J Islamabad Med Dental Coll 2022 145
Open Access
Utility of Cell Block with p53 Immunostain in Diagnosing
Urothelial Carcinoma
Alina Ali1, Nosheen Nabi2, Maryam Fatima3, Armaghana Qamar Khan4, Henna Azmat5, Ashok kumar
Tanwani6.
1Postgraduate resident, Pathology department, SZAMBU PIMS, Islamabad.
2Assistant professor, Department of Pathology, Rawal Institute of Health sciences Islamabad.
3Senior Registrar, Department of Pathology, HBS Medical and Dental College, Islamabad.
4Medical Officer, Department of Pathology, SZAMBU, PIMS, Islamabad.
5Medical officer, Pathology, Federal Government Polyclinic Hospital Islamabad.
6Head of Department, Pathology, HBS Medical and Dental College, Islamabad.
A B S T R A C T
Background: Urine cytology is an initial noninvasive screening test done in patients with suspected urothelial
carcinoma. The objective of this study was to compare the sensitivity of conventional smear cytology with cell block
technique for the diagnosis of malignant cells in urine and to assess the efficacy of p53 immuno-marker in the
diagnosis and prognosis of urinary bladder cancer.
Methodology: The study was carried out in the department of Histopathology, Pakistan Institute of Medical
Sciences(PIMS), Islamabad. Cell blocks and cytology smears were prepared from urine samples of 80 patients of
urothelial carcinoma and p53 stain was applied. Data was analyzed using SPSS version 21. Sensitivity of conventional
smear and cell block techniques were calculated for both low and high grade urothelial carcinomas.
Results:The sensitivity of detecting atypical cells using conventional smear was 31% and 70.6% for low grade and high
grade urothelial tumors respectively. The sensitivity increased to 41.4% and 84.3% for low and high grade urothelial
carcinomas respectively with cell block technique. Positive p53 stain was seen in 13.8% of cases with low grade tumors
and 66.7% cases with high grade tumors. Chi-square test was used to find the association of cytology findings with the
histologic grade.
Conclusion: Sensitivity of urine cell blocks is higher than the conventional smear method for detecting high grade
lesions. It can be used as an efficient non-invasive technique and an adjunct tool for the determination of disease
relapse and overall survival in patients.
Keywords: Carcinoma, Cytology, P53 Protein, Urine
Authors’ Contribution:
1,2Conception; Literature research;
manuscript design and drafting; 3, 4
Critical analysis and manuscript review;
5,6Data analysis; Manuscript Editing.
Correspondence:
Nosheen Nabi
Email: nosheennabi@yahoo.com
Article info:
Received: April 22, 2021
Accepted: September 6, 2022
Cite this article. Ali A, Nabi N, Fatima M, Khan Q A, Azmat H, Tanwani KA. Utility of Cell Block
with p53 Immunostain in Diagnosing Urothelial Carcinoma. J Islamabad Med Dental Coll. 2022;
11(3): 145-151
DOI: https://doi.org/10.35787/jimdc.v11i3.709
Funding Source: Nil
Conflict of Interest: NIL
Introduction
Carcinoma of urinary bladder is the 9th most
common malignancy worldwide.1 Risk factors
include, male sex, aging, tobacco, Schistosoma
haematobium infection, occupational carcinogens
and genetic alterations, p53 being the most
important.2
O R I G I N A L A R T I C L E
J Islamabad Med Dental Coll 2022 146
Urine cytology is performed as an initial evaluation
routine test for suspected bladder carcinoma
although cystoscopic examination and biopsy are
currently the gold standard method.3 It has a low
sensitivity for detecting urothelial carcinoma but still
performed as it is non-invasive and relatively
inexpensive. 4 In patients with bladder cancer, the
diagnostic accuracy is enhanced if cell blocks are
made together with the conventional smears. 5 Cell
blocks are also useful for special stains and
immunohistochemical staining.6
This study aims to determine whether urine cell
block technique can improve the diagnostic
sensitivity as compared to urine cytology alone for
detecting urothelial carcinoma and also to evaluate
the importance of p53 mutations and its diagnostic
and prognostic value in bladder cancer Some of the
previous international studies strongly support this
view while according to some authors, cell block
technique has no significant role in improving the
diagnostic utility of urine cell cytology. 7,8 We didn’t
find any local study done regarding cell block
technique in urine cytology.
P53 expression is the most common gene involved
in human cancers. P53 positivity on urine cell blocks
can be used as a useful marker for assessment of
urinary bladder cancer prognosis as P53 mutation is
associated with increased recurrence rate of bladder
cancer. 9 We also added p53 immunohistochemistry
for even better results and to check the possibility of
using cell blocks for ancillary techniques like
immunohistochemistry.
Methodology
This cross-sectional study was conducted in the
department of Histopathology, Pakistan Institute of
Medical Sciences (PIMS), Islamabad after approval
from ethical committee at Shaheed Zulfiqar Ali
Bhutto Medical University. The study duration was 4
years (2016-2019). Sample size was calculated to be
80 by using WHO sample size calculator taking 95%
confidence level and 60% anticipated population
proportion.10
Consecutive samples of 80 patients with diagnosis of
urothelial carcinoma on histopathology were
included in the study after taking informed consent
from the patients. Patients with other urinary
bladder tumors, PUNLUMP and other reactive
conditions and those with inadequate smears were
excluded. Smears with any atypical cells were
considered satisfactory as adequacy criteria for
urine cytology and blocks is not yet established so
those smears which didn’t show any atypical
urothelial cells were considered inadequate were
excluded.11
About 30 ml freshly voided samples of urine were
collected from these patients, centrifuged
immediately at 3000 revolutions per minute for ten
minutes and subjected to the conventional smear
cytology and cell block technique. From each of the
case, two urine cytology smears were selected and
each of the smear contained at least 10 atypical
cells. These two slides were subjected to
Hematoxylin and Eosin (H&E) staining.
Immunohistochemical staining for P53 was applied
on sections made from all cell blocks. Cell block
containing at least 5% positively stained nuclei were
labelled as positive P53 staining.12
Data was analyzed using SPSS software version 21.
Sensitivity of conventional smear and cell block
techniques were calculated for both high and low
grades of urothelial carcinoma. Specificity
calculation was not useful in this study since already
diagnosed cases of urothelial cancer were included
and no true negatives or false positives were present
in this study. Chi-square test was applied to calculate
the relationship between P53 expression and tumor
grade. p values were also calculated to evaluate the
statistical significance of conventional smear and
cell block technique for low and high grades of
urothelial cancer. p value of ≤0.05 was taken as
significant.
J Islamabad Med Dental Coll 2022 147
Results
Among 80 study cases, 18 (22.5%) were females and
62 (77.5%) males. Male to female ratio was 3.4:1.
The peak age was 50-70 years for both males and
females(range: 21-87 years) (Table 1).
Table: I Age Distribution of Cases
Age Range
(years)
21-30
31-40
41-50
51-60
61-70
71-80
81-90
Males
(n=62)
1(1.6%)
4(6.4%)
8 (12.9%)
17(27.4%)
22(35.4%)
8(12.9%)
2(3.2%)
Females
(n=18)
0 (0%)
0 (0%)
1 (5.5%)
7 (38.8%)
4 (22.2%)
6 (33.3%)
0 (0%)
Total
(n=80)
1(1.2%)
4(5%)
9(11.2%)
24(30%)
26(32.5%)
14(17.5%)
2 (2.5%)
Among 80 cases of urothelial cancer, 37% cases
(29/80) were low grade while 63% cases (51/80)
were diagnosed as high grade urothelial carcinoma
on surgical biopsy.
With conventional smear method, atypical cells
were detected in 31% low grade & 70.6% high grade
urothelial carcinoma cases. With cell block
technique, atypical cells were seen in 41.3% cases of
low-grade & 84.3% cases of high grade urothelial
cancer. Atypical cells were identified in 10 additional
cases by cell block method. Malignant cells were
missed in 35 (43.8%) cases by conventional smear
and 25 (31.2%) cases by the cell block method.
(Table 2).
Table II: Comparison of Conventional Smear, Cell Block and Immunohistochemistry
Conventional Smear
Cell Block
P53 staining
P
Values
Positive for
malignant
cells
Negative for
malignant
cells
Positive for
malignant
cells
Negative for
malignant
cells
Positive
Negative
LGUC
(n=29)
9
(31%)
20
(69%)
12
(41.4%)
17
(58.6%)
6
(20.7%)
23
(79.3%)
0.000
HGUC
(n=51)
36
(70.6%)
15
(29.4%)
43
(84.3%)
8
(15.7%)
34
(66.7%)
17
(33.3%)
0.000
Total
cases
(n=80)
45
(56.2%)
35
(43.8%)
55
(68.8%)
25
(31.2%)
40
(50%)
40
(50%)
(p53
staining
among
grades of
tumor)
0.006
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Figure 1: Microscopic Examination of a High-Grade Urothelial Carcinoma.
Figure 1; High grade urothelial carcinoma A, Biopsy
(H&Ex400). B, Cytology smear showing atypical
urothelial cells (H&Ex400).C, Cell block showing few
atypical cells (H&Ex400). D, P53 immunostaining
positive (x400).
Sensitivity for low grade urothelial carcinoma by
conventional smear method was 31%.
Sensitivity for high grade urothelial carcinoma by
conventional smear method was 70.6%.( 36 out of51
cases)
Sensitivity for low grade urothelial carcinoma by cell
block method was 41.4%. (12 out of 29 cases)
Sensitivity for high grade urothelial carcinoma by cell
block method was 84.3%. ( 43 out of 51 cases)
With the use of cell block technique, the sensitivity
improved.
Chi-square test was employed to find out the
significance of tumor grade with cytology results
considering p values of 0.05 or less to be statistically
significant. In our results p values were found to be
significant for both low grade (p=0.000) and high
grade (p=0.000) urothelial cancers.
P53 positivity was seen more in the cases of high
grade urothelial cancer (Table 2)
J Islamabad Med Dental Coll 2022 149
P value was calculated to determine the significance
of association of p53 immune stain expression with
different hitopathological grades of urothelial
carcinoma. Significant association was observed
between P53 expression for high grade urothelial
cancer (p=0.006) urothelial cancers. (Table 2)
Discussion
Routine cystoscopies are done at regular intervals
for follow up of patients, it costs high and about 10%
of the lesions are overlooked. 13 There is obviously a
need for a non-invasive method for timely detection
of urothelial cancer, in order to lessen the number
of cystoscopies. Conventional smear and cell block
methods save the patient from undergoing repeated
invasive procedures like cystoscopic biopsy. In a
developing country like Pakistan, it can save money
and hospital resources which can be utilized
effectively where needed.
Cytology is a widely used non-invasive test though
its use is restricted by its poor sensitivity extending
from 28-100% (median: 48%) reported by different
authors. It is useful in diagnosing high grade tumors,
however it is less sensitivity for low-grade urothelial
tumors.15
The current study demonstrates that urine cytology
and cell block methods are useful adjuvant tests in
establishing the diagnosis of urothelial cancer. Cell
block method is cheap, simple to perform and help
in better preservation of cells in comparison to
conventional smear method. 16 It also reduces the
gap between cytology and histology. Paraffin
embedded cell blocks can be handled like a biopsy
specimen and multiple sections are available for
other ancillary studies. 17
The sensitivity using conventional smear was 31%
and 70.6% for low grade and high-grade tumors
respectively. The sensitivity rates of urine cytology
reported by Yafi et al were 18.3% for low grade and
51.3% for high grade urothelial cancer.18
Santwani et al reported that cell block technique
demonstrates better architectural patterns,
increases the positive results and can be used for
application of different special stains and
immunohistochemical markers.19Qamar et al
evaluated the association of immunohistochemical
expression of P53 with grade and stage of urothelial
cancers and found that only 16% cases of low grade
carcinoma showed P53 positivity whereas 91% cases
of high grade carcinoma were P53 positive. 20. In our
study, cell blocks from low grade urothelial cancer
showed 20.7% P53 positivity and from high grade
urothelial cancer showed 66.7% P53 positivity.
Mumtaz et al found that P53 positive expression was
seen in 72.9% cases of high grade urothelial
carcinoma and only 36.2% cases of low grade
tumors.21. According to V Nassai et al ,39% cases of
high grade urothelial carcinoma while 11% of low
grade urothelial carcinoma were positive for p53
immunostain. 22 Thakur et al found p53 expression
more in the high grade urothelial carcinomas (52
cases) as compared to 32 cases of low grade. 23. This
study demonstrates a sensitivity and specificity of
cytologic diagnosis comparable to previously
reported studies. Cell block technique is clearly
superior to conventional smears for diagnosis of
atypical urothelial cells in urine. The results of both
conventional smears and cell blocks were similar to
the results found in literature. Urinary cytology and
cell block techniques can serve as non-invasive
adjuncts to biopsy for detecting high-grade lesions.
Their usefulness is limited to low-grade tumors.
The major limitation of this study is selection of
those cases already diagnosed with urolethial
carcinoma. Including those as well would have
strengthened the study. Another limitation is low
cellularity in urine samples and some conditions like
infections, calculi or presence of blood may have
masked atypical cells in the urine sample thus
affecting the results.
Conclusion
Block technique is superior to conventional smears
for diagnosis of atypical urothelial cells in urine.
Urinary cytology and cell block techniques can serve
J Islamabad Med Dental Coll 2022 150
as non-invasive adjuncts to biopsy for detecting
high-grade lesions. Their usefulness is limited to low-
grade tumors. Additionally, cell blocks can also be
used for ancillary studies like
immunohistochemistry.
Recommendations
Comparison of immunostaining on biopsy with that
on cell blocks should be done.
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