Autoantibodies as potential biomarkers for nasopharyngeal carcinoma

Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma (NPC). We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor-associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage-displayed proteins, 22 have sequence identity with known or putative tumor-associated proteins. The results of immunochemical reactivity of patients' sera with phage-expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors. The autoantibody against phage-expressed protein MAGE, HSP70, Fibronectin, and CD44 measured by ELISA had greater predictive value than that against EBNA-1, respectively. The antibody levels against MAGE in sera positively correlated with the clinical stages of NPC, and the antibody levels against other three proteins partly correlated with the clinical stages of NPC. Our studies suggested that the autoantibodies against tumor-associated antigens in the sera of NPC patients could be used as a screening test for NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy.     

Identification of circulating antibodies to tumor-associated proteins for combined use as markers of non-small cell lung cancer

Currently only a limited number of tumor markers for non-small cell lung cancer (NSCLC) are available. Antibodies to tumor-associated proteins may expand the number of available tumor markers for lung cancer and be used together in a serum profile to enhance sensitivity and specificity. In this study, we isolated 57 tumor-associated proteins from two NSCLC cDNA T7 phage libraries using biopan enrichment techniques with NSCLC patient and normal sera. Sequence analysis showed that among the 57 phage-displayed proteins 45 have sequence identity with known or putative tumor-associated proteins. Immunochemical reactivity of patient sera with phage-expressed proteins showed enrichment on the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in patient sera but not in normal sera. Antibodies to five phage-expressed proteins were measured by enzyme-linked immunosorbent assay (ELISA) to validate the concept that combinations have greater predictive value than any single antibody alone. Logistic regression analysis showed that combined measurements of five antibodies was more predictive of disease than any single antibody alone, underscoring the importance of identifying multiple potential markers. The resulting antibody profiling is a feasible diagnostic strategy for NSCLC. An inventory of corresponding proteins may have significant relevance to tumor biology, novel drug development, and immunotherapies.     

Using proteomic approach to identify tumor-associated antigens as markers in hepatocellular carcinoma

Many studies have demonstrated that intracellular proteins, which are involved in carcinogenesis, can provoke autoantibody responses. Therefore, autoantibodies can be used clinically for cancer detection and for proteomic analysis in identification of tumor-associated antigens (TAAs) that are potentially involved in malignant transformation. Liver cancer, especially hepatocellular carcinoma (HCC), is one of the most common tumors in the world. The majority of people with HCC will die within 1 year of its detection. This high case fatality rate can partially be attributed to a lack of diagnostic methods that allow early detection. In the present study, sera from 20 patients with HCC, 30 patients with chronic hepatitis (CH), and 30 patients with liver cirrhosis (LC) as well as sera from 10 normal individuals were used in a proteomic approach to identify HCC-related TAAs. Thirty-four immunoreactive protein spots were excised from the two-dimensional gel electrophoresis (2DE), digested with trypsin, and subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of 34 immunoreactive protein spots, 28 were identified. Seventeen of them were not only reactive with serum antibodies in HCC but also with antibodies in pre-HCC conditions, and 11 were only reactive with serum antibodies in HCC but not with antibodies in pre-HCC conditions. In the subsequent analysis, two representative proteins, HSP60 and HSP70, were selected as examples for the validation purpose. The results from immunoassay were consistent with the data from proteomic analysis, supporting our hypothesis that proteins identified with autoantibodies that have been present in precancer conditions may be not appropriate to use as TAA markers in cancer detection.     

Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display

Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.     

Identification of MST1/STK4 and SULF1 proteins as autoantibody targets for the diagnosis of colorectal cancer by using phage microarrays

The characterization of the humoral response in cancer patients is becoming a practical alternative to improve early detection. We prepared phage microarrays containing colorectal cancer cDNA libraries to identify phage-expressed peptides recognized by tumor-specific autoantibodies from patient sera. From a total of 1536 printed phages, 128 gave statistically significant values to discriminate cancer patients from control samples. From this, 43 peptide sequences were unique following DNA sequencing. Six phages containing homologous sequences to STK4/MST1, SULF1, NHSL1, SREBF2, GRN, and GTF2I were selected to build up a predictor panel. A previous study with high-density protein microarrays had identified STK4/MST1 as a candidate biomarker. An independent collection of 153 serum samples (50 colorectal cancer sera and 103 reference samples, including healthy donors and sera from other related pathologies) was used as a validation set to study prediction capability. A combination of four phages and two recombinant proteins, corresponding to MST1 and SULF1, achieved an area under the curve of 0.86 to correctly discriminate cancer from healthy sera. Inclusion of sera from other different neoplasias did not change significantly this value. For early stages (A+B), the corrected area under the curve was 0.786. Moreover, we have demonstrated that MST1 and SULF1 proteins, homologous to phage-peptide sequences, can replace the original phages in the predictor panel, improving their diagnostic accuracy.     

Listeria Phage and Phage Tail Induction Triggered by Components of Bacterial Growth Media (Phosphate,LiCl, Nalidixic Acid,and Acriflavine)

The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na₂HPO₄ at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na₂HPO₄ and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.     

BMI-1 autoantibody as a new potential biomarker for cervical carcinoma

BMI-1 is overexpressed in a variety of cancers, which can elicit an immune response leading to the induction of autoantibodies. However, BMI-1 autoantibody as a biomarker has seldom been studied with the exception of nasopharyngeal carcinoma. Whether BMI-1 autoantibodies can be used as a biomarker for cervical carcinoma is unclear. In this study,BMI-1 proteins were isolated by screening of a T7 phage cDNA library from mixed cervical carcinoma tissues. We analyzed BMI-1 autoantibody levels in serum samples from 67 patients with cervical carcinoma and 65 controls using ELISA and immunoblot. BMI-1 mRNA or protein levels were over-expressed in cervical carcinoma cell lines. Immunoblot results exhibited increased BMI-1 autoantibody levels in patient sera compared to normal sera. Additionally, the results for antibody affinity assay showed that there was no difference between cervical polyps and normal sera of BMI-1 autoantibody levels, but it was significantly greater in patient sera than that in normal controls (patient 0.827±0.043 and normal 0.445±0.023; P<0.001). What''s more, the levels of BMI-1 autoantibody increased significantly at stage I (0.672±0.019) compared to normal sera (P<0.001), and levels of BMI-1 autoantibodies were increased gradually during the tumor progression (stage I 0.672±0.019; stage II 0.775 ±0.019; stage III 0.890 ±0.027; stage IV 1.043±0.041), which were significantly correlated with disease progression of cervical carcer (P<0.001). Statistical analyses using logistic regression and receiver operating characteristics (ROC) curves indicated that the BMI-1 autoantibody level can be used as a biomarker for cervical carcinoma (sensitivity 0.78 and specificity 0.76; AUC = 0.922). In conclusion, measuring BMI-1 autoantibody levels of patients with cervical cancer could have clinical prognostic value as well as a non-tissue specific biomarker for neoplasms expressing BMI-1.     

Proteomic analysis of autoantibodies in patients with hepatocellular carcinoma

To detect autoantibodies that could be diagnostic markers for hepatocellular carcinoma (HCC), we analyzed serum autoantibodies comprehensively that showed immunoreactivity to proteins in tumor tissue obtained from patients with HCC. Fifteen paired samples of HCC tissue and corresponding nontumorous liver tissue as well as five normal liver tissue samples were used in the study. A combination of proteomics and SEREX (serologic analysis of recombinant cDNA expression libraries) technique was used. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified four immunoreactive spots with stronger staining intensity in tumorous tissues than in corresponding nontumorous tissues and in normal liver tissues. Matched proteins on 2-DE gels were identified by LC-MS/MS. These immunoreactive proteins were heat shock 70 kDa protein 1 (HSP70), glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and manganese superoxide dismutase (Mn-SOD). In HCC sera, occurrences of autoantibodies against these proteins were 7/15 (46.7%), 5/15 (33.3%), 5/15 (33.3%), and 6/15 (40.0%), respectively, whereas 2/20 (10.0%), 7/20 (35.0%), 0/20 (0.0%), and 2/20 (10.0%) were in control sera. Immunoblot analysis using commercially available purified proteins was performed to confirm the specificity of autoantibodies. By statistical analysis, autoantibodies against HSP70, peroxiredoxin, and Mn-SOD showed significantly high-frequency immunoreaction in HCC sera. The three antibodies were considered patient-specific antibodies in HCC and may be candidate diagnostic biomarkers for HCC.     

A distinct repertoire of autoantibodies in hepatocellular carcinoma identified by proteomic analysis

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for hepatocellular carcinoma (HCC). We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. Sera from 37 patients with HCC and 31 subjects chronically infected with HBV or HCV without HCC were investigated. Sera from 116 patients with other cancers, three patients with systemic lupus erythematosus, and 24 healthy subjects were utilized as controls. We report the identification of eight proteins, for each of which autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p < 0.05). Autoantibodies to four of these proteins were detected at a comparable frequency in sera from patients with chronic hepatitis. The other four proteins, which consisted of calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A, and F(1)-ATP synthase beta-subunit, induced autoantibodies among patients with HCC, independently of their HBV/HCV status. Calreticulin, and a novel truncated form of calreticulin (Crt32) we have identified, most commonly elicited autoantibodies among patients with HCC (27%). We conclude that a distinct repertoire of autoantibodies is associated with HCC that may have utility in early diagnosis of HCC among high risk subjects with chronic hepatitis.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

Identification of Novel Autoantibodies for Detection of Malignant Mesothelioma

Background

The malignant mesothelioma (MM) survival rate has been hampered by the lack of efficient and accurate early detection methods. The immune system may detect the early changes of tumor progression by responding with tumor-associated autoantibody production. Hence, in this study, we translated the humoral immune response to cancer proteins into a potential blood test for MM.

Methodology/Principal Findings

A T7 phage MM cDNA library was constructed using MM tumor tissues and biopanned for tumor-associated antigens (TAAs) using pooled MM patient and normal serum samples. About 1008 individual phage TAA clones from the biopanned library were subjected to protein microarray construction and tested with 53 MM and 52 control serum samples as a training group. Nine candidate autoantibody markers were selected from the training group using Tclass system and logistic regression statistical analysis, which achieved 94.3% sensitivity and 90.4% specificity with an AUC value of 0.89 in receiver operating characteristic analysis. The classifier was further evaluated with 50 patient and 50 normal serum samples as an independent blind validation, and the sensitivity of 86.0% and the specificity of 86.0% were obtained with an AUC of 0.82. Sequencing and BLASTN analysis of the classifier revealed that five of these nine candidate markers were found to have strong homology to cancer related proteins (PDIA6, MEG3, SDCCAG3, IGHG3, IGHG1).

Conclusions/Significance

Our results indicated that using a panel of 9 autoantibody markers presented a promising accuracy for MM detection. Although the results need further validation in high-risk groups, they provided the potentials in developing a serum-based assay for MM diagnosis.     

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

Background

Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS) or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity.

Results

A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv) against these five antigens were selected using antibody phage display and characterised.

Conclusion

In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.     

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.     

Development and validation of a high throughput system for discovery of antigens for autoantibody detection

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.     

Serum anti-Ku86 is a potential biomarker for early detection of hepatitis C virus-related hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most common cancers worldwide and the third most common cause of cancer-related death. Imaging studies including ultrasound and computed tomography are recommended for early detection of HCC, but they are operator dependent, costly and involve radiation. Therefore, there is a need for simple and sensitive serum markers for the early detection of hepatocellular carcinoma (HCC). In our recent proteomic studies, a number of proteins overexpressed in HCC tissues were identified. We thought if the serum autoantibodies to these overexpressed proteins were detectable in HCC patients. Of these proteins, we focused on Ku86, a nuclear protein involved in multiple biological processes and aimed to assess the diagnostic value of serum anti-Ku86 in the early detection of HCC. Serum samples were obtained prior to treatment from 58 consecutive patients with early or relatively early hepatitis C virus (HCV)-related HCC and 137 patients with HCV-related liver cirrhosis without evidence of HCC. Enzyme immunoassays were used to measure serum levels of autoantibodies. Serum levels of anti-Ku86 antibodies were significantly elevated in HCC patients compared to those in liver cirrhosis patients (0.41±0.28 vs. 0.18±0.08Abs at 450nm, P<0001). Setting the cut-off level to give 90% specificity, anti-Ku86 was positive in 60.7% of stage I solitary tumor <2cm in diameter, whereas the sensitivities of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) were 17.8% and 21.4%, respectively. The results of ROC analyses indicated the better performance of anti-Ku86 for early detection of HCC. Serum anti-Ku86 levels decreased after surgical resection of the tumors in the 12 HCC cases tested, Elevation of anti-Ku86 in solid tumors other than liver was minimal. Serum anti-Ku86 is a potential biomarker for early detection of HCV-related HCC. Further studies in a larger number of HCC patients with various etiologies are needed to further evaluate the diagnostic and pathophysiological roles of elevation of serum anti-Ku86 in early HCC.     

Complete Genomic Sequence of Bacteriophage φEcoM-GJ1, a Novel Phage That Has Myovirus Morphology and a Podovirus-Like RNA Polymerase

The complete genome of EcoM-GJ1, a lytic phage that attacks porcine enterotoxigenic Escherichia coli of serotype O149:H10:F4, was sequenced and analyzed. The morphology of the phage and the identity of the structural proteins were also determined. The genome consisted of 52,975 bp with a G+C content of 44% and was terminally redundant and circularly permuted. Seventy-five potential open reading frames (ORFs) were identified and annotated, but only 29 possessed homologs. The proteins of five ORFs showed homology with proteins of phages of the family Myoviridae, nine with proteins of phages of the family Podoviridae, and six with proteins of phages of the family Siphoviridae. ORF 1 encoded a T7-like single-subunit RNA polymerase and was preceded by a putative E. coli σ⁷⁰-like promoter. Nine putative phage promoters were detected throughout the genome. The genome included a tRNA gene of 95 bp that had a putative 18-bp intron. The phage morphology was typical of phages of the family Myoviridae, with an icosahedral head, a neck, and a long contractile tail with tail fibers. The analysis shows that EcoM-GJ1 is unique, having the morphology of the Myoviridae, a gene for RNA polymerase, which is characteristic of phages of the T7 group of the Podoviridae, and several genes that encode proteins with homology to proteins of phages of the family Siphoviridae.     

Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.     

Preliminary study of thyroid and colon cancers-associated antigens and their cognate autoantibodies as potential cancer biomarkers

Background: Autoantibodies, which are produced against tumor-associated antigens, are potential tumor markers and attract a growing interest for cancer detection, differential diagnostics and prognosis.

Objective: To evaluate the diagnostic significance of 40 antigens identified by immunoscreening of cDNA libraries from thyroid and colon cancers by allogenic screening with different tumor types patients’ sera.

Method: Plaque-spot serological assay.

Results: Increased frequency of antibody response in sera of cancer patients compared with that of healthy donors was shown toward 14 antigens, 8 of which (CG016, BTN3A3, FKBP4, XRCC4, TSGA2, ACTR1A, FXYD3 and CTSH) have revealed exclusively cancer-related serological profile.

Conclusion: Allogenic screening of 40 SEREX-antigens with sera from cancer patients and healthy donors allowed us to reveal 14 antigens with potential diagnostic significance. These antigens and their cognate autoantibodies could be considered as valuable targets for further analysis as potential cancer biomarkers.     

Correlation of zinc finger protein 2, a prognostic biomarker,with immune infiltrates in liver cancer

Purpose: The expression and clinical value of zinc finger protein 2 gene (ZIC2) in hepatocellular carcinoma (HCC) were analyzed by mining gene information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases.Methods: Gene chip data sets were retrieved from GEO and TCGA and screened for differentially expressed genes in HCC. Gene expression profile interaction analysis (GEPIA) and Kaplan–Meier curves were used to analyze the relationship between differentially expressed genes (DEGs) and survival and prognosis in patients with HCC. Moreover, the Genecards database was used to extract ZIC2-related proteins and to analyze the physiological process of protein enrichment. Furthermore, the relationships between ZIC2 gene and tumor cell immune invasion and that between immune cell infiltration and the 5-year survival rate were studied using the tumor immune evaluation resource (TIMER) database.Results: Datasets from GEO and TCGA revealed that ZIC2 was differentially expressed in HCC tissues and normal tissues (P<0.05). High ZIC2 expression was associated with overall survival (OS) and progress-free survival in HCC patients. Overall, 25 ZIC2 related proteins, including Gli3, PRKDC, and rnf180 were identified and protein enrichment analysis indicated these were associated with four types of cell components, six types of cell functions, and eight types of biological processes. ZIC2 was positively correlated with immune infiltration cells in patients with HCC, and higher expression of ZIC2 mRNA CD4+T cells is associated with a better 5-year survival.Conclusion: ZIC2 gene may be used as an immune response marker in liver cancer to predict the prognosis of HCC.     

Altered glycosylation,expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C,hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients’ sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients’ group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients’ group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients’ group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients’ groups, to the contrary high glycan branching was observed in HCC patients’ group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients’ group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.