MAGE-1 mRNA Research Articles

A new tool for early diagnosis of rheumatoid arthritis using combined biomarkers; synovial MAGE-1 mRNA and serum anti-CCP and RF.

Introductionrheumatoid arthritis (RA) is a common autoimmune disease with unknown etiology and pathogenesis. Biomarkers have the potential to aid in the clinical diagnosis of the disease, or to provide means of detecting early signs of the disease. Evaluating Melanoma associated antigen genes (MAGE-1) mRNA expression rate in synovial fluid cells and serum levels of anti-cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) for RA early diagnosis.Methodsa total of 213 subjects were enrolled in the study, 135 RA patients and 78 normal subjects with traumatic knee joints (control group). Serum RF and anti-CCP were estimated quantitatively using ELISA. MAGE-1 mRNA expression rate was analyzed by RT-PCR.Resultsa significant increase in serum levels of RF IgM and anti-CCP in RA patients compared to the controls. A positively significant correlation was found between serum anti-CCP and RF IgM. The expression rate of MAGE-1 mRNA was 100% in RA patients versus the controls (0%). The specificity and the sensitivity of the three biomarkers was 100%.Conclusionthe high expression rate of MAGE-1 in synovial fluid cells of RA patients is encouraging its utilization as a diagnostic biomarker for RA. The combined use of MAGE-1 transcript in synovial fluid cells, serum RF and anti-CCP is recommended for improving early diagnostic ability of RA.

MAGE Genes mRNAs: Potential Early Diagnostic Markers for Hepatocellular Carcinoma in HCV Saudi Patients

In recent years, several different multi-marker assays have been developed for the detection of HCC cells in the peripheral blood. This study included 89 Saudi patients with chronic HCV and 30 ages and sex matched healthy control subjects. MAGE-1 mRNA was expressed in the peripheral blood of 3(13.6%) patients with localized HCC and in 9(40.9%) patients with metastatic HCC. MAGE-3 mRNA was expressed in the peripheral blood of 3(13.6%) patients with localized HCC and in 8(42.1%) patients with metastatic HCC. Also, MAGE-4 mRNA was expressed in the peripheral blood of 4(18.1%) patients with localized HCC and in 10(52.6%) patients with metastatic HCC.All control subjects showed no expression of any MAGE mRNAs. In conclusions, MAGE-1, MAGE-3 and MAGE-4 may be promising diagnostic tools for monitoring the prognosis of HCC patients and for early detection of circulating metastasis of HCC cells.

Diagnostic value of cancer-testis antigen mRNA expression in peripheral blood of patients with small hepatocellular carcinoma

Objective To evaluate the diagnostic value of cancer-testis antigen (CTA) mRNA in peripheral blood samples from patients with small hepatocellular carcinoma (HCC).Methods Peripheral blood samples were drawn from 30 patients with small HCC before operation.The expression of the above CTA genes in the peripheral blood mononuclear cells (PBMCs) was detected by using nested reverse transcriptase-polymerase chain reaction (RT-PCR),and serum alpha-fetoprotein (AFP) levels were also determined.Results Of the 30 patients,the positive rate of MAGE-1,SSX-1 and CTp11 transcripts was 26.6％ (6/30),26.6％ (6/30) and 16.6％ (5/30) in PBMCs,respectively.The positive rate for at least one of the three CTA gene transcripts was 40.0％ (12/30) in PBMCs of these patients.MAGE-1,SSX-1 and/or CTp11 mRNA were not detected in the PBMCs of those patients from whom the resected tumor sampies were negative for MAGE-1,SSX-1 and/or CTp11 mRNA,none in the PBMCs from 15 healthy donors and 15 cirrhotic patients.Among the 30 patients,there were only 6 patients whose serum AFP met the general diagnostic standard (AFP ＞ 400 μg/L) for HCC,whereas the other patients were negative for serum AFP (AFP ≤ 20 μg/L) or AFP positive with low concentration (20 μg/L ＜ AFP≤400 pg/L).The positive rate for at least one of the three CTA gene transcripts in PBMCs from the AFP negative patients or the AFP positive patients with low concentration was 33.3％ (5/15) and 44.4％ (4/9),respectively.50.0％ (15/30) of the patients with small HCC could be diagnosed by combining the results of the nested RT-PCR and the serum AFP detection,and this diagnostic rate was significantly higher than that (20.0％)made by AFP detection alone (P ＜ 0.05).Conclusion Detecting MAGE-1,SSX-1 and CTp11 mRNA in PBMCs is va1uable for diagnosing the patients with small HCC. Key words: Carcinoma,hepatocellular; Cancer-testis antigen; Diagnosis

Qualitative Assay of Synovial MAGE-1 m-RNA as a New Trend in Diagnosis of Rheumatoid Arthritis

Rheumatoid Arthritis (RA) is the commonest inflammatory joint disease, affectingnearly 1% of the adult population worldwide. Its diagnosis depends on clinicalsymptoms, laboratory investigations and imaging. The emphasis in the managementof this disease is early diagnosis and intervention. Diagnostic specificity ofrheumatoid factor (RF) for RA is poor since it is also found in many other rheumaticand non-rheumatic diseases. Anticyclic citrullinated peptide (anti-CCP) was reportedto have a high specificity for RA diagnosis, especially in patients with early disease.However, it was found to have lower sensitivity than RF in RA diagnosis. This studyaimed to evaluate the expression of MAGE-1 mRNA by the synovial fluid cells frominflamed joints, and the serum levels of anti-CCP as biochemical markers for theearly diagnosis of RA. The study included 30 subjects of both sexes: 10 controlswith traumatic knee joint effusion and 20 rheumatoid arthritis patients. BothSerum RF and anti-CCP were determined quantitatively by enzyme-linkedimmune-sorbent assay (ELISA). The expression of MAGE-1 mRNA by the synovialfluid cells obtained from the inflamed joints is evaluated by applying reversetranscription-PCR amplification. MAGE-1 mRNA was detected in synovial fluid cellsof all patients but not in controls. There was significant increase (P < 0.01) ofboth serum RF IgM and anti-CCP in rheumatoid patients group when compared tocontrol one. There was significant positive correlation (r = 0.928; P = < 0.01)between serum RF IgM and anti-CCP. The specificity of MAGE-I, Anti-CCP and RFin RA diagnosis was 100%, and the sensitivity of both MAGE-I and Anti-CCP was100% while that of RF was 85%. Conclusively, the expression results of MAGE-1transcript in synovial fluid were 100% positive encouraging its utilization as abiochemical marker for RA diagnosis. The combined use of serum anti-CCP andMAGE-1 transcript in the synovial fluid cells is recommended for early diagnosis ofRA.

MAGE-1 and MAGE-3 mRNA expressions as molecular biomarkers in patients with hepatitis C virus-related hepatocellular carcinoma

IntroductionThe melanoma antigen (MAGE) family members are tumour-specific antigens exclusively expressed in neoplastic cells. Therefore, the present work was designed to study the expression of MAGE-1 and MAGE-3 mRNAs in...

MAGE-3 and MAGE-4 genes as possible markers for early detection of metastases in hepatitis C virus Egyptian patients complicated by hepatocellular carcinoma

The dissemination of hepatocellular carcinoma (HCC) cells into the circulation plays a critical role in post-operative recurrence and metastasis. Early detection of metastatic tumor cells is critical to identify HCC patients at high risk of relapse. MAGE-3 and -4 genes were evaluated by reverse transcription polymerase chain reaction for the possibility of using them as new markers for early detection of metastases in 160 chronic HCV Egyptian patients, 115 of them were complicated with HCC. The expressions of MAGE-3 and MAGE-4 mRNA in peripheral blood of patients with metastatic HCC were 36 and 52%, respectively. While the expressions of MAGE-3 and MAGE-4 mRNA in peripheral blood of patients with localized HCC were 12.5 and 15%, respectively. Moreover, at least one type of mRNA was found in the peripheral blood of 68% of the metastatic HCC patients and in 20% of the localized HCC patients. While neither the controls nor the cirrhotic patients show expression of MAGE-4 mRNA in their peripheral blood. MAGE-3 and MAGE-4 may be a promising diagnostic tool for monitoring the prognosis of HCC patients and early detection of occult hematogenous metastasis of HCC.

Detection of tumor cells in peripheral blood of patients with gastric cancer using mRNA of MAGE genes as markers

To 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. Peripheral 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. Of 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. MAGE-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.

Melanoma antigen‐1 mRNA combined with α‐fetoprotein mRNA levels in peripheral blood of patients with hepatocellular carcinoma: a predictor of postoperative recurrence or metastasis?

The aim of the study was to identify whether melanoma antigen (MAGE)-1 mRNA and alpha-fetoprotein (AFP) mRNA expressed in peripheral blood could be used to predict the recurrence and metastasis of hepatocellular carcinoma (HCC) after hepatectomy. One hundred and forty-two HCC patients underwent hepatectomy. The control group includes 27 patients with chronic virus hepatitis and cirrhosis and 10 healthy volunteers. Peripheral blood samples were collected on the seventh day before operation, seventh day after operation and 30th day after operation. MAGE-1 mRNA and AFP mRNA were tested by nested reverse transcription polymerase chain reaction. Median follow up was 25.5 months (range 4-40 months). Patient survival, disease-free survival and clinicopathological features were compared between patients with positive and negative MAGE-1 mRNA and/or AFP mRNA. The expression of MAGE-1 mRNA and/or AFP mRNA in peripheral blood was closely correlated to the pathological stage and the positive ratio of tumour cells in the peripheral blood (P < 0.01). There was recurrence and/or metastasis after operation in 55 of 142 HCC patients. Among the 55 patients who had recurrence or metastasis, MAGE-1 mRNA and/or AFP mRNA in peripheral blood were persistently detected after operation in 38 patients and MAGE-1 mRNA and AFP mRNA turned to positive after operation in 14 patients. In contrast, no recurrence was found in 62 patients whose MAGE-1 mRNA and/or AFP mRNA turned to negative after operation. 88.1% (52 of 59) of patients with MAGE-1 mRNA and/or AFP mRNA persistently positive after operation showed recurrence or metastasis, whereas only 3.6% patients (3 of 83) with the negative of MAGE-1 mRNA and/or AFP mRNA after operation showed recurrence or metastasis (P < 0.001). Melanoma antigen-1 mRNA combined with AFP mRNA in peripheral blood after hepatectomy is more sensitive and specific than AFP mRNA singly for predicting the recurrence and metastasis of the HCC patients, whereas preoperative transient detection is not.

Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression

The detection of isolated (circulating or disseminated) tumour cells (ITC) in patients with cancer requires very sensitive methods, as such cells are very rare. In the present study, the method that combines the negative isolation of CD45- leukocytes from the blood and bone marrow of patients with gastric cancer by flow cytometry, followed by the positive isolation of single cytokeratin-positive (CK+) cells by a Laser Capture Microdissection System for the determination of MAGE-1, -2 mRNA expression was used to detect ITC. This study shows that this method is highly sensitive as it allows to determine beta-actin-mRNA expression in a single CK+ cell. Using > or =5 CK+ cells as a cut-off level, the MAGE-1 mRNA expression was detected in 100% of CK+ cells in the peripheral blood and in 75% of bone marrow samples of patients with gastric cancer. The MAGE-2 mRNA expression was observed in 40 and 58% of samples, respectively. Furthermore, an analysis of primary tumours and locoreginal lymph nodes with respect to the mRNA expression of the two genes showed that MAGE-1 mRNA expression was detected in 88% of the primary tumours and in 67% of the lymph node samples, whereas the MAGE-2 mRNA expression was observed in 72 and 67% of the cases, respectively. Thus, the method described here allows the precise and sensitive determination of tumour-associated gene expression in single ITC present in the blood and bone marrow of patients with gastric cancer.

Decitabine (DAC) in combination with doxorubicin (DOX) and cyclophosphamide (CTX) in relapsed neuroblastoma (NBL): A Children's Oncology Group Study

9565 Background: Loss of caspase-8 expression causes resistance to apoptosis-inducing agents and potentiates metastasis in NBL. In vitro treatment of NBL cell lines with DAC results in increased caspase-8 expression and enhanced apoptosis following exposure to DOX. As a component of a pediatric phase 1 study, we evaluated low-dose DAC with DOX and CTX in a cohort of children with relapsed NBL. Promoter specific DNA methylation in peripheral blood mononuclear cells (PBMC) was studied. Methods: Patients received a 1-hour infusion of DAC, 5 mg/m2, daily on days 0–6, followed on day 7 by 45 mg/m2 of DOX (with dexrazoxane) and 1 g/m2 of CTX. All patients received G-CSF. Cycles were repeated every 28 days. Methylation-specific and quantitative PCR were used to measure promoter-specific DNA methylation in PBMC prior to and after DAC (day 7 and day 28). Results: Twelve patients with recurrent NBL (median age 9 years, 10 males) were enrolled. DAC/DOX/CTX was well tolerated with grade 4 myelosuppression being the predominant toxicity. Grade 4 neutropenia, median 11 days duration, occurred in 10 pts and grade 4 thrombocytopenia, median 10 days, in 7 patients. Eight patients had progressive disease after = 2 courses of therapy. The remaining 4 patients had stable disease with 5 to 8 courses. Methylation and subsequent demethylation (40–90% decreased methylation) of the MAGE-1 promoter was detected before and after DAC respectively, in PBMC from 6 of 9 patients analyzed to date. Re-expression of MAGE-1 mRNA was also demonstrated in post treatment samples. Caspase-8 demethylation was not detected in PBMC; nor was hypermethylation of caspase-8 detected in pre-treatment PBMC, possibly due to low tumor burden in blood. Conclusions: DAC in combination with cytotoxic chemotherapy was relatively well tolerated in heavily pre-treated children with relapsed NBL. Low-dose DAC induces demethylation and re-expression of MAGE-1 in PBMC, and may be a potential surrogate marker of demethylation within tumor cells. The use of low dose DAC in combination with other agents warrants further study in NBL. No significant financial relationships to disclose.

Effects of expression silencing of MAGE3 by RNA interference on location and metastasis of lung carcinoma cells

To construct small interfering RNA (siRNA) expression vectors targeting human MAGE3 gene and to observe the effects of gene silencing of MAGE3 by RNA interference on location and metastasis of lung carcinoma cells. MAGE3 mRNA targeted hairpin siRNA was devised and the oligonucleotide strands of DNA fragments encoding the above siRNA were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into pSUPERneoGFP, followed by amplification and DNA sequencing, then transfected into human lung carcinoma NCI-H446. The expression of MAGE3 gene mRNA and protein were examined by RT-PCR and Western blotting. Colony formation assay and Boyden chamber assay were performed to detect the effects of MAGE3 on colony formation and metastasis. The DNA fragments encoding MAGE3-targeted siRNA were cloned into the pSUPERneoGFP and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western blotting revealed a strongly decreased expression level of MAGE3. The lung carcinoma cells transfected by siRNA group was significantly lower than others, an effect on its colony formation and invasiveness. The colony formation of lung carcinoma cells transfected by siRNA in soft agar and the number of cells penetrating matrigel both reduced, there is significant difference compared with untransfected group and transfected empty vector. An siRNA vector targeting human MAGE3 gene has been successfully constructed. Expression silencing of MAGE3 by RNA interference could reduce location and metastasis of lung carcinoma cells effectively.

Expression of MAGE-A1 mRNA is associated with gene hypomethylation in hepatocarcinoma cell lines

A correlation between melanoma-associated antigen (MAGE) A1 mRNA expression and genome-wide hypomethylation has been observed in some carcinomas, but this relationship is not known in hepatocarcinoma. Total RNA and genomic DNA were prepared from ten human hepatocarcinoma cell lines in which the genetic characteristics are stable. MAGE-1 mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR), and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern-blot assay. The methylation status of the MAGE-A1 gene promoter was measured by enzyme digestion and PCR. MAGE-A1 mRNA was detected in the hepatocarcinoma cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405, which showed moderate to low levels of cell differentiation. In contrast, MAGE-A1 mRNA expression was not detected in the hepatoma cell lines HepG2215, HepG2, QGY-7701, and Huh7, which showed moderate to high levels of differentiation. The level of demethylation in MAGE-A1 mRNA-positive cell lines was much higher than that in MAGE-A1 mRNA-negative cell lines (P=0.02). The results suggest that MAGE-A1 mRNA expression in human hepatoma cell lines is associated with hypomethylation of the genome and the MAGE-A1 promoter domain. This study will be helpful to reveal the expression mechanisms of MAGE-like tumor antigens in cancer cells.

Perioperative detection of MAGE-1mRNA and AFP mRNA in peripheral blood of patients with hepatocellular carcinoma: relationships with recurrence

To detect MAGE-1 mRNA and AFP mRNA in the peripheral blood of patients with hepatocellular carcinoma (HCC) perioperatively, and explore their relationships with recurrence. Peripheral blood from 45 patients with HCC, obtained perioperatively, was tested for the presence of MAGE-1 mRNA and AFP mRNA by nested RT-PCR. All patients were followed for metastatic recurrence for average 11 months after surgery. The peripheral blood from 22 patients with hepatitis B and cirrhosis, 11 patients with hepatic hemangioma, 12 patients with metastatic liver cancer and 20 healthy volunteers served as control. Four of the twelve (33.3%) metastatic hepatic cancer patients were positive for MAGE-1 mRNA, and three of the twenty-two (13.6%) patients with hepatitis B and cirrhosis were positive for AFP mRNA while the other controls kept negative in their peripheral blood. Detection rates for MAGE-1 mRNA/AFP mRNA in the peripheral blood of patients with HCC were 42.2%/51.1% before operation, 20%/24.4% seven days after surgery, and 15.6%/22.2% 28 days later respectively. In chi-square analysis, detection of peripheral-blood MAGE-1 mRNA and AFP mRNA 28 days after surgery indicated metastatic recurrence. Furthermore, the two-marker detection had a higher specificity for prediction of metastatic recurrence than single marker detection. Combined detection of cancer-specific MAGE-1 mRNA and hepatocyte-specific AFP mRNA by nested RT-PCR in the peripheral blood of patients with HCC 28 days after surgery may be useful for the prediction of metastatic recurrence.

HER-2, gp100, and MAGE-1 Are Expressed in Human Glioblastoma and Recognized by Cytotoxic T Cells

It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens, such as HER-2, gp100, and MAGE-1. To further determine the possible utilization of these antigens for glioma immunotherapy and as surrogate markers for specific tumor antigen cytotoxicity, we characterized the presence of mRNA and protein expression in 43 primary glioblastoma multiforme (GBM) cell lines and 7 established human GBM cell lines. HER-2, gp100, and MAGE-1 mRNA expression was detected in 81.4%, 46.5%, and 39.5% of the GBM primary cell lines, respectively. Using immunoreactive staining analysis by flow cytometry, HER-2, gp100, and MAGE-1 protein expression was detected in 76%, 45%, and 38% of the GBM primary cell lines, respectively. HLA-A1-restricted epitope specific for MAGE-1 peptide (EADPTGHSY) CTL clone B07 and HLA-A2-restricted epitope specific for HER-2 peptide (KIFGSLAFL) CTL clone A05 and gp100 peptide (ITDQVPFSV) CTL clone CK3H6 were used in this study. The specificity of CTL clone was verified by HLA/peptide tetramer staining. Three CTL clones could efficiently recognize GBM tumor cells in an antigen-specific and MHC class I-restricted manner. IFN-gamma treatment can dramatically increase MHC class I expression of GBM tumor cells and significantly increase CTL recognition of tumor cells. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the mRNA expression of MAGE-1 and epitope presentation by autologous MHC. These data indicate that HER-2, gp100, and MAGE-1 could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigen-specific active immunotherapy strategies for glioma patients.

Expression of cancer-testis antigens in hepatocellular carcinoma.

To investigate the expression of cancer-testis (CT) antigens MAGE-1, SSX-1,CTp11 and HCA587 genes in hepatocellular carcinoma (HCC) and the possibility of applying these antigens as targets for specific immunotherapy for HCC. Expression levels of MAGE-1, SSX-1, CTp11 and HCA587 mRNA were detected with reverse transcription polymerase chain reaction (RT-PCR) in HCC tissues and corresponding adjacent non-cancerous tissues from 105 HCC patients, 40 samples of cirrhosis and normal liver tissues. Genes of five samples with positive PCR results were sequenced. Of 105 HCC tissues, MAGE1, SSX-1,CTp11 and HCA587 mRNA expressions were detectable in 75.2%(79/105), 72.4%(76/105), 62.9%(66/105) and 56.2%(59/105) of HCC samples, respectively. About 93.3%(98/105), 72.4%(76/105), 48.6%(51/105) and 37.1%(39/105) of HCC tissues positively expressed at least one, two, three, and four members of CT antigens, respectively. Conversely, only SSX-1 could be detectable in 2.9%(3/105) of the corresponding adjacent non-HCC tissues in which no metastatic lesion was found. Of the latter 3 patients, biopsy samples far from tumor were obtained in 2 patients and RT-PCR indicated no expression of SSX-1 mRNA in these two samples. In addition, none of 40 samples of cirrhotic and normal liver tissues expressed CT antigen gene mRNA. DNA sequences confirmed that the RT-PCR products were true target cDNA. No relationship was found between expression of CT antigens and clinico pathological indicators such as age, gender, tumor size, degree of tumor differentiation, serum alpha-fetoprotein level and infection of hepatitis B virus or hepatitis C virus (P>0.05). CT antigens genes (MAGE-1, SSX-1, CTp11 and HCA587) are expressed with high percentage and specificity in HCC and their products are promising targets for antigen-specific immunotherapy of HCC. High frequent co-expression of multiple members of CT antigens in HCC provides possibility of polyvalent vaccinations for HCC.

Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells.

The members of MAGE gene family are highly expressed in human hepatocellular carcinoma (HCC). In the present study, we tested the tumour-specific MAGE-1 and MAGE-3 transcripts in the peripheral blood of HCC patients by nested RT–PCR to detect the circulating tumour cells and evaluate their potential clinical implication. Of 30 HCC patients, the positive rate of MAGE-1 and MAGE-3 transcripts was 43.3% (13 out of 30) and 33.3% (10 out of 30) in PBMC samples, whilst the positive rate was 70% (21 out of 30) and 53.3% (16 out of 30) in the resected HCC tissue samples, respectively. The positivity for at least one MAGE gene transcript was 63.3% (19 out of 30) in PBMC samples of HCC patients and 83.3% (25 out of 30) in the resected HCC tissue samples. MAGE-1 and/or MAGE-3 mRNA were not detected in the PBMC of those patients from whom the resected HCC tissues were MAGE-1 or MAGE-3 mRNA negative, nor in the 25 PBMC samples from healthy donors. The detection of MAGE transcripts in PBMC was correlated with the advanced stages and tumour size of the HCC, being 82.4% (14 out of 17) in tumour stages III and IVa, 56.6% (five out of nine) in stage II, and null (nought out of four) in stage I. The serum α-FP in 33.3% (10 out of 30) of HCC patients was normal or slightly elevated (<40 ng ml−1). However, six of these 10 patients (α-FP <40 ng ml−1) were MAGE-1 and /or MAGE-3 mRNA positive in their PBMC. The follow-up survey of MAGE mRNA in PBMC was performed in 12 patients. Seven patients with persistent MAGE-1 and/or MAGE-3 mRNA positive or from negative turned to positive died because of metastasis and/or recurrence. In striking contrast, all four patients with MAGE-1 and/or MAGE-3 mRNA from positive turned to negative and one patient with persistent MAGE-3 transcript negative are alive after last test. Collectively, detection of MAGE transcripts with follow-up survey in PBMC is a feasible and reliable assay for the early prediction of the relapse and prognosis of the HCC patients.British Journal of Cancer (2002) 86, 110–116. DOI: 10.1038/sj/bjc/6600016 www.bjcancer.com© 2002 The Cancer Research Campaign

Induction of MAGE-3 expression in lung and esophageal cancer cells

Background. Although MAGE-3 has been detected in approximately 40% of lung and esophageal cancers, expression of this cancer testis antigen appears to be below the threshold for immune recognition in patients with these malignancies. The aim of this study was to determine if the demethylating agent, 5-Aza-2′-deoxycytidine (DAC) and if the histone deacetylase inhibitor Depsipeptide FR901228 (DP) could enhance MAGE-3 expression in lung and esophageal cancer cells. Methods. Eleven lung and esophageal cancer lines and cultured normal human bronchial epithelial (NHBE) cells were exposed to normal media (NM), DAC, DP, or combination DAC/DP at varying concentrations and exposure durations. MAGE-3 expression was evaluated by quantitative RT-PCR (TaqMan) and immunohistochemistry techniques. Trypan blue exclusion techniques were used to examine the proliferation of cancer cells after drug exposure. Results. Relative to untreated controls, MAGE-3 expression was enhanced 32-fold (range 3.9 to 110) by DAC alone (0.1 μmol/L × 72 h), 2.1-fold (0.4 to 4.2) by DP alone (25 ng/mL × 6h), and 57-fold (4.6 to 209) by sequential DAC/DP exposure. Increased MAGE-3 mRNA copy numbers coincided with enhanced protein levels in these cells. MAGE-3 expression persisted after drug exposure. Flow cytometry confirmed the presence of functional HLA class I expression in these cells. Sequential DAC/DP treatment mediated pronounced growth inhibition in cancer cells but not NHBE. Conclusions. Sequential DAC/DP treatment may be a novel strategy to simultaneously augment MAGE-3 expression and induce growth arrest in thoracic malignancies.

Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9

In an effort to define new cancer-testis (CT) genes, we investigated whether BRDT, a testis-restricted member of the RING3 family of transcriptional regulators, is also expressed in cancer. Standard RT–PCR expression analysis detected BRDT transcripts in 12 of 47 cases of non-small cell lung cancer and single cases of both squamous cell carcinoma of the head and neck (1/12) and esophagus (1/12) but not in melanoma or in cancers of the colon, breast, kidney and bladder. Typing of 33 non-small cell lung cancers for coexpression of a panel of CT antigens revealed a high incidence (60%) of MAGE-3 mRNA expression, followed by MAGE-1 (36%), CT7/MAGE-C1 (30%), CT10 (30%), SSX4 (23%), BRDT (21%), NY-ESO-1 (21%) and HOM-MEL-40/SSX2 (15%). The coexpression pattern of these antigens provides a foundation for developing a polyvalent lung cancer vaccine.

MAGE-1 and related MAGE gene expression may be associated with hepatocellular carcinoma

The possibility of tumor rejection antigen, encoded by the MAGE-1 gene, being a target for immunotherapy in hepatocellular carcinoma (HCC) patients and the cloning of the full-length cDNA of this gene for subsequent studies were explored with the aim of discovering new immunotherapeutic strategies for HCC. Expression of the MAGE-1 gene in HCC specimens and HCC cell lines was examined by the reverse transcription/polymerase chain reaction (RT-PCR) with a pair of specific primers, which gave a 421-bp fragment. Another pair of primers were then used to amplify the full-length MAGE-1 cDNA by the same method. The PCR products were then digested by restriction endonucleases and inserted into the plasmid PUC19. After primary selection of the recombinants by endonuclease digestion, the sequences of the inserted gene fragments were confirmed by DNA sequence analysis. In 45 HCC patients, MAGE-1 mRNA was expressed in 27 tumor tissues (60%) and 5 paratumor tissues (11.1%). All the four HCC cell lines positively expressed the MAGE-1 gene. Owing to the high homology of the MAGE gene family, we obtained three clones of different MAGE genes using the same pair of cloning primers. The three clones were confirmed to be a full-length MAGE-1 gene, a 750-bp fragment of the MAGE-3 gene and a fragment highly homologous to MAGE-6 and MAGE-12 but not identical to any known MAGE genes. The high expression frequency of MAGE-1 gene in HCC suggests the possibility of using it as a target for immunotherapy in HCC patients. Some MAGE genes other than MAGE-1 may also be expressed in HCC together with an unknown MAGE gene that might introduce new targets for immune attacks. The three gene clones obtained in this study can be used in further investigations and especially in the development of new liver cancer vaccines.

Prognostic significance of occult metastases detected by sentinel lymphadenectomy and reverse transcriptase-polymerase chain reaction in early-stage melanoma patients.

Detection of micrometastases in the regional tumor-draining lymph nodes is critical for accurate staging and prognosis in melanoma patients. We hypothesized that a multiple-mRNA marker (MM) reverse transcriptase-polymerase chain reaction (RT-PCR) assay would improve the detection of occult metastases in the sentinel node (SN), compared with hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC), and that MM expression is predictive of disease relapse. Seventy-two consecutive patients with clinical early-stage melanoma underwent sentinel lymphadenectomy (SLND). Their SNs were serially sectioned and assessed for MAGE-3, MART-1, and tyrosinase mRNA expression by RT-PCR, in parallel with H&E staining and IHC, for melanoma metastases. MM expression in the SNs was correlated with H&E and IHC assay results, standard prognostic factors, and disease-free survival. In 17 patients with H&E- and/or IHC-positive SNs, 16 (94%) expressed two or more mRNA markers. Twenty (36%) of 55 patients with histopathologically negative SNs expressed two or more mRNA markers. By multivariate analysis, patients at increased risk of metastases to the SN had thicker lesions (P =.03), were 60 years of age or younger (P <.05), and/or were MM-positive (P <.001). Patients with histopathologically melanoma-free SNs who were MM-positive, compared with those who were positive for one or fewer mRNA markers, were at increased risk of recurrence (P =.02). Patients who were MM-positive with histopathologically proven metastases in the SN were at greatest risk of disease relapse (P =. 01). H&E staining and IHC underestimate the true incidence of melanoma metastases. MM expression in the SN more accurately reflects melanoma micrometastases and is also a more powerful predictor of disease relapse than are H&E staining and IHC alone.