Study On The Relationship Between Autoantibodies Against Tumor-associated Antigens In Peripheral Blood And Liver Cancer

ObjectiveHepatocellular Primary liver cancer(PLC)is one of the most common malignancies and it is the third lethal cause of tumor worldwide.China accounts for about fifty percent of liver cancer cases in the world.Diagnosis of liver cancer in late stage is one of most important reasons for high mortality.Early diagnosis and early prevention is the critical point to reduce the death rate.Alpha fetal protein(AFP)is the most common diagnostic marker in clinical application.However,the AFP level in serum of twenty to thirty percent patients is not increased,regarding AFP as key diagnostic data could lead to some omitting or misjudging conditions.Therefore new tumor biomarker assisting to diagnose liver cancer is necessary.Tumor-associated antigens(TAAs)are the main targets for clinical tumor detection.Tumor-associated autoantibodies are products of host humoral immune responding to tumor-associated antigens.Many studies have found the presence of autoantibodies in the plasma of cancer patients,which have many advantages that are not existing in the current screening methods for liver cancer,such as stable structure,non-degradable,long half-life and so on.The most important point is that the autoantibodies grow in the early stage,which can be detected before clinical symptoms appear,even in the precancerous lesions.In this study,the changes of autoantibodies in the plasma of Hepatocellular carcinoma(HCC)patients were detected by designing and synthesizing the linear antigenic peptide.We aim to find one or more biomarkers that can be used in the diagnosis or prognosis assessment of liver cancer.MethodsBased on literature review,six target proteins,including CD25,Octamer-binding transcription factor 4(OCT4),p16,Baculoviral IAP repeat containing 5(BIRC5),c-myc and Annexin A1(ANXA-1)were brought into this study.According to the previous work of the research,optimizing the antigen peptide design to improve the stability of the antigen peptide and the effectiveness of the detection antibody,and designing several independent linear antigen peptides from different antigen epitopes of the same protein could help to find the key epitopes that can induce the humoral immune response.In this study,122 HCC patients who had been diagnosed for the first time without any treatment and 231 healthy controls whose gender and age were well matched with HCC were recruited.The expression levels of corresponding autoantibodies in the plasma of HCC group and healthy control group were determined by optimized enzyme-linked immunosorbent assay(ELISA).SPSS22.0 was used for data statistics and analysis to compare the differences in the expression levels of autoantibodies between the two groups,at the levels of gender,age and barcelona clinic liver cancer(BCLC)stage.Subgroup analysis of AFP expression,tumor size,vascular involvement and extrahepatic metastasis was conducted in HCC group.Receiver operating characteristic(ROC)curve was used to analyze the value of different antibodies in the diagnosis of HCC.In combination with the clinical follow-up information of HCC patients,Kaplan-Meier curve and Cox regression were used to explore the relationship between the level of autoantibodies and overall survival(OS)of HCC patients,meanwhile the factors affecting the prognosis of patients.Result(1)The expression of autoantibody of CD25.Compared with healthy control group,CD25 IgG was significantly increased in HCC patients with different genders and ages(P <0.001).Compared with control group,the expression of CD25 IgG in stage B and stage C+D of HCC patient' plasma was significantly increased(P <0.001;P <0.001),and the expression was increased with the progression of tumor.CD25 IgG of higher AFP expression group was significantly increased compared with that in normal or lower AFP expression group(P =0.004).CD25 IgG in the group(tumor size < 3cm)was significantly lower than that in the groups(tumor size =3-5 cm and >5cm)(P=0.025).The result of ROC analysis showed that the sensibility of CD25 IgG for diagnosis of HCC was 14.8%,when specificity was 95%.(2)The expression of autoantibody of OTC4.Compared with healthy control group,OCT4 IgG was significantly increased in HCC group(P <0.001).Although OCT4 IgG in male and female was increased,there was no difference between male and female.Compared with same aged healthy people,OCT4 IgG was significantly increased in HCC patients' plasma(age?50)(P <0.001);analysis in sub group showed that the expression of OCT4 IgG in positive vascular involvement group was significantly higher than negative vascular involvement group(P =0.019).The expression of OCT4 IgG in HCC patients' plasma(stage of 0+A,B or C+D)was significantly increased(P =0.031;P =0.002;P <0.001),and the expression was increased with the tumor progression.The result of ROC analysis showed that the sensibility of OCT4 IgG for diagnosis of HCC was 18.9%,when specificity was 95%.The sensibility of OCT4 IgG for diagnosis of HCC in stage C+D was 26.9%.The Kaplan-Meier survival analysis showed that the expression of OCT4 IgG in HCC patients' plasma was negatively related to the prognosis.Multivariate Cox regression analysis indicated that BCLC staging,extrahepatic metastasis,vascular involvement and OCT4 IgG level were important factors affecting the prognosis of patients with HCC.(3)The expression of autoantibody of p16.Compared with healthy control group,p16 a IgG was significantly increased in HCC group(P <0.001).Compared with healthy control group,the expression of p16 a IgG of HCC patient was increased no matter in different genders and ages.Compared with control group,the expression of p16 a IgG in HCC patients' plasma(stage of B or C+D)was significantly increased(P <0.001;P <0.001),and the expression was increased with the tumor progression.The ROC analysis showed that the sensibility of p16 a IgG for diagnosis of HCC was 17.2%,when specificity was 95%.It showed the similar result with a sensibility of 11.4%,when we tried to test p16 a through a modified method.Moreover,the trend of antigen peptides derived from the same protein in HCC patients was also different.There was no statistically significant difference in plasma level of p16 b IgG between HCC patients and healthy controls(P =0.981).(4)The expression of autoantibodies of BIRC5,c-myc and ANXA-1.There were no significant differences in the plasma levels of BIRC5 a,BIRC5b,c-myc and ANXA-1 IgG between HCC group and healthy control group(P =0.376;P =0.529;P =0.301;P =0.062).Conclusion(1)The autoantibodies levels of CD25 and p16 a are significantly increased in the plasma of HCC patients with intermediate and terminal stage.It has certain diagnostic value for HCC.Compared with AFP normal or lower expression group,the autoantibody level of CD25 is significantly increased in AFP high expression group.The detection of CD25 autoantibody can be combined with AFP to assist the diagnosis of liver cancer.(2)The expression of OCT4 autoantibody in HCC patient is significantly increased along the BCLC stage differing,and the autoantibody level of OCT4 is negatively related with prognosis.Autoantibody of OCT4 may be a potential biomarker for HCC,which trends to be developmentally valuable to the assessment of HCC prognosis.(3)The expression of autoantibodies of p16 b,BIRC5,c-myc and ANXA-1 have no significantly difference between the two groups.Our previously published studies showed that these levels of autoantibody had abnormally expression in other tumors,which indicated that the autoantibodies expression level in different tissue type were different and they're highly organ-specific.(4)The designing way of antigenic peptides and the optimized ELISA testing method show significant effect on detection of antibody.We screen out a key epitope in p16 protein structure to trigger immunity by designing multiple homologous antigen polypeptide,which would establish a base for optimizing detection of antibody and clinical application in the future.