International Urology and Nephrology 23 (1), pp. 5--11 (1991)
Immunohistochemical Detection of Beta-Human Chorionic Gonadotropin in Urothelial Carcinoma H. OZKARDE,S, A. ERGEN, H. A. 0ZEN, A. AYHAN, D. REMZI Departm;nt of Urology, Hacettepe University School of Medicine, Ankara, Turkey (Received January 25, 1990) Histologic sections from the specimens of 60 patients with urothelial carcinoma were stained immunohistochemically to search for beta-human chorionic gonadotropin (B-HCG) production. There were 6 doubtful, 5 weak and 1 strong positive B-HCG stainings among 65 sections from 60 patients. De novo acquisition of B-HCG production capability of tumour cells seemed to be predictive for early haematogenous dissemination of the disease, but the data obtained in this particular study were insufficient to suggest B-HCG as a routine prognostic marker.
The high incidence and the great inclination to recurrence of urothelial tumours have led to innumerable investigations on the aetiology, diagnosis and treatment of this malignancy. Today, nothing can obviate the proper diagnosis of a bladder tumour and the tumours of the upper urinary tract as long as the condition is suspected and the procedure is either cystoscopy or ureterorenoscopy and biopsy. However, the main challenge is in the treatment modalities of these patients since the natural history of the disease could not always be well predicted. There is no doubt that the biologic potential of transitional cell neoplasms is closely related to histologic grading [I ]. But we need more to explain the invasive nature of some low grade turnouts or the diminishing differentiation of the primary tumour in its recurrences. In 1976, beta-human chorionic gonadotropin (B-HCG) production was shown in many neoplasms including transitional cell carcinoma (TCC). Later on, B-HCG was demonstrated in the tissues, sera or urine of patients with TCC in a few more studies [2, 3, 4]. Is the B-HCG production of TCC frequent enough to deserve to be an index for natural behaviour of this malignancy ? We reviewed the histologic sections of urothelial turnouts in 60 patients and searched for evidence of B-HCG production immunohistochemically.
VSP, Utrecht Akad6miai Kiad6, Budapest
6
Ozkardes et al. : Urothelial carcinoma
Materials and methods
Sixty patients diagnosed as having urothelial malignancies between 1984 and 1988 at our institution constituted the study group. They were chosen out of 128 patients, the turnout grades of whom were reevaluated. The patients' ages ranged between 35 and 78 years (mean 58 2). Fifty-two were men and 8 were women. None of the patients had any evidence of choriocarcinoma as a second malignancy. The majority of the specimens were bladder tissues obtained by transurethral resections (TUR); there was 1 case with renal pelvis tumour and 4 were cystectomy cases, either partial or total. The review of the histologic section (haematoxylin and eosin; H. E.) was made by the same consultant pathologist (AA) without knowing previous evaluations. The turnout grades were determined according to the Mostofi classification modified by the World Health Organization [5]. The number of patients and histologic sections are given in Table 1. Some sections belong to the same patients' turnouts at different times. Table 1 Number of patients and histologic sections according to tumour grades TCC grade
No. of patients
No. o f slides
I II III
19 22 19
20 24 21
60
65
Total
New sections from the paraffin blocs of these specimens were prepared. Following deparaffinization, rehydration and fixation, the new sections were stained immunohistochemically by using Immustain | anti-Beta-HCG monoclonal antibody (mouse) and StreptAvidin-Biotin universal kit (Diagnostic Products Corporation, USA) according to the principles of the avidin-biotin immunoperoxidase technique which is defined elsewhere [2, 3, 6]. The slides were then examined again and positives for B-HCG staining were noted.
Results
The results of immunohistochemical staining for B-HCG are shown in Table 2. As seen, there is only 1 strongly positive B-HCG stained high-grade tumour in this series. The H. E. and immune stained slides of this patient are shown in Figs 1 and 2. The expression of B-HCG is almost similar to a B-HCG International Urology and Nephrology 23, 1991
O z k a r d e s et al.: Urothelial carcinoma
7
Fig. 1. Photomicrograph of a high grade (III/III) muscle invasive TCC. Note the syncytial pattern of the turnout cells (H. E., original magnification x 40) Fig. 2. The tumour in Fig. 1 after immunohistochemical staining for B-HCG. The striking pattern of dense (brownish colour) B-HCG staining is seen (Anti-B-HCG Immustain| original magnification • 100)
Ozkarde$ et aL : Urotheliat carcinoma
Table 2 Results of immunohistochemical staining for B-HCG in urothelial carcinoma TCC grade
]No. of patients
No. of slides
19 22 19
20 24 21
60
65
6
I II
III Total
Positive B-HCG staining Doubtful
Weak
Strong
1
1
--
3 2
2 2
-1
5
1
p r o d u c i n g t e s t i c u l a r t u m o u r . T h i s p a t i e n t was d i a g n o s e d in A u g u s t 1987, h a d t o t a l c y s t e c t o m y a n d ileal l o o p u r i n a r y d i v e r s i o n S e p t e m b e r 1988 a n d d i e d in F e b r u a r y 1989 w i t h massive p u l m o n a r y metastases. T h e fates o f o t h e r p a t i e n t s are given i n T a b l e 3, a n d t h e e x a m p l e s o f w e a k positive a n d d o u b t f u l s t a i n i n g s are s h o w n i n F i g s 3 a n d 4.
Table 3 Follow-up of patients with overall results of B-HCG staining Patient No.
TCC grade, stage, d a t e
TissueB-HCG expression
Type of surgery
Last follow-up time and current status of patient and disease
1
1,
A 3/85
Doubtful
TUR
1/89, Alive, NED
2
I,
A 8/88
Weak
TUR
8/89, Alive, NED
3
II,
B1 5/84
Doubtful
TUR
1/89, Dead
4
II,
B1 2/86
Weak
TUR
4/89, Alive, NED
5
II,
A 3]87
Doubtful
TUR
7/88, Alive, ?
6
II,
B1 4/88
Weak
TUR
4/89, Alive, NED
7
III,
D2 6/87
Weak
TUR
?, Dead
8
III,
C 1/88
9
III,
B2 8/87
Strong
Doubtful
TUR
6/88, Dead
Total cysteetomy
2/89, Dead
I0
III,
C 11/88
Doubtful
TUR
5/89, Dead
tl
III, B2 11/88
Doubtful
TUR
7/89, Alive, metastatic tumour
12
III, B1 12/88
Weak
Partial cystectomy 6/89, Alive, NED
NED: No evidence of disease. International Urology and Nephrology 23, 1991
Ozkardes et al.: Urothelial carcinoma
9
Fig. 3. Photomicrograph presenting weak tissue expression of B-HCG in another patient's TCC. Tumour cells with brownish coloured cytoplasm are observed infrequently throughout the section (Anti-B-HCG Immustain| original magnification X 100) Fig. 4. Doubtful tissue expression of B-HCG in low-grade TCC. The colour change is observed dispersed throughout the section but the typical cytoplasmic staining could not be demonstrated (Anti-B-HCG Immustain| original magnification • 40)
10
Ozkardes' et al.: Urothelial carcinoma
Discussion
The great variability of the natural behaviour of human urothelial malignancies has led the investigators to search for several prognostic indices and markers for the follow-up of these patients. The majority of histologic and cytologic work-up concentrated on the changes in the level of nuclear content of the cell, the lack of the expression of normally found antigens in the cells or finding new antigens that became newly expressed in tissues [6]. To name a few of these we mention chromosomal analyses, demonstration ofABO(H), T (Thomsen-Friedenreich) antigens and several others on cell surfaces and determination of cell ploidy through flow cytometry [7, 8, 9, 10, 11, 12, 13]. Beta-HCG expression in tumour cells of urothelial malignancies came to the attention of investigators since the 1970s [3]. Later on, the prognostic significance of this expression came into consideration. It is well known that B-HCG is the specific marker of gestational and nongestational choriocarcinoma. Troph oblastic cells, the physiologic producers of B-HCG, have been found in peripheral circulation without any malignant change in these tissues. This haematogenous dissemination property continues after malignant degeneration of these cells and in a sense detected as the typical haematogenous spreading pattern of chorio: carcinoma. Do the retrodifferentiated cells which acquire B-HCG production capability gain also the characteristics of the tissues that physiologically produce this marker ? If this is so, B-HCG production by a tumour could well be a sign of early haematogenous metastases and consequently an index for poor prognosis. This subject is controversial. The paucity of data in this study suggest that B-HCG production may be a feature of high-grade urothelial carcinoma and a predictor of early haematogenous metastases. But we were unable to find the tissue expression of B-HCG frequent enough to advise B-HCG as a routine marker and prognostic index for urothelial cancer. References 1. Soloway, M. S.: Evaluation and management of patients with superficial bladder cancer. Urol. Clin. North Am., 14, 771 (1987). 2. Yamase, H. T., Wurzel, R. S., Nieh, P. T., Gondos, B. : Immunohistochemical demonstration of human chorionic gonadotropin in tumours of the urinary bladder. Ann. Clin. Lab. Sci., 15, 414 (1985). 3. Shah, W. M., Newman, J., O'Brien, J. M,, Considine, J.: Ectopic beta-human ehorionic gonadotropin production by bladder urothelial neoplasia. Arch. Pathol. Lab. Med., 110, 107 (1986). 4. Dexeus, F., Logothetis, C., Hossan, E., Samuels, M. L.: Carcinoembryonic antigen and beta-human chorionic gonadotropin as serum markers for advanced urothelial malignancies. J. Urol., 136, 403 (1986). 5. Johnson, D. E., Swanson, D. A., Esehenbach, A. C.: Tumours of the Genitourinary Tract. Appleton & Lange, USA 1988. 6. Javadpour, N. : Multiple cell markers in bladder cancer. Principles and clinical applications. Urol. Clin. North Am., 11, 609 (1984). International Urology and Nephrology 23, 1991
Ozkarde~" et aL : Urothelial carc#toma
11
7. Coon, J. S., Weinstein, R. S., Summers, J. L. : Blood group precursor T-antigen expression in human bladder carcinoma. Am. Y. Clin. Pathol., 77, 692 (1982). 8. Newman, A. J., Carlton, C. E., 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). 9. Abel, P. D., Thorpe, S. J., Williams, G. : Blood group antigen expression in frozen sections of presenting bladder cancer: 3-year prospective follow-up of prognostic value. Br. J. Urol., 63, 171 (1989). 10. Richie, J. P., Blute, R. D., Waisman, J. : Immunologic indicators of prognosis in bladder cancer: the importance of cell surface antigens. J. Urol., 123, 22 (1980). 11. Gustafson, H., Tribukait, B., Esposti, P. L.: D N A profile and tumour progression in patients with superficial bladder tumours. Urol. Res., 10, 13 (1982). 12. Summers, J. L., Coon, J. S., Ward, R. M. : Prognosis in carcinoma of the urinary bladder based upon tissue blood group ABH and Thomsen-Friedenreich antigen status and karyotype of the initial turnout. Cancer Res., 43, 934 (1983). 13. Carter, H. B., Amberson, J. B., Bander, N. H. : Newer diagnostic techniques for bladder cancer. UroL Clin. North Am., 14, 763 (1987).
International Urology and Nephrology 23, 1991
Immunohistochemical Detection of Beta-Human Chorionic Gonadotropin in Urothelial Carcinoma H. OZKARDE,S, A. ERGEN, H. A. 0ZEN, A. AYHAN, D. REMZI Departm;nt of Urology, Hacettepe University School of Medicine, Ankara, Turkey (Received January 25, 1990) Histologic sections from the specimens of 60 patients with urothelial carcinoma were stained immunohistochemically to search for beta-human chorionic gonadotropin (B-HCG) production. There were 6 doubtful, 5 weak and 1 strong positive B-HCG stainings among 65 sections from 60 patients. De novo acquisition of B-HCG production capability of tumour cells seemed to be predictive for early haematogenous dissemination of the disease, but the data obtained in this particular study were insufficient to suggest B-HCG as a routine prognostic marker.
The high incidence and the great inclination to recurrence of urothelial tumours have led to innumerable investigations on the aetiology, diagnosis and treatment of this malignancy. Today, nothing can obviate the proper diagnosis of a bladder tumour and the tumours of the upper urinary tract as long as the condition is suspected and the procedure is either cystoscopy or ureterorenoscopy and biopsy. However, the main challenge is in the treatment modalities of these patients since the natural history of the disease could not always be well predicted. There is no doubt that the biologic potential of transitional cell neoplasms is closely related to histologic grading [I ]. But we need more to explain the invasive nature of some low grade turnouts or the diminishing differentiation of the primary tumour in its recurrences. In 1976, beta-human chorionic gonadotropin (B-HCG) production was shown in many neoplasms including transitional cell carcinoma (TCC). Later on, B-HCG was demonstrated in the tissues, sera or urine of patients with TCC in a few more studies [2, 3, 4]. Is the B-HCG production of TCC frequent enough to deserve to be an index for natural behaviour of this malignancy ? We reviewed the histologic sections of urothelial turnouts in 60 patients and searched for evidence of B-HCG production immunohistochemically.
VSP, Utrecht Akad6miai Kiad6, Budapest
6
Ozkardes et al. : Urothelial carcinoma
Materials and methods
Sixty patients diagnosed as having urothelial malignancies between 1984 and 1988 at our institution constituted the study group. They were chosen out of 128 patients, the turnout grades of whom were reevaluated. The patients' ages ranged between 35 and 78 years (mean 58 2). Fifty-two were men and 8 were women. None of the patients had any evidence of choriocarcinoma as a second malignancy. The majority of the specimens were bladder tissues obtained by transurethral resections (TUR); there was 1 case with renal pelvis tumour and 4 were cystectomy cases, either partial or total. The review of the histologic section (haematoxylin and eosin; H. E.) was made by the same consultant pathologist (AA) without knowing previous evaluations. The turnout grades were determined according to the Mostofi classification modified by the World Health Organization [5]. The number of patients and histologic sections are given in Table 1. Some sections belong to the same patients' turnouts at different times. Table 1 Number of patients and histologic sections according to tumour grades TCC grade
No. of patients
No. o f slides
I II III
19 22 19
20 24 21
60
65
Total
New sections from the paraffin blocs of these specimens were prepared. Following deparaffinization, rehydration and fixation, the new sections were stained immunohistochemically by using Immustain | anti-Beta-HCG monoclonal antibody (mouse) and StreptAvidin-Biotin universal kit (Diagnostic Products Corporation, USA) according to the principles of the avidin-biotin immunoperoxidase technique which is defined elsewhere [2, 3, 6]. The slides were then examined again and positives for B-HCG staining were noted.
Results
The results of immunohistochemical staining for B-HCG are shown in Table 2. As seen, there is only 1 strongly positive B-HCG stained high-grade tumour in this series. The H. E. and immune stained slides of this patient are shown in Figs 1 and 2. The expression of B-HCG is almost similar to a B-HCG International Urology and Nephrology 23, 1991
O z k a r d e s et al.: Urothelial carcinoma
7
Fig. 1. Photomicrograph of a high grade (III/III) muscle invasive TCC. Note the syncytial pattern of the turnout cells (H. E., original magnification x 40) Fig. 2. The tumour in Fig. 1 after immunohistochemical staining for B-HCG. The striking pattern of dense (brownish colour) B-HCG staining is seen (Anti-B-HCG Immustain| original magnification • 100)
Ozkarde$ et aL : Urotheliat carcinoma
Table 2 Results of immunohistochemical staining for B-HCG in urothelial carcinoma TCC grade
]No. of patients
No. of slides
19 22 19
20 24 21
60
65
6
I II
III Total
Positive B-HCG staining Doubtful
Weak
Strong
1
1
--
3 2
2 2
-1
5
1
p r o d u c i n g t e s t i c u l a r t u m o u r . T h i s p a t i e n t was d i a g n o s e d in A u g u s t 1987, h a d t o t a l c y s t e c t o m y a n d ileal l o o p u r i n a r y d i v e r s i o n S e p t e m b e r 1988 a n d d i e d in F e b r u a r y 1989 w i t h massive p u l m o n a r y metastases. T h e fates o f o t h e r p a t i e n t s are given i n T a b l e 3, a n d t h e e x a m p l e s o f w e a k positive a n d d o u b t f u l s t a i n i n g s are s h o w n i n F i g s 3 a n d 4.
Table 3 Follow-up of patients with overall results of B-HCG staining Patient No.
TCC grade, stage, d a t e
TissueB-HCG expression
Type of surgery
Last follow-up time and current status of patient and disease
1
1,
A 3/85
Doubtful
TUR
1/89, Alive, NED
2
I,
A 8/88
Weak
TUR
8/89, Alive, NED
3
II,
B1 5/84
Doubtful
TUR
1/89, Dead
4
II,
B1 2/86
Weak
TUR
4/89, Alive, NED
5
II,
A 3]87
Doubtful
TUR
7/88, Alive, ?
6
II,
B1 4/88
Weak
TUR
4/89, Alive, NED
7
III,
D2 6/87
Weak
TUR
?, Dead
8
III,
C 1/88
9
III,
B2 8/87
Strong
Doubtful
TUR
6/88, Dead
Total cysteetomy
2/89, Dead
I0
III,
C 11/88
Doubtful
TUR
5/89, Dead
tl
III, B2 11/88
Doubtful
TUR
7/89, Alive, metastatic tumour
12
III, B1 12/88
Weak
Partial cystectomy 6/89, Alive, NED
NED: No evidence of disease. International Urology and Nephrology 23, 1991
Ozkardes et al.: Urothelial carcinoma
9
Fig. 3. Photomicrograph presenting weak tissue expression of B-HCG in another patient's TCC. Tumour cells with brownish coloured cytoplasm are observed infrequently throughout the section (Anti-B-HCG Immustain| original magnification X 100) Fig. 4. Doubtful tissue expression of B-HCG in low-grade TCC. The colour change is observed dispersed throughout the section but the typical cytoplasmic staining could not be demonstrated (Anti-B-HCG Immustain| original magnification • 40)
10
Ozkardes' et al.: Urothelial carcinoma
Discussion
The great variability of the natural behaviour of human urothelial malignancies has led the investigators to search for several prognostic indices and markers for the follow-up of these patients. The majority of histologic and cytologic work-up concentrated on the changes in the level of nuclear content of the cell, the lack of the expression of normally found antigens in the cells or finding new antigens that became newly expressed in tissues [6]. To name a few of these we mention chromosomal analyses, demonstration ofABO(H), T (Thomsen-Friedenreich) antigens and several others on cell surfaces and determination of cell ploidy through flow cytometry [7, 8, 9, 10, 11, 12, 13]. Beta-HCG expression in tumour cells of urothelial malignancies came to the attention of investigators since the 1970s [3]. Later on, the prognostic significance of this expression came into consideration. It is well known that B-HCG is the specific marker of gestational and nongestational choriocarcinoma. Troph oblastic cells, the physiologic producers of B-HCG, have been found in peripheral circulation without any malignant change in these tissues. This haematogenous dissemination property continues after malignant degeneration of these cells and in a sense detected as the typical haematogenous spreading pattern of chorio: carcinoma. Do the retrodifferentiated cells which acquire B-HCG production capability gain also the characteristics of the tissues that physiologically produce this marker ? If this is so, B-HCG production by a tumour could well be a sign of early haematogenous metastases and consequently an index for poor prognosis. This subject is controversial. The paucity of data in this study suggest that B-HCG production may be a feature of high-grade urothelial carcinoma and a predictor of early haematogenous metastases. But we were unable to find the tissue expression of B-HCG frequent enough to advise B-HCG as a routine marker and prognostic index for urothelial cancer. References 1. Soloway, M. S.: Evaluation and management of patients with superficial bladder cancer. Urol. Clin. North Am., 14, 771 (1987). 2. Yamase, H. T., Wurzel, R. S., Nieh, P. T., Gondos, B. : Immunohistochemical demonstration of human chorionic gonadotropin in tumours of the urinary bladder. Ann. Clin. Lab. Sci., 15, 414 (1985). 3. Shah, W. M., Newman, J., O'Brien, J. M,, Considine, J.: Ectopic beta-human ehorionic gonadotropin production by bladder urothelial neoplasia. Arch. Pathol. Lab. Med., 110, 107 (1986). 4. Dexeus, F., Logothetis, C., Hossan, E., Samuels, M. L.: Carcinoembryonic antigen and beta-human chorionic gonadotropin as serum markers for advanced urothelial malignancies. J. Urol., 136, 403 (1986). 5. Johnson, D. E., Swanson, D. A., Esehenbach, A. C.: Tumours of the Genitourinary Tract. Appleton & Lange, USA 1988. 6. Javadpour, N. : Multiple cell markers in bladder cancer. Principles and clinical applications. Urol. Clin. North Am., 11, 609 (1984). International Urology and Nephrology 23, 1991
Ozkarde~" et aL : Urothelial carc#toma
11
7. Coon, J. S., Weinstein, R. S., Summers, J. L. : Blood group precursor T-antigen expression in human bladder carcinoma. Am. Y. Clin. Pathol., 77, 692 (1982). 8. Newman, A. J., Carlton, C. E., 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). 9. Abel, P. D., Thorpe, S. J., Williams, G. : Blood group antigen expression in frozen sections of presenting bladder cancer: 3-year prospective follow-up of prognostic value. Br. J. Urol., 63, 171 (1989). 10. Richie, J. P., Blute, R. D., Waisman, J. : Immunologic indicators of prognosis in bladder cancer: the importance of cell surface antigens. J. Urol., 123, 22 (1980). 11. Gustafson, H., Tribukait, B., Esposti, P. L.: D N A profile and tumour progression in patients with superficial bladder tumours. Urol. Res., 10, 13 (1982). 12. Summers, J. L., Coon, J. S., Ward, R. M. : Prognosis in carcinoma of the urinary bladder based upon tissue blood group ABH and Thomsen-Friedenreich antigen status and karyotype of the initial turnout. Cancer Res., 43, 934 (1983). 13. Carter, H. B., Amberson, J. B., Bander, N. H. : Newer diagnostic techniques for bladder cancer. UroL Clin. North Am., 14, 763 (1987).
International Urology and Nephrology 23, 1991
