GYNECOLOGlC
ONCOLOGY
Circulating MICHAEL Departments
5,
228-232 (1977)
Antibodies in Patients Carcinoma
with Ovarian
A. GERBER, DAVID KOFFLER, AND CARMEL J. COHEN
of Pathology and Obstetrics and Gynecology, Mount Sinai School of Medicine of The City University of New York, New York, New York 10029
Received January 25, 1977 Sera from 91 patients with ovarian malignancy were examined by the indirect and the complement fixation fluorescent antibody techniques for the presence of antibodies to ovarian carcinoma antigens using cryostat sections of four ovarian carcinomas as substrates. Four sera stained the cytoplasm of ovarian carcinoma cells, but three of these also reacted with mitochondria and the fourth with microsomes at the same or at higher titers. The staining of ovarian carcinoma by these four sera was abolished by absorption with purified rat liver mitochondria or microsomes, respectively. Thus, no ovarian carcinoma-specific antibodies were demonstrable by the fluorescent antibody technique. The overall incidence of non-organ-specific autoantibodies was increased in patients with ovarian carcinoma.
INTRODUCTION
There is considerable evidence that cellular and/or humoral immune responses occur in man to a wide variety of tumors [4, 51. In ovarian carcinoma, cellular hypersensitivity to tumor-associated antigens derived from ovarian carcinoma has been demonstrated in several studies [ 1,9]. Furthermore, some reports suggested that sera or effusions of patients with ovarian carcinoma contain antibodies reactive with extracts of ovarian carcinoma tissue cultures [8] or ovarian carcinoma cells [2, 31, but their specificity for ovarian carcinoma has not been demonstrated. In the course of studies designed to detect tumor-associated antigens in ovarian carcinoma we found four human sera which reacted by immunofluorescence with cryostat sections of ovarian carcinoma. In order to test the specificity of these sera, their reactivity with different substrates was studied and absorption experiments were performed. The present report pertains to the characterization of these and other tissue-reactive antibodies in the sera of ovarian carcinoma patients. MATERIALS
AND METHODS
Sera from 91 patients with histologically proven ovarian malignancy were collected prior to or within 2 weeks following operation before any chemotherapy was started. Sera from 67 normal females were used as controls. The sera were stored at -70°C. Tissues from three papillary serous cystadenocarcinomas and one mutinous cystadenocarcinoma of the ovary were obtained at surgery. Rat stomach and kidney and normal human liver, obtained at autopsy, were used as substrates for the determination of autoantibodies. All tissues were snap frozen in dry ice-isopentane and stored at -70°C. 228 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ANTIBODIES
IN
OVARIAN
CARCINOMA
PATIENTS
229
The indirect fluorescent antibody technique was employed to detect serum antibodies to ovarian carcinoma. Sera, at 1:2 and 1: 10 dilutions, to be examined were applied to unfixed, washed cryostat sections for 30 minutes at room temperature, washed in three changes of phosphate-buffered saline, pH 7.2 (PBS), and subsequently treated for 30 min with polyvalent fluoresceinated rabbit anti-human y-globulin. The antiserum had been prepared by immunizing rabbits with Cohn Fraction II from human plasma (E. R. Squibb & Sons, New York) and reacted with myelomatous tissue derived from patients with IgG and IgA myelomata and Waldenstrom’s macroglobulinemia, but it did not stain the ovarian tumor substrates directly. After repeated washings, the specimens were cover-slipped and examined under an Ortholux fluorescence microscope (E. Leitz GmbH, Wetzlar, Germany). Positive sera were tested to an endpoint dilution. Thirty selected sera were tested on acetone-fixed cryostat sections of three ovarian carcinomas. Antibodies to nuclei, mitochondria, microsomes, smooth muscle, and gastric parietal cells were determined using rat stomach and kidney and normal human liver as substrates. Mitochondrial antibodies were considered to be present when a serum showed both gastric parietal cell and renal tubular staining. To amplify the fluorescent antibody reactions, the complement fixation fluorescent antibody technique was employed in parallel with the indirect fluorescent antibody technique. After initial incubation with patients’ sera, fresh human serum was applied to the sections as a source of complement, followed by fluoresceinated rabbit antiserum to the C3 component prepared by immunizing rabbits with purified C3 (kindly supplied by Dr. N. Cooper, Scripps Clinic and Research Foundation, LaJolla, California). Inactivated instead of fresh human serum and sections incubated with fresh human serum without prior application of the patient’s serum, and with rabbit antiserum only, served as controls. The complement fixation fluorescent antibody technique could not be used for the detection of non-organ-specific antibodies, because of nonspecific binding of fresh human serum to rat tissue. Therefore, in addition to the indirect fluorescent antibody technique, a triple layer fluorescent antibody technique was employed. After initial incubation with the patients’ sera, rabbit anti-human y-globulin was applied to the sections followed by fluoresceinated goat anti-rabbit y-globulin prepared by immunizing goats with normal rabbit y-globulin purified by DEAESephadex column chromatography. Sections incubated with rabbit antiserum without prior application of the patient’s serum and with goat antiserum only served as controls. In order to determine the specificity of the antibodies in sera of patients with ovarian carcinoma, positive sera were absorbed two times with an equal volume of purified rat liver mitochondria or microsomes at a protein concentration of 100 mgml (kindly prepared by Dr. A. Cederbaum according to the method of Johnson and Lardy [6]). The absorptions were done at 37°C for 30 min, followed by overnight absorption at 4°C. The precipitates were removed by centrifugation at 2OOOg for 30 min. Incubation of sera with PBS instead of rat liver mitochondria or microsomes served as controls. Sera from two patients with primary biliary cirrhosis and high-titered mitochondrial antibodies were absorbed in parallel. The absorbed and control sera were tested for residual antibody activity for ovarian
230
GERBER, KOFFLER,
carcinoma by the complement indirect fluorescent antibody
AND COHEN
fixation antibody technique technique.
and on rat tissue by the
RESULTS
Reactivity with ovarian carcinoma. Antibody titers of 1:lO or higher were considered as positive inasmuch as sera at lower titers gave nonspecific fluorescence with ovarian tissue. None of 30 selected sera stained fixed cryostat sections of ovarian carcinomas. Only one out of 91 sera from patients with ovarian carcinoma showed cytoplasmic staining of a papillary serous cystadenocarcinoma of the ovary when tested by the indirect fluorescent antibody technique on unfixed cryostat sections (Table 1). This serum and three additional sera reacted with a well-differentiated papillary adenocarcinoma of the ovary at titers ranging from 1: 10 to 1:40 by the complement fixation fluorescent antibody technique (Table 2). The cytoplasm of the carcinoma cells was stained in a homogeneous or finely granular pattern. The nuclei were negative. Only one of these four sera stained the other two ovarian carcinomas, a well-differentiated papillary adenocarcinoma and a mutinous cystadenocarcinoma, as detected by the complement fixation fluorescent antibody technique. Reactivity with other tissues. When tested by the triple layer fluorescent antibody technique, 30 of 91 sera of patients with ovarian carcinoma reacted with human liver and rat kidney or stomach. Of these, twelve sera reacted with gastric parietal cells, nine with smooth muscle, none with nuclei, six with mitochondria, and one with gastric chief cells. Seven sera had more than one antibody. When tested by the indirect fluorescent antibody technique, four sera reacted with mitochondria, three with gastric parietal cells, one with chief cells, and two with hepatic nuclei. Of the four sera which stained ovarian carcinoma, three reacted with mitochondria and one with gastric chief cells at the same or at a higher titer both by the indirect and the complement fixation fluorescent antibody techniques (Table 2). The titer of the three sera with mitochondrial antibody which did not react with ovarian carcinoma was 1: 10. After absorption of the four sera which stained the ovarian carcinoma with purified rat liver mitochondria, the reactivity of three sera with ovarian carcinoma and with mitochondria was abolished. The TABLE
I
REACTIVITY OF SEW WITH OVARIAN CARCINOMAS AS DETERMINED BY THE INDIRECT FLUORESCENT
Sera Total number Reaction with ovarian carcinoma Reaction with normal tissues Gastric parietal cells Mitochondria Microsomes Nuclei Smooth muscle
AND NORMAL TISSUES ANTIBODY TECHNIQUE
Number of ovarian carcinoma sera
Number of control sera
91 4 30 12 6 1 9 9
67 0 4 3 0 0 1 0
ANTIBODIES
TITERS
Serum 1 2 3 4 5-7
IN
OVARIAN
CARCINOMA
PATIENTS
231
TABLE 2 OF SERA WITH ANTIBODIES TO MITOCHONDRIA OR MICROSOMES FROM PATIENTS WITH OVARIAN CARCINOMA Ovarian
carcinoma I:40 I:40 I:10 I:40 -
Mitochondria I:160 1:40 I:40 I:10
Microsomes I:160 -
fourth serum which was not absorbed by mitochondria reacted with gastric chief cells, and absorption of this serum with rat liver microsomes eliminated the reactivity both with gastric chief cells and ovarian carcinoma. Sera from two patients with primary biliary cirrhosis and high-titered mitochondrial antibodies also stained the cytoplasm of ovarian carcinoma cells. Their reactivity both with mitochondria and ovarian carcinoma was also abolished by absorption with rat liver mitochondria. Reactivity of control SCYU. Both the indirect and the triple layer fluorescent antibody techniques revealed binding to rat tissues of four out of 67 sera from healthy females. Three sera contained antibodies to parietal cells and one to nuclei. None reacted with a well-differentiated papillary adenocarcinoma of the ovary by the complement fixation fluorescent antibody technique. DISCUSSION Four of ninety-one sera from patients with ovarian carcinoma manifested antibodies reactive with cryostat sections of ovarian carcinoma using a sensitive in vitro complement fixation technique. These antibodies were shown to be directed against mitochondrial antigens in three sera and microsomal antigens in one serum. Only sera with moderate- or high-titered mitochondrial or microsomal antibodies reacted with tumor cells, but not low-titered mitochondrial antisera, probably because normal tissues represent a better substrate for non-organspecific antibodies than neoplastic tissues. Further investigation of ovarian carcinoma sera revealed an incidence of 33% of non-organ-specific autoantibodies, directed against nuclei (lo%), smooth muscle (IO%), gastric parietal cells (13%), mitochondria (6.6%), and microsomes. In contrast, the incidence of autoantibodies in control sera was 6%. An increased incidence of autoantibodies, particularly to nuclei and smooth muscle, has been demonstrated in patients with other tumors. Sera from patients with a variety of malignant tumors were reported to have an incidence of smooth muscle antibodies of 67.5% and anti-nuclear antibodies of 27% [ 111. Approximately one-third of patients with breast carcinoma manifested smooth muscle and anti-nuclear antibodies [IO]. A lower incidence of autoantibodies (27%) was found in children with Wilm’s tumor [7]. For a limited number of tumors, specific antibodies have been found to be directed against tumor-associated antigens, i.e., malignant melanoma and osteogenic sarcoma [4], but the presence of concomitant autoantibodies in these cases has not been evaluated. The cumulative evidence indicates, therefore, that the predominant autoantibody in the sera of tumor
232
GERBER,
KOFFLER,
AND COHEN
patients is nonspecific and similar in nature to antibodies found in the sera of patients with diseases in which immune mechanisms play a central role. Whether or not the increased incidence of autoantibodies represents a general defect in immune surveillance or is a nonspecific response to immunization with foreign tumor antigens is a subject worthy of further exploration. Studies are presently being conducted to determine if the cellular hypersensitivity found in patients with ovarian carcinoma [l] is correlated with the presence of autoantibodies. One additional consideration derives from the current study. The presence of non-organ-specific autoantibodies may interfere with the evaluation of tumorspecific antibodies when groups of tumors are uniquely rich in mitochondria or endoplasmic reticulum. Therefore, investigation of antibody activity with normal cell organelles should be included in addition to experiments using homologous and unrelated tumors for demonstration of antibody specificity in the sera of tumor patients. It cannot be ruled out that the exhaustive absorption procedures employed in this study resulted in physical loss of low-titered antibodies specific for ovarian carcinoma. More sensitive techniques are needed for the demonstration of these antibodies. In addition, antibodies to cell surface antigens of tumor cells are not detected by the techniques employed in this study. ACKNOWLEDGMENTS The expert technical assistance of Mrs. Minda Gerber is gratefully acknowledged. This work was supported by Mount Sinai Gynecological Cancer Research Fund and United States Public Health Service Grant No. AM 13 721-03. Dr. Gerber is the recipient of a Research Career Development Award, No. I K04 Al 00035-01, from the United States Public Health Service.
REFERENCES I. Chen, S. Y., Koffler, D., and Cohen, C. J. Cell-mediated immunity in patients with ovarian carcinoma, Amer. J. Obstet. Gynecol. 115, 467-470 (1973). 2. Disaia, P. J., Nalick, R. H., and Townsend, D. E. Antibody cytotoxicity studies in ovarian and cervical malignancies, Obstet. Gynecol. 42, 644-650 (1973). 3. Dorsett, B. H., Ioachim, H. L., Stolbach, L., Walker, J., and Barber, H. R. K. Isolation of tumor-specific antibodies from effusions of ovarian carcinoma, ht. J. Cancer 16, 779-786 (1975). 4. Hellstrom, K. E., and Hellstrom, I. Immunity to neuroblastomas and melanomas, Annu. Rev. Med. 23, 19-38 (1972). 5. Herberman, R. B. Cell-mediated immunity to tumor cells, Advan. Cancer Res. 19, 207-253 (1974). 6. Johnson, D., and Lardy, H. Cell fractionation. (Estabrook, R. W., and Pullman, M. E., Eds.), in Methods in enzymology, Academic Press, New York and London, Vol. 10, pp. 94-99 (1967). 7. Kumar, S., and Taylor, G. Non-organ specific and tumor-specific antibodies in children with Wilm’s tumour, ht. J. Cancer 16, 448-455 (1975). 8. Levi, M. M. Antigenicity of ovarian and cervical malignancies with a view toward possible immunodiagnosis, Amer. J. Obstet. Gynecol. 109, 689-698 (1971). 9. Melnick, H., and Barber, H. R. K. Cellular immunogenic responsiveness to extracts of ovarian epithelial tumors, Gynecol. Oncol. 3, 77-84 (1975). IO. Wasserman, J., Glas, U., and Blomgren, H. Autoantibodies in patients with carcinoma of the breast, C/in. Exp. Immunol. 19, 417-422 (1975). I I. Whitehouse, J. M. A. Circulating antibodies in human malignant disease, Bit. J. Cancer 28, 170-174 (1973).
ONCOLOGY
Circulating MICHAEL Departments
5,
228-232 (1977)
Antibodies in Patients Carcinoma
with Ovarian
A. GERBER, DAVID KOFFLER, AND CARMEL J. COHEN
of Pathology and Obstetrics and Gynecology, Mount Sinai School of Medicine of The City University of New York, New York, New York 10029
Received January 25, 1977 Sera from 91 patients with ovarian malignancy were examined by the indirect and the complement fixation fluorescent antibody techniques for the presence of antibodies to ovarian carcinoma antigens using cryostat sections of four ovarian carcinomas as substrates. Four sera stained the cytoplasm of ovarian carcinoma cells, but three of these also reacted with mitochondria and the fourth with microsomes at the same or at higher titers. The staining of ovarian carcinoma by these four sera was abolished by absorption with purified rat liver mitochondria or microsomes, respectively. Thus, no ovarian carcinoma-specific antibodies were demonstrable by the fluorescent antibody technique. The overall incidence of non-organ-specific autoantibodies was increased in patients with ovarian carcinoma.
INTRODUCTION
There is considerable evidence that cellular and/or humoral immune responses occur in man to a wide variety of tumors [4, 51. In ovarian carcinoma, cellular hypersensitivity to tumor-associated antigens derived from ovarian carcinoma has been demonstrated in several studies [ 1,9]. Furthermore, some reports suggested that sera or effusions of patients with ovarian carcinoma contain antibodies reactive with extracts of ovarian carcinoma tissue cultures [8] or ovarian carcinoma cells [2, 31, but their specificity for ovarian carcinoma has not been demonstrated. In the course of studies designed to detect tumor-associated antigens in ovarian carcinoma we found four human sera which reacted by immunofluorescence with cryostat sections of ovarian carcinoma. In order to test the specificity of these sera, their reactivity with different substrates was studied and absorption experiments were performed. The present report pertains to the characterization of these and other tissue-reactive antibodies in the sera of ovarian carcinoma patients. MATERIALS
AND METHODS
Sera from 91 patients with histologically proven ovarian malignancy were collected prior to or within 2 weeks following operation before any chemotherapy was started. Sera from 67 normal females were used as controls. The sera were stored at -70°C. Tissues from three papillary serous cystadenocarcinomas and one mutinous cystadenocarcinoma of the ovary were obtained at surgery. Rat stomach and kidney and normal human liver, obtained at autopsy, were used as substrates for the determination of autoantibodies. All tissues were snap frozen in dry ice-isopentane and stored at -70°C. 228 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ANTIBODIES
IN
OVARIAN
CARCINOMA
PATIENTS
229
The indirect fluorescent antibody technique was employed to detect serum antibodies to ovarian carcinoma. Sera, at 1:2 and 1: 10 dilutions, to be examined were applied to unfixed, washed cryostat sections for 30 minutes at room temperature, washed in three changes of phosphate-buffered saline, pH 7.2 (PBS), and subsequently treated for 30 min with polyvalent fluoresceinated rabbit anti-human y-globulin. The antiserum had been prepared by immunizing rabbits with Cohn Fraction II from human plasma (E. R. Squibb & Sons, New York) and reacted with myelomatous tissue derived from patients with IgG and IgA myelomata and Waldenstrom’s macroglobulinemia, but it did not stain the ovarian tumor substrates directly. After repeated washings, the specimens were cover-slipped and examined under an Ortholux fluorescence microscope (E. Leitz GmbH, Wetzlar, Germany). Positive sera were tested to an endpoint dilution. Thirty selected sera were tested on acetone-fixed cryostat sections of three ovarian carcinomas. Antibodies to nuclei, mitochondria, microsomes, smooth muscle, and gastric parietal cells were determined using rat stomach and kidney and normal human liver as substrates. Mitochondrial antibodies were considered to be present when a serum showed both gastric parietal cell and renal tubular staining. To amplify the fluorescent antibody reactions, the complement fixation fluorescent antibody technique was employed in parallel with the indirect fluorescent antibody technique. After initial incubation with patients’ sera, fresh human serum was applied to the sections as a source of complement, followed by fluoresceinated rabbit antiserum to the C3 component prepared by immunizing rabbits with purified C3 (kindly supplied by Dr. N. Cooper, Scripps Clinic and Research Foundation, LaJolla, California). Inactivated instead of fresh human serum and sections incubated with fresh human serum without prior application of the patient’s serum, and with rabbit antiserum only, served as controls. The complement fixation fluorescent antibody technique could not be used for the detection of non-organ-specific antibodies, because of nonspecific binding of fresh human serum to rat tissue. Therefore, in addition to the indirect fluorescent antibody technique, a triple layer fluorescent antibody technique was employed. After initial incubation with the patients’ sera, rabbit anti-human y-globulin was applied to the sections followed by fluoresceinated goat anti-rabbit y-globulin prepared by immunizing goats with normal rabbit y-globulin purified by DEAESephadex column chromatography. Sections incubated with rabbit antiserum without prior application of the patient’s serum and with goat antiserum only served as controls. In order to determine the specificity of the antibodies in sera of patients with ovarian carcinoma, positive sera were absorbed two times with an equal volume of purified rat liver mitochondria or microsomes at a protein concentration of 100 mgml (kindly prepared by Dr. A. Cederbaum according to the method of Johnson and Lardy [6]). The absorptions were done at 37°C for 30 min, followed by overnight absorption at 4°C. The precipitates were removed by centrifugation at 2OOOg for 30 min. Incubation of sera with PBS instead of rat liver mitochondria or microsomes served as controls. Sera from two patients with primary biliary cirrhosis and high-titered mitochondrial antibodies were absorbed in parallel. The absorbed and control sera were tested for residual antibody activity for ovarian
230
GERBER, KOFFLER,
carcinoma by the complement indirect fluorescent antibody
AND COHEN
fixation antibody technique technique.
and on rat tissue by the
RESULTS
Reactivity with ovarian carcinoma. Antibody titers of 1:lO or higher were considered as positive inasmuch as sera at lower titers gave nonspecific fluorescence with ovarian tissue. None of 30 selected sera stained fixed cryostat sections of ovarian carcinomas. Only one out of 91 sera from patients with ovarian carcinoma showed cytoplasmic staining of a papillary serous cystadenocarcinoma of the ovary when tested by the indirect fluorescent antibody technique on unfixed cryostat sections (Table 1). This serum and three additional sera reacted with a well-differentiated papillary adenocarcinoma of the ovary at titers ranging from 1: 10 to 1:40 by the complement fixation fluorescent antibody technique (Table 2). The cytoplasm of the carcinoma cells was stained in a homogeneous or finely granular pattern. The nuclei were negative. Only one of these four sera stained the other two ovarian carcinomas, a well-differentiated papillary adenocarcinoma and a mutinous cystadenocarcinoma, as detected by the complement fixation fluorescent antibody technique. Reactivity with other tissues. When tested by the triple layer fluorescent antibody technique, 30 of 91 sera of patients with ovarian carcinoma reacted with human liver and rat kidney or stomach. Of these, twelve sera reacted with gastric parietal cells, nine with smooth muscle, none with nuclei, six with mitochondria, and one with gastric chief cells. Seven sera had more than one antibody. When tested by the indirect fluorescent antibody technique, four sera reacted with mitochondria, three with gastric parietal cells, one with chief cells, and two with hepatic nuclei. Of the four sera which stained ovarian carcinoma, three reacted with mitochondria and one with gastric chief cells at the same or at a higher titer both by the indirect and the complement fixation fluorescent antibody techniques (Table 2). The titer of the three sera with mitochondrial antibody which did not react with ovarian carcinoma was 1: 10. After absorption of the four sera which stained the ovarian carcinoma with purified rat liver mitochondria, the reactivity of three sera with ovarian carcinoma and with mitochondria was abolished. The TABLE
I
REACTIVITY OF SEW WITH OVARIAN CARCINOMAS AS DETERMINED BY THE INDIRECT FLUORESCENT
Sera Total number Reaction with ovarian carcinoma Reaction with normal tissues Gastric parietal cells Mitochondria Microsomes Nuclei Smooth muscle
AND NORMAL TISSUES ANTIBODY TECHNIQUE
Number of ovarian carcinoma sera
Number of control sera
91 4 30 12 6 1 9 9
67 0 4 3 0 0 1 0
ANTIBODIES
TITERS
Serum 1 2 3 4 5-7
IN
OVARIAN
CARCINOMA
PATIENTS
231
TABLE 2 OF SERA WITH ANTIBODIES TO MITOCHONDRIA OR MICROSOMES FROM PATIENTS WITH OVARIAN CARCINOMA Ovarian
carcinoma I:40 I:40 I:10 I:40 -
Mitochondria I:160 1:40 I:40 I:10
Microsomes I:160 -
fourth serum which was not absorbed by mitochondria reacted with gastric chief cells, and absorption of this serum with rat liver microsomes eliminated the reactivity both with gastric chief cells and ovarian carcinoma. Sera from two patients with primary biliary cirrhosis and high-titered mitochondrial antibodies also stained the cytoplasm of ovarian carcinoma cells. Their reactivity both with mitochondria and ovarian carcinoma was also abolished by absorption with rat liver mitochondria. Reactivity of control SCYU. Both the indirect and the triple layer fluorescent antibody techniques revealed binding to rat tissues of four out of 67 sera from healthy females. Three sera contained antibodies to parietal cells and one to nuclei. None reacted with a well-differentiated papillary adenocarcinoma of the ovary by the complement fixation fluorescent antibody technique. DISCUSSION Four of ninety-one sera from patients with ovarian carcinoma manifested antibodies reactive with cryostat sections of ovarian carcinoma using a sensitive in vitro complement fixation technique. These antibodies were shown to be directed against mitochondrial antigens in three sera and microsomal antigens in one serum. Only sera with moderate- or high-titered mitochondrial or microsomal antibodies reacted with tumor cells, but not low-titered mitochondrial antisera, probably because normal tissues represent a better substrate for non-organspecific antibodies than neoplastic tissues. Further investigation of ovarian carcinoma sera revealed an incidence of 33% of non-organ-specific autoantibodies, directed against nuclei (lo%), smooth muscle (IO%), gastric parietal cells (13%), mitochondria (6.6%), and microsomes. In contrast, the incidence of autoantibodies in control sera was 6%. An increased incidence of autoantibodies, particularly to nuclei and smooth muscle, has been demonstrated in patients with other tumors. Sera from patients with a variety of malignant tumors were reported to have an incidence of smooth muscle antibodies of 67.5% and anti-nuclear antibodies of 27% [ 111. Approximately one-third of patients with breast carcinoma manifested smooth muscle and anti-nuclear antibodies [IO]. A lower incidence of autoantibodies (27%) was found in children with Wilm’s tumor [7]. For a limited number of tumors, specific antibodies have been found to be directed against tumor-associated antigens, i.e., malignant melanoma and osteogenic sarcoma [4], but the presence of concomitant autoantibodies in these cases has not been evaluated. The cumulative evidence indicates, therefore, that the predominant autoantibody in the sera of tumor
232
GERBER,
KOFFLER,
AND COHEN
patients is nonspecific and similar in nature to antibodies found in the sera of patients with diseases in which immune mechanisms play a central role. Whether or not the increased incidence of autoantibodies represents a general defect in immune surveillance or is a nonspecific response to immunization with foreign tumor antigens is a subject worthy of further exploration. Studies are presently being conducted to determine if the cellular hypersensitivity found in patients with ovarian carcinoma [l] is correlated with the presence of autoantibodies. One additional consideration derives from the current study. The presence of non-organ-specific autoantibodies may interfere with the evaluation of tumorspecific antibodies when groups of tumors are uniquely rich in mitochondria or endoplasmic reticulum. Therefore, investigation of antibody activity with normal cell organelles should be included in addition to experiments using homologous and unrelated tumors for demonstration of antibody specificity in the sera of tumor patients. It cannot be ruled out that the exhaustive absorption procedures employed in this study resulted in physical loss of low-titered antibodies specific for ovarian carcinoma. More sensitive techniques are needed for the demonstration of these antibodies. In addition, antibodies to cell surface antigens of tumor cells are not detected by the techniques employed in this study. ACKNOWLEDGMENTS The expert technical assistance of Mrs. Minda Gerber is gratefully acknowledged. This work was supported by Mount Sinai Gynecological Cancer Research Fund and United States Public Health Service Grant No. AM 13 721-03. Dr. Gerber is the recipient of a Research Career Development Award, No. I K04 Al 00035-01, from the United States Public Health Service.
REFERENCES I. Chen, S. Y., Koffler, D., and Cohen, C. J. Cell-mediated immunity in patients with ovarian carcinoma, Amer. J. Obstet. Gynecol. 115, 467-470 (1973). 2. Disaia, P. J., Nalick, R. H., and Townsend, D. E. Antibody cytotoxicity studies in ovarian and cervical malignancies, Obstet. Gynecol. 42, 644-650 (1973). 3. Dorsett, B. H., Ioachim, H. L., Stolbach, L., Walker, J., and Barber, H. R. K. Isolation of tumor-specific antibodies from effusions of ovarian carcinoma, ht. J. Cancer 16, 779-786 (1975). 4. Hellstrom, K. E., and Hellstrom, I. Immunity to neuroblastomas and melanomas, Annu. Rev. Med. 23, 19-38 (1972). 5. Herberman, R. B. Cell-mediated immunity to tumor cells, Advan. Cancer Res. 19, 207-253 (1974). 6. Johnson, D., and Lardy, H. Cell fractionation. (Estabrook, R. W., and Pullman, M. E., Eds.), in Methods in enzymology, Academic Press, New York and London, Vol. 10, pp. 94-99 (1967). 7. Kumar, S., and Taylor, G. Non-organ specific and tumor-specific antibodies in children with Wilm’s tumour, ht. J. Cancer 16, 448-455 (1975). 8. Levi, M. M. Antigenicity of ovarian and cervical malignancies with a view toward possible immunodiagnosis, Amer. J. Obstet. Gynecol. 109, 689-698 (1971). 9. Melnick, H., and Barber, H. R. K. Cellular immunogenic responsiveness to extracts of ovarian epithelial tumors, Gynecol. Oncol. 3, 77-84 (1975). IO. Wasserman, J., Glas, U., and Blomgren, H. Autoantibodies in patients with carcinoma of the breast, C/in. Exp. Immunol. 19, 417-422 (1975). I I. Whitehouse, J. M. A. Circulating antibodies in human malignant disease, Bit. J. Cancer 28, 170-174 (1973).
