A developed by hybridization of spleen cells from mice

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1 Elevation of CA 125 in Patients With Benign and Malignant Ascites JEAN-FRANCOIS BERGMANN, MD,' JEAN-MICHEL BIDART, PHD,* MARTINE GEORGE, MD,t MICHEL BEAUGRAND, MD,$ VICTOR G. LEVY, MD, AND CLAUDE BOHUON, PHD' The presence of CA 125, an ovarian cancer-associated antigen, was assessed in serum and ascites from patients with ovarian cancer (n = 47), hepatocellular carcinoma (n = 21), and liver cirrhosis (n = 40). Abnormal levels of serum CA 125 were observed in 49% of ovarian cancer patients, and in 89% of these same patients with ascites. In all cases of cirrhosis or hepatocellular carcinoma with ascites, CA 125 levels were above 35 U/ml. The specificity and sensitivity of CA 125 measurement for detecting ascites in chronic liver diseases were 73% and loo%, respectively. Furthermore, the CA 125 level was always higher in ascites than in serum. The authors conclude that CA 125 is a nonspecific marker of ascites. This result must be considered in the interpretation of CA 125 elevation in patients with ovarian cancer. Moreover, CA 125 may be of value in the diagnosis and monitoring of peritoneal diseases. Cancer 59: , MONOCLONAL ANTIBODY (oc 125) has recently been A developed by hybridization of spleen cells from mice immunized with an epithelial cell line OVCA 433 cultured from ascitic fluid of a patient bearing a papillary serous cystadenocarcinoma of the ovary. CA I25 is the antigenic determinant recognized by this monoclonal antibody.' Radioimmunoassay for seric CA 125 determination has been proposed for monitoring the course of epithelial ovarian cancer,' and elevated levels of antigen were found in sera from more than 80% of cases of surgically demonstrated ovarian carcinoma. The most marked elevations of CA 125 were observed in patients with peritoneal diffusion of ovarian carcinoma (Stages 111, IV), in patients with other abdominal malignancies, and in patients with chronic liver The common factor in these diseases was peritoneal involvement and the formation of ascites. Thus, CA 125 might be a nonspecific marker of ascites synthesized by peritoneal cells. For testing this hypothesis, we measured CA From the *Dtpartement de Biologie Clinique, tapartement de Gynicologie, Institut Gustave-Roussy, Villejuif, Service d'hcpatogastro- Entirologie, HBpital Saint-Antoine, Pans, and $Service dhipatogastro- Entirologie, HBpital Jean Verdier, Bondy, France. Address for reprints: Jean-Michel Bidart, PhD, Dipartement de Biologie Clinique, lnstitut Gustave-Roussy, Villejuif Cidex, France. The authors thank the nursing staff of the Institut Gustave-Roussy, HBpital Jean Verdier, and HBpital Saint-Antoine for collecting the sera, and Marcelle Duteil and Josiane Lecalvez for providingexcellent technical assistance. Accepted for publication August 15, in the serum of patients with various benign and malignant diseases, paying particular attention to the presence or absence of ascites. Subjects Patients and Methods Three groups of patients were studied. Group I included 47 patients with histologically confirmed ovarian carcinoma. Ages ranged from 22 to 80 years with a mean of 52 f 12. Nine patients (19%) had Stage 1-11 disease, 25 (53%) Stage 111, and 13 (28%) Stage IV disease.6 Tumor histology was as follows: 29 serous, eight endometroid, one mucinous, one clear cell, and eight undifferentiated carcinomas. All patients had clinical, echographic, and surgical evaluation at the time of the serum sample. For each patient, the presence of ascites and/or peritoneal masses was recorded. Group I1 included 2 1 patients with histologically proven hepatocellular carcinoma (HCC), with ascites in 11 cases and without ascites in 10. Group I11 included 40 patients with cirrhosis due to: alcohol (29 patients), chronic active HBsAg-positive hepatitis (1 l), primary biliary cirrhosis (four) and cryptogenetic cirrhosis (three). Twenty-four patients had ascites confirmed by paracentesis. CA 125 and protein levels were measured in all ascitic fluids. Serum samples for CA 125 were collected at the same time. Three of those patients also had pleural effusions which were punctured for CA 125 measurement. 213

2 ~ ~~ 214 CANCER January VOl. 59 TABLE 1. Results of CA 125 Levels in Serum No. of patients with CA 125 No. of patients >35 U/ml >220 U/ml tested Nb ( /.I Nb ( /.I Group I Ovarian carcinoma (21) 14 (30) Group I1 Hepatocellular carcinoma Group I11 Cirrhosis (57) 30 (75) 9 (43) 15 (37) Serum samples from three patients with acute pancreatitis and seven with pleural tuberculosis were also studied. Assay of CA 125 CA 125 measurement was performed using an immunoradiometric assay kit (ELSA-CA 125, Commissariat A l hergie atomique Saclay, France). This simultaneous sandwich solid-phase radioimmunoassay used purified OC 125 antibody coated on polystyrene beads which bind CA 125. Coated beads were incubated overnight with 100 pl of 251-labeled OC 125 and 200 p1 of specimen sera, standards or internal control. Then, serum and excess lz5ilabeled OC 125 were washed out and the activity associated with the immunosorbent was measured in a gamma counter. The results were expressed in unit per milliliter (U/ml), an arbitrary unit based on a reference standard. In our experiment, the coefficient of intra-assay and interassay variations were 8.2% and 8.6%, respectively. Since a hook effect was observed at 500 U/ml, serum samples with CA 125 levels exceeding this value were diluted at a ratio of 1 : 10. The normal upper limit was taken as 35 U/ ml. Only 1% of normal blood donors were found to have a CA 125 level above 35 U/ml and none had above 65 U/ml (n = 120). Data Analysis Statistical testing included dependent and independent means t tests, calculation of Pearson correlation coefficients and Yates-corrected chi-squared analyses. A P value less than 0.05 defined statistical significance. Results Among 47 patients with ovarian carcinoma, serum CA 125 values were above 35 U/ml in 23 patients (49%), and above 220 U/ml in 14 patients (30%) at the time of the echographic and surgical evaluation (Table 1). In 15 patients, CA 125 levels were measured before cytoreductive surgery or combination chemotherapy. Abnormal levels of CA 125 were observed in 57% of patients with HCC and in 75% of patients with cirrhosis (Table 1). Correlation of CA 125 With the Clinical State of Patients With Ovarian Carcinoma (Group I) Antigen levels were compared according to the presence or absence of ascites and peritoneal involvement. In ascitic patients, 17/19 (89%) had abnormal CA 125 levels. In patients without ascites (28 cases), only 6 (21%) had CA 125 levels above 35 U/ml. The difference was highly significant (P < 0.001). CA 125 levels above 220 U/ml were found in 13 patients with ascites but in only one patient without ascites (Fig. 1). Sensitivity and specificity of the test for the detection ascites in patients with ovarian cancer were 89% and 79%, respectively. Comparison of CA 125 levels according to the presence or absence of peritoneal carcinomatosis with peritoneal nodules showed that, in patients without carcinomatosis, only 3/13 (23%) had abnormal levels of CA 125. In patients with peritoneal nodules, 20/34 (59%) had CA 125 levels above 35 U/ml. The difference was not significant. Moreover, in 15 patients with peritoneal carcinomatosis but without ascites, 12 (80%) had normal CA 125 levels. All these patients had carcinomatosis with at least one tumor mass with a diameter greater than 2 cm. Correlation of CA 125 Levels With the Presence of Ascites in Patients With Liver Diseases (Group 11 and 111) In Group I1 (21 patients with HCC), twelve patients (57%) presented high levels of seric CA 125. For all these patients, it was possible to assess the presence of ascites by clinical and echographic examination and by paracentesis. In the group of patients without ascites, 9/10 had normal seric CA 125 levels; all patients with ascites had CA 125 levels above 100 U/ml. The difference was highly significant (P < 0.001) (Fig. 1). Mean values for CA 125 were 42.3 k 17.1 in patients with HCC without ascites and f 21.5 in ascitic patients with HCC (P < 0.001). In group I11 (40 patients with cirrhosis without HCC), CA 125 levels were abnormal in 30 cases (75%). 24 patients had ascites, and all had CA 125 levels above 35 U/ml. Among the 16 patients without ascites, 1 1 had normal CA 125 levels (P < 0.001) (Fig. 1). Mean values for CA 125 were 43.5 k 14.2 in cirrhotic patients without ascites and 29 1 f 29 in patients with ascites (P < ). Those results were similar whatever the etiology of cirrhosis. For the whole population of ascitic patients with cirrhosis, measurement of CA 125 in ascitic fluid always showed higher levels in ascites than in serum (Fig. 2). In order to demonstrate a possible correlation between CA 125 and

3 No. 2 CA 125 IN PATIENTS WITH ASCITES - Bergmann et al. 215 FIG. 1. CA 125 levels in patients with cirrhosis (n = 40), hepatocellular carcinoma (n = 21) and ovarian carcinoma (n = 47) with or without ascites. an inflammatory peritoneal process, we compared CA 125 and total protein levels in ascites. No significant correlation was found (r = NS). Specificity and sensitivity of CA 125 measurements for detecting ascites in liver diseases were 73% and loo%, respectively. One patient with ascites and elevated serum CA 125 levels (520 U/ml) had meningitis. CA 125 measurement in spinal fluid showed a high CA 125 level (100 U/ml). In one patient with acute hemorragic pancreatitis, CA 125 levels were 178 in the serum, 300 in ascites, and 6 in a pancreatic cyst. The two other patients with acute pancreatitis with abdominal necrosis also had high levels of seric CA 125. One patient had primary biliary cirrhosis with ascites. Plasmaphereses were performed for intense pruritus. CA 125 reached 290 U/ml before plasmapheresis and were normal during plasmapheresis. In the seven patients with pleural tuberculosis, CA 125 were normal in serum but weakly elevated ( U/ml) in pleural fluid. The three cirrhotic patients with pleurisy also had high levels of CA 125 in the pleural fluid (75, 180, and 350, respectively). Discussion Other tumor markers such as CEA, CA or placental alkaline phosphatase are less efficient. CA 125 levels correlate well with the progression or regression of disease in CA 125 is considered a clinically useful marker for FIG. 2. Comparison of blood and ascites concentrations of CA 125 in monitoring patients with epithelial ovarian ~ancer.~,~ cirrhotic patients (n = 24). 1ssa I.* > 111 5## I### CA 125 in Serum IU/ml)

4 216 CANCER January Vol % of But specificity of CA 125 is low due to its elevation in other malignant and benign diseases, especially in chronic liver disease^.^.^ In the current study, increased serum levels of CA 125 were found in 49% of all patients with epithelial ovarian cancers, but also in 42% of patients with HCC, and 75% of patients with cirrhosis. The most prominent common factor in ovarian cancer and liver diseases is the high frequency of ascites. In this study, we demonstrate that high CA 125 levels are closely related to the presence of ascites whatever the origin. CA 125 antigen might also be synthesized by peritoneal cells, and thus could be considered as a nonspecific marker of peritoneal involvement. In our patients with ovarian carcinoma, the low percentage of cases associated with an abnormal CA 125 seric level (49%) could be related to serial CA 125 examination in patients in complete remission of the disease. CA 125 levels are elevated in 89% of patients with ascites, but in only 2 1 % of patients without ascites. The difference is not due to the diffusion of the disease because only 59% of patients with peritoneal carcinomatosis had high CA 125 levels. Moreover, 80% of patients with surgically proven peritoneal carcinomatosis without ascites had normal CA 125 levels. Thus, CA 125 levels are well-correlated with ascites but not with tumoral mass and peritoneal diffusion of the cancer. The high percentage of abnormal seric CA 125 levels in Stage 111 or IV ovarian cancers238 could be easily explained by the fact that ascites is very frequent in these cases. Using the biotin-avidin immunoperoxidase technique, CA 125 has been detected in peritoneum and also in pleura, pericardium, the mullerian duct, and the amnion.'-'' The hypothesis that the antigen is synthesized by peritoneal cells rather than only by ovarian cancer cells is supported by the fact that, in women with ovarian cancer, ovarian venous blood concentration of CA 125 is identical or lower than in a peripheral vein." However, a "cross-reaction'' between ovarian cancer antigen and an ascitic protein remains possible and could explain why, by indirect immunofluorescence, OC 125 stains 83% of ovarian serous adenocarcinomas. l2 Nevertheless, CA 125 levels correlate well with the clinical course in patients with ovarian can~er,~.'.~,~ and its measurement presents a great advantage in the monitoring of therapy. The increased CA 125 level might simply indicate reappearance of the ascites that could be synthesized early in tumor progression, but sometimes several months before clinical recurrence of the disease. A rise in CA 125 levels may be related to cancer progression only after other causes of ascites or peritoneal inflammation have been ruled out. In patients with liver disease (Groups I1 and 111) the relationship between CA 125 elevation and the presence of ascites is well documented. Seventy-three percent of patients without ascites had a normal CA 125 level and all patients with ascites had a seric CA 125 level above 35 U/ml. Moreover, CA 125 measurement in serum and in ascites for each patient, always showed a higher level for each patient in ascitic fluid than in serum. This is quite unusual, since most serum proteins are at a lower concentration in ascites. CA 125 might be synthesized in the peritoneal cavity and pass through the peritoneum from the ascites to the serum. CA 125 levels are not correlated with total protein concentration in ascites: some tested ascites presented very high CA 125 levels and a low protein level. The nonspecificity of this antigen for the diagnosis of ovarian carcinoma is supported by other studies. An elevation in the CA 125 levels in nonovarian cancers have been already reported; CA 125 levels were elevated in disseminated cancer of the fallopian tube, endometrium, endocervix, l4 pancreas, colorectum, breast,6 and lung.2915 Concentrations higher than 220 U/ml were found in 3% of patients with cancer without metastases, but in 26% of patients with disseminated cancer.i6 The dissemination of those cancers, particularly in the peritoneum appears to be the major factor in the CA 125 increase. In contrast to the findings of Ruibal et ~l.,~ we think that CA 125 is not related to a hepatic disorder and cannot be used to diagnose cirrhosis or HCC, but can confirm nonspecific peritoneal involvement. Finally, mean values of CA 125 in the serum of ascitic patients are 261 U/ml in cases of ovarian cancer, 276 U/ml in cases of HCC and 291 U/ ml in cases of cirrhosis. These results are quite similar. It appears that CA 125 may be synthesized by peritoneal cells in the case of nonspecific imtation, such as ovarian cancer or portal hypertension with ascites, but also in the case of acute hemorrhagic pancreatitis, peritoniti~,'~ or even after abdominal surgery.i6 We have also detected CA 125 antigen in spinal fluid; in patients with pleurisy, slightly elevated levels were detected in pleural effusion fluid, but no elevation was found in serum. These latter resuits are in accordance with immunohistologic studies which detected CA 125 in peritoneum, pericardium, and pleura. '' It is concluded that CA 125 is elevated in the serum of nearly all patients with ascites, whether the cause be ovarian carcinoma, hepatocellular carcinoma, or cirrhosis. Concentrations in the ascitic fluid are always higher than in the serum, and it is suggested that CA 125, synthesized by the peritoneum, could be considered as a marker of ascites or peritoneal inflammation. Nevertheless, the measurement of CA I25 remains relevant for monitoring patients with ovarian carcinoma and is closely correlated with peritoneal extension of the disease. REFERENCES 1. Bast RC, Feeney M, Lazarus H, Nadler LM, Colvin RB, Knapp RC. Reactivity of a monoclonal antibody with human ovarian carcinoma. JCIin Invest 1981; 68:

5 No. 2 CA 125 IN PATIENTS WITH ASCITES - Bergmann et a] Bast RC, Klug TL, St John E et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian carcinoma. N Engl JMed 1983; 309: Klug TL, Bast RC, Niloff JM, Knapp RC, Zurawski VR Jr. Monoclonal antibody immunoradiometric assay for an antigenic determinant (CA-125) associated with human epithelial ovarian carcinomas. Cancer Res 1984; 44: Ruibal A, Encabo G, Martinez-Miralles E et al. CA- 125 seric levels in non malignant pathologies. Bull Cancer 1984; 71: Ricolleau G, Chatal JF, Fumoleau P, Kremer M, Douillard JY, Cortet C. Radioimmunoassay of the CA-125 antigen in ovarian carcinomas: Advantages compared with CA 19-9 and CEA. Tumour Biol 1984; 5: Young RC, Knapp RC, Perez CA. Cancer of the ovary. In: De Vita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. Philadelphia: J.B. Lippincott, 1982; Eerde Kens MW, Nouwen EJ, Pollet DE, Briers TW, De Broe ME. Placental alkaline phosphatase and cancer antigen 125 in sera of patients with benign and malignant diseases. Clin Chem 1985; 31: Canney PA, Moore M, Wilkinson PM, James RD. Ovarian cancer antigen CA-125: a prospective clinical assessment of its role as a tumour marker. Br J Cancer 1984; 50: Kabawat SE, Bast RC, Ghan AK et al. Tissue distribution of a coelomic epithelium related antigen recognized by the monoclonal antibody OC 125. Int J Gynecol Pathol 1983; 2: Bast RC, Knapp RC. Immunologic approaches to the management of ovarian carcinoma. Semin Oncol 1984; 11: Heinonen PK, Tontti K, Koivula T, Pystynen P. Tumour-associated antigen CA 125 in patients with ovarian cancer. Br Jobstet Gynecol 1985; 92:$ Kabawat SE. Bast RC. Welch WR. KnaDD RC. Colvin RB. Immunopathologic characterization of a monoclbnal antibody that recognizes common surface antigens of human ovarian tumors of serous, endometrioid and clear cell types. Am J Clin Pathol 1983; 79: Bast RC, Klug TL, Schaetzl E et al. Monitoring human ovarian carcinoma with a combination of CA-125, CA 19-9 and carcinoembryonic antigen. Am J Obstet Gynecol 1984; 149: Niloff JM, Klug TL, Schaetzl E, Zunawski VR, Knapp RC, Bast RC. Elevation of serum CA 125 in carcinomas of the fallopian tube, endometrium and endocervix. Am J Obstet Gynecol 1984; 148: Ruibal A, Encabo G, Miralles EM et al. CA 125 seric levels in human clinical practice. Our experience in 1542 cases (Poster). International Meeting on Monoclonal Antibodies in Oncology: Clinical a p plications. Nantes, France, June Ganz PA. A radioimmunoassay for ovarian cancer (letter). N Engl JMed 1983; 309:724.