Assessment of Messenger RNA of 31-#{247}4-N-Acetylgalactosaminyl- transferase as a Molecular Marker for Metastatic Melanoma’

Gangliosides GM2 [GalNAc 1-4(NeuAca2-3)Gal 14Glc M-1Cer] and GD2 [GalNAc il-4(NeuAca2 -8NeuAca23)Gal 1-4Glc il-1Cer] are cell surface tumor-associated antigens and have been demonstrated to be important markers of human malignant melanoma progression. Expression of these glycolipid antigens on melanoma tissues can be assessed by immunohistochemistry or biochemical analysis. These methodologies, however, are not logistically practical or sensitive for testing metastatic melanoma cells in blood or in tissue biopsies. In the present study, we hypothesized that the enzyme involved in GM2 and GD2 synthesis, il4-Nacetylgalactosaminyltransferase ( 14GalNac-T), can be a useful marker for detection of occult metastatic melanoma. A reverse transcription PCR and Southern blot assay to detect 1-*4GalNac-T mRNA expression was developed. I1-#{247}4GalNac-T mRNA was detected in all 13 melanoma cell lines tested. Metastatic melanoma of lymph nodes and different organ sites expressed 314GalNac-T mRNA at various levels. Detection sensitivity of the reverse transcription PCR assay was 1 ng of total RNA extracted from tumor specimens and approximately S melanoma cells in 20 million normal donor peripheral blood lymphocytes. In assessment of blood from 126 melanoma patients, fll-*4GalNac-T mRNA was more frequently found in advanced-stage melanomas and in patients showing more aggressive tumor progression. Normal donor blood samples (n = 37) were all negative for 31-s4GalNac-T mRNA expression. These results suggest that 1-+4GalNac-T mRNA is a promising molecular marker for detecting melanoma cells, characterizing antigen expression, and monitoring tumor progression. Received 7/29/97; revised 1 1/26/97; accepted I 1/26/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with I 8 U.S.C. Section 1734 solely to indicate this fact. C Supported in part by Grants P01 CA 29605 and CA 1038 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404. Fax: (310) 449-5282. INTRODUCTION Aberrant ganglioside expression on the surface of tumor cells has been associated with specific events of tumor progression (1). Neural crest-derived tumors, such as melanomas, astrocytomas, and neuroblastomas, aberrantly express a high level of sialyliated glycosphingolipids, namely gangliosides. on their surface membrane. Gangliosides play an important role in tumor growth, invasion, and metastasis ( 1 ). Melanoma is well known to express high levels of specific types of ganglioside during tumor progression (2, 3). The two most prominent gangliosides, GM23 and GD2, were previously demonstrated to be associated with melanoma tumor progression. Both of these gangliosides were originally referred to as oncofetal antigens (OFA1 and OFA-2) on melanomas (4, 5), and overexpression of these two gangliosides has been shown to contribute to the tumonigenicity of human melanoma (3). Normal mclanocytc primary cultures (transformed cell lines) express GM2 at very minimal level (6). Other human cancers, such as renal cell carcinomas, gastric carcinomas, neuroblastomas, adult leukemias, lung carcinomas, and gliomas, have also been shown to have elevated levels of GM2 and/or GD2 compared to respective normal tissue counterparts (7-13). In addition, GM2 and GD2 have been demonstrated to be highly immunogenic in humans (14, 15). Antibodies and cellular immune responses to both of these gangliosides have been detected in melanoma patients (14-17). Currently, various forms of immunotherapy targeting GM2/GD2 are under investigation (14, 15, 18, 19). The biosynthesis of gangliosides has been described to involve three major pathways, referred to as the a, b, and c series (20). The carbohydrate structures on individual gangliosides are the result of specific sequential catalytic reactions of specific glycosyltransferases. Gangliosides GM2 and GD2 are derived from precursor gangliosides GM3 and GD3, respectively. The enzyme 1-+4GalNac-T (EC 2.4.1.92) has recently been cloned, and its role in the biosynthesis of GM2/GD2 has been verified (21). 13]4GalNac-T is also commonly known as GM2/GD2 synthase, and it is detected in melanoma and neuroblastomas (21, 22). The level of enzyme activity in melanoma has been correlated with GM2 and/or GD2 expression (22). In recent reports, we have demonstrated the use of melanoma-associated antigens as mRNA markers for detection of malignant melanoma by RT-PCR and an automated Southern blot analysis (23-26). The assay is highly sensitive in the detection of occult metastatic melanoma cells in draining lymph 3 The abbreviations used are: GM2, GalNAc13l-4(NeuAca2-3)Gal13l4Glc13 1-ICer; GD2, Ga1NAc13 l-4(NeuAca2-8NeuAca2-3)Ga113 14Glc13l-lCer; GM3, NeuAca2-3Gal13l-4Glc13l-lCer: GD3, NeuAca28NeuAca2-3Ga113l-4Glc13l-lCer; AJCC, American Joint Commission on Cancer; 1 I -4GalNac-T. 131 -+4-N-acetylgalactosaminyltransferase; PBL, peripheral blood lymphocyte: RT, reverse transcription: TDLN, tumor-draining lymph node. Research. on May 28, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 412 131-*4GalNac-T mRNA as a Melanoma RT-PCR Marker nodes and blood. The RT-PCR and Southern blot assay system we have developed allows assessment of minimal amounts of tissues, blood, or cells. In this report, we describe the use of 13l4GalNac-T mRNA expression as a RT-PCR marker to detect metastatic melanoma cells in tissue and blood. Development of new molecular tumor markers will help solve the problems that arc encountered in tumor diagnosis, such as tumor heterogeneity and clonal selection during tumor progression and treatment. Furthermore, a sensitive molecular assay for the detection of these immunogenic GM2 and GD2 tumor antigen markers should be particularly valuable in monitoring tumor regression or progression following specific antigen-targeted