The Study Of Diagnostic Biomarker For Early Stage Hepatocellular Carcinoma And The Infectious Etiology Of Esophageal Cancer

Background and ObjectionHepatocellular carcinoma (HCC) is one of the most malignant tumors worldwide with extremely poor prognosis due to be diagnosed at later stages. The useful biomarkers of HCC remains incompletely developed. Its early detection and treatment is an effective way to improve patients’ survival. Alpha-fetoprotein (AFP), a widely used serological biomarker for HCC diagnosis, is limited in the early detection of HCC. Identification of new biomarkers may improve early diagnosis of HCC as well as better understanding the mechanisms underlying tumorigenesis. In our previous study, by use of a subcellular proteomic approach, we identified differentially expressed proteins in HCC cell lines. Annexin A2, one of several candidate proteins, was chosen for further validation. As a calcium-dependent phospholipid-binding protein that plays an important role in the regulation of signal transduction and cellular growth pathways, annexin A2was found over-expressed in HCC tissues compared with benign liver disease. The aim of this study is to further verification the characteristics and diagnostic value of annexin A2expression in cancerous tissues and sera of patients with HCC.Esophageal carcinoma (EC) is the eighth most prevalent malignant disease and ranks as sixth leading cause of cancer death worldwide. EC can be histologically classified into two main types:esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA). ESCC constituted the large majority (over90%) of all EC cases in the world. EC exhibits distinct geographic distribution between countries, as well as between different regions of the same country. A rise of EC has been noted in the high risk countries for EC, namely the People’s Republic of China, South Africa, Iran, Chile and Brazil et al. Epidemiological studies have suggested many factors are involved in ESCC carcinogenesis including heredity, alcohol use, tobacco consumption, nutritional deficiencies, poor oral health, high intake of nitrites, consumption of high-temperature food and infections. In recent years, more and more studies suggested that viral infections, in particular with human papillomavirus (HPV) infections, may play an important role in the etiology of ESCC. The HPV virion is a single circular double stranded DNA virus that belongs to the papillomaviridae family. To date, more than100HPV genotypes have been identified. According to their risk of causing cervical cancer, HPV have been broadly categorized into high-risk, low-risk and probable high-risk subtypes. The association between HPV and ESCC was first reported by Syrjanen in1982.Since then, many reports regarding this topic have been published, and the findings have been contradictory and inconclusive. It has been suggested that specimens obtained from low-or high-risk areas, the various analysis methods used in each study and contamination during sample preparation are likely the main source of the variability in the HPV detection rates in ESCC. Many molecular methods (such as in situ hybridization and PCR-based methods) have been developed to detect HPV-DNA and identify HPV genotypes. matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) system (PCR-MS) shows more sensitivity and specifity for detecting HPV DNA in serum/tissue from some cancers than either Hybrid Capture2or TaqMan QPCR. To definitively evaluate the association between HPV and ESCC, we examined the presence of HPV DNA in fresh frozen ESCC tissues and peripheral blood samples collected from two areas with different ESCC incidence rates in China by use of PCR-MS and general-primer-mediated PCR. Several published papers had reported some HPV positive ESCC cell lines, and they were widely used in the study of HPV-related carcinogenesis of esophagus, but these cell lines’ true identities need to be verified.As extremely useful materials, cell lines derived from ESCC tissues play an important role in study of biology of EC and have been utilized by many research groups and drug development to improve our understanding of tumor growth and treatment. Several published papers had reported some HPV positive ESCC cell lines, and they were widely used in the study of HPV-related carcinogenesis of esophagus, but these cell lines’ true identities need to be verified. Long-term in vitro cell culture is susceptible to cross-contamination by other cell lines during routine manipulation. Because of labeling errors or cross-contamination by other cell types during routine culture, many cell lines in current use may not be what they are ought to be. Although cell line cross-contamination and misidentification have received more and more attention, few ESCC cell lines have been assembled and genotyped. The aim of this study was to authenticate9ESCC cell lines widely used in China by use of short tandem repeat (STR) profiling and detect HPV in these cell lines.The causal role of HPV infection in esophageal squamous cell carcinoma (ESCC) remains unclear, in part because of a relatively low HPV copy number and infection rate in ESCC which may be explained by the decrease of HPV viral load after infection and/or a’hit and run’ mechanism. The most commonly used method for detection of serum antibodies is the enzyme-linked immunosordent assay (ELISA), but the antigen used in this method are difficult to obtain, furthermore, ELISA can not identify the epitope of antibody recognition. Peptide scan technology, design artificially synthetic polypeptide analog protein antigen, could be applied not only to detect the autoantibodies inthe sera of patients with esophageal cancer, but also to map the epitopes of the autoantibodies. Because the HPV L1antibodies are considered a life time measure of exposure to HPV infection; however, the assay is not a suitable screening measure for identifying current HPV-related tumors. Antibody response has been shown to the oncogenic viral proteins E6and/or E7of HPV-16, has been shown to provide a better, more sensitive marker for HPV-associated esophageal cancers. The E6/E7antibody response had a much higher correlation with tumor HPV DNA presence than did the L1antibodies. So our objective is to synthesize peptide array to analyze HPV16E6/E7auto-antibodies in the sera of patients with esophageal cancer.Materials and MethodsThe expression of annexin A2in a large number of195HCC tumor and paired adjacent non-tumor liver tissue samples were detected by immunohistochemistry. Serum levels of annexin A2protein expression in404samples, including95early and80late stage HCC,142non-cancerous controls (23hepatitis,51posthepatitic cirrhosis,19liver benign tumors and49healthy controls) and87other cancers were quantitatively measured by used of an established ELISA. Also, serum AFP was measured by a commercially available kit. The specificity and sensitivity of serum annexin A2and AFP measurements were represented by receiver operating characteristic curve (ROC) curves. Subsequently, the tissue and serum samples from multiple time points were collected and analyzed for annexin A2during hepatocarcinogenesis of p21-HBx gene knockin transgenic mice model.A total of74ESCC frozen tumor tissues and paired adjacent non-cancerous tissue samples, including39cases from Anyang areas with a high incidence of ESCC and35cases from Beijing with low incidence of ESCC, were collected from ESCC patients undergoing resection. In addition,239peripheral blood samples were collected from patients with ESCC and healthy controls. Careful attention has been drawn to minimize cross-contamination between samples and potential of DNA contamination by environmental HPV during specimen collection and DNA extraction and HPV DNA testing. The presence of HPV DNA was assessed by new PCR-MS platform that can detect a broader spectrum of HPV genotypes included15high-risk HPV types (16,18,31,33,35,39,45,51,52,56,58,59,68,73,82),12low-risk HPV types (6,11,40,42,43,44,54,61,70,72,81,6108) and3probable high-risk HPV types (26,53,66). Also, the same samples were analyzed by general-primer-mediated PCR with consensus MY09/MY11primer pair and HPV genotypes were identified by direct sequencing of amplified products. Genomic DNA from9ESCC cells cell lines (KYSE140, KYSE150, KYSE170, KYSE180, KYSE410, KYSE510, WHCO1, EC109and EC9706) was extracted by use of commercially available kit. Authentication was verified by performing STR profiling and the resulting genetic profiles were compared with STR profile databases of four cell banks. HPV status in these cell lines were also detected by PCR-MS platform. Peptide array which covered all the peptides of HPV16E6E7were synthesized by a synthesizer. The peptide library were18amino acids long peptides with16amino acids overlapping with the adjacent peptides immobilised on a cellulose membrane. The membrane was incubated with commercial antibodies, pooled serum of ESCC patients and healthy controls.Statistical Analysis:Data were expressed as median (range) or mean±standard deviation as appropriate. Continuous variables were compared by Mann-Whitney U or Kruskal-Wallis H test as appropriate. Independent samples t-test was used for the mean comparison. The Chi-square test was applied for categorical variables. The overall accuracy of annexinA2in HCC diagnosis was calculated using the area under receiver operating characteristics curve (ROC) and its95%confidence internal(CI). All data was analyzed using the Statistical Package for Social Science (version19.0, SPSS Inc, Chicago, IL). Any P values (two tailed) below0.05were considered statistically significant.ResultsThe overexpression of annexin A2was observed in59.5%(116/195) of HCC tissues and12.8%(25/195) of matched adjacent non-cancerous tissues, respectively.(X2test, P<0.0001). Using a self-established ELISA, we found that annexin A2significantly increased in the sera of HCC (median,24.75ng/μl) compared with the healthy controls (median,16.70ng/μl), hepatitis (median,6.48ng/μl), posthepatitic cirrhosis (median,7.39ng/μl),(Mann-Whitney test, P<0.0001), benign tumors (median,19.92ng/μl),(Mann-Whitney test, P=0.002) and other malignant tumors (median,3.75ng/μl),(Mann-Whitney test, P<0.0001).Raised concentrations of annexin A2were observed in83.2%(79/95) of early stage (median,24.32ng/μl) and78.4%(58/74) of AFP-negative (median,24.09ng/μl) patients. Measuring annexin A2and AFP in serum can improve the reciprocally holistic diagnostic value (AUC=0.86,95%confidence interval:0.81-0.90). Annexin A2alone had a better area under the ROC curve (AUC=0.79,95%confidence interval:0.73-0.85) in comparison with AFP (AUC=0.73,95%confidence interval:0.66-0.80) in detecting of early stage HCC. Combining both markers notably improved the diagnostic efficiency of early HCC with an achieved sensitivity of87.4%. During hepatocarcinogenesis, Annexin A2was strongly expressed in the HCC tissues of tumor-bearing transgenic mice compared with normal liver tissues of wild-type or adjacent non-cancerous liver tissues of transgenic mice at different times. The transgenic mice harboring HCC had significantly higher serum annexin A2than those without tumors. The results showed that annexin A2expression was substantially elevated in HCC-bearing mice, in accordance with the finding in human samples.Among148tissues DNA and239blood DNA with adequate human P-globin, none of these samples tested positive for HPV DNA by PCR-MS analysis. And all samples were negative for HPV DNA by consensus PCR. All KYSE series, EC109and WHCO1cell lines had a unique genetic profile, The9loci of KYSE series (except KYSE410) and EC109matched the STR profiles of those cell lines in the cell banks’ databases. The STR profile of WHCO1cell line has not yet been deposited but they were distinct from any cell line reported in the current STR profile databases maintained by four cell banks. The STR profile of EC9706was identical to those of HeLa and the genotype of KYSE410was identical to that of KYSE150cell in the cell bank’s databases. No HPV DNA was found in any of authenticated ESCC cell lines. Commercial HPV16E6polyclonal antibodies recognize epitopes located in MHQKRTAMFQDPQERP RKLPQLCTELQTTIHD, commercial HPV16E7monoclonal antibodies recognize epitopes on TTDLYCYE. Antibodies against HPV16E6E7in mixed serum of the healthy individuals was rare, less recognition site were found. However, ESCC patients’mixed sera had more HPV16E6E7autoantibodies which were detected by peptide array. And these HPV16E6E7autoantibodies recognized some common epitopes.ConclusionAnnexin A2may be an independent serological candidate for HCC, especially in the early stage cases with normal serum AFP. Further independent and prospective studies are needed to validate pathologic characteristics and efficiency of annexin A2in HCC diagnosis.By paying great attention to sample procedures and using the sensitive and specific techniques available, we did not detect any HPV DNA in the ESCC tissues, peripheral blood and ESCC cell lines. Study of larger numbers of samples may be required before a definite conclusion regarding. Through STR profiling, we showed that the cell line EC9706and KYSE410are misidentified. EC109and EC9706cell lines might be contaminated by other HPV-positive cell lines in some reports which claimed these cell lines harbor HPV. Investigators should be aware of the continuing problem of cell line cross-contamination. This effort will considerably reduce cell contamination and thus improve the quality of ESCC research. Through epitope scanning, we found HPV16E6/E7autoantibodies are more common in serum of ESCC than healthy controls, specific recognition some common epitopes. The discover of the epitopes of HPV16E6/E7with high frequency and the address of the these epitopes will offer new clues for the further research the association between ESCC and HPV.