Research progress on the role of MAGE-A3 in the occurrence and development of esophageal cancer / 中国肿瘤生物治疗杂志

@#[摘 要] 食管癌（EC）是临床常见的恶性肿瘤之一，近年来免疫治疗的应用使EC患者的预后得到了改善，但患者对免疫治疗的应答有着明显的异质性，且欠缺有效的疗效预测生物标志物，无法精准筛选获益人群，存在原发性耐药，因此需要寻找新型免疫治疗靶点使更多患者获益。黑色素瘤相关抗原A3（MAGE-A3）是一种癌-睾丸抗原，在正常组织中几乎不表达，而在多种恶性肿瘤组织中呈现高表达，参与肿瘤细胞的增殖、侵袭和转移等多种生物学过程。MAGE-A3基因具有限制性表达和良好的免疫原性，是良好的EC免疫治疗靶点，对EC的诊断、治疗和预后判断等具有良好的应用价值。由于MAGE-A3基因在EC组织中高表达，并与不良预后相关。目前针对MAGE-A3基因靶点的肿瘤疫苗和过继性细胞免疫治疗等疗法均展现出良好的应用前景。

Osteosarcoma of the jaw - experience at the Medical University Vienna and comparative study with international tumor registries

OBJECTIVES: Osteosarcoma of the jaw (OSAJ) is fundamentally different in clinical practice from its peripheral counterparts. Studies are difficult to conduct due to low incidence rates. The primary aim of this study was to provide for the first time a comprehensive retrospective analysis of the treatment concepts and outcome data of OSAJ patients treated at the University Hospital Vienna and to compare these with two recently published studies on OSAJ. The clinical study was accompanied by a biomarker study investigating the prognostic relevance of melanoma-associated antigen-A (MAGE-A) in OSAJ specimens. METHOD: Eighteen patients were included, and their outcomes were compared to published data. Immunohistochemistry was performed with mouse monoclonal antibodies against MAGE-A. Survival rates were estimated by the Kaplan-Meyer method. The log-rank test was used to analyze potential prognostic parameters. Fisher's exact test was performed to define the significant differences between the survival rates of the current study and the DOESAK registry. RESULTS: Disease-specific survival was 93.8% after five and 56.3% after ten years. The development of metastases (p=0.033) or relapse (p=0.037) was associated with worsened outcomes in our group as well as in the comparative group. Despite the different treatment concepts of the study groups, survival rates were comparable. MAGE-A failed to show prognostic relevance for OSAJ patients. CONCLUSIONS: Uncertainties about the optimal treatment strategies of OSAJ patients will currently remain. Thus, prospective studies of OSAJ are needed but are only feasible in a multicenter study setting, conducted over a prolonged time period.

Expressions and clinical significances of MAGE-A9, MAGE-A11 and Ki67 in laryngeal squamous cell carcinoma / 中国肿瘤生物治疗杂志

@# Objective: To explore the expressions of melanoma antigen (MAGE) -A9, -A11 and Ki67 in laryngeal squamous cell carcinoma (LSCC) tissues, and to analyze their correlation with clinicopathological features and the prognosisof LSCC patients. Methods: A total of 73 pairs of LSCC tissuesand corresponding para-cancerous tissues resected from LSCC patients, who were treated at the Fourth Hospital of Hebei Medical University from 2012 to 2014,were collected for this study. At the same time, testicular tissues from 3 patients with prostate cancer after castration were selected as positive control. The protein expressions of MAGE-A9, MAGE-A11 and Ki67 in LSCC tissues and its para-cancerous tissues were detected by immunohistochemistry. Results: The expression rates of MAGEA9, MAGE-A11 protein and Ki67 in LSCC tissues were 47.94% (35/73), 49.32% (36/73) and 46.58% (34/73) respectively, which were significantly higher than those in para-cancerous tissues. The protein expressions of MAGE-A9 and MAGE-A11 were correlated with clinical stage and lymphatic metastasis of LSCC (P<0.05). The expression of Ki67LI was correlated with tumor size, clinical stage and lymphatic metastasis of LSCC (P<0.05). The correlation analysis showed that the expressions of MAGE-A9 and MAGE-A11 were positively correlated with Ki67 (r=0.258, P＝0.027; r=0.672, P=0.001). Kaplan-Meier survival curve analysis showed that the survival rates of patients with high expression of MAGE-A9 protein (P=0.009), MAGE-A11 protein (P=0.031) and Ki67LI (P=0.040) were significantly lower than those with low expressions. And the survival time of patients with both high expressions of MAGE-A9 and Ki67LI (P=0.001) or both high expressions of MAGE-A11 and Ki67 (P=0.001) was significantly shorter than that of patients with low expression (both or single). Univariate and multivariate Cox regression analysis further indicated that MAGE-A9 protein (P=0.028) and MAGE-A11 protein (P=0.042) were independent prognostic factors for overall survival of LSCC patients. Conclusion: MAGE-A9, MAGE-A11 and Ki67 are tumor-associated antigens of LSCC, which can be used as prognostic indicators for LSCC.

Screening and identification of MAGE-A11 related genes based on DNAmicroarray / 中国肿瘤生物治疗杂志

@# Objective: To screen related genes of melanoma-associated antigen-A11 (MAGE-A11) in breast cancer cells based on highthroughput DNAmicroarray technology, and to validate from the aspects of quantity and function. Methods: DNAmicroarray was used to screen the differently-expresseddown-stream mRNAs of MAGE-A11 in breast cancercelllines (MCF-7, MDA-MB-231 and BT-549). Cluster analysis was applied on representative genes and quantitative RT-PCR was used to validate. CCK-8, scratch wound healing assay and Transwell assaywere used to detect the effect of MAGE-A11 on the proliferation,migration and invasion of breast cancer cells. Results: Over-expression of MAGE-A11 caused the differential expression of 1608 down-stream genes in 3 breast cancer cell lines, which was associated with various cell functions such as protein ubiquitination,cell proliferation and apoptosis, tumor invasion and metastasis.qRT-PCR validated that the expression of ZNF-451, CENPTJ, CDK13, API5 and LMO7, which were highly expressed in microarray, were also significantly higher than those in control group (P<0.01);in addition, SHPRH, PML, MARK2, LIMA1 and ANGPTL4, which were low-expressed in microarray, were also significantly lower than those in control group (P<0.01). MAGE-A11transfection directly increased the proliferation of breast cancer MCF-7, MDA-MB-231 and BT-549 cells at 72 h (all P<0.01); compared with control group after transfectionexhibited obvious wound healing at 48 h (P<0.05 or P<0.01) and significantly increased trans-membrane cell numbers (all P<0.01). Conclusion: Many differentially expressed genes related to ubiquitination, cell proliferation and apoptosis, tumor invasion and migration were screened in MCF-7, MDA-MB-231 and BT-549 breast cancer cells. Among them, 10 typical differentially expressed genes were identified in terms of quantity and function.

Expression and significance of MAGE-A9 in hepatocellular carcinoma / 肿瘤研究与临床

Objective To detect the expression of MAGE-A9 in human hepatocellular carcinoma (HCC), and to investigate the association between expression of MAGE-A9 and the clinicopathological characteristics of HCC.Methods Reverse transcription-polymerase chain reaction (RT-PCR), one-step quantitative-PCR (qPCR) and immunohistochemistry (IHC) methods were performed to characterize the expression of MAGE-A9 in HCC cell lines and tissues.Kaplan-Meier survival analysis and Cox regression were employed to evaluate the prognosis of 100 HCC patients.Results The expression of MAGE-A9 mRNA in HCC (4.44±0.342) was significantly higher than that in non-cancerous cells and tissues (1.73±0.178) (P ＜ 0.05).The expression level of the MAGE-A9 protein in HCC was related to the pathological grade (P =0.003), portal vein invasion (P =0.001), distant metastasis (P =0.022) and TNM stage (P =0.005).Cox regression analysis revealed that MAGE-A9 expression is an independent prognostic factor for disease-free survival (P =0.006) and overall survival (P =0.022).Conclusion MAGE-A9 is a valuable prognostic biomarker for HCC patients, and its high expression suggests unfavorable survival outcomes in HCC patients.

Detection of Rare Cancer Cells in the Blood by RNA Extraction of Filtered Mononuclear Cells and Reverse Transcription-PCR / 임상검사와정도관리

BACKGROUND: To enhance the cancer cell detection rate in blood, we tried to detect cancer cells by blood filtration, RNA extraction and reverse transcription (RT)-PCR in the filtered mononuclear cells (MNCs). METHODS: From the specimens of whole blood and filtered MNCs, RNA was extracted by the guanidinium isothiocyanate buffer method. Filtration efficiency was evaluated by measurement of leukocyte count, red cell count, and hemoglobin level. To compare the RNA extraction efficiency between whole blood and filtered MNCs, the followings were examined: (1) RNA electrophoresis, (2) hTERT, survivin, plakophilin, LunX, and MAGE A1-6 RT-PCR, and (3) the detection limit of added SNU484 cells in the blood by MAGE A1-6 RT-PCR. Finally blood specimens of 13 lung cancer patients were used to detect cancer cells by LunX and MAGE A1-6 RT-PCR with filtered MNCs. RESULTS: The filtration method revealed 0%, 92.8% and 95.1% filtration rates for leukocyte, red cell, and hemoglobin, respectively. Contamination of concentrated genomic DNA was observed in the electrophoresis of RNA extracted from whole blood. Positive rates of hTERT, survivin and plakophilin were higher in the filtered MNCs. The filtration method detected 1 SNU484 cell/mL, and the blood samples of 4 (30.8%) lung cancer patients showed positive results for RT-PCR. CONCLUSIONS: For the detection of cancer cells in the blood, the filtration method was very efficient, and LunX and MAGE A1-6 genes would be useful for the detection of blood cancer cells in patients with lung cancer.

Use of Dentritic Cells Pulsed with HLA-A2-restricted MAGE-A1 Peptide to Generate Cytotoxic T Lymphocytes against Malignant Glioma / 华中科技大学学报（医学）（英德文版）

This study developed a novel approach of targeting malignant glioma with pMAGE-A1278-286-specific cytotoxic T lymphocytes (CTLs) induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen (HLA)-A2-restricted pMAGE-A1278-286 peptide-pulsed dentritic cells. Cytotoxic assays were performed by the colorimetric CytoTox 96 assay to analyze cytotoxic activity of the induced CTLs against various target cells. The induced CTLs showed approximately 45% specific lysis against T2pMAGE-A 1278-286 (pMAGE-A 1278-286 peptide pulsed T2 cells) and U251 (HLA-A2+, MAGE-A 1 +)at an effector:target ratio of 40:1, and approximately 5% cytolysis against T2pHIV, A172 (HLA-A2,MAGE-AI+), K562 and T2 cells without being pulsed with peptide at any effector:target ratio. The specific killing activity of the induced CTLs against T2pMAGE-A1278-286 and U251 was much more obvious than in any other control group (P＜0.05). The cytotoxic activity against the T2pMAGE-A1278-286 and U251 was significantly eliminated by anti-HLA class I mAb W6/32.These results suggest that pMAGE-A1278-286 epitope may serve as a surrogate tumor antigen target of specific immunotherapy for treating HLA-A2 patients with malignant glioma.

Melanoma antigen-A family in tumor / 国际肿瘤学杂志

As a tumor-specific antigen highly expressed in various types of tumors, MACE-A does not exist in normal adult tissues, except for testis and placenta. Therefore MAGE-A antigens are regarded. tumor specific antigen,and have significant significance for cancer immunotherapy.

Detección de mamoglobina y mage-A3: en ganglios linfáticos axilares: histológicamente negativos: en cáncer de mama / Detection of MAGE-A3 and mamoglobina: axillary lymph node: histologically negative in breast cancer

La detección histológica de metástasis en los ganglios linfáticos axilares es un parámetro de mayor valor pronóstico en cáncer de mama. Un 30 por ciento de pacientes con ganglios linfáticos negativos tienen recaída en cinco años, sugiriendo que, este estudio no es suficiente para detectar todas la metástasis. En el presente estudio, se determinó la expresión de marcadores mamoglobina y MAGE-A3. en ganglios axilares histológicamente negativos de pacientes con cáncer de mama, mediante reacción en cadena polimerasa transcriptasa-reversa, a fin de detectar células tumorales aisladas o micrometástasis. La expresión mamoglobina y MAGE-A3 se detectaron en 55,6 por ciento y 33,3 por ciento, respectivamente. Se amplificó el ARN de mamoglobina y MAGE-A3 en ganglios histológicamente positivos en un 72,7 por ciento y 54,5 por ciento, respectivamente. Se relacionó la presencia o ausencia de células tumorales en ganglios negativos con tamaño tumoral, grado histológico, émbolos neoplásicos vasculares, obteniendo resultados no concluyentes, no se logró distribución equitativa de las muestras. Estos resultados muestran que la mamoglobina y MAGE-A3 son útiles para detección de células tumorales aisladas en ganglios axilares negativos.

Histological detection of metastasis in axillary lymph nodes is a current practice, and the parameter has greatest prognostic value in breast cancer. 30 per cent of negative lymph nodes patients at histology studies has disease relapse in five years, suggesting that this nethod is inadequate to identify all cases with metastasis. In this study, the expression of mammaglobin and MAGE-A3 markers were determined in histological negative axillaries lymph nodes brest cancer patients, by reverse transcriptase polymerase chain reaction. The expression mammaglobin and MAGE-A3 were detected in 55.6 per cent and 33.3 per cent, respectively. The ARN of mammaglobin and MAGE-A3 was amplified in patients with histological positive axilaries lymph nodes, obtaining an expression of 72,7 percent and 54,5 per cent, respectively. Finally, presence or absence of tumour cells detected in negative lymph nodes were related to tumour seze, histological grade, vascular tumour emboli; non conclusive results were obtaining because it wasn't possible to obtain an equitable distribution of the samples. These results show that mammaglobin and MAGE-A3 are useful for detection of isolated tumour cells in axilar node negative.

Advances in research of MAGE-A3 / 中国癌症杂志

Melanoma-associated antigen MAGE-A3 is expressed in various types of tumors,but not in normal cells except testis and placenta.MAGE-A3 can be presented by both HLA class I and HLA class II molecules after being processed into antigen peptide.MAGE-A3 has recently been widely used in the diagnosis and immunotherapy of tumors.This article reviews the applications of MAGE-A3 in the diagnosis and immunotherapy of tumors.

Expression of melanoma antigen-a mRNA in coal tar pitch-induced lung cancer tissues in mice / 第二军医大学学报

Objective: To investigate the expression of mage-a mRNA in lung cancer tissues of mice induced by coal tar pitch(CTP) fume and to discuss the above model for lung cancer immunotherapy with mage-a. Methods: Tumor tissue samples of lung cancer and paired non-tumor tissues were obtained from 8 lung cancer mice. Total RNA was extracted and cDNA was synthesized. Nested polymerase chain reaction amplification using mage-a specific primer was performed to detect the expression of mage-a. The 2 clones of mage-a mRNA positive PCR products were sequenced by DNAs sequencer (PE-377). Results: Of 29 mice in the experimental group, 8 were induced to lung cancer.Among which 5 (5/8) expressed mage-a mRNA. The expression of mage-a gene was not found in adjacent lung tissues. The DNA sequencing confirmed that the target gene fragments in 2 samples of PCR products were mage-a cDNA. Conclusion: The mage-a gene is highly expressed in lung cancer in mice induced by CTP fume, suggesting that CTP-induced lung cancer in mice may be an ideal animal model for lung cancer therapeutic experiment with MAGE-A.

Study on the evolutional reIationship among the member of MAGE-A family / 中国免疫学杂志

Abstract Objective:To explore homology in mage-A family and find some common epitopes recognized by cytotoxic T lymphocyte(CTL) to oppose tumor escape.Methods:The amino acid sequence,differents open reading frame(ORF) and the known CTL epitope of themember of MAGE-A family,were analyzed using DNAstar software, and phylogenetic tree is also constructed. Results: MAGE-A family shareidentity nucleotide 57.6% - 98.0%,the phylogenetic tree showed that they are derived from a common ancestor at different time, as well asfound some CTL epitopes high similarity. Conclusion: MAGE-A family come from common an ancestor, but they bave many differences in thepattern of expression in tumor.This study can help to find the best CTL epitope vaccine to cure tumor.

Finding and identification of a novel HLA-A2-restricted CTL epitope derived fromthe tumor antigen MAGE-A3 / 中国免疫学杂志

To identify novel HLA-A2-restricted CTL epitopes derived from the tumor antigen MAGE-A3.Methods:The CTL epitope candidates were predicted by using the motif prediction method combined with the secondary anchor residues plot. It was established that the molecule model of CIL epitope candidates bound to the HLA-A2 molecule and of the HLA-A2-peptide complex by molecule modeling. Four selected candidates were assayed by the standard 51 Cr release assay to determine their abilities of inducing the generation of specific CTLs. Results: Among them, the pepnde MAGE-A3201-209 could effectively induce specific Oils and the CITs could lyse not only the melanoma cell line LB373-MEL but also the murine mastocytoma cell line genetically modified with the human HLA-A2 gene and pulsed with MAGE-A32oi.2cs ? Conclusion; MAGE-AS201-209 is a novel CTL epitope presented by HLA-A2. This study provides experimental basis for design and study of tumor therapeutic peptide vaccines based on the tumor antigen MAGE-A3.