Original Paper U rollnt 1992:48:14-19
Department of Pathology (Prof. A. Llombart-Bosch) and Service of Urology. Hospital Clínico Universitario, University of Valencia, and Service of Preventive Medicine, La Fe Hospital, Valencia, Spain
Behavior of Epithelial Differentiation Antigens (Carcinoembryonic Antigen, Epithelial Membrane Antigen, Keratin and Cytokeratin) in Transitional Cell Carcinomas of the Bladder
Key Words
Abstract
Bladder cancer Immunohistochemistry Carcinoembryonic antigen Keratin Cytokeratin Epithelial membrane antigen
Results of an immunohistochemical study in normal urothelium and transi tional cell carcinomas of the bladder are presented. Paraffin-embedded mate rial was confronted with immunoantisera against carcinoembryonic antigen (CEA), keratin (K), cytokeratin (CK) and epithelial membrane antigen (EMA). Immunohistochemical findings confirm the changes in reactivity of dysplastic urothelium and carcinoma in situ for CEA, CK and EMA, in com parison with normal urothelium. Statistically significant differences were also found, depending upon tumor stage, in staining of transitional cell carcinomas for K and CK. Expression of CK correlated with the tumor differentiation grade: normal urothelium and well-differentiated carcinomas showed a spe cific pattern of immunostaining for the basal cells, this pattern being lost in poorly differentiated carcinomas.
Introduction
Two important objectives in the diagnosis of bladder cancer are to delimit clearly the different tumor grades and to recognize the cytological abnormalities of fiat uro thelium which are prone to progress to invasion [1-3]. Both questions are important from clinical and pathologi cal points of view [4-6]. The role of the pathologist is to identify the histological characteristics of the tumor which ultimately determine prognosis and help the clini cian in treatment selection. However, characterization of the different tumor grades and of the severity of urothelial
Received: May 27,1990 Accepted after revision: February’ 26. 1 99 1
dysplasia is influenced by subjective interpretations [7,8]. Therefore, the search for other markers for the biological malignant potential of bladder cancer has become impor tant [9, 10]. In the present study we describe the pattern of immunoreactivity of four epithelial differentiation antigens carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), keratin (K) and cytokeratin (CK) - in nor mal and dysplastic urothelium and in transitional cell car cinomas of the bladder, and we analyze these findings to see whether they correlate with tumor grade and stage.
M.C. Sdnchcz-Fcrniindcz. MD Department of Pathology, Hospital Clinico Universitario Av. Blasco Ibanez. 17 E-46010 Valencia (Spain)
© 1992 S. Kargcr AG. Basel 0042-1138/92/0481-0014 $2.75/0
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M.C. Sánchez-Fernández de Sevilla3 L. Morell-Quadrenya M. Gil-Saloinb M. Pérez-Bacetea B. Fenollosa-Entrenac A. Llombart-Boscha
Site of staining Cell membrane Cytoplasm Luminal glycocalix Type of staining Diffuse Globular
CEA
K
CK
EMA
++
+ + /-
++
+ ++
+
+
+ +
+
-
-
+
Material and Methods Tumor tissues were obtained from 104 patients with transitional cell carcinoma of the urinary bladder (79 males, 25 females, mean age 59 years, range 23-78). Ninety-eight were surgical specimens obtained by TUR (66 cases) or radical cystectomy (32 cases) and six were autopsy specimens. Seventy-nine transitional cell carcinomas were suitable for study according to tumor grade [2]: grade 1,6 cases; grade II, 25 cases; grade III, 48 cases. Sixty-one tumors were suitable for study according to tumor stage [ 11] and were grouped as superfi cial (Ta or T l, 21 cases) and invasive ones (T2, T3 or T4, 40 cases). Histologically normal urothelium (30 specimens), urothelial dyspla sia (13 specimens) and carcinoma in situ (12 specimens) were obtained from flat, macroscopically normal-looking epithelium of cystectomy specimens from the above-mentioned patients. In 6 cases with lymph node, liver and/or pulmonary métastasés, simultaneous evaluation of the primary tumor and its métastasés was performed. All materials were fixed in 10% formalin, embedded in paraffin, sec tioned at 4 pm thickness and stained with hematoxylin-eosin. Paraffin sections were cut on to slides coated with Pappas’ [12] chrome alumgelatin for immunohistochemical study. The following antibodies were used: anti-CEA (Dakopatts A 115, whole rabbit serum, dilution 1/500), anti-K (Dakopatts A 575, human callus, whole rabbit serum, dilution 1/300), anti-CK (Dako-CKl, Dakopatts M 717, mouse serum, clone LP34, molecular weight 52 kilodaltons, dilution 1/100) and anti-EMA (Dako-EMA, Dakopatts M 613. mouse serum, clone E29, dilution 1/200). Pig antirabbit immunoglobulin (Dakopatts) at 1/100 was used as second antibody for K and CEA (studied by polyclonal antibodies). A goat antirabbit immunoglobulin-biotin conjugate (Zimed) at 1/200 was used as second antibody for CK and EMA (studied by mono clonal antibodies). The third antiserum was a peroxidase-antiperoxi dase (PAP) complex obtained from rabbits in the case of polyclonal antibodies, and an avidin-biotin-peroxidase complex (ABC) elabo rated according to Hsu et al. [ 13] [2 5 mg biotin-peroxidase (Zimed) + 100 mg avidin (Sigma) + 100 ml Tris buffer] in the case of mono clonal antibodies. Peroxidase was revealed with diaminobenzidine. When CK was studied, the slides were pretreated with trypsin. Posi tive and negative controls were used in every case to assess the sensi tivity and specificity o f immunostaining. Positive or negative reactivity against the different antisera was recorded. Intensity of immunostaining was graded as negative (-), poorly positive (-/+), moderately positive (+), and strongly positive
Table 2. Intensity of staining in normal urothclium, urothelial dysplasia and carcinoma in situ
CEA
K
CK
EMA
Normal urothclium Superficial cells Intermediate cells Basal cells
+/+/+/-
+ /+ /+ /-
-
++
++ + -
Dysplastic urothelium
+
+ /-
+
+
Carcinoma in situ
++
+/-
+ /-
+ /-
(++). The sites of immunostaining (cell membrane, cytoplasm and luminal glycocalix), its type (diffuse or globular) and its extension (expressed as the percentage of stained cells) were also recorded. For statistical evaluation, y } test (with Yates correction if re quired) was used.
Results
Comparison o f Normal Urothelium with Dysplasia and Carcinoma in situ Sites and types of immunostaining in normal urothe lium are shown in table 1. Most significative findings were obtained when studying CEA. This antigen is poorly positive in normal urothelium and became progressively more positive in dysplasia and carcinoma in situ. Staining variations for EMA, K and CK were not significant. K is poorly positive in the entire urothelial thickness. CK presents a specific staining pattern in the basal urothelial layer. EMA shows reactivity in the whole urothelium except in basal cells (table 2). Comparison o f Tumor Grades Differences were significant only in the case of CK, when comparing grades I—II tumors with those of grade 111 (p < 0.05) (table 3). Percentages of stained cells were similar among the three tumor grades. The extent of immunostaining did not allow a better characterization of tumor grades (fig. 1-4). Comparison o f Tumor Stages There were more superficial than invasive tumors among the cases positive for K and CK (p < 0.05) (ta ble 4). Staining differences between primary tumors and metastases did not reach statistical significance for any of the antigens used in the study (table 5).
15
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Table 1. Sites and types of immunostaining in normal urothc-
lium
Fig. 1. Percentage o f CEA-stained cells according to tumor stage
and grade.
Fig. 2. Percentage of K-stained cells according to tumor stage and
grade.
Table 3. Distribution of stained tumors accordings to tumor
Table 4. Distribution of stained tumors according to stage
grade Tumor grade
CEA
K
CK
EMA
I 11 III
4/6 16/25 42/48 3.46 n.s.
6/6 21/25 35/48 2.98 n.s.
5/6 22/25 32/48 4.21 n.s.
5/6 19/25 40/48 0.60 n.s.
22/31 42/48 3.35 n.s.
27/31 35/48 0.43 n.s.
27/31 32/48 4.16 p < 0.05
24/31 40/48 0.43 n.s.
r
p value I + II III
X2
p value
Results are expressed as number of stained tumors/total number of tumors, n.s = Not significant.
Tumor stage
CEA
K
CK
EMA
T a-T l T 2-T 3-T 4
15/21 35/40 2.41 n.s.
20/21 28/40 5.23 p < 0.05
19/21 27/40 3.92 p < 0.05
18/21 34/40 0.01 n.s.
X2
p value
Results are expressed as number of stained tumors/total number of tumors, n.s. = Not significant.
Table 5. Quantification of stained primary tumors and their
métastasés
Primary tumors Métastasés X2
p value
CEA
K
CK
EMA
1/6 6/14 0.38 n.s.
4/6 5/14 0.62 n.s.
3/5 3/13 0.87 n.s.
2/6 4/14 0.10 n.s.
16
Sanchcz-Fernandez de Sevilla/ Morell-Quadreny/Gil-Salom/Pcrez-Baccte/ Fenollosa-Entrena/Llombart-Bosch
Epithelial Differentiation Antigens in Bladder Cancer
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Results are expressed as number o f stained cases/total number of cases, n.s. = Not significant.
Stained cells, %
Fig. 4. Percentage o f EMA-stained cells according to tumor stage
and grade.
A specific staining pattern for CK was observed in the basal cells of superficial tumors, resembling that detected in normal urothelium (fig. 5). Most invasive tumors were negative for CK, but in the few which were positive, all the cells were stained (fig. 6). CEA was positive only in superficial tumor cells (fig. 7). EMA and K showed a dif fuse reactivity in tumor cells, without a specific staining pattern (fig. 8). A higher percentage of cells was positive for K and CK in superficial tumors than in invasive ones. The percent age of positive cells for CEA and EMA was higher in inva sive tumors than in superficial ones (fig. 1-4).
A progressive increase in staining for CEA in dysplasia and carcinoma in situ was one of the most significant findings in our study. Wahren [ 19] noted that normal uro thelial cells did not express CEA, but in the vicinity of papillary tumors, CEA-positive cell populations were identified. These did not differ morphologically from nor mal CEA-negative urothelium. An increase in staining for CEA in carcinoma in situ has also been reported by others [3, 20, 21], confirming the fitness of this antigen for detecting preneoplastic changes of the urothelium. In transitional cell carcinomas, results of staining for CEA are variable [3, 22], The progressive increase in the num ber of positive tumors with higher grade and stage ob served by others [23-25] was also detected in our study, but did not reach statistical significance. Elevated urinary levels of CEA have been reported in patients with bladder cancer [26], However, a definitive correlation between urinary CEA levels and tumor immunostaining for CEA is lacking [21], K polyclonal antibody stained uniformly the entire urothelial thickness, without significant changes in dysplastic urothelium and carcinoma in situ, as reported by other authors [27-29], Monoclonal CK antibody (molec ular weight 52 kilodaltons) shows a specific staining pat-
Discussion
Several investigators have reported a correlation be tween morphologic differentiation and expression of spe cific molecules both in normal and neoplastic urothelium [14-16]. Immunohistochemical markers have been useful in our study for a more exact typing of tumor grades and a better characterization of urothelial dysplasia and carci noma in situ. The prognostic relevance of these latter le sions has been stressed by several authors [ 1,4-6, 17, 18].
17
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Fig. 3. Percentage of CK-stained cells according to tumor stage
and grade.
Fig. 7. Specific staining pattern for CEA in superficial tumor cells. PAP. X 40. Fig. 8. Invasive transitional cell carcinoma showing strong posi tivity for EMA in all cells. ABC. X 40.
tern for the basal layer of normal and dysplastic urothelium. In carcinoma in situ its distribution is scanty and irregular. Ramaekers et al. [27] found with CK 18 (molec ular weight 45 kilodaltons) a selective staining pattern for superficial cells. These authors did not report if there were significant staining changes in carcinoma in situ. In transitional cell carcinomas, both anti-K (polyclonal and monoclonal) revealed significant differences in stain ing according to tumor stage (p < 0.05). Differences in staining for CK according to tumor grade (p < 0.05) were also found. Well-differentiated carcinomas presented a specific staining pattern for the basal cells, similar as in normal urothelium, this pattern being lost in poorly dif ferentiated carcinomas. Several authors have observed the same staining distribution for K in transitional cell
carcinomas as in our study [27, 30-33], but differences according to tumor grade and stage have been reported only occasionally [34], Elevated CK 18 urinary levels have been found to correlate with advanced tumor grade and stage, and have been ascribed to high cell turnover rate followed by increased release of CK by the tumor [35]. Perhaps a loss or denaturalization of intermediate filaments from cytoskeletons may account for the nonde tection of this antigen in tissue sections of high-grade high-stage transitional cell carcinomas, this loss being detected, however, at an extracellular level in urine. Normal urothelium and transitional cell carcinomas showed immunoreactivity for EMA [22, 36, 37], with a characteristic distribution. In normal urothelium and in superficial tumors, localization of EMA was restricted to
18
Sanchez-Fernandez de Sevilla/ Morell-Quadreny/Gil-Salom/Perez-Bacete/ Fenollosa-Entrena/Llombart-Bosch
Epithelial Differentiation Antigens in Bladder Cancer
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Fig. 5. Specific staining pattern for CK in the basal cells of a superficial tumor. ABC. X 40. Fig. 6. Invasive transitional cell carcinoma showing strong cyto plasmic positivity for CK in almost all cells. ABC. X 40.
the cell membrane, while in invasive tumors staining for EMA was localized in the cell membrane and in the cyto plasm (globular type), in agreement with previous reports [38], Invasive tumors showed stronger intensity and
greater extension of immunostaining, with a mosaic ap pearance [39], than superficial ones, but differences in the number of stained tumors according to grade and stage were not significant in our study.
References 16 Hodges GM, Smolirs MA. Trejdosiewicz LK: Urothelium-spccific antibody and lectin sur face mapping o f bladder urothelium. Histo chem J 1982;14:755-756. 17 Galian PH. Desligneres S. Franc B. et al: Lé sions carcinomateuses intra-épithéliales de la vessie. Arch Anat Cytol Pathol 1979:27:187193. 18 Soto EA, Friedell GH, Tiltman AJ: Bladder cancer as seen in giant histologie sections. Can cer 1977;39:447-455. 19 Wahren B: Cellular content o f carcinoembryonic antigen in urothelial carcinoma. Can cer 1978;42:1533-1539. 20 Shevchuk MM, Fenoglio CM, Richart RM: Carcinoembryonic antigen localization in be nign and malignant transitional epithelium. Cancer 1981 ;47:899-905. 21 Wiley EL, Mendelsohn G, Droller MJ. Eggles ton JC: Immunoperoxidase detection o f carci noembryonic antigen and blood group sub stances in papillary transitional cell carcinoma o f the bladder. J Urol 1982;128:276-280. 22 Hcydcrman E, Brown BH, Richardson TC: Epithelial markers in prostatic, bladder and colorectal cancer: An immunoperoxidase study o f epithelial membrane antigen, carcinoem bryonic antigen and prostatic acid phospha tase. J Clin Pathol 1984;37:1363-1369. 23 Jautzke G, Altenaehr E: Immunohistochemical demonstration o f carcinoembryonic antigen (CEA) and its correlation with grading and staging on tissue sections o f urinary bladder carcinomas. Cancer 1982;50:2052-2056. 24 López-Beltrán A, Garcia-Calvo J, Morales C, et al: Determinación inmunohistoquimica del antígeno carcinoembrionario en carcinomas uroteliales de vejiga. Oncología 1986;9:44-48. 25 Fujioka T, Ohhori T, Lovrekovich L, et al: Investigation o f blood group antigens and car cinoembryonic antigen in urinary' bladder car cinoma. Urol Int 1986:41:397—402. 26 Hall R: Carcinoembryonic antigen in the urine o f patients with urothelial carcinoma. Br J Urol 1973;45:88-92. 27 Ramaekers F, Huysman A, Muesker D, et al: Cytokeratin expression during neoplastic pro gression o f human transitional cell carcinomas as detected by a monoclonal and a polyclonal antibody. Lab Invest 1985;52:31-38. 28 Schlegel R. Banks-Schlegel S, McLeod JA, et al: Immunoperoxidase localization o f keratin in human neoplasms. Am J Pathol 1980; 110:4149. 29 Schlegel R, Banks-Schlegel S, Pinkus GS: Im munohistochemical localization o f keratin in normal human tissues. Lab Invest 1980;42:91— 96.
30 Nadakavukaren KK, Summerhayes IC, Sal cedo BF, et al: A monoclonal antibody recog nizing a keratin filament protein in a subset of transitional and glandular epithelia. Differenti ation 1984:27:209-220. 31 Said JW: Immunohistochemical localization of keratin proteins in tumor diagnosis. Hum Pa thol 1983;14:1017-1019. 32 Steffens J. Friedmann W, Lobeck H: Immuno histochemical demonstration o f tumor-associ ated antigens with the aid o f monoclonal and polyclonal antisera in carcinoma o f the blad der! Urol Res 1985;13:55-59. 33 Sun IT , Shih CH, Green HC: Keratin cytoskeletons in epithelial cells o f internal organs. Proc Natl Acad Sci USA 1979;76:2813-2817. 34 Asamoto M, Fukushima S. Tatemoto Y, et al: Immunohistochemical expression o f keratin proteins in urinary bladder carcinoma. Pathol ResPract 1989:184:194-201. 35 Baker WC. DeVere White R. Rossitto PV, et al: Quantitative analysis o f keratin 18 in the urine o f patients with bladder cancer. J Urol 1988; 140:436-439. 36 Pinkus GS, Kurtin PJ: Epithelial membrane antigen: A diagnostic discriminant in surgical pathology: Immunohistochemical profile in epithelial, mesenchymal and hematopoietic neoplasms using paraffin sections and mono clonal antibodies. Hum Pathol 1985; 16:929940. 37 Sloane JP, Macpath BS, Ormerod MG: Distri bution of epithelial membrane antigen in nor mal and neoplastic tissues and its value in diag nostic tumor pathology. Cancer 1981 ;47:1786— 1795. 38 Takashi M, MuraseT, K injoT, etal: Epithelial membrane antigen as an immunohistochemi cal marker for transitional cell carcinoma o f the urinary bladder. Urol Int 1987;42:170— 175. 39 Asamoto M, Fukushima S. Oosumi H, et al: Immunohistochemical distribution o f epithe lial membrane antigen in bladder carcinomas as detected with a monoclonal antibody. Urol Res 1989;17:273-277.
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1 Melicow MM, Hollowell JW: Intraurothelial cancer: Carcinoma in situ, Bowen's disease of the urinary system: Discussion o f thirty cases. J Urol 1952:68:763-772. 2 Mostofi FK. Sobin LH. Torloni H: Histological Typing o f Urinary Bladder Tumors. Genève, World Health Organization, 1973. 3 Helpap B, Vogel J. Dehr P, et al: Comparison between cell kinetical and immunohistochemi cal studies on carcinoma and atypia/dysplasia o f urinary bladder mucosa. Virchows Arch 1985:406:309-322. 4 Skinner DG, Richie JP, Cooper PH. et al: The clinical significance o f carcinoma in situ o f the bladder and its association with overt carcino ma. J Urol 1974:112:68-72. 5 Koss LG. Nakanishi I, Freed SZ: Nonpapillary carcinoma in situ and atypical hyperplasia in cancerous bladders. Urology 1977;9:442-454. 6 Boccon-Gibod L, Boccon-Gibod L, Desligneres S, et al: Tumeurs de vessie. Indications et résultats des biopsies systématiques en mu queuse optiquement saine (avec étude des anti gènes de surface). Arch Anat Cytol Pathol 1986;34:224-229. 7 Murphy WM, Soloway MS: Urothelial dyspla sia. J Urol 1982:127:849-854. 8 Webb JN: The histopathology o f bladder can cer; in Zingg EJ, Wallace DMA (eds): Bladder Cancer. Berlin, Springer, 1985, pp 23-51. 9 Emmott RC, Javadpour N, Bergman SM, et al: Correlation o f the cell surface antigens with stage and grade in cancer of the bladder. J Urol 1979;121:37-39. 10 Javadpour N: Multiple cell markers in bladder cancer. Principles and clinical applications. Urol Clin North Am 1984;11:609-616. 11 Hermanek P, Subin LH: TNM: Classification des tumeurs malignes. Paris, Springer, 1988, pp 143-145. 12 Pappas PW: The use o f chrome alumgelatin rubbing solution as a general adhesive for par affin sections. Stain Technol 1971:46:121127. 13 Hsu SM, Raine L, Fanger H: Use o f avidinebiotin-peroxidase complex (ABC) in immuno peroxidase techniques: A comparison between ABC and unlabcled antibody (PAP) proce dures. J Histochem Cytochem 1981:29:577580. 14 Alroy J. Weinstein RS: Intraepithelial asym metric unit membrane plaques in mammalian urinary'bladder. Anat Rec 1980;197:75-83. 15 Hicks RM: Discussion o f morphological mark ers o f early neoplastic change in the urinary bladder. Cancer Res 1977;37:2822-2823.
Department of Pathology (Prof. A. Llombart-Bosch) and Service of Urology. Hospital Clínico Universitario, University of Valencia, and Service of Preventive Medicine, La Fe Hospital, Valencia, Spain
Behavior of Epithelial Differentiation Antigens (Carcinoembryonic Antigen, Epithelial Membrane Antigen, Keratin and Cytokeratin) in Transitional Cell Carcinomas of the Bladder
Key Words
Abstract
Bladder cancer Immunohistochemistry Carcinoembryonic antigen Keratin Cytokeratin Epithelial membrane antigen
Results of an immunohistochemical study in normal urothelium and transi tional cell carcinomas of the bladder are presented. Paraffin-embedded mate rial was confronted with immunoantisera against carcinoembryonic antigen (CEA), keratin (K), cytokeratin (CK) and epithelial membrane antigen (EMA). Immunohistochemical findings confirm the changes in reactivity of dysplastic urothelium and carcinoma in situ for CEA, CK and EMA, in com parison with normal urothelium. Statistically significant differences were also found, depending upon tumor stage, in staining of transitional cell carcinomas for K and CK. Expression of CK correlated with the tumor differentiation grade: normal urothelium and well-differentiated carcinomas showed a spe cific pattern of immunostaining for the basal cells, this pattern being lost in poorly differentiated carcinomas.
Introduction
Two important objectives in the diagnosis of bladder cancer are to delimit clearly the different tumor grades and to recognize the cytological abnormalities of fiat uro thelium which are prone to progress to invasion [1-3]. Both questions are important from clinical and pathologi cal points of view [4-6]. The role of the pathologist is to identify the histological characteristics of the tumor which ultimately determine prognosis and help the clini cian in treatment selection. However, characterization of the different tumor grades and of the severity of urothelial
Received: May 27,1990 Accepted after revision: February’ 26. 1 99 1
dysplasia is influenced by subjective interpretations [7,8]. Therefore, the search for other markers for the biological malignant potential of bladder cancer has become impor tant [9, 10]. In the present study we describe the pattern of immunoreactivity of four epithelial differentiation antigens carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), keratin (K) and cytokeratin (CK) - in nor mal and dysplastic urothelium and in transitional cell car cinomas of the bladder, and we analyze these findings to see whether they correlate with tumor grade and stage.
M.C. Sdnchcz-Fcrniindcz. MD Department of Pathology, Hospital Clinico Universitario Av. Blasco Ibanez. 17 E-46010 Valencia (Spain)
© 1992 S. Kargcr AG. Basel 0042-1138/92/0481-0014 $2.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/16/2017 4:34:38 AM
M.C. Sánchez-Fernández de Sevilla3 L. Morell-Quadrenya M. Gil-Saloinb M. Pérez-Bacetea B. Fenollosa-Entrenac A. Llombart-Boscha
Site of staining Cell membrane Cytoplasm Luminal glycocalix Type of staining Diffuse Globular
CEA
K
CK
EMA
++
+ + /-
++
+ ++
+
+
+ +
+
-
-
+
Material and Methods Tumor tissues were obtained from 104 patients with transitional cell carcinoma of the urinary bladder (79 males, 25 females, mean age 59 years, range 23-78). Ninety-eight were surgical specimens obtained by TUR (66 cases) or radical cystectomy (32 cases) and six were autopsy specimens. Seventy-nine transitional cell carcinomas were suitable for study according to tumor grade [2]: grade 1,6 cases; grade II, 25 cases; grade III, 48 cases. Sixty-one tumors were suitable for study according to tumor stage [ 11] and were grouped as superfi cial (Ta or T l, 21 cases) and invasive ones (T2, T3 or T4, 40 cases). Histologically normal urothelium (30 specimens), urothelial dyspla sia (13 specimens) and carcinoma in situ (12 specimens) were obtained from flat, macroscopically normal-looking epithelium of cystectomy specimens from the above-mentioned patients. In 6 cases with lymph node, liver and/or pulmonary métastasés, simultaneous evaluation of the primary tumor and its métastasés was performed. All materials were fixed in 10% formalin, embedded in paraffin, sec tioned at 4 pm thickness and stained with hematoxylin-eosin. Paraffin sections were cut on to slides coated with Pappas’ [12] chrome alumgelatin for immunohistochemical study. The following antibodies were used: anti-CEA (Dakopatts A 115, whole rabbit serum, dilution 1/500), anti-K (Dakopatts A 575, human callus, whole rabbit serum, dilution 1/300), anti-CK (Dako-CKl, Dakopatts M 717, mouse serum, clone LP34, molecular weight 52 kilodaltons, dilution 1/100) and anti-EMA (Dako-EMA, Dakopatts M 613. mouse serum, clone E29, dilution 1/200). Pig antirabbit immunoglobulin (Dakopatts) at 1/100 was used as second antibody for K and CEA (studied by polyclonal antibodies). A goat antirabbit immunoglobulin-biotin conjugate (Zimed) at 1/200 was used as second antibody for CK and EMA (studied by mono clonal antibodies). The third antiserum was a peroxidase-antiperoxi dase (PAP) complex obtained from rabbits in the case of polyclonal antibodies, and an avidin-biotin-peroxidase complex (ABC) elabo rated according to Hsu et al. [ 13] [2 5 mg biotin-peroxidase (Zimed) + 100 mg avidin (Sigma) + 100 ml Tris buffer] in the case of mono clonal antibodies. Peroxidase was revealed with diaminobenzidine. When CK was studied, the slides were pretreated with trypsin. Posi tive and negative controls were used in every case to assess the sensi tivity and specificity o f immunostaining. Positive or negative reactivity against the different antisera was recorded. Intensity of immunostaining was graded as negative (-), poorly positive (-/+), moderately positive (+), and strongly positive
Table 2. Intensity of staining in normal urothclium, urothelial dysplasia and carcinoma in situ
CEA
K
CK
EMA
Normal urothclium Superficial cells Intermediate cells Basal cells
+/+/+/-
+ /+ /+ /-
-
++
++ + -
Dysplastic urothelium
+
+ /-
+
+
Carcinoma in situ
++
+/-
+ /-
+ /-
(++). The sites of immunostaining (cell membrane, cytoplasm and luminal glycocalix), its type (diffuse or globular) and its extension (expressed as the percentage of stained cells) were also recorded. For statistical evaluation, y } test (with Yates correction if re quired) was used.
Results
Comparison o f Normal Urothelium with Dysplasia and Carcinoma in situ Sites and types of immunostaining in normal urothe lium are shown in table 1. Most significative findings were obtained when studying CEA. This antigen is poorly positive in normal urothelium and became progressively more positive in dysplasia and carcinoma in situ. Staining variations for EMA, K and CK were not significant. K is poorly positive in the entire urothelial thickness. CK presents a specific staining pattern in the basal urothelial layer. EMA shows reactivity in the whole urothelium except in basal cells (table 2). Comparison o f Tumor Grades Differences were significant only in the case of CK, when comparing grades I—II tumors with those of grade 111 (p < 0.05) (table 3). Percentages of stained cells were similar among the three tumor grades. The extent of immunostaining did not allow a better characterization of tumor grades (fig. 1-4). Comparison o f Tumor Stages There were more superficial than invasive tumors among the cases positive for K and CK (p < 0.05) (ta ble 4). Staining differences between primary tumors and metastases did not reach statistical significance for any of the antigens used in the study (table 5).
15
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Table 1. Sites and types of immunostaining in normal urothc-
lium
Fig. 1. Percentage o f CEA-stained cells according to tumor stage
and grade.
Fig. 2. Percentage of K-stained cells according to tumor stage and
grade.
Table 3. Distribution of stained tumors accordings to tumor
Table 4. Distribution of stained tumors according to stage
grade Tumor grade
CEA
K
CK
EMA
I 11 III
4/6 16/25 42/48 3.46 n.s.
6/6 21/25 35/48 2.98 n.s.
5/6 22/25 32/48 4.21 n.s.
5/6 19/25 40/48 0.60 n.s.
22/31 42/48 3.35 n.s.
27/31 35/48 0.43 n.s.
27/31 32/48 4.16 p < 0.05
24/31 40/48 0.43 n.s.
r
p value I + II III
X2
p value
Results are expressed as number of stained tumors/total number of tumors, n.s = Not significant.
Tumor stage
CEA
K
CK
EMA
T a-T l T 2-T 3-T 4
15/21 35/40 2.41 n.s.
20/21 28/40 5.23 p < 0.05
19/21 27/40 3.92 p < 0.05
18/21 34/40 0.01 n.s.
X2
p value
Results are expressed as number of stained tumors/total number of tumors, n.s. = Not significant.
Table 5. Quantification of stained primary tumors and their
métastasés
Primary tumors Métastasés X2
p value
CEA
K
CK
EMA
1/6 6/14 0.38 n.s.
4/6 5/14 0.62 n.s.
3/5 3/13 0.87 n.s.
2/6 4/14 0.10 n.s.
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Epithelial Differentiation Antigens in Bladder Cancer
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Results are expressed as number o f stained cases/total number of cases, n.s. = Not significant.
Stained cells, %
Fig. 4. Percentage o f EMA-stained cells according to tumor stage
and grade.
A specific staining pattern for CK was observed in the basal cells of superficial tumors, resembling that detected in normal urothelium (fig. 5). Most invasive tumors were negative for CK, but in the few which were positive, all the cells were stained (fig. 6). CEA was positive only in superficial tumor cells (fig. 7). EMA and K showed a dif fuse reactivity in tumor cells, without a specific staining pattern (fig. 8). A higher percentage of cells was positive for K and CK in superficial tumors than in invasive ones. The percent age of positive cells for CEA and EMA was higher in inva sive tumors than in superficial ones (fig. 1-4).
A progressive increase in staining for CEA in dysplasia and carcinoma in situ was one of the most significant findings in our study. Wahren [ 19] noted that normal uro thelial cells did not express CEA, but in the vicinity of papillary tumors, CEA-positive cell populations were identified. These did not differ morphologically from nor mal CEA-negative urothelium. An increase in staining for CEA in carcinoma in situ has also been reported by others [3, 20, 21], confirming the fitness of this antigen for detecting preneoplastic changes of the urothelium. In transitional cell carcinomas, results of staining for CEA are variable [3, 22], The progressive increase in the num ber of positive tumors with higher grade and stage ob served by others [23-25] was also detected in our study, but did not reach statistical significance. Elevated urinary levels of CEA have been reported in patients with bladder cancer [26], However, a definitive correlation between urinary CEA levels and tumor immunostaining for CEA is lacking [21], K polyclonal antibody stained uniformly the entire urothelial thickness, without significant changes in dysplastic urothelium and carcinoma in situ, as reported by other authors [27-29], Monoclonal CK antibody (molec ular weight 52 kilodaltons) shows a specific staining pat-
Discussion
Several investigators have reported a correlation be tween morphologic differentiation and expression of spe cific molecules both in normal and neoplastic urothelium [14-16]. Immunohistochemical markers have been useful in our study for a more exact typing of tumor grades and a better characterization of urothelial dysplasia and carci noma in situ. The prognostic relevance of these latter le sions has been stressed by several authors [ 1,4-6, 17, 18].
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Fig. 3. Percentage of CK-stained cells according to tumor stage
and grade.
Fig. 7. Specific staining pattern for CEA in superficial tumor cells. PAP. X 40. Fig. 8. Invasive transitional cell carcinoma showing strong posi tivity for EMA in all cells. ABC. X 40.
tern for the basal layer of normal and dysplastic urothelium. In carcinoma in situ its distribution is scanty and irregular. Ramaekers et al. [27] found with CK 18 (molec ular weight 45 kilodaltons) a selective staining pattern for superficial cells. These authors did not report if there were significant staining changes in carcinoma in situ. In transitional cell carcinomas, both anti-K (polyclonal and monoclonal) revealed significant differences in stain ing according to tumor stage (p < 0.05). Differences in staining for CK according to tumor grade (p < 0.05) were also found. Well-differentiated carcinomas presented a specific staining pattern for the basal cells, similar as in normal urothelium, this pattern being lost in poorly dif ferentiated carcinomas. Several authors have observed the same staining distribution for K in transitional cell
carcinomas as in our study [27, 30-33], but differences according to tumor grade and stage have been reported only occasionally [34], Elevated CK 18 urinary levels have been found to correlate with advanced tumor grade and stage, and have been ascribed to high cell turnover rate followed by increased release of CK by the tumor [35]. Perhaps a loss or denaturalization of intermediate filaments from cytoskeletons may account for the nonde tection of this antigen in tissue sections of high-grade high-stage transitional cell carcinomas, this loss being detected, however, at an extracellular level in urine. Normal urothelium and transitional cell carcinomas showed immunoreactivity for EMA [22, 36, 37], with a characteristic distribution. In normal urothelium and in superficial tumors, localization of EMA was restricted to
18
Sanchez-Fernandez de Sevilla/ Morell-Quadreny/Gil-Salom/Perez-Bacete/ Fenollosa-Entrena/Llombart-Bosch
Epithelial Differentiation Antigens in Bladder Cancer
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Fig. 5. Specific staining pattern for CK in the basal cells of a superficial tumor. ABC. X 40. Fig. 6. Invasive transitional cell carcinoma showing strong cyto plasmic positivity for CK in almost all cells. ABC. X 40.
the cell membrane, while in invasive tumors staining for EMA was localized in the cell membrane and in the cyto plasm (globular type), in agreement with previous reports [38], Invasive tumors showed stronger intensity and
greater extension of immunostaining, with a mosaic ap pearance [39], than superficial ones, but differences in the number of stained tumors according to grade and stage were not significant in our study.
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