TAG-72 in Gastric Cancer: Relationship between Preoperative Serum Levels and Tumoral Content of the Antigen

Tumor-associated glycoprotein-72 (TAG-72) is a new tumor marker that can be identified with the monoclonal antibody (MAb) B72.3, which was generated against a membrane-enriched fraction of human mammary carcinoma cells (1). Tissue expression of this high-molecularweight mucin glycoprotein has been observed in a variety of malignant epithelial tumors including breast, stomach, colon, lung, ovarian and endometrial carcinomas (2). Recent studies have shown that this tumor marker may be useful in the postoperative management of patients with gastric cancer. Several authors have demonstrated the clinical utility of TAG-72 serum levels in the early identification of recurrences (3-7). In addition, a significant correlation has been described between preoperative serum levels of this antigen and poor prognosis in gastric cancer patients (8, 9), even for resected patients when multivariate analysis was used (10). However, it is as yet unclear why the serum level of TAG-72 at the time of surgery has an influence on long-term survival. The aim of this study was to prospectively evaluate by means of an immunoradiometric assay the possible relationship between the preoperative serum levels of this antigen and its tumoral content, both membranous (TAG-72m) and cytosolic (TAG-72c), in 47 non-metastatic gastric cancer patients. In addition, the TAG-72 tissue content was also measured in non-neoplastic adjacent mucosa samples of 30 patients. Blood samples collected in the early morning five to ten days prior to surgery from a peripheral vein were centrifuged and the serum was frozen at –20°C until use. Tissue pieces were removed from the tumor, avoiding grossly necrotic tissue, and from surrounding mucosa at least 2 cm from the tumors. The mucosa surrounding the tumors was macroscopically and microscopically free from neoplastic growth. The tumor and surrounding tissue were rapidly transported on ice to the laboratory and stored at –70°C until assayed; the specimens were processed at the same time. They were pulverized with a microdismembrator (BRAUN) and homogenized with a glass-teflon potter in Tris-HCl buffer (Tris 10 mM, EDTA 1.5 mM, 10% glycerol, 0.1% monothioglycerol) at pH 7.4. The samples were centrifuged at low speed for 10 min at 800 x g and the supernatant was ultracentrifuged for 60 min at 100,000 x g. All procedures were performed at 0° to 4°C. We obtained a supernatant that contained the cytosol and a precipitate with the membranes. TAG 72 antigen was analyzed in the serum and in the cytosolic and membranous samples using a two-site solid-phase immunoradiometric assay (ELSA-CA 72-4, CIS Bio International, Gif-sur-Yvette Cedex, France). The imprecision of the assay was assessed using two samples with different concentrations (9 and 83 U/mL); they were tested either 30 times in the same series of assays or in duplicate in 10 different series, with within-run CVs of 9.3% and 5.1% and between-run CVs of 11.7% and 5.7%, respectively. The detection limit was found to be 0.8 U/mL. The serum concentrations of TAG-72 were expressed as U/mL; TAG-72 tissue content was expressed as U/mg protein. In accordance with our previous report (11), the upper cutoff value of activity for TAG-72 serum levels was defined as 6 U/mL. The TAG-72 content was expressed as mean ± standard error (SE). For data analysis the patients were subdivided into different groups based on different clinical or pathological parameters. For comparison of TAG-72 content between different groups the Mann-Whitney or Kruskal-Wallis test was used. To compare differences between percentages of TAG-72 serum positivity we used the chi-square test with Yates’ correction. Associations between the various TAG-72 contents were assessed by the Spearman rank correlation test. The results show a wide variability in preoperative serum TAG-72 levels (range: 3-357 U/mL), with concentrations above the cutoff level of 6 U/mL in 46.8% of patients. The results also show a wide variability of the TAG-72m (0.01-149 U/mg prot.) and TAG-72c (0.6-91 U/mg prot.) tumoral contents. However, as can be seen in Table I, there were no significant differences in preoperative serum levels and tumoral contents of the antigen in relation to the clinicopathologic parameters. In addition, there were no significant differences between the TAG-72 content in tumors (Table I) and paired sets of normal adjacent mucosa samples (n=30); TAG-72m: 12.5±6.8, range 0.1-208 U/mg prot. ; TAG-72c: 8.5±3.5, range 0.1-104 U/mg prot. There were also no significant differences between membranous and cytosolic TAG-72 contents either in tumors or in non-neoplastic adjacent mucosa samples. However, there was a positive and significant correlation between TAG-72m and TAG-72c contents in tumors (n=47; r sub S: 0.52; p<0.001) and in The International Journal of Biological Markers, Vol. 15 no. 2, pp. 197-199 © 2000 Wichtig Editore