Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Determining The Immunohistochemical Expression

of P53 and its Role in Grading Urothelial Carcinoma
Faiqa Mubeen*1
Amna Mehmood*2
Ayesha Sajjad3
Mehreen Mushtaq4
Faryal Javaid5
Sana Ullah Khan6**
Maria Aslam7
Usama Rehman 8
1
Consultant Histopathologist, Muhammad Medical College and teaching hospital, Peshawar, Khyber Pakhtunkhwa, Pakistan
2
Project leader, Department of Medical Physics, Martin Luther University, Halle, Germany and visiting Assistant Professor,
Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar, Pakistan
3
FCPS-2 Trainee, Rehman Medical Institute, Peshawar, Khyber Pakhtunkhwa, Pakistan
4
Assistant Professor, Histopathology, Pakistan institute of medical sciences, Islamabad, Pakistan
5
FCPS-2 Trainee, Rehman Medical Institute, Peshawar, Khyber Pakhtunkhwa, Pakistan
6
Assistant Professor, Histopathology, Lady Reading Hospital (Medical Teaching Institution),
Peshawar, Khyber Pakhtunkhwa, Pakistan
7
Consultant Pathologist, Armed forces institute of Pathology, Rawalpindi, Pakistan
8
Usama Rehman, Consultant Pathologist, Sheikh Zayed Medical college and hospital, Rahim yar khan, Punjab, Pakistan.

*Both authors have contributed equally to the manuscript.

** Corresponding author: Sana Ullah Khan

Abstract:- 49(51%) cases were low grade Urothelial carcinoma

whereas 48(49%) cases displayed high grade
 Objective morphology. p53 was found positive in 63(64.95%)
To establish the importance of patients. Among positive cases, 45 cases were high grade
immunohistochemical staining of P53 in grading of and 18 were low grade Urothelial carcinoma.
Urothelial Carcinoma.
 Conclusion
 Methodology P53 Positivity was seen in 64.95% patients with
A retrospective cross-sectional study was carried Urothelial carcinoma. P53 is an important
out at the department of Histopathology, Lady Reading immunohistochemical marker for early diagnosis and
Hospital (Medical Teaching Institution), Peshawar, from grading of Urothelial carcinoma cases.
August 2021 till February 2022. 97 Paraffin embedded
blocks of Urothelial carcinoma along with clinical record Keywords:- P53, Urothelial Carcinoma,
of these patients, from January 2018 till December 2020, Immunohistochemistry.
were retrieved from data bank of Histopathology
department and Health management information I. INTRODUCTION
system. Cutting of blocks and staining with Hematoxylin
and Eosin (H&E) stain and P53 antibody was done. Urothelial carcinoma (UC) is a common malignancy
Expression of p53 was noted by two consultant of the genitourinary tract with more than half a million new
pathologists. Nuclear immunoreactivity of strong cases, and a mortality of almost two hundred thousand
intensity was considered positive, if present in more than globally, in 2018. Histologically, UC is said to be the most
10% of tumor cells, and negative if either no staining or common urinary bladder tumor that comprises of more than
staining in less than 10% of tumor cells was noted. 90% of all cases.1 Bladder carcinoma is the 9th most
Statistical analysis (Pearsmann correlation) was used to common cancer worldwide.2 In United States, it represents
determine the correlation among various variables. 4th most common cancer in males and 10th most common
tumor in females with a male to female ratio of 3:1 and
 Results median age of diagnosis at 68 years.3 In Pakistan, a study
Out of 97 patients, the minimum age was 20 years conducted in Armed Forces Institute of Pathology,
while maximum age was found to be 90 years with mean Rawalpindi showed that UC is the7th most commonly
+ standard deviation of 64 +11 years. There were 86 occurring tumor in both men and women and represents
(88.6%) male patients and 11 (11.3) female patients. 93.4% of all bladder malignancies. 2 Smoking is the one of

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Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
most significant risk factors which has strong association management information system (HMIS) of the institution.
with bladder cancer.4 Other factors include Retrieved blocks were cut at 3-5um thickness for routine
cyclophosphamide, phenacetin, auramine, some dyes, H&E staining and p53 immunohistochemical staining.
Schistosoma and previous irradiation of urinary bladder for DAKO kit was used for immunohistochemistry according to
treatment of prostatic carcinoma.5 WHO/ISUP, in its latest guidelines of manufacturer. Non neoplastic urinary bladder
classification of tumors of urinary tract, classified Urothelial tissue was used as negative control while FFPE block of
tumors into invasive and non-invasive Urothelial neoplasms. skin tissue was used as positive control. Slides were
Among the invasive UCs, various subtypes like Nested, examined by two consultant Histopathologist, blind to the
Microcystic, Sarcomatoid, Micropapillary, Plasmacytoid, original issued reports. Histopathologic diagnoses were
Giant cell, Lymphoepithelioma like, Clear cell and poorly made for all the cases included in study and recorded in a
differentiated, are included. The non-invasive Urothelial predesigned proforma. P53 antibody stained slides were
neoplasms are subdivided into UC in situ, papilloma, than evaluated. Each case was assigned positive or negative
papillary urothelial neoplasm of low-grade malignant status, based on nuclear staining intensity and percentage
potential (PUNLMP), low grade Urothelial carcinoma score. Strong nuclear immunoreactivity in greater than 10 %
(LGUC), and high-grade Urothelial carcinoma (HGUC).6 tumor cells was regarded positive. Negative status was
Prognosis of UC is determined by various factors like recorded for a case with either no staining at all or staining
pathological tumor grade and stage, muscularis propria in fewer than 10% of tumor cells. Variables like age, gender,
invasion and patient’s age.7 Among these, grade of the grade of tumor, muscle invasion and p53 status were
tumor is the single most significant prognostic factor. The recorded. For the purpose of statistical analysis, Spearman
prognosis of LGUC is generally good, though these tumors correlation was employed. Calculation of mean and standard
show high recurrence rates. Approximately 30% of these deviation was performed for numerical variables like age.
recurrent tumors progress by invading lamina propria.8 Tumor grade, gender, age and p53 immunohistochemical
Genetics play a very crucial role in causation and expression were expressed in the form of percentages and
progression of UC. P53 is a tumor suppressor gene on frequencies. Effect modifiers including age, tumor grade and
chromosome 17p, which is a frequently mutated gene gender were controlled by stratification. Spearman
observed in carcinoma of lung, breast and urinary bladder.9 correlation was applied and p-value of less than 0.05 was
Wild-type p53 aids to inhibit tumor proliferation by averting considered significant.
the neovascularization carried by production of endogenous
vascular endothelial growth factor (VEGF) and basic III. RESULTS
fibroblast growth factor (FGF). Mutated p53 loses its crucial
regulatory role and hence, there is unchecked Among 97 cases, the minimum and maximum age of
neovascularization, promoting tumor multiplication and patient recorded was 20 years and 90 years (as shown in
advancement to progress.10 Expression of p53 has both Figure 1) with mean+ standard deviation as 64+ 11 years
diagnostic as well as prognostic importance in Urothelial respectively. There were 86 (88.6%) male patients and 11
tumors. Over expression of p53 occurs in high proportion of (11.3%) female patients which makes male to female ratio
Urothelial tumors, particularly high-grade forms and of 7.8:1. Among male patients, 42 cases of HGUC and 44
correlates well with prognosis. It is also an indicator of p53 cases of LGUC were diagnosed. Among the 11 female
mutation in neoplastic cells.9This study was intended to patients, 06 cases showed high grade morphology while 05
establish diagnostic utility of p53 antibody in classifying UC cases were classified as LGUC. Among 97 cases, LGUC
into high grade and low grade forms based on expression of was seen in 49 (51%) while sections from 48(49%) cases
p53. This classification will help in predicting prognosis and showed high grade features. Immunohistochemically p53
outcome of therapy, since LGUC carry better prognosis and was found positive in 63(65%) patients and it was negative
5-year survival rates as compared to HGUC. in 34 (35%) patients as shown in Table 1. Among the
positive cases, 45 were HGUC and 18 were LGUC. Out of
II. METHODOLOGY 34 negative cases, 03 cases showed high grade morphology
while 31 cases showed features of LGUC as shown in Table
A retrospective cross-sectional study was carried out at 1.
the Histopathology department, Lady Reading Hospital
(Medical Teaching Institution), Peshawar, from August Table 1 Expression of p53 in UC
2021 till February 2022. Using WHO sample size calculator, P53 Expression
the sample size calculation was done, keeping into account Tumor Frequency Posi Frequency P-
these parameters; Confidence level (1-a=95 %Anticipated Grade Negative (%) tive (%) Value
population proportion (P) = 49.5%Absolute precision High
required (d) = 10 %Minimum sample size (n) = 97). Grade 03 3.09 45 46.39
Formalin fixed, paraffin embedded (FFPE), blocks of 97 p<0.05
Low
cases UC were included in the study using non-probability, *
Grade 31 31.96 18 18.56
consecutive sampling technique. The biopsies which were Total 34 35.05 63 64.95
inadequate, autolyzed or showing preservation and fixation *P-Value is Significant at <0.05 Level.
artifacts were excluded from the study. Proforma of the
patients for data collection was filled with record retrieved
from data bank of Pathology department and Health

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Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Table 2 Correlation of P53 Expression and Muscle Invasion IV. DISCUSSION
Muscle Invasion
P53 Expression Present Absent P-Value With the advancement in diagnostic methods over the
Negative 04 30 last two decades, it is now common to use
Positive 54 9 p<0.05* immunohistochemical markers for assessing predictive and
Total 58 39 prognostic potential of various tumors. Numerous
*P-Value is Significant At <0.05 Level. immunohistochemical markers have been inspected in cases
of UC of urinary bladder.11 In the current study, we assessed
Among the p53 positive cases, 54 cases showed the role of p53 in grading UC. P53 is a cancer suppressor
muscle invasion while 04 cases of muscle invasive UC did gene which is strongly positive in high grade UCs. 97
not show p53 expression making significant correlation patients were included in our study with calculated mean +
between muscle invasion and p53 expression (p-value standard deviation for age as 64 + 11 years and male to
<0.05) as shown in Table 2. female ratio of 8:1. Another study showed male to female
ratio of 4:1 with the same number of participants in their
Table 3 Correlation of P53 Expression with Tumor Grade research.5 Other investigators have published male to female
P53 Expression ratio of 7.46:1, in cases of UC, which is concordant with our
Tumor Frequency Posi Frequency P- results.12 We did not find any substantial association
Grade Negative (%) tive (%) Value between age group and grading of UC. In Turkey, a study
High was conducted, over 18 years, to see the possible effect of
Grade 03 6.25 45 93.75 p<0.05 age on the expected behavior and progression of bladder
* tumor in different age groups. They found that single and
Low
small tumors were usually present in patients younger than
Grade 31 63.3 18 36.7
40 years with lower recurrence rate but with similar tumor
Total 34 63
progression rate in both young and old age groups. They
*P-Value is Significant at <0.05 Level
concluded their manuscript with remarks that invasiveness
of tumors in young patients should be cautiously evaluated,
Table 4 Correlation of P53 Expression and Patients’ Gender
and earlier intervention should be initiated for halting the
P53 Expression P-Value progression for better outcome.13 A multicenter research
Gender Negative Positive conducted in Iraq in 2018, stated that expression of p53 and
Male 27 59 P<0.05* p21 was strongest in high grade and muscle invasive UC.14
Female 7 4 These results are concordant with our findings since 54/58
Total 34 63 (93.1%) cases of muscle invasive carcinoma of both low
*P-Value is Significant at <0.05 Level. grade and high grade types displayed positive p53 staining
as shown in Table 2. The product of altered p53 gene
accumulates in tumor cells nuclei and is detected by
immunohistochemistry.15 In the current project, 63 (65%) of
the cases yielded positive results while 34(35%) cases
showed negative results for p53 overexpression on
application of p53 antibody. Among p53 positive cases, 45
cases were HGUC and 3 cases were LGUC. Among the
negative 34 cases, 31 cases were LGUC while 3 cases
showed high grade morphology on H&E stained sections.
Negative expression of p53 in these cases can be delineated
by the fact that in spite of p53 gene mutation, protein
product does not gather in the nucleus of 15% to 20% of
tumors.16 This is because point mutations in p53 result in
absence of or severe reduction in synthesis of p53 protein.
Some tumors show nuclear accumulation of p53 protein
product, in the absence of gene mutation. In such cases, it
has been proved that accumulation of some gene products
like MDM2 deactivate wild-type p53 protein, consequently
Fig 1 Distribution of Age Groups with Tumor Grade resulting in a prolonged half-life of p53 gene products.
MDM2 overexpression causing overexpression of p53 with
Expression of p53 was seen in 59 male patients and 04 no p53 gene mutation is elaborated by a research carried out
female patients while no expression was observed in 27 by Özyalvacli G et al.16 Nonetheless, p53 expression was
male patients and 7 female patients as demonstrated in Table statistically substantial in high grade urothelial cancers in
4. Among 48 cases of HGUC, 45 (93.75%) cases showed present study and the same is indicated by results of various
p53 positivity while 3(6.35%) cases did not show any p53 other research projects.15,16,17
antibody staining. 18 (36.7%) cases of LGUC showed p53
positivity while 31(63.3%) cases (p-value <0.05) as shown
in table 3.

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Volume 8, Issue 2, February – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
In a trial conducted at Egypt, it was reported that increased
p53 was seen mainly in high grade tumors as compared to
low grade tumors.23 P53 positivity in our study was similar
to this trial, since more cases of HGUC stained positive as
compared to LGUC. Roy Chowdhury and colleagues
demonstrated nuclear p53 positivity in 90% of low grade
and 100% of high grade tumors, however they concluded
their research by stating that p53 gene might be unrelated to
development of urothelial neoplasm, since cases of
PUNLMP show negative staining for p53 antibody.24 The
present findings are in contrast to that. In the present study,
intense p53 positivity was noted in cases of HGUC, weak
Fig 1 Papillary UC (a) High Grade; H&E, X 200 (b) Low staining in cases of LGUC and no staining in a High-grade
Grade; H&E, X 200 tumor that showed rhabdoid differentiation. This may
suggest that when Urothelial papillary tumor
dedifferentiates, it accumulates mutations other than p53. A
project carried out by He et al. emphasized upon RAS
pathway activation and prognostic role of RAS in UC that
are p53 deficient.25 These findings were confounded by
Zhou and colleagues, who outlined the role Fibroblast
Growth Factor3 Beta (FGR3b) in cell proliferation and
tumor progression.26 In our project, 3 cases of HGUC and
31 cases of LGUC were negative for p53 antibody. This
negativity might be due to mutations, other than p53,
involved in initiation and progression of these tumors.
Positivity of p53 correlated well with higher grade and stage
Fig 2 Papillary UC (a) High Grade; Strong P53 Positivity, X of UC.
400 (b) Low Grade; Weak P53 Positivity, X 200
V. CONCLUSION
Overexpression of p53 gene in UC has also been
investigated by Yin H et al. They applied CK20, Ki67 and UC progresses by acquiring mutations, notably
p53 on 84 cases of noninvasive papillary Urothelial mutations in p53. This mutation has a diagnostic and
neoplasms. According to their results, all benign neoplasms prognostic value. Immunohistochemical staining for p53
showed negative immunostaining for p53 antibody, with a antibody not just aid in early diagnosis but it also
significant difference between high and low-grade UCs. underscores important prognostic connotation. Strong p53
Only 21% LGUC included in their study expressed p53 positivity is seen in cases of HGUC while weak to absent
antibody.17 These findings are in accordance with our study staining is seen in cases of LGUC. Moreover, p53 negativity
since 18% of p53 negative cases and 02% of p53 positive is also required in patients with UC undergoing treatment
cases showed low grade morphologic features. Our results with bacille Calmette–Guerin. We recommend using p53
are compatible with another experiment carried out by antibody in cases of UC since it has a diagnostic and
Mumtaz et al.5 73% of their cases with High grade features prognostic value.
and 36% cases with low grade morphology showed p53
protein overexpression.5 In a study carried out at King  Conflict of Interest
Edward Medical University, Lahore, from January to The authors do not have any conflict of interest.
December 2016, p53 was positive in 91% of HGUC and
16% of LGUC.9 Our findings are similar to this study, since  Acknowledgement:
p53 positivity was seen in majority of HGUC included in This work has not been presented at any
our project. P53 staining in non-muscle invasive UC is also conference/symposium.
investigated by R. Stec et al who showed that 96.27% of
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