UROPATHOLOGY
SQUAMOUS CELL CARCINOMA-ASSOCIATED ANTIGEN IN UROEPITHELIAL CARCINOMA TAIJI TSUKAMOTO, M.D. YOSHIAKI KUMAMOTO, M.D. KIYOTAKA OHMURA, M.D. NORIOMI MIYAO, M.D.
From the Department Sapporo, Japan
AKIHITO NAMMBU, M.D. YOSHIO TAKAGI, M.D. NAOKI ITOH, M.D.
of Urology, Sapporo Medical College,
ABSTRACTWe investigated the clinical significance of squamous cell carcinoma (SCC) antigen determined by radioimmunoassay on patients with uroepithelial carcinoma. Serum SCC antigen levels and the positive rates were significantly higher in uroepithelial carcinoma having an SCC component than in normal controls, benign urologic diseases, other urogenital carcinomas, or in pure transitional cell carcinoma (TCC) of uroepithelial carcinomas. A substantial number of patients with pure TCC showed a positive serum level of SCC antigen. Immunohistochemical staining of SCC antigen on the uroepithelial carcinomas confirmed that some, not all, pure TCC, mostly grade 3, expressed SCC antigen in their cells. These results suggested a biologic characteristic of grade 3 TCC which is closely related to that of SCC. The serum levels of SCC antigen correlated well with the disease extensions in uroepithelial carcinoma containing SCC component. Changes in serum levels of SCC antigen following treatment reflected the clinical courses of patients, particularly in those with elevated pretreatment levels. The results of our study indicated that the determination of SCC antigen would be clinically useful for monitoring clinical courses of patients with uroepithelial carcinomas containing SCC component and of those with pure TCC whose pretreatment level was elevated.
Kato and Torigoe’ first identified squamous cell carcinoma-associated antigen (SCC antigen), a protein subfraction extracted and purified from squamous cell carcinoma (SCC) of uterine cervix, and developed the method for radioimmunoassay. Since then, extensive studies have been performed on its clinical availability in SCC of various organs with the exception of the urinary tract. The results so far indicate that serum level of SCC antigen is useful, particularly in monitoring the clinical course.2-4 However, there have been no studies on SCC that arises from uroepithelium. Thus, it seems worthwhile to investigate the clinical significance of SCC antigen in uroepithelial carcinoma. We present herein our results with serum levels of SCC antigen in patients with uroepithelial carcinoma, such as transitional
UROLOGY
/ NOVEMBER
1992
/ VOLUME
40, NUMBER 5
cell carcinoma (TCC) and SCC, and of immunohistochemical staining of SCC antigen on the carcinomas. Material and Methods The serum level of SCC antigen was determined in 214 patients with various urogenital diseases: 84 patients with histologically proved uroepithelial carcinoma (61 TCC, 14 mixed TCC with SCC, and 9 pure SCC), 79 with other urogenital carcinomas (renal cell, prostate, and testicular), and 51 with benign urologic diseases. Eighty-four patients with uroepithelial carcinoma comprised 65 of bladder and 19 of renal pelvic or ureteral carcinomas. Thirty-seven healthy adult volunteers served as normal controls.
477
w
Serum liseases
Cl.0
1
(ndmt)
SCC antigen
1.5
5
10
50
\lormal controls
. TCC + SCC or pure SCC cn=23)
.:., :’ ,:‘r’::g,;;;;.‘.y.;.D ,:. +m
qoa .
0.9
:'
8
Mean k SD (ng/mQ)
Positive rate (%I
,
l.lkO.2
2.7
l.lkO.2
3.8
1.5f2.4
9.8
8.7k16.1
82.6
FIGURE 1. Serum levels and positive rates of SCC antigen in normal controls, patients with uroepithelial carcinomas, benign urologic diseases, or in other urogenital carcinomas. Uroepithelial carcinomas classified into pure TCC, mixed TCC with SCC, and into pure SCC.
Blood samples were collected from all patients before treatments. Serial determinations of serum level of SCC antigen were done in 20 patients with uroepithelial carcinoma during follow-up period. Histopathologic extensions of the carcinomas were classified according to UICC-TNM classification of bladder carcinoma,5 and those of renal pelvic or ureteral carcinomas were determined according to a new classification advocated by Akaza, Koiso, and Niijimae which modified the UICC-TNM classification of bladder carcinomas. Grades of TCC or SCC were classified as grades 1, 2, and 3 or well, moderate, and poor and evaluated respectively. Radioimmunoassay Shortly after the collection, blood samples were centrifuged at 2,500 rpm for ten minutes, and the serums were stored at - 80 “C until assay. Serum levels were determined by radioimmunometric method (SCC Riabead, Dainabot Ltd., Tokyo, Japan). This method using monoclonal antibody for SCC antigen can detect serum levels from 1.0 to 150 ng/mL with 8.0 percent of mean intra-assay deviation and with 8.7 percent of inter-assay deviation. All samples were duplicately measured.
478
Immunohistochemical staining of SCC antigen on carcinoma tissue Immunohistochemical staining of SCC antigen was investigated in 9 patients with uroepithelial carcinoma using immunoperoxidase method and avidin-biotin complex staining (Vectastain ABC Kit, Vector Laboratory, Inc., Burlingame, CA). Two sections of 3 pm thick were deparaffinized and dehydrated according to standard technique. One of the sections was stained with hematoxylin and eosin for the standard histologic study. Following elimination of endogenous peroxidase with 3% hydrogen peroxide, 10 PglmL of mouse antiSCC antigen monoclonal antibody (courtesy of Dr. Hiroshi Kato, Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube, Japan, and Dainabot Ltd., Tokyo, Japan) was applied to the other section for sixty minutes, washed and stained with a biotin-conjugate anti-mouse antibody (at a dilution of 1:200). Then, the section was stained with an avidin-biotin peroxidase complex. Visualization of the reaction product was achieved with an application of 3,3 ‘-diaminobenzidine tetrahydrochloride. The staining of nucleus was performed with hematoxylin.
UROLOGY
/ NOVEMBER 1992 I
VOLUME 40, NUMBER 5
------EIYiCi:+iiii------------
see
poor (“=,,)
.,._. :..,......~_~.~.........~.~
::::::j::::::j::::::::::::::::: ,:,.__:...:._....
$:::::““:;,:1111;1;;;;;; $+..$$+= . . .. . . ,.,._ :.::::. ,.:.:.:+:.:.:.: ..;.:.:.:.:.:.:.:::j:::::::i:: ..,.,,........... .............. .
____
8
-__-__,--
_______
4.323.5
______,
72.7
FIGURE 2. Serum levels and positive rates of SCC antigen according to histopathologic grade in pure TCC and degree of dif erentiation in carcinomas associated with SCC component.
Results Serum level of SCC antigen with radioimmunoassay From results in 37 controls which showed the mean serum level of 1.1 + 0.2 (mean _+ SD) ng/mL, we determined 1.5 ng/mL, mean + 2 SD, as an upper normal limit. Thus, serum SCC antigen of more than 1.5 ng/mL was considered “positive” or “elevated.” Figure 1 demonstrates a distribution of serum levels of SCC antigen in normal subjects and in various diseases, including uroepithelial carcinomas and positive rates. Only 1 patient (2.7%) had serum SCC antigen over the upper normal limit in the control group. Mean serum level of SCC antigen and positive rate were almost the same in normal subjects, in benign urologic diseases, and in any other urogenital carcinomas. In uroepithelial carcinomas, 6 of 61 patients (10%) with pure TCC had a positive level of serum SCC antigen, although the mean level was not significantly different from that of the controls (unpaired Student’s t test). In contrast, the patients with a mixed TCC with SCC or pure SCC showed a significantly higher level of serum SCC antigen (unpaired Student’s t test, p 9'0T Z'T
T'6
3X 'Qd 30 'ON
=nd
I?LI.roupra~ 30 uo!sua~xg
TABLE II.
Immunohistochemical staining and pretreatment level of SCC antigen in uroepithelial carcinomas
Case No.
staining of SCC FIGURE 4. Immunohistochemical antigen on uroepithelial carcinoma. Grade 3 TCC hematoxylin-eosin stain (A), and positive immunohistochemical stain of SCC antigen (B). Grade 2 TCC hematoxylin-eosin stain (C), and negative immunohistochemical stain of SCC antigen (0). (Original magnijications x 118 [A] and x 235 [Cl).
range after treatment. These normal levels persisted during the subsequent follow-up period. Immunohistochemical
staining
of SCC
antigen
Immunohistochemical staining of SCC antigen was performed on 9 patients, 7 with pure TCC (2 with grade 3, 4 with grade 3) and 2 with pure SCC (Table II, Fig. 4). In 3 patients with grade 2 TCC, whose pretreatment serum level SCC antigen was normal, SCC antigen
UROLOGY
/ NOVEMBER1992
I VOLUME40,NUMBER5
Histopathol. TCC TCC TCC TCC TCC TCC TCC
gr. 2 gr. 2 gr. 2 gr. 3 gr. 3 gr. 3 gr. 3 SCC mod. SCC mod.
XC
Antigen Stain Neg. Neg. Pos. Neg. Pos. Pos. Pos. Pos.
Pos.
Serum SCC Antigen (ng/mL)
SQUAMOUS CELL CARCINOMA-ASSOCIATED ANTIGEN IN UROEPITHELIAL CARCINOMA TAIJI TSUKAMOTO, M.D. YOSHIAKI KUMAMOTO, M.D. KIYOTAKA OHMURA, M.D. NORIOMI MIYAO, M.D.
From the Department Sapporo, Japan
AKIHITO NAMMBU, M.D. YOSHIO TAKAGI, M.D. NAOKI ITOH, M.D.
of Urology, Sapporo Medical College,
ABSTRACTWe investigated the clinical significance of squamous cell carcinoma (SCC) antigen determined by radioimmunoassay on patients with uroepithelial carcinoma. Serum SCC antigen levels and the positive rates were significantly higher in uroepithelial carcinoma having an SCC component than in normal controls, benign urologic diseases, other urogenital carcinomas, or in pure transitional cell carcinoma (TCC) of uroepithelial carcinomas. A substantial number of patients with pure TCC showed a positive serum level of SCC antigen. Immunohistochemical staining of SCC antigen on the uroepithelial carcinomas confirmed that some, not all, pure TCC, mostly grade 3, expressed SCC antigen in their cells. These results suggested a biologic characteristic of grade 3 TCC which is closely related to that of SCC. The serum levels of SCC antigen correlated well with the disease extensions in uroepithelial carcinoma containing SCC component. Changes in serum levels of SCC antigen following treatment reflected the clinical courses of patients, particularly in those with elevated pretreatment levels. The results of our study indicated that the determination of SCC antigen would be clinically useful for monitoring clinical courses of patients with uroepithelial carcinomas containing SCC component and of those with pure TCC whose pretreatment level was elevated.
Kato and Torigoe’ first identified squamous cell carcinoma-associated antigen (SCC antigen), a protein subfraction extracted and purified from squamous cell carcinoma (SCC) of uterine cervix, and developed the method for radioimmunoassay. Since then, extensive studies have been performed on its clinical availability in SCC of various organs with the exception of the urinary tract. The results so far indicate that serum level of SCC antigen is useful, particularly in monitoring the clinical course.2-4 However, there have been no studies on SCC that arises from uroepithelium. Thus, it seems worthwhile to investigate the clinical significance of SCC antigen in uroepithelial carcinoma. We present herein our results with serum levels of SCC antigen in patients with uroepithelial carcinoma, such as transitional
UROLOGY
/ NOVEMBER
1992
/ VOLUME
40, NUMBER 5
cell carcinoma (TCC) and SCC, and of immunohistochemical staining of SCC antigen on the carcinomas. Material and Methods The serum level of SCC antigen was determined in 214 patients with various urogenital diseases: 84 patients with histologically proved uroepithelial carcinoma (61 TCC, 14 mixed TCC with SCC, and 9 pure SCC), 79 with other urogenital carcinomas (renal cell, prostate, and testicular), and 51 with benign urologic diseases. Eighty-four patients with uroepithelial carcinoma comprised 65 of bladder and 19 of renal pelvic or ureteral carcinomas. Thirty-seven healthy adult volunteers served as normal controls.
477
w
Serum liseases
Cl.0
1
(ndmt)
SCC antigen
1.5
5
10
50
\lormal controls
. TCC + SCC or pure SCC cn=23)
.:., :’ ,:‘r’::g,;;;;.‘.y.;.D ,:. +m
qoa .
0.9
:'
8
Mean k SD (ng/mQ)
Positive rate (%I
,
l.lkO.2
2.7
l.lkO.2
3.8
1.5f2.4
9.8
8.7k16.1
82.6
FIGURE 1. Serum levels and positive rates of SCC antigen in normal controls, patients with uroepithelial carcinomas, benign urologic diseases, or in other urogenital carcinomas. Uroepithelial carcinomas classified into pure TCC, mixed TCC with SCC, and into pure SCC.
Blood samples were collected from all patients before treatments. Serial determinations of serum level of SCC antigen were done in 20 patients with uroepithelial carcinoma during follow-up period. Histopathologic extensions of the carcinomas were classified according to UICC-TNM classification of bladder carcinoma,5 and those of renal pelvic or ureteral carcinomas were determined according to a new classification advocated by Akaza, Koiso, and Niijimae which modified the UICC-TNM classification of bladder carcinomas. Grades of TCC or SCC were classified as grades 1, 2, and 3 or well, moderate, and poor and evaluated respectively. Radioimmunoassay Shortly after the collection, blood samples were centrifuged at 2,500 rpm for ten minutes, and the serums were stored at - 80 “C until assay. Serum levels were determined by radioimmunometric method (SCC Riabead, Dainabot Ltd., Tokyo, Japan). This method using monoclonal antibody for SCC antigen can detect serum levels from 1.0 to 150 ng/mL with 8.0 percent of mean intra-assay deviation and with 8.7 percent of inter-assay deviation. All samples were duplicately measured.
478
Immunohistochemical staining of SCC antigen on carcinoma tissue Immunohistochemical staining of SCC antigen was investigated in 9 patients with uroepithelial carcinoma using immunoperoxidase method and avidin-biotin complex staining (Vectastain ABC Kit, Vector Laboratory, Inc., Burlingame, CA). Two sections of 3 pm thick were deparaffinized and dehydrated according to standard technique. One of the sections was stained with hematoxylin and eosin for the standard histologic study. Following elimination of endogenous peroxidase with 3% hydrogen peroxide, 10 PglmL of mouse antiSCC antigen monoclonal antibody (courtesy of Dr. Hiroshi Kato, Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube, Japan, and Dainabot Ltd., Tokyo, Japan) was applied to the other section for sixty minutes, washed and stained with a biotin-conjugate anti-mouse antibody (at a dilution of 1:200). Then, the section was stained with an avidin-biotin peroxidase complex. Visualization of the reaction product was achieved with an application of 3,3 ‘-diaminobenzidine tetrahydrochloride. The staining of nucleus was performed with hematoxylin.
UROLOGY
/ NOVEMBER 1992 I
VOLUME 40, NUMBER 5
------EIYiCi:+iiii------------
see
poor (“=,,)
.,._. :..,......~_~.~.........~.~
::::::j::::::j::::::::::::::::: ,:,.__:...:._....
$:::::““:;,:1111;1;;;;;; $+..$$+= . . .. . . ,.,._ :.::::. ,.:.:.:+:.:.:.: ..;.:.:.:.:.:.:.:::j:::::::i:: ..,.,,........... .............. .
____
8
-__-__,--
_______
4.323.5
______,
72.7
FIGURE 2. Serum levels and positive rates of SCC antigen according to histopathologic grade in pure TCC and degree of dif erentiation in carcinomas associated with SCC component.
Results Serum level of SCC antigen with radioimmunoassay From results in 37 controls which showed the mean serum level of 1.1 + 0.2 (mean _+ SD) ng/mL, we determined 1.5 ng/mL, mean + 2 SD, as an upper normal limit. Thus, serum SCC antigen of more than 1.5 ng/mL was considered “positive” or “elevated.” Figure 1 demonstrates a distribution of serum levels of SCC antigen in normal subjects and in various diseases, including uroepithelial carcinomas and positive rates. Only 1 patient (2.7%) had serum SCC antigen over the upper normal limit in the control group. Mean serum level of SCC antigen and positive rate were almost the same in normal subjects, in benign urologic diseases, and in any other urogenital carcinomas. In uroepithelial carcinomas, 6 of 61 patients (10%) with pure TCC had a positive level of serum SCC antigen, although the mean level was not significantly different from that of the controls (unpaired Student’s t test). In contrast, the patients with a mixed TCC with SCC or pure SCC showed a significantly higher level of serum SCC antigen (unpaired Student’s t test, p 9'0T Z'T
T'6
3X 'Qd 30 'ON
=nd
I?LI.roupra~ 30 uo!sua~xg
TABLE II.
Immunohistochemical staining and pretreatment level of SCC antigen in uroepithelial carcinomas
Case No.
staining of SCC FIGURE 4. Immunohistochemical antigen on uroepithelial carcinoma. Grade 3 TCC hematoxylin-eosin stain (A), and positive immunohistochemical stain of SCC antigen (B). Grade 2 TCC hematoxylin-eosin stain (C), and negative immunohistochemical stain of SCC antigen (0). (Original magnijications x 118 [A] and x 235 [Cl).
range after treatment. These normal levels persisted during the subsequent follow-up period. Immunohistochemical
staining
of SCC
antigen
Immunohistochemical staining of SCC antigen was performed on 9 patients, 7 with pure TCC (2 with grade 3, 4 with grade 3) and 2 with pure SCC (Table II, Fig. 4). In 3 patients with grade 2 TCC, whose pretreatment serum level SCC antigen was normal, SCC antigen
UROLOGY
/ NOVEMBER1992
I VOLUME40,NUMBER5
Histopathol. TCC TCC TCC TCC TCC TCC TCC
gr. 2 gr. 2 gr. 2 gr. 3 gr. 3 gr. 3 gr. 3 SCC mod. SCC mod.
XC
Antigen Stain Neg. Neg. Pos. Neg. Pos. Pos. Pos. Pos.
Pos.
Serum SCC Antigen (ng/mL)
