Endometrial antibodies versus CA-125 for the detection of endometriosis*

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1 FERTILITY AND STERILITY Copyright 99 The American Fertility Society Vol. 55, No., January 99 Printed on acid-free paper in U.S.A. Endometrial antibodies versus CA-5 for the detection of endometriosis* Robert A. Wild, M.D.t:j: Vani Hirisave, Ph.D. til Angela Bianco, B.S. II EdwardS. Podczaski, M.D.II Laurence M. Demers, Ph.D. 'If University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, and Hershey Medical Center of the Pennsylvania State University, Hershey, Pennsylvania Detection of endometrial antibodies using an indirect immunofluorescence method along with a well-established human endometrial carcinoma cell line was evaluated and compared with CA- 5 for detecting endometriosis. Two hundred two patient sera from the infertility, gynecological, and gynecological oncology services were evaluated. The sensitivity for antibody testing was 3.% with a specificity of 7.%, in contrast to a sensitivity of 7.3% and a specificity of.6% for CA- 5. These preliminary findings offer promise that antibody detection methods may be a useful adjunct in the diagnosis of endometriosis. Fertil Steril55:90, 99 A noninvasive method for diagnosing endometriosis is of current interest. Measurement of circulating CA-5 concentrations (the high molecular weight glycoprotein that is expressed on some derivatives of celomic epithelium) has been suggested as useful for detecting endometriosis; - 3 however, in the experience of some but not all investigators, this test lacks the appropriate clinical sensitivity to make its use practical for this purpose. Circulating endometrial antibodies have also been detected in patients with endometriosis, 4 5 and their antigenic Received June 5, 990; revised and accepted August 4, 990. * Presented at the 45th Annual Meeting of The American Fertility Society, San Francisco, California, November to 6, 99. t Supported in part by a contract from Winthrop Pharmaceuticals Division of Sterling Drug, New York City, New York. :j: Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center. Reprint requests: Robert A. Wild, M.D., University of Oklahoma Health Sciences Center, Department of Obstetrics and Gynecology, P.O. Box 690 4SP70, Oklahoma City, Oklahoma II Department of Obstetrics and Gynecology, Hershey Medical Center of the Pennsylvania State University. Department of Pathology, Hershey Medical Center of the Pennsylvania State University. sites have been identified. 6 The immunoglobulin fraction after digestion with the enzyme pepsin was immunoreactive, 7 suggesting that the low titer endometrial antibodies detected by indirect immunofluorescence (IIF) and enzyme-linked immunoassay are because of specific immunological recognition and not nonspecific binding to the immunoglobulin receptors. The purpose of this study was to determine whether antibody detection by IIF utilizing endometrial carcinoma cell line is more sensitive and/or specific for detecting endometriosis than is the measurement of circulating CA-5 levels. MATERIALS AND METHODS Two hundred two sera samples from 93 infertility patients, 36 sera from gynecology patients, and 73 sera from gynecological oncology patients were evaluated. The serum was obtained after informed consent. This study was approved by the Institutional Review Board of the Hershey Medical Center of The Pennsylvania State University. The infertility patients were evaluated by semen 90 Wild et al. Antibody versus CA-5 for endometriosis

2 Table CA-5 and IIF" Results From the Infertility Serviceb Stage of disease Results No disease Stage I Stage II Stage III Stage IV Immunofluorescence Positive 6 7 Negative 5 4 False-positive 7 (7.5)c False-negative (.) CA-5 Positive Negative 7 0 False-positive (.) False-negative 5 (54.) a IIF, indirect immunofluorescence utilizing endometrial carcinoma cell lines. b Evaluation included 93 sera from 93 patients to 40 years of age with a mean of 30.7 years. c Values in parentheses are percents. analysis, a hysterosalpingogram, and endometrial biopsy before undergoing laparoscopy /laparotomy. The infertility patients ranged in ages between and 40 years with a mean age of 30.7 years. Likewise, gynecological patient serum and oncology patient serum were obtained before surgery in which the pelvic cavity was visualized. The patients ranged in age between 9 years and 77 years from the gynecology service with a mean age of 4. years. Both preoperative and postoperative major surgery bloods were obtained from the gynecological oncology service. The mean age of these patients was 56. years with ages from to 79 years. The serum was assayed blindly by IIF utilizing monolayer cultures of endometrial carcinoma cell line by previously described methods. 6 The assays were blindly performed by a single technician (V.H.) without knowledge of the surgical findings until subsequent matching at chart review. Briefly, mono layers of a human endometrial carcinoma cell lines were obtained by growing in eight chamber glass slides. Serum dilutions were carried to :64. Optimum staining was frequently observed at :4 to :. The endometrial carcinoma cell lines were treated with patient and control serum after incubation and wash with fluorescein -conjugated sheep antihuman IgG (light and heavy chains) at a :0 dilution. Each assay contained a known positive and two negative controls (male serum known to be negative and a saline control). Fluorescence was evaluated using Nikon optics (Nikon, Inc., Garden City, NY) and was ranked according to intensity of immunofluorescence from 0 to 3+. Fluorescence of + to 3+ was considered positive for purpose of tabulation of the data. If staining was considered to be 0 to + or questionable, it was considered negative. In addition, as a control, (because the antigenic sites were previously localized to normal glandular endometrial monolayer preparations), 3 positive and 5 negative sera (by the above criteria) were also tested against epithelial monolayers derived from freshly isolated endometrial gland preparations. The findings were identical. 6 Preliminary results tested for antibody in endometriosis have been reported. Circulating CA-5 antigen concentrations were determined utilizing immunoradiometric assays obtained from CEN TOCOR, Inc. (Malvern, PA) that employ a monoclonal antibody, OC 5 to detect CA-5. We utilized an upper reference limit of 6 units/ml for CA-5. Values above that were considered positive. The presence of endometriosis and other gynecological disorders was evaluated by laparoscopy /laparotomy and review of the pathology specimens. Endometriosis was staged using the revised 95 American Fertility Society Staging System. 9 The presence of endometriosis at surgery was considered to be the gold standard. Sensitivity and specificity were calculated as previously described. 0 RESULTS The results from our infertility patients are shown in Table. Among 93 sera from infertility patients, 6 were positive and 5 were negative by IIF testing. It was noted that 77% of the time endometriosis was found at surgery and that 5 of the, Vol. 55, No., January 99 Wild et al. Antibody versus CA-5 for endometriosis 9

3 Table Immunological Results Contrasted With Surgical Results From the Gynecology Service CA-5 Immunofluorescence Negative Positive Negative Positive Intensity <6 U/mL Normal pelvis Adenomatous hyperplasia Leiomyoma (intravenous), adenomyosis Leiomyoma (intramural), adenomyosis Leiomyoma (intramural), ovarian cyst Leiomyoma, pelvic adhesions Periovarian adhesions, infarcted ovary Pelvic adhesions-multiple Papillary serous cystadenofibroma Serous cystadenoma Rheumatoid arthritis, peritubal adhesions Cervical dysplasia Endometriosis I Endometriosis I, ovarian adhesions, polyp Endometriosis II, leiomyoma, polyp Endometriosis II, leiomyoma Endometriosis I, serous cystadenoma 3! , +, (n) (c + n)' False-positive False-negative 5 (3.9) (.) (.7) 4 (.) a Immunofluorescence versus CA-5 in 36 patients 0 to 77 years of age with a mean of 4. years. b n, nuclear staining. ' c + n, cytoplasmic and nuclear staining. 93 patients had minimal or mild disease. This high percentage of patients with early disease probably reflects the nature of the referral practices of the investigators who contributed to the study. There appeared to be no relationship between the degree of intensity of fluorescence and the stage of the disease. There were 7 false-positive tests (7.5%) with IIF and (.%) of the sera were false-negative for antibody determinations. In these same 93 sera, the circulating CA-5 concentrations exceeded 6 units in, whereas 7 of the sera had normal levels. One was considered a false-positive (.% ), whereas 5 (54.%) of the serum were false-negative. Results from the gynecology service are shown in Table in which the CA-5levels were contrasted with results by IIF for the various gynecological diagnoses. Four patients had false-positive results for CA-5 when a normal pelvis was encountered and three sera were considered false-positive by IIF. Two patients had adenomyosis, both positive for CA-5 and one of the sera was positive by IIF. Five patients had endometriosis, but only one was infertile (all with early stage disease). Three of five sera were positive by IIF, and none showed elevations for CA-5. Both the false-positive rates and the false-negative rates were higher for CA-5 than for antibody detection by IIF in this group of patients. There were two patients who demonstrated atypical nuclear staining (as opposed to previously documented cytoplasmic staining 6 ), and one patient was positive for nuclear and cytoplasmic staining. Their respective ages were 77 years, 7 years, and 4 7 years. There were three false-positive results noted among the 73 sera tested from the gynecological oncology service. Atypical nuclear and nuclear and cytoplasmic staining were found frequently (0 and 7 times, respectively, from the 73 sera). Antibodies were detected in 30 of the 73 sera tested. Sensitivity and specificity comparisons between the CA -5 assay and antibody detection by IIF are contrasted in Table 3. There were too few cases of endometriosis in the gynecological oncology patient group to calculate meaningful sensitivity and specificity data. Overall, we found that endometriosis antibody detection utilizing this fluorescence-based assay was more sensitive (3.% versus 7.3%) for detecting endometriosis than was the use of CA-5. The specificity of CA -5 for detecting endometriosis in patients with infertility and in patients with various gynecological disorders was slightly higher (.6% versus 7.%). 9 Wild et al. Antibody versus CA-5 for endometriosis

4 Table 3 Results of Immunological Testing Versus Surgical Evaluation for All Three Services a Immunofluorescence versus CA-5 clinical correlation Total Patients sera tested True +veb True -vee False +ve False ve Infertile 93 6 versus Gynecological 36 3 versus 0 Gynecological cancer 73 4 versus 0 7 versus 3 7 versus 4 versus 3 versus 5 VS 5 Total 0 64 versus 4 versus 43 versus 9 3 versus 56 a Ant.ibody sensitivity and specificity was 3.% and 7.%, respectively, versus CA-5 sensitivity and specificity 7.3% and.6%, respectively. b +ve, positive. c -ve, negative. DISCUSSION We have confirmed the work of several investigators 4 ' 5 ' ' who have suggested that the serum of patients with endometriosis contain antibodies against endometrial tissue. By IIF, we found that these antibodies bind to a human endometrial cancer cell line in tissue culture. The localization of the binding was cytoplasmic. Not all patients with endometriosis demonstrate antibodies. However, the majority of false-negatives were found in those patients who had minimal to mild disease ( of false-negative results from the infertility and gynecological services). Only one false-negative serum came from a patient with stage III disease. The incidence of false-positive tests utilizing this technique was infrequent (4 of 0 samples). In older gynecological and gynecological cancer patients, nuclear staining was encountered. Of 0 patient sera tested, sensitivity of the IIF assay for the presence of endometriosis was 3%, whereas the specificity was 79%. In contrast, sensitivity of the CA- 5 assay on these same sera was 7% with a specificity of 3%. It is possible that a few infertility patients may have antibodies even though they do not have endometriosis. It is also possible that the specificity results may be related to microscopic endometriosis not perceived at laparoscopy_l3-5 Gynecological cancer patients frequently demonstrate antinuclear antibodies, and some of them show both nuclear and cytoplasmic staining. This may be because of the fact that cancer patients may have several different epithelial antigens coming from the malignant tumor that might have led to antibodies that would react with certain epithelial antigens present in the tumor cell line used for the assay. No patient with endometriosis displayed nuclear staining. Nuclear staining was limited to the older patients from the gynecology service (ages 47, 7, and 77 years). It is widely known that autoantibodies, in particular, antinuclear antibodies are noted to be more prevalent in older individuals with or without autoimmune diseases. 6 The low sensitivity of CA-5 for detecting endometriosis is in keeping with the experience of some other investigators but not all. - 3 The determination of sensitivity and specificity also depend on the patient population studied. Causes for falsepositive and false-negative results with CA-5 have been well documented. 3 This evaluation was not designed to test the assay system that is more useful for monitoring response to therapy. That question awaits further evaluation. Improved methodology for antibody detection needs to be combined with evaluations oflarger numbers of patients with and without endometriosis to determine if antibody detection will play a meaningful role in the evaluation and therapy of patients with endometriosis. REFERENCES. Barbieri RL, Niloff JM, Bast RC, Jr, Schactzl E, Kistner RW, Knapp RC: Elevated serum concentrations on CA-5 in patients with advanced endometriosis. Fertil Steril 45: 630, 96. Patton PE, Field CS, Harms RW, Coulam CB: CA-5levels in endometriosis. Fertil Steril45:770, Pittaway DE, FayezJA: The use ofca-5 in the diagnosis and management of endometriosis. Fertil Steril 46:790, Wild RA, Shiver CA: Antiendometrial antibodies in patients with endometriosis. Am J Reprod Immunol Microbiol:4, Chihal HJ, Mathur S, Holtz GL, Williamson HO: An endometrial antibody assay in the clinical diagnosis and management of endometriosis. Fertil Steril46:40, Wild RA, Satyaswaroop PG, Shivers CA: Epithelial localization of antiendometrial antibodies associated with endometriosis. Am J Reprod Immunol Microbiol3:6, 97 Vol. 55, No., January 99 Wild et al. Antibody versus CA-5 for endometriosis 93

5 7. Wild RA, Zhang R, Medders D: F(ab'h Segment is the active component of IgG autoantibody generation in patients with endometriosis. Presented at the 46th Annual Meeting of The American Fertility Society, Washington, DC, October 3 to, 990. Published by The American Fertility Society, 990, p S73. Wild RA, Hirisave V, Podczaski ES, Coulam C, Shivers A, Satyaswaroop PG: Autoantibodies associated with endometriosis: can their detection predict presence of disease? (Abstr.) Am J Reprod Immunol Microbiol6:09, 9 9. The American Fertility Society: Revised American Fertility Society classification of endometriosis: 95. Fertil Steril 43:35, Mausner and Kramer: Screening in the detection of disease. In Epidemiology, an Introductory Text, Vol.. New York, Saunders Publishers, Inc., 95, p. Gleicher N, El-Roeiy A, Contino E, Friberg J: Is endome- triosis an auto-immune disease? Obstet Gynecol 0:5, 97. Meek SC, Hodge DD, Musick JR: Autoimmunity in infertile patients with endometriosis. Am J Obstet Gynecol5: 365, 9 3. Murphy AA, Green WR, Bobbie D, dela Cruz ZC, Rock JA: Unsuspected endometriosis documented by scanning elective microscopy in visually normal peritoneum. Fertil Steril 46:5, Cornillie FJ, Vasquez G, Brosens IA: The response of female endometrial implants to the antiprogesterone steroid R 33: a histologic and ultrastructural study. Pathol Res Pract :647, Brosens IA, Cornillie FJ: Peritoneal endometriosis. Contrib Gynecol Obstet 6:5, Hallgron HM: Lymphocyte phytohemagglutination responsiveness, immunoglobulins, and autoantibodies in aging humans. J Immunol:0, Wild et al. Antibody versus CA -5 for endometriosis