Clin. exp. Immunol. (1976) 23, 436-443.
Clinical significance of antibodies to ovarian antigens; Association with cancer of the genito-urinary tract ANNE P. FORBES, J. R. LAKE & K. J. BLOCH Department of Medicine, Harvard Medical School and Endocrine, Arthritis and Clinical Immunology Units of the Medical Services, Massachusetts General Hospital, Boston, Massachusetts, U.S.A.
(Received 14 July 1975)
SUMMARY
Serum from 491 patients with cancer and from 151 patients of comparable age without recognized cancer was tested by indirect immunofluorescence for antibodies to ovarian antigens. Circulating antibody to the cytoplasm of rabbit ova was found in from 19 to 47%4 of patients with cancer of the ovary, endometrium, kidney, bladder or testis, or with lymphoma, and in only 3.300 of control patients. Antibodies to thecacell antigens were not more common in patients with cancer than in other patients, but within the group of cancer patients, such antibodies were, in all but one case, associated with cancer of the genito-urinary tract or with lymphoma. The anti-thecacell antibodies observed produced a staining pattern indistinguishable from that obtained with the serum of patients with Addison's disease but were not, in the cancer patients, associated with antibodies to adrenal tissue. Tests for antibody to the cytoplasm of ova were more frequently positive in patients with progressive cancers than in patients successfully treated by surgery or radiation, and were seen in patients in whom cancer had recurred following removal of the organ of origin. These findings suggest that the antigen or antigens that evoke antibody to the cytoplasm of ova arise from tumour cells rather than from damage to normal tissue by surgery, tumour invasion or radiation.
INTRODUCTION Circulating antibody to the cytoplasm of ova was first reported from our laboratory by Vallotton & Forbes (1966). This antibody was found in the course of testing sera by indirect immunofluorescence on a new substrate-rabbit ovary. The antibody was demonstrated in a small series of patients who had defective ovaries, premature ovarian failure, infertility, or a history of a pregnancy resulting in miscarriage or the birth of a chromosomally abnormal child. A second antibody to gonadal tissue was described by Irvine (1968) and Irvine, Chan & Scarth (1969), and by Anderson et al. (1968), who detected antibodies to theca and interstitial cells of rabbit and human ovary, interstitial cells of rabbit and human testis, and to human trophoblast, in the serum of patients with Addison's disease of non-tuberculous aetiology. The antibodies were observed in patients who had clinical or histological evidence of ovarian insufficiency in addition to adrenal disease, and they appeared to be directed to antigens shared by the steroid-producing cells of the gonads and adrenals. Correspondence: Dr Anne P. Forbes, Massachusetts General Hospital, Boston, Massachusetts 02114, U.S.A.
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437
We recently noted the association between certain cancers and antibody to an ovarian antigen. Serum from three patients who had neither gonadal nor adrenal insufficiency, but had in common a history of irradiation of the pelvis for carcinoma, was found to contain antibody to the cytoplasm of ova. This observation led us to test for the presence of anti-gonadal antibodies in patients with a variety of tumours. The study was designed to compare the incidence of anti-gonadal antibodies in patients with cancer to the incidence in patients without malignant disease, and to ascertain whether such antibodies might be associated with specific cancers. We further sought to determine whether the antibodies were related to the presence of tumour per se or to damage of normal tissue by tumour or by radiation therapy used in treatment.
CLINICAL MATERIAL Case material was obtained from the Tumour Clinic and the Radiation Therapy Clinic and from the Gynecology Service and the Urology Service of the Massachusetts General Hospital. Serum from 491 patients undergoing radiation treatment or operation for tumours was tested. In addition, serum from 200 unselected hospital patients was tested. The records of the latter patients were reviewed and patients with a history of gonadal disorder, cancer of any organ, or radiation therapy were removed from the list. There remained 151 patients suitable for a control series (category 7 below). The 642 patients tested comprised the following groups: (1) Three hundred and twenty-five patients with cancer of the male or female genital tract or urinary tract, including cancer of the ovary, endometrium, cervix, kidney, bladder, and prostate. Patients in this group were classified as having 'tumour present' if tested before, or within 2 weeks of, operation, or at the start of radiation therapy, or if spread or recurrence of their tumour was recognized. There were 209 patients with 'tumour present'. Patients were classified as 'tumour-free' if all recognized tumour tissue had been surgically removed and there was no evidence of recurrence up to the time of testing. One hundred and sixteen patients were classified as 'tumour-free'. (2) Thirty-seven patients with Hodgkin's disease or other lymphoma treated by radiation. (3) Thirty-four patients receiving pelvic radiation for cancer of the vulva, vagina, or non-genito-urinary organs. Patients with cancer of the breast receiving radiation for metastases to the pelvis were included in this category. (4) Thirty-seven patients with cancer of the breast not involving the pelvis. (5) Forty-two patients undergoing radiation for cancer of the head, neck, or lung, or for sarcoma distant from the pelvis. (6) Sixteen patients with benign ovarian tumours or cysts. Of the 325 patients with cancer in group 1, 198 had received radiation therapy before testing, whereas 127 had not. In the entire series, 58 patients were tested both before and after radiation therapy.
LABORATORY METHODS The indirect-immunofluorescence technique was employed essentially as described by Doniach, Roitt & Couchman (1964). Young, (ca. 41 months) virgin rabbits were found to have ovaries of suitable size and follicle density. Testis tissue was obtained from adult rabbits. Healthy animals were killed by the intravenous injection of air and whole ovaries, or testis slices less than 0 5 cm in thickness, were placed in stoppered tubes, frozen immediately in a dry ice-ethanol mixture and stored at -1 5'C. Frozen sections 8 pm thick were cut on the day of assay. Sera to be tested for antibody to ovary were first absorbed with rabbit liver powder. Twenty-five milligrams of the powder was added to 1 ml of a 1:10 dilution of serum and incubated for 1 hr at room temperature with frequent stirring, followed by centrifugation for 10 min at 1000g. Sera to be assayed for antibody to testicular interstitial cells were tested without absorption at a dilution of 1 :10. Tissue sections were incubated with test sera for 45 min at room temperature in a moist chamber. A second application of dilute serum was added mid-way through the incubation. The slides were rinsed and allowed to stand for 10 min with barbital buffer (pH 7-2) before being incubated for 45 min with a 1:20 dilution of fluoresceinconjugated rabbit anti-human globulin (Sylvana Products Company, Milburn, New Jersey). Each week, 1 ml of stock conjugate was absorbed twice with 100 mg of rat liver powder (Sylvana), centrifuged, and filtered through a Millipore filter (Millipore Corporation, Bedford, Massachusetts) before dilution. After a final 10-min wash with buffer, the slides were mounted in 50% glycerol-buffer mixture and examine ed under a Zeiss fluorescence microscope equipped with an FITC exciter filter. A serum known to be positive
Anne P. Forbes, J. R. Lake & K. J. Bloch
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for antibody to the cytoplasm of ova was included in each assay. The slides were numbered by a technician and not identified by the reader until after the results had been recorded. Fluorescence was characterized as strong, moderately strong, or weak. Weakly positive assays for antibody to cytoplasm of ova were repeated at least once. If not repeatedly positive, they were classified as negative. Weak fluorescence of the theca externa was a common finding and was disregarded. Sera were tested on both ovary and testis tissue. Many sera were also tested on rabbit kidney and human adrenal tissue. Human ovary was found to be unsuitable for the demonstration ofantibody to the cytoplasm of ova because of the high background fluorescence of the stroma. Two specimens of monkey ovary (Macaca mulatta) were examined. Although the background fluorescence was somewhat higher than in rabbit tissue, it was, nevertheless, possible with strongly positive sera to demonstrate reactions with the cytoplasm of ova or of theca cells. Sera that contained anti-mitochondrial antibodies produced strong staining of ovarian stroma as well as of theca and granulosa cells and of testicular germinal epithelium. Antibody specific for theca cells could therefore not be detected in the presence of anti-mitochondrial antibodies. Although comparative assays showed that fluorescein conjugated polyvalent goat antiserum to human immunoglobulin (Behring Diagnostics, Somerville, New Jersey) produced somewhat stronger fluorescence in tests performed with weakly positive human sera, rabbit anti-human globulin was used throughout, inasmuch as the control sera, assayed at the beginning of the study, had been tested with that product.
RESULTS
Antibody to cytoplasm of ova in patients with tumours Four hundred and ninety-one patients with tumours were tested. Their ages ranged from 5 to 87 years, with a mean of 58 years. Serum obtained from these patients yielded eighteen strong or moderately strong, and fifty-two weakly positive tests for antibody to the cytoplasm of ova. The staining pattern obtained with these sera (Fig. la) was indistinguishable from that previously reported. The incidence of the antibody in the series of tumour patients was 14.8% or more than four time the incidence in control patients without gonadal defects, cancer, or radiation therapy; the incidence of positive tests in the 126 patients with cancer of the genital or urinary tract who had not been treated with radiation was 12.7%; the incidence in patients who had received radiation was 8%. There was no increase in the incidence of positive tests among fifty-eight patients who were tested both before and after radiation therapy. For this reason, radiated and non-radiated groups have been combined in most categories (Table 1).
FIG. 1. (a) Section of rabbit ovary incubated with a human serum positive for antibody to the cytoplasm of ova followed by fluorescein-labelled rabbit anti-human globulin. Cytoplasm of ova is fluorescent, granulosa cells and surrounding stroma are dark. (b) Section of rabbit ovary prepared as in (a) with a serum positive for antibody to theca cells. Contents of primary follicles and Graafian follicles are unstained; theca externa and scattered interstitial cells in stroma are fluorescent.
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TABLE 1. Number and percentage of positive (pos.) tests for antibody to cytoplasm of ova in sera of patients in various disease categories Category and status of tumour
No. pts
Cancer of male and female genital tract and urinary tract 19 Cancer of ovary, tumour present 28 Cancer of ovary, 'tumour free' 29 Cancer of endometrium, tumour present 40 Cancer of endometrium, 'tumour free' 45 Cancer of cervix, tumour present 36 Cancer of cervix, 'tumour arrested by radiation' 16 Cancer of cervix, 'tumour free after aperation' 9 Cancer of kidney, tumour present 4 Cancer of kidney, 'tumour free' 42 Cancer of bladder, tumour present 12 Cancer of bladder, 'tumour free' 5 Cancer of testis, tumour present 10 Cancer of testis, 'tumour free' 24 Cancer of prostate, tumour present 6 Cancer of prostate, 'tumour free' Diverse neoplasms 37 Hodgkin's disease, other lymphomas 34 Pelvic radiation for cancer of vagina, vulva or non genitourinary organs 37 Cancer of breastt 30 Cancer of head, neck and lung 12 Sarcomat 16 Benign ovarian tumours or cysts Patients without recognized cancer All diagnoses
151
Strong or Moderate pos.
pos.*
Percentage pos.
4 0 1 0 1 1 0 1 0 4 0 1 1 2 0
9 2 10 2 4 4 1 2 0 8 0 1 1 4 0
47 7 34 5 9 11 6 22 0 19 0 20 10 17 0
2
0
9 4
24 12
0 0 0 0
5 2 0 2
14 7 0 13
0
5
Total
3-3
Includes strong, moderately strong and weak positive tests. t Excepting cases of metastases to pelvic bones treated by radiation.
*
The results of tests for antibody to cytoplasm of ova in the different tumour categories and control patients are listed in Table 1. The categories of cancer of the ovary, endometrium, cervix, kidney, bladder, testis, and prostate have been further subdivided into the subcategories, 'tumour present' and 'tumour free'. Inspection of Table 1 reveals that the strong or moderately strong positive tests occurred in patients who had 'tumour present' in the genital or urinary tract or who had lymphoma. In addition, two of thirty-six patients who appeared to have been successfully treated with radiation alone for cancer of the cervix, had strongly positive tests. The incidence of all positive tests (strong, moderately strong and weak) was 19-47% in the patients with primary tumours present in ovary, endometrium, kidney, bladder or testis. In contrast, the incidence of positive tests in similar patients who were 'tumour free' was O-10%. The incidence of positive tests in patients with lymphoma was 24%. Although the majority of the latter patients had received some radiation to the lower abdomen or groin, and many had also received cytotoxic. drugs which have been reported to cause gonadal damage (Sobrino, Levine & DeConti, 1971; Morgenfeld, 1972), two of the nine with positive tests had had radiation to the neck only, and had not had chemotherapy. The 151 control patients ranged in age from 3 to 88 years with a mean of 52 years. None of the control sera gave a strong or moderately strong test for antibody to the cytoplasm of
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ova. Five of the sera (3.30%) gave weakly positive tests. These five were from: (1) a 15year-old male with multiple operations for ulcerative colitis; (2) a 25-year-old male with rejection of a kidney transplant; (3) a 37-year-old female with cirrhosis of the liver and pancreatitis; (4) a 54-year-old female with arteriosclerosis and depression; (5) a 73-year-old female with a 'benign renal mass'.
Antibody to ovarian theca cells and testicular interstitial cells Strong or moderately strong fluorescence of ovarian theca and interstitial cells was produced by nineteen, or 40%, of the sera of patients with tumours and by eight, or 50 0, of the control sera. Seven of the twenty-seven positive sera, four of them from females, also contained antibody to testicular interstitial cells. Only one other of the 642 sera tested reacted with testicular interstitial cells. Of the twenty-seven positive sera, three yielded positive tests for antibody to cytoplasm of ova. Although there was no significant difference in incidence of antibody to theca cells between cancer patients and control patients, it is of interest that all but one of the nineteen positive tests in patients with tumours were associated with cancer of the genito-urinary tract or with lymphoma. Serum from twelve patients with Addison's disease was tested for comparison. Six contained antibody to theca cells and ovarian interstitial cells. The staining pattern in sections of rabbit ovary was indistinguishable from that obtained with the positive sera of patients with cancer and their controls (Fig. lb). All the patients with Addison's disease also had positive tests for antibody to adrenal tissue, whereas none of the patients with cancer and antitheca cell antibody had antibody to adrenal tissue. One of the patients with cancer had been known to have Addison's disease 2 years before the discovery of a testicular seminoma. This patient's serum contained antibody to theca cells and testicular interstitial cells but not to adrenal antigens. None of the other patients with cancer was suspected of adrenal insufficiency.
DISCUSSION Our tests for circulating antibodies to ovarian antigens in 491 patients with cancer and 151 patients of comparable age without recognized cancer revealed that circulating antibody to the cytoplasm of ova occurs in from 19 to 4700 of patients who harbor certain cancers of the genito-urinary organs or a lymphoma and in only 3.300 of control patients of similar age. Antibodies to theca cell antigens were not found to be more common in patients with cancer than in other patients. Nevertheless, it was interesting that their occurrence within the group of cancer patients was in all but one case associated with cancer of the genitourinary tract or lymphoma. In the course of the study, we confirmed the reports of Irvine (1968), and Irvine et al. (1969) and of Anderson et al. (1968) who observed circulating antibodies in patients with adrenal disease that react with the steroid-producing cells of male and female gonads. The presence in patients undergoing treatment for certain cancers, of an antibody that is uncommon in other patients suggests that an antigen that is not normally encountered by immunocompetent cells has been made available to them as a result of the disease. Several possible sources of antigen, or antigens giving rise to antibody to the cytoplasm of ova were considered. (1) The antigen might be a constituent of normal tissue released into the circulation as a result of their destruction by cancer. This possibility was considered unlikely since positive tests were found in some patients in whom cancer had recurred after total removal of the organ of origin. (2) The antigen might have been produced in either normal or malignant cells as a result of exposure to radiation. This hypothesis could not account for the presence of antibody
Antibodies to ovarian antigens
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to the cytoplasm of ova observed in a number of patients tested prior to radiation treatment. Furthermore, the incidence of positive tests for this antibody was not increased by treatment in the fifty-eight patients who were tested both before and after radiation. An association between radiation therapy and the appearance of antibody to theca cells, by contrast, was not excluded by our data. All but two of the nineteen patients with cancer in whom antibody to theca cells was observed had had irradiation of the pelvic organs or kidney. Only four of the nineteen patients were tested both before and after radiation; of these, three had negative tests for anti-theca cell antibody before radiation. (3) The antigen-evoking antibody to cytoplasm of ova might be a tumour antigen shared by several tumours. Tumour-associated antigens have been found in renal-cell carcinoma (Stjernsward et al., 1970; Burtin & Gendron, 1973), bladder carcinoma (Bubenik et al.,
1970), and carcinoma of the ovary (Levi, Keller & Mandl, 1969). Levi (1971) also observed circulating antibody to ovarian tumour antigens in patients with carcinoma of the ovary. Immunological cross-reactions between antigens from cervical and ovarian cancer were demonstrated by Gall (1973). An antigen common to mouse ova and certain tumours in mice was demonstrated by Baranska, Koldowski & Koprowski (1970) and by Koprowski et al. (1971), who observed fluorescence of tissue-culture cells from a variety of methylcholanthrene- or simian virus 40induced mouse tumours treated with fluorescein-labelled antiserum to unfertilized mouse ova. Some tumour-associated antigens are shared by foetal organs (Gold, 1967), and foetal antigens have been found in patients with tumours ofthe gonads (Mawas etal., 1969), kidney (Kistner & Sonnabend, 1974), a bladder (Guinan et al., 1974) and in a variety of gynaecological malignancies (Disaia, 1975). We have not tested foetal tissue for its reactivity with anti-gonadal antibodies, thus the possibility that the antigen involved is a foetal antigen has not been excluded. The anti-ovarian cytoplasm antibodies are associated with cancers that arise in gonads or structures of Wolffian- or Miillerian-duct origin, which are closely related during embryonic development. In addition to their proximity in the embryo, however, the same organs share certain characteristics in adult life, and these characteristics might involve common antigens. Thus, the epithelium of the endometrium, cervix and bladder are all oestrogen-sensitive and presumably have oestrogen receptors (Jensen & Jacobson, 1960; Baulieu et al. 1971), as do certain oestrogen-dependent tumours (Jensen & Jacobson, 1960). Similarly, kidney, prostate, and germinal epithelium share sensitivity to androgens and have androgen receptors (Mainwaring et al., 1973). Antibodies to theca cells appear to be directed to the steroid-producing cells of adrenals and gonads. These cells carry on similar metabolic activities in adult life, in addition to being of closely related embryonic origins. It seems likely that the anti-gonadal antibodies observed in the present study are directed against tumour antigens, but the characteristics of the antigens remain to be determined. No explanation for the association of anti-gonadal antibodies with Hodgkin's disease or other lymphomas is apparent. Tumour-associated antigens have been demonstrated in Hodgkin's disease (Order, Porter & Hellman, 1971; Long et al., 1973). The corresponding antibodies have been found to cross-react with foetal liver (Katz et al., 1973), but have not been tested against gonadal tissue. (4) The antigen to which antibody to the cytoplasm of ova is directed might be the product, not of tumour cells, but of an abnormal organ that had, as one defect, the production of an antigen not made by normal tissue, and, as another defect, a propensity to develop cancer. The observations that antibody to the cytoplasm of ova occurs in some patients with congenitally defective ovaries and that 'certain congenital gonadal defects predispose to malignancy would be in accord with this explanation. The presence of antibody to the cytoplasm of ova in 3.300, and of antibody to theca cells in 5°/, of hospitalized patients without recognized cancer or gonadal defect, is not surprising. Some organ-specific antibodies are common in the population at large, especially in the
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elderly (Hill, 1961; Diaz-Jouanen, Strickland & Williams, 1975); the appearance of antigonadal antibodies may also be age-related. A second cause may be the presence of unrecognized cancer in the control population. Willis (1967), found clinically unrecognized cancer in 128 of 1000 consecutive autopsies. The design of the present study did not permit a prolonged follow-up of all control patients with positive tests. Therefore, it is not known whether a positive test may indicate the presence of cancer at an early stage. This work was supported in part by grant number 1420-Cl from the Massachusetts Chapter of the American Cancer Society: USPHS grants numbers AM-03564 and AM-05067; and a grant from the Massachusetts Chapter of the Arthritis Foundation and L. H. Bendit Foundation. REFERENCES 1,297 patients without thyroid disease. Brit. ANDERSON, J.R., GOUDIE, R.B., GRAY, K. & med. J. i, 1793. STUART-SMITH, D.A. (1968) Immunological features of idiopathic Addison's disease: an IRVINE, W.J. (1968) Immunological aspects of premature ovarian failure associated with idioantibody to cells producing steroid hormones. pathic Addison's disease. Lancet, ii, 883. Clin. exp. Immunol. 3,107. BARANSKA, W., KOLDOWSKI, P. & KoPROWSKI, H. IRVINE, W.J., CHAN, M.M.W. & SCARTH, L. (1969) The further characterization of autoantibodies (1970) Antigenic study of unfertilized mouse eggs: reactive with extra-adrenal steroid-producing cross reactivity with SV 40-induced antigens. cells in patients with adrenal disorders. Clin. exp. Proc. nat. Acad. Sci. (Wash.), 67, 193. Immunol. 4, 489. BAULIEU, E.E., ALBERGA, A., JUNG, I., LEBEAU, M.C., MERCIER-BODARD, C., MILGROM, E., JENSEN, E.V. & JACOBSON, H.I. (1960) Fate of C., steroid estrogens in target tissue. Biological RAYNAUD-JAMMET, J.P., RAYNAUD, Activities of Steroids in Relation to Cancer ROCHEFORT, H., TRUONG, H. & ROBEL, P. (1971) (ed. by G. Pincus and E. P. Vollmer), p. 161. Metabolism and protein binding of sex steroids in Academic Press, New York. target organs: an approach to the mechanism of hormone action. Recent. Prog. Horni. Res. 27, KATZ, D.H., ORDER, S.E., GRAVES, M. & 351. BENACERRAF, B. (1973) Purification of Hodgkin's BUBENIK, J., PERLMANN, P., HELMSTEIN, K. & disease tumour-associated antigens. Proc. nat. MOBERGER, G. (1970) Cellular and humoral Acad. Sci. (Wash.), 70, 396. immune responses to human urinary bladder KISTNER, G.S. & SONNABEND, W. (1974) Antikorper carcinomas. Int. J. Cancer, 5, 310. gegen ein fetorenales Antigen in Hepatitis-BBURTIN, P. & GENDRON, M.C. (1973) A tumor Patienten Tragern von Nierentumoren und associated antigen in human nephroblastomas. gesunden Individuen Schweiz. Med. Wschr. 104, Proc. nat. Acad. Sci. (Wash.), 70, 2051. 485. DIAZ-JOUANEN, E., STRICKLAND, R.G. & WILLIAMS, KoPROWSKI, H., KOLDOWSKI, P., SAWICKI, W. & R.C. (1975) Studies of human lymphocytes in the BARANSKA, W. (1971) Antigenic study of unnewborn and the aged. Amer. J. Med. 58, 620. fertilized mouse eggs. Proceedings of the First Conference and Workshop on Emnbryonic and DISAIA, P.J., HAVERBACK, D.J., DYCE, B.J. & Foetal Antigens in Cancer (ed. by N. G. Anderson MORROW, C.P. (1975) Carcinoembryonic antigen and J. H. Coggin), p. 291. Distributed by: in patients with gynecologic malignancies. Amer. National, Technical Information Service, U.S. J. Obstet. Gynec. 121, 159. Dept. of Commerce, Springfield, Virginia 22151. DONIACH, D., RoITT, I.M. & COUCHMAN, K.G. (1964) Combined immunofluorescent test for LEVI, M.M., KELLER, S. & MANDL, I. (1969) Antigenicity of a papillary cystadenocardinoma thyroid, gastric and antinuclear autoantibodies tissue homogenate and its fractions. Amer. J. (ANF). Protocol of the techniques used in the Obstet. Gynec. 105, 856. Autoinununity Laboratory of the Middlesex Hospital Medical School, London. LEVI, M.M. (1971) Antigenicity of ovarian and cervical malignancies with a view toward possible GALL, S.A., WALLING, J. & PEARL, J. (1973) immunodiagnosis. Amer. J. Obstet. Gynec. 109, Demonstration of tumour-associated antigens in 689. gynecologic malignancies. Amer. J. Obstet. Gynec. 115, 387. LONG, J.C., AISENBERG, A.C., ZAMECNIK, M.V. & ZAMECNIK, P. (1973) Tumour antigen in tissue GOLD, P. (1967) Circulating antibodies against cultures derived from patients with Hodgkin's carcinoembryonic antigens of the human digestive disease. Proc. nat. Acad. Sci. (Wash.), 70, 1540. system. Cancer, 20, 1663. GUINAN, P., ABLIN, R.J., SADOUGHI, N. & BUSH, MAINWARING, W.I.P., MANGAN, F.R., WILCE, P.A. & MILROY, E.G.P. (1973) A review of current I.M. (1974) Carcinoembryonic-like antigen in research on the binding and mechanism of action urine of patients with carcinoma of bladder and of androgenic steroids, notably 5-a-dihydrotestonormal controls. J. Surg. Oncol. 6, 127. sterone. Receptors for Reproductive Hormones. HILL, O.W. (1961) Thyroglobulin antibodies in
Antibodies to ovarian antigens Advances in Experimental Medicine and Biology, volume 36. Plenum Press, New York. MAWAS, C., KOHEN, M., LEMERLE, J., BUFFE, D., SCHWEISGUTH, 0. & BURTIN, P. (1969) Serum alpha-l-fetoprotein (fetuin), in children with malignant ovarian or testicular teratomas, Int. J. Cancer, 4, 76. MORGENFELD, M.C., GOLDBERG, V., PARISIER, H, & BUGNARD, S.C. (1972) Ovarian lesions due to cytostatic agents during treatment of Hodgkin's disease. Surg. Gynecol. Obstet. 134, 826. ORDER, S.E., PORTER, M. & HELLMAN, S. (1971) Hodgkin's disease: evidence for a tumourassociated antigen. N. Engl. J. Med. 285, 471.
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SOBRINO, L.J., LEVINE, R.A. & DECONTI, R.C. (1971) Amenorrhea in patients with Hodgkin's disease treated with anti-neoplastic agents. Amer. J. Obstet. Gynec. 109, 135. STJERNSWARD, J., ALMGARD, L.E., FRANZEN, S., VON SCHREEB, T. & WADSTR6M, L.B. (1970) Tumor distinctive cellular immunity to renal carcinoma. Clin. exp. Immunol. 6, 963. VALLOTTON, M.B. & FORBES, A.P. (1966) Antibodies to cytoplasm of ova. Lancet, ii, 264. WILLIS, R.A. (1967) Pathology of Tumours. Butterworth, London.
Clinical significance of antibodies to ovarian antigens; Association with cancer of the genito-urinary tract ANNE P. FORBES, J. R. LAKE & K. J. BLOCH Department of Medicine, Harvard Medical School and Endocrine, Arthritis and Clinical Immunology Units of the Medical Services, Massachusetts General Hospital, Boston, Massachusetts, U.S.A.
(Received 14 July 1975)
SUMMARY
Serum from 491 patients with cancer and from 151 patients of comparable age without recognized cancer was tested by indirect immunofluorescence for antibodies to ovarian antigens. Circulating antibody to the cytoplasm of rabbit ova was found in from 19 to 47%4 of patients with cancer of the ovary, endometrium, kidney, bladder or testis, or with lymphoma, and in only 3.300 of control patients. Antibodies to thecacell antigens were not more common in patients with cancer than in other patients, but within the group of cancer patients, such antibodies were, in all but one case, associated with cancer of the genito-urinary tract or with lymphoma. The anti-thecacell antibodies observed produced a staining pattern indistinguishable from that obtained with the serum of patients with Addison's disease but were not, in the cancer patients, associated with antibodies to adrenal tissue. Tests for antibody to the cytoplasm of ova were more frequently positive in patients with progressive cancers than in patients successfully treated by surgery or radiation, and were seen in patients in whom cancer had recurred following removal of the organ of origin. These findings suggest that the antigen or antigens that evoke antibody to the cytoplasm of ova arise from tumour cells rather than from damage to normal tissue by surgery, tumour invasion or radiation.
INTRODUCTION Circulating antibody to the cytoplasm of ova was first reported from our laboratory by Vallotton & Forbes (1966). This antibody was found in the course of testing sera by indirect immunofluorescence on a new substrate-rabbit ovary. The antibody was demonstrated in a small series of patients who had defective ovaries, premature ovarian failure, infertility, or a history of a pregnancy resulting in miscarriage or the birth of a chromosomally abnormal child. A second antibody to gonadal tissue was described by Irvine (1968) and Irvine, Chan & Scarth (1969), and by Anderson et al. (1968), who detected antibodies to theca and interstitial cells of rabbit and human ovary, interstitial cells of rabbit and human testis, and to human trophoblast, in the serum of patients with Addison's disease of non-tuberculous aetiology. The antibodies were observed in patients who had clinical or histological evidence of ovarian insufficiency in addition to adrenal disease, and they appeared to be directed to antigens shared by the steroid-producing cells of the gonads and adrenals. Correspondence: Dr Anne P. Forbes, Massachusetts General Hospital, Boston, Massachusetts 02114, U.S.A.
436
Antibodies to ovarian antigens
437
We recently noted the association between certain cancers and antibody to an ovarian antigen. Serum from three patients who had neither gonadal nor adrenal insufficiency, but had in common a history of irradiation of the pelvis for carcinoma, was found to contain antibody to the cytoplasm of ova. This observation led us to test for the presence of anti-gonadal antibodies in patients with a variety of tumours. The study was designed to compare the incidence of anti-gonadal antibodies in patients with cancer to the incidence in patients without malignant disease, and to ascertain whether such antibodies might be associated with specific cancers. We further sought to determine whether the antibodies were related to the presence of tumour per se or to damage of normal tissue by tumour or by radiation therapy used in treatment.
CLINICAL MATERIAL Case material was obtained from the Tumour Clinic and the Radiation Therapy Clinic and from the Gynecology Service and the Urology Service of the Massachusetts General Hospital. Serum from 491 patients undergoing radiation treatment or operation for tumours was tested. In addition, serum from 200 unselected hospital patients was tested. The records of the latter patients were reviewed and patients with a history of gonadal disorder, cancer of any organ, or radiation therapy were removed from the list. There remained 151 patients suitable for a control series (category 7 below). The 642 patients tested comprised the following groups: (1) Three hundred and twenty-five patients with cancer of the male or female genital tract or urinary tract, including cancer of the ovary, endometrium, cervix, kidney, bladder, and prostate. Patients in this group were classified as having 'tumour present' if tested before, or within 2 weeks of, operation, or at the start of radiation therapy, or if spread or recurrence of their tumour was recognized. There were 209 patients with 'tumour present'. Patients were classified as 'tumour-free' if all recognized tumour tissue had been surgically removed and there was no evidence of recurrence up to the time of testing. One hundred and sixteen patients were classified as 'tumour-free'. (2) Thirty-seven patients with Hodgkin's disease or other lymphoma treated by radiation. (3) Thirty-four patients receiving pelvic radiation for cancer of the vulva, vagina, or non-genito-urinary organs. Patients with cancer of the breast receiving radiation for metastases to the pelvis were included in this category. (4) Thirty-seven patients with cancer of the breast not involving the pelvis. (5) Forty-two patients undergoing radiation for cancer of the head, neck, or lung, or for sarcoma distant from the pelvis. (6) Sixteen patients with benign ovarian tumours or cysts. Of the 325 patients with cancer in group 1, 198 had received radiation therapy before testing, whereas 127 had not. In the entire series, 58 patients were tested both before and after radiation therapy.
LABORATORY METHODS The indirect-immunofluorescence technique was employed essentially as described by Doniach, Roitt & Couchman (1964). Young, (ca. 41 months) virgin rabbits were found to have ovaries of suitable size and follicle density. Testis tissue was obtained from adult rabbits. Healthy animals were killed by the intravenous injection of air and whole ovaries, or testis slices less than 0 5 cm in thickness, were placed in stoppered tubes, frozen immediately in a dry ice-ethanol mixture and stored at -1 5'C. Frozen sections 8 pm thick were cut on the day of assay. Sera to be tested for antibody to ovary were first absorbed with rabbit liver powder. Twenty-five milligrams of the powder was added to 1 ml of a 1:10 dilution of serum and incubated for 1 hr at room temperature with frequent stirring, followed by centrifugation for 10 min at 1000g. Sera to be assayed for antibody to testicular interstitial cells were tested without absorption at a dilution of 1 :10. Tissue sections were incubated with test sera for 45 min at room temperature in a moist chamber. A second application of dilute serum was added mid-way through the incubation. The slides were rinsed and allowed to stand for 10 min with barbital buffer (pH 7-2) before being incubated for 45 min with a 1:20 dilution of fluoresceinconjugated rabbit anti-human globulin (Sylvana Products Company, Milburn, New Jersey). Each week, 1 ml of stock conjugate was absorbed twice with 100 mg of rat liver powder (Sylvana), centrifuged, and filtered through a Millipore filter (Millipore Corporation, Bedford, Massachusetts) before dilution. After a final 10-min wash with buffer, the slides were mounted in 50% glycerol-buffer mixture and examine ed under a Zeiss fluorescence microscope equipped with an FITC exciter filter. A serum known to be positive
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for antibody to the cytoplasm of ova was included in each assay. The slides were numbered by a technician and not identified by the reader until after the results had been recorded. Fluorescence was characterized as strong, moderately strong, or weak. Weakly positive assays for antibody to cytoplasm of ova were repeated at least once. If not repeatedly positive, they were classified as negative. Weak fluorescence of the theca externa was a common finding and was disregarded. Sera were tested on both ovary and testis tissue. Many sera were also tested on rabbit kidney and human adrenal tissue. Human ovary was found to be unsuitable for the demonstration ofantibody to the cytoplasm of ova because of the high background fluorescence of the stroma. Two specimens of monkey ovary (Macaca mulatta) were examined. Although the background fluorescence was somewhat higher than in rabbit tissue, it was, nevertheless, possible with strongly positive sera to demonstrate reactions with the cytoplasm of ova or of theca cells. Sera that contained anti-mitochondrial antibodies produced strong staining of ovarian stroma as well as of theca and granulosa cells and of testicular germinal epithelium. Antibody specific for theca cells could therefore not be detected in the presence of anti-mitochondrial antibodies. Although comparative assays showed that fluorescein conjugated polyvalent goat antiserum to human immunoglobulin (Behring Diagnostics, Somerville, New Jersey) produced somewhat stronger fluorescence in tests performed with weakly positive human sera, rabbit anti-human globulin was used throughout, inasmuch as the control sera, assayed at the beginning of the study, had been tested with that product.
RESULTS
Antibody to cytoplasm of ova in patients with tumours Four hundred and ninety-one patients with tumours were tested. Their ages ranged from 5 to 87 years, with a mean of 58 years. Serum obtained from these patients yielded eighteen strong or moderately strong, and fifty-two weakly positive tests for antibody to the cytoplasm of ova. The staining pattern obtained with these sera (Fig. la) was indistinguishable from that previously reported. The incidence of the antibody in the series of tumour patients was 14.8% or more than four time the incidence in control patients without gonadal defects, cancer, or radiation therapy; the incidence of positive tests in the 126 patients with cancer of the genital or urinary tract who had not been treated with radiation was 12.7%; the incidence in patients who had received radiation was 8%. There was no increase in the incidence of positive tests among fifty-eight patients who were tested both before and after radiation therapy. For this reason, radiated and non-radiated groups have been combined in most categories (Table 1).
FIG. 1. (a) Section of rabbit ovary incubated with a human serum positive for antibody to the cytoplasm of ova followed by fluorescein-labelled rabbit anti-human globulin. Cytoplasm of ova is fluorescent, granulosa cells and surrounding stroma are dark. (b) Section of rabbit ovary prepared as in (a) with a serum positive for antibody to theca cells. Contents of primary follicles and Graafian follicles are unstained; theca externa and scattered interstitial cells in stroma are fluorescent.
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Antibodies to ovarian antigens
TABLE 1. Number and percentage of positive (pos.) tests for antibody to cytoplasm of ova in sera of patients in various disease categories Category and status of tumour
No. pts
Cancer of male and female genital tract and urinary tract 19 Cancer of ovary, tumour present 28 Cancer of ovary, 'tumour free' 29 Cancer of endometrium, tumour present 40 Cancer of endometrium, 'tumour free' 45 Cancer of cervix, tumour present 36 Cancer of cervix, 'tumour arrested by radiation' 16 Cancer of cervix, 'tumour free after aperation' 9 Cancer of kidney, tumour present 4 Cancer of kidney, 'tumour free' 42 Cancer of bladder, tumour present 12 Cancer of bladder, 'tumour free' 5 Cancer of testis, tumour present 10 Cancer of testis, 'tumour free' 24 Cancer of prostate, tumour present 6 Cancer of prostate, 'tumour free' Diverse neoplasms 37 Hodgkin's disease, other lymphomas 34 Pelvic radiation for cancer of vagina, vulva or non genitourinary organs 37 Cancer of breastt 30 Cancer of head, neck and lung 12 Sarcomat 16 Benign ovarian tumours or cysts Patients without recognized cancer All diagnoses
151
Strong or Moderate pos.
pos.*
Percentage pos.
4 0 1 0 1 1 0 1 0 4 0 1 1 2 0
9 2 10 2 4 4 1 2 0 8 0 1 1 4 0
47 7 34 5 9 11 6 22 0 19 0 20 10 17 0
2
0
9 4
24 12
0 0 0 0
5 2 0 2
14 7 0 13
0
5
Total
3-3
Includes strong, moderately strong and weak positive tests. t Excepting cases of metastases to pelvic bones treated by radiation.
*
The results of tests for antibody to cytoplasm of ova in the different tumour categories and control patients are listed in Table 1. The categories of cancer of the ovary, endometrium, cervix, kidney, bladder, testis, and prostate have been further subdivided into the subcategories, 'tumour present' and 'tumour free'. Inspection of Table 1 reveals that the strong or moderately strong positive tests occurred in patients who had 'tumour present' in the genital or urinary tract or who had lymphoma. In addition, two of thirty-six patients who appeared to have been successfully treated with radiation alone for cancer of the cervix, had strongly positive tests. The incidence of all positive tests (strong, moderately strong and weak) was 19-47% in the patients with primary tumours present in ovary, endometrium, kidney, bladder or testis. In contrast, the incidence of positive tests in similar patients who were 'tumour free' was O-10%. The incidence of positive tests in patients with lymphoma was 24%. Although the majority of the latter patients had received some radiation to the lower abdomen or groin, and many had also received cytotoxic. drugs which have been reported to cause gonadal damage (Sobrino, Levine & DeConti, 1971; Morgenfeld, 1972), two of the nine with positive tests had had radiation to the neck only, and had not had chemotherapy. The 151 control patients ranged in age from 3 to 88 years with a mean of 52 years. None of the control sera gave a strong or moderately strong test for antibody to the cytoplasm of
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ova. Five of the sera (3.30%) gave weakly positive tests. These five were from: (1) a 15year-old male with multiple operations for ulcerative colitis; (2) a 25-year-old male with rejection of a kidney transplant; (3) a 37-year-old female with cirrhosis of the liver and pancreatitis; (4) a 54-year-old female with arteriosclerosis and depression; (5) a 73-year-old female with a 'benign renal mass'.
Antibody to ovarian theca cells and testicular interstitial cells Strong or moderately strong fluorescence of ovarian theca and interstitial cells was produced by nineteen, or 40%, of the sera of patients with tumours and by eight, or 50 0, of the control sera. Seven of the twenty-seven positive sera, four of them from females, also contained antibody to testicular interstitial cells. Only one other of the 642 sera tested reacted with testicular interstitial cells. Of the twenty-seven positive sera, three yielded positive tests for antibody to cytoplasm of ova. Although there was no significant difference in incidence of antibody to theca cells between cancer patients and control patients, it is of interest that all but one of the nineteen positive tests in patients with tumours were associated with cancer of the genito-urinary tract or with lymphoma. Serum from twelve patients with Addison's disease was tested for comparison. Six contained antibody to theca cells and ovarian interstitial cells. The staining pattern in sections of rabbit ovary was indistinguishable from that obtained with the positive sera of patients with cancer and their controls (Fig. lb). All the patients with Addison's disease also had positive tests for antibody to adrenal tissue, whereas none of the patients with cancer and antitheca cell antibody had antibody to adrenal tissue. One of the patients with cancer had been known to have Addison's disease 2 years before the discovery of a testicular seminoma. This patient's serum contained antibody to theca cells and testicular interstitial cells but not to adrenal antigens. None of the other patients with cancer was suspected of adrenal insufficiency.
DISCUSSION Our tests for circulating antibodies to ovarian antigens in 491 patients with cancer and 151 patients of comparable age without recognized cancer revealed that circulating antibody to the cytoplasm of ova occurs in from 19 to 4700 of patients who harbor certain cancers of the genito-urinary organs or a lymphoma and in only 3.300 of control patients of similar age. Antibodies to theca cell antigens were not found to be more common in patients with cancer than in other patients. Nevertheless, it was interesting that their occurrence within the group of cancer patients was in all but one case associated with cancer of the genitourinary tract or lymphoma. In the course of the study, we confirmed the reports of Irvine (1968), and Irvine et al. (1969) and of Anderson et al. (1968) who observed circulating antibodies in patients with adrenal disease that react with the steroid-producing cells of male and female gonads. The presence in patients undergoing treatment for certain cancers, of an antibody that is uncommon in other patients suggests that an antigen that is not normally encountered by immunocompetent cells has been made available to them as a result of the disease. Several possible sources of antigen, or antigens giving rise to antibody to the cytoplasm of ova were considered. (1) The antigen might be a constituent of normal tissue released into the circulation as a result of their destruction by cancer. This possibility was considered unlikely since positive tests were found in some patients in whom cancer had recurred after total removal of the organ of origin. (2) The antigen might have been produced in either normal or malignant cells as a result of exposure to radiation. This hypothesis could not account for the presence of antibody
Antibodies to ovarian antigens
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to the cytoplasm of ova observed in a number of patients tested prior to radiation treatment. Furthermore, the incidence of positive tests for this antibody was not increased by treatment in the fifty-eight patients who were tested both before and after radiation. An association between radiation therapy and the appearance of antibody to theca cells, by contrast, was not excluded by our data. All but two of the nineteen patients with cancer in whom antibody to theca cells was observed had had irradiation of the pelvic organs or kidney. Only four of the nineteen patients were tested both before and after radiation; of these, three had negative tests for anti-theca cell antibody before radiation. (3) The antigen-evoking antibody to cytoplasm of ova might be a tumour antigen shared by several tumours. Tumour-associated antigens have been found in renal-cell carcinoma (Stjernsward et al., 1970; Burtin & Gendron, 1973), bladder carcinoma (Bubenik et al.,
1970), and carcinoma of the ovary (Levi, Keller & Mandl, 1969). Levi (1971) also observed circulating antibody to ovarian tumour antigens in patients with carcinoma of the ovary. Immunological cross-reactions between antigens from cervical and ovarian cancer were demonstrated by Gall (1973). An antigen common to mouse ova and certain tumours in mice was demonstrated by Baranska, Koldowski & Koprowski (1970) and by Koprowski et al. (1971), who observed fluorescence of tissue-culture cells from a variety of methylcholanthrene- or simian virus 40induced mouse tumours treated with fluorescein-labelled antiserum to unfertilized mouse ova. Some tumour-associated antigens are shared by foetal organs (Gold, 1967), and foetal antigens have been found in patients with tumours ofthe gonads (Mawas etal., 1969), kidney (Kistner & Sonnabend, 1974), a bladder (Guinan et al., 1974) and in a variety of gynaecological malignancies (Disaia, 1975). We have not tested foetal tissue for its reactivity with anti-gonadal antibodies, thus the possibility that the antigen involved is a foetal antigen has not been excluded. The anti-ovarian cytoplasm antibodies are associated with cancers that arise in gonads or structures of Wolffian- or Miillerian-duct origin, which are closely related during embryonic development. In addition to their proximity in the embryo, however, the same organs share certain characteristics in adult life, and these characteristics might involve common antigens. Thus, the epithelium of the endometrium, cervix and bladder are all oestrogen-sensitive and presumably have oestrogen receptors (Jensen & Jacobson, 1960; Baulieu et al. 1971), as do certain oestrogen-dependent tumours (Jensen & Jacobson, 1960). Similarly, kidney, prostate, and germinal epithelium share sensitivity to androgens and have androgen receptors (Mainwaring et al., 1973). Antibodies to theca cells appear to be directed to the steroid-producing cells of adrenals and gonads. These cells carry on similar metabolic activities in adult life, in addition to being of closely related embryonic origins. It seems likely that the anti-gonadal antibodies observed in the present study are directed against tumour antigens, but the characteristics of the antigens remain to be determined. No explanation for the association of anti-gonadal antibodies with Hodgkin's disease or other lymphomas is apparent. Tumour-associated antigens have been demonstrated in Hodgkin's disease (Order, Porter & Hellman, 1971; Long et al., 1973). The corresponding antibodies have been found to cross-react with foetal liver (Katz et al., 1973), but have not been tested against gonadal tissue. (4) The antigen to which antibody to the cytoplasm of ova is directed might be the product, not of tumour cells, but of an abnormal organ that had, as one defect, the production of an antigen not made by normal tissue, and, as another defect, a propensity to develop cancer. The observations that antibody to the cytoplasm of ova occurs in some patients with congenitally defective ovaries and that 'certain congenital gonadal defects predispose to malignancy would be in accord with this explanation. The presence of antibody to the cytoplasm of ova in 3.300, and of antibody to theca cells in 5°/, of hospitalized patients without recognized cancer or gonadal defect, is not surprising. Some organ-specific antibodies are common in the population at large, especially in the
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elderly (Hill, 1961; Diaz-Jouanen, Strickland & Williams, 1975); the appearance of antigonadal antibodies may also be age-related. A second cause may be the presence of unrecognized cancer in the control population. Willis (1967), found clinically unrecognized cancer in 128 of 1000 consecutive autopsies. The design of the present study did not permit a prolonged follow-up of all control patients with positive tests. Therefore, it is not known whether a positive test may indicate the presence of cancer at an early stage. This work was supported in part by grant number 1420-Cl from the Massachusetts Chapter of the American Cancer Society: USPHS grants numbers AM-03564 and AM-05067; and a grant from the Massachusetts Chapter of the Arthritis Foundation and L. H. Bendit Foundation. REFERENCES 1,297 patients without thyroid disease. Brit. ANDERSON, J.R., GOUDIE, R.B., GRAY, K. & med. J. i, 1793. STUART-SMITH, D.A. (1968) Immunological features of idiopathic Addison's disease: an IRVINE, W.J. (1968) Immunological aspects of premature ovarian failure associated with idioantibody to cells producing steroid hormones. pathic Addison's disease. Lancet, ii, 883. Clin. exp. Immunol. 3,107. BARANSKA, W., KOLDOWSKI, P. & KoPROWSKI, H. IRVINE, W.J., CHAN, M.M.W. & SCARTH, L. (1969) The further characterization of autoantibodies (1970) Antigenic study of unfertilized mouse eggs: reactive with extra-adrenal steroid-producing cross reactivity with SV 40-induced antigens. cells in patients with adrenal disorders. Clin. exp. Proc. nat. Acad. Sci. (Wash.), 67, 193. Immunol. 4, 489. BAULIEU, E.E., ALBERGA, A., JUNG, I., LEBEAU, M.C., MERCIER-BODARD, C., MILGROM, E., JENSEN, E.V. & JACOBSON, H.I. (1960) Fate of C., steroid estrogens in target tissue. Biological RAYNAUD-JAMMET, J.P., RAYNAUD, Activities of Steroids in Relation to Cancer ROCHEFORT, H., TRUONG, H. & ROBEL, P. (1971) (ed. by G. Pincus and E. P. Vollmer), p. 161. Metabolism and protein binding of sex steroids in Academic Press, New York. target organs: an approach to the mechanism of hormone action. Recent. Prog. Horni. Res. 27, KATZ, D.H., ORDER, S.E., GRAVES, M. & 351. BENACERRAF, B. (1973) Purification of Hodgkin's BUBENIK, J., PERLMANN, P., HELMSTEIN, K. & disease tumour-associated antigens. Proc. nat. MOBERGER, G. (1970) Cellular and humoral Acad. Sci. (Wash.), 70, 396. immune responses to human urinary bladder KISTNER, G.S. & SONNABEND, W. (1974) Antikorper carcinomas. Int. J. Cancer, 5, 310. gegen ein fetorenales Antigen in Hepatitis-BBURTIN, P. & GENDRON, M.C. (1973) A tumor Patienten Tragern von Nierentumoren und associated antigen in human nephroblastomas. gesunden Individuen Schweiz. Med. Wschr. 104, Proc. nat. Acad. Sci. (Wash.), 70, 2051. 485. DIAZ-JOUANEN, E., STRICKLAND, R.G. & WILLIAMS, KoPROWSKI, H., KOLDOWSKI, P., SAWICKI, W. & R.C. (1975) Studies of human lymphocytes in the BARANSKA, W. (1971) Antigenic study of unnewborn and the aged. Amer. J. Med. 58, 620. fertilized mouse eggs. Proceedings of the First Conference and Workshop on Emnbryonic and DISAIA, P.J., HAVERBACK, D.J., DYCE, B.J. & Foetal Antigens in Cancer (ed. by N. G. Anderson MORROW, C.P. (1975) Carcinoembryonic antigen and J. H. Coggin), p. 291. Distributed by: in patients with gynecologic malignancies. Amer. National, Technical Information Service, U.S. J. Obstet. Gynec. 121, 159. Dept. of Commerce, Springfield, Virginia 22151. DONIACH, D., RoITT, I.M. & COUCHMAN, K.G. (1964) Combined immunofluorescent test for LEVI, M.M., KELLER, S. & MANDL, I. (1969) Antigenicity of a papillary cystadenocardinoma thyroid, gastric and antinuclear autoantibodies tissue homogenate and its fractions. Amer. J. (ANF). Protocol of the techniques used in the Obstet. Gynec. 105, 856. Autoinununity Laboratory of the Middlesex Hospital Medical School, London. LEVI, M.M. (1971) Antigenicity of ovarian and cervical malignancies with a view toward possible GALL, S.A., WALLING, J. & PEARL, J. (1973) immunodiagnosis. Amer. J. Obstet. Gynec. 109, Demonstration of tumour-associated antigens in 689. gynecologic malignancies. Amer. J. Obstet. Gynec. 115, 387. LONG, J.C., AISENBERG, A.C., ZAMECNIK, M.V. & ZAMECNIK, P. (1973) Tumour antigen in tissue GOLD, P. (1967) Circulating antibodies against cultures derived from patients with Hodgkin's carcinoembryonic antigens of the human digestive disease. Proc. nat. Acad. Sci. (Wash.), 70, 1540. system. Cancer, 20, 1663. GUINAN, P., ABLIN, R.J., SADOUGHI, N. & BUSH, MAINWARING, W.I.P., MANGAN, F.R., WILCE, P.A. & MILROY, E.G.P. (1973) A review of current I.M. (1974) Carcinoembryonic-like antigen in research on the binding and mechanism of action urine of patients with carcinoma of bladder and of androgenic steroids, notably 5-a-dihydrotestonormal controls. J. Surg. Oncol. 6, 127. sterone. Receptors for Reproductive Hormones. HILL, O.W. (1961) Thyroglobulin antibodies in
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