. These results suggest that the anti-FOXP3IgG may also be a useful immunomarker for diagnosis in ESCC.③Thelevels of anti-TGF-β1IgG were no diagnostic meaning in NSCLC and ESCCthrough ROC analysis. The change of anti-TGF-β1IgG was the risk factors inNSCLC stageⅡ（P=0.008）.It was the positive correlation between anti-CD25IgGand anti-FOXP3IgG (r=0.327, P=0.327),but the levels of anti-TGF-β1IgGwere no relevance either anti-FOXP3IgG or anti-CD25IgG．2．The tumor associated mocules：（1）The peptides：here are the sequences ofthe four peptides：P16：H-NSYGRLVVLHRAGARLDVRDAWGRLPVDLA-OH；TP53：H-LIRVEGNLRVEYPGTRVRAMAIYKQSQHMTE-OH；CAGE：H-FKIKNNMVGVVIGYSGSKIKDLQHSTNTKIE-OH；SOX2：H-LQYNSMTSSQTYMNGSPTYSMNAFMVWSRGQRR-OH．（2）The levels of autoantibodies：①Thelevels of anti-P16IgG in lung adenocarcinoma,especially in female paitents,werehigher than healthy group,but only a little contribution to the diagnosis. The positiverate in patients with NSCLC was higher than healthy group（P=0.038），the positiverate decline slowly at stageⅠ-Ⅲ，but rise up at stage Ⅳ.The result suggested thatthe levels of anti-P16IgG were no contribution to diagnosis in NSCLC，but theraised levels of anti-P16IgG,especially at early stage(stageⅠ)，were the risk factorof NSCLC（P<0.001）. The AUC of anti-P16IgG was0.614，the sensitivitywas33.3%，specificity was91.0%in female patients with ESCC. It suggested thatlevels of anti-P16IgG had only a little diagnostic importance in female patients with ESCC.The raised levels of anti-P16IgG were the risk factors of stageⅡ of ESCC（P<0.001）。 The levels of anti-P16IgG were positive correlation withanti-CD25IgG（r=0.537，P<0.001） in healthy group,but the levels of anti-P16IgGwere positive correlation with anti-CD25IgG and anti-FOXP3IgG（r=0.535，P<0.001；r=0.169，P=0.002）.②The levels of anti-CAGEIgG in male patients withlung squamous carcinoma were significantly higher than that in healthy mengroup(P=0.017), while the levels of anti-CAGEIgG in female patients with lungsquamous carcinoma were lower than healthy women (P=0.048). The levels of anti-CAGEIgG in male patients with NSCLC were higher than that in female patientswith NSCLC. The positive rate of anti-CAGE IgG in patients with NSCLC in different stages had no statistical difference compared with healthy group (P>0.05).The levels of anti–CAGEIgG in male and female patients with ESCC and that ofhealthy group were no difference(P>0.05). The anti-CAGEIgG in different genderpatients with NSCLC and ESCC were all no diagnostic meaning.The levels ofanti-CAGEIgG in tumor were positive correlation with anti-CD25IgG (r=0.202,P=0.202) and anti-FOXP3IgG (r=0.141, P=0.141), but were negative correlationwith anti-TGF-beta1IgG (r=0.157, P=0.027). The changes of levels ofanti-CAGEIgG were not the risk factors in different stages of NSCLC and/orESCC(P>0.05).③The levels of circulating anti-SOX2IgG in patients with lungsquamous carcinoma and adenocarcinoma had no statistical difference comparedwith healthy group (P>0.05); anti-SOX2IgG was no diagnostic meaning to lungcancer through the analysis of ROC curve.The levels of circulating in patients withESCC were higher than healthy group (P=0.029), the levels of anti-SOX2IgG infemale ESCC patients were obviously higher than healthy women group(P=0.037),the AUC of female patients with ESCC was0.680, sensitivity was38.5%, specificitywas93.7%; The AUC, the sensitivity and specificity in female patients were higherthan in male patients with ESCC.The levels of anti-SOX2IgG in female patientswith ESCC had diagnostic value in a certain. The levels of anti-SOX2IgG in tumorwere negative correlation with anti-CD25IgG (r=0.164, P=0.164), positiveassociation with anti-FOXP3IgG (r=0.214, P=0.214) in healthy group, but no anycorrelation with anti-TGF-beta1IgG. The changes of levels of anti-SOX2IgGwere not the risk factors in different stages of NSCLC and/or ESCC（P>0.05）.④The levels of circulating anti-TP53IgG in male patients with NSCLC weresignificantly higher than healthy men group(P=0.009), the AUC was0.636, thesensitivity was20.3%, specificity was90.2%. Anti-TP53IgG in male patients withNSCLC maybe had diagnostic meaning in a certain, but the sensitivity was a littlelow. The levels of circulating anti-TP53IgG in patients with ESCC were nosignificant difference compared with healthy group (P=0.701). The positive rate ofanti-TP53IgG in patients with NSCLC or ESCC were low. Anti-TP53IgG was no correlation with anti-CD25IgG or anti-FOXP3IgG or anti-TGF-beta1IgG．Thechanges of levels of anti-TP53IgG were not the risk factor in different stages ofNSCLC and/or ESCC（P>0.05）.Moreover, there were significant correlations between circulating levels ofautoantibodies for CD25, FOXP3and p16. In patients with NSCLC, the coefficientof correlation (r) was0.537between anti-CD25IgG and anti-p16IgG (P<0.001),0.169between FOXP3IgG and anti-p16IgG (P=0.002) and0.205betweenanti-CD25IgG and anti-FOXP3IgG (P=0.001).Conclusion（1）Circulating anti-CD25IgG and anti-FOXP3IgG are very likely to serve asthe novel markers for NSCLC and ESCC.（2）Circulating anti-P16IgG showed a weak association with both NSCLCand ESCC. Further investigation is needed to confirm whether anti-p16IgG canserve as a potential marker for thoracic cancers.（3） Circulating autoantibodies for TGF-β1，P53，SOX2and CAGE did notshow a significant change in patients either with NSCLC or with ESCC, althoughinitial work from a number of studies have their increase in circulating levels in lungcancer. Possibly, further mapping of HLA restricted antigens is needed in futurestudy.（4）anti-CD25IgG is correlation with anti-FOXP3IgG，and anti-CD25IgG andanti-FOXP3respectively are correlation with anti-P16IgG、anti-CAGE IgG、anti-SOX2IgG. It shows that CD25+FOXP3+Treg cells can regulate the function oftumor associated factors.（5）The present work was undertaken to develop an in-house ELISAtest usingthe HLA restricted antigens derived from IRMs and TAAs. This is a novel methodand may a very useful for detection of circulating antibodies as biomarkers forcancer.