Characterization of tumor-associated antigens of ovarian carcinoma

GYNECOLOGIC

ONCOLOGY

4, 167-175 (1976)

Characterization

of Tumor-Associated Ovarian Carcinoma’

Antigens

of

SUZANNE KNAUF, M.S., AND GERALD I. URBACH, M.D.2 The Department of Obstetrics and Gynecology, University of Toronto, The Wellesley Hospital,

Toronto,

Ontario,

Canada

Received February 4, 1976 The distribution of four tumor-associated antigens of human ovarian carcinoma has been investigated by immunoelectrophoretic studies of perchloric acid extracts of ovarian tumors using selected rabbit anti-tumor antisera. The tumor antigens have been fractionated by chromatography on Sephadex G-200 and Sepharose 4B. Protein was eluted from the Sephadex G-200 columns in peaks I, II, and III at elution volume/void volume (VJV,) of 1.0, 1.7, and 3.0 and from Sepharose 4B columns in peaks IV, V, and VI at VJV, of 1.0, 2.4, and 2.8, respectively. Peaks I, II, IV, and V have material which exhibits P-like mobility in immunoelectrophoretic analysis (barbital buffer, pH 8.6). Antigens exhibiting y- and a-like mobility are eluted in peaks I, II, V, and VI and peaks II, III, and VI, respectively. As previously described by this laboratory [Knauf, S., and Urbach, G. I., Amer. J. Obstet. Gynecol. 123, 302-304 (1975)3, the two p antigens are located at R, < 0.05 in 5.6% SDSacrylamide gels. Antigens with y- and a-like mobility have been tentatively identified in 5.6% acrylamide gels at RI = 0.30 to 0.42 and Rf = 0.42 to 0.46 with apparent molecular weights of 7-9 x IO4 and 5.5-7 x lo4 daltons, respectively. of ovarian cancer is, at present, largely a matter of chance. By is made, the tumor has reached the inoperable stage in over 60% of patients. It is therefore imperative that an assay for the detection of ovarian cancer in asymptomatic women be developed if the prognosis for the ovarian cancer patient is to improve. The demonstration of tumor antigens which are associated with the malignant state and which are present in the serum of patients with tumors provides a Early

detection

the time a firm diagnosis

promising approach to early cancer recognition. For example, carcinoembryonic antigen (CEA), which was originally derived from human colonic cancer [l], has been found in the serum of patients with several different tumors, including ovarian malignancies [2-51. However, since elevated serum CEA levels have been demonstrated in only 25% of patients with known ovarian tumors [4], the CEA assay appears ill-suited to screening for ovarian neoplasm. Tumor-associated antigens (TAA’s) of human ovarian cancer have been detected in ovarian tumor cell cultures and homogenates [5-151. This paper reports the attempt to separate and partially purify the four TAA’s of human ovarian cancer previously described in this laboratory [I21 so that a more specific serum assay for the diagnosis of ovarian malignancy in asymptomatic women may be developed. 1 Supported by The Ontario Cancer Treatment and Research Foundation Project No. 275 and 324. 2 Address for reprints: Gerald I. Urbach, M.D., No. 204Jones Building, The Wellesley Hospital, 160 Wellesley Street East, Toronto, Ontario, Canada M4Y 1J3. 167 Copyright @I 1976 by Academic Press, Inc. All rights of reproduction m any form reserved

168

KNAUF

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URBACH

MATERIALS AND METHODS Human ovarian tissue was collected and stored at -70°C. Specimens were classified as normal, mutinous or serous cystadenocarcinoma (MCA or SCA) or solid epidermoid carcinoma (SEC) by histological examination. Preparation of perchloric acid (PCA) extracts, immunization of rabbits, absorption of antisera to remove all antibodies against normal components, immunodiffusion, and immunoelectrophoresis were carried out as previously described 1121. Column chromatography. PCA extract (IO mg) was fractionated on a Sephadex G-200 column (2.6 x 40 cm) or a Sepharose 4B column (1.6 x 70 cm) in 0.1 M Tris, pH 8.0, containing 0.2 M sodium chloride and 0.02% sodium azide. Flow rates were adjusted to 15 and 8 ml/hr for the Sephadex G-200 and Sepharose 4B, respectively. Fractions were collected and the ODzso ,,,,, of each fraction was determined. Peak fractions were pooled and concentrated to approximately 0.1 ml by ultrafiltration in an Amicon filter unit with a UMlO filter or by dialysis against distilled water, followed by dialysis against polyethylene glycol 20,000. Protein determinations were carried out by the modified micro-biuret method [16]. The void volume (V,) was determined with dextran blue 2000. The elution volume (V,) and the V,/V,, of each peak was measured. Gel electrophoresis. Sodium dodecyl sulphate-acrylamide disc gel electrophoresis (SDS PAGE) was performed using 5.6% gels according to the method of Fairbanks et al. [ 171.Samples were solubilized in dithiothreitol and SDS at 90°C for 60 min or in a boiling-water bath for 5 min. Gels were prepared in 16 cm tubes, and samples were electrophoresed at 8 mA/tube until the pyronin Y marker dye was 1 cm from the end of the tube. Gels were stained for glycoprotein with periodic acid-Schiff reagent (PAS) and/or protein with Coomassie brilliant blue. Molecular weight standards were serum albumin, catalase, ovalbumin, alcohol dehydrogenase, pepsin, and cytochrome c. The relative mobility (Rf) of each protein or glycoprotein band was calculated relative to pyronin Y. RESULTS Identijcation of antigens. Four different anti-tumor antisera were obtained by immunization of rabbits with the PCA extracts of four tumors (two SCA, one SEC, and one MCA). The antisera were absorbed with normal human serum and ovarian tissue to remove antibodies against normal human components. Each of ten tumor extracts and six normal ovarian extracts was tested against each of the four absorbed anti-tumor antisera. The results are summarized in Table 1. TAA’s are named with reference to the four immunoelectrophoretic precipitation arcs of tumor extract from patient MC and antiserum prepared against this extract (anti-MC). Antigen A exhibits (Y-, B and C exhibit /3-, and D exhibits y-like mobility in barbital buffer at pH 8.6. A and D share at least some common antigenic determinants with C [ 123. Antigens B and C form concentric arcs at the origin. If only one of these arcs and neither antigen A nor D is present, it is impossible to distinguish antigen B from C. In such cases, the arc is designated “B or C.” Antiserum prepared against tumor extract from patient DT (anti-DT) contains antibodies against antigens A and C, antiserum against tumor extract from patient MH (anti-MH) contains anti-A, -B, and -C, and antiserum against

OVARIAN

TUMOR-ASSOCIATED

TABLE IDENTIFICATION

OF TAA’S

Extract

169

ANTIGENS

1

BY IMMUN~ELECTR~PHORESI~~

TAA’s detectable*

Class

Patient

Anti-MC

Anti-DT

Anti-MH

Anti-MS

All TAA’s detectable

SCA

MC DT NY EP MD

ABCD B or C B or C CD A

AC AC A B or C NTd

ABC -= AC NT

BC NT

ABCD AC ABorC ACD A

SEC

MS DS MM

BC CD -

B or C AC -

A A

BC B or C -

BC ACD A

MCA

MH LA

AC A

AC -

ABC ABC

-

ABC ABC

o Perchloric acid extract (100 pg) and 100 yl of absorbed antiserum were used for immunoelectrophoresis. * Refers to arcs of tumor extract from patient MC vs antiserum against this extract (anti-MC). ’ (--), No detectable immunoelectrophoretic pattern. d NT, not tested.

tumor extract from patient MS (anti-MS) contains anti-B and -C. It should be noted that there appears to be a variation in the immunological activity of extracts from different patients and/or the potency of the antibodies in each antiserum directed against a particular antigen or antigenic family, as evidenced by differences in the intensity of the precipitation arcs. Antigen A appears to be the most common antigen and was observed in tumor extracts from all patients except MS. Antigens B and C were found in four extracts, while either B or C was found in all but two extracts. Antigen D was the least common antigen, detected in only three extracts, none of which was from MCA tissue. Evidence that the antigen A, B, C, or D in one tumor is the same antigen or family of antigens as that in other tumor extracts was confirmed by double diffusion. Column chromatography. An attempt was made to separate and purify the four TAA’s by column chromatography. Nine tumor and two normal perchloric acid extracts were fractionated on Sephadex G-200 as shown in Fig. 1. Protein was eluted from the column in peaks I, II, and III at VJV, of 1.0, 1.7, and 3.0, respectively. These peaks contained greater than 85% of the applied protein, with the remainder of the protein in the interpeak fractions. Two different protein (OD2& elution profiles, type i and type ii, were observed. Five tumor extracts exhibited a type i pattern similar to that presented in Fig. 1 for the tumor extract from patient MM. Four tumor extracts and the two normal extracts exhibited type ii behavior as shown in Fig. 1 for the tumor extract from patient DT. Two of four SEC and three of six SCA extracts chromatographed with type i patterns, while all MCA and normal extracts tested had type ii patterns. Pooled peak fractions were concentrated and tested for antigenic activity by immunodiffusion and immunoelectrophoresis against absorbed anti-MC an-

170

KNAUF AND URBACH I F&O

PEAKS ANTIGENS w

II ABaCD

III A ,

’ TYPE i PATIENT

MM

sPI 8 0.4

TYPE ii PATIENT DT

EFFLUENT

VGLIJM

Id

FIG. 1. Sephadex G-200 chromatography of tumor perchloric acid extracts from patients MM and DT. The range of the percentages of total protein applied to the column which eluted in each peak is given for all extracts with each elution pattern (see Results). Tumor antigens detectable by immunoelectrophoresis of concentrated tumor peak fractions against absorbed anti-MC antiserum are also given.

tiserum. Antigens B, C, and D were eluted in peak I; antigens A, B or C, and D in peak II; and antigen A in peak III. Antigens B, C, and D always eluted from the Sephadex G-200 columns before antigen A, regardless of the GDzB,,elution profile. Since antigens B, C, and D were detected in the void volume (peak I) of Sephadex G-200 columns, further fractionation of these antigens was attempted on Sepharose 4B. Seven tumor and two normal extracts were chromatographed on Sepharose 4B. Peak fractions were combined, concentrated, and tested for antigenic activity with absorbed anti-MC antiserum. The results were presented in Fig. 2. There are three major peaks (IV, V, and VI) at V,No of 1.0, 2.4, and 2.8 which contain 80% or more of the applied protein. The relatively broad interpeak IV-V region contained less than 1% of the applied protein. Two distinct protein elution profiles, type ia and type iia, were observed. When tested on Sepharose 4B, all tumor extracts which exhibited type i behavior on Sephadex G-200 were found to be type ia. That is, over 50% of the protein was excluded from both Sephadex G-200 and Sepharose 4B. Type iia extracts, on the other hand, contained less than 1% of the applied protein in peak IV. All type ii (three tumor and two normal) extracts tested on Sepharose 4B exhibited type iia elution profiles. Antigens B or C and D were detectable in the interpeak IV-V region in spite of the fact that there was very little protein as indicated by absorbance at 280 nm.

OVARIAN PEAKS ANTIGENS

TUMOR-ASSOCIATED IV M

INTERPEAK WV BorC.0

ANTIGENS

V CD

171

VI A,0 s

TYPE ia PATIENT

NY

Y

EFFLUENT

VOLUME

(ml)

FIG. 2. Sepharose 4B chromatography of tumor perchloric acid extracts from patients NY and MH. The range of the percentages of total protein applied to the column which eluted in each peak is given for all extracts with each elution pattern (see Results). Tumor antigens detectable by immunoelectrophoresis of concentrated tumor peak fractions against absorbed anti-MC antiserum are also given.

Scans of the material obtained by gel filtration from 200 to 600 nm indicated no correlation between optical density and antigenic activity. Gel electrophoresis. Previous studies in our laboratory indicated that, at least in the case of antigens B and C, SDS PAGE was useful for detecting TAA’s [13]. To further characterize the TAA’s separated by column chromatography, SDS PAGE was carried out on unfractionated PCA extracts and the material eluted in the peak fractions and inter-peak IV-V region from Sephadex G-200 and Sepharose 4B columns. SDS gels of unfractionated normal and tumor PCA extracts stained for protein and glycoprotein are shown in Fig. 3. Glycoprotein bands which stain with PAS but not with Coomassie blue were found near the top of the gel. One of these bands has been designated GP-1 and has been previously described by this laboratory [ 131. GP- 1 was originally believed to be a single glycoprotein band at Rf = 0.01 on 5.6% gels. Immunological studies of GP-I material have indicated that the GP-1 band actually contains two antigens, antigens B and C. One antigen is tumor associated while the other is present in normal tissue at a level one-tenth that in tumor tissue. More recent experiments have indicated that there are in reality one or two bands in the area Rf < 0.05 on 5.6% gels, depending upon the particular extract applied to the gel. These glycoprotein bands are detectable only in tumor and not in normal extracts when 200 ,ug (by weight) of PCA extract is applied per gel.

172

KNAUF AND UKBACH

FIG. 3. SDS PAGE of one normal and six tumor PCA extracts. Protein (200 pccgby weight) was applied per gel. The gels on the left were stained with the Schiff reagent. The gels on the right are the same gels, superstained with Coomassie blue. The normal extract is on the gel farthest to the left of each group of gels. India ink marks the position of the pyronin Y (PY) tracking dye. A scale of mobilities relative to the PY is on the left.

Although the amount (by weight) of normal or tumor PCA extract per gel was equal, tumor extracts appeared to have a greater number of darkly staining protein bands, especially in the regionRf = 0.45 to 0.52. In subsequent experiments with a large number of other normal and tumor extracts, the same results were obtained. Depending upon the extract and quantity of protein applied to the gel, either one very diffuse or two distinct glycoprotein bands were detected at Rf = 0.45 to 0.52. Unlike the glycoprotein bands at R, < 0.05 which stained only with PAS, the

OVARIAN

TUMOR-ASSOCIATED

173

ANTIGENS

glycoprotein bands in the middle of the gels stained with both PAS and Coomassie blue. In addition to the glycoprotein bands, there were also two other Coomassie blue-staining bands in the region Rf = 0.45 to 0.52. To characterize these bands, peak fractions from the Sepharose 4B columns were examined by SDS PAGE. Table 2 lists the differences in the acrylamide-gel staining patterns of normal and tumor peak fractions. TABLE 2 COMPAR~SONOFPROTEINANDGLYCOPROTEINBANDSOFSEPHAROSE 4B FRACTIONSFROMNORMAL AND TUMOR EXTRACTSDETECTABLE BY SDS PAGE ON 5.6% GELS Sepharose 4B fraction9 Stainb

Band (RI)

Interpeak IV-V

Peak IV

PAS

co.05

Tumor

PAS + CB

0.45-0.52

Tumor(ia)d

Tumor (Normal)’ Tumor(iia)d

CB

0.30-0.42 0.42-0.46

-

-

Peak V

Peak VI

-

-

Tumor Normal Turnore -

Tumor Normal Turnore Tumor

a “Tumor” or “normal” indicates that the band was detectable in the fractions of tumor or normal extracts, respectively. (-) Indicates the band was not present. b Gels were stained for glycoprotein with the Schiff reagent (PAS) or for protein with Coomassie blue (CB). c Pink coloration at top of gel. Not a true band. d (ia) and (iia) refer to Sepharose 4B chromatographic patterns (see Fig. 2). e Present only in five of seven tumor fractions.

Apparent molecular weights of the tumor proteins have been determined by comparing the relative acrylamide-gel mobilities of the proteins with those whose molecular weights are known. The glycoproteins at Rf = 0.45 to 0.52 have apparent molecular weights of 4-5.5 x lo4 daltons. Proteins between Rf = 0.30 to 0.42 and R, = 0.42 to 0.46 have apparent molecular weights of 7-9 x 104and 5.5-7 x lo4 daltons, respectively. DISCUSSION Four TAA’s of human ovarian cancer, antigens A, B, C, and D, have been detected in ten tumor extracts and have been fractionated on Sephadex G-200 and Sepharose 4B columns. A summary of the immunological, chromatographic, and acrylamide-gel data for the TAA’s is presented in Table 3. Antigens B and C with p-like mobility in immunoelectrophoresis were the largest antigens separated. They were excluded by Sephadex G-200 and Sepharose 4B but were also present in the second peaks of both columns, indicating considerable heterogeneity. These two antigens were previously shown to be present in the GP-1 band of PAS-stained 5.6% acrylamide gels [12]. One antigen appears to be tumor associated, and the second antigen is present in normal tissue at levels which are approximately one-tenth of those in tumor tissue. It is not yet known which

174

KNAUF

AND

URBACH

TABLE 3 SEPARATION OF OVARIAN TAA’s

Sephadex G-200 fractions Sepharose 4B fractions Acrylamide-gel bands (Rf) Apparent molecular weight (daltons)”

BY COLUMN CHROMATOGRAPHY AND SDS PAGE

A

B or C

D

II, III VI 0.42-0.46 5.5-7 x 104

I, II IV, IV-V, v <0.05 >20 x 104

I, II IV-V, v, VI 0.30-0.42 7-9 x 104

a Determined by SDS PAGE. antigen is C and which is B. Glycoprotein at Rf < 0.05 was observed in gels of all tumor extracts, even though immunodiffusion and immunoelectrophoresis were too insensitive to detect B and C in two of the tumors tested. The glycoproteins at Rf = 0.45 to 0.52 are eluted from Sepharose 4B columns first in type ia tumors, then type iia tumors, and finally in normals. These glycoproteins did not appear to be tumor-associated antigens, although there may be subtle differences in the glycoproteins of normals and tumors, as indicated by their different Sepharose 4B elution patterns. The extracts from patients DS and MH which contained peak VI antigen A in amounts detectable by immunoelectrophoresis also contained at least one Coomassie blue-stained band at Rf = 0.42 to 0.46 (apparent molecular weight = 5.5-7 x lo4 daltons). This indicates that this band probably contains antigen A, or at least a protein or protein fragment which contains some of the antigenic determinants of antigen A. In addition, peak VI of all tumor extracts fractionated (all of which contained antigen A) had at least one band in this region. Similarly, extracts from patients MC, EP, and DS, which contained antigen D detectable by immunoelectrophoresis, and extracts from patients NY, EP, MC, DT, and DS, which contained peak V antigen D, had a Coomassie blue-staining band at Rf = 0.30 to 0.42 (apparent molecular weight = 7-9 x lo4 daltons). MCA extracts, which contained no antigen D demonstrable by immunoelectrophoresis, did not have a Coomassie blue band at R, = 0.30 to 0.42. This investigation employs a nondenaturing system (column chromatography) and a denaturing system (SDS PAGE) to purify and characterize the ovarian TAA’s. The apparent molecular weights of the TAA’s obtained by SDS PAGE are of the order of lo4 to lo5 daltons. The same antigens are eluted from the Sephadex G-200 and Sepharose 4B columns with a much larger apparent molecular weight, probably because of aggregation of proteins and glycoproteins in the nondenaturing system. Largely as a result of this phenomenon, the combination of the two systems was useful for separating the TAA’s. Because the acrylamide-gel staining patterns contained so many overlapping bands in the area Rf = 0.3 to 0.5, it would have been impossible to identify antigens A and D without first fractionating the extracts by column chromatography. Antigens B and C have previously been shown to be universally distributed in tumor extracts 1133,and the data presented here confirm this result. However, one of these j3 antigens is not absolutely tumor specific and we have not yet succeeded in separating the two j3 antigens from each other. It may therefore prove worth-

OVARIAN

TUMOR-ASSOCIATED

ANTIGENS

175

while to examine the other antigens in greater detail, particularly since antigen A is present in all tumors except the tumor from patient MS and antigen D is present in all tumors except the two MCA tumors studied. Now that antigens A and D have been identified on acrylamide gels, it should be possible to elute these antigens from the gels and to prepare antiserum against each antigen in a manner similar to that already described for antigens B and C [ 131.If either or both of the antigens is absolutely tumor specific, they may be used to develop an immunological assay for the early detection of ovarian malignancy. ACKNOWLEDGMENTS The authors wish to thank Drs. R. Sheinin and P. Knauf for reviewing the manuscript.

REFERENCES 1. Gold, P., and Freedman, S. 0. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques, J. Exp. Med. 121,439-462 (1965). 2. Khoo, S. K., and Mackay, E. V. Carcinoembryonic antigen by radioimmunoassay in the detection of recurrence during long-term follow-up of female genital cancer, Cancer 34, 542-548 (1974). 3. Pletsch, Q., and Goldenberg, D. M. Molecular size of carcinoembryonic antigen in the plasma of patients with malignant disease, J. Nat. Cancer Inst. 53, 1201-1204 (1974). 4. Sepp&& M., Pihko, H., and Ruoslahti, E. Carcinoembryonic antigen and alpha fetoprotein in malignant tumors of the female genital tract, Cancer 35, 1377-1381 (1975). 5. Van Nagell, J. R., Pfetsch, Q. A., and Goldenberg, D. M. A study of cyst fluid and plasma carcinoembryonic antigen in patients with cystic ovarian neoplasms, Cancer Res. 35, 14331437 (1975). 6. Bhattacharya, M., and Barlow, J. J. An immunologic comparison between serous cystadenocarcinema of the ovary and other human gynecologic tumors, Amer. J. Obstet. Gynecol. 117, 849-853 (1973). 7. Bhattacharya, M., and Barlow, J. J. Immunologic studies of human serous cystadenocarcinoma of the ovary. Demonstration of tumor-associated antigens, Cancer 31, 588-595 (1973). 8. Bhattacharya, M., Barlow, J. J., Chu, T. M., and Piver, M. S. Tumor-associated antigen(s) from granulosa cell carcinomas of the ovary, Cancer Res. 34, 818-822 (1974). 9. Burton, R. M., Hope, N. J., and Lubbers, L. M. A thermostable antigen of human ovarian cancer (Abstract), Fed. Proc. 34, 1036 (1975). 10. Dorsett, B. H., and Ioachim, H. L. Common antigenic component in ovarian carcinomas: Demonstration by double diffusion and immunofluorescence techniques, Immtmof. Commun. 2, 173-184 (1973). 1I. Gall, S. A., Walling, J., and Pearl, J. Demonstration of tumor-associated antigens in human gynecologic malignancies, Amer. J. Obsret. Gynecol. 115, 387-393 (1973). 12. Knauf, S., and Urbach, G. I. Ovarian tumor-specific antigens, Amer. J. Obstet. Gynecol. 119, 966-970 ( 1974). 13. Knauf, S., and Urbach, G. I. Quantitation of antigens in normal and malignant ovarian tissue, Amer. J. Obstet. Gynecol. 123, 302-304 (1975). 14. Levi, M. M., Keller, S., and Mandl, I. Antigenicity of a papillary serous cystadenocarcinoma tissue homogenate and its fractions, Amer. J. Obstet. Gynecol. 105, 856-861 (1969). 15. Levi, M. M., Parshley, M. S., and Mandl, I. Antigenicity of papillary serous cystadenocarcinema tissue culture cells, Amer. J. Obsret. Gynecol. 102, 433-439 (1968). 16. Munkres, K. D., and Richards, F. M. The purification and properties of Neurospora malate dehydrogenase, Arch. Biochem. Biophys. 109, 466-479 (1965). 17. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biochemistry 10, 26&2617 (1971).