Identification Of Autoantibodies And Tumor-related Candidate Peptides In Serum Of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC) with 25-40%5-year survival rate is one of the common malignant tumors in China.Early detection is one of the most feasible means of improving outcomes.The identification of tumor associated antigens(TAAs) and autoantibody would improve the early diagnosis of cancer and develop novel immunotherapies.Serological analysis of recombinant cDNA expression libraries(SEREX) and serological proteome analysis(SERPA) based on 2-dimmensional electrophoresis(2-DE) are the main strategies for TAA identification. As one of important blood components,serum is easy to be obtained and containing abundant disease information.SELDI-TOF MS is the strategy broadly used for serum protein and peptide profiles.Firstly,TAAs in cytosolic fraction of ESCC cell line EC0156 were identified using modified serological proteome analysis and the biological characteristics of these antigens were investigated in advance. Autoantibody against these antigens in sera of ESCC patients and healthy controls was validated and evaluated.There were eight non redundant TAAs identified in this study.The four antigens phosphoglycerate kinase 1(PGK1),105kDa heat shock protein(HSP105),triosephosphate isomerase 1(TIM) andα-enolase(ENO1) were over-expressed in ESCC tissue and located in cytoplasma and nucleus in cancer cells. Different isoforms and degradated fragments of these four antigens were found in cytosolic fraction.Autoantibody against TIM or ENO1 in ESCC serum mainly reacted with its glycosylated protein.Autoantibody against PGK1,HSP105,TIM and ENO1 was detected in sera from 26.9%(7/26),39.1%(9/23),34.8%(8/23) and 78.3% (18/23) patients with ESCC,respectively.Autoantibody against PGK1 and ENO1 was detected in sera from 5.5%(1/18) and 25%(5/20) healthy controls(HC),respectively; whereas autoantibody against HSP105 or TIM was not detected in sera of healthy controls.These four antigens and autoandibodies may be the potential biomarkers of ESCC.In another research work,we compared the protein/peptide profiles of sera from ESCC with those from healthy controls based on previous study in our laboratory.The one classifier for discriminating ESCC and HC were established.The peak 4097Da and(or) 7774Da in ESCC serum was identified as fragment ofα-enolase using antibody capture method.The concentration ofα-enolase in serum of ESCC was significantly higher than that in pancreatic cancer,hepatocellular carcinoma and HC.The proteinα-enolase may be a potential biomarker for ESCC diagnosis.Hepatocellular carcinoma(HCC) is one worldwide malignant tumor and its 5-year survival rate is less than 10%.Alpha-fetoprotein(AFP) is only an available serologic biomarker for HCC diagnosis,whereas about 30%of HCC patients with low level of serum AFP(＜25ng/ml).ClinProt technology is a novel strategy to research clinical proteome and protein/peptide biomarkers.This method was employed to uncover the different serum protein/peptide profiles of patients with AFP negative hepatocellular carcinoma(HCC) and liver cirrhosis.Five protein peaks were selected to construct the HCC classifier with 92.86%(26/28) sensitivity and 93.94%(31/33) specificity in training cohort.The pattern was able to classify the blind test cohort with 95.6% (43/45) sensitivity and 90.91%(20/22) specificity,of which the positive rate of AFP~-HCC and AFP~+ HCC is 91.67%(22/24) and 100%(21/21),respectively.The pattern may be significant for auxiliary diagnosis of HCC.Two of protein peaks 7758Da and 9278Da were purified and identified as 13 non-redundant proteins by peptide mapping.Proteoglycan-4(PRG4) was over-expressed in HCC tissue and serum,and mainly located on hepatocellular cytoplasma.PRG4 could be served as potential biomarkers of HCC.Pancreatic cancer(PC) has the lowest survival rate for any solid cancer and its 5-year survival is only 1-5%.We compared the protein/peptide profiles of sera from PC patients with those from healthy controls.The one classifier consisting of four peaks for discriminating PC and HC were established.The peak 5344Da was identified as platelet factor 4(PF4) using antibody capture method,and its expression level in serum of PC need to be further validated.