duction one would not expect AFP secretion by trophoblastic tumors; se- rum AFP ... nontrophoblastic gynecologic neoplasms, of which 53 were ovarian can-.
10 Ovarian and Uterine Cancer Markers Markku Seppala Department of Obstetrics and Gynecology, University Central Hospital, Helsinki, Finland
Laboratory tests have long been an integral part of the diagnosis and management of gynecological cancer. Cytology permits easy detection of premalignant and early malignant lesions of the cervix, and as a result new cases are now diagnosed earlier and can be more successfully managed. Research on biochemical tumor markers is directed to the same goal. The classic example is choriocarcinoma, which can be diagnosed and monitored by measurement of chorionic gonadotropin. But many gynecologic tumors are still fatal. Thus, ovarian cancer has defied all attempts at early diagnosis, and treatment of advanced cases remains unsatisfactory. Potential markers for gynecologic cancer include oncodevelopmental antigens, enzymes, hormones, and other proteins. This review summarizes the current state of knowledge on tumor markers for the management of trophoblastic and nontrophoblastic gynecologic cancer.
1. Oncodevelopmental Antigens
1. 1. Alphafetoprotein 1.1.1. Trophoblastic Disease Alphafetoprotein (AFP) is synthesized by the fetal liver, yolk sac, and the gastrointestinal tract (Gitlin et aI., 1972). AFP reappears in most cases of primary liver cancer and yolk sac tumors, and very occasionally in gastric cancer (see Abelev, 1974). Considering the embryonic sites of AFP pro233
S. Sell et al. (eds.), Human Cancer Markers © HUMANA Press Inc. 1982
234
MARKKU SEPpALA
duction one would not expect AFP secretion by trophoblastic tumors; serum AFP levels are usually not elevated in patients with choriocarcinoma (Seppala et al., 1972), but may be in some patients with hydatidiform mole (Seppala and Ruoslahti, 1974). Vesicular fluid from hydatidiform moles may also contain AFP at high concentrations (Grudzinskas et al., 1977). However, measurement of serum AFP does not contribute to the management of patients with gestational trophoblastic disease.
1.1.2. Nontrophoblastic Tumors Nontrophoblastic gynecologic neoplasms rarely secrete AFP in increased amounts detectable in serum. In a study of 92 patients with nontrophoblastic gynecologic neoplasms, of which 53 were ovarian cancers, Seppala and coworkers (1975) found an elevated serum AFP level in only one patient (ovarian carcinoma with liver metastases). Germ cell tumors may secrete AFP (Abelev et aI., 1967; Ballas, 1972; Talerman and Haije, 1974). Germ cell tumors differentiate along either embryonic or extraembryonic pathways (Teilum, 1965), or along both; dysgerminomas proliferate without differentiation. Extraembryonic differentiation may yield yolk sac or trophoblastic tissue, the former secreting AFP and the latter chorionic gonadotropin (HCG). Embryonic differentiation yields teratomas that usually do not secrete AFP. Many germ cell tumors have mixed components and virtually all teratocarcinomas are mixed tumors (see Fox, 1980). Cells that secrete AFP or HCG may be unrecognized by routine histopathology unless specific immunohistochemical staining is used. In pure yolk sac tumors, AFP can be identified in cells lining the endodermal sinuses as well as in intra- and extracytoplasmic PAS-positive hyaline globules (Teilum et aI., 1974). In patients with yolk sac tumors, circulating AFP levels reflect the activity of the tumor. The normal half-life of AFP is 4-5 days (Seppala and Ruoslahti, 1972), and postoperative estimation of half-life permits early determination of the completeness of surgical removal. The use of radiolabeled anti-AFP antibodies has been exploited for in vivo localization of AFP-producing tumors (e.g., a Sertoli-Leydig cell tumor of both ovaries) (Goldenberg et aI., 1980b). For example, a "secondlook" operation was performed on a patient with a yolk sac tumor who had a rising AFP level despite chemotherapy. Small nodules of yolk sac tumor were removed from the peritoneal cavity, but this had no effect on the serum AFP level. Radioimmunodetection was attempted in order to localize tumor deposits. Scanning revealed an accumulation of radioactivity in the peritoneal cavity and retroperitoneal space, but the liver and the lungs were free of apparent tumor tissue. The absence of remote metastases was confirmed at a third operation, and by later autopsy. Previous experimental (Primus et aI., 1973; Mach et aI., 1974) and clinical studies (Goldenberg et aI., 1978; Dykes et aI., 1980; Goldenberg et aI., 1980) in-
OVARIAN AND UTERINE CANCER MARKERS
235
dicate that radioactive antibody scanning can show tumor deposits. Although, unlike CEA, AFP is not a cell surface marker, the local antigen concentration appears to be sufficient for successful radio localization (Goldenberg et aI., 1980b). AFP from yolk sac tumors differs from fetal serum AFP in that a greater proportion of it does not bind to concanavalin A (Ruoslahti et aI. , 1978). In the patient with yolk sac tumor described above, 40--60% of circulating AFP did not bind con A, suggesting yolk sac origin, and reflecting differences in glycosylation between AFP from fetal liver and yolk sac (Ruoslahti and Seppalii, 1979). The difference does not, however, affect the immunoreactivity of AFP (Ruoslahti et aI., 1978). In summary, AFP is an excellent marker for yolk sac tumors. Demonstration of AFP can be used for histopathological classification, localization of metastases in the body, and for monitoring of treatment. Pregnancy-related disorders and liver disease may also cause elevated serum AFP levels (Brock, 1977; Seppala, 1977), but these can be easily recognized and cause no major problems in differential diagnosis.
1.2. Carcinoembryonic Antigen 1.2.1. Trophoblastic Tumors CEA levels are usually normal in patients with trophoblastic tumors (Seppala et aI., 1976). 1.2.2. Nontrophoblastic Tumors CEA has been demonstrated in premalignant lesions and malignant tumors of the female reproductive system (Lindgren et al., 1979; Marchand et al., 1975; Goldenberg et aI., 1976; Rutanen et aI., 1978) as well as in mucinous cyst fluid (van Nagell et aI., 1975b), ascitic fluid (SeppaHi et aI., 1975), and serum of such patients (Khoo and Mackay, 1973; Seppala et aI., 1975; van Nagell et aI., 1975a; DiSaia et aI., 1975). CEA from gynecological cancers appears to be immunologically identical with colon cancer CEA (Seppala et aI., 1975), but CEA from some ovarian cancers has been reported to have a higher molecular weight (PIetsch and Goldenberg, 1974). 1.2.2.1. Expression of CEA in Premalignant Lesions and Invasive Cancer. CEA has been identified in tissue from early premalignant lesions. Using immunoperoxidase staining for epithelial lesions of the uterine cervix, Lindgren and coworkers (1979) found that normal epithelium was CEA-negative, whereas 25% of early dysplasias were CEApositive, principally in the keratinized surface layers. The incidence of CEA-positivity increased from premalignant to malignant lesions (Fig. 1). Increased CEA levels may reflect malignant potential in premalignant lesions.
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1.2.2.2. The Relationship of CEA to Prognosis of Patients with Gynecologic Cancer. In patients with ovarian cancer, elevated serum CEA levels are associated with a poor prognosis (Levin et al., 1976). This has been ascribed to the greater volume of such tumors rather than any inherent aggressiveness of CEA-positive lesions. Studies by Lindgren et al. . (1979) have demonstrated that the invasive properties of squamous cell carcinoma of the cervix are not related to the presence or absence of CEA, since patients with CEA-positive and CEA-negative lesions had similar la-year survival rates within tumors of the same clinical stage (Figs. 2 and 3).
1.2.2.3. CEA in Histopathological Classification of Gynecologic Tumors. Immunoperoxidase staining for CEA in tissue may improve the histopathologic classification of certain tumors of uterus and ovary. For example, the histological variability of endocervical and endometrial adenocarcinomas has created problems of differential diagnosis, in part because specimens obtained by endocervical curettage of the endocervix may contain endometrial tissue. Recent studies suggest that diagnosis can be improved by the demonstration of CEA in tissue (Fig. 4). Thus, in a study by Wahlstrom et al. (1979), tissue from 131 of 163 patients (80%) with endocervical adenocarcinoma, but only 11 of 137 patients (8%) with endometrial adenocarcinoma, was CEA-positive. The commonest exceptions were endocervical mesonephroid adenocarcin-
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OVARIAN AND UTERINE CANCER MARKERS
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omas (which were CEA-negative) and endometrial adenosquamous carcinomas (which were CEA-positive). After exclusion of these on simple morphological criteria, 86 of 107 endocervical adenocarcinomas (80%) were CEA-positive , and all endometrial adenocarcinomas were CEA-
Fig. 4. Immunoperoxidase staining of CEA in endocervical adenocarcinoma. (a) Hematoxylin--eosin; (b) anti-CEA antiserum; (c) anti-CEA antiserum absorbed with purified CEA. By courtesy of Dr. T. Wahlstrom, Helsinki.
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negative. Although the qualitative interpretation of immunoperoxidase slides is unambiguous when appropriate controls are included, the difference between positive and negative results must also be quantitative because the immunoperoxidase test can only detect CEA concentrations of 3-5 J.1g/g or greater in formalin-fixed specimens (Goldenberg et aI., 1976). However, not all workers agree that a clear distinction can be made. In a smaller series of endocervical adenocarcinomas, van Nagell et al. (1979) reported a lower frequency (36%) of CEA-positivity. The usefulness of CEA-staining has also been questioned on the basis of the relative incidence of endometrial and endocervical adenocarcinomas (Dufour and Stock, 1980). Since endometrial adenocarcinoma is over 20 times more common than endocervical adenocarcinoma, endometrial adenocarcinoma would contribute more CEA-positives than would endocervical adenocarcinomas in a random sample of adenocarcinomas. However, if tumors with squamous elements are rigorously excluded, 99% of CEAnegative adenocarcinomas would be endometrial. This information can be of great diagnostic value. Immunohistochemical staining of CEA may also be useful in some ovarian tumors. A study of tissue CEA content of 82 ovarian epithelial neoplasms showed that mucinous tumors contain more CEA than serous tumors (Heald et al., 1979). There was only partial correspondence between the degree of malignancy of mucinous tumors, as assessed histologically, and their content of CEA. From these studies it was postulated that examination of tissue CEA might sharpen the morphological distinction of these neoplasms and thus allow for a more precise grading of their degree of malignancy (Heald et al., 1979). 1.2.2.4. Circulating CEA Levels. Elevated serum CEA levels have been noted in 13-80% of patients with gynecologic cancer. The highest frequency is seen with endocervical cancer, and lower frequencies in endometrial adenocarcinoma. Elevated levels are also commoner and more striking in patients with advanced disease (SeppaHi et aI., 1975; Kjorstad and Orjaseter, 1977; Rutanen et aI., 1978), though they are occasionally seen even in patients with benign mucinous cystadenoma (Fig. 5). The great variation in the reported frequencies of elevated serum CEA levels results from variability in cutoff levels and on differences in assay protocols and reagents. All workers agree, however, that in gynecologic cancer elevated CEA levels are not as high as in colorectal cancer. Most studies show that circulating CEA levels correlate with the spread of disease (Seppala et aI., 1975; DiSaia et aI., 1975; Rutanen et aI., 1978). Elevated levels have been reported in early stages, e.g., carcinoma in situ (Khoo and Mackay, 1976), but most would agree that this is uncommon (Stone et aI., 1977; Seppala et aI., 1975; Kjorstad and Orjaseter, 1977; Rutanen et al., 1978).
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In gynecologic cancer, immunoperoxidase staining of tumor tissue is more sensitive than measurement of serum CEA levels for the identification of CEA-positive tumors. Thus, when tumors are CEA-positive, the serum level may be either normal or elevated, but when the tumor is CEAnegative, the serum CEA level is usually normal (Fig. 6). Exceptions to the latter have been observed among patients with advanced disease, liver disease, infectious disease, and in heavy smokers. Some studies (Khoo and Mackay, 1974) suggest that, in gynecologic cancer, reelevation of serum CEA level precedes clinical symptoms and clinical tumor by an average of 10.8 weeks. However, this does not improve prognosis since little can be done following radical surgery and full radiotherapy. Persistence or decrease of elevated CEA levels may also be used to determine the completeness of surgery.
1.2.2.5. Radioimmunodetection of Gynecologic Tumors by Anti-CEA Antibodies and External Photoscanning. Although the first attempt to identify human neoplasms with radiolabeled anti-CEA was unsuccessful (Reif et al., 1974), subsequent studies have been more promising. Goldenberg et al. (1978) described 18 patients with various cancers
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in whom all but 6 out of 38 tumor deposits were revealed using a subtraction technique. The studies were expanded to include ovarian cancer. 131I-Iabeled goat immunoglobulin G (IgG) against CEA was administered to patients with ovarian cancer (average dose 1.0 mCi; 180--250 j.1g IgG protein). The primary cancer was localized in all 13 patients and the metastases in 6 out of 9 cases (van Nagell et aI., 1980). More conventional diagnostic techniques such as computer-assisted tomography, ultrasonography, and angiography were less efficient. However, lesions smaller than 2 cm in diameter could not be detected. Tumors containing a CEA concentration above 115 ng/g could be localized with radioactive anti-CEA antibodies, and these included a benign neoplasm (van Nagell et aI., 1980). Thus, primary and secondary ovarian cancers could be detected in 100 and 67% of cases, respectively. Although these preliminary results are promising, CEA is not a specific marker for ovarian cancer but merely serves as a target for immunolocalization. The same principle applies to all tumors or tissues containing more CEA than the surrounding tissue. In studies on colonic cancer, radio-
242
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activity in the tumor has been 1.5-5.3 times higher than in the adjacent tissue (Mach et al., 1979), and successful radioimmunodetection has been achieved even in the presence of high circulating CEA concentrations (Goldenberg et al., 1978; Dykes et al., 1980). It is obvious that both specificity and purity of anti-CEA antibody are prerequisites, and demand the use of affinity-purified antibodies. Nonetheless, the method has great potential value in the immunodetection of cancer (van Nagell et aI., 1980; Dykes et al., 1980; Mach et al., 1980).
1.3. Chorionic Gonadotropin and Subunits of Glycoprotein Hormones 1.3.1. Trophoblastic Tumors Chorionic gonadotropin is synthesized by the placental syncytiotrophoblast and trophoblastic tumors (Midgley and Pierce, 1962). Of all tumor markers, HCG is the most fully investigated with regard to physiocochemical properties, physiology, and clinical application. The estimated secretion rate of HCG by one trophoblastic cell for a 10-day old human placenta is 1.4 x 10- 2 IU/day (Braunstein et al., 1973a), and the estimated production rate of HCG by malignant trophoblast in vivo is 5 x 10- 5 IU/celllday (Bagshawe, 1969) and in vitro 5 x 10- 6_10- 7 IV/cell/day (Kohler and Bridson, 1971). Evidence from in vitro and in vivo studies indicates that the amount of HCG produced is proportional to the number of viable tumor cells (Bagshawe, 1969; Lewis et al., 1969). In addition to HCG the normal placenta also contains subunits of HCG, the free alpha subunit being in excess of the beta subunit (Vaitukaitis, 1974). In choriocarcinoma the proportion of free alpha secretion is less than in normal pregnancy (Vaitukaitis and Ebersole, 1976; Rutanen, 1978), and it has been suggested that free alpha secretion is related to an unfavorable prognosis. Treatment of patients with choriocarcinoma relies heavily on information about circulating HCG levels (Hertz et aI., 1961). HCG becomes detectable in blood when as few as 104 _10 5 tumor cells are present in the body (Bagshawe, 1975), whereas 109_10 12 cells are required before tumor becomes demonstrable by other clinical measures. The measurement of serum HCG levels by radioimmunoassay (Vaitukaitis et aI., 1972) has become vital for the patient because effective treatment can be offered. The prognosis of these patients has improved to near 100% survival rate following improvements in early diagnosis and treatment. 1.3.2. HCG in Nontrophoblastic Gynecologic Cancer Studies using radioimmunoassays specific to the HCG beta subunit have revealed the presence of HCG-like immunoreactivity in a number of nontrophoblastic tumors, including gynecologic cancer (Braunstein et aI., 1973b). Although the clinical significance of HCG measurement is now
OVARIAN AND UTERINE CANCER MARKERS
243
Table 1 Elevated Serum Levels of HCG in Gynecological Cancer before Treatmenta Site Vulva Vagina Cervix Endometrium Ovary All patients
Elevated/total
%
2/8 2/6 231111 171125 5/26 49/276
25 33 21 14 19 18
"From Rutanen and Seppala, 1978.
generally accepted for the management of choriocarcinoma and germ cell tumors (Bagshawe, 1979), its application to nontrophoblastic gynecologic cancer is less apparent. Rutanen and Seppala (1978) examined the circulating levels of HCG by radioimmunoassay specific to HCG beta subunit in 380 patients with nontrophoblastic gynecologic disease. HCG-like immunoradioactivity was found in 49 of 276 cancer patients (18%) (Table 1) and in 15 of 104 patients (14%) with nonmalignant conditions. In malignant disease, HCG values were higher than in nonmalignant conditions. No difference was observed between the mean age of HCG-positive and HCG-negative cancer patients, but HCG-positive patients in the nonmalignant group were older. The frequency of HCG-positives was not related to clinical stage (Tables 2 and 3) or histological differentiation (Tables 4 Table 2 Elevated Serum Levels of HCG in Cervical Cancer before Treatmenta Clinical stageb
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% 33 25 29
III
2/6 114 13/45 1121 3111 3/20
IV All patients
23/111
o
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0/4
5
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21
aFrom Rutanen and Seppala, 1978.
bFIGO classification. Figures for adenocarcinoma of the cervix were 4/23 (17%).
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Table 3 Elevated Serum Levels of HCG in Endometrial Cancer before Treatmenta Clinical stageb
Elevated/total
%
141104 3113 0/6 0/2 171125
13 23 0 0 14
I II III IV All patients
aFrom Rutanen and Seppala, 1978. bFIGO classification.
Table 4 Elevated Serum Levels of HCG in Relation to Histological Degree of Differentiation of Cervical Cancer" Degree of differentiation
Elevated/total
%
3112 7128 5/34
25 25 22 15 21
High Medium Low Not stated Total
5/34 231111
HCG concentration, mIU/mL (mean ± SEM) 16.2 16.6 14.6 13.9
± 3.3
± 3.4 ± 3.0 ± 2.8
aFrom Rutanen and Seppala, 1978.
Table 5 Elevated Serum Levels of HCG in Relation to Histological Differentiation of Endometrial Cancer" Degree of differentiation
Elevated/total
%
7/39
18 4 26 19 14
High Medium Low Not stated Total aFrom Rutanen and Seppala, 1978.
2/51 5119 3116 171125
HCG concentration, mIU/mL (mean ± SEM) 25.7 ± 10.9 9.4 11.4 ± 0.8 9.6 ± 1.3
OVARIAN AND UTERINE CANCER MARKERS
245
and 5). After radical surgery 12 of 17 HCG-positive cancer patients became HCG-negative (71 %), while 5 remained HCG-positive. Remarkably enough, 49 initially negative patients transiently became HCG-positive after radical surgery, which included oophorectomy, and 42 remained negative. It was also observed that high concentrations of the crude pituitary HFSH/HLH* reference preparation LER 907 cross-reacted in the HCG assay. These and other results (Chen et aI., 1976) suggest the possible existence of a pituitary HCG-like substance, and HCG-like immunoreactivity has recently been demonstrated in many normal tissues (Braunstein et al., 1979). Oophorectomy is included in the radical surgery for gynecologic cancer, and this may cause supraphysiological HLH levels to interfere with the HCG assay unless the cutoff level is set above the level of HLH crossreaction. The above observations raise questions regarding the value of HCG measurement in the demonstration of residual HCG-producing cells in patients with nontrophoblastic neoplasms. Eutopic and ectopic secretion of subunits of pituitary and placental glycoprotein hormones have been shown both in vivo and in vitro (Vaitukaitis, 1973; Rosen and Weintraub, 1974; Hussa, 1977), and free alpha subunits are detectable in the serum of pregnant and postmenopausal women (Franchimont et aI., 1972). HeLa cells also release HCG and free alpha subunit in culture (Lieblich et aI., 1976), but patients with cancer of the cervix do not have elevated levels of alpha subunit. Rutanen (1978) examined serum samples from 101 patients with nontrophoblastic gynecologic cancer including cervical cancer (43 cases), endometrial cancer (40 cases), ovarian cancer (11 cases), carcinoma of the vulva (3 cases), and of the vagina (4 cases), and found that the levels of alpha subunit were no different from those of controls in corresponding age groups.
1.4. Pregnancy-Specific Beta-1-Glycoprotein (SPI) 1.4.1. Trophoblastic Tumors Pregnancy specific beta-I-glycoprotein (Bohn, 1971) has been identified by immunofluorescence methods in normal human placenta and choriocarcinoma (Tatarinov et aI., 1976; Home et aI., 1976), and also in the serum of patients with malignant trophoblastic disease (Tatarinov et aI., 1974). Subsequent studies using highly sensitive radioimmunoassays have shown that SPI is detectable in the serum of virtually all patients with untreated choriocarcinoma, and the levels approximately parallel those of HCG (Fig. 7). Isolated secretion of SPI (without HCG) has been observed in some patients with choriocarcinoma (Searle et aI., 1978; Seppiilii et aI., 1978). The significance of this observation was examined by Rutanen *Human follicle-stimulating hormone/human luteinizing hormone (HFSH1-ILH).
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