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

Cancer-testis (CT) antigens are expressed in a variety of malignant tumors, but in normal adult tissue, they are only expressed in testicular germ cells. Owing to this tumor-associated expression pattern, these antigens are of major interest as potential targets for immunotherapy and possibly for diagnostic purposes. This study was performed to analyze the expression of four CT antigens, NY-ESO-1, MAGE-A3, MAGE-A4, and CT7/MAGE-C1, in endometrial carcinoma using immunohistochemistry, and to correlate expression with histologic subtypes, grade, and expression of WT1 and p53. Formalin-fixed paraffin-embedded tissues of 130 endometrial carcinomas of the following types and grades were analyzed using a tissue microarray: 85 endometrioid carcinomas (FIGO grade 1, 39; grade 2, 11; and grade 3, 35), 18 papillary serous carcinomas, 12 clear cell carcinomas, 13 malignant mixed mullerian tumors, one mucinous adenocarcinoma, and one undifferentiated carcinoma. The following anti-CT monoclonal antibodies/antigens were studied by immunohistochemistry: monoclonal antibody ES121/NY-ESO-1, monoclonal antibody M3H67/MAGE-A3, monoclonal antibody 57B/MAGE-A4, and monoclonal antibody CT7-33/CT7. The CT expression data were compared to WT1 and p53 protein expression as analyzed in a previous study. Positive staining with anti-CT monoclonal antibodies was graded as follows: focal, <5% positive cells; 1+, 5-25% cells; 2+, 26-50% cells; 3+, 51-75%; and 4+, >75% cells. The 3+ and 4+ staining patterns were considered homogeneous patterns of potential clinical significance and were scored positive for statistical analysis. In low-grade tumors, the most immunoreactivity was seen with mAb M3H67 but little labeling was observed with the other monoclonal antibodies. In high-grade tumors, monoclonal antibodies M3H67 (25%), 57B (23%), and CT7-33 (20%) showed the highest reactivity, while ES121 showed the lowest immunoreactivity (6%). The staining pattern was mostly heterogeneous. Statistical significance was found solely for the correlation of monoclonal antibody 57B staining and p53 expression. No correlation was found for any anti-CT monoclonal antibody staining and clinical stage or for anti-CT staining and WT1 expression. CT antigens CT7, MAGE-A3 and MAGE-A4, but not NY-ESO-1, are expressed in high-grade endometrial carcinomas, and expression of MAGE-A4 is correlated with the presence of overexpressed p53.

Expression of cancer/testis (CT) antigens MAGE-A1, MAGE-A3, MAGE-A4, CT-7, and NY-ESO-1 in malignant gammopathies is heterogeneous and correlates with site, stage and risk status of disease.

Cancer/testis (CT) antigens are expressed in several malignant tumors, but not in normal tissues except for testicular germ cells. The expression of CT antigenic proteins in malignant gammopathies has not been characterized. We examined the expression of a panel of CT antigenic proteins in 29 patients with malignant gammopathies by immunohistochemistry using the following monoclonal antibodies (mAbs): mAb MA454 to MAGE-A1, mAb M3H67 to MAGE-A3, mAb 57B to MAGE-A4, mAb CT7-33 to CT7/MAGE-C1 and mAb ES121 to NY-ESO-1. We could detect at least one CT antigen in tumors from 27 of 29 patients. The expression pattern of MAGE-A1, -A3, -A4 and NY-ESO-1 is heterogeneous in most cases, revealing staining in <25% of the tumor cells. Monoclonal antibodies CT7-33 and M3H67 show the highest incidence of immunoreactivity. Importantly, CT-7 can also be detected on the surface of some myeloma cells by flow cytometry, and in one plasmacytoma case by immunohistochemistry. Expression of CT antigens is greater in patients with stage III extramedullary plasmacytoma or high-risk myeloma relative to other cohorts. These data suggest that CT antigens may have important biological implications in malignant gammopathies and that CT-7 may be a suitable target for T cell-based and possibly antibody-mediated immunotherapy of myeloma.

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with DeltaEGFR, a deletion mutation lacking exons 2-7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6(DeltaEGFR) (mouse fibroblast line NR6 transfected with DeltaEGFR). mAb 806 with selective reactivity for NR6(DeltaEGFR) in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-DeltaEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with DeltaEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without DeltaEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to DeltaEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that DeltaEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.

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

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 presents a new serologic reagent for the analysis of CT7 on a protein level and confirms what is known with regard to the expression pattern of other CT antigens in tumors: frequent heterogeneity of antigen expression.

MAGE antigen expression in monophasic and biphasic synovial sarcoma.

Synovial sarcomas are high-grade malignant mesenchymal tumors carrying a pathognomonic cytogenetic alteration t(X;18) involving the SYT gene on chromosome 18 and either SSX1 or SSX2 on chromosome X. Morphologically, biphasic (BSS) and monophasic (MSS) variants can be distinguished. Cancer/testis (CT) antigens are expressed in a variety of malignant tumors, but not in normal tissues except in germ cells, primarily of the testis. Anti-MAGE monoclonal antibody (mAb) 57B previously showed a high incidence and homogenous reactivity pattern in a preliminary analysis of synovial sarcomas. This study was performed to analyze the expression of MAGE by immunohistochemistry with mAb 57B in 25 synovial sarcomas (12 monophasic, 13 biphasic), which were typed for the t(X;18)-derived fusion transcript by reverse transcriptase polymerase chain reaction (19 SYT-SSX1, 6 SYT-SSX2). 57B immunoreactivity was present in 22 of 25 (88%) cases, and antigen expression was homogeneous in 14 of 22 57B-positive cases. Both morphological variants and both translocation types were immunoreactive; three SYT-SSX1 tumors (one MSS, two BSS) were 57B negative. Our study demonstrates that MAGE is frequently and homogeneously expressed in synovial sarcomas of both morphological variants and both translocation types, making these tumors an attractive target for MAGE antigen-based immunotherapy.