Characterization and clinical evaluation of tumor-associated antigen CA54/61 identified by monoclonal antibodies MA54 and MA61 in epithelial ovarian cancer

GYNECOLOGIC ONCOLOGY 47, 328-336 (1992)

Characterization and Clinical Evaluation of Tumor-Associated Antigen CA54/61 Identified by Monoclonal Antibodies MA54 and MA61

in Epithelial Ovarian Cancer

HIROSHI KOBAYASHI,’ HIDEKAZU OHI, MOTOI SUGIMURA, HIROMITSU SHINOHARA, AND TOSHIHIKO TERAO

Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3400, Hamamatsu, Shizuoka, 431-31, Japan

Received January 29, 1992

Monoclonal antibodies (moABs) MA54 and MA61, directed toward the O-linked mucin-type glycoprotein, have been established and showed highly specific reactivity with human ovarian cancer. Fetal intestinal and colonic mucosal cells expressed this antigen and meconium staining was also frequently positive. To investigate the characteristic of an epitopic carbohydrate recognized by these moABs, the reactivity of each moAB with meconium extract was monitored by solid-phase enzyme-linked immunosorbent assay with mono-, di-, and oligosaccharides. MA54 and MA61 react with meconium extract and the reactivities of these moABs are neuraminidase sensitive. Ovine submaxillary mucin had a strong inhibitory activity toward the reaction between meconium extract and MA54 as well as MA61, suggesting that these moABs recognize NeuAc 2-6GalNAc epitope in meconium. The second aim of this study is to investigate the possible ap plication of moABs to diagnose ovarian cancer and to compare these levels with those of the CA125 antigen. While serum CA54/61 antigen levels were elevated in 44.4% of ovarian cancer cases and serum CA125 antigen levels were elevated in 86.7% of the same population, the use of both assays indicated a sensitivity of detection of 97.8% (44 of 45 patients) in the population studied. 0 1992 Academic Press, Inc.

INTRODUCTION

Elevation of various circulating serum tumor markers has been reported to correlate with the occurrence of malignance in cancer [ 1,2]. Monoclonal antibodies (moABs)2 that recognize the carbohydrate structure of cell-surface glycoconjugates of cancer cells have been re- garded as useful tools for cancer diagnosis through as- saying of antigenic glycoproteins secreted into the blood-

’ To whom reprint requests should be addressed. ’ Abbreviations: BSA, Bovine serum albumin; ELISA, enzyme-

linked immunosorbent assay; moAB, monoclonal antibody; SD, standard deviation.

stream and as assessed from immunohistochemical studies [3-51.

Recently, new moABs recognizing a core structure of mucin-type carbohydrate glycoconjugate have been studied by Nozawa et al. [6]. MoABs MA54 and MA61, were generated by immunization with culture supernatants of lung adenocarcinoma cell line C1509. CA54/61 was ini- tially described as an antigen reacted with MA54 and MA61. CA54 reportedly has NeuAccu2-6galactose in the terminal residue [6] and CA54/61 was frequently found in the sera of ovarian cancer patients [6,7].

MoAB TKH-2 directed to the tumor-associated O- linked sialyl 2-6-N-acetylgalactosaminyl (NeuAc 2-6-Gal- NAcal-0-Ser/Thr: sialyl Tn) epitope were generated by immunization with ovine submaxillary mucin (OSM) [8,9]. MoAB TKH-2 reacts with OSM and meconium extract. The reactivity of TKH-2 is sialidase sensitive and the reactivity of TKH-2 with meconium extract was only inhibited by OSM. Also, moABs B72.3 [lo-121 and MSL102 [ 131, directed toward the sialyl Tn structure, have been established after immunization with human meta- static breast cancer and a colonic cancer cell line, re- spectively, which show highly specific reactivity with various human cancers and restricted reactivity with normal tissues. Mucin-type glycoproteins are major secretory products of the colon and contain O-linked oligosaccharides synthesized on a protein backbone [3]. In the colon and the ovary, the sialyl Tn antigen is an oncodevelop- mental cancer-associated antigen, since fetal colonic mucosal cells also expressed this antigen, particularly in goblet cell mucin [3].

We found that moABs MA54 and MA61 react with human meconium glycoproteins and the fact that these moABs recognize the sialyl Tn moiety on cancer cell surface was considered. The aim of this study is (1) to

328 @390-&X258/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

CANCER-ASSOCIATED ANTIGEN CA54/61 AND OVARIAN CANCER 329

characterize the epitopic structures recognized by moABs MA54 and MA61, in comparison with the CA125 antigen recognized by OC125, and (2) to evaluate the usefulness of CA54/61 as a new tumor marker in epithelial ovarian cancer.

MATERIALS AND METHODS

Monoclonal Antibodies

The binding specificities of the moABs MAS4, MA61, and TKH-2, directed toward the O-linked mucin-type glycoprotein, and moAB OC125, recognizing cancer-associated CA125 glycoprotein [14], to meconium extract were investigated.

The immunoglobulin class of MA54 and MA61 proved to be IgM(K) [6]. The antigen recognized by MA54 (CA54) or MA61 (CA61) proved to be a carbohydrate chain on a high molecular weight mucin-type glycoprotein, and CA54 has, reportedly, NeuAccu2-6galactose in the terminal residue [6]. MoABs MA54 and MA61 were donated by Mochida Pharmaceutical Co. Ltd., Tokyo, Japan. MoAB TKH-2 (IgGl) was raised against OSM glycoprotein [8] and was donated by Otsuka Assay Lab- oratories, Tokushima, Japan. Murine moAB OC125 against CA125 was donated by Toray Fuji Bionics, To- kyo. All other reagents were of analytical grade.

Meconium Extract

Meconium was subjected to perchloric acid precipita- tion (0.6 M, 4”C), homogenized and centrifuged, and then the acid-soluble fraction was neutralized (1 N NaOH) and dialyzed. This sample was applied on a Sephacry S-300 column. Meconium mucin was mainly eluated in the void volume of gel chromatography. The void volume fraction (partially purified meconium solution, meconium extract) was pooled and coated on 96-well Costar ELISA plate (Cambridge, MA; meconium plate).

Tissue Staining

Specimens of fetal intestine and colon were obtained at immediate autopsy from three third-trimester intra- uterine fetal deaths without any colonic disease as ap- proved by the Committee on Human research and according to the protocol established at the Department of Pathology, Hamamatsu University School of Medicine, and kindly provided by Dr. Nakamura. All tissues used in this study were fixed in formalin, embedded in paraffin, and cut into 5-pm serial sections for immunohistochemical staining.

Working concentrations of TKH-2 [3], MA54, and MA61 are 1.0 pg/ml. The streptavidin-peroxidase tech- nique of immunohistochemistry was performed as described previously [15]. Fetal intestine and colon sections

were incubated with fresh 3% hydrogen peroxide in meth- anol (30 min, 23°C) and washed with phosphate-buffered saline (PBS). Ten percent normal goat serum in PBS was applied (30 min, 23°C). Primary moABs were incubated (16 hr, 4°C) and washed. Biotinylated second antibody (7.0 pg/ml in PBS) was added (20 min, 23°C) and washed three times with PBS. Then, streptavidin-peroxidase (10 pug/ml) was added (10 min, 23°C) and washed three times with PBS. Finally, slides were reacted with 3% 3-amino- 9-ethylcarbazol substrate (5 min, 23°C DAKO) rinsed with tap water, and mounted.

Reactivity of moABs with Meconium Extract (direct enzyme-linked immunosorbent assay (ELISA)

For direct ELISA, meconium extract was coated on 96-well microtiter plates in a humid chamber (16 hr, 4°C). After three successive washes with Tris-buffered saline containing 0.05% Tween 20 (TBST), the remaining protein-binding sites were blocked by incubation with TBS- 2% bovine serum albumin (TBS-BSA) (30 min, 23°C). Plates were washed three times with TBST, followed by incubation with various concentrations of moABs (TKH- 2, MA54, MA61, and OC125; O-4.0 pg/ml). After incubation the plates were washed seven times with TBST, followed by incubation with biotinylated goat anti-mouse antibody (1.5 pg/ml, DAKO; 1 hr, 23°C). Avidin-peroxidase (DAKO) was diluted 1: 1000 in TBST containing 0.5 M NaCl. The microtiter plates were incubated (1 hr, 23°C) and then washed seven times with TBST. One hundred microliters of a stock solution of 10 mg 3,3’- 5,5’tetramethylbenzidine/ml dimethylsulfoxide was added to 10 ml 0.1 M sodium acetate/citrate buffer, pH 6.0, and then mixed thoroughly. Immediately before the reaction mixture was filled into the plates, 15 ~1 of 3% of H202 was added. One hundred microliters was filled into each well. Ten minutes later the enzyme reaction was terminated by addition of 2 N H,SO, and the ab- sorption was measured in an EIA reader (Model 2550, Bio-Rad, Richmond, California) at 450 nm.

The meconium plate was treated with neuraminidase (0.05 U/ml, 3 hr, 37°C; from Chlostridium perfuringens, type X, Sigma). The reactivities of moABs were investigated as described above.

Reactivity of Glycosaccharides with moA Bs

Specificities of each moAB to meconium extract were determined by measuring their ability to inhibit the binding of moABs by several glycosaccharides (Sigma). We used monosaccharides (D( +)glucose, D( +)galactose, D( + )fucose [6-deoxy-o-galactopyranose], N-acetyl-glu- cosamine [2-Acetamido-2-deoxy-n-glucose], N-acetylgalactosamine [2-Acetamido-2-deoxy-n-galactose], N-acetylneuraminic acid, and D( + )mannose), disaccharides (a-

330 KOBAYASHI ET AL.

lactose [4-0-P-n-galactopyranosyl-o-o-glucose] and maltose [4-O-a+D-glucopyranosyl-D-glucose]), and oligosaccharides (N-Acetylneuramine-lactose [N-Acetylneurami- nyl(2-3) and (2-6)-P-D-galactopyranosyl(l-4)-D-glucopyra- nose] and OSM) as competitors. To determine if glycosaccharides are involved in the reactivity to moABs, the inhibitory activities of these glycosaccharides were assayed. After preincubation of glycosaccharides and each moAB (1 hr, 23”(Z), the reaction mixture (100 ~1) was transferred to ELISA plates coated with meconium extract and further incubated (1 hr, 23°C). Thereafter, the procedure was as described above. Of each moAB 0.66 pg/ml was used for inhibition assays.

Human Serum Samples

Serum samples were obtained from 55 healthy nonpregnant women and 100 patients with benign gynecologic disease including 8 uterine myoma, 16 adenomyosis, 76 benign ovarian tumors, and 45 patients with histologically proven epithelial ovarian cancer (see Table 1). Diagnoses of all patients were confirmed by review of operative reports and pathology reports. Staging of ovarian cancer according to the International Federation of Gynecology and Obstetrics (FIGO) classification showed 12 patients with stage I, 10 with stage II, 14 with stage III, and 9 with stage IV. Serum samples were obtained within 2 weeks before therapy and stored at -20°C until use.

Determination of Serum CA54161 and CA125 Antigen Levels

Circulating serum CA54/61 antigen concentrations (U/ml) were determined by the sandwich enzyme immunoassay kit, supplied by Mochida Pharmaceutical Co., Ltd., which uses moABs MA54 and MA61 in a two- step procedure. Technical characteristics of the assay are described elsewhere [6]. Levels of more than 15.2 U/ml (normal mean + 2 SD) were considered elevated deter- minations. All samples were assayed in duplicate.

Serum CA125 assays were performed in duplicate using the kit provided by Centocor, Inc. (Malvern, PA). A CA125 level of 335 U/ml was defined as elevated.

Statistical Analysis

The significance in the difference between groups of subjects was determined by the Student t test [16,17].

RESULTS

Immunohistochemical Staining

In fetal intestinal and colonic mucosa, goblet cells and cell vacuoles stained with MA54, MA61, and TKH-2. In general, the staining intensity was strong with TKH-2 and

was moderate with MA54 and MA61 (Fig. 1). Meconium staining or the extracellular mucin lakes were frequently positive with all three reagents. This moAB staining was essentially absent from esophageal and gastric mucosal cells (not shown). Meconium (luminal secretions) was used as a reference for further immunochemical studies.

Specificity of moA Bs Binding to Glycosaccharides

MoAB MA54, MA61, and TKH-2 react with meconium extract in a dose-dependent manner, while OC125 does not react (Fig. 2). Neuraminidase treatment of meconium extract abolished the reactivity of these moABs (Fig. 3).

Reactivity of glycoproteins inhibiting the binding of TKH-2 to meconium extract was confined to OSM (Fig. 4A). OSM also had a significant inhibitory activity toward the reaction between meconium extract and MA54 as well as MA61 (Figs. 4B and 4C). TKH-2 reactivity to meconium extract was inhibited specifically by OSM, indicating that this moAB is clearly directed to sialyl Tn antigen, NeuAca2dGalNAc. Unlike TKH-2, the binding of MA54 was inhibited by NeuAc, GalNAc, or other sugars to some extent. Comparing OSM with other sugars, however, OSM gives rise to more than 65,000-fold enhancement in the reactivity with MA54 as assessed from the concentration, giving 50% inhibition of the antigen-antibody reaction in our immunoassay system. The binding of MA61 with meconium extract was, in part, inhibited by NeuAc.

Serum CA54161 and CA125 Antigens in Ovarian Cancer

Sera from patients with ovarian cancer at various stages, benign disease, and healthy controls were assayed for CA54/61 and CA125 antigen levels. These data are presented in Table 1. The cutoff value of CA54/61 was arbitrarily set at mean + 2 SD (15.2 U/ml) of healthy controls to define the positive rate. The mean value and its SD was 7.8 + 3.7 U/ml. The levels were elevated in 1 case of healthy controls, with a false-positive rate of 1.8%. Only 2 (2.0%) of the 100 patients with benign gynecologic disease had CA54/61 antigen levels of more than 15.2 U/ml. Elevated levels of serum CA54/61 antigen were seen in patients with epithelial ovarian cancer when compared with those in patients with benign disease and normal controls. The difference in CA54/61 antigen levels between cancer patients and controls or benign disease was significant (P < 0.05). Twenty (44.4%) of the 45 patients with ovarian cancer showed CA54/61 levels over the cutoff value; 4 patients with stage I disease (33.3%) 3 with stage II (30.0%), 8 with stage III (57.1%), and 5 with stage IV (55.6%) had elevated antigen levels. Stages I and II cases showed a significantly low frequency of elevation and low values compared to more advanced cases.

FIG. 1. Immunoperoxidase staining of formalin-fixed paraffin-embedded fetal intestinal mucosa using monoclonal antibodies MA54, MA61, and TKH-2. Globlet cells were stained with all three reagents ( x 25). In contrast to fetal colonic mucosa, intestinal goblet cell binding of these moABs was more prevalent and more intense. Immunoperoxidase staining of formalin-fixed paraffin-embedded fetal intestinal mucosa using TKH-2 (A), MA54 (B), and MA61 (C) monoclonal antibody.

331


2.0 1

/ .------------” 1.5

P i 0.5 1. lc / / 0 0.01% 0.0625 0.25 0 5 1 2 4

moA6 concentration (pg/d)

FIG. 2. Reactivity of monoclonal antibodies to solid-phase meconium extract. Differential reactivities of TKH-2 (O), MA54 (0), MA61 (m), and OC125 (0) to solid-phase meconium extract.

Significant correlation was seen between elevation of serum CA54/61 antigen levels and the size of the primary tumor (data not shown). These results indicate that serum CA54/61 antigen levels correlate with the stage of disease.

The relationship between the CA54/61 antigen levels and histology for the patients with cancer are shown in Table 2. The significant elevation was observed in sera of the mutinous type (X3%, 7/12), the serous-type (42.9%, 9/21), the clear-cell type (40.0%, 2/5), and the endometrioid type (28.6%) 2/7). Serum CA54/61 testing showed the highest sensitivity in mutinous-type ovarian cancer. The combined characteristics of two antigens, CA54/61 and CA125, were confirmed by data from nonmalignant subjects (Tables 1 and 3). Normal control subjects had a serum level of 49 with normal CA125 values, and an additional 6 subjects had elevated CA125 levels. False-positive CA125 results were observed in 29 of 100 patients with benign disease. The combination assay using CA54/61 and CA125 produced 37 false-positive results (23.8%) in nonmalignant subjects. The high false-positive rate of the combination assay is attributable to that of CA125 for adenomyosis and endometrial cyst in particular. However, the CA54/61 test has a low false-positive result in these benign diseases. Consequently, the false- positive rate in the combination assay is increased by 1.2%, compared with that in the CA125 test alone.

The findings of elevated CA54/61 and CA125 antigen levels in patients with ovarian cancer prompted an analysis of whether the use of both assays would be comple- mentary in evaluating patients with this malignancy. While CA125 antigen levels were elevated in 86.7% of patients with ovarian cancer and CA54/61 antigen levels were elevated in 44.4% of the same population, the use of both assays indicated the sensitivity of detection in 97.8% of the population studied (Table 3). The combination of CA54/61 and CA125 tests was a more sensitive

indicator than either test alone. A comparison of the serum levels of CA54/61 and CA125 in ovarian cancer showed no correlation between the two (r = 0.09, data not shown). Table 4 shows the clinical data for five patients who were positive for the CA54/61 antigen and negative for the CA125 test. Of interest is the observation that four of five patients had mutinous-type ovarian cancer.

DISCUSSION

Tumor-associated antigens identified by moABs have recently been used for serological detection of malignan- cies. Many of these tumor markers have the structure of carbohydrate antigens [18]. Two moABs, MA54 and MA61, were established and a double-determinant sandwich enzyme immunoassay system was developed by using these two moABs [6]. The antigen (CA54/61) recognized by MA54 (CA54) or MA61 (CA61) proved to be a carbohydrate chain on a high molecular weight mucin-type glycoprotein [6]. In the present study, we investigated the characteristics of the CA54/61 antigen. Furthermore, we have monitored the levels of circulating serum CA54/61 antigen in patients with epithelial ovarian cancer and compared the levels to those of the CA125 antigen.

Colonic adenocarcinoma and ovarian cancer stained with moABs MA54 and MA61 as well as TKH-2 [6]. Most often, colon cancer expressed T (Gal/313-Gal- NAcal-0-Ser/Thr), Tn (GalNAcal-0-Ser/Thr), and sialyl Tn (NeuAca2-6GalNAccul-0-Ser/Thr) antigens al- though there was heterogeneity in cellular location and staining intensity [3]. Fetal intestinal and colonic mucosa was examined immunohistochemically with an aim toward determining if these carbohydrate structures recognized by moABs MA54, MA61, and TKH-2 are oncofetal an-

moAB concentration (rg/d)

FIG. 3. Reactivity of monoclonal antibodies to solid-phase neuraminidase-treated meconium extract. Differential reactivities of TKH-2 (O), MA54 (0) MA61 (W), and OC12.5 (Cl) to solid-phase neuraminidase-treated meconium extract.

CANCER-ASSOCIATED ANTIGEN CA54/61 AND OVARIAN CANCER

0 0 0.0002 0.004 0.06 I 0.977 15.63 250

Comperttors (mM)

333

0 0.0002 0.004 0.061 0.977 15.63 250 0 0 (002 oio4 0061 0 977 1563 250

competitors (mM) Competitors (mM)

FIG. 4. Inhibition of moAB binding activity to meconium extract with different glycosaccharides. (A) Inhibition of TKH-2 binding by monosaccharides D( +)glucose (0), D( +)gaIactose (O), D( + )fucose (A), N-acetylglucosamine (A), N-acetylgalactosamine (Cl), N-acetylneuraminic acid (v), and D( +)mannose (W); disaccharides a-lactose (V) and maltose (*); and oligosaccharides N-acetylneuramin-lactose ( x ) and OSM (0) as a competitor. (B) Inhibition of MA54 binding by glycosaccharides. (C) Inhibition of MA61 binding by glycosaccharides.

tigens. A striking finding was the strong and prevalent goblet cell staining with TKH-2, MA54, and MA61. These moABs bound to cell membranes and cytoplasm, as well as to luminal contents. Meconium staining was frequently positive with all three reagents. This is a rationale that meconium was used as a reference for the present biochemical study. Also, we have detected the MA54, MA61, and TKH-2 staining in tissues of the normal fetal and adult respiratory tract. Staining was observed in the bronchial and bronchiole cells, but not in alveolar structures (not shown). Intestine and colon mucin and respiratory tract mucin are thought to be known sources for the CA54/61 antigen in the serum of normal nonpregnant individuals. In contrast, the CA125 determinant was not detected in the mucin of intestinal and colonic goblet cells. However, some investigators have evaluated OC125 binding to tissues of the normal adult lung and respiratory tract [19].

To clarify the carbohydrate structure of CA54/61 an-

tigen, the cross-reactivities of MA54 or MA61 with various kinds of glycosaccharides were investigated by a competitive inhibition assay. Both moABs react strongly with meconium extract, and the reactivities were abolished by sialidase treatment. CA54 is reported to have NeuAccu 2-bgalactose in the terminal residue [6], but the binding of MA54 and MA61 with meconium extract was com- pletely inhibited by OSM, suggesting that MA54 and MA61 react with the NeuAccuZ6GalNAc epitope. How- ever, the possibility that CA54/61 recognizes or cross- reacts with some carbohydrate moiety other than NeuActu2-6GalNAc epitope could not be ruled out, be- cause, unlike TKH-2, a competitive inhibition assay revealed that the reaction of MA54 was inhibited by NeuAc, GalNAc, or galactose to some extent.

OC125 does not react with meconium extract or OSM. MA54 and MA61 do not inhibit 125-I OC125 binding to the CA125 antigen in the conventional CA125 immu- noradiometric assay (data not shown). As determined by


TABLE 1 Distribution of Serum CA54/61 and CA125 Antigen Levels

CA54161 CA125

Patient population

Healthy controls Benign disease

Uterine myoma Adenomyosis Serous cyst of ovary Mutinous cyst of ovary Dermoid cyst Chocolate cyst

Ovarian cancer I II III IV

No. of patients

55 100

8 16 35 15 14 12

45 12 10 14 9

Mean value +-SD (U/ml)

7.8 f 3.7 8.1 + 4.0

42.9 + 127.9

No. of positive cases

(“ro)

1 ( 1.8) 2 ( 2.0) 0 ( 0.0) 2 (12.5) 0 ( 0.0) 0 ( 0.0) 0 ( 0.0) 0 ( 0.0)

20 (44.4) 4 (33.3) 3 (30.0) 8 (57.1) 5 (55.6)

Mean value +SD (U/ml)

18.3 +- 8.4 29.6 2 31.0

963.2 f 710.8

No. of positive cases

(%)

6 (10.9) 29 (29.0)

1 (12.5) 8 (50.0)

10 (28.6) 2 (13.3) 3 (21.4) 5 (41.7)

39 (86.7) 9 (75.0) 8 (80.0)

13 (92.9) 9 WV

Note. SD, standard deviation.

Davis et al. [20], the most highly purified glycopeptides expressing the CA125 determinant were not “mucin- like”. These results indicate that the CA54/61 determinant is distinct from the antigen reacting with OC125 antibody.

The determination of the serum levels of CA125 [14] 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 mutinous cancer and its high false-positive rate in benign gynecologic disease, endometriosis in particular [21]. If antigens that recognize a carbohydrate portion are used in a combination assay with other antigens that recognize a protein portion, such as CA125, their usefulness as a biochemical marker for ovarian cancer may be enhanced.

We next examined the clinical usefulness of the management of the serum CA54/61 antigen levels for the diagnosis of patients with ovarian cancer. The positive rate (> mean + 4 SD) of CA54/61 was, reportedly, 68%

TABLE 2 Serum CA54/61 Antigen Levels and Histology for Patients

with Ovarian Cancer

No. of patients No. of Mean value with positive

Histology patients (U/ml) CA54/61 (%)

Serous 21 40.1 9 (42.9) Mutinous 12 53.9 7 (58.3) Endometrioid 7 37.8 2 (28.6) Clear cell 5 35.4 2 (40.0)

Note. The cutoff value was set at mean + 2 SD (15.2 U/ml) of healthy controls. Figures in parentheses indicate percentages.

(28/41) in ovarian cancer [6]. In the present study, the CA54/61 antigen was detected in approximately 44% of ovarian cancer sera examined. In contrast, CA54/61 detection in normal and nonmalignant sera is highly restricted, suggesting that this antigen has an extremely high specificity [7] in contrast to CA125, which carries a high false-positive rate in adenomyosis and endometrial cyst. Ovarian cancer patients with stages I and II showed a significantly low frequency of elevation and low values compared to more advanced cases. The correlation between the positivity of the serum CA54/61 test and the stage seems to be entirely based on the extent of disease. In addition, the present results suggest that CA54/61 should provide increased diagnostic precision for muci- nous-type ovarian cancer. Consequently, the use of both CA54/61 and CA125 assays should further increase the ability to detect ovarian cancer. The frequent expression of the antigen in sera of cancer patients suggests that this structure may be a cancer-associated antigen and may be in the process of being further glycosylated, or the ap-

TABLE 3 Summary of Serum CA54/61 and CA125 Antigen Levels

Elevated

CA54161

Clinical status CA54161 % CA125 % C::25 %

Epithelial ovarian cancer 20/45 44.4 39145 86.7 44145 97.8 Control 31155 1.9 351155 22.6 371155 23.8

a Includes 55 healthy subjects and 100 with benign gynecologic disease.

CANCER-ASSOCIATED ANTIGEN CA54/61 AND OVARIAN CANCER 335

TABLE 4 Clinical Data of Five Patients That Were Positive for CA54/61 and Negative for CA125

Patient No.

1 2 3 4 5

Stage Histology

I Mutinous I Mutinous II Mutinous III Mutinous II Clear cell

Size of the greatest

tumor mass (4

5 6 8 7 6

CA54161 CA125 Value value

(U/ml) W/ml)

38 16 42 12 44 <8 64 24 40 31

pearance of core-region antigens provides evidence for incomplete glycosylation in cancer cells [3]. The consid- erable expression of CA54/61 antigen represents another example of precursor accumulation in cancer cells [3]. The abnormal expression of sialyl transferase in cancer cells seems to be the most feasible cause of this phenom- enon. These facts indicate that the CA54/61 antigen in- creases in quantity during the course of malignant changes in ovarian cancer. We applied MA54 and MA61 to flow cytometry to compare their expressions on cancer cell surfaces among undifferentiated and differentiated U937 promyeloid leucemia cells in order to investigate CA54/61 expression on their cell surface [22]. Our study revealed that undifferentiated cells had a higher reactivity to antibodies than had differentiated cells (unpublished data). The mechanism of the abnormal expression of this carbohydrate moiety in cancer cells needs to be resolved. Recently, we have reported that positive sialyl Tn (TKH- 2 reactive) antigen levels in sera were an independent predictor of poor prognosis in ovarian cancer [23]. There were many points of similarity in data among these antigens (sialyl Tn [24] and CA54/61). These antigens may have an identical or similar antigenic determinant epitope, NeuAccu2-6GalNAc. This finding seems to explain why the results of the present study of CA54/61 are similar to those of our previous reported studies of sialyl Tn. It will be necessary to clinically confirm the differences between CA54/61 and sialyl Tn by measuring their serum levels in the same samples from a greater number of subjects.

ACKNOWLEDGMENTS

We are greatly indebted to the personnel at Mochida Pharmaceutical Co. Ltd. (the manager, Mr. T. Katamine, and Mr. T. Kudoh, Tokyo, Japan) for the extensive assistance provided to us in the measurement of serum CA54/61 antigen levels. We thank Dr. K. Sumimoto for the statistical analysis and for his valuable advice.

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Characterization and clinical evaluation of tumor-associated antigen CA54/61 identified by monoclonal antibodies MA54 and MA61 in epithelial ovarian cancer