Clinical Evaluation of Circulating Serum Sialyl Tn Antigen Levels in Patients With Epithelial Ovarian Cancer By Hiroshi Kobayashi, Toshihiko Teroo, and Yoshiro Kawashima Sialyl Tn antigen (NeuAc a 2 - 6GalNac a 1 - 0-Ser/ Thr [STN]) with antigenic specificity in the core structure of mucin-type carbohydrate chains has been determined. In the present study, we evaluated the clinical significance of this new carbohydrate antigen, STN, in patients with epithelial ovarian cancer. With the use of a radioimmunoassay developed to detect STN antigen in serum, elevated (> 32.6 U/mL) antigen levels were observed in 50.0% of patients with ovarian cancer. In contrast, 3.8% of healthy individuals had STN antigen levels > 32.6 U/mL. In 9.6% of patients with benign gynecologic diseases and 0% of pregnant women, there were elevated levels of STN antigen. There was a significant difference (P < .001) in STN antigen levels between patients with ovarian cancer and patients with benign gynecologic diseases, pregnant women, or the controls. The mean - SD for all evaluated samples of ovarian cancer was 109.2 -
CARBOHYDRATE
antigens available as cir-
culating tumor markers for clinical evalua-
tion of malignancy include CA 19-9 and CA 50, which belong to type 1 chain carbohydrate antigens, and sialyl Lex -i (SLX), which belongs to type 2 chain carbohydrate antigens.' Recently, new monoclonal antibodies recognizing a core structure of mucin-type carbohydrate chain have 2 been made by Kjeldsen et al. These monoclonal antibodies (TKH1 and TKH2) directed to the tumor-associated 0-linked sialyl 2 -- 6-a-Nacetylgalactosaminyl (sialyl Tn [STN]) epitope were generated by immunization with ovine submaxillary mucin. The STN antigen belongs to neither type 1 nor to type 2 carbohydrate antigens. We have measured the antigen recognized by TKH2. In the present study, we examined its clinical usefulness in the management of the serum STN antigen levels for the diagnosis of patients with epithelial ovarian cancer. Furthermore, we have studied whether the use of the combination assay of STN and CA 125 would be complementary in evaluating patients with this malignancy. MATERIALS AND METHODS
Subjects Serum samples that had been randomly drawn from 130 patients with ovarian cancer and from 386 women who were
146.8 U/mL. Both the mean values and the positive rate increased as the stage advanced. Classified according to the histologic type, the highest positive rate (61.0%) was observed in mucinous adenocarcinoma. The usefulness of STN antigen as a circulating tumor marker in ovarian cancer was estimated as follows: sensitivity 50.0%, specificity 93.5%, positive predictive value 72.2%, negative predictive value 84.7%, and diagnostic value 46.8%. Serum STN antigen levels were elevated in 12 of 33 patients with ovarian cancer who had serum CA 125 antigen levels less than 35 U/mL. While CA 125 antigen levels were elevated in 74.6% and STN antigen levels were elevated in 50.0% of the same population, the use of both assays indicated the sensitivity of detection of 83.8% in the population studied. J Clin Oncol 9:983-987. o 1991 by American Society of Clinical Oncology. healthy, pregnant, or possessed benign gynecologic diseases were obtained from a bank of sera preserved at the Department of Gynecology in Hamamatsu University School of Medicine Shizuoka, Japan. Blood collection was performed within protocols approved by the members of the Gynecological Cancer Committee of this institute. The control subjects included 182 healthy nonpregnant women, 74 patients with uterine myoma, 79 patients with endometriosis (28 with adenomyosis, 21 with pelvic endometriosis, and 30 with a combination of adenomyosis and pelvic endometriosis), 35 patients with benign ovarian tumors (18 with serous cystadenoma, 10 with mucinous cystadenoma, and seven with dermoid cyst), and 16 pregnant women without complications (in the ninth to 38th week of gestation). Diagnosis was confirmed by a review of the operative and pathology reports. Staging of ovarian cancer according to the International Federation of Gynecology and Obstetrics (FIGO) classification showed 34 patients with stage I disease, 27 with stage II, 55 with stage III, and 14 with stage IV. Classified according to histologic type, 66 patients had serous adenocarcinoma, 41 had mucinous adenocarcinoma, 10 had clear-cell carcinoma, and 13 had endometrioid
From the Departmentof Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan. Submitted October 25, 1989; acceptedNovember 28, 1990. Address reprintrequests to HiroshiKobayashi, MD, Department of Obstetrics and Gynecology, Hamamatsu University School ofMedicine, Handa-cho 3600, Hamamatsu, Shizuoka, 431-31, Japan. © 1991 by American Society of Clinical Oncology. 0732-183X/91/0906-0019$3.00/0
Journal of Clinical Oncology, Vol 9, No 6 (June), 1991: pp 983-987
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983
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KOBAYASHI ET AL
carcinoma. All patients with ovarian cancer initially underwent surgery and were treated with combination chemotherapy including cisplatin, doxorubicin, and cyclophosphamide. Serum samples were obtained from patients with ovarian cancer and from those with benign gynecologic diseases before surgery. Sera were stored at -80 0 C until analysis could be performed.
Serum Immunoassays Serum STN antigen levels were measured by an immunoradiometric competitive inhibition assay using an STN Otsuka kit (Otsuka Pharmaceutical Co, Ltd, Tokushima, Japan). The assay required a 50 .LLsample. Beads, which were coated with sialyl Tn antigen purified from meconium, were incubated with both 50 [pL of serum and 200 ý±L of iodine-125 (1lI)-labeled TKH-2 antibody solution for 90 minutes. Radioactivity bound to beads was measured by a gamma counter. All samples were assayed in duplicate. Based on this study, an STN antigen level of > 32.6 U/mL was defined as elevated. Serum CA 125 assays were performed in duplicate using kits provided by Centocor, Inc (Malvern, PA). A CA 125 level of > 35 U/mL was defined as elevated.'
StatisticalAnalysis The significance in the difference between groups of subjects was determined by the Student's t-test. Diagnostic value was calculated as sensitivity x specificity.
RESULTS
The distribution of serum STN antigen levels is shown in Table 1. The mean ± SD concentration in healthy individuals was 19.4 ± 6.6 U/mL. The cutoff value (mean + 2 SD) was calculated as 32.6 U/mL. Only seven of the 182 women who were normal had STN antigen levels > 32.6 U/mL. Serum STN antigen levels (mean ± SD) according to age were as follows: < 29 years old (n = 38), 18.9 ± 7.0 U/mL; 30 to 39 years (n = 15), 17.8 ± 5.6 U/mL; 40 to 49 years (n = 89), 19.5 + 7.3 U/mL; 50 to 59 years (n = 18), 22.4 ± 6.3 U/mL; and > 60 (n = 22), 18.5 ± 4.2 U/mL. The relation-
ship between the serum STN antigen levels and menstrual cycle has been evaluated in 90 women who had regular menstrual cycles. Serum levels of this antigen were not affected by menstrual cycle. We assayed STN antigen levels in serum from patients with benign gynecologic diseases and in pregnant women (Table 1). Only 18 (8.8%) of 204 patients had elevated antigen levels. No significant difference was found between STN antigen values from these subjects and those from normal individuals. These results clearly indicate that STN is a
Table 1. Distribution of Serum STN Antigen Levels
No. of Patients
Clinical Status
Healthy controls Benign diseases Uterine myoma Endometriosis Benign ovarian tumors Pregnancy Ovarian cancer Stage I Stage II Stage III Stage IV
Patients With Elevated STN No.
STN Antigen Value*
%
182
7
3.8
19.4 + 6.6
74 79 35 16
6 8 4 0
8.1 10.1 11.4 0
20.7 22.7 21.5 17.9
34 27 55 14
9 10 35 11
26.5 37.0 63.6 78.6
34.5 44.5 144.4 277.9
+ 6.8 + 9.8
t 9.1 + 6.9 : ±
40.8 52.7 158.4 289.4
NOTE. Serum samples from 182 women who were healthy, 188 patients with benign gynecologic diseases, 16 pregnant women, and 130 patients with epithelial ovarian cancer were assayed for radioimmunoassay STN antigen levels. The cutoff value was set at 32.6 U/mL (mean + 2 SD). *Mean U/mL - SD.
tumor marker with a low false-positive rate (25 of 386, 6.5%). Serum samples from 130 patients with ovarian cancer were assayed for STN antigen values. Sixty-five (50.0%) of 130 patients were found to have STN antigen levels > 32.6 U/mL (mean _t SD; 109.2 ± 146.4 U/mL). The difference in STN antigen levels between patients with ovarian cancer and controls was significant (P < .001). The mean values and positive rate increased as the stage advanced. Furthermore, mean serum STN antigen levels increased with the diameter of the greatest tumor mass (Table 2). Classified according to histologic type, STN antigen levels were increased in 43.9% (29 of 66), 61.0% (25 of 41), 50.0% (five of 10), and 46.2% (six of 13) of patients with serous, mucinous, clear cell, and endometrioid carcinoma, respectively. Serum STN testing showed the highest sensitivity in mucinous adenocarcinoma. Table 2. Correlation of Serum STN Antigen Levels With Dimensions of Tumor Mass Diameter of Greatest Tumor Mass at Surgery < 2cm
No. of patients* Mean values of serum STN antigen levels (U/mL)
2-5cm
35
24
30.6
39.1
5-10cm
> 10cm
22
9
167.0
218.3
*The patients with diffuse abdominal disease (n = 40) were excluded.
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985
SIALYL TN ANTIGEN/OVARIAN CANCER
To evaluate the usefulness of STN as a tumor marker, the following five parameters were calculated: sensitivity 50.0%, specificity 93.5%, positivepredictive value 72.2%, negative-predictive value 84.7%, and diagnostic value 46.8%. The combined characteristics of two antigens, STN and CA 125, were confirmed by data from nonmalignant subjects. Normal control subjects had a serum level of 172 with normal CA 125 values, and an additional 10 subjects had elevated CA 125 levels. False-positive CA 125 results were observed in 10 of 74 patients with uterine myoma, in 48 of 79 patients with endometriosis, in 10 of 35 patients with benign ovarian tumors, and in two of 16 pregnant women. The combination assay using STN and CA 125 produced 13 false-positive results in normal control subjects. The combination test also showed the false-positive results in 72 of 188 patients with benign gynecologic diseases and in two of 16 pregnant women. There is a falsepositive rate of 22.5% for the combined test among controls (Table 3). The high false-positive rate of the combination assay is attributable to that of CA 125 for endometriosis in particular. However, of interest is the observation that the STN test has a low false-positive result in endometriosis. Consequently, the false-positive rate in the combination assay is increased by 1.8%, compared with that in the CA 125 test alone. The findings of elevated STN and CA 125 antigen levels in patients with ovarian cancer prompted an analysis of whether the use of both assays would be complementary in evaluating patients with this malignancy. While CA 125 antigen levels were elevated in 74.6% of patients with ovarian cancer and STN antigen levels were elevated in 50.0% of the same population, the use of both assays indicated the sensitivity of detection in 83.8% of the population studied. The combination of CA 125 and STN tests was a more sensitive
indicator than either test alone. The data shown in Fig 1 and Table 3 indicate that serum STN antigen levels were elevated in 12 of 33 patients with ovarian cancer who had serum CA 125 antigen levels less than 35 U/mL. Table 4 shows the clinical data for 12 patients who were positive for the STN antigen and negative for the CA 125 test. The relationship between serum STN and CA 125 antigen levels in patients with ovarian cancer (Fig 1) showed that there was a weak correlation between the serum STN and CA 125 antigen levels in ovarian cancer as judged from regression line Y = 0.51 X + 178.757 (X, serum STN antigen level; Y, serum CA 125 antigen level) and the correlation coefficient of .40674 (P < .05). DISCUSSION Oncogenic transformation is often associated with changes in glycosylation in either glycolipids or glycoproteins in the cell membrane. The abnormalities of glycosylation, such as the incomplete synthesis of carbohydrate chains in cancer cells, have attracted attention.2 Carbohydrate antigens with changes in glycosylation have recently been used as tumor markers. The determination of the serum levels of CA 125, a glycoprotein antigen recognized by the monoclonal antibody OC 125 reported by Bast et al, 4 has provided a useful role in diagnosing patients with ovarian cancer. However, the one shortcoming of this antigen is its relatively low positive rate in mucinous cancer and its high false-positive rate in benign gynecologic diseases, endometriosis in particular.5 If antigens that recognize a carbohydrate portion are used in a combination assay with other antigens that recognize a protein portion, such as CA 125,6 their usefulness as a biochemical marker for ovarian cancer may be enhanced. In the present study, we evaluated the significance of the new carbohydrate antigen, STN,
Table 3. Summary of Serum STN and CA 125 Antigen Levels Elevated
Elevated
Elevated
Clinical Status
STN
%
CA 125
%
STN or CA 125
%
Epithelial ovarian cancer Control*
65/130 25/386
50.0 6.5
97/130 80/386
74.6 20.7
109/130 87/386
83.8 22.5
*Includes 182 healthy subjects, 74 patients with uterine myoma, 79 with endometriosis, 35 with benign ovarian tumors, and 16 pregnant women. The combination assay using STN and CA 125 showed false-positive results in 13 of 182 healthy controls, in 13 of 74 patients with uterine myoma, in 48 of 79 patients with endometriosis, in 11 of 35 patients with benign ovarian tumors, and in two of 16 pregnant women.
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986
KOBAYASHI ET AL
500"
LU·
1
...
1.5
· '·L
400 Q, C E
Table 4. Clinical Data of 12 Patients That Were Positive for STN Antigen and Negative for CA 125 Patient No.
300
U0
· '.
·
·
200 r
E
· · ·
100
·
· · · ·
·
·
·· · ·
0
100
200
300
400
Serum STN antigen (U/mE)
Fig 1. Relationship between serum STN and CA 125 antigen levels in patients with ovarian cancer. Dotted lines represent arbitrary levels distinguishing elevated values for the respective assays (STN Ž 32.6 U/mL, CA 125 2 35 U/mL). Correlation coefficient = .40674, P < .05. Regression line y = ax + b. gradient a, 0.51; section b, 178.757.
in patients with epithelial ovarian cancer and compared the levels with those of the CA 125 antigen. The percentage of patients with ovarian cancer who had elevated STN antigen levels was 50.0%, while STN showed a low false-positive rate in contrast to CA 125, which carries a high falsepositive rate in endometriosis. Serum STN antigen levels appear to be directly correlated with tumor extension. In addition, in regard to histologic type, the sensitivity of STN antigen is high in patients with mucinous adenocarcinoma. According to Kjeldsen et al,2 the immunohistochemical assay of STN yielded no staining in normal tissues except a weak positive reaction in the Leydig cell of the testis, the goblet cell of the large intestine, and the parietal cell of the stomach. However, strong positive staining was obtained in various types of adenocarcinoma. Further trials will be required to investigate the immunohistochemical staining in various types of adenocarcinoma of the ovary. Recently, we have measured serum CA72-4
1 2 3 4 5 6 7 8 9 10 11 12
Stage
Histology
Size of the Greatest Tumor Mass
1
Serous Mucinous Mucinous Mucinous Mucinous Mucinous Mucinous Mucinous Clear cell Clear cell Endometrioid Endometrioid
4 cm 4 cm 3 cm 6 cm 4.5 cm 8 cm Diffuse 7 cm 4 cm Diffuse 5.5 cm 5 cm
I I I I II III III II III II II
STN Value (U/mL)
CA 125 Value (U/mL)
48.9 34.0 42.6 48.0 57.4 63.8 420 480 38.3 159.6 44.7 144.7
34 23 8 8 34 8 12 21 19 24 26 31
antigen levels by a radioimmunoassay using the murine monoclonal antibodies B72.3 and CC49 in patients with ovarian cancer.7 There were many points of similarity in these data between CA72-4 and STN antigen. Comparing the antigenic properties of B72.3 and STN antigen, Springer et als pointed out that Tn antigen (GalNac al - Ser/ Thr) is the antigenic determinant of CA72-4. However, Kjeldsen et a12 have recently reported that B72.3 and STN antigen have identical antigenic determinant epitopes, NeuAc a2 - 6GalNac al -- Ser/Thr. This finding seems to explain why the results of the present study of STN are similar to those of our previously reported study of CA72-4. It will be necessary to clinically confirm the differences between STN and CA72-4 by measuring their serum levels in the same samples from a greater number of subjects. ACKNOWLEDGMENT We are deeply grateful to the doctors of the Gynecological Cancer Committee of the Japan Gynecological Society in Shizuoka Prefecture (Dr Makoto Koda, Director). We are also greatly indebted to the personnel at Otsuka Assay Laboratories (Dr Sadahito Shin, Manager, Tokushima, Japan) for the extensive assistance provided to us in the measurement of serum STN and CA 125 antigen levels.
REFERENCES 1. Fukushi Y, Kannagi R, Hakomori S, et al: Location 3 and distribution of difucoganglioside (VI3 NeuAc V III3 Fuc2 nLc6) in normal and tumor tissues defined by its monoclonal antibody FH-6. Cancer Res 45:3711-3717, 1985 2. Kjeldsen T, Clausen H, Hirohashi S, et al: Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl 2 - 6-a-N-acetylgalactosaminyl (sialosyl Tn) epitope. Cancer Res 48:22112220, 1988
3. Klug TL, Bast RC Jr, Niloff JM, et al: A monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 125) associated with human epithelial ovarian carcinomas. Cancer Res 44:1048-1053, 1984 4. Bast RC Jr, Feeney M, Lazarus H, et al: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 68:1331-1337, 1981 5. Kobayashi H, Kanayama N, Hayata T, et al: Usefulness of measurement of CA 125 levels in the diagnosing and
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987
SIALYL TN ANTIGEN/OVARIAN CANCER treating endometriosis. Acta Obstet Gynec Jpn 39:10541060, 1987 6. Davis HM, Zurawsky VR, Bast RC Jr, et al: Characterization of the CA 125 antigen associated with human epithelial ovarian carcinoma. Cancer Res 46:6143-6151, 1986 7. Kobayashi H: The clinical usefulness of serum CA72-4
analysis in patients with ovarian cancer. Acta Obstet Gynec Jpn 41:585-587, 1989 8. Springer GF, Desai PR, Robinson MK, et al: The fundamental and diagnostic role of T and Tn antigens in breast carcinoma at earliest histologic stage and throughout, in Dao T, Brodie A, Ip C (eds): Tumor Markers and Their Significance in the Management of Breast Cancer. New York, NY, Liss, 1986, pp 47-70
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CARBOHYDRATE
antigens available as cir-
culating tumor markers for clinical evalua-
tion of malignancy include CA 19-9 and CA 50, which belong to type 1 chain carbohydrate antigens, and sialyl Lex -i (SLX), which belongs to type 2 chain carbohydrate antigens.' Recently, new monoclonal antibodies recognizing a core structure of mucin-type carbohydrate chain have 2 been made by Kjeldsen et al. These monoclonal antibodies (TKH1 and TKH2) directed to the tumor-associated 0-linked sialyl 2 -- 6-a-Nacetylgalactosaminyl (sialyl Tn [STN]) epitope were generated by immunization with ovine submaxillary mucin. The STN antigen belongs to neither type 1 nor to type 2 carbohydrate antigens. We have measured the antigen recognized by TKH2. In the present study, we examined its clinical usefulness in the management of the serum STN antigen levels for the diagnosis of patients with epithelial ovarian cancer. Furthermore, we have studied whether the use of the combination assay of STN and CA 125 would be complementary in evaluating patients with this malignancy. MATERIALS AND METHODS
Subjects Serum samples that had been randomly drawn from 130 patients with ovarian cancer and from 386 women who were
146.8 U/mL. Both the mean values and the positive rate increased as the stage advanced. Classified according to the histologic type, the highest positive rate (61.0%) was observed in mucinous adenocarcinoma. The usefulness of STN antigen as a circulating tumor marker in ovarian cancer was estimated as follows: sensitivity 50.0%, specificity 93.5%, positive predictive value 72.2%, negative predictive value 84.7%, and diagnostic value 46.8%. Serum STN antigen levels were elevated in 12 of 33 patients with ovarian cancer who had serum CA 125 antigen levels less than 35 U/mL. While CA 125 antigen levels were elevated in 74.6% and STN antigen levels were elevated in 50.0% of the same population, the use of both assays indicated the sensitivity of detection of 83.8% in the population studied. J Clin Oncol 9:983-987. o 1991 by American Society of Clinical Oncology. healthy, pregnant, or possessed benign gynecologic diseases were obtained from a bank of sera preserved at the Department of Gynecology in Hamamatsu University School of Medicine Shizuoka, Japan. Blood collection was performed within protocols approved by the members of the Gynecological Cancer Committee of this institute. The control subjects included 182 healthy nonpregnant women, 74 patients with uterine myoma, 79 patients with endometriosis (28 with adenomyosis, 21 with pelvic endometriosis, and 30 with a combination of adenomyosis and pelvic endometriosis), 35 patients with benign ovarian tumors (18 with serous cystadenoma, 10 with mucinous cystadenoma, and seven with dermoid cyst), and 16 pregnant women without complications (in the ninth to 38th week of gestation). Diagnosis was confirmed by a review of the operative and pathology reports. Staging of ovarian cancer according to the International Federation of Gynecology and Obstetrics (FIGO) classification showed 34 patients with stage I disease, 27 with stage II, 55 with stage III, and 14 with stage IV. Classified according to histologic type, 66 patients had serous adenocarcinoma, 41 had mucinous adenocarcinoma, 10 had clear-cell carcinoma, and 13 had endometrioid
From the Departmentof Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan. Submitted October 25, 1989; acceptedNovember 28, 1990. Address reprintrequests to HiroshiKobayashi, MD, Department of Obstetrics and Gynecology, Hamamatsu University School ofMedicine, Handa-cho 3600, Hamamatsu, Shizuoka, 431-31, Japan. © 1991 by American Society of Clinical Oncology. 0732-183X/91/0906-0019$3.00/0
Journal of Clinical Oncology, Vol 9, No 6 (June), 1991: pp 983-987
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983
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KOBAYASHI ET AL
carcinoma. All patients with ovarian cancer initially underwent surgery and were treated with combination chemotherapy including cisplatin, doxorubicin, and cyclophosphamide. Serum samples were obtained from patients with ovarian cancer and from those with benign gynecologic diseases before surgery. Sera were stored at -80 0 C until analysis could be performed.
Serum Immunoassays Serum STN antigen levels were measured by an immunoradiometric competitive inhibition assay using an STN Otsuka kit (Otsuka Pharmaceutical Co, Ltd, Tokushima, Japan). The assay required a 50 .LLsample. Beads, which were coated with sialyl Tn antigen purified from meconium, were incubated with both 50 [pL of serum and 200 ý±L of iodine-125 (1lI)-labeled TKH-2 antibody solution for 90 minutes. Radioactivity bound to beads was measured by a gamma counter. All samples were assayed in duplicate. Based on this study, an STN antigen level of > 32.6 U/mL was defined as elevated. Serum CA 125 assays were performed in duplicate using kits provided by Centocor, Inc (Malvern, PA). A CA 125 level of > 35 U/mL was defined as elevated.'
StatisticalAnalysis The significance in the difference between groups of subjects was determined by the Student's t-test. Diagnostic value was calculated as sensitivity x specificity.
RESULTS
The distribution of serum STN antigen levels is shown in Table 1. The mean ± SD concentration in healthy individuals was 19.4 ± 6.6 U/mL. The cutoff value (mean + 2 SD) was calculated as 32.6 U/mL. Only seven of the 182 women who were normal had STN antigen levels > 32.6 U/mL. Serum STN antigen levels (mean ± SD) according to age were as follows: < 29 years old (n = 38), 18.9 ± 7.0 U/mL; 30 to 39 years (n = 15), 17.8 ± 5.6 U/mL; 40 to 49 years (n = 89), 19.5 + 7.3 U/mL; 50 to 59 years (n = 18), 22.4 ± 6.3 U/mL; and > 60 (n = 22), 18.5 ± 4.2 U/mL. The relation-
ship between the serum STN antigen levels and menstrual cycle has been evaluated in 90 women who had regular menstrual cycles. Serum levels of this antigen were not affected by menstrual cycle. We assayed STN antigen levels in serum from patients with benign gynecologic diseases and in pregnant women (Table 1). Only 18 (8.8%) of 204 patients had elevated antigen levels. No significant difference was found between STN antigen values from these subjects and those from normal individuals. These results clearly indicate that STN is a
Table 1. Distribution of Serum STN Antigen Levels
No. of Patients
Clinical Status
Healthy controls Benign diseases Uterine myoma Endometriosis Benign ovarian tumors Pregnancy Ovarian cancer Stage I Stage II Stage III Stage IV
Patients With Elevated STN No.
STN Antigen Value*
%
182
7
3.8
19.4 + 6.6
74 79 35 16
6 8 4 0
8.1 10.1 11.4 0
20.7 22.7 21.5 17.9
34 27 55 14
9 10 35 11
26.5 37.0 63.6 78.6
34.5 44.5 144.4 277.9
+ 6.8 + 9.8
t 9.1 + 6.9 : ±
40.8 52.7 158.4 289.4
NOTE. Serum samples from 182 women who were healthy, 188 patients with benign gynecologic diseases, 16 pregnant women, and 130 patients with epithelial ovarian cancer were assayed for radioimmunoassay STN antigen levels. The cutoff value was set at 32.6 U/mL (mean + 2 SD). *Mean U/mL - SD.
tumor marker with a low false-positive rate (25 of 386, 6.5%). Serum samples from 130 patients with ovarian cancer were assayed for STN antigen values. Sixty-five (50.0%) of 130 patients were found to have STN antigen levels > 32.6 U/mL (mean _t SD; 109.2 ± 146.4 U/mL). The difference in STN antigen levels between patients with ovarian cancer and controls was significant (P < .001). The mean values and positive rate increased as the stage advanced. Furthermore, mean serum STN antigen levels increased with the diameter of the greatest tumor mass (Table 2). Classified according to histologic type, STN antigen levels were increased in 43.9% (29 of 66), 61.0% (25 of 41), 50.0% (five of 10), and 46.2% (six of 13) of patients with serous, mucinous, clear cell, and endometrioid carcinoma, respectively. Serum STN testing showed the highest sensitivity in mucinous adenocarcinoma. Table 2. Correlation of Serum STN Antigen Levels With Dimensions of Tumor Mass Diameter of Greatest Tumor Mass at Surgery < 2cm
No. of patients* Mean values of serum STN antigen levels (U/mL)
2-5cm
35
24
30.6
39.1
5-10cm
> 10cm
22
9
167.0
218.3
*The patients with diffuse abdominal disease (n = 40) were excluded.
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985
SIALYL TN ANTIGEN/OVARIAN CANCER
To evaluate the usefulness of STN as a tumor marker, the following five parameters were calculated: sensitivity 50.0%, specificity 93.5%, positivepredictive value 72.2%, negative-predictive value 84.7%, and diagnostic value 46.8%. The combined characteristics of two antigens, STN and CA 125, were confirmed by data from nonmalignant subjects. Normal control subjects had a serum level of 172 with normal CA 125 values, and an additional 10 subjects had elevated CA 125 levels. False-positive CA 125 results were observed in 10 of 74 patients with uterine myoma, in 48 of 79 patients with endometriosis, in 10 of 35 patients with benign ovarian tumors, and in two of 16 pregnant women. The combination assay using STN and CA 125 produced 13 false-positive results in normal control subjects. The combination test also showed the false-positive results in 72 of 188 patients with benign gynecologic diseases and in two of 16 pregnant women. There is a falsepositive rate of 22.5% for the combined test among controls (Table 3). The high false-positive rate of the combination assay is attributable to that of CA 125 for endometriosis in particular. However, of interest is the observation that the STN test has a low false-positive result in endometriosis. Consequently, the false-positive rate in the combination assay is increased by 1.8%, compared with that in the CA 125 test alone. The findings of elevated STN and CA 125 antigen levels in patients with ovarian cancer prompted an analysis of whether the use of both assays would be complementary in evaluating patients with this malignancy. While CA 125 antigen levels were elevated in 74.6% of patients with ovarian cancer and STN antigen levels were elevated in 50.0% of the same population, the use of both assays indicated the sensitivity of detection in 83.8% of the population studied. The combination of CA 125 and STN tests was a more sensitive
indicator than either test alone. The data shown in Fig 1 and Table 3 indicate that serum STN antigen levels were elevated in 12 of 33 patients with ovarian cancer who had serum CA 125 antigen levels less than 35 U/mL. Table 4 shows the clinical data for 12 patients who were positive for the STN antigen and negative for the CA 125 test. The relationship between serum STN and CA 125 antigen levels in patients with ovarian cancer (Fig 1) showed that there was a weak correlation between the serum STN and CA 125 antigen levels in ovarian cancer as judged from regression line Y = 0.51 X + 178.757 (X, serum STN antigen level; Y, serum CA 125 antigen level) and the correlation coefficient of .40674 (P < .05). DISCUSSION Oncogenic transformation is often associated with changes in glycosylation in either glycolipids or glycoproteins in the cell membrane. The abnormalities of glycosylation, such as the incomplete synthesis of carbohydrate chains in cancer cells, have attracted attention.2 Carbohydrate antigens with changes in glycosylation have recently been used as tumor markers. The determination of the serum levels of CA 125, a glycoprotein antigen recognized by the monoclonal antibody OC 125 reported by Bast et al, 4 has provided a useful role in diagnosing patients with ovarian cancer. However, the one shortcoming of this antigen is its relatively low positive rate in mucinous cancer and its high false-positive rate in benign gynecologic diseases, endometriosis in particular.5 If antigens that recognize a carbohydrate portion are used in a combination assay with other antigens that recognize a protein portion, such as CA 125,6 their usefulness as a biochemical marker for ovarian cancer may be enhanced. In the present study, we evaluated the significance of the new carbohydrate antigen, STN,
Table 3. Summary of Serum STN and CA 125 Antigen Levels Elevated
Elevated
Elevated
Clinical Status
STN
%
CA 125
%
STN or CA 125
%
Epithelial ovarian cancer Control*
65/130 25/386
50.0 6.5
97/130 80/386
74.6 20.7
109/130 87/386
83.8 22.5
*Includes 182 healthy subjects, 74 patients with uterine myoma, 79 with endometriosis, 35 with benign ovarian tumors, and 16 pregnant women. The combination assay using STN and CA 125 showed false-positive results in 13 of 182 healthy controls, in 13 of 74 patients with uterine myoma, in 48 of 79 patients with endometriosis, in 11 of 35 patients with benign ovarian tumors, and in two of 16 pregnant women.
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986
KOBAYASHI ET AL
500"
LU·
1
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Table 4. Clinical Data of 12 Patients That Were Positive for STN Antigen and Negative for CA 125 Patient No.
300
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· '.
·
·
200 r
E
· · ·
100
·
· · · ·
·
·
·· · ·
0
100
200
300
400
Serum STN antigen (U/mE)
Fig 1. Relationship between serum STN and CA 125 antigen levels in patients with ovarian cancer. Dotted lines represent arbitrary levels distinguishing elevated values for the respective assays (STN Ž 32.6 U/mL, CA 125 2 35 U/mL). Correlation coefficient = .40674, P < .05. Regression line y = ax + b. gradient a, 0.51; section b, 178.757.
in patients with epithelial ovarian cancer and compared the levels with those of the CA 125 antigen. The percentage of patients with ovarian cancer who had elevated STN antigen levels was 50.0%, while STN showed a low false-positive rate in contrast to CA 125, which carries a high falsepositive rate in endometriosis. Serum STN antigen levels appear to be directly correlated with tumor extension. In addition, in regard to histologic type, the sensitivity of STN antigen is high in patients with mucinous adenocarcinoma. According to Kjeldsen et al,2 the immunohistochemical assay of STN yielded no staining in normal tissues except a weak positive reaction in the Leydig cell of the testis, the goblet cell of the large intestine, and the parietal cell of the stomach. However, strong positive staining was obtained in various types of adenocarcinoma. Further trials will be required to investigate the immunohistochemical staining in various types of adenocarcinoma of the ovary. Recently, we have measured serum CA72-4
1 2 3 4 5 6 7 8 9 10 11 12
Stage
Histology
Size of the Greatest Tumor Mass
1
Serous Mucinous Mucinous Mucinous Mucinous Mucinous Mucinous Mucinous Clear cell Clear cell Endometrioid Endometrioid
4 cm 4 cm 3 cm 6 cm 4.5 cm 8 cm Diffuse 7 cm 4 cm Diffuse 5.5 cm 5 cm
I I I I II III III II III II II
STN Value (U/mL)
CA 125 Value (U/mL)
48.9 34.0 42.6 48.0 57.4 63.8 420 480 38.3 159.6 44.7 144.7
34 23 8 8 34 8 12 21 19 24 26 31
antigen levels by a radioimmunoassay using the murine monoclonal antibodies B72.3 and CC49 in patients with ovarian cancer.7 There were many points of similarity in these data between CA72-4 and STN antigen. Comparing the antigenic properties of B72.3 and STN antigen, Springer et als pointed out that Tn antigen (GalNac al - Ser/ Thr) is the antigenic determinant of CA72-4. However, Kjeldsen et a12 have recently reported that B72.3 and STN antigen have identical antigenic determinant epitopes, NeuAc a2 - 6GalNac al -- Ser/Thr. This finding seems to explain why the results of the present study of STN are similar to those of our previously reported study of CA72-4. It will be necessary to clinically confirm the differences between STN and CA72-4 by measuring their serum levels in the same samples from a greater number of subjects. ACKNOWLEDGMENT We are deeply grateful to the doctors of the Gynecological Cancer Committee of the Japan Gynecological Society in Shizuoka Prefecture (Dr Makoto Koda, Director). We are also greatly indebted to the personnel at Otsuka Assay Laboratories (Dr Sadahito Shin, Manager, Tokushima, Japan) for the extensive assistance provided to us in the measurement of serum STN and CA 125 antigen levels.
REFERENCES 1. Fukushi Y, Kannagi R, Hakomori S, et al: Location 3 and distribution of difucoganglioside (VI3 NeuAc V III3 Fuc2 nLc6) in normal and tumor tissues defined by its monoclonal antibody FH-6. Cancer Res 45:3711-3717, 1985 2. Kjeldsen T, Clausen H, Hirohashi S, et al: Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl 2 - 6-a-N-acetylgalactosaminyl (sialosyl Tn) epitope. Cancer Res 48:22112220, 1988
3. Klug TL, Bast RC Jr, Niloff JM, et al: A monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 125) associated with human epithelial ovarian carcinomas. Cancer Res 44:1048-1053, 1984 4. Bast RC Jr, Feeney M, Lazarus H, et al: Reactivity of a monoclonal antibody with human ovarian carcinoma. J Clin Invest 68:1331-1337, 1981 5. Kobayashi H, Kanayama N, Hayata T, et al: Usefulness of measurement of CA 125 levels in the diagnosing and
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SIALYL TN ANTIGEN/OVARIAN CANCER treating endometriosis. Acta Obstet Gynec Jpn 39:10541060, 1987 6. Davis HM, Zurawsky VR, Bast RC Jr, et al: Characterization of the CA 125 antigen associated with human epithelial ovarian carcinoma. Cancer Res 46:6143-6151, 1986 7. Kobayashi H: The clinical usefulness of serum CA72-4
analysis in patients with ovarian cancer. Acta Obstet Gynec Jpn 41:585-587, 1989 8. Springer GF, Desai PR, Robinson MK, et al: The fundamental and diagnostic role of T and Tn antigens in breast carcinoma at earliest histologic stage and throughout, in Dao T, Brodie A, Ip C (eds): Tumor Markers and Their Significance in the Management of Breast Cancer. New York, NY, Liss, 1986, pp 47-70
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