OBJECTIVETo detect tumor cells in the peripheral blood of patients with hepatocellular carcinoma (HCC) by using the mRNA of the MAGE-1 and MAGE-3 genes as specific tumor markers.

METHODSPeripheral blood was obtained from 25 HCC patients and 20 healthy volunteers. The mRNA of the MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR. The MAGE-1 and MAGE-3 transcripts in the tumor tissues of these HCC patients were also detected by RT-PCR.

RESULTSOf the 25 HCC patients, MAGE-1 and MAGE-3 mRNA were positive in 44% (11/25) and 36% (9/25) of PBMCs respectively, and in 68% (17/25) and 56% (14/25) of HCC tissues respectively. In the PBMCs of the 25 HCC patients, 16 (64%) samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMCs from the patients whose tumors did not express the MAGE genes, nor in the PBMCs from the 20 healthy donors. The positive rate of MAGE mRNA in the PBMCs was closely correlated with the TNM stages and the diameter of tumors, but there was no correlation between the positive rate of MAGE mRNA in PBMCs and tumor differentiation degree or serum alpha-FP level. Of 9 HCC patients whose serum alpha-FP was normal or slightly elevated (< 50 ng/ml), 6 were MAGE-1 and/or MAGE-3 mRNA positive in their PBMCs.

CONCLUSIONMAGE-1 and MAGE-3 mRNA could be specifically detected with high percentage in the PBMCs of HCC patients by our method. They can be used as specific tumor markers for the detection of the circulating HCC cells, and the detection results may be helpful to evaluate the prognosis of HCC patients.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; Humans ; Leukocytes, Mononuclear ; Liver Neoplasms ; genetics ; Melanoma-Specific Antigens ; Neoplasm Proteins ; RNA, Messenger ; genetics

OBJECTIVETo investigate the expression of MAGE-B genes in hepatocellular carcinoma (HCC) in order to find new targets for immunotherapy.

METHODSThe expression of MAGE-B1, B2, A1 and A3 mRNA was detected using RT-PCR in HCC tissues and the corresponding adjacent non-HCC tissues from 47 HCC patients, 30 samples of cirrhosis and normal liver tissues. Four samples selected randomly from MAGE-B1 or B2 with positive RT-PCR results were sequenced to confirm the results of RT-PCR. The relationship between the expression of MAGE-B and some clinicopathological parameters was analyzed.

RESULTSMAGE-B1 mRNA and MAGE-B2 mRNA were detected in 44.7% (21/47) and 61.7% (29/47) of HCC samples, respectively, while neither MAGE-B1 nor MAGE-B2 could be detected in the corresponding adjacent non-HCC liver tissues. In addition, none of 30 samples of cirrhosis and normal liver tissues was shown to express both MAGE-B genes. The DNA sequence confirmed that the RT-PCR products were truly target cDNA. The frequency of the expression of MAGE-A1 and A3 was 74.5% (35/47) and 44.7% (21/47), respectively. There was significant correlation between the expression of MAGE-B and MAGE-A (P < 0.05). However, the positive expression of MAGE-B was observed in 5 out of 12 HCC tissues without expression of MAGE-A1 and/or A3. When all four MAGE genes were examined, the positive rate of expression of one, two, three and four genes was 83.0% (39/47), 55.3% (26/47), 48.9% (23/47), and 38.3% (18/47) of 47 HCC tissues, respectively. No correlation was found between the expression of MAGE-B and clinical parameters such as age, sex, tumor size, degree of tumor differentiation, serum alpha-fetoprotein level and hepatitis B virus or hepatitis C virus infection (P > 0.05).

CONCLUSIONMAGE-B genes are expressed with relatively high frequency and specificity in HCC. Most HCC patients with positive expression of at least one member of MAGE-B or MAGE-A gene family are adequate candidates to receive specific immunotherapy. Frequent co-expression of multiple members of MAGE-B and MAGE-A subfamilies provides the possibility of using polyvalent vaccines to achieve more effective immunotherapeutic results.

Antigens, Neoplasm ; genetics ; Carcinoma, Hepatocellular ; genetics ; Gene Expression ; Humans ; Liver Neoplasms ; genetics ; Melanoma-Specific Antigens ; Neoplasm Proteins ; genetics ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo determine at the mRNA level whether the MAGE-A1 and -A3 genes are expressed in cancer cell lines from salivary glands and relevant clinical carcinomas, and thus to distinguish cancerous tissues from normal tissues and benign tumors in salivary glands.

METHODSThe expression of the MEGE-A1 and MEGE-A3 genes at the mRNA level was determined by reverse transcription and polymerase chain reaction (RT-PCR) in 2 cell lines of human adenoid cyst carcinomas (ACC-2 and ACC-M), in 18 malignant tumors and 9 benign salivary gland tumors, and in 10 samples of normal salivary gland tissues.

RESULTSBoth MAGE-A1 and -A3 genes were expressed in ACC-2 and ACC-M cell lines. None of the 9 benign tumors or the 10 normal tissue samples of salivary glands expressed the genes. The MAGE-A1 and -A3 genes were expressed in 9 (50%) and 11 (61%) of 18 salivary gland carcinomas, respectively, and at least 1 of the 2 genes was expressed in 14 (78%) of them. Of the 18 salivary gland carcinomas, 6 low-differentiated carcinomas (33%) expressed both genes, whereas 4 high-differentiated carcinomas (22%) expressed neither gene.

CONCLUSIONSThe MAGE-A1 and -A3 genes can be expressed in cancer cell lines of salivary glands and relevant clinical carcinomas in addition to other malignant tumors of various histological origins. The results suggest the possibility of immunotherapy against salivary gland carcinomas by using MAGE-gene-encoded products as target antigens for tumor-rejection.

Adult ; Aged ; Antigens, Neoplasm ; genetics ; Female ; Gene Expression ; Humans ; Male ; Melanoma-Specific Antigens ; Middle Aged ; Neoplasm Proteins ; genetics ; Salivary Gland Neoplasms ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVE(1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression.

METHODSThe cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes.

RESULTSThe MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05).

CONCLUSIONSWith a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.

Antigens, Neoplasm ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; genetics ; immunology ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; Liver Neoplasms ; genetics ; immunology ; Male ; Melanoma-Specific Antigens ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo explore the possibility of using melanoma antigen (MAGE)-1 and MAGE-3 gene encoding proteins as an index of potential target for immunotherapy in intrahepatic cholangiocarcinoma (IHCC) patients.

METHODSThe expressions of MAGE-1 and MAGE-3 genes in tumor tissues and tumor adjacent non-IHCC liver tissues were examined by RT-PCR method. The relationship between positive expression rates of MAGE-1 and MAGE-3 genes and clinical data including sex, age, tumor diameters, tumor envelope, tumor nodules number, and hepatitis B virus surface antigen were determined.

RESULTSThe positive expression rates of MAGE-1 (35%) and MAGE-3 genes (45%) were significantly higher in the tumor tissues than in tumor adjacent tissues (0) (P<0.01). The positive expression rates of MAGE-1 and MAGE-3 genes had no relationship with the clinical data (P >0.05), except the morphology of tumor (P <0.05).

CONCLUSIONThe high expression rates of MAGE-1 and MAGE-3 genes in IHCC suggests the MAGE-1 and MAGE-3 gene may be a target for immunotherapy in IHCC patients.

Adult ; Aged ; Antigens, Neoplasm ; genetics ; Bile Duct Neoplasms ; genetics ; Bile Ducts, Intrahepatic ; pathology ; Cholangiocarcinoma ; genetics ; Female ; Humans ; In Vitro Techniques ; Liver Neoplasms ; genetics ; Male ; Melanoma-Specific Antigens ; Middle Aged ; Neoplasm Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the expression of MAGE, GAGE and BAGE genes in human hepatocellular carcinoma (HCC) cell lines.

METHODSThe expression levels of MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE mRNAs in HCC cell lines SMMC-7721, QQY-7701, BEL-7402 were studied by reverse transcription polymerase chain reaction and were compared with those in biopsied liver tissues.

RESULTSMAGE-1 and BAGE mRNAs were expressed in SMMC-7721 cells. MAGE-3 and BAGE mRNAs were expressed in QQY-7701 cells. MAGE-1 and GAGE1-2 mRNAs were expressed in BEL-7402 cells. None of these genes was expressed in biopsied liver tissues.

CONCLUSIONSMAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE are expressed in hepatocellular carcinoma cell lines. These tumor-specific antigens can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC.

Antigens, Neoplasm ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; pathology ; Melanoma-Specific Antigens ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo develop a sensitive and specific RT-PCR assay using the mRNA of MAGE-1 and MAGE-3 genes as specific tumor markers for the detection of the tumor cells in the peripheral blood of patients with gastric cancer.

METHODSPeripheral blood was obtained from 40 patients with gastric cancer and from 20 healthy volunteers. The mRNA of MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMC) was detected by RT-PCR. The expressions of MAGE-1 and MAGE-3 mRNA in the tumor tissues of these gastric cancer patients were also detected by RT-PCR. Meanwhile,CEA expression by nested RT-PCR in PBMC of 40 gastric cancer patients was also detected.

RESULTSOf 40 gastric cancer patients, MAGE-1 and MAGE-3 mRNA were positive in 47.5% (19/40) and 25% (10/40) of PBMC respectively, and in 62.5% (25/40) and 30% (12/40) of gastric cancer tissues respectively. As a whole, in the PBMC of 40 gastric cancer patients, 25 (62.5%) samples were found to express at least one type of MAGE mRNA. In the patients whose tumors did not express MAGE-1 and/or MAGE-3 genes, the corresponding MAGE mRNA was also undetected in their PBMC. There was no expression of MAGE-1 or MAGE-3 gene in the PBMC from the 20 healthy donors. The positive rate of MAGE mRNA in PBMC was closely correlated with the tumor stage and lymph node metastasis (P <0.05). Positive rate of CEA gene expression was 32.5% (13/40) in the PBMC of 40 gastric cancer patients, 29 (72.5%)samples were detected to express at least one type of MAGE gene and CEA gene mRNA.

CONCLUSIONSMAGE-1, MAGE-3 and CEA mRNA are specifically detected with high percentage in the PBMC of gastric cancer patients by RT-PCR. They could be used as specific tumor markers for the detection of the circulating gastric cancer cells, and the detection results may be helpful to evaluate the prognosis of gastric cancer patients.

Adult ; Aged ; Antigens, Neoplasm ; blood ; genetics ; Biomarkers, Tumor ; blood ; Carcinoembryonic Antigen ; blood ; Female ; Humans ; Male ; Melanoma-Specific Antigens ; Middle Aged ; Neoplasm Proteins ; blood ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Stomach Neoplasms ; blood ; genetics ; pathology

OBJECTIVESTo investigate the correlation between MAGE-A1 mRNA expression and genic demethylation in hepatoma cell lines.

METHODSTotal RNA and genomic DNA were prepared from 10 human hepatoma cell lines. MAGE-1 mRNA expression was determined with RT-PCR and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern blot assay. The genomic DNA was digested by HpaII, then the promoter of MAGE-A1 gene was amplified with primers CDS21, EDP4 and CDS20. EDP4 and the PCR products were further hybridized with a probe to detect the methylation in the promoter of the MAGE-A1 gene. HLA-A locus was typed using SSP kit.

RESULTSIn cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405, MAGE-A1 mRNA expression was positive and cell differentiation was moderate or low. In cell lines of HepG2 2.2.15, HepG2, QGY-7701 and Huh7, MAGE-A1 mRNA expression was negative and cell differentiation was well or moderate. The level of genomic demethylation in MAGE-A1 mRNA positive cell lines was much higher than that in MAGE-A1 mRNA negative cell lines (t = 2.896, P = 0.02). The methylation analysis showed that methylation in the promoters of MAGE-A1 gene of HepG2 2.2.15, HepG2, QGY-7701 and Huh7 was high, and that methylation in those of SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405 was low.

CONCLUSIONThe results suggest that MAGE-A1 mRNA expression in the human hepatoma cell lines is associated with genic hypomethylation.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; immunology ; metabolism ; DNA Methylation ; Humans ; Liver Neoplasms ; genetics ; immunology ; metabolism ; Melanoma-Specific Antigens ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured

OBJECTIVESTo investigate the expression of melanoma-associated antigen 1 (MAGE-1) in Chinese hepatocellular carcinoma (HCC) patients and to determine the existence and distribution of single nucleotide polymorphisms (SNP) of MAGE-1 gene.

METHODSTotal RNA was extracted from cancer tissues and adjacent tissues from 19 HCC patients and the expression of MAGE-1 mRNA was examined by using RT-PCR. The PCR products were sequenced to analysis the gene variation. Genomic DNA was extracted from cancer tissues, adjacent tissues and peripheral blood cells of 19 HCC patients, as well as from peripheral blood cells of 23 healthy donors. The PCR product of MAGE-1 DNA was sequenced to determine the existence and distribution of SNPs of MAGE-1 gene.

RESULTS9 of 19 (47.4%) tumor tissues and none of adjacent tissue from HCC patients expressed MAGE-1 mRNA. There were three kinds of gene variations of cDNA in 9 MAGE-1 mRNA positive patients. One type was named type I including 1 patient, which sequence is as same as that of the GenBank M77481. The other was named TGA type including 5 patients, which involved three nucleotide changes (C159T, A272G and G393A) and result in two amino acid changes (T32A and R72Q). Another one was named GTG type including 3 patients, which involved three nucleotide changes (A272G, C991T, A1125G) and result in only one amino acid changes (T32A). According to the analysis of genomic DNA, above three types were not specific mutations of tumor tissue, but were SNPs. These SNPs types were distributed in HCC patients and normal donors with the frequencies of 26.3% (5/19) and 60.9% (14/23) for type I, 57.9% (11/19) and 47.8% (11/23) for TGA type, and 21.1% (4/19) and 21.7% (5/23) for GTG type, respectively. The sequences of two new SNPs had been deposited in GenBank with the accession numbers of AF463515 and AY148486. In male population, the distributions of SNPs were not correlated to HCC suffering or MAGE-1 expression. Several new HLA-restricted epitopes were probably resulted from SNPs existing. The three-dimensional models of MAGE-1 proteins of type I and TGA type was established by using computer software.

CONCLUSIONThe expression rate of MAGE-1 gene in Chinese HCC patients is high. Three SNP types of MAGE-1 gene exist in Chinese population. The three-dimensional models of MAGE-1 proteins were obtained by computer processing. These results will be helpful to developing MAGE-1 peptide-vaccine for HCC immunotherapy.

Adult ; Aged ; Amino Acid Sequence ; Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; Female ; Humans ; Liver Neoplasms ; genetics ; Male ; Melanoma-Specific Antigens ; Middle Aged ; Molecular Sequence Data ; Neoplasm Proteins ; genetics ; Polymorphism, Single Nucleotide ; RNA, Messenger ; analysis ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo investigate the possibility of using melanoma antigen-1 (MAGE-1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC).

METHODSThe expressions of MAGE-1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT-PCR. The type of human leucocyte antigen I(HLA I) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE-1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay.

RESULTSThe expressions of both MAGE-1 and HLA-A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE-1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE-1 nor HLA-A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28-day culture. The ratio of CD3(+) T cells increased by 16% (from 54% to 70%), and the ratio of CD8(+) T cells increased by 20% (from 36% to 56%) during 28-day culture. When the ratio of effector cells to target cells was 10:1, effector cells exhibited 62.5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE-1 antigen, 40.25% cytotoxicity against BEL7405 cells, compared with 17.88% cytolysis observed against autogenous lymphoblasts, 19.55% against HLE cells, and 1.6% against QGY7701 cells. When the ratio of effector cells to target cells was 3.3:1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53.6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15.6%, 13% and 1%, respectively.

CONCLUSIONThe results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE-1 and HLA-A24 could be successfully induced by the MAGE-1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA-A24 HCC patients.

Adult ; Antigens, Neoplasm ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; immunology ; HLA-A Antigens ; analysis ; HLA-A24 Antigen ; Humans ; Liver Neoplasms ; immunology ; Male ; Melanoma-Specific Antigens ; Neoplasm Proteins ; genetics ; immunology ; RNA, Messenger ; analysis ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured