An initial analysis of preoperative serum CA 125 levels in patients with early stage ovarian carcinoma

GYNECOLOGIC

ONCOLOGY

30,

7-14 (1988)

An Initial Analysis of Preoperative Serum CA 125 Levels in Patients with Early Stage Ovarian Carcinoma VINCENT R. ZURAWSKI, JR., *J ROBERT C. KNAPP,“‘? NINA EINHORN,~ PETER KENEMANS,~ RODRIGUE MORTEL,~~ KAZUO OHMI,# ROBERT C. BAST, JR.,** ROY E. RITTS, JR. ,tt AND GEORGE MALKASIANSS *The Gynecologic Oncology Laboratories, the Brigham and Women’s Hospital, Harvard Medical School and the fDana-Farber Cancer Institute, Boston, Massachusetts 02115; jtGynecologica1 Department of the Radiumhemmet, Karolinska Hospital and Institute, Stockholm, Sweden; Olnstitute of Obstetrics and Gynecology, St. Radboud Hospital, University of Nijmegen, The Netherlands; /IThe Milton S. Hershey Medical Center,Hershey, Pennsylvania 17033; #Department of Gynecology, National Cancer Center Hospital, Tokyo, Japan; **Department of Medicine, Duke University School of Medicine, Durham, North Carolina 27710; and the VMicrobiology Research Laboratory and .UDepartment of Obstetrics and Gynecology, the Mayo Clinic, Rochester, Minnesota 55905 Received March 25, 1987 Preoperative serum CA 125 levels were determined for 36 patients with Stage I and II ovarian carcinoma. Levels ranged from 9 to 1962 U/ml with a mean of 216 U/ml. In Stage I patients, CA 125 levels averaged 133 U/ml and in Stage II patients 382 U/ml. Nine of 24 Stage I (38%) and 9 of 12 Stage II patients (75%) had CA 125 levels in excess of 65 U/ml in a population somewhat overrepresented in mutinous tumors. Patients with nonmutinous neoplasms had CA 125 elevations more often-in 75% of the cases-than those with mutinous tumors. A larger study will be required to more precisely estimate the fraction of early stage patients with elevated preoperative serum CA 125 levels; however, this investigation demonstrates an assay sensitivity minimally adequate to initiate a pilot evaluation of serum CA 125 levels in a population at risk for ovarian carcinoma. 0 1988 Academic

Press, Inc.

INTRODUCTION

Ovarian cancer is the fourth largest cause of cancer mortality in women in the world, largely because the majority of cases are diagnosed after the disease has become disseminated in the body, when it is mostly refractory to current therapeutic approaches. An immunoassay has been developed which measures serum levels of the antigenic determinant CA 125 [1,2]. Elevated levels of this determinant have been found in approximately 80% of women with epithelial ovarian cancer [l]; however, nearly all the cases of ovarian carcinoma reported to date with such ’ To whom reprint requests should be addressed at the Brigham and Women’s Hospital, 75 Francis Street. Boston MA 02115. 7 0090-8258/88$1SO Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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elevations have had Stage III or IV disease. It has also been reported [3] that serum CA 125 levels are infrequently elevated in women at risk for ovarian cancer, indicating that it may be worthwhile to explore the possible utility of the CA 125 immunoassay as a component of a strategy for the early detection of ovarian carcinoma. Therefore, we initiated a multiinstitutional evaluation of preoperative serum CA 125 levels in women with Stage I and II disease, since it would be unlikely that CA 125 levels would be elevated in women with occult, nondisseminated disease if a significant fraction of those with clinically diagnosed early stage disease did not have elevated preoperative levels. The present report describes the initial results of this investigation. MATERIAL AND METHODS Subjects and Sample Collection From June 1983 to April 1986, preoperative serum samples were obtained consecutively at six institutions from 36 women who were suspected of having ovarian cancer and were scheduled for laparotomy, and for whom ultimate diagnoses of Stage I or II ovarian carcinoma were obtained. Staging laparotomies were performed at each institution; staging was accomplished according to guidelines of the International Federation of Gynecology and Obstetrics (FIGO). No woman with a Stage I or II diagnosis at any institution was excluded from this study unless a staging laparotomy was not performed or unless clear evidence of a double-primary tumor with one of nonovarian origin existed. Patients with tumors of borderline malignant potential were evaluated but will be the subject of a separate report. Ten women from the Mayo Clinic (Rochester, MN), nine from the Radiumhemmet and Karolinska Hospital (Stockholm, Sweden), six from St. Radboud Hospital and the Unversity of Nijmegen (Nijmegen, The Netherlands), five from the Brigham and Women’s Hospital (Boston, MA), and three each from the Hershey Medical Center (Hershey, PA) and the National Cancer Center Hospital (Tokyo, Japan) were included. Operative notes and pathology reports were collected from each site, transcribed to English when necessary and reviewed in aggregate by two of us (R.C.K. and V.R.Z., Jr.,) blinded both from CA 125 results and from diagnoses asssigned at each institution. FIG0 stages and histopathologic and grading diagnoses were corroborated during this review. Twenty-four of the subjects had Stage I and 12 had Stage II epithelial ovarian carcinoma, including 14 tumors with mutinous, 8 with serous, 6 with endometrioid, 4 with clear cell, and 2 with mixed histologic types. Two other patients were diagnosed with unclassified adenocarcinomas but these 2 were considered to have nonmucinous pathologies for the purpose of analyses. Of those with Stage I disease approximately 70% comprised the group classified with Stage Ia disease, a fraction not dissimilar to that found in larger populations [4]. A higher than normal percentage of patients, however, were diagnosed with mutinous rather than other histologic tumor types. Ten of 17 (59%) who had Stage Ia diagnoses also had mutinous tumors, contrasted to the 28% reported by Pettersson [4].

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125 IN

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The size of the tumors was also available and extracted from the medical records in all but two cases. Products of the two largest tumor dimensions were computed and used for comparative analysis. In six cases the dimensions of a cyst containing a smaller tumor volume were used for comparison. All of these cysts measured more than ten centimeters in their largest dimension. Excepting these six cases only one tumor of the group measured less than 6 cm in its largest dimension. This case was in a patient with a highly differentiated serous tumor and a serum CA 125 level of 16.4 U/ml. Overall, two-dimensional products ranged from 2.2 to 900 cm2 with a mean of 276 2 195 cm2 (SD). Concurrent disorders were also noted among these patients, including cardiovascular disease, hypertension, hypothyroidism, osteoporosis, arthritis, microcystic anemia, and esophageal hiatal hernia. One patient was a recovered alcoholic but with no present signs of active liver disease. Presenting symptoms included abdominal discomfort or swelling, apparent ascites, irregular menses, or urinary stress incontinence. One woman presented with a case of cholelithiasis. The median age of the patients was 55 years. Patient data from all institutions were pooled for analysis. Although patient referral patterns may not have been identical at each site, the characterization of these patients by surgical staging (see above) and histopathological analysis was accomplished using uniform FIG0 classifications. Sample and Statistical Analyses Serum CA 125 levels were determined at each site using immunoradiometric assay kits that were manufactured at Centocor, Inc. (Malvern, PA) according to the manufacturer’s instructions. Performance characteristics of this assay have been reported [2]. Results of these determinations were transmitted to one of us (V.R.Z., Jr.) for further analysis. Statistical analyses were performed on an IBM AT computer equipped with an SAS PC program (SAS Institute, Inc., Cary, NC). The CA 125 level, as a dependent variable, was compared among tumors of different size, grade, stage, histopathologic type, and concurrent diagnoses as independent variables, using a one-way nonparametric procedure [5]. In addition, the association between CA 125 levels categorized by 35 and 65 U/ml reference values [1,2] and stage, grade, histopathologic type, and patient’s age (~50 years or 350 years) was tested by categorical analysis using a single-tailed Fisher’s exact test [6]. In all cases, P values less than 0.05 were considered significant. Because the Mayo Clinic used a four-point grading system [7] and all other institutions used a threepoint system [8], Mayo data was converted to a three-point system to facilitate analysis. Broders’ grades 3 and 4 [7] were grouped together as poorly differentiated (Grade 3); Broders’ grades 1 and 2 were retained as grade 1 (well differentiated) and grade 2 (moderately well differentiated), respectively. RESULTS Preoperative serum CA 125 levels for patients with Stage I and II ovarian carcinoma are presented in Fig. 1. Clearly, there is a wide variation in the distribution of CA 125 levels observed, which ranged from 9 to 1962 U/ml (Table 1). Moreover, it appeared that individuals with mutinous tumors had lower CA

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FIG. 1 Serum CA 125 levels versus stage of ovarian carcinoma. Nonmucinous tumors are indicated with filled circles; mutinous tumors are indicated with open circles. CA 125 levels are plotted on a logarithmic scale. The dashed line indicates the 65 U/ml reference value and the solid line indicates the 35 U/ml reference value.

TABLE 1 SERUM CA 125 LEVELS IN EARLY STAGE OVARIAN CARCINOMA PATIENTS

CA 125 level (U/ml) Group”

Mean -C SD

All (36)

216 + 383

67.0

9.0-1962

All Stage I (24) All Stage II (12)

133 k 206 382 k 577

36.6 154.0

9.0-847 16.4-1962

All 350 years (24) All ~50 years (12)

237 ? 419 173 f 313

86.5 46.5

9.0-1962 12.0-1080

All nonmucinous (22) Nonmucinous Stage I (10) Nonmucinous Stage II (12) Mucinousb (14)

332 k 456 272 k 261 382 2 577 33.2 + 18.1

146.0 143.0 154.0 28.5

9.0-1962 9.0-841 16.4-1962 12.0-73.0

Nonmucinous 350 years (16) Nonmucinous <50 years (6)

337 2 481 319 k 403

161.0 136.0

9.0-1962 16.4-1080

Median

L?Number in parentheses represents the number of women in a specified group. b All 14 of the mutinous tumors were in Stage I patients.

Range

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CARCINOMA

TABLE 2 FREQUENCY OF ELEVATED SERUM CA 125 LEVELS IN EARLY STAGEOVARIAN CARCINOMA PATIENTS

Number of subjects” Group All 250 years <50 years Stage I Stage II Nonmucinous Nonmucinous Stage I Mutinous

<35 U/ml

23X65

U/ml

365 U/ml

Total

14 (39)

4 (11)

I8 (50)

36

8 (33)

6 (50)

3 (13) 1 ( 8)

I3 (54) 5 (42)

24 12

I2 (50) 2 (17)

3 (12) 1 ( 8)

9 (38) 9 (75)

24 I2

4 (18)

1 ( 5) 0 ( 0) 3 (21)

I7 (77)

22 IO I4

2 cm IO (71)

8 (80) 1 ( 7)

0 Number in parentheses represents the percentage of women in each category.

125 levels than those with nonmucinous neoplasms. It is important to note that all mutinous tumors occurred in Stage I patients in this population. Mean CA 125 levels of 133 and 382 U/ml for Stage I and II disease, respectively, were considerably higher than those seen in normal populations, which have been reported to be closer to 10 U/ml [l-3]. Median levels were similarly increased. It can be seen in Table 1 that there is an apparent difference between the CA 125 levels for those patients with mutinous and nonmucinous tumors. This difference was significant, even comparing mutinous and nonmucinous tumors with Stage I disease only (Kruskal-Wallis test, P = 0.007). Differences between CA 125 levels in Stage I and II tumors were not significant in this study population when comparing only those with nonmucinous tumors (P = 0.896). Mean CA 125 levels for nonmucinous Stage I and II disease were 272 and 382 U/ml and median levels 143 and 154 U/ml, respectively. Serum CA 125 levels were also classified as elevated or nonelevated using the reference values 35 and 65 U/ml reported by Bast et al. [l] and Klug et al. [2] (Fig. 1, Table 2). No clear association between age or stage of disease and elevated CA 125 levels could be found. In contrast, using both the 35 and the 65 U/ml reference values, a significant association between the frequency of elevation and the histologic type of disease-mutinous or nonmucinous-was observed using the Fisher exact test (P = 0.018 and P < 0.0001, respectively). Grades of the tumors were not uniformly distributed among stage or histopathologic type. Therefore, although analysis of the data suggested a potentially significant direct association between tumor grade and elevated or increased CA 125 levels, this study population was insufficient to validate this possibility. There was no correlation of CA 125 levels with concurrent diagnoses or with tumor size. DISCUSSION This study was undertaken to provide an initial evaluation of preoperative serum CA 125 levels in patients with early stage epithelial ovarian cancer. Pre-

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operative samples collected consecutively from 36 ovarian carcinoma patients at six institutions around the world were evaluated. More than 75% of all the patients with nonmucinous disease in this study had elevated CA 125 levels, irrespective of whether a 35 or a 65 U/ml reference value was used to categorize these patients (Fig. 1, Table 2). In contrast, the subset of women with mutinous tumors, all of whom had Stage I disease, rarely had elevated levels. This is consistent with the report of Kabawat et al. [9], who indicated that none of eight frozen sections of mutinous tumors stained positive using a sensitive biotin-avidin immunoperoxidase technique, and with that of Shishi et al. [lo], who observed only nonuniform immunoperoxidase staining in 3 of 13 Pronase-treated paraffin sections of mutinous ovarian neoplasms. Elevated serum CA 125 levels have been observed in a few cases of patients with advanced mutinous ovarian carcinoma, but it is not clear whether this high level of CA 125 is due to shedding of the antigenic determinant from the tumor or from serosal epithelium containing disseminated cancer. Kivinen et al. [Ill have suggested that preoperative serum CA 125 levels are directly correlated with stage of disease among women with ovarian carcinoma. This result was not supported by the present investigation when comparing Stage I and II patients only, even though the large fraction of Stage I mutinous tumors (58%) present in this study population would certainly have biased any analysis comparing the different stages of disease in favor of this conclusion. Similarly, a disproportionate number of high-grade tumors was observed among the Stage II patients in this study relative to those with Stage I disease. This would obviate any conclusions with regard to the observed increased CA 125 levels apparently associated with higher grade. It cannot be ruled out, however, that the grade of the tumor is a primary correlate of CA 125 level, since, as in other series [12], the mutinous carcinomas tended to be low stage and low grade. Clearly, an evaluation of a larger number of cases, also including those with advanced disease, will be needed to delineate precisely any potential associations of CA 125 levels with stage, grade, and specific histopathologic type. Nonetheless, it is clear that a significant proportion of patients with Stage I and II disease in this population did have substantially elevated serum CA 125 levels. In fact, 38% of all the Stage I patients had serum CA 125 levels exceeding 65 U/ml and 50% had levels exceeding 35 U/ml. Of the 17 patients with Stage Ia disease, preoperative serum CA 125 levels were greater than 35 U/ml in 7 cases (41%) and greater than 65 U/ml in 6 cases (35%). The results of the present investigation are also noteworthy when it is recognized that the Stage Ia population evaluated was overrepresented in mutinous carcinomas by more than twice that normally reported [4], and that patients with these mutinous neoplasms had CA 125 levels less than 35 U/ml in 90% of the cases evaluated. The evident lack of sensitivity of the CA 125 immunoradiometric assay for early stage mutinous tumors does indicate that another serum assay complementary to the CA 125 immunoassay may need to be developed, possibly enabling early detection of these mutinous neoplasms. Interestingly, however, whereas approximately 25% of patients with Stage I ovarian carcinomas are diagnosed with mutinous disease, only about 9% each with Stage II and III disease and 7%

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with Stage IV disease are thus diagnosed [4]. Consequently, the need to increase the proportion of early stage diagnoses is most acute among patients with nonmutinous cancers. This study was initiated as a part of a larger effort aimed at determining the potential utility of the CA 125 immunoassay in the early detection of ovarian carcinoma. We were encouraged to find that the majority of patients with early stage disease had elevated preoperative serum CA 125levels. It must be emphasized, however, that even if sensitivity for early disease, particularly nonmucinous disease, remains high as more data are accumulated, these results could serve only as a preamble to a very large study aimed at detection of occult disease in a population at risk for ovarian cancer. Bast et al. [13] have identified one case in retrospect where CA 125 levels were elevated nearly a year prior to a diagnosis of nonmucinous ovarian carcinoma. Initial results regarding the specificity of the CA 125 assay in an at-risk population have also been reported, which appear encouraging [3]. Therefore, the CA 125 immunoassay remains a candidate to be a component of a strategy for the early detection of ovarian carcinoma. ACKNOWLEDGMENTS The authors are indebted to Ms. Susan Broderick and Mr. David Martin for their assistance in organizing and analyzing this data and to Ms. Damella Resetar for her help in preparing the manuscript. This work was supported in part by grants from the USPHS, NIH No. 1 R43 CA 20945-01 (to V.R.Z., Jr.) and from the Dutch Prevention Fund, Praev. Funds 28-1248 (to P.K.).

REFERENCES 1. Bast, R. C., Jr., Klug, T. L., St. John, E., Jenison, E., Niloff, J. M., Lazarus, H., Berkowitz, R. S., Leavitt, T., Griffiths, T., Parker, L., Zurawski, V. R., Jr., and Knapp, R. C. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer, N. Engl. .Z. Med. 309, 883-887 (1983). 2. Klug, T. L., Bast, R. C., Jr., Niloff, J. M., Knapp, R. C., and Zurawski, V. R., Jr. Monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 125) associated with human epithelial ovarian carcinomas, Cancer Res. 44, 1048-1053 (1984). 3. Zurawski, V. R., Jr., Broderick, S. F., Pickens, P., Knapp, R. C., and Bast, R. C., Jr. Serum CA 125 levels in a large group of nonhospitalized women: Relevance for the early detection of ovarian cancer, Obstet. Gynecol. 69, 606-611 (1987). 4. Pettersson, F. Annual report on the results of treatment in gynecological cancer, International Federation of Gynecology and Obstetrics, Stockholm, Sweden Vol. 19 (1985). 5. Kruskal, W. H., and Wallis, W. A. Use of ranks in one-criterion analysis of variance. J. Amer. Stat. Assoc. 47, 583-621 (1952). 6. Kendall, M., and Stuart, A. The advanced theory of statistics, Macmillan, New York, Vol. 2, pp. 580-585 (1979). 7. Broders, A. C. Carcinoma: Grading and practical application, Arch. P&ho/. 2, 376-382 (1926). 8. Day, T. G., Gallager, H. S., and Rutledge, F. N. Epithelial carcinoma of the ovary: prognostic importance of histologic grade, Natl. Cancer Inst. Monogr. 42, 15-20 (1975). 9. Kabawat, S. E., Bast, R. C., Jr., Welch, W. R., Knapp, R. C., and Calvin, R. B. Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumors of serous, endometrioid and clear cell types. Amer. .Z. C/in. Pathol. 79, 98104 (1983). 10. Shishi, J., Ghazizadeh, M., Oguro, T., Aihara, K., and Araki, T., Immunohistochemical localization of CA 125antigen in formalin-fixed paraffin sections of ovarian tumors with the use of PronaseR, Amer. J. C/in. Patho/. 85, 595-598 (1986).

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11. Kivinen, S., Kuoppala, T., Leppilampi, M., Vuori, J., and Kauppila, A., Tumor-associated antigen CA 125 before and during the treatment of ovarian carcinoma, Ubstet. Gynecol. 67, 468-472 (1986). 12. Malkasian, G. D., Jr., Melton, L. J., III, O’Brien, P. C., and Greene, M. H. Prognostic significance of histologic classification and grading of epithelial malignancies of the ovary, Amer. J. Obstet. Gynecol. 149, 274-282 (1984). 13. Bast, R. C., Jr., Siegal, F. P., Runowicz, C., Klug, T. L., Zurawski, V. R., Jr., Schonholz, D., Cohen, C. J., and Knapp, R. C. Elevation of serum CA 125 prior to diagnosis of an epithelial ovarian carcinoma, Gynecol. Gncol. 22, 115-120 (1985).