Expression of cancer-testis (CT) antigens in placenta.

Besides their variable presence in fetal and adult germ cells, CT antigens have occasionally been detected in placental tissue. However, these data are scarce and solely based on mRNA analyses; nothing is known about their presence at the protein level. Here, we analyzed the expression of various CT antigens in placental tissues from gestational age week 5 to week 42 using monoclonal antibodies to various antigens of the MAGE-A and -C families, NY-ESO-1, as well as GAGE. We show that CT antigen expression in placenta varies widely for the various antigens, ranging from completely negative to abundant. Since little is known about the function and biology of CT antigens, interpretation of this highly variable expression pattern is purely speculative. However, our data indicate that the various CT antigens have different functions during placental development.

Glycoprotein A34, a novel target for antibody-based cancer immunotherapy.

To identify novel, tissue-restricted cell surface proteins in cancer which can serve as targets for antibody-based diagnostics and therapeutics, a translated version of the expressed sequence tag database (tblastn) was mined for transcripts with similarity to the glycoprotein A33 (GPA33) colon cancer antigen. A novel human transcript, termed A34, was identified which encoded a putative cell surface protein, GPA34, which is approximately 30% identical to GPA33 and other members of the junctional adhesion molecule (JAM) family. Conventional end-point and quantitative real-time RT-PCR showed that A34 mRNA expression is highly tissue-restricted, as it is expressed predominantly in stomach and testis. A34 mRNA was also detected in 6/19 (31%) gastric cancers, 8/16 (50%) esophageal carcinomas, and 4/17 (23%) ovarian cancers, but not in lung, breast or colon carcinomas. A murine monoclonal antibody (mAb A34) was generated to the extracellular domain of the A34 protein and used to biochemically and immunohistochemically characterize the A34 antigenic system. The mAb A34 specifically recognized glycoproteins ranging in apparent size from 55-70 kDa, present in normal gastric mucosa and in COS-7 cells transfected with A34 cDNA. Of 31 different normal tissues examined by immunohistochemistry, GPA34 protein expression was detected primarily in normal stomach mucosa and testicular germ cells, and in the tumor cells of 5/17 (29%) gastric cancers, 7/11 (63%) esophageal cancers, and 2/21 (9%) ovarian cancers, in agreement with gene expression results. The A34 antigen and monoclonal antibody may be of considerable value for immunotherapy of different types of cancer.

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.

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.

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.

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.