Document jm3rX5zRY9XxZGXVvkx07g9gQ

18 IMMUNOPATHOLOGIC OBSERVATIONS IN LIVER ANGIOSARCOMA Enrique Espinosa, M.D. Department of Pathology University of Louisville School of Medicine Louisville, Kentucky I. INTRODUCTION In hepatic fibrosis and angiosarcoma associated with vinyl chloride exposure of industrial workers, manifestations of the disease could not be detected in most cases until the process was far advanced (1). Normal values of liver function tests were reported in a case with significant vinyl chloride hepa tic fibrosis (2), and only a small percentage of workers of a plant unit where seven cases of liver angiosarcoma were diag nosed had abnormal blood screening tests (3). Thus, conven tional liver function tests do not appear to be sensitive in dicators of vinyl chloride liver disease. Development of more sensitive methods for detecting the disease in early stages would be of great importance. An approach to this may be pro vided by immunologic studies. In such a study the question arises whether the fibrotic and angiosarcomatous livers contain antigens that are different from those present in normal tis sue and whether such changes could stimulate an immunologic response. The purpose of this study was to search for anti genic changes in the angiosarcomatous tissue and to test for possible presence of an antibody response in the host. II. PROCEDURES AND MATERIALS USED A. Patients' Sera and Tissue Specimens Serum samples from B.F. Goodrich Co. workers with histor ies of vinyl chloride exposure of several years included sam ples from two individuals with liver angiosarcoma, ten with liver dysfunction with fibrosis and ten with normal liver 'function tests. The patients with angiosarcoma died and the diagnosis was confirmed at autopsy and portions of tumor and neighboring liver tissues were obtained at autopsy. Patients with liver dysfunction with fibrosis included individuals with 927 CMA 052989 abnormalities in liver function tests and fibrosis detected at biopsy. Tissues were also obtained from coroner's autopsies of healthy individuals a few hours after death by gunshot wounds. B. Tissue Extracts and Antisera Liver angiosarcoma and adjacent liver tissue and post-mor tem tissues considered to be normal were frozen and stored at -70*C until used. Portions were cut, thawed and homogenized in 2-3 volumes of distilled water in a Potter-Elvehjem grinder in an ice bath until a smooth suspension was obtained. After centrifugation at 20,000 x G for 30 min, the supernatant fluid containing the aqueous extract was lyophilized. Albino rab bits were immunized with the tumor or normal liver extracts in Freund's complete adjuvant and sera collected and stored fol lowing procedures detailed elsewhere (4). Reaction of these immune sera with human serum or plasma was eliminated by ab sorption with 100 mg of lyophilized, pooled normal human serum /ml antiserum. Antisera were routinely absorbed in this man ner prior to use. Additional absorption with tissue extracts was carried out with 100 mg lyophilized extract/ml antiserum. Absorptions followed a procedure described previously (5). C. Treatment of Tissue Extracts Enzymatic treatment of tissue extracts was carried out with Pronase and trypsin as previously described (6). Periodate oxidation was done according to Rajam et al. (7). Ammonium sulfate and cold ethanol fractionations were carried out as detailed (8). D. Immunodiffusion and Immunofluorescence Double immunodiffusion was carried out in 0.8% agarose in phosphate-buffered saline pH 7.2 (PBS) containing 0.1% sodium azide. Circular wells, 2 mm in diameter, 3 mm apart were used. Immunoelectrophoresis was performed according to Schei- . .degger (9) using 0.8% agarose in 0.025 M Veronal buffer at pH 82 In immunofluorescent studies cryostat sections of rat kid ney, stomach or intestine and liver (4 microns) were used as substrate for antimitochondrial, antismooth muscle and anti nuclear antibodies. Liver sections from rats exposed 4, 8 and 14 days to 1-2% vinyl chloride for 4 hr/day were also used. The sections were covered with dilutions of patients' sera for 45 min at room temperature, washed twice in PBS for 10 min and then covered with fluorescein conjugated IgG fraction of rab bit anti-human immunoglobulins serum (Cappel Laboratories, Inc.) for 45 min and washed as before prior to examination. Cryostat sections of liver angiosarcoma and liver tissue con-, sidered to be normal (4 microns) were washed twice in PBS for 10 min to wash off nonfixed immunoglobulins. After drying. 928 CMA- 052990 sections were stained with fluorescein conjugated TgG frac tions ci rst-jit. s.-.:i-hu.T.an IgS sertt a.-.d goat anti-huir.au law serum (Cappel Laboratories, Inc.). Sections were washed as above and examined under the fluorescent microscope. Represen tative frozen sections were stained with hematoxylin and ecsin to allow correlation between immunofluorescence and the histo logical findings. The antigenic preservation of the tissues was indicated by the demonstration of their staining by anti nuclear factor according to the indirect immunofluorescent procedure. E. Elution of Tumor-bound IgG The tumor and liver tissues were extracted five times with PBS to wash off nonfixed immunoglobulins and then extracted at pH 2.5 to release bound IgG according to a procedure applied in the elution of renal-bound antibody (10). F. Circulating Tissue Antigens Liver-specific antigen LSA (8), tissue antigens of wide organ distribution (4) and bile antigens (11) were tested in the patients' sera by immunodiffusion as described previously. III. RESULTS A. Angiosarcoma-related Antigen To test for presence of new antigens appearing in liver angiosarcoma, antiangiosarcoma serum was absorbed with human sarum and liver extract and tested by immunodiffusion with ex tracts of both normal liver and angiosarcoma tumor at varying concentrations. This absorption eliminated all reactivity with liver extracts prepared from five normal individuals but not with the angiosarcoma extracts where one line of precipi tation remained (Fig. 1). This line of precipitation could still be seen after additional absorption of the antiserum with kidney extract. In contrast, absorption with the tumor extracts eliminated completely the angiosarcoma-related line of precipitation. Absorption with spleen and lung extracts also eliminated this line of precipitation. Thus, the antigen appeared to be of restricted tissue distribution and not an giosarcoma specific. The antigen was inactivated by trypsin and Pronase and thus appeared to be a protein or closely asso ciated to protein. Incubation of the tumor extracts for 1 hr at 4C in citrate buffer, pH 2.5, resulted in inactivation of the antigen whereas incubation in phosphate buffer, pH 5.0, neutral or alkaline pH up to pH 10.0, did not affect it. The antigen was shown to be relatively thermolabile. Incubation of the tumor extracts for 30 min in PBS at 25 and 56"C did not affect the antigen whereas incubation at 70C and higher 929 CMA 052991 completely inactivated it. The antigen precipitated mainly at 20-30% saturated ammonium sulfate and at ethanol concentra tions of 30-70% (Table I). B. Absence of a Normal Tissue Antigen in Angiosarcoma Antiliver serum absorbed with human serum gave several arcs of precipitation in iimnunoelectrophoresis with extracts of normal liver and angiosarcoma tissue (Fig. 2a). These lines could not be seen following additional absorption of the anti serum with normal liver. In contrast, absorption with liver angiosarcoma extract failed to eliminate one of the arcs of precipitation (Fig. 2b). Thus, the tissue antigen related to this arc of precipitation appeared to be absent in the angiosarcomatous tissue whereas the antigens corresponding to the other arcs were present. The absent antigen in angiosarcoma was shown to be present in kidney and lung extracts in addi tion to liver by absorption and direct inmmnodiffusion tests. Physicochemical characterization studies indicated this anti gen to be unaffected by Pronase and trypsin and inactivated by periodate treatment. The antigen was relatively thermostable withstanding incubation at 70*C for 30 min in PBS. The anti gen was destroyed following incubation of the liver extract in citrate buffer at pH 2.S or lower for 1 hr at 4*C; incubation at pH S.O or higher (up to pH 10.0) did not affect it. This antigen precipitated over a wide range of ammonium sulfate and ethanol concentrations (Table I). C. Angiosarcoma-bound IgG IgG fluorescent staining appeared in a linear pattern in the peripheral portion of the tumor cells suggesting in vivo binding by the tumor of the immunoglobulin. This staining is illustrated in Fig. 3. Fig. 4 shows the angiosarcomatous cells surrounding irregular vascular spaces. Relatively coarse, linear fluorescence was also present along some hepa tic cords and strands of connective tissue. There was no ev idence of IgM. Control post-mortem liver tissue did not show any significant fluorescence of bound IgG. Staining for IgG of the tumor sections did not change after several washings at pH 7.2 indicating that the IgG was firmly bound to the tumor. In contrast, sections showed marked diminution of staining after washing at pH 2.5. Elution of bound IgG from salineextracted tumor homogenates was thus attempted at acid pH. With the five successive saline extractions the amount of sa line soluble IgG gradually diminished to nondetectable levels; and at acid pH bound IgG was released from the homogenate. Similar treatment of liver homogenates did not demonstrate presence of bound IgG (Table II). D. Circulating Autoantibodies and Tissue Antigens Serum autoantibodies to nuclei, mitochondria and smooth muscle were negative in all patients examined. In addition. 930 CMA 052992 serum from these patients did not show reactivity with liver from rats exposed to vinyl chloride. Liver-specific antigen LSA (8), bile antigens (11) and other tissue antigens (4) as sociated with liver damage were not detected in these pa tients. IV. DISCUSSION The immunologic characteristics of cancer have been under intense investigation during recent years, and antigenic dif ferences between normal and malignant tissue are considered to be fundamental factors in the immunologic approach to cancer therapy and diagnosis. Liver angiosarcomatous tissue was thus analyzed in this work for presence of neoantigens, normal tis sue antigens and tumor-bound immunoglobulins. Several normal tissue antigens were found by immunodiffusion to be present in the tumor, but one antigen of rather wide organ distribution was not detected. These findings are in agreement with obser vations in other tumors indicating that tumor cells contain many of the antigens of their original hosts and lack some normal tissue antigens. For example, immunohistochemical studies have shown the loss of kidney antigens in stilbestroland x-ray-induced kidney tumors (12), of skin antigen in 3methylcholanthrene-induced mouse squamous cell carcinoma (13) and of certain muscle antigens in 20-methylcholanthrene-induced rat rabdomyosarcoma (14). By far the most extensively studied class of tumors are the chemically induced hepatomata where deletion of liver antigens have been shown in tumors in duced with 4-dimethylaminoazobenzene (15, 16), diethylnitrosamine (15) and 2-acetomidofluorene in the rat (15, 17) and oaminoazotoluene in the mouse (18). In human carcinoma, loss of. antigens have been reported in squamous cell carcinoma (12, 19), loss o| t)ie ABH blood group isoantigens in some > solid tumors (2D, 21) and of HL-A isoantigen in lymphoma (22). In addition, it has been well documented that as cells trans form from a normal state to malignancy they may gain new anti genic specificities. Tumor-specific transplantation antigens have beet)jjdemonstrated in a number of experimentally induced tumors (23-26) as well as in spontaneous tumors in man (27-' ' ' 29). In the present report immunodiffusion analyses of liver angiosarcoma and other human tissues with rabbit antiangiosar coma serum did not indicate the presence of a tumor-specific antigen but rather of an antigen found in lung and spleen but not in liver and kidney. This antigen is being further char acterized in our laboratory. Of particular interest is the demonstration of tumor-bound IgG by immunofluorescence and elution experiments. This find ing must however be interpreted with caution and should be confirmed in biopsy specimens. The tumor-bound IgG may repre sent specific antitumor antibody, antibody fixed by the tumor tissue "nonspecifically* or part of both. Further speculation is premature until it has been shown that the staining pattern 931 CMA 052993 is due to the deposition of a specific antibody, that the elu ted antibody is specific or until the relevant antigen has been identified. Work is in progress to determine the precise significance of the finding of IgG in the tumor. V. SUMMARY Immunodiffusion analyses of human liver angiosarcoma asso ciated with vinyl chloride exposure indicated presence in the tumor of an antigen not detected in normal liver and kidney but found to be present in lung and spleen. This antigen was shown to be a protein, inactivated by Pronase and trypsin, relatively susceptible to heating and to acid pH and precipi tated mainly at 20-30t saturated ammonium sulfate and at 3070% ethanol concentrations. The tumor was shown to contain several antigenic constituents of normal tissue but one normal tissue antigen was not detected. This antigen was character ised as a substance unaffected by Pronase and trypsin and in activated by periodate. It was relatively thermostable, af fected by acid pH and precipitated over a wide range of ammon ium sulfate and ethanol concentrations. Tumor specimens ob tained at autopsy contained bound IgG as shown by immunofluor escence and elution experiments suggesting possible in vivo binding of IgG to the tumor. Acknowledgements: The author wishes to thank Drs. W. M. Christopherson, G. R. Schrodt and P. H. Carstens for fruitful discussion and advice about the histologic sections and Drs. C. Tamburro and L. Maak for providing serum samples and au topsy material, i also wish to thank Miss M. VanBraun and Mrs. V. Petrey for skilled technical assistance. This work was supported in part by a grant from B. P. Goodrich, Co. 932 CMA 052994 TABLE I. Angiosarcoma-related Antigen and Antigen Absent from the Tumor in Ammonium Sulfate and Ethanol Fractions a Presence of Fraction tested Angiosarcoma-related b antigen Antigen absent from c Angiosarcoma Ammonium sulfate: 0-20% saturation 20-30% saturation 30-50% saturation 50-70% saturation - + +++ ++ Ethanol: 0-20% - ++ 20-30% - +-H- 30-50% 50-70% d SN M* H * +-M++ + a +++, ++, + indicate strength of double diffusion reaction in dilution assay, b Detected in angiosarcoma fractions, c Detected in liver fractions, d SN supernate of the 70% ethanol precipitation, dialyzed and lyophilized- 933 CMA. 052995 TABLE lx. igG in Saline and Acid Extracts of Angiosarcoma and Liver Tissues Weight solid extracted from a Preparation tested 1 gm (wet weight) tissue Presence of IgG (mg) Angiosarcoma: Saline extract 1 29.8 Saline extract 2 8.6 ++ Saline extract 3 6.2 + Saline extract 4 5.6 - Saline extract 5 6.1 - Acid extract 6.1 ++ Livers Saline extract 1 47.4 +++ Saline extract 2 14.5 +++ Saline extract 3 8.6 Saline extract 4 7.4 - Saline extract 5 6.4 - Acid extract 9.0 a Tested by immunodiffusion at concentrations of the eluates ranging up to 2%. Present at concentrations 0.05-0.1% (+++) 0.2-0.51 (++); 1-2% (+); negative at 2% (-). 934 CMA. 052996 FIG. 1. Demonstration of angiosarcoma-relaxed antigen. Peripheral wells have 2-fold serial dilutions of liver angiosarcoma extract (a) and normal liver extract (b). Dilutions are clockwise and start at 100 ag/ml in the upper right well. Central wells in each plate contain rabbit antiangio sarcoma serum absorbed with normal human serum and liver extract. FIG. 2. Demonstration of tissue antigen absent in angiosarcoma, (a) Trough contains rabbit antihuman liver serum absorbed with normal human serum, (b) Trough contains the antiliver serum additionally absorbed with angiosarcoma extract. In both plates top wells have 10* solution of liver extract and lower wells angiosarcoma extract. Anode is to the right. 9 33 CMA 052997 FIG. 3. Immunofluorescent staining of liver angiosarcoma by fluorescein conjugated IgG fraction of rabbit antihuman IgG serum (x 400}. 936 CbiA 052998