Characterization of four novel epithelial ovarian cancer cell lines

Dear Editor: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. EOCs originate from either the surface epithelium itself or from the crypts or inclusion cysts on the surface epithelium of the ovary (Whitman et al., 1993). EOCs are designated according to their cell type: serous, mucinous, endometrioid, clear cell, Brenner, or undifferentiated (Serov et al., 1973). They are graded according to degree of differentiation: borderline or low malignant potential represent minimal deviation from their benign counterpart while well differentiated tumors are grade 1, moderately differentiated are grade 2, and poorly differentiated are grade 3 carcinomas. At surgery EOCs are also classified according to stage, grade and amount of residual disease based on criteria established by the International Federation of Gynecology and Obstetrics (FIGO). A common behavior of EOC (in one-third of all cases) is seeding of the peritoneal fluid leading to subsequent implantation over peritoneal surfaces with ascites formation (Morrow and Curtin, 1998). EOCs can arise sporadically and in rarer instances in association with familial cancer syndromes (Easton et al., 1993). It is presently unclear whether the underlying molecular events in the development of all EOCs are similar or whether distinct events are associated with particular subtypes. We have previously described an efficient and rapid technique for the establishment of primary cultures from EOC (Lounis et al., 1994). While these primary cultures are ideally suited to certain studies, the major drawback is the inability to maintain these cultures over extended periods for long-term experiments and for tumor assays. Most human ovarian cancer cell lines described were derived from ascites or pleural effusion (DiSaia et al., 1975; Fogh and Trcmpe, 1975; Sinna et al., 1979; Bast et al,, 1981; Hamilton et al., 1983; Langdon et al., 1988; Golombick et al., 1990; Wong et al., 1990; Golombick and Bezwda, 1991; Yamada et al., 1991; Grunt et al., 1993; Provencher et al., 1993; Hirte et al., i994; Buller et al., 1995; Alama et al., 1996), with only a few cell lines derived from primary ovarian solid tumors (Woods et al., 1979; Langdon et al., 1988; Crickard et al., 1989) or metastases (Buick et al., 1985). In addition, most cell lines originate from tumor material obtained following adjuvant therapy such as chemotherapy or radiation therapy, which could introduce confounding genetic events. Following the derivation of primary EOC cultures (Lounis et al., 1994), we were able to establish four independent spontaneously immortalized epithelial ovarian cancer cell lines from patients who were never exposed to chemotherapy or radiation therapy. The cell lines were derived from ovarian malignant tumors (TOV-21G, TOV-81D, and TOV-112D) and from an ovarian malignant ascites (OV-90). The ovarian tumor cell lines are derived from different histopathologies of ovarian tumors: a clear cell carcinoma (TOV-21G), a papillary serous adenocarcinoma (TOV-81D), an endometrioid carcinoma (TOV-112D), and an adenocarcinoma (OV-90). The clinical information is summarized in Table 1. All patients were diagnosed with advanced disease. Three tumors were grade 3 while one, TOV-81D, was classified as grade 1-2. In Canada, the average age at diagnosis is 54 yr (NCIC, 1999) and therefore TOV112D was derived from a patient with early age of onset ovarian cancer (42 yr at the time of diagnosis). This patient survived less then 3 mo despite an optimal cytoreduetive surgical procedure and platinol-based chemotherapy. TOV-81D is particularly interesting since it was derived from a patient with a familial history of breast and ovarian cancer. In this patient the disease was rather indolent with relapse occurring greater than 5 yr later. Since all the cell lines described here were derived from women of French-Canadian descent they were screened for mutations found to occur in this population (Tonin et al., 1998). Through these studies it was possible to identify a germline BRCA2 mutation, a nucleotide 8765delAG mutation in exon 20, in TOV-81D. All cell lines expressed BRCA1 and BRCA2 as detected by reverse transcriptasepolymerase chain reaction (RT-PCR). Three of the cell lines grow as monolayer cultures on a solid surface (TOV-21G, TOV-81D, and OV-90) while the TOV-112D cell line is loosely adherent and the cells have a tendency to compact and form foci. TOV-81D cells have a very flat morphology and are large surface cells with abundant cytoplasm (Fig. 1D). The epithelial morphology is highly similar to the morphology of cell cultures derived from normal ovarian epithelium (Lounis et al., 1994) and resembles the morphology of a previously described ovarian cell line SKOV-3 (Fogh and Trempe, 1975) which was established from the ascites of a patient with an ovarian adenocarcinoma (Fig. 1E). In contrast, the cellular morphology of the TOV-21G (Fig. 1A), TOVl12D (Fig. 1B), and OV-90 (Fig. 1C) differ from SKOV-3, with ceils being generally smaller and more refractile (Fig. 1). In particular, the OV-90 cells maintain a classic morphology, characterized by ruffled membranes (Lounis et al., 1994), which have been observed in primary cultures from ovarian ascites. The expression of epithelial specific keratins was verified (Table 2). The TOV-21G, OV-90, and TOV-112D cell lines show strong immunofluorescence against the keratin specific CAM5.2 antibody while the TOV-81D cell line reacted weakly with the CAM5.2 antibody. Staining was as reported previously for OV and TOV cultures (Lounis et al., 1994) and was consistent with a staining pattern associated with transformed calls. In addition, we have assessed the expression of antigens MH99 (Mattes et al., 1983, 1987) and B72.3 whose expression correlates with epithelial ovarian carcinomas (Thor et al.~ 1986). All cell lines showed strong positive immunofluorescent staining for both MH99 and B72.3, and this staining appeared stronger than the staining pattern observed for SKOV-3 (Table 2). Staining for CAM5.2, MH99, and B72.1 were negative in the NIH3T3 fibroblast control cell line (Table 2). Finally, the expression of the HER2/NEU oncogene, has been associated with poor prognosis in ovarian cancer (Slamon et al., 1989), was determined. All four ovarian cell lines express neu to some extent