Purpose: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. Experimental Design: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. Results: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1and was undetectable by postoperative day 7. Conclusions: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.Mesothelin is a cell surface protein present on normal mesothelial cells lining the body cavities. It is highly expressed in several cancers, including mesotheliomas, ovarian and pancreatic cancers, and some squamous cell carcinomas (1 -4). Human mesothelin is made as a 69 kDa polypeptide with a hydrophobic sequence at the carboxyl end that is removed and replaced by phosphatidylinositol. This glycosyl-phosphatidylinositol linkage anchors mesothelin to the cell membrane (1,5). After glycosylation at one or more of its four putative glycosylation sites, it is probably cleaved by the protease furin to yield a 32 kDa soluble protein called megakaryocyte potentiating factor (MPF) and a 40 kDa cell membrane bound protein called mesothelin. However, the proteolytic cleavage of mesothelin by furin has not been clearly shown for human tumors. MPF has been shown to potentiate megakaryocyte proliferation in mouse bone marrow cultures in the presence of interleukin-3 (6, 7). No data is available to show if MPF has megakaryocyte potentiating activity against human bone marrow precursors. The normal biological function of cell membrane -bound mesothelin is not known and mice with a knockout of the mesothelin gene have no obvious phenotype (8). A recent study presented evidence that mesothelin binds CA125 and may, therefore, play a role in the dissemination of ovarian cancer in...
Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296 -359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296 -359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors.
Purpose: Mesothelin is a glycosyl-phosphatidylinositol^anchored glycoprotein present on the cell surface. Mesothelin is a differentiation antigen that is highly expressed on mesothelioma, ovarian cancer, and pancreatic cancer. The existence of a spontaneous humoral immune response to mesothelin in humans has not been fully studied. Here we addressed the issue of whether mesothelin elicits a humoral immune response in patients with mesothelioma and ovarian cancer. Experimental Design: Using an ELISA, we analyzed immunoglobulin G antibodies specific for mesothelin in sera from patients with mesothelioma and epithelial ovarian cancer. Tumor specimens were examined by immunohistochemistry for mesothelin protein expression. Results: Elevated levels of mesothelin-specific antibodies were detected in the sera of 39.1% of patients with mesothelioma (27 of 69 patients) and 41.7% with epithelial ovarian cancer (10 of 24 patients) when compared with a normal control population (44 blood donors; P < 0.01 for both mesothelioma and ovarian cancer). We also found that 53% to 56% of patients with mesothelin immunostaining-positive mesothelioma and ovarian cancer had antibodies specific for mesothelin, whereas only 0% to 8% of patients with negative mesothelin immunostaining had detectable mesothelin-specific antibodies (c 2 test: P < 0.01for mesothelioma and P = 0.025 for ovarian cancer). Conclusions: Our findings indicate that mesothelin is a new tumor antigen in patients with mesothelioma and ovarian cancer and the immunogenicity of mesothelin is associated with its high expression on the tumor cells. Mesothelin represents an excellent target for immunebased therapies.
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