|Year : 2018 | Volume
| Issue : 3 | Page : 826-833
Immunohistochemical expression of cytokeratin 14 and association with the extent of squamous differentiation in urothelial carcinoma
Alyaa A Moselhy, Hayam A Aiad, Hala S El Rebey, Shereen F Mahmoud
Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Menoufia, Egypt
|Date of Submission||31-May-2017|
|Date of Acceptance||07-Aug-2017|
|Date of Web Publication||31-Dec-2018|
Alyaa A Moselhy
Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Menoufia
Source of Support: None, Conflict of Interest: None
Evaluation of the diagnostic utility of cytokeratin 14 (CK14) immunostaining for squamous differentiation in urothelial carcinoma (UC).
UC with squamous differentiation usually presents at an advanced stage than pure UC. Moreover, increased extent of squamous differentiation in UC tends to be associated with poor survival. Sometimes, immunohistochemical marker may be needed for diagnosis of squamous differentiation in UC.
Materials and methods
This retrospective cross-sectional study included 58 cases of radical cystectomy and 16 cases of cystoscopic biopsy diagnosed with bladder cancer. The cases included 16 cases of squamous cell carcinoma (SCC), 26 of pure UC, 31 of UC with squamous differentiation, and one case of adenocarcinoma. They were subjected to immunohistochemical staining for CK14. CK14 expression and its association with some clinicopathologic parameters were evaluated.
CK14 showed positive expression in 74.2% of UC with squamous differentiation, 19.2% of pure UC, and all cases of SCC. It showed sensitivity of 74% and specificity of 81% for detecting squamous differentiation in UC. Its expression has significant association with the extent of squamous differentiation in UC (P = 0.001), perineural invasion (P = 0.03), and grade of SCC (P = 0.0001).
CK14 is a specific and sensitive marker in diagnosis of SCC and squamous differentiation in UC, and its expression is associated with poor prognostic parameters in bladder cancer.
Keywords: cytokeratin 14, perineural invasion, squamous differentiation, urothelial carcinoma
|How to cite this article:|
Moselhy AA, Aiad HA, El Rebey HS, Mahmoud SF. Immunohistochemical expression of cytokeratin 14 and association with the extent of squamous differentiation in urothelial carcinoma. Menoufia Med J 2018;31:826-33
|How to cite this URL:|
Moselhy AA, Aiad HA, El Rebey HS, Mahmoud SF. Immunohistochemical expression of cytokeratin 14 and association with the extent of squamous differentiation in urothelial carcinoma. Menoufia Med J [serial online] 2018 [cited 2019 Jun 16];31:826-33. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/826/248744
| Introduction|| |
Bladder cancer is the ninth prominent cancer worldwide. In Egypt, it is the third common malignancy, accounting for 8.7% of total malignancies in both sex according to National Cancer Registry, 2014, and it is the most prevalent cancer among Egyptian men.
Urothelial carcinoma (UC) is the most common histologic type of bladder cancer. The ability to give variable morphologic variants that have worse prognosis is an important feature of UC. So, it is important to detect variant histologies to guide the therapeutic process. Of these variants, UC with squamous differentiation is the most prevalent one, which could be encountered in 40% of UC cases.
Prognostic significance of squamous differentiation in UC is debatable; however, many studies have reported its aggressive behavior owing to association with advanced tumor stage and nodal metastasis, as well as poor response to chemotherapy, radiotherapy, and immunotherapy.
Some studies reported that increased extent of squamous differentiation in UC is associated with increased lymph node metastasis and decreased patient's survival. So, it is important to detect presence as well as extent of squamous differentiation in UC and including it in histopathologic report.
Squamous differentiation in UC can be detected histologically by presence of intercellular bridges, individual cell keratinization, and keratin pearl formation, which may be missed in focal or nonkeratinizing cases.
It may be difficult to differentiate high-grade UC from high-grade squamous cell carcinoma (SCC) as high-grade UC may have squamous appearance. Thus, immunohistochemical marker diagnostic of SCC and squamous differentiation in UC is needed.
Cytokeratins (CKs) are multigene family formed of 20 members of related polypeptides from CK1 to CK20. They are the major group of intermediate filaments that present in epithelial cells. CKs retain expression even in transformed cells in different combinations. This property helps in phenotyping of human carcinomas. CKs are divided in to two types: type I acidic and type II basic.
Cytokeratin 14 (CK14) is a type I (acidic) human intermediate filament protein, which usually pairs with CK5 type II (basic) CK.
CK14 is expressed in the basal compartment of all stratified squamous epithelia. It is a useful marker in the identification of basal cell epithelium in prostate and myoepithelium in breast and has also been shown to be a marker of SCC of the lung, and it is not expressed in normal urothelium. So the expression of CK14 in UC may represent squamous phenotypic switch.
This study aims to investigate the diagnostic utility of CK14 for SCC and squamous differentiation in UC as well as to assess association of its expression with some clinicopathologic parameters.
| Materials and Methods|| |
The protocol of this study was approved by the ethical committee of Faculty of Medicine, Menoufia University.
This retrospective cross-sectional study was conducted on 74 cases of bladder cancer (58 cases of radical cystectomy and 16 cases of cystoscopic biopsy), including 16 cases of SCC, 26 cases of pure UC, 31 cases of UC with squamous differentiation, and one case of adenocarcinoma. The cases were retrieved from the archives of Pathology Departments, Faculty of Medicine, Menoufia University, and Cairo University, spanning the period between January 2014 and January 2017. They were selected based on histopathologic diagnosis. All tissue blocks were subjected to CK14 immunostaining.
Clinical data were collected from patients' medical reports and includes age, sex, and type of specimen (radical cystectomy or cystoscopic biopsy).
We have re-evaluated the hematoxylin and eosin-stained sections to confirm the diagnosis and to determine the available prognostic and predictive features, including grade, tumor stage, nodal metastasis, squamous metaplasia, extent of squamous differentiation in UC (classified as <50%, 50–90%, and >90%), lymphovascular invasion (LVI), and perineural invasion (PNI). Grading was done according to WHO/ISUP grading criteria and staging according to American Joint Committee on Cancer staging system.
The method used for immunostaining was streptavidin–biotin amplified system. The slides were submitted to subsequent steps of deparaffinization, rehydration, and blocking of endogenous peroxidase activity. Antigen retrieval was done by boiling in citrate buffer saline (pH = 6) followed by cooling at room temperature. The primary antibody was incubated overnight at room temperature, and then secondary antibody was applied with diaminobenzidine as a chromogen substrate and Mayer's hematoxylin as a counter stain. CK14 purified mouse monoclonal antibody (GTX 62683; GeneTex, CA, USA) was received as 100 μl of ready-to-use diluted solution. Positive controls for CK14 (normal skin tissue) together with negative control (omitting of primary antibody step) were included in each run of staining.
CK14 immunostain appears as brown cytoplasmic staining.
The assessment included the following: total immunostaining score (TIS) was calculated by multiplying percentage score (PS), and intensity score (IS):
PS: 0 = no positive cells, 1 = any positive cell up to 10%, 2 = 10–50%, 3 = 51–80%, and 4 = more than 80%.
IS: 0 = no color reaction, 1 = mild intensity, 2 = moderate intensity, and 3 = strong intensity.
TIS: 0–1 = negative, 2–3 = mild, 4–8 = moderate, and 9–12 = strong.
Data were collected, tabulated, and statistically analyzed using a personal computer with ‘statistical package for the social sciences’ version 16 programs (SPSS Inc., Chicago, Illinois, USA). χ2-Test and Fisher's exact tests were used for comparison between qualitative variables. P value up to 0.05 was considered significant.
Statistical calculations were done to assess the diagnostic values of qualitative data as accuracy, sensitivity, specificity, positive predictive value, and negative predictive value.
| Results|| |
Clinicopathologic characteristics of all studied groups are summarized in [Table 1].
Association between the extent of squamous differentiation and some clinicopathologic parameters
Studying association between percentage of squamous differentiation and certain clinicopathologic parameters that may have prognostic significance (tumor stage, nodal metastasis, and vascular and PNI) revealed no significant statistical association [Table 2].
|Table 2: Association between percentage of squamous differentiation and clinicopathologic parameters|
Click here to view
Descriptive results of cytokeratin 14 expression among all studied groups
CK14 staining of 74 cases showed positive cytoplasmic staining in 39 (52.7%) cases. It was positive in 23/31 (74.2%) cases of UC with squamous differentiation, 5/26 (19.2%) cases of pure UC, and in all cases (16/16) of SCC [Figure 1]a and [Figure 1]b. It was negative in the case of adenocarcinoma [Figure 1]c as well as foci of glandular and sarcomatoid differentiation of UC.
|Figure 1: (a) A case of well-differentiated squamous cell carcinoma (hematoxylin and eosin, ×40). (b) Positive cytoplasmic expression of cytokeratin 14 in squamous cell carcinoma (immunoperoxidase method, ×200). (c) Positive cytokeratin 14 cytoplasmic expression in squamous metaplasia (immunoperoxidase method, ×40). (d) Negative cytokeratin 14 expression in bladder adenocarcinoma (immunoperoxidase method, ×40).|
Click here to view
Non-neoplastic bladder mucosa (normal urothelium), benign proliferative urothelial lesions (Von Brunn's nest, cystitis cystica, and glandularis), and stromal cells did not express CK14 [Figure 2]b and [Figure 2]d; however, it was positive in all six (100%) cases of squamous metaplasia, showing strong expression in five (83.33%) cases and moderate expression in one (16.33%) case [Figure 1]d.
|Figure 2: (a) A case of urothelial carcinoma with areas of squamous differentiation showing individual cell keratinization and intercellular bridges (hematoxylin and eosin, ×400). (b) A case of urothelial carcinoma with areas of squamous differentiation showing extensive inflammatory infiltrate (hematoxylin and eosin, ×200). (c) Positive cytoplasmic expression of cytokeratin 14 in areas of squamous differentiation with negative expression in areas of urothrlial carcinoma (immunoperoxidase method, ×400). (d) Positive cytoplasmic expression of cytokeratin 14 in areas of squamous differentiation with negative expression in stromal and inflammatory cells (immunoperoxidase method, ×200).|
Click here to view
In all CK14-positive cases of UC with squamous differentiation, CK14 staining appeared only in areas of squamous differentiation and not in areas of UC [Figure 2]a and [Figure 2]c. It showed strong expression in 15 (48.4%) cases and moderate expression in eight (25.8%) cases.
Diagnostic value of cytokeratin 14
CK14 showed positive expression in 23/31 (74.2%) cases for areas of squamous differentiation, and it was negative in 8/31 (25.8%) cases for areas of squamous differentiation, and it showed positivity in 5/26 (19.2%) cases of pure UC.
CK14 showed sensitivity of 74%, specificity of 81%, positive predictive value of 82% for areas of squamous differentiation, and negative predictive value of 72% for pure UC, and accuracy of 77%.
Association between cytokeratin 14 expression and studied clinicopathologic parameters
CK14 showed significant statistical association with presence of squamous differentiation (P = 0.001); grade of SCC (P = 0.0001), with more expression in grade I and II than grade III; increased percentage of squamous differentiation (P = 0.001); and presence of PNI (P = 0.03) [Table 3].
|Table 3: Relationship between cytokeratin 14 expression and clinicopathological data among studied group|
Click here to view
There was no significant statistical association between CK14 expression and other studied clinicopathologic parameters.
| Discussion|| |
Bladder cancer is significant cause of morbidity and mortality all over the world. It is the ninth common cancer worldwide, accounting for 450 000 newly diagnosed cases in 2012. It is more prevalent in developing countries than developed ones and is responsible for half of all 16 500 bladder cancer deaths there.
In Egypt, it is the third most common malignancy and is the most prevalent malignancy among Egyptian males,.
The most important prognostic factor of bladder cancer is pathological stage. Muscle-invasive bladder cancer accounts for 20–30% of all cases that present to seek medical care. Unfortunately, these cases have a 5-year survival rate of 50% even after postsurgical aggressive treatment. So, new additional pathologic prognostic parameters need to be assessed.
UC is the most prevalent histologic type of bladder cancer, which represents 90% of bladder malignancies, followed by SCC, representing 5%, and all other histologic types, representing collectively the remaining 5%. The ability to give variable morphologic variants is a characteristic feature of UC. These variants have worse prognosis than conventional UC. So, it will be valuable to detect variant histologies to guide individualized therapeutic treatment.
UC with squamous differentiation is the most prevalent mixed histologic variant that could be encountered in 40% of UC cases,.
The exact prognostic significance of squamous differentiation in UC is still a matter of debate; however, many studies have reported its aggressive behavior owing to association with advanced tumor stage and nodal metastasis, as well as poor response to chemotherapy, radiotherapy, and immunotherapy.
Moreover, the extent of squamous differentiation may has prognostic value as some studies reported that increased percentage of squamous differentiation in UC is associated with increased nodal positivity and reduced patient's survival.
In our study, we analyzed the association between percentage of squamous differentiation and clinicopathologic parameters that may have prognostic effect such as vascular and PNI, tumor stage, and nodal metastasis. We classified the extent of squamous differentiation into either focal (<50%), extensive (50–90%), or more extensive (>90%). No significant statistical association was detected, and this was against the results of Mitra et al. and Liu et al., which may be because of the different nature of bladder cancer in Egypt.
In our study, CK14 showed positive cytoplasmic staining in 23/31 (74.2%) cases of UC with squamous differentiation and (5/26) (19.2%) cases of pure UC, which may be represented by foci of squamous differentiation not evident on histopathologic assessment. CK14 expression showed significant correlation with squamous differentiation in UC, which is in agreement with Gulmann et al. who showed CK14 expression in 87% of cases with squamous differentiation, and Hammam et al., who reported positive expression in 58% of cases. It showed sensitivity of 74%, specificity of 81%, positive predictive value of 82% for areas of squamous differentiation, and negative predictive value of 72% for pure UC. It was expressed in areas of squamous differentiation only and not in areas of UC.
Previous studies have investigated the diagnostic utility of MAC87 for squamous differentiation in UC and proved to have high sensitivity and specificity, but neutrophil, macrophages, monocytes, and histiocytes also showed positive expression. Inflammation was a common finding in our specimens, so immune reactivity of inflammatory cells can cause misdiagnosis of squamous differentiation, but fortunately CK14 was not expressed in either stromal cells or inflammatory cells.
P63 and CK5/6, which are markers of systemic SCC, are no useful in detecting squamous differentiation in UC because they are expressed in both UC and SCC.
Regarding SCC, CK14 showed positive cytoplasmic expression in all cases. This results agreed with Mostafa et al., Hammam et al., and Gulmann et al., who also showed positive CK14 expression in all cases of SCC. Moreover, Gaisa et al. reported that CK14 was expressed in 95.8% of cases of SCC.
Its expression showed association with grade of SCC (P = 0.0001), with reduced expression from grade I to grade III. It retained expression even in cases of poorly differentiated SCC, which is in agreement with Chu et al. who reported CK14 expression in SCC of any organ regardless of the grade. CK14 showed negative expression in the studied case of adenocarcinoma, which is in agreement with Chu et al.. It also showed negative expression in areas of glandular and sarcomatoid differentiation in UC. Therefore, CK14 will be helpful marker in differentiating poorly differentiated SCC from poorly differentiated UC and adenocarcinoma.
CK14 showed positive cytoplasmic expression in all six cases of squamous metaplasia. It was expressed neither in normal urothelium nor in benign proliferative urothelial lesions, so it can be used for early detection of squamous metaplasia in the covering urothelium that may act as precursor lesion for bladder SCC. Squamous metaplasia may be the origin of squamous differentiation in UC, but this remains debatable. Fadl-Elmula et al. found that squamous element in UC with squamous differentiation has the same cytogenetic changes as urothelial element.
In our study, CK14 expression showed significant statistical association with presence of PNI (P = 0.03) and increased percentage of squamous differentiation in UC (P = 0.001).
There was controversy regarding the prognostic value of extent of squamous differentiation in UC, LVI, and PNI. Hong and colleagues reported that LVI and PNI are related to poor prognosis on univariate analysis, unlike Kucuk et al. who reported no significant statistical correlation Hong et al. and Mitra et al., found that extensive squamous differentiation is associated with poor prognosis. So, LVI, PNI, and increased percentage of squamous differentiation may be indicators of poor prognosis.
Therefore, positive CK14 staining in UC may be indicator of poor prognosis.
| Conclusion|| |
Ck14 is an immunohistochemical marker for squamous differentiation in UC, and its expression is associated with presence of poor prognostic parameters.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Globocan. Cancer incidence and mortality worldwide IARC Cancer Base No. 11. International Agency for Research on Cancer. 2012; Available from: http://globocan.iarc.fr
. [Last acceded 2017 Mar].
Elshikh E, Anan I, Ebaid A, Al Gamal D, Hamed M, Metry M. The National Cancer Registry in Egypt: a retrospective cross sectional epidemiological study, in ISPOR
ed. Milano, Italy: Annual European Congress; 2015. 7–11.
Amin MB, Gomez JA, Young RH. Urothelial transitional cell carcinoma with endophytic growth patterns: a discussion of patterns of invasion and problems associated with assessment of invasion in 18 cases. Am J Surg Pathol 1997:21
Sakano S, Matsuyama H, Kamiryo Y, Hayashida S, Yamamoto N, Kaneda Y, et al
. Impact of variant histology on disease aggressiveness and outcome after nephroureterectomy in Japanese patients with upper tract urothelial carcinoma. Int J Clin Oncol 2015; 20
Hansel DE, Zhang Z, Petillo D, Teh BT. Gene profiling suggests a common evolution of bladder cancer subtypes. BMC Med Genomics 2013; 6
Liu Y, Bui MM, Xu B. Urothelial carcinoma with squamous differentiation is associated with high tumor stage and pelvic lymph-node metastasis. Cancer Control 2017; 24
Gofrit ON, Yutkin V, Shapiro A, Pizov G, Zorn KC, Hidas G, et al
., The response of variant histology bladder cancer to intravesical immunotherapy compared to conventional cancer. Front Oncol 2013; 6
Mitra AP, Bartsch CC, Bartsch G Jr, Miranda G, Skinner EC, Daneshmand S. Does presence of squamous and glandular differentiation in urothelial carcinoma of the bladder at cystectomy portend poor prognosis? An intensive case–control analysis. Urol Oncol 2014; 32
Lopez-Beltran A, Martin J, Garcia J, Toro M. Squamous and glandular differentiation in urothelial bladder carcinomas. Histopathology, histochemistry and immunohistochemical expression of carcinoembryonic antigen. Histol Histopathol 1988; 3
Chang A, Amin A, Gabrielson E, Illei P, Roden RB, Sharma R, et al
. 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 2012; 36
Moll R. Cytokeratins as markers of differentiation. Expression profiles in epithelia and epithelial tumors. Veroff Pathol 1993; 142
Walid M, Osborne T, Robinson J. Primary brain sarcoma or metastatic carcinoma? Indian J Cancer 2009; 46
Xue LY, Zou SM, Zheng S, Xie YQ, Wen P, Liu XY, et al
. Expression of fascin and CK14 in different histopathologic types of cancer and its differential diagnostic significance. Zhonghua Zhong Liu Za Zhi 2010; 32
Amin MB, McKenney JK, Paner GP, Hansel DE, Grignon DJ, Montironi R, et al
. ICUD-EAU international consultation on bladder cancer 2012: pathology. Eur Urol 2013; 63
Fong D, Seeber A, Terracciano L, Kasal A, Mazzoleni G, Lehne F, et al
. Expression of EpCAM(MF) and EpCAM(MT) variants in human carcinomas. J Clin Pathol 67; 67
Khaled HM, Hamza MR, Mansour O, Gaafar R, Zaghloul MS. A phase II study of gemcitabine plus cisplatin chemotherapy in advanced bilharzial bladder carcinoma. Eur J Cancer 2000; 36
Cheng L, Neumann RM, Nehra A, Spotts BE, Weaver AL, Bostwick DG. Cancer heterogeneity and its biologic implications in the grading of urothelial carcinoma. Cancer 2000; 88
Antunes AA, Nesrallah LJ, Dall'oglio MF, Maluf CE, Camara C, Leite KR, et al
. The role of squamous differentiation in patients with transitional cell carcinoma of the bladder treated with radical cystectomy. Int Braz J Urol 2007; 33
Sakamoto N, Tsuneyoshi M, Enjoji M. Urinary bladder carcinoma with a neoplastic squamous component: a mapping study of 31 cases. Histopathology 1992; 21
Lagwinski N, Thomas A, Stephenson AJ, Campbell S, Hoschar AP, El-Gabry E, et al
. Squamous cell carcinoma of the bladder: a clinicopathologic analysis of 45 cases. Am J Surg Pathol 2007; 31
Gulmann C, Paner GP, Parakh RS, Hansel DE, Shen SS, Ro JY, et al
. Immunohistochemical profile to distinguish urothelial from squamous differentiation in carcinomas of urothelial tract. Hum Pathol 2013; 44
Hammam O, Wishahiz M, Khalil H, El Ganzouri H, Badawy M, Elkhquly A, et al
. Expression of cytokeratin 7, 20, 14 in urothelial carcinoma and squamous cell carcinoma of the Egyprian urinary bladder cancer. J Egypt Soc Parasitol 2014; 44
Hayashi T, Sentani K, Oue N, Anami K, Sakamoto N, Ohara S, et al
. Desmocollin 2 is a new immunohistochemical marker indicative of squamous differentiation in urothelial carcinoma. Histopathology 2011; 59
Mostafa MG, Fadil SH, Ahmed AN, Eltaher AM. CK-14 unequivocally distinguishes squamous cell carcinoma of the urinary bladder. AAMJ 2004; 2
Gaisa NT, Braunschweig T, Reimer N, Bornemann J, Eltze E, Siegert S, et al
. Different immunohistochemical and ultrastructural phenotypes of squamous differentiation in bladder cancer. Virchows Arch 2011; 458
Chu PG, Lyda MH, Weiss LM. Cytokeratin 14 expression in epithelial neoplasms: a survey of 435 cases with emphasis on its value in differentiating squamous cell carcinomas from other epithelial tumours. Histopathology 2001; 39
Fadl-Elmula A, Gorunova L, Lundgren R, Mandahl N, Forsby N, Mitelman F, et al
. Chromosomal abnormalities in two bladder carcinomas with secondary squamous cell differentiation. Cancer Genet Cytogenet 1998; 102
Hong SK, Kwak C, Jeon HG, Lee E, Lee SE. Do vascular, lymphatic, and perineural invasion have prognostic implications for bladder cancer after radical cystectomy? Urology 2005 65
Kucuk U, Pala EE, Cakr E, Sezer O, Bayol U, Divrik RT, et al
. Clinical, demographic and histopathological prognostic factors for urothelial carcinoma of the bladder. Cent European J Urol 2015; 68
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]