Alina Ali 1 , Nosheen Nabi 2 , Maryam Fatima 3 , Armaghana Qamar 4 , Henna Azmat 5 , Ashok kumar Tanwani 6 .
1Postgraduate resident, Pathology department, SZAMBU PIMS, Islamabad.
2Assistant professor, Pathology department, Rawal Institute of Health sciences, Islamabad.
3Senior Registrar Histopathology, HBS Medical and Dental College, Islamabad.
4Medical Officer, Pathology department, SZAMBU, PIMS, Islamabad.
5Medical officer, Pathology department, Federal Government Polyclinic Hospital Islamabad.
6Head of Pathology Department, HBS Medical and Dental College, Islamabad.
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: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
Carcinoma of urinary bladder is the 9th most
common malignancy worldwide. 1 Risk factors
include, male sex, aging, tobacco, Schistosoma
haematobium infection, occupational carcinogens
ORIGINAL ARTICLE
and genetic alterations, p53 being the most
important. 2
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.
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.
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 |
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) 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)
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. 19 Qamar 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.
Block technique is superior to conventional smears for diagnosis of atypical urothelial cells in urine. 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. Additionally, cell blocks can also be used for ancillary studies like immunohistochemistry.
Comparison of immunostaining on biopsy with that on cell blocks should be done.
An Official Publication of
Islamabad Medical & Dental College
Volume 11 Issue 3
Nosheen Nabi
Email:
nosheennabi@yahoo.com
Cite this article.Ali A, Nabi N, Fatima M, Qamar 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