Largely due to late diagnosis, ovarian cancer is the leading cause of death from gynecological malignancies. After complete treatment of the disease confined in the peritoneal cavity, many patients show disease progression and recurrence, suggesting that hematogenous micrometastases have a role, as has been proposed for other epithelial malignancies. However, obtaining reliable numbers of circulating cancer cells (CCC) emigrating from solid cancers remains technically challenging due to their extreme rarity, i.e., these cells represent less than 1 cell / 1,000,000 of cells in blood. We have utilized a cell separation technology for the purpose of enriching, identifying and enumerating viable and invasive CCC that employs a cell adhesion matrix (CAM), which mimics the tumor microenvironment. Viable tumor cells and tumor-associated leukocytes/monocytes (<0.1% of the total leukocyte/monocyte population) attach to the CAM with great avidity but normal cells (including red blood cells) and dead or dying tumor cells do not. When composed of a fluorescently labeled collagen polymer film coated with blood-borne adhesion molecules, the resulting CAM film readily identifies invasive cancer cells by their ability to digest and ingest labeled-collagen fragments. In this assay system, the identification of cancer cells is based on their ability to ingest and digest TRITC-collagen fragments, a behavior that normal cells do not express. In addition, conventional pathological methods for identifying cancer cells were used. Ultimately, six criteria were validated to classify and count viable CCC: (1) positive immunohistochemical detection for the ovarian epithelial-specific marker CA125 or epithelial membrane antigen EMA (Epi+); (2) positive red fluorescence from ingested and concentrated rhodamine-labeled collagen fragments (Co+); (3) negative blue fluorescence (Hoechst-labeling of nuclei; Hs-) due to CCC´s high resistance to staining by apoptotic indicator dyes versus circulating endothelial cells and leukocytes; (4) negative immunohistochemical detection for the leukocyte common antigens CD45 or monocyte/macrophage antigen CD68 (Leu-); (5) positive immunohistochemical detection (green fluorescence) for the endothelial specific markers CD31 and van Willebrand factor (vWF; Endo+); (6) cells cultured ex vivo exhibit pathological tumor cytology. Blood specimens from 38 ovarian cancer patients [FIGO stage I-IV], 35 normal donors (negative controls), and 25 samples from patients with other diseases or undergoing chemotherapy were studied. All 35 of the negative controls and the 25 samples from the latter patients had no detectable viable CCC per ml of blood. Twenty-one of the 32 (66%) samples from patients with stage I-III ovarian cancer demonstrated viable CCC cells, ranging from 41 to 3,700 counted circulating cancer cells per ml of blood. Of particular note, one of five patients with stage IV serous adenocarcinoma exhibits over 7,500 CCC per ml of blood. Thus, ovarian carcinoma cells can be detected in the blood from a significant percentage of ovarian cancer patients. This technique could be used in large prospective studies of patients with ovarian cancer to learn whether the detection of rare carcinoma cells is a useful predictive or prognostic factor for metastasis.
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