Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target.

Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in colorectal cancer (CRC) to determine its potential as a prognosis and immunotherapeutic target. The expression of MAGE-D4 mRNA and protein was determined by RT-PCR and immunohistochemistry (IHC) in CRCs with paired adjacent non-tumor tissues, colorectal adenomas and normal colorectal tissues, respectively. Sera from 64 CRC patients were tested for MAGE-D4 antibody by ELISA. MAGE-D4 mRNA was more frequently expressed in CRCs (76.7%, 46/60) than in adjacent non-tumor tissues (15.0%, 9/60). MAGE-D4 protein was detected in all the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; P=0.050). Humoral immunity to MAGE-D4 was detected in 12 of 64 (18.8%) CRC patients' sera but not in 77 healthy donors. There was no correlation between MAGE-D4 expression, serum antibody and clinicopathological parameters. These findings suggest MAGE-D4 may serve as a potentially prognostic biomarker and an attractive target of immunotherapy in CRC.

High expression and frequently humoral immune response of melanoma-associated antigen D4 in glioma.

MAGE-D4 is a novel member of MAGE super-family. It has preliminarily been demonstrated that MAGE-D4 mRNA is not expressed in majority of normal tissues except for brain and ovary in which only trace amount of MAGE-D4 mRNA can be detected, but predominantly expressed in glioma. MAGE-D4 protein expression and its immunogenicity in glioma have not been elucidated well. This study was designed to analyze MAGE-D4 expression both at mRNA and protein level, characteristic of humoral immune response, and their relationships with glioma patients' clinicopathological parameters. Recombinant MAGE-D4 protein and antiserum were generated. Quantitative RT-PCR analysis revealed that MAGE-D4 mRNA expression was overall up-regulated in 41 glioma specimens compared with that in 14 normal brain tissues. Immunohistochemistry analysis showed that 78% (21/27) glioma tissues expressed MAGE-D4 protein, which was predominantly located in the cytoplasm of tumor cells, but absent in any neuroglia cell of normal brain tissues. ELISA analysis demonstrated that humoral response against MAGE-D4 was detected in 17% (7/41) of glioma patients' sera but not in 77 healthy donors. No apparent correlation was observed between the expression and immunogenicity of MAGE-D4 with clinicopathological parameters of glioma. In summary, these results indicate that MAGE-D4 is highly expressed in glioma and can develop specifically humoral response in glioma patients, which supports that it may be a promising biomarker for glioma diagnosis and immunotherapy.

Cancer testis antigen OY-TES-1 expression and serum immunogenicity in colorectal cancer: its relationship to clinicopathological parameters.

Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because their expression is restricted in normal germ line tissues but frequently detected in variety of tumors. OY-TES-1 is identified as a member of CT antigens. Current knowledge about OY-TES-1 expression in colorectal cancer (CRC) is solely based on mRNA analysis. None of previous researches has studied OY-TES-1 at protein level. In this study, OY-TES-1 polyclonal antibody was generated. The expression of OY-TES-1 mRNA and protein was detected by RT-PCR and immunohistochemistry in 60 CRC and paired adjacent non-tumor tissues, 24 colorectal adenoma and 3 normal colon tissues, respectively. Sera from 73 CRC patients were also tested for OY-TES-1 antibody by ELISA. Our results showed that the frequency of OY-TES-1 mRNA expression was statistically higher in CRC (73.3%, 44/60) than that in adjacent non-tumor tissue (55.0%, 33/60) and colorectal adenoma (45.8%, 11/24). For the first time, OY-TES-1 protein expression was found in (43.3%, 26/60) of CRC tissues, but absent in any of adjacent non-tumor and colorectal adenoma tissues. No OY-TES-1 expression was found in normal colon by either RT-PCR or immunohistochemistry. Furthermore, OY-TES-1 protein expression was correlated with tumor invasion stage (P=0.004) and histological grade (P=0.040). Anti-OY-TES-1 antibody was detected in (9.6%, 7/73) of CRC patients' sera but not in 76 healthy donors. This finding demonstrates that OY-TES-1 is frequently expressed in CRC and is able to induce humoral immune response spontaneously in CRC patients, suggesting that it might be a promising immunotherapy target for CRC.

Soluble expression of recombinant human SMP30 for detecting serum SMP30 antibody levels in hepatocellular carcinoma patients.

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen, was earlier shown by our research group to be highly expressed in HCC paracancerous tissues, but have low levels in HCC tissues. In order to detect anti-SMP30 antibody in serum of HCC patients, we established pET30a-SMP30 and pColdIII-SMP30 expression systems in Escherichia coli. However, the expression product was mainly in the form of inclusion bodies. In this research, we used several combinations of chaperones, four molecular chaperone plasmids with pET30a-SMP30 and five molecular chaperone plasmids with pColdIII-SMP30 to increase the amount of soluble protein. Results showed that co-expression of HIS-SMP30 with pTf16, combined with the addition of osmosis-regulator, and a two-step expression resulted in the highest enhancement of solubility. A total of 175 cases of HCC serum were studied by ELISA to detect anti- SMP30 antibody with recombinant SMP30 protein. Some 22 were positive and x2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

[Construction of eukaryotic expression vector encoding ACRBP and its expression in hepatocarcinoma cells].

AIM: To construct the eukaryotic expression vector pEGFP-N1/ACRBP and stably express ACRBP in human hepatocarcinoma cells, providing functional clues for ACRBP. METHODS: A recombinant plasmid pMAL-C2/ACRBP was used as a template to amplify ACRBP cDNA. The PCR product was ligated into an eukaryotic expression vector pEGFP-N1 to construct a recombinant plasmid pEGFP-N1/ACRBP. Then the pEGFP-N1/ACRBP was transfected by Fugene HD into ACRBP-negative HepG2 cells. The stably transfected clones were selected by G418. RT-PCR and immunohistochemistry were used to detect the expression of ACRBP in HepG2 cells. RESULTS: The eukaryotic expression vector pEGFP-N1/ACRBP was constructed and confirmed by sequencing. The stably transfected HepG2 cells expressed ACRBP. CONCLUSION: The eukaryotic expression vector pEGFP-N1/ACRBP has been successfully constructed and transfected into HepG2 cells, resulting in stable expression of ACRBP.