©1990 S. Karger AG. isel 0378-7346/90/0301-0052$: 5/0
Gynecol Obstet Invest 1990;30:52-58
Clinical Usefulness of Serum Sialyl SSEA-1 Antigen Levels in Patients with Epithelial Ovarian Cancer
Comparative Effectiveness of Sialyl SSEA-1 and CA 125 Hiroshi Kohayashi, Yoshiro Kawashima
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan
Key Words. Sialyl SSEA-1 • CA 125 • Ovarian cancer • Endometriosis
CA 125, a glycoprotein antigen recognized by the monoclonal antibody OC 125 reported by Bast et al. [1], has by far been the most useful antigen for detecting and following up patients with ovarian cancer. High scrum concentrations were found in more than 90% of patients with epithelial nonmucinous-type ovarian cancer, and changes in the serum CA 125 level during treatment cor related well with the clinical course [2], Although the sen sitivity of CA 125 is high, the specificity is not so high, because the serum CA 125 antigen levels are elevated by benign gynecologic diseases [3] as well as in pregnancy [4], Namely, the greatest shortcoming of this antigen is its relatively low positive rate in mucinous-type ovarian can cer and its high false-positive rate in benign gynecologic diseases, endometriosis in particular. Sialyl SSEA-1 antigen [5], recognized by its mono clonal antibody FH-6. belongs to the type 2 chain carbo hydrate antigens, and its structure was clearly identified
as VI3 NeuAc V3 III3 FuC2 nLc6- SSEA-1 antigen and its derivatives have been detected in various human cancer tissues [6] and of which sialyl SSEA-1 antigen has proved to be the best circulating tumor marker, for lung adeno carcinoma in particular. In the present study, to confirm the clinical significance of the sialyl SSEA-1 antigen as a circulating tumor marker in patients with ovarian can cer, the serum sialyl SSEA-1 antigen levels were evalu ated in healthy individuals and in pregnant women as well as in patients with various benign gynecologic dis eases and ovarian cancer. The serum sialyl SSEA-1 anti gen levels were compared with the clinical course to determine the prognostic value and were evaluated to determine whether this assay may be useful in monitor ing patients after treatment. We specifically investigated whether the determination of sialyl SSEA-1 antigen in combination with CA 125 antigen could raise the detec tion rate in patients with ovarian cancer.
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Abstract. The serum levels of sialyl SSEA-1 antigen, a type 2 chain carbohydrate antigen detected using the monoclonal antibody FH-6, were elevated in 47.2% of patients with epithelial ovarian cancer, with the percent positivity increasing with the clinical stage. Of the histological type, it is interesting to note the relatively high sensitivity in patients with mucinous adenocarcinoma and clear cell carcinoma in contrast with the CA 125 antigen levels. Although the percentage of patients with ovarian cancer who had elevated sialyl SSEA-1 antigen levels is lower than that observed with elevated CA 125 antigen levels, the false-positive rate is significantly low in the sialyl SSEA-1 test. Serial sialyl SSEA-1 antigen levels obtained during follow-up were strong predictors of clinical outcome. The combined determination possible with sialyl SSEA-1 and CA 125 did not markedly increase the detection rate because of the overlap in the positivity. However, increased levels of both serum sialyl SSEA-1 antigen and CA 125 antigen indicated the presence of malignancies in pregnant women associated with ovarian tumors.
Serum Sialyl SSEA-1 Levels in Ovarian Cancer
Serum samples were obtained from 127 healthy nonpregnant women and 188 pregnant women without complications whose ges tational ages were between the 4th and the 40th week. Sera from 5 women after delivery (1-day puerperium) were also obtained. Serum samples from 90 patients with benign gynecologic diseases including 31 patients with uterine myoma, 43 with benign ovarian tumors and 16 with endometriosis were used. Diagnosis of all patients with benign gynecologic diseases were confirmed by review of operative reports and pathology reports. Two hundred and three patients with histologically proved epithelial ovarian cancers were studied. The selection criteria and patient characteristics of the 203 patients with ovarian cancer included in this study are described in table 1. Serum samples from 89 patients with ovarian cancer were obtained before treatment. Diagnoses were confirmed by a review of operative reports and pathology reports. Judgement of disease progression was based on chart review and evaluation of objective intraopera tive observation. All patients were initially treated with surgical pro cedures and combination chemotherapy including cisplatin. Clini cal staging of ovarian cancers according to the International Feder ation of Gynecology and Obstetrics (FIGO) classification showed 19 patients with stage I (including 3 pregnant women with ovarian can cer; 10th, 13th and 16th gestational week), 16 with stage II (includ ing 1 pregnant woman with ovarian cancer whose gestational age was the 11th week), 36 with stage III and 18 with stage IV before treatment. Serum sialyl SSEA-1 antigen levels were traced in 114 patients with ovarian cancer after induction therapy (surgical procedures and cisplatin-based combination chemotherapy). Serial marker as says were performed at a median interval of about I month. Tumors were absent at second-look operations in 60 individuals and de tected at second-look operations and/or clinical image diagnosis including computed tomography or ultrasonography in 54 ovarian cancer patients. All specimens wree stored at -80°C until analy sis. Circulating sialyl SSEA-1 antigen concentrations (U/ml) were detected by sandwich radioimmunoassay using an FH-6 Otsuka kit. The CA 125 antigen concentrations (U/ml) were measured by simul taneous sandwich solid-phase radioimmunoassay using a Centocor CA 125 radioimmunoassay kit (Centocor, Malvern, Pa., USA). Lev els of more than 38 U/ml and more than 35 U/ml for sialyl SSEA-1 and CA 125 antigen, respectively, were considered elevated deter minations. All samples were assayed in duplicate. The significance in the differences between groups of subjects was determined by Student’s t test.
Results We have assayed sialyl SSEA-1 antigen levels in the sera from various groups of subjects. These data are pre sented in figure 1. One group included 127 healthy wom en. A second group included patients with benign gyne cologic diseases and the third included pregnant women without complications (fig. 2). The fourth included pa tients with epithelial ovarian cancer. Serum samples
Table 1. Patient population Number of patients Patients before treatment 1 FIGO stage II III IV serous Histological findings mucinous endometrioid clear cell Patients without residual tumor at second-look operation Patients with residual tumor at second-look operation and/or found by image diagnosis Total
89 19(21.3) 16 (18.0) 36 (40.4) 18 (20.2) 41 (46.1) 32 (36.0) 9 (10.1) 7 (7.9) 60 54 203
Figures in parentheses indicate percentages. Patients before treatment had a median age of 57 years (range 36-79 years).
from 127 controls were assayed for sialyl SSEA-1 antigen levels. The mean value and its standard deviation (SD) was 21.0 ±8.5 U/ml. The levels were elevated in 5 cases, with a false-positive rate of 3.9%, including 2 cases of postmenopausal women and 2 cases examined during menstruation. This group consisted of 111 subjects with normal CA 125 values and an additional 16 selected sub jects (12.6%) with elevated CA 125 levels. When the two tests were combined to evaluate the control population, a false-positive rate of 14.2% was present. Fifteen of the 90 patients with nonmalignant gynecologic tumors (16.7%) had sialyl SSEA-1 antigen values of more than 38 U/ml. The mean sialyl SSEA-1 antigen levels and the positivity in patients with uterine myoma were 28.5 U/ml and 16.1%, respectively; those in patients with benign ovarian tumors were 30.0 U/ml and 11.6%, respectively. In one case diagnosed as a combination of serous cystadenoma and pelvic endometriosis, the level of this antigen was markedly elevated (207 U/ml). The mean value and the positivity in patients with endome triosis were 33.2 U/ml and 31.3%, respectively, and the false-positive rate was slightly higher in patients with endometriosis than in controls. Thirty-six of 90 patients with benign gynecologic disease (40.0%) had elevated levels of CA 125 antigen. High percentages (62.5%) of serum CA 125 > 35 U/ml were observed in patients with endometriosis in particular, while 31.3% of endometrio sis patients had elevated sialyl SSEA-1 antigen levels.
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Materials and Methods
53
Kobayashi/Kawashima
54 S eru m S ialyl S SE A -1 levels
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Table 2. Sialyl SSEA-1 antigen levels and histology for patients with ovarian cancer Histology
Patients, n
Elevated sialyl SSEA-1
Serous Mucinous Endometrioid Clear cell
41 32 9 7
17 (41.4) 17(53.1) 3 (33.3) 5(71.4)
Figures in parentheses indicate percentages.
Additionally, serum samples from 89 patients with epithelial ovarian cancer were assayed for sialyl SSEA-1 antigen levels and CA 125 antigen levels. Forty-two of the 89 patients (47.2%) were found to have sialyl SSEA-1 antigen levels of > 38 U/ml. Four patients with stage I disease, 7 with stage II, 18 with stage III and 13 with stage IV had elevated preoperative serum sialyl SSEA-1 levels. The serum sialyl SSEA-1 levels and positivity in patients with stage I were 28.9 U/ml and 20.1 %, respec tively; those in patients with stage II, III and IV were 45.3 U/ml and 43.8%, 182.3 U/ml and 50.0% and 63.2 U/ml and 72.2%, respectively. The positivity of this antigen gradually increased as the clinical stage of ovar
•
•
7 5 .9
Fig. 1. Distribution of serum sialyl SSEA-1 antigen levels. Control = nonpregnant healthy women; n = number of patients. Clinical staging was performed according to the systems adopted by the F1GO. During follow-up, tumors were absent at second-look operation in 60 pa tients (without residual tumor), and residual tumors were de tected at second-look operation and/or clinical image diagnosis including computed tomography or ultrasonography in 54 pa tients (with residual tumor). a p < 0.01, bp < 0.001 compared with control group. 1Serum CA 125 antigen levels of more than 35 U/ml in a patient with ovar ian cancer.
ian cancer advanced, but the mean serum sialyl SSEA-1 antigen levels did not always correlate with the clinical stage. Stage I cases showed a significantly low frequency of elevation compared to more advanced cases. The dif ference in sialyl SSEA-1 antigen levels between patients with ovarian cancer and controls was significant (p < 0. 001).
The relationship between the sialyl SSEA-1 antigen levels and histology for the patients with ovarian cancer are shown in table 2. Seventeen of 41 patients with serous adenocarcinoma, 17 of 32 patients with mucinous adenocarcinoma, 3 of 9 with endometrioid carcinoma and 5 of 7 with clear cell carcinoma of the ovary had elevated sialyl SSEA-1 antigen levels. Of particular inter est is the relatively high sensitivity in patients with muci nous adenocarcinoma and clear cell carcinoma. Serum CA 125 antigen levels were also determined in the same serum samples obtained from 89 patients with ovarian cancer. Serum CA 125 antigen levels were elevated in 74.2% of ovarian cancer patients as indicated in fig ure 1. The relationship between the elevated sialyl SSEA-1 antigen levels during follow-up and the subsequent clin ical outcome are found in figure 1. Forty-one of the 42 patients (97.6%) with an elevated value had residual dis-
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*
C o n t r o l'
50
,
Serum Sialyl SSEA-1 Levels in Ovarian Cancer
55
eases or recurrence, whereas only 13 of 72 patients (18.1 %) with normal levels had residual tumors or recur rence. The sialyl SSEA-1 levels were significantly lower in patients without residual tumors at second-look oper ation than in those with residual tumors (p < 0.001). Thus, monitoring assay levels during follow-up after induction treatment were strongly correlated with the clinical outcome. For all 114 patients, the sensitivity and specificity of this antigen for detecting residual tumors was 96.7 and 81.9%, respectively. We investigated whether the combined determination of these two markers, sialyl SSEA-1 and CA 125, could raise the rate of detection of ovarian cancer (fig. 3). Data obtained by the determination of these two markers in combination were compared with those obtained for each marker separately. The data indicate that while CA 125 antigen levels are elevated in 74.2% of patients with ovarian cancer and sialyl SSEA-1 antigen is elevated in 47.2% of the same population, the use of both assays indicated a sensitivity of detection of 75.3% in the pop ulation studied. Forty-one patients were indicated as positive by both the sialyl SSEA-1 test and CA 125 test. Only 1 patient was positive for sialyl SSEA-1 and nega tive for CA 125, whereas 23 patients were negative for sialyl SSEA-1 and positive for CA 125. Only 1 patient with stage III, clear cell carcinoma, was newly detected by the combination test. Thus, measurement of sialyl SSEA-1 antigen levels may not be useful in the popula tion of ovarian cancer patients with nonelevated levels of CA 125. Overall, the combination of CA 125 and sia-
Fig. 3. Correlation between serum concentration of sialyl SSEA1 antigen and CA 125 antigen in patients with ovarian cancer. Sam ples were obtained before treatment. | = Serum CA 125 antigen levels of more than 300 U/ml; = serum sialyl SSEA-1 antigen levels of more than 200 U/ml. A cutoff value of 38 U/ml was used in the sialyl SSEA-1 test, whereas that of 35 U/ml was used in the CA 125 test. Only 1 patient with stage III, clear cell carcinoma, was positive for the sialyl SSEA-1 and negative for the CA 125 test.
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Fig. 2. Distribution of serum sialyl SSEA-1 antigen levels dur ing pregnancy. Samples were obtained from 188 healthy preg nant women (•) whose gesta tional ages were between the 4th and 40th week and from 5 women on the first day after de livery (postpartum). ® - Preg nant women with ovarian can cer; o = pregnant women with benign ovarian tumors.
Kobayashi/Kawashima
56
Fig. 4. Distribution of serum CA 125 antigen levels during pregnancy. Samples were obtained from 188 healthy pregnant women (•) whose gestational ages were between the 4th and 40th week and from 5 women on the first day after delivery (postpartum). Pregnant women with ovarian cancer; o = pregnant women with benign ovarian tumors.
early pregnant stage from the 4th to the 18th gestational week. The antigen levels were dispersed from 5 to 926 U/ml. After the 19th gestational week, the serum CA 125 antigen levels showed < 35 U/ml. The findings of ele vated CA 125 antigen in pregnant women with ovarian tumors prompted an analysis of whether the use of this assay would be useful in evaluating patients with this malignancy. Although the CA 125 test results were posi tive for all 4 pregnant women with ovarian cancer, 11 of 14 pregnant women with benign ovarian tumors also presented a positive CA 125 test result. The sialyl SSEA1 test is considered to be more useful than the CA 125 test for distinguishing malignancies from nonmalignant ovarian tumors during pregnancy. Discussion Tumor-associated antigens identified by monoclonal antibodies have recently been used for serological detec tion of malignancies. Many of these antigens have the structure of carbohydrate antigens [7], In the present study, we have monitored the levels of circulating sialyl
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lyl SSEA-1 antigen levels was not indicated to be a more sensitive indicator than either test alone. The serum sialyl SSEA-1 antigen levels were mea sured in 188 pregnant women without complication dur ing the course of pregnancy, in 5 women after delivery, in 4 pregnant women with ovarian cancer, and in 14 pregnant women with benign ovarian tumors to deter mine whether ovarian cancer can be distinguished from benign ovarian tumors by this test. As shown in figure 2, the serum levels were elevated in 15 cases (positivity 8.0%) during the early pregnant stage, especially from the 5th to the 9th gestational week. The antigen levels were raised to within 84 U/ml. After the 10th gestational week, the serum sialyl SSEA-1 antigen levels showed < 38 U/ml and did not change with the further progress of pregnancy. The sialyl SSEA-1 test results were positive in 3 of the 4 pregnant women with ovarian cancer, whereas all 14 pregnant women with benign ovarian tumors had negative test results. As shown in figure 4, the serum CA 125 antigen levels were also simultaneously measured in the same samples in pregnant women. The serum CA 125 antigen levels were elevated in 47 cases (positivity 25.0%) during the
SSEA-1 antigen in patients with ovarian cancer. This antigen belongs to the type 2 chain carbohydrate anti gens and is defined by its monoclonal antibody FH-6 [5]. The sialyl SSEA-1 antigen has been proved to be useful in the management of lung adenocarcinoma and pan creatic cancer. Fukushi et al. [8] reported that this anti gen is detected in the serum of more than 66% of patients with lung cancer, pancreatic cancer and hepato ma. Imura et al. [9] detected positive serum sialyl SSEA1 antigen levels in 44.9% of patients with lung adenocar cinoma, 62.3% of patients with pancreatic cancer and 57.1 % of patients with ovarian cancer. We examined the clinical usefulness of the manage ment of the serum sialyl SSEA-1 antigen levels for the diagnosis of patients with epithelial ovarian cancer and compared the levels to those of CA 125 antigen. The results showed that 47.2% of patients with surgically demonstrated ovarian cancer had elevated sialyl SSEA-1 antigen levels. In contrast, only 5 of 127 healthy women (3.9%) had sialyl SSEA-1 antigen levels > 38 U/ml. Sim ilarly, only 15 of 90 patients ( 16.7%) with nonmalignant gynecologic diseases had elevated levels of this antigen. The difference between patients with ovarian cancer and controls was significant (p < 0.001). The mean value ± SD and positivity in healthy women resembled the data reported by Imura et al. [10]. The serum sialyl SSEA-1 antigen levels are reported [10] to be higher in persons over 60 years of age than in those under 60 years and slightly higher in males than in females. These findings suggest that the serum sialyl SSEA-1 antigen levels, the distribution of which was different from that of the serum CA 125 antigen levels [4], depend on sex and age. Among patients with benign gynecologic diseases, el evated levels of serum sialyl SSEA-1 antigen as well as of CA 125 were observed in patients with endometriosis. An extremely high concentration of sialyl SSEA-1 anti gen as well as CA 125 [11, 12] was found in both the fluid content of chocolate cysts and amniotic fluid (data not shown). Fukushi et al. [8] reported that FH-6-positive loci in normal adult tissues were limited to the prox imal convoluted tubuli in the kidneys and granulocytes. More recently, Inoue et al. [13] reported a weak positive reaction in human eutopic endometrium by immunostaining. The elevated levels of serum sialyl SSEA-1 antigen in patients with endometriosis and in the fluid content of chocolate cysts may reflect the nature of the immunohistochemical analysis, that is, positive staining at the endometrium. Another source of this antigen in addition to the endometrium is also indicated, because
57
the levels of this antigen are often elevated in postmeno pausal women. Preoperative serum samples from patients with ovar ian cancer revealed positivity in 20.1% of those with stage I disease, 43.8% of those with stage II and 50.0 and 72.2% of those with stage III and IV. respectively. Inoue et al. [13] reported a positivity in 69% of patients with ovarian cancer which is higher than the rate we obtained. Our results are similar to those reported by Imura et al. [9], The correlation between the positivity of the serum sialyl SSEA-1 test and clinical stage seems to be entirely based on the volume of the tumor mass. Of the histolog ical type, it is interesting to note the relatively high sen sitivity in patients with mucinous adenocarcinoma and clear cell carcinoma in contrast with the CA 125 antigen levels. The low positive predictive value makes the assay unsuitable as a screening test for the detection of ovarian cancer in a general population. The values of the sialyl SSEA-1 antigen assay as an aid in diagnosing malignant disease would be greater, if elevated levels persisted in patients with ovarian tumors. Serial measurement of cir culating sialyl SSEA-1 antigen was monitored in patients with ovarian cancer who were followed over a period of time. Elevated antigen levels during follow-up were asso ciated with the disease progression. A significant in crease in this antigen level was observed in 41 of 54 (75.9%) patients with recurrence or residual tumor at second-look operation, whereas the antigen level nor malized in 59 of 60 (98.3%) patients without residual tumor at second-look operation. Consequently, this anti gen is confirmed to be useful in monitoring the effect of therapy for determining the prognosis. However, the sia lyl SSEA-1 test may not be of use for the detection of ovarian cancer in the early stage. The combined assay of multiple tumor markers may provide both higher sensitivity and lower specificity than a single test. Therefore, we must consider the incre ment of the false-positive rate in nonmalignant disease when evaluating the combination test. The findings of elevated sialyl SSEA-1 and CA 125 levels in patients with ovarian cancer prompted an analysis whether the use of both assays would be complementary in evaluat ing patients with this malignancy. The combined deter mination of sialyl SSEA-1 and CA 125 revealed only one additional patient with ovarian cancer compared with the determination of CA 125 alone. The results suggest that this combination test was not useful for increasing the sensitivity because of the overlap in the positivity. The determination of a predictive value for this combi
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Serum Sialyl SSEA-1 Levels in Ovarian Cancer
Kobayashi/Kawashima
nation test will require the investigation of a more exten sive population of patients. The serum sialyl SSEA-1 antigen levels as well as the CA 125 levels were slighlty elevated in the first trimester of pregnancy [4], However, the duration and the distri bution of the elevated serum sialyl SSEA-1 antigen levels were shorter and more restricted than those of the serum CA 125 antigen levels. This difference would probably be more appropriate to evaluate pregnant women associ ated with ovarian cancer. The serum sialyl SSEA-1 anti gen levels are considered useful for distinguishing malig nancies from nonmalignant ovarian tumors. Conse quently, one of the characteristics of the sialyl SSEA-1 test in contrast with the CA 125 test is the possibility of detecting ovarian cancer in pregnant women. Although the percentage of patients with ovarian can cer who had elevated sialyl SSEA-1 antigen levels is lower than that observed with elevated CA 125 antigen levels, this sialyl SSEA-1 test has a higher specificity, because this test has a significantly low false positivity for nonneoplastic diseases except for endometriosis, in which cases the CA 125 levels are often elevated. How ever, the use of both assays, sialyl SSEA-1 and CA 125, may not be beneficial in increasing sensitivity in ovarian cancer patients. In the future, combinations with other tumor markers such as type 1 chain carbohydrate antigens will be stud ied to determine the possibility of detecting early stages of occult ovarian cancer. Acknowledgements The authors would like to thank Dr. Sadahito Shin for the mea surement of sialyl SSEA-1 antigen and are grateful to Dr. Toshihiko Terao for his valuable advice.
References 1 Bast RC, Feeney M, Lazarus H, et al: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981 ;68: 1331-1337. 2 Schilthus MS, Aalders JG, Bouma J, et al: Serum CA 125 levels in epithelial ovarian cancer: Relationship with findings at sec ond-look operations and their role in the detection of tumor recurrence. Br J Obstet Gynaecol 1987;94:202-207.
3 Takahashi K, Kijima S, Yoshino K, et al: Differential diagnosis between uterine myoma and endometriosis using CA 125 as a new tumor marker of ovarian carcinoma. Asia-Oceania J Obstei Gynaecol 1986;11:99-103. 4 Kuzuya K, Nozaki M, Chihara T, et al: Evaluation of CA 125 as a circulating tumor marker for ovarian cancer. Acta Obstei Gynaecol Jpn 1986:38:949-957. 5 Fukushi Y, Nudelman E, Levery SB, el al: Novel fucolipids accu mulating in human adenocarcinoma. III. A hybridoma antibody (FH-6) defining a human cancer-associated difucoganglioside (VPNeuAc VMlPFucj nLc6). J Biol Chem 1984;259:1051110517. 6 Hakomori S, Nudelman E, Levery SB, et al: Novel fucolipids accumulating in human adenocarcinoma. I. Glycolipids with dior trifucosylated type 2 chain. J Biol Chem 1984;259:4672— 4680. 7 Kannagi R: Biochemistry and clinical evaluation of cancer-asso ciated carbohydrate antigen: An approach to the diagnosis using monoclonal antibody cocktails. Rinsyo Byori 1986; 11:1247— 1264. 8 Fukushi Y. Kannagi R. Hakomori S. et al: Location and distri bution of difucoganglioside (VPNeuAc VMlI 'Fuc: nLc6) in nor mal and tumor tissues defined by its monoclonal antibody FH-6. Cancer Res 1985;45:3711-3717. 9 Imura H, Endo J, Ohkura H, et al: Initial basic and clinical eval uation of a solid phase immunoradiometric assay for sialyl SSEA-1 antigen. 2. Evaluation of clinical significance. Jpn J Cancer Chemother 1987; 14:1322-1331. 10 Imura H, Endo J, Ohkura H. et al: Initial basic and clinical eval uation of a solid phase immunoradiometric assay for sialyl SSEA-1 antigen. 1. Evaluation of assay conditions and normal values. Jpn J Cancer Chemother 1987;14:1315-1321. II Kobayashi H, Miyake W, Yamashita M, et al: The mechanism of the elevation of serum CA 125 levels in patients with endome triosis. Acta Obstet Gynaecol Jpn 1988:40:467-472. 12 O’Brien TJ, Hardin JW, Bannon GA, et al: CA 125 antigen in human amniotic fluid and fetal membranes. Am J Obstet Gyne col 1986;155:50-55. 13 Inoue M, Shimizu C, Sasagawa T, et al: The clinical value of sialyl SSEA-1 antigen in patients with gynecologic tumors. Acta Obstet Gynaecol Jpn 1987;39:2120-2124.
Received: May 22, 1989 Accepted after revision: December 5, 1989 Hiroshi Kobayashi, MD Department of Obstetrics and Gynecology Hamamatsu University School of Medicine Handa-cho 3600, Hamamatsu Shizuoka 431-31 (Japan)
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58
Gynecol Obstet Invest 1990;30:52-58
Clinical Usefulness of Serum Sialyl SSEA-1 Antigen Levels in Patients with Epithelial Ovarian Cancer
Comparative Effectiveness of Sialyl SSEA-1 and CA 125 Hiroshi Kohayashi, Yoshiro Kawashima
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan
Key Words. Sialyl SSEA-1 • CA 125 • Ovarian cancer • Endometriosis
CA 125, a glycoprotein antigen recognized by the monoclonal antibody OC 125 reported by Bast et al. [1], has by far been the most useful antigen for detecting and following up patients with ovarian cancer. High scrum concentrations were found in more than 90% of patients with epithelial nonmucinous-type ovarian cancer, and changes in the serum CA 125 level during treatment cor related well with the clinical course [2], Although the sen sitivity of CA 125 is high, the specificity is not so high, because the serum CA 125 antigen levels are elevated by benign gynecologic diseases [3] as well as in pregnancy [4], Namely, the greatest shortcoming of this antigen is its relatively low positive rate in mucinous-type ovarian can cer and its high false-positive rate in benign gynecologic diseases, endometriosis in particular. Sialyl SSEA-1 antigen [5], recognized by its mono clonal antibody FH-6. belongs to the type 2 chain carbo hydrate antigens, and its structure was clearly identified
as VI3 NeuAc V3 III3 FuC2 nLc6- SSEA-1 antigen and its derivatives have been detected in various human cancer tissues [6] and of which sialyl SSEA-1 antigen has proved to be the best circulating tumor marker, for lung adeno carcinoma in particular. In the present study, to confirm the clinical significance of the sialyl SSEA-1 antigen as a circulating tumor marker in patients with ovarian can cer, the serum sialyl SSEA-1 antigen levels were evalu ated in healthy individuals and in pregnant women as well as in patients with various benign gynecologic dis eases and ovarian cancer. The serum sialyl SSEA-1 anti gen levels were compared with the clinical course to determine the prognostic value and were evaluated to determine whether this assay may be useful in monitor ing patients after treatment. We specifically investigated whether the determination of sialyl SSEA-1 antigen in combination with CA 125 antigen could raise the detec tion rate in patients with ovarian cancer.
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Abstract. The serum levels of sialyl SSEA-1 antigen, a type 2 chain carbohydrate antigen detected using the monoclonal antibody FH-6, were elevated in 47.2% of patients with epithelial ovarian cancer, with the percent positivity increasing with the clinical stage. Of the histological type, it is interesting to note the relatively high sensitivity in patients with mucinous adenocarcinoma and clear cell carcinoma in contrast with the CA 125 antigen levels. Although the percentage of patients with ovarian cancer who had elevated sialyl SSEA-1 antigen levels is lower than that observed with elevated CA 125 antigen levels, the false-positive rate is significantly low in the sialyl SSEA-1 test. Serial sialyl SSEA-1 antigen levels obtained during follow-up were strong predictors of clinical outcome. The combined determination possible with sialyl SSEA-1 and CA 125 did not markedly increase the detection rate because of the overlap in the positivity. However, increased levels of both serum sialyl SSEA-1 antigen and CA 125 antigen indicated the presence of malignancies in pregnant women associated with ovarian tumors.
Serum Sialyl SSEA-1 Levels in Ovarian Cancer
Serum samples were obtained from 127 healthy nonpregnant women and 188 pregnant women without complications whose ges tational ages were between the 4th and the 40th week. Sera from 5 women after delivery (1-day puerperium) were also obtained. Serum samples from 90 patients with benign gynecologic diseases including 31 patients with uterine myoma, 43 with benign ovarian tumors and 16 with endometriosis were used. Diagnosis of all patients with benign gynecologic diseases were confirmed by review of operative reports and pathology reports. Two hundred and three patients with histologically proved epithelial ovarian cancers were studied. The selection criteria and patient characteristics of the 203 patients with ovarian cancer included in this study are described in table 1. Serum samples from 89 patients with ovarian cancer were obtained before treatment. Diagnoses were confirmed by a review of operative reports and pathology reports. Judgement of disease progression was based on chart review and evaluation of objective intraopera tive observation. All patients were initially treated with surgical pro cedures and combination chemotherapy including cisplatin. Clini cal staging of ovarian cancers according to the International Feder ation of Gynecology and Obstetrics (FIGO) classification showed 19 patients with stage I (including 3 pregnant women with ovarian can cer; 10th, 13th and 16th gestational week), 16 with stage II (includ ing 1 pregnant woman with ovarian cancer whose gestational age was the 11th week), 36 with stage III and 18 with stage IV before treatment. Serum sialyl SSEA-1 antigen levels were traced in 114 patients with ovarian cancer after induction therapy (surgical procedures and cisplatin-based combination chemotherapy). Serial marker as says were performed at a median interval of about I month. Tumors were absent at second-look operations in 60 individuals and de tected at second-look operations and/or clinical image diagnosis including computed tomography or ultrasonography in 54 ovarian cancer patients. All specimens wree stored at -80°C until analy sis. Circulating sialyl SSEA-1 antigen concentrations (U/ml) were detected by sandwich radioimmunoassay using an FH-6 Otsuka kit. The CA 125 antigen concentrations (U/ml) were measured by simul taneous sandwich solid-phase radioimmunoassay using a Centocor CA 125 radioimmunoassay kit (Centocor, Malvern, Pa., USA). Lev els of more than 38 U/ml and more than 35 U/ml for sialyl SSEA-1 and CA 125 antigen, respectively, were considered elevated deter minations. All samples were assayed in duplicate. The significance in the differences between groups of subjects was determined by Student’s t test.
Results We have assayed sialyl SSEA-1 antigen levels in the sera from various groups of subjects. These data are pre sented in figure 1. One group included 127 healthy wom en. A second group included patients with benign gyne cologic diseases and the third included pregnant women without complications (fig. 2). The fourth included pa tients with epithelial ovarian cancer. Serum samples
Table 1. Patient population Number of patients Patients before treatment 1 FIGO stage II III IV serous Histological findings mucinous endometrioid clear cell Patients without residual tumor at second-look operation Patients with residual tumor at second-look operation and/or found by image diagnosis Total
89 19(21.3) 16 (18.0) 36 (40.4) 18 (20.2) 41 (46.1) 32 (36.0) 9 (10.1) 7 (7.9) 60 54 203
Figures in parentheses indicate percentages. Patients before treatment had a median age of 57 years (range 36-79 years).
from 127 controls were assayed for sialyl SSEA-1 antigen levels. The mean value and its standard deviation (SD) was 21.0 ±8.5 U/ml. The levels were elevated in 5 cases, with a false-positive rate of 3.9%, including 2 cases of postmenopausal women and 2 cases examined during menstruation. This group consisted of 111 subjects with normal CA 125 values and an additional 16 selected sub jects (12.6%) with elevated CA 125 levels. When the two tests were combined to evaluate the control population, a false-positive rate of 14.2% was present. Fifteen of the 90 patients with nonmalignant gynecologic tumors (16.7%) had sialyl SSEA-1 antigen values of more than 38 U/ml. The mean sialyl SSEA-1 antigen levels and the positivity in patients with uterine myoma were 28.5 U/ml and 16.1%, respectively; those in patients with benign ovarian tumors were 30.0 U/ml and 11.6%, respectively. In one case diagnosed as a combination of serous cystadenoma and pelvic endometriosis, the level of this antigen was markedly elevated (207 U/ml). The mean value and the positivity in patients with endome triosis were 33.2 U/ml and 31.3%, respectively, and the false-positive rate was slightly higher in patients with endometriosis than in controls. Thirty-six of 90 patients with benign gynecologic disease (40.0%) had elevated levels of CA 125 antigen. High percentages (62.5%) of serum CA 125 > 35 U/ml were observed in patients with endometriosis in particular, while 31.3% of endometrio sis patients had elevated sialyl SSEA-1 antigen levels.
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Materials and Methods
53
Kobayashi/Kawashima
54 S eru m S ialyl S SE A -1 levels
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150
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100
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12 7
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43
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2 0 .1
16
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5 0 .0
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without residual tumor
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Table 2. Sialyl SSEA-1 antigen levels and histology for patients with ovarian cancer Histology
Patients, n
Elevated sialyl SSEA-1
Serous Mucinous Endometrioid Clear cell
41 32 9 7
17 (41.4) 17(53.1) 3 (33.3) 5(71.4)
Figures in parentheses indicate percentages.
Additionally, serum samples from 89 patients with epithelial ovarian cancer were assayed for sialyl SSEA-1 antigen levels and CA 125 antigen levels. Forty-two of the 89 patients (47.2%) were found to have sialyl SSEA-1 antigen levels of > 38 U/ml. Four patients with stage I disease, 7 with stage II, 18 with stage III and 13 with stage IV had elevated preoperative serum sialyl SSEA-1 levels. The serum sialyl SSEA-1 levels and positivity in patients with stage I were 28.9 U/ml and 20.1 %, respec tively; those in patients with stage II, III and IV were 45.3 U/ml and 43.8%, 182.3 U/ml and 50.0% and 63.2 U/ml and 72.2%, respectively. The positivity of this antigen gradually increased as the clinical stage of ovar
•
•
7 5 .9
Fig. 1. Distribution of serum sialyl SSEA-1 antigen levels. Control = nonpregnant healthy women; n = number of patients. Clinical staging was performed according to the systems adopted by the F1GO. During follow-up, tumors were absent at second-look operation in 60 pa tients (without residual tumor), and residual tumors were de tected at second-look operation and/or clinical image diagnosis including computed tomography or ultrasonography in 54 pa tients (with residual tumor). a p < 0.01, bp < 0.001 compared with control group. 1Serum CA 125 antigen levels of more than 35 U/ml in a patient with ovar ian cancer.
ian cancer advanced, but the mean serum sialyl SSEA-1 antigen levels did not always correlate with the clinical stage. Stage I cases showed a significantly low frequency of elevation compared to more advanced cases. The dif ference in sialyl SSEA-1 antigen levels between patients with ovarian cancer and controls was significant (p < 0. 001).
The relationship between the sialyl SSEA-1 antigen levels and histology for the patients with ovarian cancer are shown in table 2. Seventeen of 41 patients with serous adenocarcinoma, 17 of 32 patients with mucinous adenocarcinoma, 3 of 9 with endometrioid carcinoma and 5 of 7 with clear cell carcinoma of the ovary had elevated sialyl SSEA-1 antigen levels. Of particular inter est is the relatively high sensitivity in patients with muci nous adenocarcinoma and clear cell carcinoma. Serum CA 125 antigen levels were also determined in the same serum samples obtained from 89 patients with ovarian cancer. Serum CA 125 antigen levels were elevated in 74.2% of ovarian cancer patients as indicated in fig ure 1. The relationship between the elevated sialyl SSEA-1 antigen levels during follow-up and the subsequent clin ical outcome are found in figure 1. Forty-one of the 42 patients (97.6%) with an elevated value had residual dis-
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*
C o n t r o l'
50
,
Serum Sialyl SSEA-1 Levels in Ovarian Cancer
55
eases or recurrence, whereas only 13 of 72 patients (18.1 %) with normal levels had residual tumors or recur rence. The sialyl SSEA-1 levels were significantly lower in patients without residual tumors at second-look oper ation than in those with residual tumors (p < 0.001). Thus, monitoring assay levels during follow-up after induction treatment were strongly correlated with the clinical outcome. For all 114 patients, the sensitivity and specificity of this antigen for detecting residual tumors was 96.7 and 81.9%, respectively. We investigated whether the combined determination of these two markers, sialyl SSEA-1 and CA 125, could raise the rate of detection of ovarian cancer (fig. 3). Data obtained by the determination of these two markers in combination were compared with those obtained for each marker separately. The data indicate that while CA 125 antigen levels are elevated in 74.2% of patients with ovarian cancer and sialyl SSEA-1 antigen is elevated in 47.2% of the same population, the use of both assays indicated a sensitivity of detection of 75.3% in the pop ulation studied. Forty-one patients were indicated as positive by both the sialyl SSEA-1 test and CA 125 test. Only 1 patient was positive for sialyl SSEA-1 and nega tive for CA 125, whereas 23 patients were negative for sialyl SSEA-1 and positive for CA 125. Only 1 patient with stage III, clear cell carcinoma, was newly detected by the combination test. Thus, measurement of sialyl SSEA-1 antigen levels may not be useful in the popula tion of ovarian cancer patients with nonelevated levels of CA 125. Overall, the combination of CA 125 and sia-
Fig. 3. Correlation between serum concentration of sialyl SSEA1 antigen and CA 125 antigen in patients with ovarian cancer. Sam ples were obtained before treatment. | = Serum CA 125 antigen levels of more than 300 U/ml; = serum sialyl SSEA-1 antigen levels of more than 200 U/ml. A cutoff value of 38 U/ml was used in the sialyl SSEA-1 test, whereas that of 35 U/ml was used in the CA 125 test. Only 1 patient with stage III, clear cell carcinoma, was positive for the sialyl SSEA-1 and negative for the CA 125 test.
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Fig. 2. Distribution of serum sialyl SSEA-1 antigen levels dur ing pregnancy. Samples were obtained from 188 healthy preg nant women (•) whose gesta tional ages were between the 4th and 40th week and from 5 women on the first day after de livery (postpartum). ® - Preg nant women with ovarian can cer; o = pregnant women with benign ovarian tumors.
Kobayashi/Kawashima
56
Fig. 4. Distribution of serum CA 125 antigen levels during pregnancy. Samples were obtained from 188 healthy pregnant women (•) whose gestational ages were between the 4th and 40th week and from 5 women on the first day after delivery (postpartum). Pregnant women with ovarian cancer; o = pregnant women with benign ovarian tumors.
early pregnant stage from the 4th to the 18th gestational week. The antigen levels were dispersed from 5 to 926 U/ml. After the 19th gestational week, the serum CA 125 antigen levels showed < 35 U/ml. The findings of ele vated CA 125 antigen in pregnant women with ovarian tumors prompted an analysis of whether the use of this assay would be useful in evaluating patients with this malignancy. Although the CA 125 test results were posi tive for all 4 pregnant women with ovarian cancer, 11 of 14 pregnant women with benign ovarian tumors also presented a positive CA 125 test result. The sialyl SSEA1 test is considered to be more useful than the CA 125 test for distinguishing malignancies from nonmalignant ovarian tumors during pregnancy. Discussion Tumor-associated antigens identified by monoclonal antibodies have recently been used for serological detec tion of malignancies. Many of these antigens have the structure of carbohydrate antigens [7], In the present study, we have monitored the levels of circulating sialyl
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lyl SSEA-1 antigen levels was not indicated to be a more sensitive indicator than either test alone. The serum sialyl SSEA-1 antigen levels were mea sured in 188 pregnant women without complication dur ing the course of pregnancy, in 5 women after delivery, in 4 pregnant women with ovarian cancer, and in 14 pregnant women with benign ovarian tumors to deter mine whether ovarian cancer can be distinguished from benign ovarian tumors by this test. As shown in figure 2, the serum levels were elevated in 15 cases (positivity 8.0%) during the early pregnant stage, especially from the 5th to the 9th gestational week. The antigen levels were raised to within 84 U/ml. After the 10th gestational week, the serum sialyl SSEA-1 antigen levels showed < 38 U/ml and did not change with the further progress of pregnancy. The sialyl SSEA-1 test results were positive in 3 of the 4 pregnant women with ovarian cancer, whereas all 14 pregnant women with benign ovarian tumors had negative test results. As shown in figure 4, the serum CA 125 antigen levels were also simultaneously measured in the same samples in pregnant women. The serum CA 125 antigen levels were elevated in 47 cases (positivity 25.0%) during the
SSEA-1 antigen in patients with ovarian cancer. This antigen belongs to the type 2 chain carbohydrate anti gens and is defined by its monoclonal antibody FH-6 [5]. The sialyl SSEA-1 antigen has been proved to be useful in the management of lung adenocarcinoma and pan creatic cancer. Fukushi et al. [8] reported that this anti gen is detected in the serum of more than 66% of patients with lung cancer, pancreatic cancer and hepato ma. Imura et al. [9] detected positive serum sialyl SSEA1 antigen levels in 44.9% of patients with lung adenocar cinoma, 62.3% of patients with pancreatic cancer and 57.1 % of patients with ovarian cancer. We examined the clinical usefulness of the manage ment of the serum sialyl SSEA-1 antigen levels for the diagnosis of patients with epithelial ovarian cancer and compared the levels to those of CA 125 antigen. The results showed that 47.2% of patients with surgically demonstrated ovarian cancer had elevated sialyl SSEA-1 antigen levels. In contrast, only 5 of 127 healthy women (3.9%) had sialyl SSEA-1 antigen levels > 38 U/ml. Sim ilarly, only 15 of 90 patients ( 16.7%) with nonmalignant gynecologic diseases had elevated levels of this antigen. The difference between patients with ovarian cancer and controls was significant (p < 0.001). The mean value ± SD and positivity in healthy women resembled the data reported by Imura et al. [10]. The serum sialyl SSEA-1 antigen levels are reported [10] to be higher in persons over 60 years of age than in those under 60 years and slightly higher in males than in females. These findings suggest that the serum sialyl SSEA-1 antigen levels, the distribution of which was different from that of the serum CA 125 antigen levels [4], depend on sex and age. Among patients with benign gynecologic diseases, el evated levels of serum sialyl SSEA-1 antigen as well as of CA 125 were observed in patients with endometriosis. An extremely high concentration of sialyl SSEA-1 anti gen as well as CA 125 [11, 12] was found in both the fluid content of chocolate cysts and amniotic fluid (data not shown). Fukushi et al. [8] reported that FH-6-positive loci in normal adult tissues were limited to the prox imal convoluted tubuli in the kidneys and granulocytes. More recently, Inoue et al. [13] reported a weak positive reaction in human eutopic endometrium by immunostaining. The elevated levels of serum sialyl SSEA-1 antigen in patients with endometriosis and in the fluid content of chocolate cysts may reflect the nature of the immunohistochemical analysis, that is, positive staining at the endometrium. Another source of this antigen in addition to the endometrium is also indicated, because
57
the levels of this antigen are often elevated in postmeno pausal women. Preoperative serum samples from patients with ovar ian cancer revealed positivity in 20.1% of those with stage I disease, 43.8% of those with stage II and 50.0 and 72.2% of those with stage III and IV. respectively. Inoue et al. [13] reported a positivity in 69% of patients with ovarian cancer which is higher than the rate we obtained. Our results are similar to those reported by Imura et al. [9], The correlation between the positivity of the serum sialyl SSEA-1 test and clinical stage seems to be entirely based on the volume of the tumor mass. Of the histolog ical type, it is interesting to note the relatively high sen sitivity in patients with mucinous adenocarcinoma and clear cell carcinoma in contrast with the CA 125 antigen levels. The low positive predictive value makes the assay unsuitable as a screening test for the detection of ovarian cancer in a general population. The values of the sialyl SSEA-1 antigen assay as an aid in diagnosing malignant disease would be greater, if elevated levels persisted in patients with ovarian tumors. Serial measurement of cir culating sialyl SSEA-1 antigen was monitored in patients with ovarian cancer who were followed over a period of time. Elevated antigen levels during follow-up were asso ciated with the disease progression. A significant in crease in this antigen level was observed in 41 of 54 (75.9%) patients with recurrence or residual tumor at second-look operation, whereas the antigen level nor malized in 59 of 60 (98.3%) patients without residual tumor at second-look operation. Consequently, this anti gen is confirmed to be useful in monitoring the effect of therapy for determining the prognosis. However, the sia lyl SSEA-1 test may not be of use for the detection of ovarian cancer in the early stage. The combined assay of multiple tumor markers may provide both higher sensitivity and lower specificity than a single test. Therefore, we must consider the incre ment of the false-positive rate in nonmalignant disease when evaluating the combination test. The findings of elevated sialyl SSEA-1 and CA 125 levels in patients with ovarian cancer prompted an analysis whether the use of both assays would be complementary in evaluat ing patients with this malignancy. The combined deter mination of sialyl SSEA-1 and CA 125 revealed only one additional patient with ovarian cancer compared with the determination of CA 125 alone. The results suggest that this combination test was not useful for increasing the sensitivity because of the overlap in the positivity. The determination of a predictive value for this combi
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Serum Sialyl SSEA-1 Levels in Ovarian Cancer
Kobayashi/Kawashima
nation test will require the investigation of a more exten sive population of patients. The serum sialyl SSEA-1 antigen levels as well as the CA 125 levels were slighlty elevated in the first trimester of pregnancy [4], However, the duration and the distri bution of the elevated serum sialyl SSEA-1 antigen levels were shorter and more restricted than those of the serum CA 125 antigen levels. This difference would probably be more appropriate to evaluate pregnant women associ ated with ovarian cancer. The serum sialyl SSEA-1 anti gen levels are considered useful for distinguishing malig nancies from nonmalignant ovarian tumors. Conse quently, one of the characteristics of the sialyl SSEA-1 test in contrast with the CA 125 test is the possibility of detecting ovarian cancer in pregnant women. Although the percentage of patients with ovarian can cer who had elevated sialyl SSEA-1 antigen levels is lower than that observed with elevated CA 125 antigen levels, this sialyl SSEA-1 test has a higher specificity, because this test has a significantly low false positivity for nonneoplastic diseases except for endometriosis, in which cases the CA 125 levels are often elevated. How ever, the use of both assays, sialyl SSEA-1 and CA 125, may not be beneficial in increasing sensitivity in ovarian cancer patients. In the future, combinations with other tumor markers such as type 1 chain carbohydrate antigens will be stud ied to determine the possibility of detecting early stages of occult ovarian cancer. Acknowledgements The authors would like to thank Dr. Sadahito Shin for the mea surement of sialyl SSEA-1 antigen and are grateful to Dr. Toshihiko Terao for his valuable advice.
References 1 Bast RC, Feeney M, Lazarus H, et al: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 1981 ;68: 1331-1337. 2 Schilthus MS, Aalders JG, Bouma J, et al: Serum CA 125 levels in epithelial ovarian cancer: Relationship with findings at sec ond-look operations and their role in the detection of tumor recurrence. Br J Obstet Gynaecol 1987;94:202-207.
3 Takahashi K, Kijima S, Yoshino K, et al: Differential diagnosis between uterine myoma and endometriosis using CA 125 as a new tumor marker of ovarian carcinoma. Asia-Oceania J Obstei Gynaecol 1986;11:99-103. 4 Kuzuya K, Nozaki M, Chihara T, et al: Evaluation of CA 125 as a circulating tumor marker for ovarian cancer. Acta Obstei Gynaecol Jpn 1986:38:949-957. 5 Fukushi Y, Nudelman E, Levery SB, el al: Novel fucolipids accu mulating in human adenocarcinoma. III. A hybridoma antibody (FH-6) defining a human cancer-associated difucoganglioside (VPNeuAc VMlPFucj nLc6). J Biol Chem 1984;259:1051110517. 6 Hakomori S, Nudelman E, Levery SB, et al: Novel fucolipids accumulating in human adenocarcinoma. I. Glycolipids with dior trifucosylated type 2 chain. J Biol Chem 1984;259:4672— 4680. 7 Kannagi R: Biochemistry and clinical evaluation of cancer-asso ciated carbohydrate antigen: An approach to the diagnosis using monoclonal antibody cocktails. Rinsyo Byori 1986; 11:1247— 1264. 8 Fukushi Y. Kannagi R. Hakomori S. et al: Location and distri bution of difucoganglioside (VPNeuAc VMlI 'Fuc: nLc6) in nor mal and tumor tissues defined by its monoclonal antibody FH-6. Cancer Res 1985;45:3711-3717. 9 Imura H, Endo J, Ohkura H, et al: Initial basic and clinical eval uation of a solid phase immunoradiometric assay for sialyl SSEA-1 antigen. 2. Evaluation of clinical significance. Jpn J Cancer Chemother 1987; 14:1322-1331. 10 Imura H, Endo J, Ohkura H. et al: Initial basic and clinical eval uation of a solid phase immunoradiometric assay for sialyl SSEA-1 antigen. 1. Evaluation of assay conditions and normal values. Jpn J Cancer Chemother 1987;14:1315-1321. II Kobayashi H, Miyake W, Yamashita M, et al: The mechanism of the elevation of serum CA 125 levels in patients with endome triosis. Acta Obstet Gynaecol Jpn 1988:40:467-472. 12 O’Brien TJ, Hardin JW, Bannon GA, et al: CA 125 antigen in human amniotic fluid and fetal membranes. Am J Obstet Gyne col 1986;155:50-55. 13 Inoue M, Shimizu C, Sasagawa T, et al: The clinical value of sialyl SSEA-1 antigen in patients with gynecologic tumors. Acta Obstet Gynaecol Jpn 1987;39:2120-2124.
Received: May 22, 1989 Accepted after revision: December 5, 1989 Hiroshi Kobayashi, MD Department of Obstetrics and Gynecology Hamamatsu University School of Medicine Handa-cho 3600, Hamamatsu Shizuoka 431-31 (Japan)
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