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CA 15-3 as a tumor marker in gynecological malignancies
GYNECOLOGIC ONCOLOGY 30,
265-273 (1988)
CA 15-3 as a Tumor Marker in Gynecological Malignancies G. SCAMBIA, P. BENEDETTI PANICI, G. BAIOCCHI, L. PERRONE, S. GREGGI, AND S. MANCUSO Department
of Gynecology,
Catholic
University,
Rome, Italy
Received August 17, 1987 Serum levels of CA 15-3 were measured in 778 samples from 270 patients with benign and malignant gynecological conditions. Malignant tumors were present in 180 patients including 58 cases with cancer of the ovary, 47 of the endometrium, 61 of the cervix, and 14 of the vulva. The 90 cases with benign conditions included 24 patients with ovarian tumors, 28 with fibromyomatosis, 18 with endometriosis, and 20 with endometrial hyperplasia. Of 180 cancer patients, CA 15-3 serum levels were elevated (>30 U/ml) in 74 cases (41%) and the frequency of abnormal marker values increased with clinical stage. Of 90 patients with benign conditions, high CA 15-3 levels were found in 5 cases (6%) with benign ovarian tumors. Elevated levels of the marker were most commonly seen in ovarian cancer patients (71%). In endometrial, cervical, and vulvar cancer abnormal CA 15-3 values occurred in 32, 26, and 14%, respectively. In endometrial cancer the percentage of positive marker levels increased with more infiltrating and/or less differentiated tumors. A positive correlation was found between residual tumor after surgery and CA 15-3 levels. Serial measurements in sera of patients who underwent chemotherapy showed a good correlation with response to treatment. CA 15-3 values were correlated with clinical course of disease in 87% of cases. 0 1988 Academic Press, Inc.
INTRODUCTION Tumor-associated antigens in the serum of cancer patients may prove to be useful in the detection of the disease itself and the monitoring of therapy. CA 125, carcinoembryonic antigen (CEA), cr-fetoprotein (a-FP) and the /3 subunit of human choriogonadotropin (P-HCG) are currently used as tumor markers in gynecological cancer patients [l-3]. The CA 125 assay has been reported to have a useful role in monitoring patients with epithelial ovarian carcinoma. but lacks specificity and sensitivity in the presence of small residual tumors [4,5]. P-HCG and a-FP are valuable markers for trophoblastic tumors and endodermal sinus tumors, respectively [6-71. However, these tumors comprise only a small proportion of gynecological malignancies. CEA is probably the best known in gynecological oncology. However, the sensitivity and the specificity of CEA are rather poor so that its use for gynecological malignancies is only of limited value [8,9]. Recently, the development of several monoclonal antibodies has made it possible to identify new tumor-associated antigens, among these, a cell surface antigen 265 0090-8258/88$1.50 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ET AL.
termed MAM-6 which is expressed by human breast cancer cells. This antigen is an epithelial membrane antigen present on the apical side of epithelial cells lining normal ductal and alveolar structures [lO,ll]. Hilkens et al. [12] reported that MAM-6 is present in the serum of a high percentage of breast cancer patients and is well correlated with the clinical course of the disease. Preliminary findings by the same authors suggested that this antigen is also detectable in the serum of many ovarian and cervical cancer patients [12]. The CA 15-3 assay has been developed to detect the presence of MAM-6 antigen in the serum. Two monoclonal antibodies are used for this assay, 115D8 as catcher antibody and DF3 as tracer antibody. The data presented in this study may throw some light on the potential application of CA 15-3 assay in the screening and monitoring of gynecological cancer patients. PATIENTS AND METHODS Seven hundred and seventy-eight serum samples from 270 patients with benign and malignant gynecological conditions were studied. These specimens were collected in our institution from January 1986 to April 1987. Diagnosis of each lesion was histologically confirmed. Malignant tumors were present in 180 patients including 58 cases with cancer of the ovary, 47 of endometrium, 61 of the cervix, and 14 of the vulva. The cancers were staged according to FIG0 criteria. Of the ovarian cancer patients, 11 had stage I disease, 34 stage III, and 13 stage IV. Histologically, 30 tumors were serous, 10 mutinous, 14 endometrioid, 1 clear cells, and 3 undifferentiated. Thirty patients with endometrial adenocarcinoma were at stage I, 9 at stage II, and 8 at stage III. For these tumors, the degree of cell differentiation and the depth of myometrial infiltration were also determined. Well-differentiated tumors infiltrating the inner third of the myometrium were defined as Gl and Ml, respectively. Tumors with more severe pathologic features were classified as >Gl and >Ml. Serum determinations were also performed on 12 and 49 patients with noninvasive and invasive cervical cancer, respectively. These cancer patients had stage I (13 cases), stage II (19 cases), and stage III (17 cases). Histologically all specimens were squamous cell tumors, except two adenocarcinomas. Of patients with squamous cell vulvar cancer, 2 were at stage I, 7 at stage II, and 5 at stage III. The median age of these patients was 59 years (range 28-75). The first serum sample was taken before therapy in all cases. Usually a postoperative sample was obtained 4-6 weeks after surgery or just before the first cycle of chemotherapy. Serial CA 15-3 measurements were performed in patients undergoing chemotherapy and/or during follow-up. Stability, progression, and regression of the disease were defined according to UICC criteria. The 90 cases with benign gynecological conditions included 24 patients with ovarian tumors, 28 with uterine fibromyomatosis, 18 with endometriosis, and 20 with endometrial hyperplasia. The median age of these patients was 31 years (range 15-49). Venous blood samples for CA 15-3 determinations were separated by centrifugation and aliquots were stored at -20°C until assayed. CA 15-3 assay was performed within 1 month of collection using a commercially available kit (CIS,
CA
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TABLE 1 CA 15-3 LEVELS
Type of sera Malignant conditions Ovarian cancer Endometrial cancer Cervical cancer in situ invasive Vulvar cancer Benign conditions Ovarian tumors Fibromyomatosis Endometriosis Endom. hyperplasia
IN SERA OF PATIENTS
WITH MALIGNANT
AND BENIGN
CONDITIONS
No. of sera tested
Median levels (Units/ml)
Range
% of sera >30 U/ml
180 58
18 45
8-250 12-250
41 71
47 61 12 49 14 90 24 28 18
18 18 16 20 22 15 19 15 13
8-200 12-150 12- 33 12-150 12-133 8- 46 12- 46 IO- 25 8- 27
32 26 8 31 14 5 21 -
20
14
8- 25
-
Compagnie ORIS Industrie S.A.). The upper normal limit was taken as 30 U/ml. This level corresponded to the mean value + 2 SD of 50 serum samples from apparently healthy women. CA 15-3 titers were defined as increasing or decreasing when changes were greater than 50% of the previous value. For statistical analysis Student’s t test, Fisher’s test, and the x2 square were used. RESULTS Pretreatment evaluation. Table 1 shows the median level, range, and percentage positivity of CA 15-3 serum levels in patients with malignant and benign gynecological conditions. CA 15-3 concentrations were found to be above 30 U/ml in 74 of 180 (41%) cancer patients. Abnormally high marker values were most frequently seen in patients with ovarian cancer. Elevated levels occurred in 41 of 58 cases (71%). Fifteen of 47 (32%) patients with endometrial adenocarcinoma had positive CA 15-3 values. Abnormal levels were also found in 15 of 49 (31%) and 2 of 14 (14%) patients with cervical and vulvar cancer respectively. Five out of 24 (21%) patients with benign ovarian tumors had increased CA 15-3 serum levels. However, the serum concentrations in these benign conditions were significantly lower (mean 18.4, SD 12.9) than in ovarian cancer (mean 63.8, SD 60.4) (P < 0.01). All patients with fibromyomatosis, endometriosis, and endometrial hyperplasia showed CA 15-3 values within the normal range. The difference in incidence of abnormal marker values between malignancies and benign conditions was significant (x2 = 32.01, P < 0.001). Table 2 shows the behavior of CA 15-3 levels determined in 58 sera from ovarian cancer patients in relation to stage and histotype of disease. Twentyseven percent (3/11) stage I patients, 79% (27/34) stage III patients, and 85%
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TABLE 2 CA 15-3 SERUM LEVELS IN OVARIAN CANCER ACCORDINGTO STAGE AND HISTOTYPE No. of sera tested
Median level (Units/ml)
Range
% of sera >30 U/ml
II 34 I3
I2 60 92
l2- 47 12-200 15-250
21 79 85
30 IO I4 3 I
65 28 65 55 25
12-250 l4- 65 12-120 20-130 -
71 50 79 66 -
Stage of disease I III IV Hystologic type Serous Mutinous Endometrioid Undifferentiated Clear cell
(11/13) stage IV patients had abnormal CA 15-3 serum levels. Moreover, serum concentrations of marker in advanced stages(mean 86.1; SD 60.3) were significantly higher than those in early stages (mean 22.4; SD 13.8) (P < 0.01). CA 15-3 serum levels were increased in 77% (23/30), 50% (5/10), and 79% (11/14) of patients with serous, mutinous, and endometrioid tumors, respectively. Two out of three patients with undifferentiated tumors had elevated CA 15-3 levels. The results relative to endometrial cancer patients are shown in Table 3. Again, a tendency toward higher marker values in advanced stages was evidenced by a 13% incidence in stage I in contrast to an 87% incidence in stage III. Moreover, the percentage of positive CA 15-3 levels increased in those patients with >Ml and/or >Gl tumors. This percentage increase was significant (P < 0.01 and P = 0.01). Table 4 shows that only 1 of 12 patients (8%) with in situ carcinoma of the TABLE 3 CA 15-3 SERUM LEVELS IN END~METRIAL CANCER ACCORDING TO STAGE, MYOMETRIAL INVASION AND HISTOLOGIC GRADE
Stage of disease
No. of sera tested
Median level (Units/ml)
Range
% of sera >30 U/ml
I II III Ml >Ml Cl Xi1
30 9 8 27 20 I5 32
16 27 71 16 30 13 22
12- 44 8- 52 15-200 l2- 44 8-200 8- 27 12-200
I3 44 87 7* 65 -** 46
* P > 0.01. ** P = 0.01 by Fisher’s exact test.
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15-3 IN GYNECOLOGICAL
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MALIGNANCIES
TABLE 4 CA 15-3 SERUM Stage of disease Cervix in situ I II III Vulva I II III
LEVELS
IN CERVICAL
AND VULVAR
CANCER ACCORDING
TO STAGE
No. of sera tested
Median level (Units/ml)
Range
% of sera >30 U/ml
12 13 19 17
16 16 17 40
12- 33 12- 87 12- 71 12-150
8 15 26 47
2 7 5
20 16 20
15- 25 12- 48 12-133
14 20
cervix was found to have positive CA 15-3 levels. However, this value was only slightly above normal (33 U/ml). Also in invasive cervical cancer there was a progressive increase in the percentage of patients with abnormal values as stage increased (from 15% in stage I to 47% in stage III). Elevated values of CA 15-3 were found in two patients with squamous cell vulvar cancer, 1 case at stage II and 1 at stage III. Postsurgery evaluation. All patients with normal preoperative CA 15-3 values showed normal marker levels. Of the 74 patients with high preoperative values, 43 returned to normal. Of the latter patients, 34 were disease-free after surgery and none experienced a recurrence of tumor during a median follow-up period of 8 months (range 214). Among the remaining 9 patients with ovarian cancer, residual tumor <2 cm and >2 cm was present in 5 and 4 cases, respectively. Hence, the CA 15-3 assay gave a false negative rate of 20% (9/43). Abnormal postoperative CA 15-3 levels were detected in 31 cases. Among these patients, 21 with ovarian cancer had macroscopic residual disease. The remaining 10 patients (4 with endometrial cancer and 6 with cervical cancer) were disease-free. However, 9 of these patients were found to have lymph node involvement (Table 5). In contrast, of 33 patients with negative nodes, only 1 TABLE 5 CA 15-3 SERUM LEVELS AFTER
SURGERY
Pathologic status Levels
No. of sera tested
No residual disease
Residual disease
43 31
34 10”
9 21
CA 15-3 ~30 U/ml CA 15-3 >30 U/ml L?Nine patients had positive nodes.
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TABLE 6 VARIATION OF CA 15-3 LEVELS ACCORDING TO CHEMOTHERAPYRESPONSE
Concordance rate
Clinical status” CA 15-3 values Fall Stable Rise Total
No. of sera tested
CR
PR
NC
P
Cases
m
22 7 10 39
11 2 0 13
10 2 0 12
1 3 0 4
0 0 10 10
21122 317 lO/lO 34139
(95) (43) (100) (87)
o CR, complete remission; PR partial remission; NC, no change; P, progression.
(3%) with a stage Ib “barrel” cervical carcinoma had slightly above abnormal CA 15-3 values (33 U/ml). CA 15-3 in relation to chemotherapy. Fifty-two patients received chemotherapy. Twenty-five of these patients had residual ovarian tumor. Twenty-four patients with locally advanced cervical cancer underwent neo-adjuvant chemotherapy in a pilot study carried out at our institution. Three patients had endometrial cancer. Of these 52 patients 39 had CA 153-releasing tumors. During chemotherapy, serial measurements of CA 15-3 were performed in these patients and correlated with clinical behavior of the disease. Table 6 shows the changes of CA 15-3 levels according to chemotherapy response. Overall, the relationship between clinical behavior of the disease and variation of marker was 87% (34/39). Three examples of good correlation between serial changes in CA 15-3 levels and clinical course of the disease are shown in Fig. 1. The titers of CA 15-3 were determined monthly for over 6 months. One patient with endometrial cancer experienced a good response to chemotherapy and marker values fell rapidly. Progressively increasing CA 15-3 levels were found in the second (endometrial cancer) and third (cervical cancer) cases, with progression of disease. In these progressed cases the rise in antigen level preceded clinical progression of disease by 4 and 5 weeks. DISCUSSION The clinical importance of CA 15-3 was first shown by Hilkens et al. [12], who reported this antigen to be increased mostly in patients with advanced stages of breast cancer and well correlated with clinical course of the disease. In this study, elevated CA 15-3 serum values were found in 41% of patients with gynecological malignancies. In contrast, none of the patients with gynecological benign disease except a limited percentage (21%) of those with benign ovarian tumors showed abnormal CA 15-3 values. This is particularly worth noting since elevated CA 125 values often have been found in endometriosis patients [4,13]. In any type of tumor, the distribution of positive CA 15-3 levels was such that the percentage of patients with elevated values increased with stage. CA 15-3 concentrations would therefore appear to be directly correlated with tumor extention.
CA
15-3 IN GYNECOLOGICAL MALIGNANCIES
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C
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180
120 DAYS
FIG. 1. Serial CA 15-3 levels in three patients during the course of chemotherapy. Arrows indicate the time of clinical progression the disease. The dotted line shows the upper limit of normal (30 U/ml).
The highest incidence of abnormal CA 15-3 values was observed in ovarian malignancies. This finding is in agreement with the preliminary results of Hilkens et al. [12], who reported increased serum levels of the MAM-6 antigen in 78% (18/23) of patients with advanced ovarian carcinoma. No significant correlation between CA 15-3 values and histologic type was seen in the malignant group of ovarian tumors examined. However, the finding of increased marker levels in 5 of 10 mutinous ovarian cancers appears to be, relevant. CA 15-3, in fact, is predominantly expressed by serous cancer [14,15]. Therefore the CA 15-3 assay should permit a better detection and monitoring of nonserous ovarian malignancies. In other neoplasms the sensitivity of the CA 15-3 assay was low, varing from 14% in vulvar cancer to 26 and 32% in cervical and endometrial cancer, respectively. On the other hand, this sensitivity rate is comparable to that demonstrated by other tumor markers such as CEA, CA 125, and WFP [2,3,9,16]. Therefore, because of low sensitivity, CA 15-3 assay alone has no place in screening for nonovarian gynecological malignancies. Its combined use with other tumor markers should be assessed in view of obtaining a higher sensitivity. Nevertheless more than other markers [3,8,9], CA 15-3 appears to be related to prognostic factors in endometrial cancer. A positive correlation was also found between residual tumor after surgery and CA 15-3 levels. The sensitivity of the assay was 70% (21/30). False negative results were constituted by patients with residual ovarian cancers. A similar false negative rate has been reported by various authors for the CA 125 assay in ovarian cancer [6,17,18]. Therefore, at present, a residual tumor cannot be excluded when negative marker values are found. Interestingly, 90% (9110) of cases with false positive results after surgery had unfavorable prognostic factors, such as
272
SCAMBIA ET AL.
lymph node involvement. Since lymph nodal status is a major prognostic parameter in both endometrial and cervical cancer [19,20,21] this observation further suggests that CA 15-3 levels might well be correlated with the prognosis of disease. Serial measurement of CA 15-3 in sera of patients who underwent chemotherapy showed a good correlation with response to treatment. CA 15-3 values and clinical course of the disease were correlated in 87% of the cases. In conclusion, these findings suggest that CA 15-3 may prove to be a useful tumor marker in ovarian cancer. As far as the other gynecological malignancies are concerned, although CA 15-3 assay seems to have a lower sensitivity, it is revealed to be a useful indicator of the clinical course of the disease. Furthermore preliminary evidence suggests that CA 15-3 levels could be related to prognostic factors and thus provide information on the biologic behavior of these malignancies. REFERENCES 1. Bast, R. C., Klug, T. L., St. John, E., Jenison, E. Niloff, J. M., Lazarus, H., Berkowitz, R. S., Leavih, T., Gtiffiths, C. T., Parker, L., Zurawski, V. R., and Knapp, R. C. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N. Engl. J. Med. 309, 883-887 (1983). 2. Fritsche, H. A., Freedman, R. S., Liu, F., Aconc, L. D., and Collinsworth, W. L. A survey of tumor markers in patients with squamous cell carcinoma of the uterine cervix. Gynecol. Oncol. 14, 230-235 (1982). 3. Donaldson, E. S., Van Nagell, J. R., Pursell, S., Gay, E. C., Meeker, W. R., Kashimiri, R., and van de Voorde, J. Multiple biochemical markers in patients with gynecologic malignancies. Cancer 45, 948-953 (1980). 4. Pittaway, D. E., and Fayer, J. A. The use of CA 125 in the diagnosis and management of endometriosis. Fertil. Steril. 46, 790-795 (1986). 5. Schilthuis, M. S., Aalders, J. G., Bouma, J., Kooi, H., Fleuren, G. J., Willemse, P.H.B., and De Bruijn, H. W. A. Serum CA 125 levels in epithelial ovarian cancer: Relation with findings at second-look operations and their role in the detection of tumor recurrence. &it. J. Obstet. Gynecol. 94, 202-207 (1987). 6. Schlaerth, J. B., Morrow, P., Kletzky, A., Nalick, R. H., and D’Ablaing, A. Prognostic characteristics of serum human chorionic gonadotropin titer regression following molar pregnancy. Obstet. Gynecol. 58, 478-482 (1981). 7. Wagener, C., Menzel, B., Breuer, H., Weissbach, L., Tschubel, K., Henkel, K., and Gedigk, P. Immunohistochemical localization of alpha-fetoprotein (AFP) in germ cell tumors: Evidence of AFP production by tissues different from endodermal sinus tumor. Oncology 38, 236-239 (1981). 8. Di Saia, P. J., Morrow, C. P., Haverback, B. J., and Dyce, B. J. Carcinoembtyonic antigen in cancer of the female reproductive system. Cancer 39, 2365-2370 (1977). 9. Rutanen, E. M., Lindgren, J., Sipponen, P., Stenman, U. H., Saksela, E., and Seppala, M. Carcinoembryonic antigen in malignant and nonmalignant gynecologic tumors. Cancer 42,581590 (1978). 10. Hilkens, J., Hilgers, J., Buijs, F., Hageman, P. H., Schol, D., Van Doornewoard, G., and Van Der Tweel, J. Monoclonal antibodies against human milkfat globule membranes useful in carcinoma research. In Protides of the biological fluids. Pkoceedings of the 31st colloquium, pp. 1009-1015 (1983). 11. Hilkens, J., Kroezen, V., Bonfrer, J. M. G., Bruning, P. F., Hilgers, J., and Van Eijkeren, M. A sandwich-radioimmunoassay for a new antigen (MAM-6) present in the serum of patients with carcinomas. In Protides of the biological fluids. Proceedings of the 32nd Colloquium, pp. 651-653 (1984). 12. Hilkens, J., Kroezen, V., Bonfrer, J. M. G., De Jong-Bakker, M., and Brtming, P. F. MAM-6 antigen, a new serum marker for breast cancer monitoring. Cancer Res. 46, 2582-2587 (1986).
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MALIGNANCIES
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13. Malkasian, G. D., Podratz, K. C., Stanhope, C. R., Ritts, R. E., and Zurawski, V. R. CA 125 in gynecologic practice. Amer. J. Obstet. Gynecol. 155, 515-518 (1986). 14. Kivinen, S., Kuoppila, T., Leppilampi, M., Vuori, J., and Kauppila, A. Tumor-associated antigen CA 125 before and during the treatment of ovarian carcinoma. Obstet. Gynecol. 67, 468-472 (1986). 15. Krebs, H. B., Goplerud, D. R., Kilpatrick, S. J., Myers, M. B., and Hunt, A. A role of CA 125 as tumor marker in ovarian carcinoma. Obstet. Gynecol. 67, 473-477 (1986). 16. Niloff, J. M., Klug, T. L., Schaetzl, E., Zurawski, V. R., Knapp, R. C., and Bast, R. C. Elevation of serum CA 125 in carcinomas of the fallopian tube, endometrium and endocervix. Amer. J. Obstet. Gynecol. 148, 1057-1058 (1984). 17. Berek, J. S., Knapp, R. C., Malkasian, G. D., Lavin, P. T., Whitney, C., Niloff, J. M., and Bast, R. C. CA 125 serum levels correlated with second-look operations among ovarian cancer patients. Obstet. Gynecol. 67, 685-689 (1986). 18. Brioschi, P. A., Irion, O., Bischof, P., Bader, M., Fomi, M., and Krauer, F. Serum CA 125 in epithelial ovarian cancer. A longitudinal study. Brit. J. Obstet. Gynecol. 94, 1%-201 (1987). 19. Hsu, C. T., Cheng, H. S., and Su, S. C. Prognosis of uterine cervical cancer with extensive lymph node metastases. Amer. J. Obstet. Gynecol. 114, 954-960 (1972). 20. Morrow, C. P., Di Saia, P. J., and Towsend, D. J. Current management of endometrial carcinoma. Obstet. Gynceol. 42, 399-405 (1973). 21. Ballon, S. C., Berman, M. L., and Lagasse, L. D. Survival after extraperitoneal pelvic and para-aortic lymphadenectomy and radiation therapy in cervical carcinoma. Obstet. Gynecol. 57, 90-95 (1981).
265-273 (1988)
CA 15-3 as a Tumor Marker in Gynecological Malignancies G. SCAMBIA, P. BENEDETTI PANICI, G. BAIOCCHI, L. PERRONE, S. GREGGI, AND S. MANCUSO Department
of Gynecology,
Catholic
University,
Rome, Italy
Received August 17, 1987 Serum levels of CA 15-3 were measured in 778 samples from 270 patients with benign and malignant gynecological conditions. Malignant tumors were present in 180 patients including 58 cases with cancer of the ovary, 47 of the endometrium, 61 of the cervix, and 14 of the vulva. The 90 cases with benign conditions included 24 patients with ovarian tumors, 28 with fibromyomatosis, 18 with endometriosis, and 20 with endometrial hyperplasia. Of 180 cancer patients, CA 15-3 serum levels were elevated (>30 U/ml) in 74 cases (41%) and the frequency of abnormal marker values increased with clinical stage. Of 90 patients with benign conditions, high CA 15-3 levels were found in 5 cases (6%) with benign ovarian tumors. Elevated levels of the marker were most commonly seen in ovarian cancer patients (71%). In endometrial, cervical, and vulvar cancer abnormal CA 15-3 values occurred in 32, 26, and 14%, respectively. In endometrial cancer the percentage of positive marker levels increased with more infiltrating and/or less differentiated tumors. A positive correlation was found between residual tumor after surgery and CA 15-3 levels. Serial measurements in sera of patients who underwent chemotherapy showed a good correlation with response to treatment. CA 15-3 values were correlated with clinical course of disease in 87% of cases. 0 1988 Academic Press, Inc.
INTRODUCTION Tumor-associated antigens in the serum of cancer patients may prove to be useful in the detection of the disease itself and the monitoring of therapy. CA 125, carcinoembryonic antigen (CEA), cr-fetoprotein (a-FP) and the /3 subunit of human choriogonadotropin (P-HCG) are currently used as tumor markers in gynecological cancer patients [l-3]. The CA 125 assay has been reported to have a useful role in monitoring patients with epithelial ovarian carcinoma. but lacks specificity and sensitivity in the presence of small residual tumors [4,5]. P-HCG and a-FP are valuable markers for trophoblastic tumors and endodermal sinus tumors, respectively [6-71. However, these tumors comprise only a small proportion of gynecological malignancies. CEA is probably the best known in gynecological oncology. However, the sensitivity and the specificity of CEA are rather poor so that its use for gynecological malignancies is only of limited value [8,9]. Recently, the development of several monoclonal antibodies has made it possible to identify new tumor-associated antigens, among these, a cell surface antigen 265 0090-8258/88$1.50 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
266
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ET AL.
termed MAM-6 which is expressed by human breast cancer cells. This antigen is an epithelial membrane antigen present on the apical side of epithelial cells lining normal ductal and alveolar structures [lO,ll]. Hilkens et al. [12] reported that MAM-6 is present in the serum of a high percentage of breast cancer patients and is well correlated with the clinical course of the disease. Preliminary findings by the same authors suggested that this antigen is also detectable in the serum of many ovarian and cervical cancer patients [12]. The CA 15-3 assay has been developed to detect the presence of MAM-6 antigen in the serum. Two monoclonal antibodies are used for this assay, 115D8 as catcher antibody and DF3 as tracer antibody. The data presented in this study may throw some light on the potential application of CA 15-3 assay in the screening and monitoring of gynecological cancer patients. PATIENTS AND METHODS Seven hundred and seventy-eight serum samples from 270 patients with benign and malignant gynecological conditions were studied. These specimens were collected in our institution from January 1986 to April 1987. Diagnosis of each lesion was histologically confirmed. Malignant tumors were present in 180 patients including 58 cases with cancer of the ovary, 47 of endometrium, 61 of the cervix, and 14 of the vulva. The cancers were staged according to FIG0 criteria. Of the ovarian cancer patients, 11 had stage I disease, 34 stage III, and 13 stage IV. Histologically, 30 tumors were serous, 10 mutinous, 14 endometrioid, 1 clear cells, and 3 undifferentiated. Thirty patients with endometrial adenocarcinoma were at stage I, 9 at stage II, and 8 at stage III. For these tumors, the degree of cell differentiation and the depth of myometrial infiltration were also determined. Well-differentiated tumors infiltrating the inner third of the myometrium were defined as Gl and Ml, respectively. Tumors with more severe pathologic features were classified as >Gl and >Ml. Serum determinations were also performed on 12 and 49 patients with noninvasive and invasive cervical cancer, respectively. These cancer patients had stage I (13 cases), stage II (19 cases), and stage III (17 cases). Histologically all specimens were squamous cell tumors, except two adenocarcinomas. Of patients with squamous cell vulvar cancer, 2 were at stage I, 7 at stage II, and 5 at stage III. The median age of these patients was 59 years (range 28-75). The first serum sample was taken before therapy in all cases. Usually a postoperative sample was obtained 4-6 weeks after surgery or just before the first cycle of chemotherapy. Serial CA 15-3 measurements were performed in patients undergoing chemotherapy and/or during follow-up. Stability, progression, and regression of the disease were defined according to UICC criteria. The 90 cases with benign gynecological conditions included 24 patients with ovarian tumors, 28 with uterine fibromyomatosis, 18 with endometriosis, and 20 with endometrial hyperplasia. The median age of these patients was 31 years (range 15-49). Venous blood samples for CA 15-3 determinations were separated by centrifugation and aliquots were stored at -20°C until assayed. CA 15-3 assay was performed within 1 month of collection using a commercially available kit (CIS,
CA
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267
MALIGNANCIES
TABLE 1 CA 15-3 LEVELS
Type of sera Malignant conditions Ovarian cancer Endometrial cancer Cervical cancer in situ invasive Vulvar cancer Benign conditions Ovarian tumors Fibromyomatosis Endometriosis Endom. hyperplasia
IN SERA OF PATIENTS
WITH MALIGNANT
AND BENIGN
CONDITIONS
No. of sera tested
Median levels (Units/ml)
Range
% of sera >30 U/ml
180 58
18 45
8-250 12-250
41 71
47 61 12 49 14 90 24 28 18
18 18 16 20 22 15 19 15 13
8-200 12-150 12- 33 12-150 12-133 8- 46 12- 46 IO- 25 8- 27
32 26 8 31 14 5 21 -
20
14
8- 25
-
Compagnie ORIS Industrie S.A.). The upper normal limit was taken as 30 U/ml. This level corresponded to the mean value + 2 SD of 50 serum samples from apparently healthy women. CA 15-3 titers were defined as increasing or decreasing when changes were greater than 50% of the previous value. For statistical analysis Student’s t test, Fisher’s test, and the x2 square were used. RESULTS Pretreatment evaluation. Table 1 shows the median level, range, and percentage positivity of CA 15-3 serum levels in patients with malignant and benign gynecological conditions. CA 15-3 concentrations were found to be above 30 U/ml in 74 of 180 (41%) cancer patients. Abnormally high marker values were most frequently seen in patients with ovarian cancer. Elevated levels occurred in 41 of 58 cases (71%). Fifteen of 47 (32%) patients with endometrial adenocarcinoma had positive CA 15-3 values. Abnormal levels were also found in 15 of 49 (31%) and 2 of 14 (14%) patients with cervical and vulvar cancer respectively. Five out of 24 (21%) patients with benign ovarian tumors had increased CA 15-3 serum levels. However, the serum concentrations in these benign conditions were significantly lower (mean 18.4, SD 12.9) than in ovarian cancer (mean 63.8, SD 60.4) (P < 0.01). All patients with fibromyomatosis, endometriosis, and endometrial hyperplasia showed CA 15-3 values within the normal range. The difference in incidence of abnormal marker values between malignancies and benign conditions was significant (x2 = 32.01, P < 0.001). Table 2 shows the behavior of CA 15-3 levels determined in 58 sera from ovarian cancer patients in relation to stage and histotype of disease. Twentyseven percent (3/11) stage I patients, 79% (27/34) stage III patients, and 85%
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ET AL.
TABLE 2 CA 15-3 SERUM LEVELS IN OVARIAN CANCER ACCORDINGTO STAGE AND HISTOTYPE No. of sera tested
Median level (Units/ml)
Range
% of sera >30 U/ml
II 34 I3
I2 60 92
l2- 47 12-200 15-250
21 79 85
30 IO I4 3 I
65 28 65 55 25
12-250 l4- 65 12-120 20-130 -
71 50 79 66 -
Stage of disease I III IV Hystologic type Serous Mutinous Endometrioid Undifferentiated Clear cell
(11/13) stage IV patients had abnormal CA 15-3 serum levels. Moreover, serum concentrations of marker in advanced stages(mean 86.1; SD 60.3) were significantly higher than those in early stages (mean 22.4; SD 13.8) (P < 0.01). CA 15-3 serum levels were increased in 77% (23/30), 50% (5/10), and 79% (11/14) of patients with serous, mutinous, and endometrioid tumors, respectively. Two out of three patients with undifferentiated tumors had elevated CA 15-3 levels. The results relative to endometrial cancer patients are shown in Table 3. Again, a tendency toward higher marker values in advanced stages was evidenced by a 13% incidence in stage I in contrast to an 87% incidence in stage III. Moreover, the percentage of positive CA 15-3 levels increased in those patients with >Ml and/or >Gl tumors. This percentage increase was significant (P < 0.01 and P = 0.01). Table 4 shows that only 1 of 12 patients (8%) with in situ carcinoma of the TABLE 3 CA 15-3 SERUM LEVELS IN END~METRIAL CANCER ACCORDING TO STAGE, MYOMETRIAL INVASION AND HISTOLOGIC GRADE
Stage of disease
No. of sera tested
Median level (Units/ml)
Range
% of sera >30 U/ml
I II III Ml >Ml Cl Xi1
30 9 8 27 20 I5 32
16 27 71 16 30 13 22
12- 44 8- 52 15-200 l2- 44 8-200 8- 27 12-200
I3 44 87 7* 65 -** 46
* P > 0.01. ** P = 0.01 by Fisher’s exact test.
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MALIGNANCIES
TABLE 4 CA 15-3 SERUM Stage of disease Cervix in situ I II III Vulva I II III
LEVELS
IN CERVICAL
AND VULVAR
CANCER ACCORDING
TO STAGE
No. of sera tested
Median level (Units/ml)
Range
% of sera >30 U/ml
12 13 19 17
16 16 17 40
12- 33 12- 87 12- 71 12-150
8 15 26 47
2 7 5
20 16 20
15- 25 12- 48 12-133
14 20
cervix was found to have positive CA 15-3 levels. However, this value was only slightly above normal (33 U/ml). Also in invasive cervical cancer there was a progressive increase in the percentage of patients with abnormal values as stage increased (from 15% in stage I to 47% in stage III). Elevated values of CA 15-3 were found in two patients with squamous cell vulvar cancer, 1 case at stage II and 1 at stage III. Postsurgery evaluation. All patients with normal preoperative CA 15-3 values showed normal marker levels. Of the 74 patients with high preoperative values, 43 returned to normal. Of the latter patients, 34 were disease-free after surgery and none experienced a recurrence of tumor during a median follow-up period of 8 months (range 214). Among the remaining 9 patients with ovarian cancer, residual tumor <2 cm and >2 cm was present in 5 and 4 cases, respectively. Hence, the CA 15-3 assay gave a false negative rate of 20% (9/43). Abnormal postoperative CA 15-3 levels were detected in 31 cases. Among these patients, 21 with ovarian cancer had macroscopic residual disease. The remaining 10 patients (4 with endometrial cancer and 6 with cervical cancer) were disease-free. However, 9 of these patients were found to have lymph node involvement (Table 5). In contrast, of 33 patients with negative nodes, only 1 TABLE 5 CA 15-3 SERUM LEVELS AFTER
SURGERY
Pathologic status Levels
No. of sera tested
No residual disease
Residual disease
43 31
34 10”
9 21
CA 15-3 ~30 U/ml CA 15-3 >30 U/ml L?Nine patients had positive nodes.
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SCAMBIA ET AL.
TABLE 6 VARIATION OF CA 15-3 LEVELS ACCORDING TO CHEMOTHERAPYRESPONSE
Concordance rate
Clinical status” CA 15-3 values Fall Stable Rise Total
No. of sera tested
CR
PR
NC
P
Cases
m
22 7 10 39
11 2 0 13
10 2 0 12
1 3 0 4
0 0 10 10
21122 317 lO/lO 34139
(95) (43) (100) (87)
o CR, complete remission; PR partial remission; NC, no change; P, progression.
(3%) with a stage Ib “barrel” cervical carcinoma had slightly above abnormal CA 15-3 values (33 U/ml). CA 15-3 in relation to chemotherapy. Fifty-two patients received chemotherapy. Twenty-five of these patients had residual ovarian tumor. Twenty-four patients with locally advanced cervical cancer underwent neo-adjuvant chemotherapy in a pilot study carried out at our institution. Three patients had endometrial cancer. Of these 52 patients 39 had CA 153-releasing tumors. During chemotherapy, serial measurements of CA 15-3 were performed in these patients and correlated with clinical behavior of the disease. Table 6 shows the changes of CA 15-3 levels according to chemotherapy response. Overall, the relationship between clinical behavior of the disease and variation of marker was 87% (34/39). Three examples of good correlation between serial changes in CA 15-3 levels and clinical course of the disease are shown in Fig. 1. The titers of CA 15-3 were determined monthly for over 6 months. One patient with endometrial cancer experienced a good response to chemotherapy and marker values fell rapidly. Progressively increasing CA 15-3 levels were found in the second (endometrial cancer) and third (cervical cancer) cases, with progression of disease. In these progressed cases the rise in antigen level preceded clinical progression of disease by 4 and 5 weeks. DISCUSSION The clinical importance of CA 15-3 was first shown by Hilkens et al. [12], who reported this antigen to be increased mostly in patients with advanced stages of breast cancer and well correlated with clinical course of the disease. In this study, elevated CA 15-3 serum values were found in 41% of patients with gynecological malignancies. In contrast, none of the patients with gynecological benign disease except a limited percentage (21%) of those with benign ovarian tumors showed abnormal CA 15-3 values. This is particularly worth noting since elevated CA 125 values often have been found in endometriosis patients [4,13]. In any type of tumor, the distribution of positive CA 15-3 levels was such that the percentage of patients with elevated values increased with stage. CA 15-3 concentrations would therefore appear to be directly correlated with tumor extention.
CA
15-3 IN GYNECOLOGICAL MALIGNANCIES
I
I
C
6C
271
180
120 DAYS
FIG. 1. Serial CA 15-3 levels in three patients during the course of chemotherapy. Arrows indicate the time of clinical progression the disease. The dotted line shows the upper limit of normal (30 U/ml).
The highest incidence of abnormal CA 15-3 values was observed in ovarian malignancies. This finding is in agreement with the preliminary results of Hilkens et al. [12], who reported increased serum levels of the MAM-6 antigen in 78% (18/23) of patients with advanced ovarian carcinoma. No significant correlation between CA 15-3 values and histologic type was seen in the malignant group of ovarian tumors examined. However, the finding of increased marker levels in 5 of 10 mutinous ovarian cancers appears to be, relevant. CA 15-3, in fact, is predominantly expressed by serous cancer [14,15]. Therefore the CA 15-3 assay should permit a better detection and monitoring of nonserous ovarian malignancies. In other neoplasms the sensitivity of the CA 15-3 assay was low, varing from 14% in vulvar cancer to 26 and 32% in cervical and endometrial cancer, respectively. On the other hand, this sensitivity rate is comparable to that demonstrated by other tumor markers such as CEA, CA 125, and WFP [2,3,9,16]. Therefore, because of low sensitivity, CA 15-3 assay alone has no place in screening for nonovarian gynecological malignancies. Its combined use with other tumor markers should be assessed in view of obtaining a higher sensitivity. Nevertheless more than other markers [3,8,9], CA 15-3 appears to be related to prognostic factors in endometrial cancer. A positive correlation was also found between residual tumor after surgery and CA 15-3 levels. The sensitivity of the assay was 70% (21/30). False negative results were constituted by patients with residual ovarian cancers. A similar false negative rate has been reported by various authors for the CA 125 assay in ovarian cancer [6,17,18]. Therefore, at present, a residual tumor cannot be excluded when negative marker values are found. Interestingly, 90% (9110) of cases with false positive results after surgery had unfavorable prognostic factors, such as
272
SCAMBIA ET AL.
lymph node involvement. Since lymph nodal status is a major prognostic parameter in both endometrial and cervical cancer [19,20,21] this observation further suggests that CA 15-3 levels might well be correlated with the prognosis of disease. Serial measurement of CA 15-3 in sera of patients who underwent chemotherapy showed a good correlation with response to treatment. CA 15-3 values and clinical course of the disease were correlated in 87% of the cases. In conclusion, these findings suggest that CA 15-3 may prove to be a useful tumor marker in ovarian cancer. As far as the other gynecological malignancies are concerned, although CA 15-3 assay seems to have a lower sensitivity, it is revealed to be a useful indicator of the clinical course of the disease. Furthermore preliminary evidence suggests that CA 15-3 levels could be related to prognostic factors and thus provide information on the biologic behavior of these malignancies. REFERENCES 1. Bast, R. C., Klug, T. L., St. John, E., Jenison, E. Niloff, J. M., Lazarus, H., Berkowitz, R. S., Leavih, T., Gtiffiths, C. T., Parker, L., Zurawski, V. R., and Knapp, R. C. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N. Engl. J. Med. 309, 883-887 (1983). 2. Fritsche, H. A., Freedman, R. S., Liu, F., Aconc, L. D., and Collinsworth, W. L. A survey of tumor markers in patients with squamous cell carcinoma of the uterine cervix. Gynecol. Oncol. 14, 230-235 (1982). 3. Donaldson, E. S., Van Nagell, J. R., Pursell, S., Gay, E. C., Meeker, W. R., Kashimiri, R., and van de Voorde, J. Multiple biochemical markers in patients with gynecologic malignancies. Cancer 45, 948-953 (1980). 4. Pittaway, D. E., and Fayer, J. A. The use of CA 125 in the diagnosis and management of endometriosis. Fertil. Steril. 46, 790-795 (1986). 5. Schilthuis, M. S., Aalders, J. G., Bouma, J., Kooi, H., Fleuren, G. J., Willemse, P.H.B., and De Bruijn, H. W. A. Serum CA 125 levels in epithelial ovarian cancer: Relation with findings at second-look operations and their role in the detection of tumor recurrence. &it. J. Obstet. Gynecol. 94, 202-207 (1987). 6. Schlaerth, J. B., Morrow, P., Kletzky, A., Nalick, R. H., and D’Ablaing, A. Prognostic characteristics of serum human chorionic gonadotropin titer regression following molar pregnancy. Obstet. Gynecol. 58, 478-482 (1981). 7. Wagener, C., Menzel, B., Breuer, H., Weissbach, L., Tschubel, K., Henkel, K., and Gedigk, P. Immunohistochemical localization of alpha-fetoprotein (AFP) in germ cell tumors: Evidence of AFP production by tissues different from endodermal sinus tumor. Oncology 38, 236-239 (1981). 8. Di Saia, P. J., Morrow, C. P., Haverback, B. J., and Dyce, B. J. Carcinoembtyonic antigen in cancer of the female reproductive system. Cancer 39, 2365-2370 (1977). 9. Rutanen, E. M., Lindgren, J., Sipponen, P., Stenman, U. H., Saksela, E., and Seppala, M. Carcinoembryonic antigen in malignant and nonmalignant gynecologic tumors. Cancer 42,581590 (1978). 10. Hilkens, J., Hilgers, J., Buijs, F., Hageman, P. H., Schol, D., Van Doornewoard, G., and Van Der Tweel, J. Monoclonal antibodies against human milkfat globule membranes useful in carcinoma research. In Protides of the biological fluids. Pkoceedings of the 31st colloquium, pp. 1009-1015 (1983). 11. Hilkens, J., Kroezen, V., Bonfrer, J. M. G., Bruning, P. F., Hilgers, J., and Van Eijkeren, M. A sandwich-radioimmunoassay for a new antigen (MAM-6) present in the serum of patients with carcinomas. In Protides of the biological fluids. Proceedings of the 32nd Colloquium, pp. 651-653 (1984). 12. Hilkens, J., Kroezen, V., Bonfrer, J. M. G., De Jong-Bakker, M., and Brtming, P. F. MAM-6 antigen, a new serum marker for breast cancer monitoring. Cancer Res. 46, 2582-2587 (1986).
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13. Malkasian, G. D., Podratz, K. C., Stanhope, C. R., Ritts, R. E., and Zurawski, V. R. CA 125 in gynecologic practice. Amer. J. Obstet. Gynecol. 155, 515-518 (1986). 14. Kivinen, S., Kuoppila, T., Leppilampi, M., Vuori, J., and Kauppila, A. Tumor-associated antigen CA 125 before and during the treatment of ovarian carcinoma. Obstet. Gynecol. 67, 468-472 (1986). 15. Krebs, H. B., Goplerud, D. R., Kilpatrick, S. J., Myers, M. B., and Hunt, A. A role of CA 125 as tumor marker in ovarian carcinoma. Obstet. Gynecol. 67, 473-477 (1986). 16. Niloff, J. M., Klug, T. L., Schaetzl, E., Zurawski, V. R., Knapp, R. C., and Bast, R. C. Elevation of serum CA 125 in carcinomas of the fallopian tube, endometrium and endocervix. Amer. J. Obstet. Gynecol. 148, 1057-1058 (1984). 17. Berek, J. S., Knapp, R. C., Malkasian, G. D., Lavin, P. T., Whitney, C., Niloff, J. M., and Bast, R. C. CA 125 serum levels correlated with second-look operations among ovarian cancer patients. Obstet. Gynecol. 67, 685-689 (1986). 18. Brioschi, P. A., Irion, O., Bischof, P., Bader, M., Fomi, M., and Krauer, F. Serum CA 125 in epithelial ovarian cancer. A longitudinal study. Brit. J. Obstet. Gynecol. 94, 1%-201 (1987). 19. Hsu, C. T., Cheng, H. S., and Su, S. C. Prognosis of uterine cervical cancer with extensive lymph node metastases. Amer. J. Obstet. Gynecol. 114, 954-960 (1972). 20. Morrow, C. P., Di Saia, P. J., and Towsend, D. J. Current management of endometrial carcinoma. Obstet. Gynceol. 42, 399-405 (1973). 21. Ballon, S. C., Berman, M. L., and Lagasse, L. D. Survival after extraperitoneal pelvic and para-aortic lymphadenectomy and radiation therapy in cervical carcinoma. Obstet. Gynecol. 57, 90-95 (1981).