Documente Academic
Documente Profesional
Documente Cultură
By
Dr. DILIP KUMAR
Dissertation Submitted to the
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
In partial fulfillment of the requirements for
the degree of
MD
IN
PATHOLOGY
DEPARTMENT OF PATHOLOGY,
SRI KRISHNA MEDICAL COLLEGE,
MARPUR
2019
1
2
3
4
5
ACKNOWLEDGEMENT
His blessings.
teacher and guide Dr. Shivakumar S., M.D., D.C.P, Professor and Head, Department of
Pathology, Mandya Institute of Medical Sciences, Mandya, for his timely guidance,
M.D., Associate Professor, Dr. B.G.Malathi M.D., Associate Professor, Dr. B.Kala M.D.,
Associate Professor and Dr. Y. Muralidhar Bhat M.D., Associate Professor for their
guidance.
Dr. Manjunath M.R, Assistant Professor and Dr. Shoba K.L, Assistant Professor,
and all the other technical staff of Department of Pathology for their regular and
timely help.
I express my sincere thanks to the statistician Mr. Nagaraj for his kind and
timely help.
6
7
LIST OF ABBREVIATIONS USED
AR Antigen Retrieval
ADC Adenocarcinoma
IHC Immunohistochemistry
Rb Retinoblastoma
Hr High risk
8
CDK Cyclin dependent kinase
Id Inhibitors of differentiation
9
ABSTRACT
Background
worldwide. Various screening methodologies have been developed for early detection
Recently many biomarkers have been used for diagnostic and prognostic purpose in
carcinoma cervix. The p53 protein is useful in differentiating neoplastic from non-
neoplastic and CIN from microinvasive carcinoma. The p53 expression is also a
prognostic parameters.
Objectives
epithelium.
cervical carcinomas.
carcinomas.
Methodology
department of pathology, MIMS, Mandya from January 2012 to December 2012 were
study the p53 expression in normal and neoplastic cervical epithelium. The p53
10
Results
positivity. The predominant histologic type of carcinoma cervix positive for p53 was
was p53 negative. The single case of CIN III showed p53 positive cells in all the
association with the microscopic type of carcinoma cervix (p value=0.044). The LCK
association was observed between the histologic subtype of SCC and p53 positivity (p
value=0.884).
The p53 positivity increased with the age, parity, clinical stage and grade of
the disease. However no statistically significant association was observed between the
Conclusion
normal cervical epithelium due to high proliferative index and p53 marker can be
used to differentiate these. The p53 expression was greater in invasive cervical
carcinoma and among them, the expression was more intense with higher grade and
pathogenesis of cervical cancer. Our study indicates that p53 is a powerful prognostic
marker.
11
Few cases of carcinoma cervix were p53 negative suggesting that other factors
may be involved in the carcinogenesis. Therefore, further HPV studies and other
Key words
parameters.
12
TABLE OF CONTENTS
SL. PAGE
PARTICLUARS
NO. NO.
1. INTRODUCTION 1
2. OBJECTIVES 3
3. REVIEW OF LITERATURE 4
4. METHODOLOGY 27
5. RESULTS 34
6. DISCUSSION 69
7. CONCLUSION 79
8. SUMMARY 81
9. BIBLIOGRAPHY 84
10. ANNEXURES
a. PERFORMA 91
b. MASTERCHART 97
13
LIST OF TABLES
Table Page
Title
No. No.
17. Correlation between p53 score and histologic type of cervical Carcinoma 53
14
20. Relation between p53 score and Broder’s grading 56
28. Table showing correlation of clinical stage with p53 positivity between 76
various studies
29. Table showing correlation of Broder’s grade with p53 positivity between 78
various studies
15
LIST OF FIGURES
Table Page
Title
No. No.
15. Figure showing relation between p53 score and FIGO staging 51
17. Figure showing correlation between p53 score and histologic type of 53
cervical carcinoma
18. Figure showing correlation between p53 positivity and Broder’s grading 54
19. Figure showing correlation between p53 grade and Broder’s grading 55
20. Figure showing relation between p53 score and Broder’s grading 56
21. Figure showing relation between p53 score with histologic subtypes of 58
SCC
16
22. Figure displaying the relation of p53 score with histologic subtype of 59
SCC
17
INTRODUCTION
Cervical carcinoma is the leading cancer in India, although breast cancer is the
leading cancer site globally. In India, the incidence of cervical carcinoma has
increased from 0.11 million in the year 2000 to 0.16 million cases in 2010.1 Over 80%
of the cases of the cervical carcinoma present at a fairly advanced stage2 and around
80,000 deaths are reported due to cervical carcinoma in India. For carcinoma cervix,
cytology screening is not feasible in India in view of the technical and financial
alternative to cytology in India. The other alternatives like HPV DNA testing have
been evaluated in other countries but not in India as it is still being used as a research
biomarker. There are many biomarkers of cervical cancer, some of which have a
primary marker being HPV DNA and secondary markers like p53, c-fos, p50, fra 1,
p16, notch 1, rb and telomerase. Out of these markers the primary marker has been
widely studied in India and not many studies have been done in India on secondary
markers.1 The secondary marker p53 has been studied at various centres in the world
18
p53 monitors the integrity of the genome.3 DNA repair process is initiated by
the activation of the p53 gene through DNA protein kinase during DNA damage.4
Mechanisms of inactivation or loss of the wild type of p53 gene can be by either of
the following processes- mutation within the genome, virally encoded p53 binding
protein, mutant p53 forms an oligomeric complex with wild type of p53 which turns
out to be functionless and mutant p53 gains a new oncogenic function that overcomes
p53 with the histopathological type, grade of carcinoma cervix and clinico-
19
AIMS AND OBJECTIVES
epithelium.
carcinomas.
carcinomas.
20
REVIEW OF LITERATURE
Cervical carcinoma is the leading cancer in India, although breast cancer is the
leading cancer site globally.1 The estimated annual incidence of cases and deaths in
the low- and middle-income countries (LMIC) is more than 450,000 and 240,000
respectively. More than 88% of deaths are estimated to occur in these LMIC and this
percentage is predicted to rise to at least 91.5% by 2030. 4 In 2008 in India, the annual
incidence and mortality from cervical carcinoma was 134,420 cases (age standardized
rate: 27/100,000) and 72,825 deaths (age standardized rate: 15.2/100,000).4 It has
been estimated that 100,000 new cases of cancer of cervix occur in India every year
and 70% or more of these are stage III or more at diagnosis.2 But in the recent years
HISTORY
In 400 BC, the Greek physician Hippocrates identified warts and wrote about
AD, Aulus Cornelius Celsus identified distinct types of warts: accrochordon (skin
Aretaeus, one of the most celebrated of the ancient Greek physicians, who
probably practised in 2nd or 3rd century BC, described uterine cancer as superficial and
deep ulcers, which would later infiltrate the uterus. He also described another cancer
21
type which doesn’t present as ulcer, but which is rather a growth in uterus. He
distinguished between the two lesions and acknowledged that the symptoms and
interest in epidemiology and he studied the death certificates of women dying from
cervical carcinoma. He observed that uterine cancer was rare in celibate nuns.9
common in female sex workers, it was also more common in women whose husbands
had a high number of sexual partners or were regular customers of prostitutes and it
In 1976, zur Hausen, Giesmann and their co-workers identified Human papilloma
virus (HPV) 16 in precursor lesions of genital cancer. In 1985, they demonstrated the
presence of HPV DNA in cervical carcinoma cells. The findings of these scientists
created a basis for subsequent studies, which led to the development of two preventive
technique by Papanicolau, the launch of pap screening by Papanicolau and Traut and
the invention of a specific spatula by Ayre in 1946 to scrape the cervix. Another
22
ETIOLOGY
HPV has been linked to many types of cervical disease, ranging from the
unique among human cancers by being the first found to be virtually solely
More than 80 HPV types have been identified and about 40 of them can infect
the genital tract.11 Genital HPV types have been subdivided into low-risk types, which
are found mainly in genital warts and high–risk types, which are frequently associated
with invasive cervical carcinoma. The most common low-risk types are HPV 6, 11
and high risk types are HPV 16, 18, 31, 45 and found in cervical squamous cell
carcinoma.12
HPV is the greatest risk factor in the development of cervical carcinoma and its role
established.12
when virions gain access to the cells of basal layer, probably through microwounds.
The viral genome is maintained in these cells as a stable episome at low copy number
23
The early HPV genes E1 and E2 support viral DNA replication and its
segregation such that infected cells can be maintained in the lesion for a long period.
As the viral DNA replication depends almost totally on host replication factors
(except for viral helicase E1), the other early genes E6 and E7 are required to co-
ordinate the host cell environment so that it is suitable for viral DNA replication. E6
and E7 induce unscheduled re-entry of the cell into S-phase of cell cycle, activating
the host replication machinery needed for amplification of viral genomes. E7 protein
drives the cell into S-phase by associating with and causing degradation of members
of Rb family. For the high risk HPV types, this includes pRb and p130 protein. As a
result E7 disrupts the association between pRb and E2F family of transcription factors
The function of the viral E6 protein complements that of E7 and in the high
risk HPV types, the two proteins are expressed together from a single polycistronic
mRNA species. A key function of the high risk HPV types is to bind to the cellular
p53 tumor suppressor protein and cause its degradation via the ubiquitin pathway,
thereby inhibiting its apoptotic activity. Integration of the high risk genomes is
Risk factors13
The risk factors for cervical carcinoma are related to both host and viral
24
1. Multiple sexual partners
4. High parity
6. Immunosuppression
9. Use of nicotine
clinical stage with dismal five-year survival rates of less than 40%.14, 15
The main goal of screening is to reduce the incidence and mortality of cervical
carcinoma. In the case of carcinoma cervix, it has been shown to be effective for
identifying pre-neoplastic stage early, thereby reducing mortality. There are different
methods of screening, but the most effective has been Pap smear.1
vaginal smears and it became apparent that abnormal cells could be found in several
25
The other methods used for screening are as follows1
marker.
5. Depth of invasion.
6. Endometrial extension: The presence of this feature decreases the survival rate
by a factor of 10-20%.
26
10. Microscopic type: Some authors have found a better prognosis with large cell
non-keratinizing type and a worse prognosis with the small cell type but others
found no correlation.
associated with improved survival in one study and poor survival in other.
13. Cell proliferation index: High S-phase rates as determined by flow cytometry
are correlated with both a poorly differentiated histologic type and decreased
15. HPV: It has been claimed that HPV is a major determinant of the course of
cervical cancer.
16. Others: Stromal infiltration by S-100 protein positive Langerhans cells, allelic
HPV DNA
27
High risk (hr) HPV DNA is present in virtually all cervical carcinomas and hence,
copy numbers differ between patients and could be used as a prognostic cell biologic
marker. For hr-HPV DNA copy number, no relation with survival has been found;
while high levels HPV E6/E7 mRNA expression, high levels of HPV-16 E6 mRNA
Angiogenesis markers
factor 1-α (HIF-1α), vascular endothelial growth factor (VEGF) and angiogenesis
HIF-1α expression is associated with poor overall and disease free survival. VEGF is
involved in neo-vascularisation of tumours and in few studies high VEGF has been
related to poor overall survival whereas one study failed to illustrate such a
correlation. The angiogenesis inhibitor TSP-1 has not been related to survival.7
Apoptosis markers
The various markers in this category which have been studied are Bax, Bcl-2,
Bcl-xl, MDM2 and p53. The protein Bcl-2 enhances cell survival and can be inhibited
protein which is able to inactivate tumour suppressor gene p53, thereby enhancing
cell survival. In one study, a relation between Bcl-2 positive staining and better
28
Cell cycle regulation markers
activity of which is regulated by several activators (cyclins) and inhibitors (Ink4, Cip,
expression is related to better disease specific survival but for p21 or p27 no relation
to survival has been found. Inhibitors of differentiation (Id) are transcription factors
involved in cell cycle regulation, apoptosis and angiogenesis. Three different types of
Id proteins (Id-1, 2 and 3) have also been investigated. In one study strong and
moderate Id-1 expression was related to poor overall and disease free survival.7
DNA characteristics
DNA ploidy and DNA index may have diverse biological behaviour and therefore
DNA ploidy and DNA index did not show a relation with survival while
proliferation index, defined as the percentage of G2M plus the percentage of S-phase
fraction has been related to poor survival with S-phase fraction being an independent
prognostic marker.7
29
Epidermal growth factor receptor (EGFR) pathway
EGFR and HER2/neu are tyrosine kinase receptors that belong to the
as well as positive C-erbB-2 immunostaining and poor survival has been observed by
some studies whereas certain other studies deny the presence of any such relation.7
Metastasis markers
molecules. Several isoforms of CD44 are known like CD44v1 to CD44v10. In few
studies positive CD44v6 immunostaining has been related to poor overall survival,
kinase and a reduction in nm23 gene expression has been associated with high
for poor survival and almost all metastasis markers appear to be related to survival in
cervical carcinoma.7
Proliferation markers
therefore cell biological markers reflecting the proliferation rate of tumours have been
30
Serum tumour markers
normal squamous epithelial cells, but only in limited amounts in sera from healthy
controls. Serum SCC-Ag levels have been investigated using enzyme immunoassays
in various studies and a strong relation between high pre-operative serum SCC-Ag
levels and poor survival has been observed. Serum cancer antigen 125 (CA-125) and
the combination of CA-125 with SCC-Ag, also called double tumour marker (DTM)
index were evaluated and a relation with poor disease specific survival has been
observed.7
activator inhibitor type1) and FHIT (fragile histidine triad) has shown no relation with
Review of IHC
seeks to exploit the specificity provided by the binding of an antibody with its antigen
at a light microscopic level. IHC has a long history, extending more than half a
detect corresponding antigens in frozen tissue sections. However, only since the early
1990s has the method found general application in surgical pathology. A series of
31
technical developments led eventually to the wide range of IHC applications in use
was related to the need to achieve greater sensitivity. Greater sensitivity would
facilitate staining of FFPE tissues from a simple one- step direct conjugate method to
avidin-biotin conjugate [ABC], and biotin streptavidin [B-SA] methods and would
polymer- based labeling systems. As the IHC method has evolved, its use in
diagnostic pathology has expanded such that the use of one or more IHC stains is
classification. IHC has also been adapted to the identification and demonstration of
to unmask some antigens that had been altered by formalin fixation. The antigen-
paraffin sections at high temperature before IHC staining procedures. The intensity of
32
various modifications of the AR technique have been described. Worldwide
application of AR- IHC in pathology has validated the feasibility of AR-IHC and
expanded its use in molecular morphology, while raising some basic questions and
Review of p53
The discovery of p53 protein in 1979 was the culmination of two subtypes of
studies involving a well known virologic approach and a less known serologic
approach.19
Virologic approach: Studies in 1979 by many authors like Chang et al, Kress
et al, Lane and Crawford, Linzer and Levine, Meleo et al on SV-40 transformed cells
showed that a 55-kDa protein is co-precipitated with the large T-antigen. This
association was shown to be the result of an in-vivo association between the two
proteins (Lane and Crawford, 1979). It has been postulated that this protein could be
Linzer and Levine in 1979 found that the 54-kDa protein was over expressed
It has been postulated that SV40 infection or transformation of mouse cells stimulates
mice to some methylcholanthrene-induced tumour cell lines was directed toward the p53
protein. Later, Kress et al and Melero et al in 1979 and Rotter et al in 1980 found that
animals bearing several types of tumours elicited an immune response specific for p53.19
33
The p53 has been given many adjectives like guardian of the genome (Lane,
1992), Death star (Vousden, 2000), Good and bad cop (Sharpless and DePinho,
2002), an acrobat in tumorigenesis (Moll and Schramm, 1998) and molecule of the
potentially neoplastic, cells in two distinct ways: by causing a pause in the cell cycle,
and by promoting exit from the cell cycle altogether (programmed cell death, or
the two sequelae of DNA damage: Either genome integrity is restored by DNA repair,
in which case cells can be released from transient cell cycle arrest, or when damage
persists, cells can be permanently eliminated from the population by apoptosis. This
function of p53 as “guardian of the genome” may extend to a role in initial monitoring
and repair of DNA damage in addition to direct control of cell growth and death. It is
not known what cellular signals decide between these alternatives or convert cell
strand breakage and rejoining. Increasing genetic disorder in the form of aneuploidy
and detrimental genetic recombination events with loss of genetic material (LOH) can
34
The inability to delay cell division processes increases the probability that
DNA damage will remain uncorrected during DNA replication, leading to neoplastic
progression. Between 30 and 70% of malignant tumours of almost every organ and
histologic subtype have a point mutation in one of the two p53 gene copies and loss of
the other allele. In addition, loss of p53 function by other mechanisms may be
important in some of the cancers that do not have p53 allele loss or mutation.19
Most tumor mutations are missense base substitutions in the p53 coding
sequence that change a single amino acid in the core domain, which governs
conformation and specific interactions with DNA. The Li-Fraumeni (L-F) syndrome
brain, and adrenocortical tumours, sarcomas, and leukemia. Many have germline p53
mutations, and the carrier has a 90% risk of developing cancer by age 70. Most L-F
germline mutations are missense mutations in the core domain of the p53 gene.19
functional tests. IHC is a rapid preliminary indication of p53 status in tumours and
This approach is feasible because mutant p53 in tumor cells usually adopts a
conformation more resistant to degradation than the wild-type, increasing the protein
half-life. The correlation between the presence of a p53 missense mutation and
cancer types, for example in head and neck tumours, in lung cancers of various cell
subtypes, and in colorectal cancer. Frequent nuclear staining without gene structural
mutation has been reported for skin melanomas and preinvasive prostate neoplasia.
The p53 protein may be biochemically altered by a different mechanism than gene
35
Inactivation of p53 represents a key step in cervical carcinogenesis, similarly
to other human cancers, in which the p53 gene is frequently mutated. A common p53
proline residue, Pro72 (p53pro) or an arginine residue, Arg72 (p53arg). Both forms
have wild-type biological activity. It was recently proposed that the two p53 variants
cancer. Storey et al. in 1998 showed that the p53arg variant is more efficiently
inactivated by the viral oncoprotein E6 of the high risk HPV types than the p53pro
variant. In addition, they analyzed cervical specimens for the distribution of the two
p53 variants in healthy women and women with cancer. Their findings suggest that
women with the arg/arg allele type are at higher risk of HPV-associated cervical
cancer than pro/pro or heterozygotes. The functional data reported by Storey and
colleagues show that both p53 variants are targeted by E6 protein, although the
p53arg is a better substrate for E6 than the p53pro. It is possible that the different
carcinogenesis and, therefore, only a weak association between the p53arg allele and
gradient gel electrophoresis (DGGE), the mutational status of the four ‘hotspot’
36
regions of the p53 gene in 47 primary cervical carcinomas was observed. The study
concluded that somatic mutation in the hotspot regions of p53 gene occurs
which several host genetic and environmental factors may be involved, could explain
the discrepancy of the different studies. The p53 appears to be more frequently
mutated in HPV-negative tumours. The frequency of mutation is rather low, less than
lung or colon carcinomas. These mutations in HPV-negative tumour has lent support
interaction of HPV E6 viral protein with p53 protein and by which they could achieve
cervical carcinomas for p53 gene mutations by PCR and SSCP analysis of exons 5-8,
which have been reported to be the most common sites of mutations in this gene. Of the 3
implications for protein conformation. Moreover, these amino acid substitutions map to a
region important for p53 DNA-binding activity, implying an eventual loss of function.23
37
In 1982, Crawford et al first described antibodies against human p53 protein in
9% of the breast cancer patient sera. Canon de Fromentel et al found that such
antibodies were present in sera of children with a wide variety of cancer in 1987.19
Monoclonal antibodies directed against the p53 protein have been invaluable
tools for both clinical and basic research. In clinical lab, the use of various p53
monoclonal antibodies has led to an extensive series of IHC analyses for rapid
The p53 is a 53-kDa phosphoprotein produced by p53 gene. This 20-kb human
expressed in small amounts with short half life. The primary functions are growth
The p53 protein contains three main functional domains: an N-terminal acidic
oligomerisation domain. All the three required for efficient binding of p53 to the
recognition sites. The vast majority of tumor associated p53 missense mutations occur
More than 95% of alterations in p53 are point mutation that produces the
mutant p53 proteins which loses its transactivational activity. Thus, mutated p53 has a
changed conformation, a longer half life (increased stability) and disordered function.
IHC analyses in many kinds of tumor have demonstrated a good co-relation between
p53 gene mutation and overexprression of p53 protein. Such a correlation has also
38
Nuclear staining of the majority of tumor cells accompanied by absence of
of HPV along with expression of p53 and Ki-67 in 47 cases of invasive carcinoma, 10
cases of cervical intraepithelial neoplasia (CIN) III and 10 normal cervical specimens.
He observed that normal cervix was negative for p53 expression, 10% cases of CIN
III and 50% cases of invasive carcinoma especially keratinising-type showed positive
p53 expression.25
carcinoma. They took 40 consecutive samples from 17 patients and observed that
cases with stable or decreased expression had the shortest time for progression (32.3
months) whereas no expression of p53 was associated with a longer progression time
(113.9 months). This observation led them to conclude that p53 can be used to predict
progression.26
prognostic markers in 76 patients with invasive carcinoma cervix (stage IIb/III) who
were treated with RT only at Chandigarh. Variable expression of p53 was observed
and was positive in 53.9% cases. The p53 positivity correlated with poor survival
39
Ne Win et al in Myanmar did a study on p53 expression in 2004. The study
labelled goat anti-mouse IgG as secondary antibody. In this study they observed that
p53 expression was seen in 32/40 (80%) of carcinoma cervix patients and in all
associated with early clinical stages, with adenocarcinoma and appears to be related to
p53 mutation.28
cervix. The percentage positivity of p53 was higher in squamous cell carcinoma
significant (p=0.02). The p53 positivity in squamous cell carcinoma was associated
expression of p53 and its role as early biomarker in carcinoma cervix. The study was
lesions (LSIL=20, HSIL=20, SCC Grade I=20, SCC Grade II=20 and SCC Grade
III=20) and 20 control samples. They observed that p53 was negative in normal
samples and 35% positivity in LSIL, 80% positivity in HSIL and 100% positivity in
SCC. They concluded that p53 could be used as an early biomarker for carcinoma
cervix.30
40
Geok Chin et al in 2008 studied 25 cases with CIN III and 36 cases with
distinguishing CIN III from SCC of cervix. They found that p53 was positive in 72%
of CIN III and 94.4% squamous cell carcinoma of cervix. They also observed that p53
localised in nuclei of dysplastic and carcinoma cells. They concluded that these
markers may serve as useful adjuncts in differentiating CIN III from SCC in difficult
situations.31
before and after RT. The high p27 expression and low p53 expression in carcinoma
cells before RT were regarded as predictive factors for good prognosis of patients
Kurshid Anwar et al in 1996 studied the association of HPV infection and p53
overexpression in female genital tract. They studied 41 cases of cervical cancer (CIN-
12, SCC-21 and adenocarcinoma-8) and found that p53 was positive in 68% (CIN-6,
SCC-17 and adenocarcinoma-5) of the cases. Twenty six cases of cervical cancer
were positive for both p53 and hr-HPV. They didn’t find any significant association
between the presence of specific type of HPV and detectable levels of p53.33
p53 and proliferating cell nuclear antigen (PCNA) in pre-neoplastic and neoplastic
cervical lesions. They found that p53 was positive in 22.2% cases of CIN and 45.4 %
cases of carcinoma. They also found that expression of p53 increased from CIN to
carcinoma. They concluded that these markers were of importance in low grade CIN
41
Florina Vasilescu et al in 2009 studied 26 cases (CIN-6, Grade 1 SCC-3,
Grade 2 SCC-8, and Grade 3 SCC-9) to evaluate p53, p63 and Ki-67 in HPV-induced
cervical neoplasia. They found that p53 was positive in 53.86% of cervical cancer.
They concluded that p53 is a prognostic factor for aggressiveness of tumour, when
of the bcl-2 apoptotic family of proteins in primary and recurrent cervical cancer.
expression of bcl-2, mcl-1, bax and p53 in 46 patients with primary and 28 with
recurrent cervical carcinoma. Kaplan-Meier survival analysis was performed using the
log-rank test for differences between groups. In the primary disease group, positive
staining for p53 was associated with a survival disadvantage (p53 +ve, 4-year survival
38% vs p53 -ve, 4-year survival 78%, p=0.02) and positive staining for bcl-2 was
associated with a better 5 year survival (bcl-2 +ve, 84% vs bcl-2 -ve, 53%, p=0.03).36
and HPV DNA with clinical outcome in early cervical carcinoma. They found that
p53 was positive in 40.8% of cervical cancer. The p53 expression correlated with age
(p=0.02), hormonal status (p=0.01), FIGO staging (p=0.03) and recurrence (p=0.01).
They didn’t find any significant correlation between p53 and lymph node metastasis,
42
A study done in India in 2006 by Rajaram S et al assessed the expression of
p53 using immunohistochemistry with mouse monoclonal antibody and HPV DNA
using hybrid capture II technology in 30 consecutive patients. The result showed that
high risk HPV was universally present in all cases of carcinoma cervix and the viral
load was associated with stage and tumor volume while p53 protein was expressed in
43
METHODOLOGY
Pathology, MIMS, Mandya for histopathological evaluation during the study period of
January 2012 to December 2012 were studied. Of these, 30 cases were cervical
biopsy/hysterectomy done for the clinical diagnosis of cervical cancer and 30 were
Clinical data was obtained from the patient’s outpatient and inpatient records
the department, the specimens were adequately fixed in 10% neutral buffer formalin
following which the evaluation of gross features was done. The gross details of
specimens submitted for evaluation of malignancy were observed and recorded based
on the protocol for evaluation of cervical malignancy. The hysterectomies done for
other gynaecologic causes were grossed based on the routine protocol for
hysterectomy specimen.
Then the representative tissue from hysterectomy specimen and entire tissue
from cervical biopsy specimen was subjected to routine processing for paraffin
embedding. Four to five micron thick sections were taken from paraffin embedded
blocks, stained with haematoxylin and eosin [H and E] stain and studied as per the
performa.
44
Procedure for H and E staining
The tumours were typed according to the WHO classification system. The
modified Broder’s grading was used to grade the tumours. The important microscopic
Nuclear Mitotic
Differentiation
pleomorphism figures
Well differentiated, abundant intracellular
Grade 1 bridging, cytoplasmic keratinisation, keratin Minimal <2/HPF
pearls
Moderately differentiated, individual cell Up to
Grade 2 Moderate
keratinisation present 4/HPF
Poorly differentiated, immature tumor cells
Grade 3 Marked >4/HPF
with scant cytoplasm present
45
Squamous cell carcinoma was further divided into histological subtypes as
pattern, cellular characteristics and stromal reaction and includes the following:
Keratinising type
46
Table – 4 : Characteristics of Small cell-type Squamous Cell Carcinoma
Stromal
The stroma consists of loose connective tissue
reaction
All the 60 cases were subjected to IHC study for p53. The 30 cases of
hysterectomies done for other gynaecologic causes were used to study the expression
of p53 in normal cervical epithelium. The polymer based IHC kit of BioGenex RTU
was used.
Preparation of reagents
2. Wash buffer
NaCl 8 grams
47
Procedure for IHC staining for p53 antibody
Incubate for 37o C for one day and further incubate at 58o C for overnight.
peroxidase enzyme.
Treatment with primary antibody for p53 for 2 hours to identify the tumour
48
Treatment with SS polymer (secondary antibody) for 30 minutes to elongate
Wash in TBS buffer (pH-7.6) three times for 5 minutes each to wash unbound
antibodies.
Treatment with DAB working solution for 5-8 minutes to give brown colour to
antigens.
Nuclear staining either as coarse or fine granular dots was considered positive.
The intensity of staining and the number or percentage of positive cells was assessed.
49
Table – 6 : Percentage of positivity of p53 staining28
The score of p53 is the sum of intensity and percentage of positive cells. The
The collected data was entered in excel sheet and analysed using Epiinfo
software and the descriptive statistics, Chi-square test, Student’s t-test, McNemar’s
test and other applicable statistical tests were applied for the data as applicable. The p
50
RESULTS
2012 to December 2012. Of the 60 cases, 30 were of carcinoma cervix and other 30
were of hysterectomies done for other gynaecologic causes which were used to study
In the present study, the age of patients of carcinoma cervix ranged from 31 to
70 years with a mean age of 48.37 + 11.08 years. The peak incidence of carcinoma of
In the present study, all the cases were married and had child birth. Of the 30
and 2 patients (6.7%) were of parity more than 5. The mean parity of cases of cervical
carcinoma was 3.50 + 1.925. Most women had parity of 3 to 5 (53.3%). (Table 7,
Figure 2)
In the present study, equal number of pre- and post-menopausal women was
present. (Table 7)
51
Table – 7 : Distribution of clinical parameters of carcinoma cervix patients (age,
menopausal status, parity)
14
12
10
No. OF PATIENTS
8 31-40
41-50
6
51-60
4 61-70
AGE
52
Figure – 2 : Distribution of parity in cases of carcinoma cervix
>5
CASES
3 TO 5
0 TO 2
0 5 10 15 20
presented with white discharge per vagina (WDPV), 9 cases (30%) presented with
(PMB), 6 cases (20%) presented with lower abdominal pain (LAP), 2 cases (6.7%)
presented with dysfunctional uterine bleeding (DUB) and 1 case (3.3%) presented
53
Table – 8 : Distribution of frequency of presenting complaints between
premenopausal and postmenopausal cases
Pre-Menopausal Post-Menopausal
Presenting complaint
(cases=15) (cases=15)
No. % No. %
WDPV 8 53.3 6 40.0
LAP 2 13.3 4 26.7
CB 7 46.7 2 13.3
IMB 1 6.70 0 0.00
PMB 0 0.00 9 60.0
DUB 2 13.3 0 0.00
25
20 DUB
PMB
15 IMB
CB
10 LAP
WDPV
0
CASES (PREMENOPAUSAL) CASES (POSTMENOPAUSAL)
Family history
In the present study, none of the cases had positive family history of
carcinoma cervix.
54
Past history
In the present study, none of the cases had past history of cervical carcinoma
Clinical finding
lesion which bleeds on touch; 7 (23.3%) had ulcerative lesions and 2 (6.7%) cases had
cases (16.7%) were of Stage 2a, 8 cases (26.7%) were of Stage 2b, 7 cases (23.3 %)
were of Stage 3a and 6 cases (20 %) were of Stage 3b. Clinical stage was not
Number (%)
Clinical stage (FIGO)
(Total No=27)
1 01 (3.30)
2a 05 (16.7)
2b 08 (26.7)
3a 07 (23.3)
3b 06 (20.0)
55
Figure – 4 : Figure showing the clinical staging of the tumor
8
7
6 1
NO OF CASES
5 2a
4 2b
3a
3
3b
2
1
0
stage
Gross examination
carcinoma cervix were received in the department of pathology, MIMS, Mandya. The
parametrial and paracervical tissue. The adnexae were unremarkable. Twenty pelvic
Microscopic examination
The tumours were typed according to the WHO classification system. The
modified Broder’s grading was used to grade the tumours. (Table 1) The important
56
In the present study, of the 30 cases of carcinoma cervix, 26 cases were
carcinoma (6.7%) (Figure 30), 1 case was adenocarcinoma (3.3%) (Figure 25) and 1
case (3.3%) (Figure 23) of CIN III. Of the 26 cases of SCC, 2 were microinvasive
SCC (7.8%) (Figure 24) and others were invasive SCC (92.2%). (Table 10, Figure 5)
were well differentiated (Grade I), 25 cases (86.2%) were moderately differentiated
(Grade II) and 2 cases (6.9%) were poorly differentiated (Grade III) carcinoma.
(90%), unifocal invasion was seen in two cases (6%) and one case was CIN III. In-
situ component associated with squamous cell carcinoma was present in 6 patients
The cases of SCC were further subtyped into LCNK, LCK and small cell type.
were large cell non-keratinising type and 10 (38.5%) were large cell keratinising type. No
cases of small cell non-keratinising SCC type were found. (Table 10, Figure 8, 28, 29)
57
Table – 10: Table showing distribution of carcinoma cervix according to histologic
type, Broder’s grade, pattern of invasion and subtypes of SCC
30
25 24
20
CIN III
MI SCC
15
SCC
ADSQ
10
AD
5
2 2
1 1
0
HISTOLOGIC TYPE
58
Figure – 6 : Figure showing distribution of Broder’s grade of tumour
Broder's grade
GRADE 1
GRADE 2
GRADE 3
Focus of Invasion
UF
MF
27
59
Figure – 8 : Figure showing microscopic subtypes of SCC
Subtypes of SCC
10
LCNK
16
LCK
The grade and intensity of p53 expression was studied in all the 30 cases of
Among the 30 cases, p53 was expressed in 26 cases (86.7%) of carcinoma cervix. It
other gynaecologic causes showing normal cervical epithelium (ecto- and endo-
these cases 27 (90%) were negative for p53 expression and 3 (10%) showed p53
While correlating the p53 positivity with age of 30 cases of carcinoma cervix, 8
out 9 cases (88.9%) in the 3rd decade, 8 out of 11 cases (72.7%) in 4th decade, 7 out of 7
cases (100%) in 5th decade and 3 out of 3 cases (100%) in 6th decade were p53 positive.
Maximum positivity was observed in 5th and 6th decade. (Table 11, Figure 10)
60
In the present study, 9 out of 12 cases (75%) with a parity of 2 showed p53
and both the cases (100%) with parity more than 5 showed p53 positivity.(Table 11,
Figure 9)
While correlating p53 positivity with menopausal status, equal p53 positivity
(50% each) was seen in pre- and post-menopausal women. (Table 11)
Table – 11: Table showing correlation of p53 positivity with clinical parameters
(parity, age, menopausal status)
Age Group
Menopausal Status
61
Figure – 9 : Figure showing relationship of p53 positivity with parity
16
14
12
10
8 p53 +VE
p53 -
6
0
0 TO 2 3 TO 5 MORE THAN 5
Figure – 10: Figure showing relationship of p53 positivity with age group
5
p53 +
4 p53 -
0
31-40 41-50 51-60 61-70
62
While correlating p53 score with age, among the 9 cases in 3rd decade, 1
patient (11.1%) showed a low p53 score, 5 patients (55.6%) showed moderate p53
score and 3 patients (33.3%) showed high p53 score,. Among the 11 cases in 4th
decade, 3 cases (27.3%) showed a low p53 score, 7 cases (63.7%) showed a moderate
p53 score and 1 case (9%) showed high p53 score. Among the 7 cases in 5th decade, 1
case (14.3%) showed a low p53 score, 2 cases (28.6%) showed a moderate p53 score
and 4 cases (57.1%) showed a high p53 score. Among the 3 cases in 6th decade, 1 case
each (33.3% each) showed low, moderate and high p53 score. (Table 12, Figure 11)
While correlating parity with p53 score, of the 12 patients with parity 2, low
p53 score (0-2) was seen in 3 patients (25%), a moderate p53 score (3-5) was seen in
7 patients (58.3%) and a high p53 score (6-8) was seen in 2 patients (16.7%). Among
the 16 cases with the parity of 3 to 5, a high p53 score was seen in 6 patients (37.5%),
a moderate p53 score was seen in 7 patients (43.7%) and a low p53 score was seen in
3 patients (18.8%). Among the cases with the parity more than 5, a high score was
seen in 1 patient (50%) and a moderate p53 score was seen in 1 (50%). This
difference was not statistically significant (p value=0.542). (Table 12, Figure 12)
premenopausal women 2 (13.3%) had a low p53 score, 8 (53.3%) had a moderate p53
score and 5 (33.4%) had a high p53 score. Among the 15 postmenopausal women, 4
(26.7%) had a low p53 score, 7 (46.6%) had a moderate p53 score and 4 (26.7%) had
63
Table – 12: Table showing correlation of p53 score with clinical parameters
(parity, age, menopausal status)
p53 SCORE
PARITY Total
0-2 3-5 6-8
0-2 03 (25.0%) 07 (58.3%) 02 (16.7%) 12 (100%)
3-5 03 (18.8%) 07 (43.7%) 06 (37.5%) 16 (100%)
>5 00 (0.00%) 01 (50.0%) 01 (50.0%) 02 (100%)
AGE GROUP
31-40 01 (11.1%) 05 (55.6%) 03 (33.3%) 09 (100%)
41-50 03 (27.3%) 07 (63.7%) 01 (9.00%) 11 (100%)
51-60 01 (14.3%) 02 (28.6%) 04 (57.1%) 07 (100%)
61-70 01 (33.3%) 01 (33.3%) 01 (33.4%) 03 (100%)
MENOPAUSAL STATUS
Premenopausal 02 (13.3%) 08 (53.3%) 05 (33.4%) 15 (100%)
Postmenopausal 04 (26.7%) 07 (46.7%) 04 (26.6%) 15 (100%)
Figure – 11: Figure showing comparison of p53 score in various age groups
5
0 to 2
4
3 to 5
3 >5
0
31-40 41-50 51-60 61-70
64
Figure – 12: Figure showing comparison of p53 score with parity
4 0 to 2
3 to 5
3 >5
0
0 to 2 3 to 5 >5
In the present study, single case (100%) in clinical stage I showed p53
positivity. The p53 expression was positive in 3 out of 5 cases (60%) of clinical stage
2a, 7 out of 8 cases (87.5%) of clinical stage 2b, 7 out of 7 cases (100%) of 3a and 5
out of 6 cases (83.3%) of clinical stage 3b. No statistically significant association was
noted between clinical stage and p53 positivity (p value=0.437). (Table 13, Figure 13)
65
Figure – 13: Figure showing distribution of p53 positivity in various FIGO Stages
4
p53 +
p53-
3
0
1 2a 2b 3a 3b
While correlating the grade of p53 positivity with the clinical stage of 23
cases, one case (100%) of stage 1 showed grade 3 positivity (Figure 36). Among the 3
p53 positive cases of clinical stage 2a, one case each (33.3% each) showed grade 1
(Figure 32), grade 3 and grade 5 positivity (Figure 38). Among the 7 p53 positive
cases of clinical stage 2b, 3 cases (42.9%) showed grade1 positivity, 3 cases (42.9%)
showed grade 3 positivity and 1 case (14.2%) showed grade 5 positivity. Of the 7 p53
positive cases of clinical stage 3a, 3 cases (42.9%) showed grade 2 positivity (Figure
34), 2 cases (28.7%) showed grade 3 positivity, 1 case (14.2%) showed grade 4
positivity and 1 case (14.2%) showed grade 5 positivity. Among the 5 p53 positive
cases of clinical stage 3b, 1 case (20%) showed grade 2 positivity, 2 cases (40%)
statistically significant association was noted between clinical stage and p53 positivity
66
Table – 14 : Correlation between p53 grade and FIGO staging
0 0 0 3 1
6-25 (Grade 2)
(0.00%) (0.00%) (0.00%) (42.9%) (20.0%)
1 1 3 2 0
26-50 (Grade 3)
(100%) (33.3%) (42.9%) (28.7%) (0.00%)
0 0 0 1 2
50-75 (Grade 4)
(0.00%) (0.00%) (0.00%) (14.2%) (40.0%)
0 1 1 1 2
>75 (Grade 5)
(0.00%) (33.4%) (14.2%) (14.2%) (40.0%)
Total 1 3 7 7 5
Figure - 14: Figure showing relationship of p53 grade with FIGO stage
3.5
2.5
1
2
2a
2b
1.5
3a
3b
1
0.5
0
grade1 grade2 grade3 grade4 grade5
67
While correlating cumulative p53 score with clinical staging, 4 out of 9 cases
with high p53 score were of clinical stage 3b, 5 out of 15 cases with moderate p53
score were of clinical stage 2b and 3a. Among the cases with low p53 score, 3 out 6
cases were of stage 2a. No statistically significant association was noted between
clinical stage and p53 score (p value=0.168). (Table 15, Figure 15)
p53 SCORE
FIGO STAGE
0-2 3-5 6-8
1 0 (0.00%) 1 (7.70%) 0 (0.00%)
2A 3 (50.0%) 1 (7.70%) 1 (12.5%)
2B 2 (33.3%) 5 (38.5%) 1 (12.5%)
3A 0 (0.00%) 5 (38.5%) 2 (25.0%)
3B 1 (16.7%) 1 (7.70%) 4 (50.0%)
Total 6 13 8
Figure – 15 : Figure showing relation between p53 score and FIGO staging
4.5
3.5
1
3 2a
2.5 2b
2 3a
3b
1.5
0.5
0
0 to 2 3 to 5 6 to 8
68
While correlating the over expression of p53 with histologic type of 30 cases
of carcinoma cervix, the one case (100%) of CIN III and both cases (100%) of
microinvasive squamous cell carcinoma were p53 positive, 22 out of 24 cases (91.7%)
of invasive squamous cell carcinoma were positive and 1 out of 2 (50%) cases of
microscopic type and p53 positivity (p value=0.044). (Table 16, Figure 16)
Table – 16: p53 protein over expression in various histologic types of cervical
carcinoma
Figure – 16: Figure showing p53 protein over expression in various histologic
types of cervical carcinoma
25
20
0
CIN III MI SCC SCC ADSC ADC
69
While correlating p53 score with 30 cases of carcinoma cervix, the one case
(100%) of CIN III showed a moderate p53 score. Both the cases (100%) of
microinvasive carcinoma showed a moderate p53 score. Among the 24 p53 positive
cases of SCC, 3 (12.5%) showed a low score, 12 cases (50.0%) showed a moderate
score and 9 cases (37.5%) showed a high p53 score. Both the cases of adenosquamous
carcinoma and single case of adenocarcinoma showed a low p53 score (100%). A
statistically significant relation was seen between p53 score and microscopic type of
Table – 17: Correlation between p53 score and histologic type of cervical
carcinoma
p53 SCORE
HISTOLOGIC TYPE p value
0-2 3-5 6-8
CIN III (n=1) 0 (0.00%) 1 (100%) 0 (0.00%)
Microinvasive SCC (n=2) 0 (0.00%) 2 (100%) 0 (0.00%)
Squamous Cell Carcinoma (n=24) 3 (12.5%) 12 (50.0%) 9 (37.5%) 0.041
Adenosquamous (n=2) 2 (100%) 0 (0.00%) 0 (0.00%)
Adenocarcinoma (n=1) 1 (100%) 0 (0.00%) 0 (0.00%)
Figure – 17: Figure showing correlation between p53 score and histologic type
of cervical carcinoma
30
25
20
0 TO 2
15 3 TO 5
6 TO 8
10 TOTAL NO OF CASES
0
CIN III MI SCC SCC ADSC ADC
70
While correlating the overexpression of p53 with Broder’s grading, 1 out of 2
were p53 positive. No statistically significant relation was noted between Broder’s
BRODER’S
TOTAL NO p53 +VE p53 –VE p value
GRADE
Figure – 18: Figure showing Correlation between p53 positivity and Broder’s
Grading
25
20
15
p53 +
p53 -
10
0
grade 1 grade 2 grade 3
71
While correlating the grade of positivity of p53 with the Broder’s grade, one
case (100%) of well differentiated carcinoma showed grade 3 positivity. Among the
positivity, 4 cases (18.2%) showed grade 2 positivity, 8 cases (36.4%) showed grade 3
positivity, 3 cases (13.7%) showed grade 4 positivity and 3 cases (13.7%) showed
grade 5 positivity. Both the cases (100%) of poorly differentiated carcinoma showed
grade and p53 positivity grade (p value=0.237). (Table 19, Figure 19)
Figure – 19: Figure showing correlation between p53 grade and Broder’s grading
6
p53 grade1
5
p53 grade2
4 p53 garde3
p53 grade4
3
p53 grade5
2
0
grade1 grade2 grade3
72
While correlating the Broder’s grade with p53 score, out of 2 well
differentiated carcinoma 1 case (50%) showed low score and other case (50%)
(20%) showed a low score, 13 (52%) showed moderate score and 7 cases (28%)
showed a high score. In poorly differentiated carcinomas, both the cases (100%)
showed high score. No statistically significant relation was noted between Broder’s
p53 score
BRODER’S GRADE p value
0-2 3-5 6-8
GRADE I (n=2) 1 (50.0%) 01 (50.0%) 0 (0.00%)
GRADE II (n=25) 5 (20.0%) 13 (52.0%) 7 (28.0%) 0.301
GRADE III (n=2) 0 (0.00%) 00 (0.00%) 2 (100%)
14
12
10
8 0 to 2
3 to 5
6 6 to 8
0
grade1 grade2 grade3
73
While correlating the histologic subtypes of squamous cell carcinoma with
over expression of p53, 14 out of 16 (87.5%) cases of LCNK type and 10 out of 10
Table – 21: Relation between p53 positivity and histologic subtypes of SCC
While correlating the histologic subtype of SCC with p53 score, among 16
cases of LCNK type, 6 showed a high p53 score, 8 cases showed a moderate score
and 2 cases showed a low score. Among the 10 cases of LCK type, 3 cases showed
high p53 score, 6 cases showed moderate score and 1 case showed low score. No
statistically significant relation was noted between histologic subtype of SCC and p53
2 8 6
Large Cell Non-Keratinising Type 16
(12.5%) (50.0%) (37.5%)
1 6 3
Large Cell Keratinising Type 10
(10.0%) (60.0%) (30.0%)
74
Figure – 21 : Relation between p53 score with histologic subtypes of SCC
5 0 TO 2
4 3 TO 5
6 TO 8
3
0
LCNK LCK
While correlating p53 grade with histologic subtype of SCC, among 14 p53
positive cases of LCNK, 2 cases (14.3%) showed grade 1 positivity, 2 cases (14.3%)
showed grade 2 positivity, 4 cases (28.6%) showed grade 3 positivity, 1 case (7.1%)
showed grade 4 positivity and 5 cases (35.7%) showed grade 5 positivity. Among the
10 p53 positive cases of LCK, 1 case (10%) showed grade 1 positivity, 2 cases (20%)
showed grade 2 positivity, 5 cases (50%) showed grade 3 positivity and 2 cases (20%)
Table – 23: Relation between p53 grade and histologic subtype of SCC
75
Figure – 22: Figure displaying the relation of p53 score with Histologic subtype
of SCC
3 LCNK
LCK
0
P53 GRADE1 P53 GRADE2 P53 GRADE3 P53 GRADE4 P53 GRADE5
76
Figure – 23 : Microphotograph showing CIN III (H & E, x40)
77
Figure – 25 :Microphotograph showing well differentiated adenocarcinoma (H&E, x40)
78
Figure – 27 : Microphotograph showing poorly differentiated SCC (H & E, x40)
79
Figure – 29 : Microphotograph showing LCNK subtype of SCC (H & E, x20)
80
Figure – 31 : Microphotograph showing basal staining in normal cervical
epithelium (DAB, x20)
81
Figure – 33 : Microphotograph showing 1+ intensity of p53 positivity in SCC
(DAB, x20)
82
Figure – 35 : Microphotograph showing grade 2 p53 positivity in all the layers
of lining epithelium of CIN III (DAB, x40)
83
Figure – 37 : Microphotograph showing grade 4 positivity of p53 in SCC (DAB, x20)
84
Figure – 39 : Microphotograph showing grade 5 positivity of p53 in SCC (DAB, x20)
85
DISCUSSION
In our study of 30 cases of carcinoma cervix, the mean age of patients was
48.4 ± 11.08. The peak incidence was seen in the 4th decade. The findings in the
present study are similar to the study done by Rajaram S et al35 (52.1 ± 12.46 years),
W. A Tjalma et al37 (52 years), Geok Chin Tan et al28 (51.1 years), Tan GC et al38
In the present study most of the patients of carcinoma cervix were of parity 3
to 5 with a mean parity of 3.50 ± 1.93. This finding is in concordance with the study
done by Rajaram S et al35 in Delhi (5.23 ± 2.34). However, in the study done by
Annika K.Lindstrom et al26 in Sweden most of the carcinoma cervix patients were of
Of the 30 cases of carcinoma cervix in our study, equal number of women was
In the present study, the most common clinical feature was WDPV (46.7%)
followed by PMB (30%) and CB (30%). The most common clinical presentation in
86
In the present study most women presented with exophytic growth (70%)
followed by ulcerative lesions (23.3%). This finding is similar to the study conducted
by Rajaram S et al35 where the most common clinical finding was exophytic growth
In the present study most patients presented later in course of the disease. This
finding matched with the findings of Ne Win et al25, Rajaram S et al35, Abeer A.
K.Lindstrom26 et al had maximum patients presenting in stage 1. (Table 10, Table 24,
Figure 4)
Table – 24: Table showing comparison of clinical stage between various studies
adenocarcinoma (3.30%) and 1 case CIN III (3.30%). This finding is in concordance
with Ne Win et al25, W. A Tjalma et al37, Kurshid Anwar et al30, Geok Chin Tan et
al28, Tan GC et al38 and RAF Crawford et al33. (Table 10, Table 25, Figure 5)
87
Table – 25: Table showing comparison of histologic types of carcinoma cervix
between various studies
(86.2%), similar to the study done by Ne Win et al25 (75.9%). In the study done by
Tjalma WA et al37 and Florina et al32, the most common grade was moderately
(46.8% and 40% respectively) and poorly differentiated (42.7% and 45%
Table – 26: Table showing comparison of Broder’s grade between various studies
Of the 26 cases of SCC, most of the cases were of large cell non-keratinising
subtype (61.5%) followed by large cell keratinising subtype (38.5%). This finding is
similar to the studies done by Kuniyuki et al29 (LCNK-55.8%) and Abeer A.Bahnassy
88
In the present study p53 expression was assessed in 30 cases of normal
cervical epithelium and in 90% of the normal cervix cases the squamous epithelium
al40, Krishnan Baskaran et al27, Nair et al48, Grace et al49 and Carrilho et al22. In the
10% cases of normal cervix which showed p53 expression, the p53 positive nuclei
were restricted to the basal layers of squamous epithelium similar to the study done by
Florina et al32. All the 30 cases of normal cervix studied showed no p53 expression in
In our study, the one case of CIN III was positive for p53. In comparison to
the normal cervical epithelium the pattern of staining was different. In CIN III the p53
positive cell were present throughout the layers of lining epithelium as compared to
normal cervical epithelium wherein the p53 positivity was present in only basal layers
et al51. This marker in the tissue section can be used as an adjunct to definitely
diagnose preneoplastic and neoplastic lesions in the cervix. (Table 16, Figure 16)
In our study it was noted that the positivity was present in the dysplastic nuclei and
with the increase in pleomorphism of the nuclei the intensity of p53 expression
increased. Similar finding was observed by Geok Chin et al28. These findings suggest
that the p53 marker may be useful to study the proliferative activity of the epithelial
cell which may further help in identifying dysplastic lesions and progression of
and Abeer A.Bahnassy et al40, a gradual increase in p53 positivity was observed as the
lesion progressed from CIN to invasive SCC. This finding has been utilised by Singh
89
et al53 in cytology smears where they found that abnormal expression of p53 was
noted in cervical dysplasia and it varied with different cytological grades. Madhumati
et al31 concluded in their study that p53 could be used as an important marker for low
grade CIN lesions showing high proliferative index. The p53 overexpression can be
used as a marker to differentiate difficult cases of CIN III from microinvasive SCC.31
While correlating p53 overexpression with age of the cervical carcinoma patients, p53
positivity was high in the patients of 5th and 6th decade and low in patients of 4th
decade. Highest p53 score was observed in 5th decade. Similar finding of high p53
positivity with increasing age was observed by Madhumati et al31 where women less
than 30 years of age showed 20% positivity as compared to women more than 30
years of age showed 47.6% p53 positivity. Therefore the p53 expression increased
with increasing age of the patients. In a study done by Ikuta et al34 a statistically
correlation was observed between age and p53 overexpression in carcinoma cervix (p
While correlating p53 positivity with parity of patients with carcinoma cervix,
a high p53 expression was seen in women with high parity (93.7% in parity 3 to 5,
100% in parity of more than 5) as compared to only 75% in patients with parity 2.
This finding suggests that expression of p53 increased in women with high parity,
however, no statistically significant association was observed between parity and p53
carcinoma cervix showed p53 positivity. This is in contrast with the study done by
association was found between menopausal status and p53 overexpression in our
90
study (p value=0.337). Although in the study done by Ikuta et a34l a significant relation
(Table 11)
The p53 was expressed in 86.7% of the cases of carcinoma cervix in our study.
The p53 expression in various studies ranged from 25.2% to 87.5%. The varying
range in different studies may be because of the fixation and AR methods. In various
studies done by Jain et al24 (p=0.029), Tjalma WA et al37 (p=0.066), Crawford RAF et
al33 (p=0.02), Suzuki et al41 and Chen HY et al42 (p=0.0315) p53 positivity was
associated with poor prognosis and reduced survival. The p53 positivity also has
91
While correlating grade of p53 positivity, a semi quantitative method was
used. In our study 26.7% cases showed p53 positivity in more than 50% of tumour
cells. Similarly studies done by Abeer A.Bahnassy et al40 and Tan GC et al38 57.9%
and 65.2% cases displayed p53 positivity in more than 50% of the tumour cells.
Florina et al32 in their study concluded that p53 was a prognostic factor for the
aggressiveness of the tumour when more >30% positivity was seen in tumour nuclei.
intensity of p53 expression (50%) similar to the study done by Ne Win et al25 (37.5%).
In our study a high percentage of p53 positivity was seen in the later stages of
the disease with 87.5% positivity in stage 2b, 100% positivity in stage 3a and 83.3%
positivity in stage 3b. (Table 13, Figure 13) These findings were similar to the studies
done by Abeer A.Bahnassy et al40 and Madhumati et al31. It is possible that p53
expression might increase in the more advanced stages of cervical carcinoma due to
incidence of p53 mutation.31 Abeer A.Bahnassy et al, in their study of 110 cases of
SCC and CIN concluded that aberrations of p27, cyclin E, CDK4 and p16 INK4A are
early events in HPV 16 and 18 associated cervical carcinoma, whereas cyclin D1 and
p53 pathway abnormalities are considered late events.40 In contrast Tjalma WA et al37
Even though high percentage of p53 positivity was seen in advanced stage of
carcinoma cervix, no significant association was found between clinical stage and p53
overexpression in our study (p value=0.437). (Table 15, Figure 15) In contrast to our
92
observation, a significant relation between p53 and clinical stage has been observed
et al49 (p value=0.00001). Ikuta et al34 observed that p53 expression was an indicator
While correlating p53 grade with clinical stage in our study, highest positivity
was seen in 3b. (Table 14, Figure 14) No significant association was found between
clinical stage and p53 score in our study (p value=0.168).This is in contrast to highest
Table – 28: Table showing correlation of clinical stage with p53 positivity
between various studies
p53 positivity. Similar positivity pattern has been seen in various studies done by
Tjalma et al37 (83%), Geok Chin et al28 (94.4%) and Tan GC et al38 (72.2%). Of the
two cases of adenosquamous cell carcinoma one showed p53 positivity and this was
restricted to the squamous part of the tumour. In our study the single case of
adenocarcinoma was p53 negative. (Table 16, Figure 16) Although in various studies
a low positivity has been detected in adenocarcinoma but due to a small sample size
93
adenocarcinoma. A statistically significant relation was found between the
microscopic type and p53 positivity (p value=0.044) in our study. Similar significant
relation was also observed by Annika K.Lindstrom et al26 (p=0.02), Surapan et al50
While correlating p53 score with microscopic type, our study observed a
(Table 17, Figure 17) In contrast, Ne Win et al25 observed a higher p53 score in
adenocarcinoma. In our study a statistically significant relation was found between the
In the present study the p53 positivity increased as the grade progressed from
grade 1 (50%) to grade 3 (100%). (Table 17, Figure 17) This finding was similar to
the study done by Florina et al32 and Tjalma WA et al37. In contrast Ne Win et al25
significant relation was found between the Broder’s grade and p53 positivity in the
present study (p value=0.419). (Table 19, Figure 19) This was in contrast to the
studies done by Tjalma WA et al37 (p=0.027) and Nair et al48 (p=0.0001). (Table 17,
Figure 17)While correlating p53 grade with the Broder’s grade, in the present study
moderately differentiated tumours showed a higher grade. This finding was similar to
the study done by Ne Win et al25 and Nair et al48. However in our study no
statistically significant relation was observed between tumour grade and p53 score (p
94
Table – 29: Table showing correlation of Broder’s grade with p53 positivity
between various studies
Of the 26 cases of SCC in our study, all the cases of LCK and 87.5% of
LCNK were positive for p53 overexpression. (Table 22, Table 23, Figure 21, Figure
22) This finding was similar to the study done by Carrilho C et al22 wherein it was
observed that p53 was positive for invasive SCC especially keratinising type. In
95
CONCLUSION
Our study evaluated the p53 expression in 30 cases with normal cervical
the p53 expression in normal cervical epithelium, CIN and invasive carcinoma of
cervix was done. The p53 expression was correlated with the various clinical and
p53 positivity (p value=0.044) and p53 score (p value=0.041) with microscopic type
of carcinoma cervix.
The p53 positivity increased with the increase in age and parity of the patients;
however the difference was not statistically significant. Similar increase of grade and
score of p53 expression was seen with increase in the stage and grade of tumour, but
wild type p53 activity is associated with increasing grades of dysplasia and is thought
96
In conclusion the expression of p53 was high in premalignant cervical lesion
compared to normal cervical epithelium due to high proliferative index and p53
marker can be used to differentiate these. The p53 expression was greater in invasive
cervical carcinoma and among them, the expression was more intense with higher
grade and stage of tumour suggesting expression of p53 is a late phenomenon in the
pathogenesis of cervical cancer. Our study indicates that p53 is a powerful prognostic
marker.
cervix and correlating p53 expression with the clinicopathological parameters with
follow up of the patients. This will provide an opportunity for development of p53
Few cases of carcinoma cervix were p53 negative suggesting that other factors
may be involved in the carcinogenesis. Therefore, further HPV studies and other
97
SUMMARY
1. In the present study, 30 of cases carcinoma cervix (29 cervical biopsies and 1
hysterectomy specimens for other gynaecologic causes were used to study the
2. All these cases were subjected to IHC for p53, the results of which were
cervix.
(70%).
and 3.3% of CIN III. Of the 26 cases of SCC, 2 were microinvasive SCC (7.8%)
carcinoma.
9. Most of the SCC were large cell non keratinising type (61.5%) followed by
98
10. Other histologic feature like multifocal invasion was present in 90%, unifocal
carcinoma cervix.
11. The p53 expression was present in 86.7% of cases of carcinoma cervix.
13. A statistically significant association of p53 score was seen with microscopic
14. The p53 positivity was high in patients with high parity, however no statistically
significant association of p53 positivity with parity was seen in the present study
(p value=0.542).
15. No statistically significant association of p53 score with menopausal status was
16. The p53 positivity increased in later stages of the disease (stage 3a-100% p53
with clinical stage of carcinoma cervix was seen in the present study (p
value=0.437).
17. The grade of p53 positivity was high in stage 3b (40%) but no statistically
significant association of p53 grade with clinical stage of carcinoma cervix was
18. In later stages of the disease a high p53 score was observed (3b- 50% cases had
clinical stage of carcinoma cervix was seen in the present study (p value=0.168).
99
19. Although p53 positivity was increased with the worsening of the grade (grade
with Broder’s grade of carcinoma cervix was seen in the present study (p
value=0.419).
20. Increase in grade was associated with increased number of cells showing p53
p53 grade with Broder’s grade of carcinoma cervix was seen in the present study
(p value=0.237).
21. Although the worsening of grade was associated with increase in the score
association of p53 score with Broder’s grade of carcinoma cervix was seen in
22. The keratinising subtype of SCC showed a 100% positivity for p53, but no
100
BIBLIOGRAPHY
4. Vogelstein B, Ingler W. p53 mutation and dysfunction. Cell 1992; 70: 523.
5. Levine AJ. p53, the cellular gatekeeper, growth and division. Cell 1997; 88: 323.
based screening, early diagnosis and treatment strategy of cervical cancer for
10. Rosai J. Cervix. Rosai and Ackerman’s Surgical Pathology. 10th Ed: Missouri.
11. Nubia Munoz, F.Xavier Bosch, Silvia de Sanjose, Rolando Herrero, Xavier
348: 518-27.
101
12. Grm HS, Bergant M, Banks L. Human papillomavirus infection, cancer &
13. Lora Hedrick Ellenson, Edyta C.Pirog. The Female Genital Tract. In: Kumar V,
Abbas AK, Fausto N, Astor JC editors. Robbins and Cotran pathologic Basis of
DM, Editors. Lyon: IARC Press; 1998 (IARC Scientific Publications No. 145).
Delhi, 2006.
16. Diagnostic cytology: its origins and principles. Koss Leopold G, Melamed
Myron R, Editors. Koss’ Diagnostic Cytology and Its Histopathologic Bases. 5th
23-35.
18. Taylor CR, Shi SR, Barr NJ. In: Dabbs D editor. Diagnostic
19. Thierry Soussi. 2013. The TP53 website. [online] Available at: http://p53.
20. David Sidransky, Monica Hollstein. Clinical implications of the p53 gene. Annu
21. Massimo Tommasino, Rosita Accardi, Sandra Caldeira, Wen Dong, Ilaria
102
22. Busby-Earle RM, Steel CM, Williams AR, Cohen B, Bird CC. p53 mutations in
of p53 gene and human papilloma virus infection in cervical carcinoma patients.
Methods for Light and Electron Microscopy. Kean University Union: Kluwer
in cervix cancer of Mozambican women. Pathol Res Pract 2003; 199: 303-311.
26. Jian-Liu Wang, Bi-Ying Zheng, Xi-Dan Li, Tord Ångstrom, Mikael S.
the Progression of Cervical Cancer. Clin Cancer Res 2004; 10: 2407-2414.
27. Jain D, Srinivasan R, Patel FD, Kumari Gupta S. Evaluation of p53 and Bcl-2
28. Ne Win, Thein Myint Thu, Aaing Naing Tun, Thi Thi Aye, Soe Soe, Kyaw
29. Annika K.Lindström, Tibor Tot, Ulf Stendahl, Stina Syrjänen, Kari Syrjänen,
103
30. Krishnan Baskaran, Santha Karunanithi, Inmozhi Sivakamasundari, Junior
role as early biomarker in carcinoma od uterine cervix. Int J Res Pharm Sci
31. Geok Chin Tan1, Noor Akmal Sharifah1, Mohd Sidik Shiran3, Shuib Salwati2,
Ahmad Zailani Hatta2, Hock Oon Paul-Ng. Utility of Ki-67 and p53 in
32. Kuniyuki Oka, Yoshiyuki Suzuki, Takashi Nakano. Expression of p27 and p53
33. Khurshid Anwar, Kaiuya Nakakuki, Hanac lrnai, Taizo Shiraishi . Infection of
34. Madhumati Goel, Kavita Somani, Anju Mehrotra, Uma Singh, Raj Mehrotra.
(PCNA) and p53 Protein in Cervical Cancer. The Journal of Obstetrics and
50(3): 357–361.
36. Crawford RAF, Caldwell C, Iles RK, Lowe D, Shepherd JH, Chard T.
recurrent cervical cancer. British Journal of Cancer 1998 July; 78(2): 210-14.
104
37. Ikuta A, Saito J, Mizokami T, Nakamoto T, Yasuhara M, Nagata F, Nakajima
squamous cell carcinoma cervix. Indian Journal of Cancer 2006; 43: 156.
39. Ng ABP, Atkin NB. Histological cell type and DNA value in the prognosis of
40. Tjalma WA., Weyler JJ, Bogers JJ, Pollefliet C, Baay M, Goovaerts GC,
Buytaert PM. The importance of biological factors (bcl-2, bax, p53, PCNA, MI,
41. Tan GC, Sharifah NA, Salwati S, Shiran MS, Hattaaz Ng HO.
correlates with higher incidence of mortality. Int J Cancer 2007; 121: 106-110.
Manar Moneir, Osama Shawki. The possible role of cell cycle regulators in
Pathology 2007; 7: 4.
44. Yoshiyuki Suzuki, Takashi Nakano, Shingo Kato, Tatsuya Ohno, Hirohiko
105
proteins in adenocarcinoma of the uterine cervix treated with radiotherapy
45. Chen HY, Hsu CT, Lin WC, Tsai HD, Chang WC. Prognostic value of p53
expression in stage IB1 cervical carcinoma. Gynecol Obstet Invest 2000; 49(4):
266-71.
46. Gabriela Haenrgen, Ulf Krause, Axel Becker, Peter Stadler, Christine
47. Horn LC, Kristin Linder, Grit Szepankiewicz, Jeanett Edelmann, Bettina
Hentschel, Andrea Tannapfel et al. p16, p14, p53 and cyclin D1 expression and
HPV analysis in small cell carcinoma of the uterine cervix. International Journal
48. Ngan HYS, Tsao SW, Liu SS, Stanley M. Abnormal expression in cervical
cancer-a study at protein, RNA and DNA levels. Genitourinary Medicine 1997;
73: 54.
49. Bitiren M, Cakmak EA, Gocmen A, Inaloz SS, Sari I, Karakok M, Aydin A. The
cervix squamous cell carcinoma. Eur J Gynaecol Oncol 2003; 24(5): 411-2.
and molecular factors with the stage of cervical cancerin a Brazilian cohort.
106
51. Prapid Nair, M Krishnan Nair, Puthuveetil GJ, M Radhakrishna Pillai. Decreased
programmed cell death in the uterine cervix associated with high risk human
52. Berlin Grace VM, Veronica Shalini J, Sreelekha TT, Niranjali Devaraj S,
91(1): 51-58.
9: 48-52.
cervical lesions and cervical cancer. Eur J Gynaecol Oncol. 2007; 28(4): 290-3.
30(5-6): 276-85.
107
PROFORMA
DATE : OCCUPATION:
ADDRESS:
PRESENTING COMPLAINTS
Duration of symptom
Colour
Appearance
MENSTRUAL HISTORY
Age of menarche
Age of menopause
No. of children
108
H/o irregular menses
Intermittent bleeding
Contact bleeding
Postmenopausal bleeding
OTHER INVESTIGATIONS DONE– pap smear: if yes specify the report findings
PATHOLOGICAL FACTORS
109
Section 1: Reporting proforma for cervical cancer in excisional cervical biopsies
Description of specimen and core macroscopic items
INVASIVE MALIGNANCY
Type
Squamous carcinoma
Adenosquamous carcinoma
Adenocarcinoma
Neuroendocrine carcinoma
Other
(specify................................................................... )
Differentiation/grade
Well/Grade 1
Moderate/Grade 2
Poor/Grade 3
Not assessable/GX
N/A
Unifocal
Multifocal
110
Other features
Grade
CIN 1
CIN 2
CIN 3
CGIN
Present
Absent
Grade:
Low
High
Present
Absent
p53 +/-
Intensity
Grade
p53 score
111
Section 2: Reporting proforma for cervical cancer in hysterectomy specimens
Description of specimen and core macroscopic items
Vaginal cuff:
P
A
Length…..…cm diameter… ........ cm
Dimensions of uterus: length…….cm transverse……cm anteroposterior ....... cm
Adnexa:
P
Differentiation/grade
Well/Grade 1
Moderate/Grade 2
Poor/Grade 3
Not assessable/GX
Not applicable
112
Tumour size: Maximum horizontal dimension ...................................................... cm
Thickness/depth of invasion (delete as appropriate)… ................ cm
Minimum thickness of uninvolved cervical stroma (minimum tumour-free
rim):...............cm
Position of this:………………...................................................................................
Closest radial resection margin (include paracervical tissue thickness): ................. cm
Position of this:………………..................................................................................
Pelvic nodes: (pelvic group includes obturator, internal, external and common iliac nodes)
Right
Left
Total number
Number involved
Para-
113
MASTER CHART
M
E P P
P
N M H 53 P 53
W A F U P
A S L M O I T G 53 S
Sl. IP/OP D R I S B F/ 53
Name G E B.NO A H P C S R I C
No. NO. P I G P G M P/
E X P A R C A N O
V T O F N
U O C D T R
Y
S E E
E
1 Ningamma 70 F 712415 39/12 Y N N Y 5 II A CB ADSC II - MF P 1 1 2
2 Varalakshmi 45 F 73606 62/12 N N IMB N 2 IIIA CB MI SCC II LCNK UF P 2 2 4
3 Gowramma 30 F 73625 71/12 Y N Cb N 4 NM CB CIN III -- - - P 2 2 4
4 Channajamma 55 F 718216 75/12 N N DUB N 3 IIB CB SCC III LCNK MF P 5 2 7
5 Gowramma 50 F 738917 149/12 N N PMB+Cb Y 2 IIB CB SCC II LCNK MF P 1 2 3
6 Shivamma 56 F 731555 150/12 N Y PMB Y 4 IIIA CB SCC II LCNK MF P 4 2 6
7 Lakshmamma 50 F 742624 199/12 Y N N Y 5 IIIA CB SCC II LCNK MF P 3 2 5
8 Boramma 70 F 759956 231/12 N N PMB Y 7 IIIA CB SCC II LCK MF P 2 2 4
9 Sushilamma 60 F 769496 261/12 N N Cb Y 3 IIB CB SCC II LCK MF P 3 2 5
10 Puttasiddamma 50 F 772587 277/12 N N DUB N 4 IIIB CB SCC II LCK MF P 4 3 7
11 Chikkalamma 42 F 777167 294/12 N Y N N 2 IIA CB SCC II LCNK MF P 3 2 5
12 Kalyanamma 60 F 778896 309/12 N N PMB Y 5 IIB CB SCC II LCK MF P 1 1 2
13 Gowramma 50 F 795189 387/12 N Y PMB Y 5 IIIB CB SCC II LCNK MF N N N 0
14 Subbamma 55 F 84275 438/12 N Y PMB Y 3 IIIB CB SCC II LCNK MF P 5 3 8
15 Puttasiddamma 40 F 822771 535/12 Y N N N 2 NM CB SCC II LCK MF P 3 3 6
16 Lakshmamma 50 F 824090 539/12 Y Y N Y 4 IIIB CB SCC II LCK MF P 2 1 3
97
17 Bhagyamma 35 F 818163 557/12 N Y N N 2 IIB CB ADC II - MF N N N 0
18 Gowramma 30 F 835068 598/12 Y N Cb N 4 IIIB CB SCC II LCK MF P 4 2 6
19 Channamma 50 F 859329 710/12 Y N N Y 2 IIA CB SCC II LCNK MF N 0 0 0
20 Sannathayamma 38 F 860981 730/12 Y N Cb N 2 IIA CB SCC II LCNK MF P 5 2 7
21 Nagamma 45 F 876920 813/12 N N PMB Y 3 IIIA CB SCC II LCNK MF P 2 1 3
22 Anjali 42 F 860869 823/12 N N Cb N 2 IIA CB ADSC I - MF N N N 0
23 Kalamma 65 F 881094 831/12 Y N N Y 4 IIIB CB SCC II LCNK MF P 5 3 8
24 Gowramma 40 F 889278 873/12 Y N N N 2 IIB CB SCC II LCNK MF P 1 2 3
25 Shivamma 42 F 895981 900/12 Y N Cb N 4 IIB CB SCC II LCK MF P 3 2 5
26 Rathnamma 35 F 898936 907/12 N N Cb N 2 IIIA CB SCC II LCK MF P 3 2 5
27 Siddamma 36 F 911577 974/12 Y N N N 2 IIB CB SCC II LCNK MF P 3 2 5
28 Shivamma 40 F 916273 979/12 Y N Cb N 2 IB2 H SCC I LCK MF P 3 1 4
29 Shanthamma 60 F 946275 1101/12 N N PMB Y 11 IIIA CB SCC III LCNK MF P 5 2 7
30 Ningamma 60 F 997965 1314/12 Y N PMB Y 3 NM CB MISCC II LCNK UF P 3 2 5
31 Shivamma 45 F 1017121 284/12 N Y Mn N 2 NA H CC+L NA NA NA N 0 0 0
32 Sarojamma 58 F 1047223 293/12 N N Mn Y 2 NA H CC NA NA NA N 0 0 0
33 Sharanamma 45 F 1042971 295/12 N N DUB N 3 NA H CC NA NA NA N 0 0 0
34 Savithramma 45 F 1046594 296/12 Y N Mn N 3 NA H CC+L NA NA NA N 0 0 0
35 Javaramma 60 F 1045102 300/12 N N Mn Y 3 NA H CC NA NA NA N 0 0 0
36 Channamma 45 F 1052613 317/12 N N Mn N 2 NA H CC+L NA NA NA N 0 0 0
37 Puttamma 50 F 1052471 377/12 N N DUB Y 2 NA H CC NA NA NA N 0 0 0
38 Suman 35 F 1075638 411/12 N N DUB N 2 NA H CC NA NA NA N 0 0 0
39 Kasthuri 30 F 1004350 418/12 N N Mn N 2 NA H CC NA NA NA N 0 0 0
40 Jayalakshmi 42 F 112645 420/12 N N Mn N 3 NA H CC+L NA NA NA N 0 0 0
41 Kanthamani 35 F 1091470 430/12 N N Mn N 2 NA H CC+L NA NA NA N 0 0 0
98
42 Kempamma 30 F 1096318 483/12 N N Mn N 2 NA H CC+A NA NA NA N 0 0 0
43 Chikkolamma 35 F 1141232 703/12 N N Mn N 3 NA H CC+L NA NA NA N 0 0 0
44 Gowramma 40 F 1147273 715/12 N N Mn N 2 NA H CC+L NA NA NA N 0 0 0
45 Lakshmamma 40 F 1150424 728/12 N N Mn N 3 NA H CC+L NA NA NA P 0 0 0
46 Chandramma 45 F 1151015 731/12 N N Mn N 3 NA H CC+L NA NA NA P 0 0 0
47 Bhagyamma 42 F 1152221 766/12 N N Mn N 3 NA H CC NA NA NA N 0 0 0
48 Lakshmamma 45 F 1151113 795/12 N N Mn N 2 NA H CC+L NA NA NA N 0 0 0
49 Jayasheela 35 F 1165473 796/12 N N Mn N 3 NA H CC NA NA NA N 0 0 0
50 Kala 42 F 1165967 798/12 N N Mn N 2 NA H CC NA NA NA N 0 0 0
51 Basammani 38 F 925301 806/12 N N Mn N 2 NA H CC+A NA NA NA N 0 0 0
52 Javaramma 35 F 1175588 830/12 N N N N 2 NA H CC+A NA NA NA N 0 0 0
53 Rathnamma 44 F 1172756 832/12 Y N Mn N 2 NA H CC NA NA NA N 0 0 0
54 Savithramma 50 F 1178985 847/12 N Y N Y 4 NA H CC NA NA NA N 0 0 0
55 Ningamma 45 F 1196421 958/12 N Y Mn N 2 NA H CC+A NA NA NA N 0 0 0
56 Lakshmamma 45 F 125986 978/12 N N Mn N 2 NA H CC+L NA NA NA N 0 0 0
57 Kalavathi 45 F 1189594 1013/12 Y N DUB N 2 NA H CC NA NA NA P 0 0 0
58 Lakshmamma 40 F 1224698 1016/12 N N Mn N 3 NA H CC+L NA NA NA N 0 0 0
59 Padmamma 37 F 1223804 1045/12 N N Mn N 3 NA H CC+L NA NA NA N 0 0 0
60 Siddamma 40 F 1239235 1076/12 N N Mn N 3 NA H CC+L NA NA NA N 0 0 0
99
KEY TO MASTERCHART
MH Menstrual history
Cb Contact bleeding
Mn Mennorhagia
CB Cervical biopsy
H Hystrectomy
Y Presence of feature
N Absence of feature
Sp Specimen type
BG Broder’s grade
UF Unifocal invasion
MF Multifocal invasion