Relationship of Carbohydrate Antigen 19-9 and Lewis Antigens in Pancreatic Cancer1

Carbohydrate antigen (CA) 19-9 identified by a murine monoclonal antibody against a colorectal carcinoma antigen is thought to be a sialylated Lewis (Le)* blood group antigen and occurs in high concentra tion in serum of patients with pancreatic carcinoma. This study was designed to identify the relationship between Lewis antigens and CA 199 in patients with pancreatic cancer. The following analyses were per formed in 20 pancreatic cancer patients: Le* and Le'1antigen phenotype in saliva (modified enzyme-linked immunosorbent assay) or on red cells (hemagglutination); CA 19-9 levels (radioimmunoassay) in serum; and CA 19-9 and Le*and Leb expression (immunoperoxidase assay) on tumor tissue. Le*~b~patients based on salivary phenotype failed to express CA 19-9 in tumor tissue and had normal or low levels of CA 19-9 (<37 units/ ml) in serum (P = 0.0011, versus Le**"and Le-"* patients). Eightyeight % of Le**11" and Le*"11*patients had elevated serum CA 19-9 levels (>37 units/ml). All Le*+b~and Le*~"*patients expressed both Le* and IA-''antigens in tumor tissue. These results support the view that I ,e" '' pancreatic cancer patients cannot manufacture CA 19-9. Surprisingly, Le'-positive patients express Lo1' antigen in tumor tissue; in this subgroup, Leb antigen may be a tumor-specific biomarker. INTRODUCTION CA3 19-9 is a tumor-associated antigen which is now known to be a sialylated Le" antigen (1). This antigen was originally defined by a monoclonal antibody produced by a hybridoma prepared from spleen cells of a mouse immunized with human colorectal carcinoma cell line SW1116 (2). It is now known that CA 19-9 is normally present in salivary mucus and in physiological exocrine pancreatic secretions (3, 4). Elevated CA 19-9 levels (>37 units/ml) have been described in a variety of gastrointestinal malignancies (5), particularly in pancreatic adenocarcinoma (6-10). In various series, elevated CA 19-9 expression is found in 69 to 92% of pancreatic cancer patients. Koprowski et al. (11) have hypothesized that patients with a Lea~b~phenotype should be unable to synthesize CA 199 since normal individuals with a Le"~b~ phenotype do not express CA 19-9 in their secretions (3). Confirmation of this relationship would better define the clinical utility of this biomarker in pancreatic cancer. In this study, MoAbs recognizing CA 19-9 (MoAb 19-9) and Le antigens a and b (MoAb Le" and Leb) were used to determine Le antigen phenotype in saliva, serum CA 19-9 levels, and Le antigen and CA 19-9 expression in malignant tissue in patients with pancreatic adenocarcinoma. PATIENTS AND METHODS Tumor tissue (primary or metastatic) was obtained from patients with adenocarcinoma of the pancreas undergoing clinical evaluation at Received 4/27/87; revised 7/24/87; accepted 7/24/87. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by National Cancer Institute Laboratory Cancer Research Center Support Grant CA36727. 2To whom requests for reprints should be addressed, at Department of Internal Medicine, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105. 3The abbreviations used are: CA, carbohydrate antigen; Le, Lewis; MoAb, monoclonal antibody; PBS, phosphate-buffered saline. the University of Nebraska Medical Center. Sections of paraffin-em bedded formalinor Bouin-fixed tissue were prepared and stained using an immunoperoxidase assay as previously described (12) with murine MoAb 19-9 and CO-514 and CO-431 (MoAb Le" and Leb) with speci ficity for Le" and Leb antigens, respectively (13). Tissue was examined by light microscopy and graded according to the percentage of cells expressing surface antigen. Serum CA 19-9 was quantitated with a radioimmunoassay kit (cour tesy of Centocor, Malvern, PA) using the appropriate monoclonal antibody. Established normal values of 6 to 37 units/ml were used in interpreting results. When possible, a fasting saliva specimen was obtained. Le antigens in saliva were qualitatively demonstrated using an enzyme-linked im munosorbent assay with MoAb Le" or Leb. In brief, heat-inactivated saliva (100 n\) diluted with carbonate-bicarbonate buffer (pH 9.6) was applied to microtiter plate wells and incubated at 4°Covernight. After washing in PBS, 100 n\ of bovine serum albumin in PBS were added and incubated one h at room temperature. After further washings in PBS, 100 ¡i\ of 1:5 diluted culture supernatant containing MoAbs Le* or Leb followed by 100 n\ of 1:100 diluted peroxidase-conjugated affinity-purified goat anti-mouse immunoglobulin and 100 //I of peroxidase substrate (4 mg of o-phenylenediamine:40 ^1 of 30% H2O2 in 10 ml of phosphate-citrate buffer, pH 5.0) were sequentially added to the wells. Brown staining of the wells indicated a positive reaction. In those patients who could not provide a saliva specimen, RBC-associated Le antigens were determined using a standard hemagglutination assay with commercially available polyclonal antiserum. Data were analyzed by x2 analysis.