Ingegerd Hellstrom1, Yuen Yee Yip2, Martin Darvas2, Elizabeth Swisher3, Karl Erik Hellstrom2. 1. Department of Pathology, University of Washington, Harborview Medical Center Seattle, 325 9th Ave, Box 359939, Seattle, WA 98104, United States. Electronic address: ihellstr@uw.edu. 2. Department of Pathology, University of Washington, Harborview Medical Center Seattle, 325 9th Ave, Box 359939, Seattle, WA 98104, United States. 3. Department of Gynecology, University of Washington, Harborview Medical Center Seattle, 325 9th Ave, Box 359939, Seattle, WA 98104, United States.
Abstract
BACKGROUND: The gene encoding HE4 undergoes alternative splicing to yield multiple protein isoforms. We investigated anti-HE4 mAbs which recognize epitopes on the C (2H5 and 3D8) or N (12A2 and 14E2) terminals. METHODS: A Luminex assay was applied to determine mAb affinity. Binding of mAbs to sections from formaline fixed ovarian carcinomas was determined by immunohistology, binding to cultured ovarian carcinoma cells was tested by flow cytometry and immunocytochemistry, and HE4 secretion to patient serum or supernatants of cultured ovarian carcinoma was assayed by ELISA. RESULTS: mAb 12A2 bound to formalin-fixed sections from 18 of 19 ovarian carcinomas, while 14E2, 2H5 and 3D8 bound to sections from 14, 7 and 4 patients, respectively. The mAbs bound independently of each other, i.e. a sample bound one mAb most strongly while another sample with similar affinity strongest bound another mAb. The intensity of binding to sections did not significantly correlate with the serum level of HE4 except for mAb 14E2, but there was a significant correlation between HE4 expression and its detection in supernatants of cultured ovarian carcinomas. CONCLUSIONS: HE4 epitopes are expressed independently of each other and their expression of HE4 by cultured ovarian carcinoma cells correlates with the release of HE4 into culture supernatants. he epitope recognized by mAb 14E2 was significantly more expressed by platinum resistant tumors. IMPACT: Expression of HE4 by most ovarian carcinomas makes it an excellent biomarker.. Further studies are needed to investigate the clinical relevance of overexpression of a particular epitope.
BACKGROUND: The gene encoding HE4 undergoes alternative splicing to yield multiple protein isoforms. We investigated anti-HE4 mAbs which recognize epitopes on the C (2H5 and 3D8) or N (12A2 and 14E2) terminals. METHODS: A Luminex assay was applied to determine mAb affinity. Binding of mAbs to sections from formaline fixed ovarian carcinomas was determined by immunohistology, binding to cultured ovarian carcinoma cells was tested by flow cytometry and immunocytochemistry, and HE4 secretion to patient serum or supernatants of cultured ovarian carcinoma was assayed by ELISA. RESULTS: mAb 12A2 bound to formalin-fixed sections from 18 of 19 ovarian carcinomas, while 14E2, 2H5 and 3D8 bound to sections from 14, 7 and 4 patients, respectively. The mAbs bound independently of each other, i.e. a sample bound one mAb most strongly while another sample with similar affinity strongest bound another mAb. The intensity of binding to sections did not significantly correlate with the serum level of HE4 except for mAb 14E2, but there was a significant correlation between HE4 expression and its detection in supernatants of cultured ovarian carcinomas. CONCLUSIONS:HE4 epitopes are expressed independently of each other and their expression of HE4 by cultured ovarian carcinoma cells correlates with the release of HE4 into culture supernatants. he epitope recognized by mAb 14E2 was significantly more expressed by platinum resistant tumors. IMPACT: Expression of HE4 by most ovarian carcinomas makes it an excellent biomarker.. Further studies are needed to investigate the clinical relevance of overexpression of a particular epitope.