Study On The Relationship Between The Levels Of Antibodies Against Tumor-associated Antigens In Peripheral Blood And The Early Diagnosis And Prognosis Of Non-small Cell Lung Cancer

Objective:At present,tumor-associated antigens(TAAs)are the main targets for clinical tumor detection.Tumor-associated autoantibodies are products of the body's humoral immune response to tumor-associated antigens.Compared with TAAs,its structure is stable and not easily hydrolyzed by proteases,and can be stably existed in the blood for a long time.Duo to the immunoamplification effect,it can exist in the blood at a higher level even when the concentration of tumor antigen is very low.Several studies have reported that some autoantibodies can be detected in the blood of lung cancer patients before clinical diagnosis.Therefore,it is believed that tumor-associated autoantibodies have an advantage in the early diagnosis of cancer compared to TAAs.Tumor development is a complex and multi-step process in which many genes involved in maintaining normal physiological activities and cell functions are mutated due to genomic instability.Therefore,the changes of their protein products may help us to understand tumor progression.At present,there are many methods for autoantibody detection.However,they are not suitable for large-scale clinical application because of the cumbersome testing procedures,high cost,and high technical requirements.In the early stage,our research team used online tools to design and synthesize antigen peptides,and verified the detection efficiency of these antigen peptides by detecting serum autoantibodies in the plasma of cancer patients such as esophageal cancer and liver cancer.At the same time,some drawbacks of the antigenic peptide have been found.For example,the antigenic peptide is easy to form crosslinks,and the solid phase is not firmly bonded.Therefore,based on previous studies,our research team selected 5 tumor-associated antigens or immunomodulatory molecules related to cancer development,namely immunomodulatory molecule CD25,FOXP3,and apoptosis inhibitory gene Survivin.(BIRC5),tumor suppressor gene p16 and annexin-A1(ANXA-1).In order to improve the efficiency of autoantibody detection,based on the preliminary work,this study carried on appropriate modification of antigenic peptides and synthesizes multiple linear antigenic peptides for different epitopes in the same protein to screen for the key epitope that could induce humoral immune response.Using a site-specific enzyme-linked immunosorbent assay(ELISA)to detect the expression of autoantibodies against tumor-associated antigens or immunoregulatory molecules in the plasma of patients with non-small cell lung cancer,combined with the pathological staging,histological type and overall survival of non-small cell lung cancer,we comprehensive analyze the relationship between the levels of autoantibodies and the early diagnosis and prognosis of lung cancer,with a view to discovering serological markers that can be used for the early diagnosis and prognosis evaluation of lung cancer.Methods:Plasma samples,demographic and clinical information,follow-up data were collected from 211 first-episode(untreated)NSCLC patients and 200 healthy subjects matched by gender,age and smoking history.Based on the TNM(tumor,node and metastasis)staging information,these 211 patients were classified into four subgroups: group I for stage T1N0M0,group II for stage T1N1M0+T2N0M0,group III for stage T2N1M0+T3N0M0 and group IV for stage ?.In this study,10 antigenic peptides were designed and synthesized,namely CD25 a,CD25b,CD25 c,FOXP3,BIRC5 a,BIRC5b,p16 a,p16b,p16 c and ANXA-1.Using healthy subject as control,a site-specific ELISA method was established to detect the levels of autoantibodies in the plasma of non-small cell lung cancer patients and healthy controls.By analyzing the correlation between plasma autoantibody levels and clinical data and follow-up information of patients with non-small cell lung cancer,the value of biological markers for early diagnosis and prognosis evaluation of NSCLC were assessed.SPSS22.0 statistical software was used for data management and statistical analysis and study the relationship between the levels of autoantibodies and early diagnosis and prognosis of NSCLC.The differences of autoantibody levels between the patient group and the control group was compared from gender,age,histological type and clinical stage.Receiver operating characteristic(ROC)curve was performed to assess the value of different autoantibodies for early diagnosis of lung cancer;Linear correlation analysis was used to explore the correlation between autoantibody levels and total Ig G;chi-square test was used to analyze the correlation between autoantibody levels and clinical parameters of NSCLC patients;Kaplan-meier curve and Cox regression were used to explore the relationship between the levels of autoantibodies and overall survival(OS)and the factors that affecting the prognosis of NSCLC patients.Results:(1)Compared with healthy controls,plasma levels of CD25 a,FOXP3,BIRC5 a,p16a,ANXA-1 Ig G in NSCLC patients were significantly higher(P < 0.001).plasma levels of CD25 b Ig G in NSCLC patients were significantly lower(P < 0.001).In addition,the expression pattern of autoantibodies against different linear antigen peptide derived from the same protein in NSCLC patients was also different.Through the subgroup analysis for different gender,age,histopathological type and tumor stage,We found that the expression of CD25 b Ig G decreased significantly in < 60 years old female patients with adenocarcinoma(P < 0.001),and the alteration mainly occurred in the early stage of NSCLC(? + ? group);The expression of FOXP3,BIRC5 a and ANXA-1 Ig G increased in>60 years old female patients with squamous cell carcinoma and this alteration mainly occurred in the middle and late stage of NSCLC.The levels of CD25 a and p16 a Ig G in NSCLC patients of different gender,age and type was significantly higher than that in healthy controls and the expression increased gradually with the progression of lung cancer.(2)ROC curve analysis showed that the sensitivity of 14.2 against a specificity of 95.0% for anti-CD25 a and anti-CD25 b Ig G assay,the sensitivity of 24.2 for anti-p16 a Ig G assay when the specificity was 95.0%.The sensitivity of CD25 b Ig G assay for early diagnosis of lung cancer reached 25%.The sensitivity of p16 a Ig G for NSCLC patients in group?-? was higher than that of patients in group I(T1N0M0).(3)Chi-square test showed that the levels of CD25 b Ig G were significantly associated with differentiation grade of NSCLC,but the correlation was disappeared after Bonferroni correction.Kaplan-meier survival analysis showed that the expression of plasma BIRC5 b Ig G was positively correlated with the prognosis of NSCLC patients.COX regression analysis found that subgroup that classified based on the TNM staging,tumor differentiation grade and BIRC5 b Ig G were independent predictors of the prognosis of NSCLC patients.However,only the subgroup was significantly associated with the prognosis of NSCLC after Bonferroni correction.Conclusion:(1)Our study established a novel protein epitope-based peptide design method and optimized ELISA method for autoantibody detection,which laid a foundation for the detection of target antibodies and clinical applications.(2)The plasma levels of CD25 a,FOXP3,BIRC5 a,p16a and ANXA-1 Ig G significantly increased in the plasma of NSCLC.They may have certain value for the diagnosis and prognosis evaluation of lung cancer,but the sensitivity of a single autoantibody can not meet the requirement of clinical diagnosis.(3)The expression of CD25 b Ig G significantly decreased in NSCLC patients of group I,which may contribute to the development of lung cancer.The sensitivity of CD25b Ig G for NSCLC patients in group I was higher than that for patients in other groups.Thus,CD25 b Ig G may be a biomarker for early diagnosis of NSCLC patients.(4)Subgroup classified based on the TNM staging is an important indicator for prognosis evaluation of NSCLC.The levels of BIRC5 b Ig G was positively correlated with the prognosis of NSCLC patients,the expression of BIRC5b autoantibodies may be a protective mechanism that attempting to prevent the progression of lung cancer by neutralizng the BIRC5 protein Thus,BIRC5b IgG may be a potential biomarker to evaluate the prognosis of NSCLC.However,it is still necessary to expand the sample size to further confirm.