Department of Obstetrics and Gynaecology WHO Collaborating Centre in Human Reproduction, University Hospital of Geneva, Switzerland
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Abstract |
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Key words: CA125/cell culture/myometrium/ovarian cancer/peritoneum
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Introduction |
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CA125 is immunolocalized in the amnion and derivatives of fetal coelomic epithelia, decidua and placenta (Kabawat et al., 1983). In adult tissues it is found in the female genital tract epithelium except on the surface of the ovary (Kabawat et al., 1983
; de Bruijn et al., 1986
). The epithelium of the pancreas, colon, gall bladder, stomach, lung, kidney, breast (Dietel et al., 1986
; Nouwen et al., 1987
), and endometriotic lesions is also positive for CA125 (Fedele et al., 1988
). Many body fluids such as milk, amniotic fluid, peritoneal or pleural fluid, cervical mucus and seminal fluid contain CA125 (de Bruijn et al., 1986
; O'Brien et al., 1986
; Bergmann et al., 1987
; Jacobs et al., 1988
; Schwartz et al., 1989
; Meisser et al., 1996
). Four sites of CA125 synthesis have been studied so far. Endometrial, decidual, amniotic and peritoneal cells have been shown to produce CA125 in vitro (Bischof et al., 1986
; Barbati et al., 1990
; Weintraub et al., 1990
; Zeimet et al., 1997
, 1998
). The normal ovary does not seem to be an important source of CA125 production, since after ovarian stimulation CA125 concentrations do not increase and there is no CA125 concentration gradient from the ovaries to the peripheral circulation (Bischof, unpublished data). However, primary cultures of human ovarian surface epithelial cells secrete detectable but modest concentrations of CA125 (Zeimet et al., 1998
). Different studies suggest that the peritoneum is an important source of CA125. The antigen can be detected immunohistochemically in the mesothelial cells of peritoneum, pleura and pericardium (Kabawat et al., 1983
). Two studies (Zeillemaker et al., 1994
; Zeimet et al., 1998
) demonstrated CA125 secretion by monolayers of mesothelial cells in culture. The role of peritoneal inflammation in the increase of the marker is also suggested by pathological conditions such as ruptured ectopic pregnancy (Bischof et al., 1989
), pelvic inflammatory disease and endometriosis (Johansson et al., 1998
). Direct in-vitro evidence demonstrating the effects of inflammatory cytokines such as interleukin-1ß (IL-1ß) or tumour necrosis factor-
(TNF-
) on human peritoneal cells is controversial (Zeillemaker et al., 1994
; Zeimet et al., 1998
). Furthermore, elevated CA125 concentrations have been found after abdominal surgery: Redman et al. (Redman et al., 1988
) observed an elevation in peritoneal fluid but not in the sera, Talbot et al. (Talbot et al., 1989
) found a postoperative CA125 increase after laparotomy in the sera of patients with normal preoperative concentration, while a third study (Yedema et al., 1993
) showed a significant CA125 increase in the serum after laparotomy for benign diseases and non-ovarian malignancies.
The purpose of the present study was to investigate in vivo and in vitro if the peritoneum is a source of CA125 and if it contributes to circulating CA125. In practical terms, we wanted to see if the increase in CA125 was higher after a peritoneal surgery and if the proportion of patients having an increased CA125 concentration was higher in patients having undergone a peritoneal surgery compared with non-peritoneal surgery.
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Materials and methods |
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After removal, the tissue pieces were minced with scissors, washed and placed in HBSS (Hanks' balanced salt solution, Sigma, St Louis, MO, USA) containing 200 IU/ml penicillin (Hoechst, Darmstadt, Germany), 200 µg/ml streptomycin (Hoechst) and 2.5 µg/ml fungizone (Gibco, Basle, Switzerland).
Explants of uterine and abdominal peritoneum, and myometrium were cultured in RPMI 1640 (Merck, Darmstaadt, Germany) containing 10% FCS (fetal calf serum) (Animed, Basel, Switzerland), 100 ng/ml streptomycine, 0.1 IU/ml penicillin and 2.5 ng/ml fungizone (referred to as complete RPMI hereafter). To mimic bacterial infection, complete RPMI was supplemented in some experiments by 100 µg/ml lipopolysaccharides (LPS; Sigma, Buchs, Switzerland). In other experiments, ascitic fluid (30% v/v) was added to complete RPMI to mimic cancer. Finally, complete RPMI was also supplemented sometimes with cycloheximide (10 µg/ml, Sigma) to inhibit protein synthesis.
Media were collected on days 2, 5 and 8 and CA125 concentration determined in duplicate in the supernatants by an immunoradiometric assay kit (CIS Biointernational, Saclay, France) according to the instructions of the manufacturer. Total proteins were measured in each supernatant with the protein Biorad kit (Biorad, Munchen, Germany) using bovine serum albumin as the standard. The concentration of CA125 in media supplemented with ascitic fluid but in absence of tissue was subtracted from the CA125 concentrations found in the culture supernatants supplemented with ascitic fluid and in presence of tissue.
Results were expressed as units of CA125 per mg total protein. Statistical analyses were performed on log transformed values by analysis of variance (ANOVA) and paired t-test (when appropriate) using the Statview program (Abacus, Berkeley, CA, USA).
Prospective study
In a prospective study we analysed a population of 40 patients admitted for surgery under general anaesthesia. The first group included nine men and 10 women undergoing abdominal surgery with peritoneum opening. Cases with endometriosis or ovarian cancer were excluded. The second group included 11 men and 10 women undergoing surgical intervention elsewhere. Data concerning age, sex, preoperative diagnosis, type of surgery, anatomicopathological diagnosis, length of the anaesthesia and the operation were recorded. A 5 ml blood specimen on heparin was obtained 2448 h preoperatively and 48 h postoperatively from each patient. After centrifugation (10 min at 1500 g), plasma samples were stored at 20°C until assayed. CA125 concentration was determined in duplicate by the same immunoradiometric assay as described above. The change between pre- and postoperative CA125 concentrations was calculated and log transformed. Statistical analyses were performed by Student's t and 2 tests.
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Results |
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Discussion |
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Our observations seem to exclude the possibility that these tissues actively produce CA125 in vitro. Different inflammatory situations such as pelvic inflammatory disease, endometriosis (especially with adhesions), malignant pathologies with ascites and peritonitis are known to induce high circulating concentrations of CA125. In order to mimic cancer or an inflammation, peritoneal explant cultures were performed in medium supplemented with ascitic fluid or LPS, speculating that these two substances could stimulate potential CA125 production. Even under these conditions, we do not find a significant effect on the CA125 release. This observation is in contrast with previous results (Zeillemaker et al., 1994). These last authors cultured mesothelial cells for 6 h and observed that the secretion of CA125 by cells grown in medium with LPS, IL-1ß or TNF-
was statistically higher than in unstimulated cells. However, the kinetics of the two studies are different: we measured CA125 after 28 days. This could perhaps explain the different results. The difference in the culture medium and the use of antibiotics certainly does not explain our negative results.
Among the 40 patients included in the prospective study, seven had a preoperative CA125 >35 IU/ml associated with malignant (pancreas, lymphoma, gastric and breast) and benign (uterine fibroid, thyroid) conditions. This underlines the lack of specificity of this marker for ovarian cancer. We excluded these cases from further analysis in order to avoid a possible decrease in postoperative CA125 due to tumour removal, as shown previously (Yedema et al., 1993). Although our sample size was limited, we observed a significant difference in the proportion of patients who had a postoperative increase of CA125 when patients with abdominal and extra-abdominal surgery were compared. This suggests that, in vivo, the peritoneum might contribute to circulating CA125 concentrations. These results confirm earlier studies (Talbot et al., 1989
; Yedema et al., 1993
) in which a postoperative increase of CA125 after laparotomy was observed. In both studies, CA125 measurements were performed between 4 and 28 days after surgery and the maximal rise in CA125 was detected 24 weeks postoperatively (Talbot et al., 1989
). Our postoperative measurements were done after 48 h and an increased CA125 concentration, although not statistically significant, could already be observed. This marginal increase is taken as indirect evidence of the peritoneal contribution to circulating CA125 concentration. It must be admitted, however, that in the five cases with abdominal tumours, the contribution of tumour manipulation to increased postoperative CA125 concentrations cannot be excluded.
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Acknowledgments |
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Notes |
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References |
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Submitted on December 14, 1999; accepted on March 15, 2000.