Cytokeratin 5/6 expression in bladder cancer: association with clinicopathologic parameters and prognosis
Hashmi et al. BMC Res Notes
Cytokeratin 5/6 expression in bladder cancer: association with clinicopathologic parameters and prognosis
Atif Ali Hashmi 2
Zubaida Fida Hussain 2
Muhammad Irfan 2
Muhammad Muzzammil Edhi 1
Sarah Kanwal 2
Naveen Faridi 2
Amir Khan 0
0 Kandahar University , Kandahar , Afghanistan
1 Brown University , Providence, RI , USA
2 Liaquat National Hospital and Medical College , Karachi , Pakistan
Objectives: Well differentiated keratinized squamous component as a part of urothelial carcinoma can be easily appreciated; however non-keratinizing squamous differentiation closely resembles urothelial differentiation. In addition prognostic significance of CK 5/6 expression in the absence of apparent squamous differentiation is still unclear. Therefore, in the present study we aimed to evaluate the frequency of CK 5/6 expression in 127 cases of urothelial carcinoma and its prognostic significance in loco-regional population. Results: Positive CK5/6 expression was noted in 6.3% (8 cases) and 13.4% (17 cases) revealed focal positive CK 5/6 expression. On the other hand, 80.3% (102 cases) showed negative CK5/6 staining. Significant association of CK5/6 expression was noted with tumor grade and muscularis propria invasion, however no significant association was noted with overall and disease free survival. On the basis of the results of our study we can conclude that CK5/6 is an independent prognostic biomarker in urothelial carcinoma and therefore can be used in the prognostic stratification of the patients with bladder cancer.
Urothelial carcinoma; Bladder cancer; Cytokeratin 5/6; CK5/6; Deep muscle invasion
Bladder cancer is among one of the most common
malignancy in males worldwide and its incidence is even
higher in developing countries owing to certain endemic
]. Urothelial carcinoma is the most
common histologic subtype of bladder cancer, while
Squamous cell carcinoma is seen in association with bladder
stones and schistosomiasis. Muscle invasion is one of the
most important prognostic factors in bladder cancer; as
it necessitates radical therapy and poor 5 year disease
free survival [
]. While, well differentiated keratinized
squamous component as part of urothelial carcinoma
can be easily appreciated; non-keratinizing squamous
differentiation closely resembles urothelial
differentiation. On the other hand, WHO/ISUP don’t recommend
routine use of immunohistochemical markers to identify
squamous differentiation in urothelial carcinoma.
Conversely, markers of squamous differentiation like p63,
p40, CK5/6 can be positive in urothelial carcinoma [
addition prognostic significance of CK 5/6 expression in
the absence of apparent squamous differentiation is still
unclear. Therefore, in the present study we aimed to
evaluate the frequency of CK 5/6 expression in urothelial
carcinoma and its prognostic significance in loco-regional
Total 240 diagnosed cases of urothelial carcinoma
specimens were selected from records of pathology
department. All patients underwent surgeries at
Liaquat National hospital, Karachi from January 2010 till
December 2014 over a period of 5 years. The study was
approved by research and ethical review committee of
Liaquat National Hospital and informed written consent
was taken from all patients at the time of surgery.
Hematoxylin and eosin stained slides and paraffin blocks of all
cases were retrieved and new sections were cut when
necessary. Slides of all cases were reviewed by two senior
histopathologists and pathologic characteristics like
histologic type, tumor grade, lamina propria invasion,
muscularis propria invasion were evaluated. Clinical records
of 61 patients were available and are thus reviewed from
institutional records to evaluate history of radiation and
chemotherapy and recurrence status. Moreover,
representative tissue blocks of 127 cases were available for
CK5/6 IHC was performed by using FLEX Monoclonal
Mouse Anti-human Cytokeratin 5/6, clone D5/16 B4 (Lot
No. 20042129) by DAKO envision method according to
manufacturers protocol on 127 cases of urothelial
carcinoma (on representative tissue blocks). Results of IHC
staining were interpreted by two senior
histopathologists with more than 5 years experience of reporting
histopathology and immunohistochemistry and they were
blinded by other histopathological features of the tumors.
For quantification, at least 1000 cells were counted in
10 HPFs (40×). Intermediate to strong cytoplasmic and
membranous staining in more than 10% of tumor cells
was considered positive. Weak to intermediate staining
in < 10% was taken as focal positive, while no staining was
considered as negative (Fig. 1).
Recurrence status and follow-up were evaluated by
reviewing hospital medical records. Overall survival
was taken as time from surgical excision till death or last
follow-up and disease free survival was defined as time
between surgical excision and local recurrence or distant
metastasis, death or last follow-up.
All cases of primary urothelial carcinoma were
included in the study. Cases of squamous cell
carcinoma or those cases of urothelial carcinoma showing
divergent differentiation (including squamous
differentiation) were excluded from the study.
Statistical package for social sciences (SPSS 21)
was used for data compilation and analysis. Mean
and standard deviation were calculated for
quantitative variables. Frequency and percentage were
calculated for qualitative variables. Chi square was applied
to determine association. Student t test or Mann
Witney test were applied to compare difference in
means among groups. Survival curves were plotted
using Kaplan–Meier method and the significance of
difference between survival curves were determined
using log-rank ratio. P value ≤ 0.05 was taken as
Mean age of patients was 63.23 + 13.9 years with male
to female ratio of 3:1. 95.8% specimens were of
transurethral resections. 50.8% (122 cases) were of high
grade morphology, whereas 49.2% (118 cases) showed
low grade histology. Lamina propria invasion was seen
in 30.4% (73 cases), while muscularis propria
invasion was noted in 22.9% (55 cases). Mean follow up of
patients involved in the study was 22.0 + 13.74 months
and recurrence was seen in 45.9% (28 cases) as
presented in Table 1.
Positive CK5/6 expression was noted in 6.3% (8
cases) and 13.4% (17 cases) revealed focal positive CK
5/6 expression. On the other hand, 80.3% (102 cases)
showed negative CK5/6 staining. Significant
association of CK5/6 expression was noted with tumor
grade and muscularis propria invasion, however no
significant association was noted with lamina propria
invasion and disease free survival (Table 2 and Figs. 2
In the present study we found that CK5/6 expression
is low in urothelial carcinoma in our set up; however, its
positivity signifies adverse prognostic features like higher
tumor grade and muscularis propria invasion.
CK5/6 is a basal cytokeratin which normally
expresses in squamous epithelium and in squamous
cell carcinoma. Although diagnosis of squamous cell
carcinoma in bladder is restricted to those tumors
which show pure squamous differentiation in the
absence of any urothelial component. Conversely,
advanced urothelial carcinoma can show divergent
differentiation (including squamous component) in up to
50% of cases and is associated with poor disease
]. Morphologic diagnosis of squamous
differentiation in urothelial carcinoma is based on the
presence of either intercellular bridges or presence of
keratinization in the form of keratin pearls or
individual cell keratinization; however non-keratinizing or
poorly differentiated squamous component can closely
resemble urothelial carcinoma and therefore can’t be
readily apparent. Gaisa et al. [
] performed IHC
markers of squamous differentiation including CK5/6 and
CK4/14; and found squamous differentiation in a high
proportion of urothelial carcinoma without
morphologic evidence of squamous differentiation. Langer
et al. evaluated the prognostic value of keratin
subtyping in urothelial carcinoma and revealed the
prognostic impact of various cytokeratin staining in urothelial
carcinoma including CK5/6.
One of the major limitations of our study was that we
performed only single biomarker of squamous
differentiation in our study; use of multiple markers like CK5/14
and CK4/14 could increase the sensitivity of the study.
However, on the basis of the results of our study we can
conclude that CK5/6 is an independent prognostic
biomarker in urothelial carcinoma and therefore can be used
in the prognostic stratification of the patients with
IHC: immunohistochemistry; WHO: World Health Organization; ISUP:
International Society of Urological Pathology.
AAH and ZFH: main author of manuscript, have made substantial
contributions to conception and design of study. MI, MME and SK: have been involved
in requisition of data. NF AND AK have been involved in analysis of the data
and revision of the manuscript. All authors revise the manuscript. All authors
read and approved the final manuscript.
We gratefully acknowledge all staff members of Pathology, Liaquat National
Hospital, Karachi, Pakistan for their help and cooperation.
The authors declare that they have no competing interests.
Availability of data and materials
Please contact author, Atif Ali Hashmi () for data
Consent to publish
Ethical approval and consent to participate
Ethics committee of Liaquat National Hospital, Karachi, Pakistan approved
the study. Written informed consent was obtained from the patients for the
There was no funding available for this manuscript.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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