Bladder cancer is the eighth most frequent carcinoma by incidence in men worldwide and the 16th most common cancer in women (Ferlay et al., 2004). In Western countries like Europe, urothelial carcinomas (UC) represent up to 95–97%, whereas other histological types are rare (i.e. prevalence of squamous carcinoma <5%) (Parkin, 2008). White Caucasians in the USA display the same distribution [96.2% UC, 0.8% squamous cell carcinomas (SCCs)], whereas African American individuals show a slightly higher prevalence of SCC (90.2% UC, 2.5% SCCs). Throughout Africa, UC is still the most frequent type of bladder cancer, but SCC prevalence is enhanced due to the close pathogenetic relation of endemic Schistosoma haematobium infection and a course of disease with this type of bladder cancer (Algeria, SCC 11.4%; Uganda, SCC 6.7%; Zimbabwe, SCC 37.8%; and Egypt, SCC 19.9%) (Hammam et al., 2014).
UC can demonstrate a broad spectrum of morphology as a result of its propensity for divergent differentiation (Amin, 2009). Approximately 60% of urothelial tumours exhibit squamous differentiation (Black et al., 2009). The actual frequency varies, as this information is not consistently recorded by pathologists. To designate squamous differentiation, one must see a clear-cut evidence of squamous production (intracellular keratin, intercellular bridges or keratin pearls), and the degree of squamous differentiation, when present, parallels the grade of the UC. In general, UC have a relatively nondescript appearance, which when viewed in isolation cannot be differentiated readily from poorly differentiated carcinomas of other types.
Immunohistochemical markers are among the diagnostic tools investigated as aids in making such distinctions (Chang et al., 2012); however, individual antibodies commonly used to support the diagnosis of UC (e.g. p63, high-molecular-weight cytokeratin) are often not especially helpful in these situations, as both are expressed in SCC and UC.
A pure histology of squamous should ideally be designated as a pure squamous of the bladder. Typically, well-differentiated or moderately differentiated SCC can be recognized easily at haematoxylin–eosin stains with their mainly exophytic or solid appearance, varying necrotic proportions and accompanying squamous epithelial changes (Lagwinski et al., 2007).
However, for a subset of poor or mixed differentiation, immunohistological verification might be helpful (Lopez-Beltran et al., 2007).
GATA3-binding protein is a transcription factor of the GATA family. These nuclear proteins recognize G-A-T-A nucleotide sequences in target gene promoters and activate or repress those genes (Chou et al., 2010). GATA3 function is known to be important in the regulation of genes such as MUC1/EMA involved in the luminal differentiation of breast epithelium 3 and genes related to T-cell development (Yagi et al., 2011). Other known functions include gene regulation in the development or maintenance of the skin, especially hair shafts (Sellheyer and Krahl, 2010), trophoblast (Home et al., 2009), and some endothelial cells, especially in the great vessels (Song et al., 2009). Constitutional allelic loss of GATA3 (haploinsufficiency) causes a syndrome characterized by hypoparathyroidism, sensorineural deafness and renal malformations, abbreviated as HDR on the basis of these manifestations. GATA3 has been thus far explored in surgical pathology as a marker for breast and UC. Most primary and metastatic mammary carcinomas express GATA3 (80–90%), but the expression is reportedly lower in triple-negative tumours (67%) (Cimino-Mathews et al., 2013). In one study, 82% of female breast carcinomas but only 32% of male breast carcinomas were GATA3-positive. GATA3 expression seems to be more extensive in lower-grade tumours and positive cases, and cases with high expression may have a better survival (Gonzalez et al., 2013). However, none of those studies have assessed the specificity of GATA3 for breast cancer. In another study, a strong expression of GATA3 (in >50% tumour cells) was detected only in mammary (94%), urothelial (86%) and endometrial (2%) adenocarcinomas but in none of the pulmonary, pancreatic, colonic, prostatic, and ovarian carcinomas in a relatively small sample (Liu et al., 2012).
GATA3 was discovered as a UC marker using CDNA expression microarrays (Higgins et al., 2007) and found downregulated in invasive bladder cancers (Miyamoto et al., 2012).
However, thus far, no larger evaluation of routinely used immunohistochemical markers and GATA3 for squamous differentiation has been performed.
The purpose of this study was to investigate the expression of a selected panel of antibodies (GATA3, CK14, CK7 and CK20) in SCC, and investigate its utility to aid in the diagnosis of urinary bladder SCC and UC with squamous differentiation.
Materials and methods
The present study was carried out on formalin-fixed paraffin-embedded tissue sections including five control cases and 75 cases with urinary bladder tumour that were retrieved consecutively from the archives of Professor Elia Anis Ishak Laboratory Pathology Centre (Cairo, Egypt), during the period from January to June 2014. All subsequent biopsies were obtained through radical cystectomies and the control cases were obtained from normal mucosa. The histological diagnosis was performed according to the WHO criteria (Travis et al., 2004).
Hematoxylin and eosin (H&E) stain was used to evaluate and grade urinary bladder carcinoma biopsy samples. Sections of 4 mm thickness were prepared from the formalin-fixed paraffin-embedded tissue. The samples were fixed in 10% buffered formalin and processed with H&E stain.
The 80 patients were categorized into the following groups:
- Group 1 included five control cases.
- Group 2 included 55 cases of SCC (keratinizing and nonkeratinizing).
- Group 3 included 10 cases of poorly differentiated UC with squamous differentiation.
- Group 4included 10 cases of conventional UC.
On the basis of histopathological grading, urinary bladder cancer was categorized as follows:
- Low-grade SCC (keratinizing and nonkeratinizing).
- High-grade SCC (keratinizing and nonkeratinizing).
- Poorly differentiated UC (transitional cell carcinoma) with squamous differentiation.
- Low-grade UC (transitional cell carcinoma).
- High-grade UC (transitional cell carcinoma).
Immunohistochemical staining was performed on formalin-fixed paraffin-embedded sections of 4 mm thickness using Anti-Cytokeratin 7 ready-to-use antibodies (Ventana, Export, Pennsylvania, USA), GATA3 (Cell Marque, Rocklin, California, USA), anti-Cytokeratin-14 (Cell Marque) and anti-Cytokeratin-20 (Ventana). Antigen retrieval was performed in all cases by steam-heating the slides in 1 mmol/l solution (pH 9.0) for 45 min. After blocking of endogenous biotin, staining was performed using an automated immunostainer (Dako, Glostrup, Denmark), followed by detection using a streptavidin–biotin detection system (Dako). Positive and negative control sections were used for each assay according to the company protocol.
Statistical analysis of the measurements was performed using SPSS for Windows, v.13 (SPSS Inc., Chicago, Illinois, USA). Numeric data were reported as mean±SD. Categorical data were reported as frequency and percentage. The χ 2-test was used to compare categorical variables between the groups. A P-value less than 0.05 was considered statistically significant for all tests.
Seventy-five patients with bladder cancer were included in this study in addition to five control specimens that were taken from urinary bladder mucosa.
Biopsy specimens were divided into four groups (Table 1):
- Group 1 included five control cases.
- Group 2 included 55 cases of SCC (keratinizing and nonkeratinizing):
- Group 2a included 40 cases of well-differentiated SCC.
- Group 2b included 15 cases of poorly differentiated SCC.
- Group 3 included 10 cases of poorly differentiated UC with squamous differentiation.
- Group 4 included 10 cases of conventional UC.
Squamous cell carcinoma (n=55, including 40 well-differentiated SCC and 15 poorly differentiated SCC)
Pure SCCs (well-differentiated or poorly differentiated) (Table 1, Diagram 1 and Fig. 2) were strongly positive for CK14 (100%) (Fig. 6) showing a significant difference from control cases and conventional UC (P=0.001) and were constantly negative for CK20 (100%), with a significant difference from control cases, UC with squamous differentiation and conventional UC (P=0.001) (Fig. 1).
An overall 5% of the well-differentiated SCCs were positive for GATA3, whereas none of the poorly differentiated cases (0%) showed any positivity for GATA3 (Fig. 4), with a significant difference from controls, UC with squamous differentiation and conventional UC (P=0.001).
An overall 52.5% of the well-differentiated SCCs were positive for CK7 (Fig. 5) and 73.3% of the poorly differentiated SCCs were positive for CK7, with a significant difference from controls, UC with squamous differentiation and conventional UC (P=0.01).
Conventional urothelial carcinoma (conventional UC) (n=10)
Pure UC had an opposite pattern and were positive for GATA3 (100%), with a significant difference from well-differentiated and poorly differentiated SCC (P=0.001), and were negative for CK14 (100%), with a significant difference from well-differentiated and poorly differentiated SCC (P=0.001); they were strongly positive for CK7, with a significant difference from well-differentiated and poorly differentiated SCC (P=0.01) and positive for CK20 (100%), with a significant difference from well-differentiated and poorly differentiated SCC (P=0.001) (Table 1, Diagram 1 and Fig. 3).
Urothelial carcinoma with marked squamous differentiation (n=10)
UC with squamous differentiation (Table 1, Diagram 1 and Fig. 7) expressed urothelial and squamous-associated markers (Fig. 8). GATA3 showed 70% positivity, with a significant difference from well-differentiated and poorly differentiated SCC (P=0.001) and conventional UC (P=0.01). CK14 showed 100% positivity (Fig. 9), with a significant difference from conventional UC (P=0.001); CK7 showed 100% positivity (Fig. 8), with a significant difference from well-differentiated and poorly differentiated SCC (P=0.01); and CK20 showed 70% positivity (Fig. 10), with a significant difference from well-differentiated and poorly differentiated SCC (P=0.001), and a significant difference from conventional UC (P=0.01).
Classifying UC can be difficult in poorly differentiated tumours, as up to 60% display squamous features and as the pathologist is often faced with the task of making a diagnosis from biopsy specimens with limited tumour sampling (Amin et al., 2012). Urinary bladder SCC and UC with squamous differentiation are often high-grade and high-stage tumours that are thought to be associated with a poorer prognosis and response to therapy compared with UC without divergent differentiation (Huang et al., 2013).
GATA-binding protein 3 (GATA3) is a novel immunohistochemical marker for UC; however, a few studies have investigated the role of GATA3 as a marker for urinary bladder SCC.
In the present study, conventional UC were positive for GATA3 (100%), whereas UC with marked squamous differentiation showed lower positivity (70%). Clark et al. (2014) found that 95% of UC studied expressed GATA3 (mean H-score of 102).
GATA3 expression was present in 70% (72/103) of conventional bladder UC and was highly concordant between matched primary and metastatic UC (Liang et al., 2014).
In another study, 78% of primary invasive UC and 23% of pulmonary SCCs were positive, indicating a lesser specificity (Gruver et al., 2012). Moreover, transitional cell lesions of ovaries, such as Brenner tumours, have been found to be GATA3 positive (Esheba et al., 2009).
As regards the expression of GATA3 in SCC, 5% of the well-differentiated SCCs were positive for GATA3, whereas none of the poorly differentiated cases (0%) showed any positivity for GATA3, with a significant difference from controls, UC with squamous differentiation and conventional UC (P=0.001).
This is in concordance with the findings of Liang et al. (2014), who found that only 7% of SCCs (1/14) expressed GATA3, and it was also significantly lower than that in conventional UC (P<0.001).
An overall 52.5% of the well-differentiated SCCs were positive for CK7 and 73.3% of the poorly differentiated SCCs were positive for CK7, with a significant difference from controls, UC with squamous differentiation and conventional UC (P=0.01).
The present study showed that conventional UC and UC with squamous differentiation were strongly positive for CK7 (100%). As regards CK20, all conventional UC were positive (100%), whereas UC with squamous differentiation showed 70% positivity.
Chu et al. (2000) found that CK7 was expressed in the majority (88%) of UC, and CK20 was positive in more than 75% of UC (Moll et al., 1988).
Our results confirmed that pure SCCs were strongly positive for CK14 (100%), showing a significant difference from control cases and conventional UCs (P=0.001), and were constantly negative for CK20 (100%), with a significant difference from control cases, UC with squamous differentiation and conventional UC (P=0.001).
This was reported for CK14 and CK20 by Gaisa et al. (2010) as well. In addition, they could show that CK14 detected even clusters of squamous phenotype cells in 47% of tumours without any morphological evidence of squamous differentiation. They claimed that CK14 is an early marker of squamous differentiation because CK14 and its obligatory heterodimer CK5 are the earliest markers expressed in still nonstratified epidermis in postgastrulation mouse embryo skin. Because of a partial positivity of CK14 in UCs, they also proposed the combination of CK14 positivity and CK20 negativity for immunohistochemical characterization of SCCs. The importance of CK20 negativity in combination with varying expression of other CKs for the diagnosis of squamous carcinomas was investigated by Suo et al. (1993), who found CK20 negative for all 42 cases of SCC from various locations. Furthermore, two other studies on SCCs and UCs of bladder confirmed the lack of expression of CK20 in SCCs (Gee et al., 2003).
The present study showed that 52.5% of the well-differentiated SCCs were positive for CK7 and 73.3% of the poorly differentiated SCCs were positive for CK7.
In conclusion, GATA3 can be used for differentiating UC from SCCs. The present study confirmed that CK14 is expressed in SCC and in UC with squamous differentiation.
CK14 positivity as well as CK20 negativity are diagnostic markers for pure squamous differentiation in bladder tumours.
Our study could successfully define a highly significant (P<0.01) immunohistochemical marker panel for precise detection of squamous differentiation in bladder cancer specimens (GATA3, CK14 and CK20).
Conflicts of interest
There are no conflicts of interest.
Amin MB, Srigley JR, Grignon DJ, et al. (2003). Updated protocol for examinations of specimen from patients with carcinoma of urinary bladder, ureter, renal pelvis, Arch pathol lab med 127:1263–1279.
Amin MB, Delahunt B, Bochner B, et al. (2012). Updated protocol for examinations of specimen from patients with carcinoma of urinary bladde. Available from college of American pathologist Web site.
Black PC, Brown GA, Dinney CP (2009). The impact of variant histology on the outcome of bladder cancer treated with curative intent. Urol Oncol 27:3–7.
Chang A, Amin A, Gabrielson E, Illei P, Roden RB, Sharma R, Epstein JI (2012). Utility of GATA3 immunohistochemistry in differentiating urothelial carcinoma from prostate adenocarcinoma and squamous cell carcinomas of the uterine cervix, anus, and lung. Am J Surg Pathol 36:1472–1476.
Chou J, Provot S, Werb Z (2010). GATA3 in development and cancer differentiation: cells GATA have it!. J Cell Physiol 222:42–49.
Chu P, Wu E, Weiss LM (2000). Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol 13:962–972.
Cimino-Mathews A, Subhawong AP, Illei PB, Sharma R, Halushka MK, Vang R, et al. (2013). GATA3 expression in breast carcinoma: utility in triple-negative, sarcomatoid, and metastatic carcinomas. Hum Pathol 27:3–7.
Clark BZ, Beriwal S, Dabbas DJ, Bhargava R (2014). Semiquantitative GATA-3 immunoreactivity in breast, bladder, gynecologic tract, and other cytokeratin 7- positive carcinomas. Am J Clin Pathol 142:64–71.
Esheba GE, Longacre TA, Atkins KA, Higgins JP (2009). Expression of the urothelial differentiation markers GATA3 and placental S100 (S100P) in female genital tract transitional cell proliferations. Am J Surg Pathol 33:347–353.
Ferlay J, Bray F, Pisani P, Parkin DM (2004). GLOBOCAN 2002 cancer incidence, mortality and prevalence worldwide IARC Cancer Base No 5, version 20. Lyon: IARC.
Gaisa NT, Braunschweig T, Reimer N, Bornemann, Eltze E, Siegert S, et al. (2010). Different immunohistochemical and ultrastructural phenotypes of squamous differentiation in bladder cancer. Virchows Arch 10:1017–1022.
Gee JR, Montoya RG, Khaled HM, Sabichi AL, Grossman HB (2003). Cytokeratin 20, AN43, PGDH, and COX-2 expression in transitional and squamous cell carcinoma of the bladder. Urol Oncol 21:266–270.
Gonzalez RS, Wang UJ, Kraus T, Sullivan H, Adams AL, Cohen C (2013). GATA-3 expression in male and female breast cancers: comparison of clinic-pathologic parameters and prognostic relevance. Hum Pathol 44:1065–1070.
Gruver AM, Amin MB, Luthringer DJ, Westfall D, Arora K, Farver CF, et al. (2012). Selective immunohistochemical markers to distinguish between metastatic high-grade urothelial carcinoma and primary poorly differentiated invasive squamous cell carcinoma of the lung. Arch Pathol Lab Med 13:1339–1346.
Hammam O, Wishahiz M, Khalil H, El Ganzouri H, Badawy M, Elkhquly A, Elesaily K (2014). Expression of cytokeratin 7, 20, 14 in urothelial carcinoma and squamous cell carcinoma of the Egyptian urinary bladder cancer. J Egypt Soc Parasitol 44:733–740.
Higgins JP, Kaygusuz G, Wang L, Montgomery K, Mason V, Zhu SX, et al. (2007). Placental S100 (S100P) and GATA3: markers for transitional epithelium and urothelial carcinoma discovered by complementary DNA microarray. Am J Surg Pathol 31:673–680.
Home P, Ray S, Dutta D, Bronshteyn I, Larson M, Paul S (2009). GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression. J Biol Chem 284:28729–28737.
Huang W, Williamson SR, Rao Q, Lopez-Beltran A, Montironi R, Eble JN, et al. (2013). Novel markers of squamous differentiation in the urinary bladder. Hum Pathol 44:1989–1997.
Lagwinski N, Thomas A, Stephenson AJ, Campbell S, Hoschar AP, Eö-Gabry E, et al. (2007). Squamous cell carcinoma of the bladder: a clinicopathological analysis of 45 cases. Am J Surg Pathol 31:1777–1787.
Liang Y, Heitzman J, Kamat AM, Dinney CP, Czerniak B (2014). Differential expression of GATA-3 in urothelial carcinoma variants. Hum Pathol 45:1466–1472.
Liu H, Shi J, Wilkerson ML, Lin F (2012). Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol 138:57–64.
Lopez-Beltran A, Requena MJ, Alvarez-Kindelan J, Quintero A, Blanca A, Montironi R (2007). Squamous differentiation in primary urothelial carcinoma of the urinary tract as seen by MAC387 immunohistochemistry. J Clin Pathol 60:332–335.
Miyamoto H, Izumi K, Yao JL, Li Y, Yang Q, McMahon LA, et al. (2012). GATA binding protein 3 is down-regulated in bladder cancer yet strong expression is an independent predictor of poor prognosis in invasive tumor. Hum Pathol 43:2033–2040.
Moll R, Achtstätter T, Becht E, Balcarova-Ständer J, Ittenson M, Franke WW (1988). Cytokeratins in normal and malignant transitional epithelium. Maintenance of expression of urothelial expression features in transitional cell carcinomas and bladder carcinoma cell culture lines. Am J Pathol 132:123–144.
Parkin DM (2008). The global burden of urinary bladder cancer. Scand J Urol Nephrol 42 (Suppl 218):12–20.
Sellheyer K, Krahl D (2010). Expression pattern of GATA-3 in embryonic and fetal human skin suggests a role in epidermal and follicular morphogenesis. J Cutan Pathol 37:357–361.
Song H, Suehiro J, Kanki Y, Kawai Y, Inoue K, Daida H, et al. (2009). Critical role for GATA3 in mediating Tie2 expression and function in large vessel endothelial cells. J Biol Chem 284:29109–29124.
Suo Z, Holm R, Nesland JM (1993). Squamous cell carcinomas. An immunohistochemical study of cytokeratins and involucrin in primary and metastatic tumours. Histopathology 23:45–54.
Travis P, Bennett S, Haines A, Pang T, Bhutta Z, Hyder AA, et al. (2004). Overcoming health-systems constraints to achieve the Millennium Development Goals. Lancet 364:900–906.
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Yagi R, Zhu J, Paul WE (2011). An updated view on transcription factor GATA3-mediated regulation of Th1 and Th2 cell differentiation. Int Immunol 23:415–420.