CT7 (MAGE‐C1) antigen expression in normal and neoplastic tissues

Abstract

CT7 (MAGE-C1) is a member of the cancer testis (CT)antigen family. The present study describes the generation of CT7-33, a monoclonal antibody (MAb) to CT7, and the preliminary protein expression analysis of CT7 in normal tissues and in a limited number of neoplastic lesions. CT7-33 was effective in frozen as well as formalin-fixed, paraffin-embedded tissues, and immunohistochemistry/reverse transcriptase polymerase chain reaction (RT-PCR) co-typing demonstrated antibody specificity. CT7-33 immunoreactivity in normal adult tissues is restricted to testicular germ cells. In neoplastic lesions, CT7-33 immunostaining is confined to tumor cells, and the frequency of CT7 protein expression mostly parallels previous mRNA analyses. Whereas colorectal and renal cell carcinomas, as well as sarcomas, exhibit poor or no CT7-33 staining, carcinomas of the mammary gland and ovary, nonsmall cell lung carcinoma and metastatic melanomas exhibit a high incidence of CT7 protein expression. However, as seen in previous analyses of other CT antigens, the expression pattern is mostly heterogeneous, and tumors with more than 50% of tumor staining are only infrequently encountered. In summary, our study Key words: CT7; MAGE-C1; heterogeneous antigen expression; monoclonal antibody CT7-33Cancer testis (CT) antigens are expressed in a variety of malignant neoplasms but not in normal adult tissues except for germ cells of the testis. 1,2 Due to their expression pattern, CT antigens appear to be ideal targets for immunotherapeutic strategies against malignant neoplasms. Since the isolation of the prototype CT gene family, MAGE, the number of CT antigens has steadily increased. 3 Whereas autologous T-cell epitope cloning was used for the initial identification of CT antigens such as MAGE and BAGE, 4,5 others have been isolated by SEREX (serological analysis of recombinant cDNA expression libraries) analysis. SEREX employs the immunologic screening of cDNA expression libraries from human tumors with autologous patient sera. 6 -9 By using the SEREX approach, we recently identified CT7, 10 a new member of the CT family. The same gene was isolated independently by Lucas et al. 11 using representational difference analysis (RDA) and termed MAGE-C1. A preliminary RT-PCR analysis revealed the presence of CT7 (MAGE-C1) in normal testis and in a variety of neoplasms, such as melanoma, and tumors of the urinary bladder, breast and lung. To assess CT7 as a potential target for immunotherapy, analysis of its actual antigen expression is essential. The present study describes the generation of the monoclonal antibody (MAb) CT7-33 to CT7 (MAGE-C1) and an immunohistochemical analysis of CT7 (MAGE-C1) expression in normal and tumorous tissues. MATERIAL AND METHODS Production of recombinant CT7 proteinA truncated CT7 product was chosen for the expression in E. coli. This truncated form encoded 350 of 1,124 amino acids in the CT7 antigen, namely positions 552-901. The purpose of this truncation was to exclude the C-terminus, which shares significant homolo...

Expression of cancer–testis antigens in endometrial carcinomas using a tissue microarray

“…The present study employs mAbs, which were previously generated by our group and others such as M3H67 to MAGE-A3 (manuscript in preparation), 57B to MAGE-A4, 16 CT7-33 to CT7, 37 and ES121 to NY-ESO-1. 23 This CTantigen panel has not been previously analyzed in endometrial cancer.…”

“…This expression pattern (ie, common expression of antigens in the MAGE family and low expression of NY-ESO-1) has been found in metastatic melanomas and in carcinomas of the head and neck, breast, and lung in previous studies at the protein level. 15,23,37,42,43 There is debate regarding the association of CT antigen expression on a protein level and tumor grade. A relationship between MAGE expression and tumor grade has been shown in transitional cell carcinomas of the urinary bladder 44 and in certain types of brain tumors.…”

“…16,21,23,30,[37][38][39] Commercially available antibodies to WT1 (Santa Cruz; Santa Cruz, CA, USA) and p53 (Clone D07 (DAKO; Carpinteria, CA, USA)) were used. A heat-based antigen-retrieval method was used for all antibodies (vegetable steamer (Black and Decker; Denver, CO, USA); 901C, 30 min).…”

“…In the current study, this panel of endometrial carcinomas was used to analyze the expression of four CT antigens by immunohistochemistry employing tissue microarray technology and previously generated mAb ES121 to NY-ESO-1, mAb 57B to MAGE-A4, mAb M3H67 to MAGE-A3, and mAb CT7-33 to CT7. 15,16,23,37 We also correlated CT antigen expression with clinical stage, histologic subtype, tumor grade, and expression of WT1 and p53, as previously studied. 36 …”

Immunohistochemical analysis of the expression of FATE/BJ-HCC-2 antigen in normal and malignant tissues

“…Hence, EDTA should be used as the antigen retrieval solution in the staining of paraffin sections to reduce or remove nonspecific reactivity. 11 …”