International Uroloyy and Nephroloyy 24 (3), pp. 255-- 264 (1992)
Detection of Blood Group Surface Antigens of Urinary Bladder Tumours Using Monoclonal Antibodies with the Avidin-Biotin Complex Technique L.-B. TAN,* C. CHIANG, C.-H. HUANG, L.-M. LIN + +Departments of Urology and Oral Pathology, Provincial Tainan Hospital; *Kaohsiung Medical College Hospital, Taiwan, Republic of China (Accepted July 17, 1991) We examined 8 normal bladder transitional epithelia and 65 transitional cell carcinomas of the urinary bladder of various stages and grades for the presence of ABO(H) blood group surface isoantigens using the immunoperoxidase staining via the Avidin-Biotin Complex (ABC) methods. It was found that 27~ of patients with grade I tumours, 50 % with grade II tumours and 82 ~ with grade III turnours had loss of cell surface isoantigens. When correlated with the clinical stage the tumours showed no surface isoantigens in stage D, while 65 % of patients with stage A tumours were positive for surface antigens. From among 37 patients (57 %), 28 (43 %) survived for more than five years. Our results suggest that surface antigens of transitional cell carcinoma of the urinary bladder tended to disappear as the histologic changes of the tumour progressed. It also was noted that a loss of ABH(O) surface isoantigens was a bad prognostic sign.
Introduction Blood group substance is a kind of glycosamines attached to the surface membrane of cells. Under normal conditions, it exists in erythrocytes, endothelial cells, epithelial cells of the bladder, lung, cervix, gastrointestinal tract, Fallopian tube and pancreas [1 ]. Once these organs undergo malignant changes, adherence o f blood group substance on these normal epithelia decreases and subsequently disappears [1-6]. The loss of such surface antigen substance is closely related to the pathological grade as well as the clinical stage o f the tumour [2-7]. Catalona [8] has found that 66% of turnouts deprived of surface antigen underwent invasive recurrence, whereas of those with positive surface antigens only 4 % showed recurrence. Consequently, emphasis was gradually put on the detection of ABO isoantigens. In the past, many researchers adopted the SRCA (Specific Red Cell Adherence) method to detect these surface isoantigens [1-8]. However, this technique had a lot of drawbacks. For example, interpretation of the results of experiments was too subjective, the rate of false negative reactivity was too high and unpredictable [9], and the sections failed to be fixed and preserved permanently [10]. At present, investigators are using anti-A, anti-B and anti-H blood group monoclonal antibodies with the immunohistochemical staining technique to detect transiVSP, ,Utrecht Akad$miai Kiad6, Budapest
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tional epithelium in both normal and malignant bladder tumours, which is found much better than the SRCA method [11, 12]. The purpose of this study has been to observe the surface isoantigens on transitional cell carcinomas of the bladder by using anti-A, anti-B and anti-H blood group monoclonal antibodies with the immunohistochemical staining technique.
Materials and methods
In the period 1978 to 1983, the Department of Urology of Kaohsiung Medical College Hospital staff had performed surgical treatment in 65 patients with transitional cell carcinoma of the urinary bladder. The tissue specimens were fixed with formalin and embedded in paraffin blocks. Of these 65 cases, 26 belonged to O type, 20 to A type and 19 to B type blood groups. All patients underwent follow-up examination for more than five years. The paraffin blocks of the tissue specimens were cut into 5 #m thick slices and placed on slides as a routine histologic process. Each paraffin block was cut into at least three sections, one of which was used for haematoxylin and eosin staining, the other two for immunoperoxidase staining. At the same time, we selected the transitional epithelia of eight normal urinary bladders as the control group, of which 5 were blood type A and 3 were type B. The tissue processing and staining procedures were as follows: 1. The serial sections were deparaffinized, washed and dehydrated. 2. The slides were placed in absolute methyl alcohol-hydrogen peroxidase solution for 25 min in order to block endogenous peroxidase activity. 3. The slides were washed in three changes with 0.05 M Tris buffer solution (TBS) pH 7.6 for 15 rain, 5 rain each time. 4. According to suggestions of Vectastain ABC Kit (Vector Laboratories, Burlingame, Calif.) non-blood group antigens were neutralized by adding blocking antibodies (non-specific goat antihuman antibody in this study) to the slide for 20 rain. 5. The slides were washed again in three changes with TBS pH 7.6 for 15 rain, 5 min each time. 6. Mouse monoclonal anti-A, anti-B, and anti-H antibodies were used as primary antibody (prepared by DAKO) and added to the slides for 24 hours. 7. The slides were washed in three changes with TBS pH 7.6 for 15 min, 5 rain each time. 8. In conformity with the instructions of Vectastain ABC Kit (Vector Laboratories, Burlingame, Calif.), the biotinylated goat IgG antibodies were put on the slides for 30 min. 9. The slides were washed again in three changes with TBS pH 7.6 for 15 rain, 5 min each time. Then the Avidin Biotin-peroxidase complex was added to the slides for 60 min. Then, 3-amino-9-ethyl carbazole (Sigma Chemical Co., St. Louis, Mo.) was added to the slide for 5 min in order to identify the site of peroxidase localization. International Urology and Nephrology 24, 1992
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Fig. 1. Papillary transitional celt carcinoma of the urinary bladder. After application of monoclonal antibody and Avidin-Biotin Complex staining technique, the whole tumorous epithelium bore positive reaction, so did the vascular endothelium which was regarded as internal positive control
Fig. 2. Another papillary transitional cell carcinoma of the urinary bladder. The use of monoclonal antibody and Avidin-13iotin Complex staining technique revealed only about 3 0 ~ of cancerous epithelium reacting positively International Urology and Nephrology 24, 1992
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Fig. 3. Another papillary transitional cell carcinoma of the urinary bladder. By monoelonal antibody and Avidin-Biotin Complex staining technique the entire cancerous epithelium assumed negative reactivity, whereas the vascular endothelium showed positive reaction, which was regarded as internal positive control
Fig. 4. Immunoperoxidase staining of normal bladder epithelium of a blood group by monoclonal anti-A antibody with the Avidin-Biotin Complex technique. The entire epithelium and vessel endothelium assumed positive reaction International Urology and Nephrolo#y 24, 1992
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10. The slides were washed with tap water for: 5 min, counterstained with Mayer's haematoxylin, dehydrated and mounted. Two pathologists who were not aware of the patients' condition carried out blind examinations to be interpreted independently. The tumours were staged according to the classification described by Jewett and Strong [13] and classified according to the Armed Forces Institute of Pathology grading system as grade 1 = well differentiated, grade II = moderately differentiated, grade 11I = poorly differentiated. Immunohistochemical staining and interpretation of blood group isoantigen reactivity of tissues were based on the percentage of tumour cells in the entire tumour liable to staining as well as on the intensity of staining of the tumour cells. If 50 % or more of the malignant mucosa was stained, the reaction was interpreted as definitely positive (Fig. 1) and weakly positive if 30-50 % of the malignant mucosa was stained (Fig. 2). The reaction was regarded as negative if 30 % or less o f the malignant mucosa was stained (Fig. 3). Definitely and weakly positive reactions were considered to be positive for surface antigens. In addition, we used the normal transitional cell epithelium of the bladder as the external positive control (Fig. 4,) the vascular endothelium as the internal positive control and connective tissue and muscle as the negative internal control.
Results
The surface isoantigens on eight normal transitional epithelia and vascular endothelia were all positive. The reaction of these 8 normal specimens were neither false negative nor false positive. It is shown in Table 1 that most of the patients with transitional cell carcinoma of the urinary bladder were O blood type. As regards the rate of loss of surface antigens in these three groups, blood type O was 46%, type A 50% and type B 74%. On an average, 55% of patients Table 1 Relationship between blood type and antigenicity Antigen Blood type
O(H) A B Total
4
Positive
Intermediate
Negative
No. (%)
No. (%)
No. (%)
8 (31) 4 (20) 3 (16)
6 (23) 6 (30) 2 (11)
12 (46) 10 (50) 14 (74)
15 (23)
14 (22)
36 (55)
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t e n d e d to lose the surface antigens as s o o n as they d e v e l o p e d c a r c i n o m a o f the urinary bladder. T a b l e 2 indicates the r e l a t i o n s h i p between the r e a c t i o n o f surface a n t i g e n a n d p a t h o l o g i c a l grade. Seeing t h a t 27 70 o f patients w i t h g r a d e I, 50 70 w i t h g r a d e Table 2 Relationship between blood group isoantigen and tumour grade Pathological grade Antigen expression
I
II
In
No.(%)
No.(%)
No.(%)
7 (46) 4 (27) 4 (27)
7 (25) 7 (25) 14 (50)
1 (4.5) 3 (13.5) 18 (8.2)
28
22
Positive Intermediate Negative Total
15
Table 3 Relationship between blood group isoantigen and tumour stage Clinical stage Antigen expression
Positive Intermediate Negative Total
A
B
C
D
No. (%)
No. (%)
No. (%)
No.(%)
11 (32) 10 (29) 13 (38)
3 (19) 3 (19) 10 (63)
1 (8) I (8) 10 (83)
0 (0) 0 (0) 3 (100)
34
16
12
3
Table 4 Relationship between blood group isoantigen and five-year survival rate Survival Antigen expression
Positive Intermediate Negative Total
< 5 years
> 5 years
No.(%)
No.(%)
5 (33) 6 (43) 26 (72)
10 (67) 8 (57) 10 (28)
37 (57)
28 (43)
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II and 82 % with grade III tumour lost the surface antigens, it was suggested that the deletion of surface antigen was closely correlated with the pathological grade. Table 3 demonstrates the relationship between surface antigen reaction and clinical stage. It shows that 38 % of stage A, 63 % of stage B, 83 % of stage C and 100 % of stage D patients lost the surface antigens, which inferred that the deletion of surface antigens was closely correlated with the clinical stage. Table 4 demonstrates the correlation between antigen response and fiveyear survival rate. It was found that 10 of 15 patients (67 %) with positive surface antigens, 8 of 14 patients (57 %) with intermediate surface antigens and 10 of 36 patients (28 %) with negative surface antigens survived for more than five years. Discussion
Many reports in the literature verified that the blood group isoantigens on well-differentiated and non-invasive transitional cell carcinoma of the urinary bladder can be detected by means of the specific red cell adherence (SRCA) method. However, in the more invasive, poorly differentiated tumours or those which were originally well differentiated, but liable to invasive potential subsequently, the surface isoantigens would be nonexistent [8, 14-17]. So far, the SRCA test is still not accepted by the majority of physicians, neither can it be taken for reference to the clinical treatment of bladder tumours. It can be only confined to the study of stage of experiment, because its process or techniques of performance and the interpretation of experimental results were still involved in a lot of unconquerable conundrums [10]. For instance, by using the SRCA test to detect the surface isoantigens of transitional cell carcinoma of the urinary bladder, about 90 % of blood type O tumours would result in false negative reactions [18]. Unfortunately, of the patients with urinary bladder tumour the majority had type O blood. Therefore, it would be incorrect to interpret the results this way. In order to avoid some defects of the SRCA test, we used the specific and individual reagent-blood group monoclonal antibodies, accompanied by the staining method of indirect immunoperoxidase to detect the blood group isoantigens in urinary bladder cancers. In order to prevent false positive results, first of all we used goat anti-human antibody as the blocking agent. The non-blood group antigens were removed first; then mouse monoclonal antihuman blood group antibody was added to the A, B and O(H) blood group antigens, respectively, for the purpose of specific binding. Then, the biotinylated goat antimouse antibody was combined with the portion of mouse antibodies which were connective on the blood group antigen. Subsequently the Avidin Biotin-peroxidase complex was joined with biotinylated anti-mouse antibody. Since Avidin has very high affinity to Biotin and can simultaneously be united with 4 Biotin molecules, even a small quantity of surface isoantigen can be detected under the microscope. The use of monoclonal antibodies with the indirect immunoperoxidase staining technique offered many advantages over the SRCA method: (1) The 4*
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techniques and process of performance were simpler and easily reproducible. The results would not turn out to be different as performed and interpreted by different people. (2) Because SRCA technique used red cells as the marker to detect antigens, it was liable to result in superimposition, failing to detect the antigen site as precisely as possible. On the contrary, using monoclonal antibodies the blood group isoantigen can be localized exactly and would not lead to false positive or false negative results. (3) Immunoperoxidase was permanently stained, so it could be retained for retrospective studies. (4) Instead of the eurax extract used in the SRCA test, anti-H monoclonal antibodies were employed in this study for detecting the surface antigens in blood type O tumours so as to enhance the precision rate of its diagnosis. The fixation of tissue specimens with formalin damages the antigen determinant of tissue so seriously that surface antigens cannot be detected, especially when the fixative tended to be acidic [19]. Besides, Seal and associates [12] also revealed that by fixing the fresh specimens to the buffered neutral formalin for a minimal time, the effect of detection of isoantigens would be better than the tissue exposure to formalin for a longer time. Accordingly, they thought that deletion of antigens was related rather to the time of tissue fixation than to cell differentiation. As a result of this report, it was suggested that 55 ~ of patients with bladder tumour had the inclination to lose surface antigens. Did it infer that those epithelial cells of the bladder without surface antigens were more liable to undergo tumorous changes or the tissue specimens were fixed so long as to damage the antigen determinant? In this study, we could not get a positive answer because we did not use fresh tissue specimens as the reference group. In a study of 124 patients with transitional cell carcinoma of the urinary bladder, Davidsohn et al. [2] found that histologic grade had a close connection with the results of the SRCA test. That is to say, most of the high grade tumours belonged to the SRCA negative group. Javadpour [20] reported on 70 patients with bladder tumour and found that the surface antigens of 70 ~ of grade I tumour patients could be detected whereas none of 26 grade III ones retained surface antigens. Meanwhile, he found that 67 ~ of stage A tumours retained the surface antigens. However, when the tumour cells invaded the muscle layer, none of the patients preserved surface antigens [25]. Emott et al. [14] had brought forth the same results. According to the indirect immunohistochemical staining method by use of monoclonal antibodies to detect surface isoantigens of the bladder tumours, Fujioka et al. found that expression o f blood group antigens was relevant to the pathological grade and clinical stage of tumour [21]. This report also indicated that pathological grade and clinical stage of tumour had relevance to the detection o f blood group isoantigens. Kay and Wallace found that the prognosis o f patients with tumour antigen deletion would be worse than of those with positive antigen [22]. Decenzo et al. [16] also revealed that patients with isoantigen negative tumour would be more liable to invasive recurrence than those with isoantigen positive one. Their theory has been corroborated by other investigators later on [17, 23-25]. If this theory International Urology and Nephrology 24, 1992
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was to be recognized as irrefutable, the five-year survival rate of patients with deletion of surface antigens would be worse than of those who retained surface antigens. As this study had suggested, the expression of blood group antigens had a correlation with pathological grade, clinical stage and survival rate in patients with bladder carcinoma. However, the expression of blood group antigens cannot be regarded as the only indicator for treating the bladder tumour. Some other factors must be taken into consideration, such as turnout stage, grade and forms of tumours, etc. Further studies are needed on this aspect conducted by more investigators so that the detection of surface antigens as a useful marker could be applied to the clinical treatment of urological tumours. References 1. Davidsohn, I. : Early immunologic diagnosis and prognosis of carcinoma. Am. J. Clin. Pathol., 57, 715 (1972). 2. Davidsohn, I., Stejskal, R., Lill, P.: The loss of isoantigens A, B, and H in carcinoma of the urinary bladder. Lab. Invest., 28, 382 (1972). 3. Davidsohn, I., Ni, L. Y. : Loss of isoantigens A, B, and H in carcinoma of the lung. Am. J. PathoL, 57, 307 (1969). 4. Davidsohn, I., Kovarik, S., Ni, L. Y. : Isoantigens A, B, and H in benign and malignant lesions of the cervix. Arch. Pathol., 87, 306 (1969). 5. Davidsohn, I., Ni, L. Y., Stejskal, R. : Tissue isoantigens A, B, and H in carcinoma of the stomach. Arch. PathoL, 92, 456 (1971). 6. England, M. D., Davidsohn, 5. : Isoantigens A, B, and H in carcinoma of the fallopian tube. Arch. PathoL, 96, 350 (1973). 7. Davidsohn, I., Ni, L. Y., Stejskal, R. : Tissue isoantigens A, B, and H in carcinoma of the pancreas. Cancer Res., 31, 1244 (1971). 8. Catalona, W. J. : Practical utility of specific red cell adherence test in bladder cancer. Urology, 18, 113 (1981). 9. Coon, J. S., Weinstein, R. S. : Variability in the expression of the O(H) antigen in human transitional epithelium. J., UroL, 125, 301 (t981). 10. Hammon, J. C., Fella, C., Vacant, J., Maiolini, R., Masseyeff, R. : Improvement of the demonstration of A, B, H surface antigens in bladder tumors by a simple immunofluorescence technique. J. UroL, 128, 1183 (1982). 11. MeAlpine, Lt. R. G., Javadpour, N., Vafier, Lt. J. A., Worsham, C. G. F., O'Connell, C. K. J.: Immunoperoxidase versus specific red cell adherence in detection of ABO (H) antigens on normal urothelium. Urology, 24, 153 (1984). 12. Seal, G. M., Rowland, R. G., Thomalla, J. V., Rudolph, R. A., Pfaff, D. S., Kamer, M., Eble, J. N. : A, B and H antigens in normal urothelium: An immunohistochemical study using monoclonal antibodies with the Avidin-Biotin Complex technique, or. UroL, 133, 513 (1985). 13. Jewelt, H. J., Strong, G. H.: Infiltrating carcinoma of the bladder: Relation of death of penetration of the bladder wall to incidence of local extension and metastasis. J. UroL, 55, 366 (1946). 14. Emott, R. C., Javadpour, N., Bergman, S. M., Soares, T.: Correlation of the cell surface antigens with stage and grade in cancer of the bladder. J. UroL, 121, 37 (1979). 15. Karlsen, S., Jordfold, G., Svaar, H. : A, B and O (H) isoantigens in tumor of the urinary bladder. UroL Int., 39, 150 (1984). 16. Decenzo, J. M., Howard, P., Irish, C. E.: Antigenic detection and prognosis of patients with stage A transitional cell bladder cancer. J. UroL, 114, 874 (1975).
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17. Lange, P. H., Limas, C., Fraley, E. E. : Tissue blood group antigens and prognosis in low stage transitional cell carcinoma of the bladder. J. Urol., 119, 52 (1978). 18. Askari, A., Colmenares, E., Saberi, A., Jarman, W. D.: Red cell surface antigen and its relationship to survival of patients with transitional cell carcinoma of the bladder. J. UroL, 125, 182 (1981). 19. Robinson, G. : Immunohistochemistry. In: Bancroft, J. D., Stevens, A. (eds): Theory and Practice of Histological Techniques. Churchill Livingstone, London 1982, pp. 406-427. 20. Javadpour, N. : Tumor markers in urologic cancer. Urology, 2, 127 (1980). 21. Fujioka, T., Ohhori, T., Lovrekovich, L., Dekernion, J. B. : Investigation of blood group antigens and carcinoembryonic antigen in urinary bladder carcinoma. UroL Int., 41, 397 (1986). 22. Kay, H. E. M., Wallace, D. M. : A and B antigens of tumors arising from urinary epitheliums. J. Nat. Cancer Inst., 26, 1349 (1961). 23. Young, A. K., Hammond, E., Middleton, A. W. Jr. : The prognostic value of cell surface antigens in low grade, non-invasive, transitional cell carcinoma of the bladder. J. UroL, 122, 462 (1979). 24. Richie, J. P., Blute, R. D., Waisman, J. : Immunologic indicators of prognosis in bladder cancer: The importance of cell surface antigens. J. Urol., 124, 22 (1980). 25. Newman, A. J. Jr., Carlton, C. E. Jr., Johnson, S. : Cell surface A, B or O (H) blood group antigens as an indicator of malignant potential in stage A bladder carcinoma. J. Urol., 124, 27 (1980).
International Urology and Nephrology 24, 1992
Detection of Blood Group Surface Antigens of Urinary Bladder Tumours Using Monoclonal Antibodies with the Avidin-Biotin Complex Technique L.-B. TAN,* C. CHIANG, C.-H. HUANG, L.-M. LIN + +Departments of Urology and Oral Pathology, Provincial Tainan Hospital; *Kaohsiung Medical College Hospital, Taiwan, Republic of China (Accepted July 17, 1991) We examined 8 normal bladder transitional epithelia and 65 transitional cell carcinomas of the urinary bladder of various stages and grades for the presence of ABO(H) blood group surface isoantigens using the immunoperoxidase staining via the Avidin-Biotin Complex (ABC) methods. It was found that 27~ of patients with grade I tumours, 50 % with grade II tumours and 82 ~ with grade III turnours had loss of cell surface isoantigens. When correlated with the clinical stage the tumours showed no surface isoantigens in stage D, while 65 % of patients with stage A tumours were positive for surface antigens. From among 37 patients (57 %), 28 (43 %) survived for more than five years. Our results suggest that surface antigens of transitional cell carcinoma of the urinary bladder tended to disappear as the histologic changes of the tumour progressed. It also was noted that a loss of ABH(O) surface isoantigens was a bad prognostic sign.
Introduction Blood group substance is a kind of glycosamines attached to the surface membrane of cells. Under normal conditions, it exists in erythrocytes, endothelial cells, epithelial cells of the bladder, lung, cervix, gastrointestinal tract, Fallopian tube and pancreas [1 ]. Once these organs undergo malignant changes, adherence o f blood group substance on these normal epithelia decreases and subsequently disappears [1-6]. The loss of such surface antigen substance is closely related to the pathological grade as well as the clinical stage o f the tumour [2-7]. Catalona [8] has found that 66% of turnouts deprived of surface antigen underwent invasive recurrence, whereas of those with positive surface antigens only 4 % showed recurrence. Consequently, emphasis was gradually put on the detection of ABO isoantigens. In the past, many researchers adopted the SRCA (Specific Red Cell Adherence) method to detect these surface isoantigens [1-8]. However, this technique had a lot of drawbacks. For example, interpretation of the results of experiments was too subjective, the rate of false negative reactivity was too high and unpredictable [9], and the sections failed to be fixed and preserved permanently [10]. At present, investigators are using anti-A, anti-B and anti-H blood group monoclonal antibodies with the immunohistochemical staining technique to detect transiVSP, ,Utrecht Akad$miai Kiad6, Budapest
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tional epithelium in both normal and malignant bladder tumours, which is found much better than the SRCA method [11, 12]. The purpose of this study has been to observe the surface isoantigens on transitional cell carcinomas of the bladder by using anti-A, anti-B and anti-H blood group monoclonal antibodies with the immunohistochemical staining technique.
Materials and methods
In the period 1978 to 1983, the Department of Urology of Kaohsiung Medical College Hospital staff had performed surgical treatment in 65 patients with transitional cell carcinoma of the urinary bladder. The tissue specimens were fixed with formalin and embedded in paraffin blocks. Of these 65 cases, 26 belonged to O type, 20 to A type and 19 to B type blood groups. All patients underwent follow-up examination for more than five years. The paraffin blocks of the tissue specimens were cut into 5 #m thick slices and placed on slides as a routine histologic process. Each paraffin block was cut into at least three sections, one of which was used for haematoxylin and eosin staining, the other two for immunoperoxidase staining. At the same time, we selected the transitional epithelia of eight normal urinary bladders as the control group, of which 5 were blood type A and 3 were type B. The tissue processing and staining procedures were as follows: 1. The serial sections were deparaffinized, washed and dehydrated. 2. The slides were placed in absolute methyl alcohol-hydrogen peroxidase solution for 25 min in order to block endogenous peroxidase activity. 3. The slides were washed in three changes with 0.05 M Tris buffer solution (TBS) pH 7.6 for 15 rain, 5 rain each time. 4. According to suggestions of Vectastain ABC Kit (Vector Laboratories, Burlingame, Calif.) non-blood group antigens were neutralized by adding blocking antibodies (non-specific goat antihuman antibody in this study) to the slide for 20 rain. 5. The slides were washed again in three changes with TBS pH 7.6 for 15 rain, 5 min each time. 6. Mouse monoclonal anti-A, anti-B, and anti-H antibodies were used as primary antibody (prepared by DAKO) and added to the slides for 24 hours. 7. The slides were washed in three changes with TBS pH 7.6 for 15 min, 5 rain each time. 8. In conformity with the instructions of Vectastain ABC Kit (Vector Laboratories, Burlingame, Calif.), the biotinylated goat IgG antibodies were put on the slides for 30 min. 9. The slides were washed again in three changes with TBS pH 7.6 for 15 rain, 5 min each time. Then the Avidin Biotin-peroxidase complex was added to the slides for 60 min. Then, 3-amino-9-ethyl carbazole (Sigma Chemical Co., St. Louis, Mo.) was added to the slide for 5 min in order to identify the site of peroxidase localization. International Urology and Nephrology 24, 1992
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Fig. 1. Papillary transitional celt carcinoma of the urinary bladder. After application of monoclonal antibody and Avidin-Biotin Complex staining technique, the whole tumorous epithelium bore positive reaction, so did the vascular endothelium which was regarded as internal positive control
Fig. 2. Another papillary transitional cell carcinoma of the urinary bladder. The use of monoclonal antibody and Avidin-13iotin Complex staining technique revealed only about 3 0 ~ of cancerous epithelium reacting positively International Urology and Nephrology 24, 1992
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Fig. 3. Another papillary transitional cell carcinoma of the urinary bladder. By monoelonal antibody and Avidin-Biotin Complex staining technique the entire cancerous epithelium assumed negative reactivity, whereas the vascular endothelium showed positive reaction, which was regarded as internal positive control
Fig. 4. Immunoperoxidase staining of normal bladder epithelium of a blood group by monoclonal anti-A antibody with the Avidin-Biotin Complex technique. The entire epithelium and vessel endothelium assumed positive reaction International Urology and Nephrolo#y 24, 1992
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10. The slides were washed with tap water for: 5 min, counterstained with Mayer's haematoxylin, dehydrated and mounted. Two pathologists who were not aware of the patients' condition carried out blind examinations to be interpreted independently. The tumours were staged according to the classification described by Jewett and Strong [13] and classified according to the Armed Forces Institute of Pathology grading system as grade 1 = well differentiated, grade II = moderately differentiated, grade 11I = poorly differentiated. Immunohistochemical staining and interpretation of blood group isoantigen reactivity of tissues were based on the percentage of tumour cells in the entire tumour liable to staining as well as on the intensity of staining of the tumour cells. If 50 % or more of the malignant mucosa was stained, the reaction was interpreted as definitely positive (Fig. 1) and weakly positive if 30-50 % of the malignant mucosa was stained (Fig. 2). The reaction was regarded as negative if 30 % or less o f the malignant mucosa was stained (Fig. 3). Definitely and weakly positive reactions were considered to be positive for surface antigens. In addition, we used the normal transitional cell epithelium of the bladder as the external positive control (Fig. 4,) the vascular endothelium as the internal positive control and connective tissue and muscle as the negative internal control.
Results
The surface isoantigens on eight normal transitional epithelia and vascular endothelia were all positive. The reaction of these 8 normal specimens were neither false negative nor false positive. It is shown in Table 1 that most of the patients with transitional cell carcinoma of the urinary bladder were O blood type. As regards the rate of loss of surface antigens in these three groups, blood type O was 46%, type A 50% and type B 74%. On an average, 55% of patients Table 1 Relationship between blood type and antigenicity Antigen Blood type
O(H) A B Total
4
Positive
Intermediate
Negative
No. (%)
No. (%)
No. (%)
8 (31) 4 (20) 3 (16)
6 (23) 6 (30) 2 (11)
12 (46) 10 (50) 14 (74)
15 (23)
14 (22)
36 (55)
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t e n d e d to lose the surface antigens as s o o n as they d e v e l o p e d c a r c i n o m a o f the urinary bladder. T a b l e 2 indicates the r e l a t i o n s h i p between the r e a c t i o n o f surface a n t i g e n a n d p a t h o l o g i c a l grade. Seeing t h a t 27 70 o f patients w i t h g r a d e I, 50 70 w i t h g r a d e Table 2 Relationship between blood group isoantigen and tumour grade Pathological grade Antigen expression
I
II
In
No.(%)
No.(%)
No.(%)
7 (46) 4 (27) 4 (27)
7 (25) 7 (25) 14 (50)
1 (4.5) 3 (13.5) 18 (8.2)
28
22
Positive Intermediate Negative Total
15
Table 3 Relationship between blood group isoantigen and tumour stage Clinical stage Antigen expression
Positive Intermediate Negative Total
A
B
C
D
No. (%)
No. (%)
No. (%)
No.(%)
11 (32) 10 (29) 13 (38)
3 (19) 3 (19) 10 (63)
1 (8) I (8) 10 (83)
0 (0) 0 (0) 3 (100)
34
16
12
3
Table 4 Relationship between blood group isoantigen and five-year survival rate Survival Antigen expression
Positive Intermediate Negative Total
< 5 years
> 5 years
No.(%)
No.(%)
5 (33) 6 (43) 26 (72)
10 (67) 8 (57) 10 (28)
37 (57)
28 (43)
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II and 82 % with grade III tumour lost the surface antigens, it was suggested that the deletion of surface antigen was closely correlated with the pathological grade. Table 3 demonstrates the relationship between surface antigen reaction and clinical stage. It shows that 38 % of stage A, 63 % of stage B, 83 % of stage C and 100 % of stage D patients lost the surface antigens, which inferred that the deletion of surface antigens was closely correlated with the clinical stage. Table 4 demonstrates the correlation between antigen response and fiveyear survival rate. It was found that 10 of 15 patients (67 %) with positive surface antigens, 8 of 14 patients (57 %) with intermediate surface antigens and 10 of 36 patients (28 %) with negative surface antigens survived for more than five years. Discussion
Many reports in the literature verified that the blood group isoantigens on well-differentiated and non-invasive transitional cell carcinoma of the urinary bladder can be detected by means of the specific red cell adherence (SRCA) method. However, in the more invasive, poorly differentiated tumours or those which were originally well differentiated, but liable to invasive potential subsequently, the surface isoantigens would be nonexistent [8, 14-17]. So far, the SRCA test is still not accepted by the majority of physicians, neither can it be taken for reference to the clinical treatment of bladder tumours. It can be only confined to the study of stage of experiment, because its process or techniques of performance and the interpretation of experimental results were still involved in a lot of unconquerable conundrums [10]. For instance, by using the SRCA test to detect the surface isoantigens of transitional cell carcinoma of the urinary bladder, about 90 % of blood type O tumours would result in false negative reactions [18]. Unfortunately, of the patients with urinary bladder tumour the majority had type O blood. Therefore, it would be incorrect to interpret the results this way. In order to avoid some defects of the SRCA test, we used the specific and individual reagent-blood group monoclonal antibodies, accompanied by the staining method of indirect immunoperoxidase to detect the blood group isoantigens in urinary bladder cancers. In order to prevent false positive results, first of all we used goat anti-human antibody as the blocking agent. The non-blood group antigens were removed first; then mouse monoclonal antihuman blood group antibody was added to the A, B and O(H) blood group antigens, respectively, for the purpose of specific binding. Then, the biotinylated goat antimouse antibody was combined with the portion of mouse antibodies which were connective on the blood group antigen. Subsequently the Avidin Biotin-peroxidase complex was joined with biotinylated anti-mouse antibody. Since Avidin has very high affinity to Biotin and can simultaneously be united with 4 Biotin molecules, even a small quantity of surface isoantigen can be detected under the microscope. The use of monoclonal antibodies with the indirect immunoperoxidase staining technique offered many advantages over the SRCA method: (1) The 4*
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techniques and process of performance were simpler and easily reproducible. The results would not turn out to be different as performed and interpreted by different people. (2) Because SRCA technique used red cells as the marker to detect antigens, it was liable to result in superimposition, failing to detect the antigen site as precisely as possible. On the contrary, using monoclonal antibodies the blood group isoantigen can be localized exactly and would not lead to false positive or false negative results. (3) Immunoperoxidase was permanently stained, so it could be retained for retrospective studies. (4) Instead of the eurax extract used in the SRCA test, anti-H monoclonal antibodies were employed in this study for detecting the surface antigens in blood type O tumours so as to enhance the precision rate of its diagnosis. The fixation of tissue specimens with formalin damages the antigen determinant of tissue so seriously that surface antigens cannot be detected, especially when the fixative tended to be acidic [19]. Besides, Seal and associates [12] also revealed that by fixing the fresh specimens to the buffered neutral formalin for a minimal time, the effect of detection of isoantigens would be better than the tissue exposure to formalin for a longer time. Accordingly, they thought that deletion of antigens was related rather to the time of tissue fixation than to cell differentiation. As a result of this report, it was suggested that 55 ~ of patients with bladder tumour had the inclination to lose surface antigens. Did it infer that those epithelial cells of the bladder without surface antigens were more liable to undergo tumorous changes or the tissue specimens were fixed so long as to damage the antigen determinant? In this study, we could not get a positive answer because we did not use fresh tissue specimens as the reference group. In a study of 124 patients with transitional cell carcinoma of the urinary bladder, Davidsohn et al. [2] found that histologic grade had a close connection with the results of the SRCA test. That is to say, most of the high grade tumours belonged to the SRCA negative group. Javadpour [20] reported on 70 patients with bladder tumour and found that the surface antigens of 70 ~ of grade I tumour patients could be detected whereas none of 26 grade III ones retained surface antigens. Meanwhile, he found that 67 ~ of stage A tumours retained the surface antigens. However, when the tumour cells invaded the muscle layer, none of the patients preserved surface antigens [25]. Emott et al. [14] had brought forth the same results. According to the indirect immunohistochemical staining method by use of monoclonal antibodies to detect surface isoantigens of the bladder tumours, Fujioka et al. found that expression o f blood group antigens was relevant to the pathological grade and clinical stage of tumour [21]. This report also indicated that pathological grade and clinical stage of tumour had relevance to the detection o f blood group isoantigens. Kay and Wallace found that the prognosis o f patients with tumour antigen deletion would be worse than of those with positive antigen [22]. Decenzo et al. [16] also revealed that patients with isoantigen negative tumour would be more liable to invasive recurrence than those with isoantigen positive one. Their theory has been corroborated by other investigators later on [17, 23-25]. If this theory International Urology and Nephrology 24, 1992
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was to be recognized as irrefutable, the five-year survival rate of patients with deletion of surface antigens would be worse than of those who retained surface antigens. As this study had suggested, the expression of blood group antigens had a correlation with pathological grade, clinical stage and survival rate in patients with bladder carcinoma. However, the expression of blood group antigens cannot be regarded as the only indicator for treating the bladder tumour. Some other factors must be taken into consideration, such as turnout stage, grade and forms of tumours, etc. Further studies are needed on this aspect conducted by more investigators so that the detection of surface antigens as a useful marker could be applied to the clinical treatment of urological tumours. References 1. Davidsohn, I. : Early immunologic diagnosis and prognosis of carcinoma. Am. J. Clin. Pathol., 57, 715 (1972). 2. Davidsohn, I., Stejskal, R., Lill, P.: The loss of isoantigens A, B, and H in carcinoma of the urinary bladder. Lab. Invest., 28, 382 (1972). 3. Davidsohn, I., Ni, L. Y. : Loss of isoantigens A, B, and H in carcinoma of the lung. Am. J. PathoL, 57, 307 (1969). 4. Davidsohn, I., Kovarik, S., Ni, L. Y. : Isoantigens A, B, and H in benign and malignant lesions of the cervix. Arch. Pathol., 87, 306 (1969). 5. Davidsohn, I., Ni, L. Y., Stejskal, R. : Tissue isoantigens A, B, and H in carcinoma of the stomach. Arch. PathoL, 92, 456 (1971). 6. England, M. D., Davidsohn, 5. : Isoantigens A, B, and H in carcinoma of the fallopian tube. Arch. PathoL, 96, 350 (1973). 7. Davidsohn, I., Ni, L. Y., Stejskal, R. : Tissue isoantigens A, B, and H in carcinoma of the pancreas. Cancer Res., 31, 1244 (1971). 8. Catalona, W. J. : Practical utility of specific red cell adherence test in bladder cancer. Urology, 18, 113 (1981). 9. Coon, J. S., Weinstein, R. S. : Variability in the expression of the O(H) antigen in human transitional epithelium. J., UroL, 125, 301 (t981). 10. Hammon, J. C., Fella, C., Vacant, J., Maiolini, R., Masseyeff, R. : Improvement of the demonstration of A, B, H surface antigens in bladder tumors by a simple immunofluorescence technique. J. UroL, 128, 1183 (1982). 11. MeAlpine, Lt. R. G., Javadpour, N., Vafier, Lt. J. A., Worsham, C. G. F., O'Connell, C. K. J.: Immunoperoxidase versus specific red cell adherence in detection of ABO (H) antigens on normal urothelium. Urology, 24, 153 (1984). 12. Seal, G. M., Rowland, R. G., Thomalla, J. V., Rudolph, R. A., Pfaff, D. S., Kamer, M., Eble, J. N. : A, B and H antigens in normal urothelium: An immunohistochemical study using monoclonal antibodies with the Avidin-Biotin Complex technique, or. UroL, 133, 513 (1985). 13. Jewelt, H. J., Strong, G. H.: Infiltrating carcinoma of the bladder: Relation of death of penetration of the bladder wall to incidence of local extension and metastasis. J. UroL, 55, 366 (1946). 14. Emott, R. C., Javadpour, N., Bergman, S. M., Soares, T.: Correlation of the cell surface antigens with stage and grade in cancer of the bladder. J. UroL, 121, 37 (1979). 15. Karlsen, S., Jordfold, G., Svaar, H. : A, B and O (H) isoantigens in tumor of the urinary bladder. UroL Int., 39, 150 (1984). 16. Decenzo, J. M., Howard, P., Irish, C. E.: Antigenic detection and prognosis of patients with stage A transitional cell bladder cancer. J. UroL, 114, 874 (1975).
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17. Lange, P. H., Limas, C., Fraley, E. E. : Tissue blood group antigens and prognosis in low stage transitional cell carcinoma of the bladder. J. Urol., 119, 52 (1978). 18. Askari, A., Colmenares, E., Saberi, A., Jarman, W. D.: Red cell surface antigen and its relationship to survival of patients with transitional cell carcinoma of the bladder. J. UroL, 125, 182 (1981). 19. Robinson, G. : Immunohistochemistry. In: Bancroft, J. D., Stevens, A. (eds): Theory and Practice of Histological Techniques. Churchill Livingstone, London 1982, pp. 406-427. 20. Javadpour, N. : Tumor markers in urologic cancer. Urology, 2, 127 (1980). 21. Fujioka, T., Ohhori, T., Lovrekovich, L., Dekernion, J. B. : Investigation of blood group antigens and carcinoembryonic antigen in urinary bladder carcinoma. UroL Int., 41, 397 (1986). 22. Kay, H. E. M., Wallace, D. M. : A and B antigens of tumors arising from urinary epitheliums. J. Nat. Cancer Inst., 26, 1349 (1961). 23. Young, A. K., Hammond, E., Middleton, A. W. Jr. : The prognostic value of cell surface antigens in low grade, non-invasive, transitional cell carcinoma of the bladder. J. UroL, 122, 462 (1979). 24. Richie, J. P., Blute, R. D., Waisman, J. : Immunologic indicators of prognosis in bladder cancer: The importance of cell surface antigens. J. Urol., 124, 22 (1980). 25. Newman, A. J. Jr., Carlton, C. E. Jr., Johnson, S. : Cell surface A, B or O (H) blood group antigens as an indicator of malignant potential in stage A bladder carcinoma. J. Urol., 124, 27 (1980).
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