A STUDY OF EXPRESSION OF p53 IN

CERVICAL CARCINOMA AND NORMAL

CERVICAL EPITHELIUM

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

Under the guidance of

Dr.Manoj Kumar Sinha
Professor and HOD

DEPARTMENT OF PATHOLOGY,
SRI KRISHNA MEDICAL COLLEGE,
MARPUR
2019

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 ACKNOWLEDGEMENT

It is most appropriate to begin by expressing my gratitude to the Almighty for

His blessings.

I express my deep sense of gratitude and humble regards to my beloved

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,

suggestions and constant inspiration that enabled me to complete this dissertation.

I express my sincere thanks to my respected teachers Dr. M.S.Siddegowda

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.

I am thankful to my teachers Dr. Manjula Manchale, Assistant Professor,

Dr. Manjunath M.R, Assistant Professor and Dr. Shoba K.L, Assistant Professor,

Department of Pathology for their valuable support.

I owe my humble thanks to the technicians, Mr. Devegowda, Mr. Praveen

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.

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 LIST OF ABBREVIATIONS USED

AR Antigen Retrieval

CIN Cervical intraepithelial neoplasia

SCC Squamous cell carcinoma

ADSC Adenosquamous carcinoma

ADC Adenocarcinoma

LCK Large cell keratinising

LCNK Large cell non- keratinising

FIGO International Federation of Gynaecology and Obstetrics

IHC Immunohistochemistry

H&E Haematoxylin and eosin

HPV Human papilloma virus

Rb Retinoblastoma

DNA Deoxyribonucleic acid

RNA Ribonucleic acid

HLA Human leukocyte antigen

TATE Tumour associated tissue eosinophilia

Hr High risk

HIF-1α Hypoxia-inducible factor 1-α

VEGF Vascular endothelial growth factor

TSP-1 Angiogenesis inhibitor thrombospondin-1

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CDK Cyclin dependent kinase

Id Inhibitors of differentiation

EGFR Epidermal growth factor receptor

SCC-Ag Squamous cell carcinoma antigen

CA-125 Carcinoma antigen-125

DTM Double tumour marker

PD-ECGF Platelet derived endothelial cell growth factor

uPA Urokinase-type plasminogen activator

PAI-1 Plasminogen activator inhibitor type1

FHIT Fragile histidine triad

FFPE Formalin fixed paraffin- embedded

PAP Peroxidase antiperoxidase

ABC Avidin-biotin conjugate

BSA Biotin streptavidin

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 ABSTRACT

Background

Cervical carcinomas are one of the leading causes of mortality in women

worldwide. Various screening methodologies have been developed for early detection

of cervical carcinoma which includes clinical, pathological and molecular methods.

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 marker for cervical carcinoma as it correlates with other clinicopathological

prognostic parameters.

Objectives

1. Study of expression of p53 in cervical carcinomas and normal cervical

epithelium.

2. Comparison of the expression of p53 among various histologic types of

cervical carcinomas.

3. Correlation of p53 expression with the histopathological grade of cervical

carcinomas.

Methodology

Thirty cases of cervical biopsy/hysterectomy specimens of carcinoma cervix

and 30 cases of hysterectomy specimens for other gynaecologic causes received at

department of pathology, MIMS, Mandya from January 2012 to December 2012 were

examined for gross and microscopic features. Immunohistochemistry was used to

study the p53 expression in normal and neoplastic cervical epithelium. The p53

expression was correlated with various clinicopathological prognostic parameters.

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Results

Of the 30 cases of carcinoma cervix, 86.7% of the cases showed p53

positivity. The predominant histologic type of carcinoma cervix positive for p53 was

moderately differentiated squamous cell carcinoma (SCC). One of the cases of

adenosquamous carcinoma (ADSC) and the single case of adenocarcinoma (ADC)

was p53 negative. The single case of CIN III showed p53 positive cells in all the

layers of squamous epithelium. The p53 positivity showed a statistically significant

association with the microscopic type of carcinoma cervix (p value=0.044). The LCK

subtype of SCC showed a higher p53 positivity but no statistically significant

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

p53 positivity and age, parity (p value=0.542), menopausal status (p value=0.337),

clinical stage (p value=0.437) and grade (p value=0.419) of carcinoma cervix.

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.

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 Few cases of carcinoma cervix were p53 negative suggesting that other factors

may be involved in the carcinogenesis. Therefore, further HPV studies and other

markers of carcinoma cervix are indicated.

Key words

Immunohistochemistry; p53; cervical carcinoma; clinicopathological prognostic

parameters.

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 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

c. KEY TO MASTERCHART 100

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 LIST OF TABLES

Table Page
Title
No. No.

1. Modified Broder’s grading system 28

2. Characteristic of large cell non-keratinising Squamous Cell Carcinoma 29

(LCNK-SCC)

3. Characteristics of Keratinising-type Squamous Cell Carcinoma (LCK-SCC) 29

4. Characteristics of Small cell-type Squamous Cell Carcinoma 30

5. Grading of intensity of p53 staining pattern 32

6. Percentage of positivity of p53 staining 33

7. Distribution of clinical parameters of carcinoma cervix patients (age, 35

menopausal status, parity)

8. Distribution of frequency of presenting complaints between premenopausal 37

and postmenopausal cases

9. Table showing the distribution of clinical staging of the tumour 38

10. Table showing distribution of carcinoma cervix according to histologic 41

type, Broder’s grade, pattern of invasion and subtypes of SCC

11. Table showing correlation of p53 positivity with clinical parameters 44

(parity, age, menopausal status)

12. Table showing correlation of p53 score with clinical parameters 47

(parity, age, menopausal status)

13. Table showing correlation of p53 positivity with FIGO staging 48

14. Correlation between p53 grade and FIGO staging 50

15. Relationship between p53 score and FIGO staging 51

16. p53 protein over expression in various histologic types of cervical 52

carcinoma

17. Correlation between p53 score and histologic type of cervical Carcinoma 53

18. Correlation between p53 positivity and Broder’s grading 54

19. Correlation between p53 grade and Broder’s grading 55

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20. Relation between p53 score and Broder’s grading 56

21. Relation between p53 positivity and histologic subtypes of SCC 57

22. Relation between p53 score with histologic subtypes of SCC 57

23. Relation between p53 grade and histologic subtype of SCC 58

24. Table showing comparison of clinical stage between various Studies 70

25. Table showing comparison of histologic types of carcinoma cervix 71

between various studies

26. Table showing comparison of Broder’s grade between various Studies 71

27. Table showing comparison of p53 overexpression between various 74

studies by IHC

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

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 LIST OF FIGURES

Table Page
Title
No. No.

1. Figure showing comparison of age at presentation in cases of carcinoma 35

cervix

2. Figure showing distribution of parity in cases of carcinoma cervix 36

3. Figure showing distribution of frequency of presenting complaints 37

between premenopausal and postmenopausal cases

4. Figure showing the clinical staging of the tumor 39

5. Figure showing distribution of various histologic types of carcinoma cervix 41

6. Figure showing distribution of Broder’s grade of tumour 42

7. Figure showing distribution of lesions based on focus of invasion 42

8. Figure showing microscopic subtypes of SCC 43

9. Figure showing relationship of p53 positivity with parity 45

10. Figure showing relationship of p53 positivity with age group 45

11. Figure showing comparison of p53 score in various age groups 47

12. Figure showing comparison of p53 score with parity 48

13. Figure showing distribution of p53 positivity in various FIGO stages 49

14. Figure showing relationship of p53 grade with FIGO stage 50

15. Figure showing relation between p53 score and FIGO staging 51

16. Figure showing p53 protein overexpression in various histologic types 52

of cervical carcinoma

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

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22. Figure displaying the relation of p53 score with histologic subtype of 59
SCC

23. Microphotograph showing CIN III (H & E, x40) 60

24. Microphotograph showing microinvasive SCC (H & E, x10) 60

25. Microphotograph showing well differentiated adenocarcinoma (H&E, x40) 61

26. Microphotograph showing moderately differentiated SCC (H & E, x20) 61

27. Microphotograph showing poorly differentiated SCC (H & E, x40) 62

28. Microphotograph showing LCNK subtype of SCC (H & E, x40) 62

29. Microphotograph showing LCK subtype of SCC (H & E, x20) 63

30. Microphotograph showing ADSC (H & E, x20) 63

31. Microphotograph showing basal staining in normal cervical epithelium 64

(DAB, x20)

32. Microphotograph showing grade 1 p53 positivity in SCC (DAB, x20) 64

33. Microphotograph showing 1+ intensity of p53 positivity in SCC (DAB, x20) 65

34. Microphotograph showing 2+ intensity of p53 positivity in CIN III 65

(DAB, x40)

35. Microphotograph showing grade 2 p53 positivity in all the layers of 66

lining epithelium of CIN III (DAB, x40)

36. Microphotograph showing grade 3 and 3+ intensity of p53 positivity in 66

SCC (DAB, x40)

37. Microphotograph showing grade 4 positivity of p53 in SCC (DAB, x20) 67

38. Microphotograph showing grade 5 positivity of p53 in SCC (DAB, x20) 67

39. Microphotograph showing grade 5 positivity of p53 in SCC (DAB, x40) 68

40. Microphotograph showing negative p53 expression in adenocarcinoma 68

(DAB, x40)

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 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,

screening has been shown to be an effective method for identifying pre-neoplastic

stage early, thereby, reducing the mortality.1

National consultations on cervical carcinoma control have concluded that

cytology screening is not feasible in India in view of the technical and financial

constraints. In this context, visual inspection strategies have been evaluated as an

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

differential expression in different types of cervical cancers.1

There are primary and secondary biomarkers in cervical carcinoma. The

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

and has been found to be deregulated in cervical cancers.1

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 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

the negative regulation by small quantities of the wild type p53.5

This study aims at comparison of expression of p53 in carcinoma cervix and

normal cervical epithelium. The study also aims at correlation of immunopositivity of

p53 with the histopathological type, grade of carcinoma cervix and clinico-

pathological prognostic parameters.

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 AIMS AND OBJECTIVES

1. Study of expression of p53 in cervical carcinomas and normal cervical

epithelium.

2. Comparison of the expression of p53 among various histologic types of cervical

carcinomas.

3. Correlation of p53 expression with the histopathological grade of cervical

carcinomas.

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 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

due to introduction of population based screening programs in developed countries

the incidence of cervical carcinoma is declining, while proportionately more patients

are being diagnosed with early stage cervical carcinoma.5

HISTORY

In 400 BC, the Greek physician Hippocrates identified warts and wrote about

cervical carcinoma. He even attempted to treat it with the procedure known as

trachelectomy, although he found that he could do nothing to treat the cancer. In 25

AD, Aulus Cornelius Celsus identified distinct types of warts: accrochordon (skin

tags), thymion (genital warts) and myremecia (non-genital warts).8

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

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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

prognosis of cancer with ulcers was the most negative.8

Rigoni-Stern, a surgeon in Padua in the mid 19th century had an amateur

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

In early 20th century, epidemiologists noted that cervical carcinoma was

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

was less in Jews.9

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

vaccines- Gardasil and Cervarix, which were approved by FDA in 2006.9

Other important milestones in the prevention strategies’ of cervical carcinoma

were: the invention of colposcope by Hinselmann in 1925, the development of pap

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

important achievement was the standardization of screening results by Bethesda

system in 1988 and further improvisation in 2001.9

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ETIOLOGY

HPV has been linked to many types of cervical disease, ranging from the

condyloma accuminatum to invasive squamous cell carcinoma. Cervical carcinoma is

unique among human cancers by being the first found to be virtually solely

attributable to the effects of an infectious agent.10

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

Epidemiological evidence has convincingly demonstrated that infection with

HPV is the greatest risk factor in the development of cervical carcinoma and its role

in the progression of the precursor lesions to invasive cervical carcinoma is well

established.12

HPV are exclusively epitheliotropic and their replication is intimately linked

to differentiation process of the host cells. Productive papillomavirus infection begins

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

and forms the reservoir for the development of a productive wart.12

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 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

and subsequent expression of proteins required for DNA replication. E7 also

associates with histone deacetylase, components of the AP1 transcription complex

and the cyclin-dependent kinase inhibitors p21 and p27.12

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

believed to represent a significant event in the pathogenesis of cervical carcinoma

associated with progression from pre-neoplastic lesions to invasive cancer.12

Risk factors13

The risk factors for cervical carcinoma are related to both host and viral

characteristics such as HPV exposure, viral oncogenecity, insufficiency of immune

response and presence of co-carcinogens. These include the following:

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 1. Multiple sexual partners

2. A male partner with multiple previous or current sexual partners

3. Young age at first intercourse

4. High parity

5. Persistent infection with a high oncogenic risk HPV

6. Immunosuppression

7. Certain HLA subtypes

8. Use of oral contraceptives

9. Use of nicotine

Screening of cervical carcinoma

More than 80% of invasive carcinoma cervix are diagnosed at an advanced

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

In 1939, Papanicolau and Herbert Traut started systematic evaluation of

vaginal smears and it became apparent that abnormal cells could be found in several

of this asymptomatic patients.16

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The other methods used for screening are as follows1

1. Unaided visual inspection

2. Visual inspection after application of acetic acid (VIA)

3. VIA with magnification

4. Visual inspection after application of Lugol’s iodine

5. HPV DNA testing

Prognostic markers in cervical carcinoma

The prognosis of cervical carcinoma is related to the following parameters: 10

1. Clinical stage: It is the most important prognostic determinator.

2. Nodal status: It is a crucial predictor found to be an independent prognostic

marker.

3. Size of the largest involved node and number of positive nodes.

4. Size of the primary tumor as determined by measurement of the tumour’s

greatest diameter or by volumetric techniques.

5. Depth of invasion.

6. Endometrial extension: The presence of this feature decreases the survival rate

by a factor of 10-20%.

7. Parametrial involvement detected microscopically.

8. Blood vessel invasion.

9. Microscopic grade: Whether the degree of tumour differentiation as evaluated

in routinely stained sections correlated with survival independently from

staging is a controversial issue. The two types of grading used is Reagen-Ng

and Broder’s method.

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 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.

11. Tumor associated tissue eosinophilia (TATE): Presence of numerous mature

eosinophils in the inflammatory infiltrate of cervical carcinoma has been

associated with improved survival in one study and poor survival in other.

12. Keratin profile: No predictive value seems to be attached.

13. Cell proliferation index: High S-phase rates as determined by flow cytometry

are correlated with both a poorly differentiated histologic type and decreased

short term survival.

14. Angiogenesis: There is no evidence of a correlation between microvessel

density and prognosis.

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

loss of chromosome 1, expression of HER2/neu, RAS oncogene and Tn

antigen have all been found to related to an unfavourable outcome.

Biomarkers in cervical carcinoma

Different types of biomarkers have been evaluated in cervical carcinoma,

some of which have a differential expression in different types of cervical carcinomas.

HPV DNA

Direct detection of HPV DNA in cervical carcinoma specimens may offer an

alternative or complementary approach to the population-based cytological screening.

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High risk (hr) HPV DNA is present in virtually all cervical carcinomas and hence,

seems not to be useful as a prognostic marker. However, hr-HPV type or hr-HPV

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

expression and HPV 18 are related to poor overall survival.7

Angiogenesis markers

Various angiogenesis markers have been evaluated like hypoxia-inducible

factor 1-α (HIF-1α), vascular endothelial growth factor (VEGF) and angiogenesis

inhibitor thrombospondin-1 (TSP-1). HIF-1α expression indicates HIF-1 activity,

which is known to have an essential role in oxygen homeostasis. Strong expression of

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

by Bax, although Bax is also directly involved in apoptosis. MDM2 is a cellular

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

survival has been found.7

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Cell cycle regulation markers

Cell cycle control is regulated by cyclin dependent kinases (CDKs), the

activity of which is regulated by several activators (cyclins) and inhibitors (Ink4, Cip,

Kip inhibitors such as p16, p21 and p27).

Constitutive and deregulated CDK or cyclin activator as well as loss of CDK

inhibitors might contribute to carcinogenesis and tumour proliferation. High p16

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

Tumours containing malignant cells with specific DNA characteristics such as

DNA ploidy and DNA index may have diverse biological behaviour and therefore

DNA characteristics may have prognostic impact.

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

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Epidermal growth factor receptor (EGFR) pathway

EGFR and HER2/neu are tyrosine kinase receptors that belong to the

epidermal growth factor receptor family. A relation between moderate/strong EGFR

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

CD44 is a transmembrane protein belonging to the family of adhesion

molecules. Several isoforms of CD44 are known like CD44v1 to CD44v10. In few

studies positive CD44v6 immunostaining has been related to poor overall survival,

while another study found no such relation.7

The product of the nm23 gene is identical to nucleoside diphosphate (NDP)

kinase and a reduction in nm23 gene expression has been associated with high

metastatic potential of tumour cells. Positive nm2/NDP kinase expression is related to

poor survival in early cervical carcinoma. Metastasizing disease is a clinical indicator

for poor survival and almost all metastasis markers appear to be related to survival in

cervical carcinoma.7

Proliferation markers

Proliferation of tumour cells is an important characteristic behaviour and

therefore cell biological markers reflecting the proliferation rate of tumours have been

investigated in various malignancies. However, for proliferation markers like BM28

and Ki67, no relation with survival has been observed.7

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Serum tumour markers

Squamous cell carcinoma antigen [SCC-Ag] can be detected in cytosol of

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

Miscellaneous cell biological markers

Overexpression of c-Met appeared to be related to poor survival. Other

investigated cell biological markers such as PD-ECGF (platelet derived endothelial

cell growth factor), uPA (urokinase-type plasminogen activator), PAI-1 (plasminogen

activator inhibitor type1) and FHIT (fragile histidine triad) has shown no relation with

survival in cervical carcinoma patients.7

Review of IHC

Immunohistochemistry (IHC) or immunocytochemistry is a method for

localizing specific antigens in tissues or cells based on antigen antibody reaction. It

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

century from 1940, when Coons developed an immunofluorescence technique to

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

today. The enzymatic label (horseradish peroxidase), developed by Avrameas and by

Nakane and colleagues, allowed visualization of the labelled antibody by light

microscopy in the presence of a suitable colourogenic substrate system.17

In Oxford, Taylor and Burns in 1974 developed the first successful

demonstration of antigens in routinely processed formalin fixed paraffin- embedded

[FFPE] tissues. Critical issue in the development of immunoperoxidase techniques

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

multiple- step detection techniques such as the peroxidase antiperoxidase (PAP),

avidin-biotin conjugate [ABC], and biotin streptavidin [B-SA] methods and would

eventually lead to amplification methods (such as tyramide) and highly sensitive

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

routine in surgical pathology, especially with respect to tumour diagnosis and

classification. IHC has also been adapted to the identification and demonstration of

both prognostic and predictive markers.17

Enzyme digestion was introduced by Huang as a pre-treatment to IHC staining

to unmask some antigens that had been altered by formalin fixation. The antigen-

retrieval (AR) technique, based on a series of biochemical studies by Fraenkel- Conrat

and coworkers was developed by Shi et al in 1991. In contrast to enzyme digestion,

the AR technique is a simple method that involves heating routinely processed

paraffin sections at high temperature before IHC staining procedures. The intensity of

IHC staining was increased dramatically after AR pre-treatment. Subsequently

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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

practical issues that are subject to ongoing evolution of the method.18

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

encoded by cellular genome.19

Linzer and Levine in 1979 found that the 54-kDa protein was over expressed

in murine SV-40 transformed cells as well as uninfected embryonic carcinoma cells.

It has been postulated that SV40 infection or transformation of mouse cells stimulates

the synthesis or stability of a cellular 54-kDa protein.19

Serologic approach: In 1979, DeLeo et al showed that the humoral response of

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

year attributed by Science, in 1993 (Harris, 1993).19

Normal (wild-type) p53 suppresses outgrowth of genetically damaged, hence

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

apoptosis). This dichotomy is thought to allow an appropriate biological response to

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

arrest to cell death.19

Cells deficient in functional p53 are genetically unstable and become

permissive for inopportune gene amplification or chromosome loss through DNA

strand breakage and rejoining. Increasing genetic disorder in the form of aneuploidy

and detrimental genetic recombination events with loss of genetic material (LOH) can

also accumulate in the cell population when unrepaired, damage to nuclear

macromolecules persists through the stages of cell division.19

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

is a rare family cancer syndrome typically characterized by an increased risk of breast,

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

Assessment of p53 function can be done by gene sequencing, IHC and

functional tests. IHC is a rapid preliminary indication of p53 status in tumours and

can be performed by immunohistochemical detection of nuclear p53 accumulation.

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

immunohistochemical staining of a tumor with certain p53 antibodies is high in some

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

mutation, possibly involving other p53-binding inhibitory proteins.19

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

polymorphism at amino acid 72 has been observed (Pro72Arg), resulting in either a

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

at codon 72 might contribute differently to the development of invasive cervical

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

degradation efficiencies of the p53 polymorphic forms marginally impact on cervical

carcinogenesis and, therefore, only a weak association between the p53arg allele and

cervical cancer could be detected.20

Studies done in 1998 by Helland et al, Hildesheim et al, Josefsson et al,

Lanham et al; in 2000 by Agorastos et al, Makni et al found an enrichment of the

Arg72 allele in cervical cancers in comparison to the healthy controls.21

In another study done on p53 mutation in cervical carcinogenesis by R M

Busby et al in 1994 using polymerase chain reaction (PCR) followed by denaturing

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

infrequently in cervical carcinoma and immunocytochemically detectable levels of

p53 are also infrequent.22

The fact that the development of cervical cancer is a multi-step process, in

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

10%, in cervical carcinoma when compared to mutations in other tumours, such as

lung or colon carcinomas. These mutations in HPV-negative tumour has lent support

to the interpretation that mechanism implicated is likely to be a consequence of the

interaction of HPV E6 viral protein with p53 protein and by which they could achieve

a biological situation resembling a partial p53 defect. Overexpression of p53 may be

found in cases of cervical carcinoma negative for HPV.20

Mehrnaz et al in 2012 screened 25 formaldehyde- fixed paraffin-embedded

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

point mutations detected, 2 corresponded to missense mutations that may have

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

Prevalence of p53 mutation in cervical cancer as detected by SSCP was

42.11% by Helland et al in 1998, 26.9% by Ishikawa et al in 2001, 13.51% by Tenti et

al in 1998 and 11.76% by Limpaiboon et al in 2000.

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

identification of p53 alteration (Dowell et al 1994).19

The p53 is a 53-kDa phosphoprotein produced by p53 gene. This 20-kb human

gene consisting of 11 exons is located on the chromosome 17p13.1. Normally it is

expressed in small amounts with short half life. The primary functions are growth

arrest, apoptosis, senescence, differentiation and antiangiogenesis.24

The p53 protein contains three main functional domains: an N-terminal acidic

transactivation domain, a central DNA binding domain and a C-terminal homo-

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

within the core domain.24

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

been detected directly by DNA sequencing (Furihata et al, 1995).24

38
 Nuclear staining of the majority of tumor cells accompanied by absence of

reactivity in surrounding uninvolved tissue or stroma is the most commonly observed

pattern characteristic of the presence of a missense p53 mutation.24

In 2003, Carrilho C et al performed a study to assess the distribution of types

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

Wang JL et al conducted a study in China during 2004 to assess the clinical

significance of p53, p16INK4a, p14ARF and PCNA in tumour progression in cervical

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

In 2003 Jain et al performed a study to evaluate p53 and Bcl-2 expression as

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

(p=0.029) at the end of 5 year follow-up period.27

39
 Ne Win et al in Myanmar did a study on p53 expression in 2004. The study

included 40 unrelated patients. The method used was indirect immunohistochemical

method employing mouse anti-human p53 monoclonal antibody and peroxidase

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

different stages and histopathological grades. The expression was significantly

associated with early clinical stages, with adenocarcinoma and appears to be related to

p53 mutation.28

Annika K.Lindstrom et al in 2009 performed a study in Sweden to assess the

differential expression of p53 in various histologic subtypes of cervical cancer. They

selected 129 cases of squamous cell carcinoma and 31 cases of adenocarcinoma of

cervix. The percentage positivity of p53 was higher in squamous cell carcinoma

(60.6%) as compared with adenocarcinoma (33.3%) which was statistically

significant (p=0.02). The p53 positivity in squamous cell carcinoma was associated

with better survival (p=0.01).29

In 2013, Krishnan Baskaran et al did a study at Tanjavur to assess over

expression of p53 and its role as early biomarker in carcinoma cervix. The study was

conducted in 100 cases of various stages of precancerous and cancerous cervical

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

squamous cell carcinoma of cervix to evaluate utility of p53 and Ki-67 in

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

Kuniyuki Oka et al in 2000 studied 202 biopsy specimens from 77 patients

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

with cervical squamous cell carcinoma treated with RT alone.32

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

Madhumati Goel et al in 2012 studied 36 cases to evaluate IHC expression of

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

lesions showing high proliferative index.34

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

exceeds 30% positivity in tumour cells.35

Crawford RAF et al performed a study in 1998 on the prognostic significance

of the bcl-2 apoptotic family of proteins in primary and recurrent cervical cancer.

They used immunohistochemistry to determine the prognostic significance of the

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

Ikuta et al in 2005 studied 49 cases to evaluate correlation of p53 expression

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,

parametrium involvement and risk of death.37

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

50% of cases (p value <0.001) suggesting deregulation.38

43
 METHODOLOGY

Sixty cases of cervical biopsy/hysterectomy submitted to the department of

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

hysterectomies done for other gynaecologic causes like dysfunctional uterine

bleeding, uterine fibroids etc with normal cervical epithelium.

Clinical data was obtained from the patient’s outpatient and inpatient records

and requisition forms accompanying the specimens to the department. On arrival to

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

 Deparaffinise in Xylene – 2 changes – 5 minutes each.

 Wash in absolute alcohol – 1 change – 3 minutes.

 Wash in water for 3-5 minutes.

 Stain with haematoxylin for 5 minutes.

 Place in running tap water for bluing for 3-5 minutes.

 Dip in acid alcohol – 1dip.

 Wash in water for 3-5 minutes.

 Stain with eosin for 1 – 2 dips.

 Wash in water for 1-2 dips.

 Dip in alcohol – 1dip.

 Blot, dry and mount in DPX.

The tumours were typed according to the WHO classification system. The

modified Broder’s grading was used to grade the tumours. The important microscopic

subtypes of squamous cell carcinoma, presence of additional in-situ component and

pattern of infiltrative margins of invasive component were studied.

Table – 1 : Modified Broder’s grading system39

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

recommended by Wentz and Reagan (1959).39 This classification is based on growth

pattern, cellular characteristics and stromal reaction and includes the following:

 Large cell non-keratinising type

 Keratinising type

 Small cell type

Table – 2 : Characteristic of large cell non-keratinising Squamous Cell

Carcinoma (NK-SCC)

Growth pattern Masses/ small nests of cells with blunt/rounded borders

Moderately large, round to oval to polygonal with moderate

Cellular amount of homogeneous cytoplasm. Nucleus is centrally placed
characteristics with irregularly clumped chromatin and occasional nucleoli.
Keratin pearls are absent.

Desmoplastic reaction and mild to moderate monocytic

Stromal reaction
inflammatory infiltrate. Focal central necrosis in large masses

Table – 3 : Characteristics of Keratinising-type Squamous Cell Carcinoma

(K-SCC)

Irregular masses of cells with sharp or irregular infiltrating

Growth pattern
margins.

Size of the cells as NK-SCC with pleomorphism being evident

along with bizarre cells. Moderate homogeneous, eosinophilic
Cellular
cytoplasm, enlarged hyperchromatic nucleus with coarse clumped
characteristics
chromatin. Keratin pearl formation and isolated cell keratinisation
are characteristic. Few mitotic figures seen

Stromal Marked desmoplastic reaction and moderate monocytic infiltrate.

reaction Focal necrosis is common.

46
Table – 4 : Characteristics of Small cell-type Squamous Cell Carcinoma

Diffuse pattern as synctial masses/nests with poorly defined

Growth pattern
boundaries

Uniformly small cells with poorly defined cytoplasmic outlines,

high N:C ratio. Round to oval to elongated cells with basophilic
Cellular
cytoplasm. The nuclei are large, oval or elongated,
characteristics
hyperchromatic with frequent nucleoli. Mitotic figures are
frequent. Absence of epithelial pearl formation

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

1. Antigen retrieval Buffer

Tri-sodium citrate buffer: pH – 6 to 6.2

Tri-sodium citrate – 2.94 grams

Dissolve in 1000 ml of water.

Adjust pH with 1N Hcl.

2. Wash buffer

Triss Buffered saline: pH 7.4 to 7.6

Triss buffer  0.6 grams

NaCl  8 grams

Dissolve in 1000 ml of water.

Adjust pH with 1N Hcl.

47
Procedure for IHC staining for p53 antibody

 Cut the sections at approximately 2-3 microns.

 Float on to the positive charged slides.

 Incubate for 37o C for one day and further incubate at 58o C for overnight.

 Two changes of xylene of 15 minutes each for deparaffinization.

 Two changes of absolute alcohol of 1 minute each for rehydration.

 One change of 95% alcohol of 1 minute for rehydration.

 One change of 70% alcohol of 1 minute for rehydration.

 Wash in tap water for 10 minutes.

 Rinse in distilled water for 5 minutes.

 Antigen retrieval by heat, using pressure cooker for 4 whistles.

 Cooling of sections to room temperature.

 Rinse in distilled water for 5 minutes.

 Wash in TBS buffer (pH-7.6) two times for 5 minutes each.

 Treatment with peroxide block for 10-15 minutes to block endogenous

peroxidase enzyme.

 Wash in TBS buffer (pH-7.6) three times for 5 minutes each.

 Treatment with power block for 15 minutes to block non-specific reaction

with the other tissue.

 Drain the excess block.

 Treatment with primary antibody for p53 for 2 hours to identify the tumour

markers by antigen- antibody reaction.

 Wash in TBS buffer (pH-7.6) three times for 5 minutes each.

 Treatment with enhancer for 20 minutes.

 Wash in TBS buffer (pH-7.6) three times for 5 minutes each.

48
  Treatment with SS polymer (secondary antibody) for 30 minutes to elongate

chain and also label the enzyme.

 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.

 Wash in TBS buffer (pH-7.6) three times for 5 minutes each.

 Wash in tap water for 5 minutes.

 Counter stain with Harris haematoxylin for 1 minute.

 Was in tap water for 5 minutes to wash away excess stain.

 Two changes of xylene for 15 minutes each.

 Two changes of absolute alcohol for 1 minute each for dehydration.

 One change of 90% alcohol of 2 minutes for dehydration.

 Two changes of absolute alcohol of 2 minutes each for dehydration.

 Clearing with xylene for two minutes.

 Mount with DPX.

Assessment of expression of p53

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.

Table – 5 : Grading of intensity of p53 staining pattern28

Staining pattern Grading of intensity

Absent 0
Mild 1+
Moderate 2+
Severe 3+

49
Table – 6 : Percentage of positivity of p53 staining28

Percentage of cells showing positivity Grade

(in 10 HPF)
1-5% of the tumor cells 1
6-25% of the tumor cells 2
26-50% of the tumor cells 3
51-75% of the tumor cells 4
>75% of the tumor cells 5

The score of p53 is the sum of intensity and percentage of positive cells. The

correlation of this score was done with the clinico-pathological parameters.

Plan of data analysis

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

value of < 0.05 was considered statistically significant.

50
 RESULTS

In the present study conducted in the department of pathology, MIMS,

Mandya, 60 cases of cervical biopsy/hysterectomies were evaluated from January

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

p53 expression in normal cervical epithelium.

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

cervix was seen in the fourth decade. (Table 7, Figure 1)

In the present study, all the cases were married and had child birth. Of the 30

cases, 12 patients (40%) were of parity 2, 16 patients (53.3%) were of parity 3 to 5

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)

AGE GROUP NUMBER PERCENTAGE

31-40 09 30.0%
41-50 11 36.7%
51-60 07 23.3%
61-70 03 10.0%
MENOPAUSE
Premenopausal 15 50.0%
Postmenopausal 15 50.0%
PARITY
0-2 12 40.0%
3-5 16 53.3%
>5 02 6.70%

Figure – 1 : Comparison of age at presentation in cases

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

In the present study, of the 30 cases of carcinoma cervix, 14 cases (46.7%)

presented with white discharge per vagina (WDPV), 9 cases (30%) presented with

contact bleeding (CB), 9 cases (30%) presented with postmenopausal bleeding

(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

with inter-menstrual bleeding (IMB). The most common clinical presentation in

premenopausal women was WDPV and CB and in postmenopausal women it was

PMB and WDPV. (Table 8, Figure 3)

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

Figure – 3 : Distribution of frequency of presenting complaints between

premenopausal and postmenopausal cases

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

i.e. all cases were of primary malignancy.

Clinical finding

On clinical examination, 21 cases (70%) had an exophytic cauliflower-like

lesion which bleeds on touch; 7 (23.3%) had ulcerative lesions and 2 (6.7%) cases had

unhealthy cervix with no growth.

Among the 30 cases of carcinoma cervix, 1 case (3.3 %) was of Stage I, 5

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

mentioned in three cases. (Table 9, Figure 4)

Table – 9 : Table showing the distribution of clinical staging of the tumour

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

CLINICAL STAGE (FIGO)

8
7

6 1
NO OF CASES

5 2a

4 2b
3a
3
3b
2
1

0
stage

Gross examination

In the present study, 29 cervical biopsies and 1 Wertheim’s hysterectomy of

carcinoma cervix were received in the department of pathology, MIMS, Mandya. The

Wertheim’s hysterectomy specimen received consisted of uterus with cervix

measuring 10 x 5.5 x 3.5 cms. The cervix showed an ulceroproliferative growth

measuring 3 x 3 x 2 cms. There was no macroscopic involvement of the vagina,

parametrial and paracervical tissue. The adnexae were unremarkable. Twenty pelvic

lymph nodes were isolated.

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

microscopic subtypes of squamous cell carcinoma, presence of additional in-situ

component and pattern of infiltrative margins of invasive component were studied.

56
 In the present study, of the 30 cases of carcinoma cervix, 26 cases were

squamous cell carcinoma (86.7%) (Figure 26), 2 cases were adenosquamous

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)

In the 29 cases of various invasive carcinoma of cervix studied, 2 cases (6.9%)

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.

(Table 10, Figure 6, 25, 26, 27)

Of the 30 cases of carcinoma cervix multifocal invasion was seen in 27 cases

(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

(23.1%). (Table 10, Figure 7)

The cases of SCC were further subtyped into LCNK, LCK and small cell type.

(Table 2, 3, 4) Among the 26 cases of squamous cell carcinoma subtypes, 16 (61.5%)

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

HISTOLOGIC TYPE (n=30) Number (%)

CIN III 01 (3.30)
MICROINVASIVE SCC 02 (6.70)
SQUAMOUS CELL CARCINOMA 24 (80.0)
ADENOSQUMAOUS 02 (6.70)
ADENOCARCINOMA 01 (3.30)
BRODER’S GRADE (n=29)
GRADE 1 02 (6.90)
GRADE 2 25 (86.2)
GRADE 3 02 (6.90)
FOCUS OF INVASION (n=30)
UNIFOCAL INVASION 02 (6.70)
MULTIFOCAL INVASION 27 (90.0)
NO INVASION 01 (3.30)
TYPES OF SCC (n=26)
LARGE CELL NON-KERATINISING TYPE 16 (61.5)
LARGE CELL KERATINISING TYPE 10 (38.5)

Figure – 5 : Figure showing distribution of various histologic types of carcinoma

cervix

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

Figure – 7 : Figure showing distribution of lesions based on focus of invasion

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

carcinoma cervix in the present study in a semi-quantitative manner (Table 5, 6).

Among the 30 cases, p53 was expressed in 26 cases (86.7%) of carcinoma cervix. It

was negative in the other 4 cases (13.3%). (Table 13)

The expression of p53 was studied in 30 cases of hysterectomies done for

other gynaecologic causes showing normal cervical epithelium (ecto- and endo-

cervical epithelium) to assess p53 expression in normal cervical epithelium. Among

these cases 27 (90%) were negative for p53 expression and 3 (10%) showed p53

positivity in basal layer of cervical squamous epithelium. (Figure 31)

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

positivity, 15 out of 16 cases (93.7%) with a parity of 3 to 5 showed p53 positivity

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)

Parity Total No p53 +ve (%) p53 –ve (%)

0-2 12 09 (75.0%) 03 (25.0%)

3-5 16 15 (93.7%) 01 (6.30%)

>5 02 02 (100%) 00 (0.00%)

Age Group

31-40 09 08 (88.9%) 01 (11.1%)

41-50 11 08 (72.7%) 03 (27.3%)

51-60 07 07 (100%) 00 (0.00%)

61-70 03 03 (100%) 00 (0.00%)

Menopausal Status

Premenopausal 15 13 (86.7%) 02 (13.3%)

Postmenopausal 15 13 (86.7%) 02 (13.3%)

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)

While correlating p53 score with menopausal status, among the 15

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

a high p53 score. (Table 12)

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)

Table – 13 : Table showing correlation of p53 positivity with FIGO staging

TOTAL NO (%) p53 +VE (%) p53 –VE (%)

CASES
30 26 4
STAGE
1 1 1(100%) 0(0.00%)
2A 5 3(60.0%) 2(40.0%)
2B 8 7(87.5%) 1(12.5%)
3A 7 7(100%) 0(0.00%)
3B 6 5(83.3%) 1(16.7%)

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%)

showed grade 4 positivity and 2 cases (40%) showed grade 5 positivity. No

statistically significant association was noted between clinical stage and p53 positivity

grade (p value=0.315). (Table 14, Figure 14)

66
 Table – 14 : Correlation between p53 grade and FIGO staging

Grade of p53 FIGO STAGE

expression (% of
cells positive 1 2A 2B 3A 3B
/10HPF)
0 1 3 0 0
1-5 (Grade 1)
(0.00%) (33.3%) (42.9%) (0.00%) (0.00%)

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)

Table – 15 : Relationship between p53 score and FIGO staging

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

adenosquamous carcinoma were positive. The single case of adenocarcinoma was

negative (Figure 40). A statistically significant association was noted between

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

Total No of cases p53 +VE p53 –VE

30 26 4 p value
Microscopic Type
CIN III 1 01 (100%) 0 (0.00%)
Microinvasive SCC 2 02 (100%) 0 (0.00%)
SCC 24 22 (91.7%) 2 (8.30%) 0.044
Adenosquamous 2 01 (50.0%) 1 (50.0%)
Adenocarcinoma 1 00 (0.00%) 1 (100%)

Figure – 16: Figure showing p53 protein over expression in various histologic
types of cervical carcinoma

25

20

15 TOTAL NO. OF CASES

p53 +
10 p53 -

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

carcinoma cervix (p value=0.041). (Table 17, Figure 17)

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

cases (50%) of well differentiated carcinoma, 22 out of 25 cases (88%) of moderately

differentiated carcinoma and both cases (100%) of poorly differentiated carcinoma

were p53 positive. No statistically significant relation was noted between Broder’s

grade and p53 positivity (p value=0.419). (Table 18, Figure 18)

Table – 18 : Correlation between p53 positivity and Broder’s grading

BRODER’S
TOTAL NO p53 +VE p53 –VE p value
GRADE

GRADE I 02 01(50.0%) 01(50.0%)

GRADE II 25 22(88.0%) 03(12.0%) 0.419

GRADE III 02 02(100%) 00(0.00%)

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

22 positive moderately differentiated carcinomas, 4 cases (18.2%) showed grade 1

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 5 positivity. No statistically significant relation was noted between Broder’s

grade and p53 positivity grade (p value=0.237). (Table 19, Figure 19)

Table – 19: Correlation between p53 grade and Broder’s grading

GRADE OF p53 BRODER’S GRADING (n=25)

EXPRESSION Grade I Grade II Grade III
1-5 (Grade 1) 0 (0.00%) 4 (18.2%) 0 (0.00%)
6-25 (Grade 2) 0 (0.00%) 4 (18.2%) 0 (0.00%)
26-50 (Grade 3) 1 (100%) 8 (36.4%) 0 (0.00%)
50-75 (Grade 4) 0 (0.00%) 3 (13.7%) 0 (0.00%)
>75 (Grade 5) 0 (0.00%) 3 (13.7%) 2 (100%)
Total 1 22 2

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%)

showed a moderate score. Of the 25 moderately differentiated carcinomas, 5 cases

(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

grade and p53 score (p value=0.301). (Table 20, Figure 20)

Table – 20 : Relation between p53 score and Broder’s grading

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%)

Figure – 20 : Relation between p53 score and Broder’s grading

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

(100%) cases of LCK type showed p53 positivity. (Table 21)

Table – 21: Relation between p53 positivity and histologic subtypes of SCC

p53 positivity TOTAL No p53 +VE p53 –VE

14 2 p value
Large Cell Non-Keratinising Type 16
(87.5%) (12.5%)
= 0.884
10 0
Large Cell Keratinising Type 10
(100%) (0.00%)

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

score (p value=0.884). (Table 22)

Table – 22 : Relation between p53 score with histologic subtypes of SCC

p53 score Total 0-2 3-5 6-8

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%)

showed grade 4 positivity. (Table 23, Figure 22, Figure )

Table – 23: Relation between p53 grade and histologic subtype of SCC

GRADE OF p53 HISTOLOGIC SUBTYPE OF SCC

EXPRESSION LCNK TYPE (n=14) LCK TYPE (n=10)
1-5 (Grade 1) 02 (14.3%) 01 (10.0%)
6-25 (Grade 2) 02 (14.3%) 02 (20.0%)
26-50 (Grade 3) 04 (28.6%) 05 (50.0%)
50-75 (Grade 4) 01 (07.1%) 02 (20.0%)
>75 (Grade 5) 05 (35.7%) 00 (0.00%)

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)

Figure – 24 : Microphotograph showing microinvasive SCC (H & E, x10)

77
Figure – 25 :Microphotograph showing well differentiated adenocarcinoma (H&E, x40)

Figure – 26 : Microphotograph showing moderately differentiated SCC (H&E, x20)

78
Figure – 27 : Microphotograph showing poorly differentiated SCC (H & E, x40)

Figure – 28 : Microphotograph showing ADSC (H & E, x20)

79
Figure – 29 : Microphotograph showing LCNK subtype of SCC (H & E, x20)

Figure – 30 : Microphotograph showing LCK subtype of SCC (H & E, x20)

80
Figure – 31 : Microphotograph showing basal staining in normal cervical
epithelium (DAB, x20)

Figure – 32 : Microphotograph showing grade 1 p53 positivity in SCC (DAB, x20)

81
Figure – 33 : Microphotograph showing 1+ intensity of p53 positivity in SCC
(DAB, x20)

Figure – 34 : Microphotograph showing 2+ intensity of p53 positivity in CIN III

(DAB, x40)

82
Figure – 35 : Microphotograph showing grade 2 p53 positivity in all the layers
of lining epithelium of CIN III (DAB, x40)

Figure –36 : Microphotograph showing grade 3 and 3+ intensity of p53

positivity in SCC (DAB, x40)

83
Figure – 37 : Microphotograph showing grade 4 positivity of p53 in SCC (DAB, x20)

Figure – 38 : Microphotograph showing grade 5 positivity of p53 in SCC (DAB, x40)

84
Figure – 39 : Microphotograph showing grade 5 positivity of p53 in SCC (DAB, x20)

Figure – 40 : Microphotograph showing negative p53 expression in adenocarcinoma

(DAB, x40)

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

(50.3 years) and Astrid et al39 (45 years). (Table 7, Figure 1)

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

parity 2.7. (Table 8, Figure 2)

Of the 30 cases of carcinoma cervix in our study, equal number of women was

in premenopausal and postmenopausal age group. In a similar study done by

Madhumati et al31, there were 19 postmenopausal women and 17 premenopausal

women in the study. (Table 8)

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

premenopausal women was WDPV and CB and in postmenopausal women it was

PMB and WDPV. (Table 9, figure 3)

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

(60%) followed by ulcerative growth (33.3%).

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.

Bahnassy et al40 and Kuniyuki et al29. In contrast study done by Annika

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

Clinical Stage (FIGO)

Study name
Stage 1 Stage 2 Stage 3 Stage 4
Kuniyuki et al29 (2000) 03 21 48 05
Ne Win et al25 (2004) 12 23 04 01
Rajaram S et al35 (2006) 02 17 10 11
Abeer A. Bahnassy et al40(2007) 17 26
Annika Landstorm et al26 (2009) 54 32 35 07
Present study 01 13 13 00

Of the 30 cases of carcinoma cervix majority were squamous cell carcinoma

(86.7%) followed by 2 cases adenosquamous cell carcinoma (6.70%), 1 case of

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

Study name SCC AD ADSC CIN

Kurshid Anwar et al30 (1996) 21 00 00 25
RAF Crawford et al33 (1998) 36 09 08 00
W A Tjalma et al37 (2001) 39 02 05 00
Ne Win et al25 (2004) 29 10 00 00
Tan GC et al38 (2007) 36 03 07 25
Present study (2013) 26 01 02 01

In our study most of the carcinoma of cervix were moderately differentiated

(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%

respectively). (Table 10, Table 26, Figure 6)

Table – 26: Table showing comparison of Broder’s grade between various studies

Study name Grade 1 Grade 2 Grade 3

Tjalma WA et al37 (2001) 05 22 05
Ne Win et al25 (2004) 02 22 20
Florina et al 32(2009) 03 08 09
Present study (2013) 02 25 02

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

et al40 (LCNK-60.5%). (Table 10, Figure 8)

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

showed negative expression of p53 similar to studies done by Abeer A.Bahnassy et

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

the endocervical epithelium. (Table 13)

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

of the squamous epithelium. Similar pattern of positivity was observed by Jeffers MD

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

disease in patients suffering for cervical cancer.

In the studies done by Krishnan Baskaran et al35, Turkcuogh I et al52 (p=0.002)

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

value=0.02). (Table 11, Figure 11)

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

score (p=0.542). (Table 11, Figure 12)

In our study equal number of premenopausal and postmenopausal women with

carcinoma cervix showed p53 positivity. This is in contrast with the study done by

Madhumati et al31 where the p53 overexpression was higher in postmenopausal

women (52.6%) as compared to premenopausal women (17.6%). No significant

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

was observed between p53 overexpression and hormonal status (p value=0.01).

(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

therapeutic implications as the low positivity prior to radiotherapy was associated

with better prognosis.29 (Table 27)

Table – 27: Table showing comparison of p53 overexpression between various

studies by IHC

Study (year) Incidence of p53 expression (%)

Kuniyuki et al29 (2000) 52.1%
Haensgen G et al43 (2001) 85.7%
Horn LC44 (2001) 63.8%
45
Ngan HY et al (2001) 25.2%
37
WA Tjalma et al (2001) 42.0%
Bitiren M et al46 (2003) 43.3%
22
Carrilho C et al (2003) 50.0%
Ne Win et al25 (2004) 80.0%
34
Ikuta et al (2005) 40.8%
38
Tan GC et al (2007) 76.0%
28
Geok Chin et al (2008) 85.2%
26
Annika Lindstorm et al (2009) 60.6%
Florina et al32 (2009) 53.9%
31
Madhumati Goel et al (2012) 45.5%
Sergio et al47 (2012) 49.4%
27
Krishnan Baskaran et al (2013) 83.0%

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.

In present study, majority of cases of carcinoma cervix showed moderate

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

increased abnormality in control of p53 expression or degradation or an increased

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

observed higher p53 positivity in stage 1b, 1a and 2a.

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

by Ikuta et al34 (p value=0.03), Abeer A.Bahnassy et al40 (p value=0.003) and Grace

et al49 (p value=0.00001). Ikuta et al34 observed that p53 expression was an indicator

of unfavourable prognosis in patients of 1b1 SCC. (Table 28)

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

p53 grade observed by Ne Win et al25 in stage 1b.

Table – 28: Table showing correlation of clinical stage with p53 positivity
between various studies

p53 positivity in various clinical stage

Study name
1 2a 2b 3a 3b 4
Abeer A. Bahnassy et al40 (2007) Low stage-11.7% High stage-65.4%
Tjalma WA et al37 (2001) 11% 38% 34% 8.5% - 8.5%
Madhumati Goel et al31 (2012) 44.4% 16.6% 50% 50%
Present study2013 100% 60% 87.5% 100% 83.3% -

In our study, 24 out of 26 cases (92.3%) of squamous cell carcinoma showed

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

of adenocarcinoma in our study, it is difficult to comment on the positivity of p53 in

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

(p<0.001) and Abeer A.Bahnassy et al40 (p=0.01).

While correlating p53 score with microscopic type, our study observed a

higher score in squamous cell carcinoma as compared to other microscopic types.

(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

microscopic type and p53 score (p value=0.041).

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

found a higher positivity in grade 1 (100%) and grade 3 (100%). No statistically

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

value=0.301) (Table 29)

94
Table – 29: Table showing correlation of Broder’s grade with p53 positivity
between various studies

p53 positivity in various grade of

Study name carcinoma cervix
Grade 1 Grade 2 Grade 3
WA Tjalma et al37 (2001) 11% 47% 42%
Ne Win et al25 (2004) 100% 71.4% 100%
Florina et al32 (2009) 11.5% 30.8% 34.6%
Present study (2013) 50% 88% 100%

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

contrast Abeer A.Bahnassy et al showed higher p53 positivity (57.7%) in LCNK as

compared to LCK (7.6%). In our study no statistically significant relation was

observed between histologic subtypes of SCC and p53 score (p value=0.884).

95
 CONCLUSION

Our study evaluated the p53 expression in 30 cases with normal cervical

epithelium and 30 cases of various types of carcinoma cervix. A comparison between

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

histopathologic parameters in cases of carcinoma cervix. The p53 positivity was

assessed by a semi quantitative method. The number of cells showing positivity

(grade) and the intensity of p53 were also studied.

According to our data a statistically significant correlation was seen between

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

with no statistical significance. The absence of statistical significance may be due to

the small sample size of our study.

Inactivation of p53 is an important step in cervical carcinogenesis. Loss of

wild type p53 activity is associated with increasing grades of dysplasia and is thought

to be due to late event in tumour progression.

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.

In future it is necessary to carry out studies with large samples of carcinoma

cervix and correlating p53 expression with the clinicopathological parameters with

follow up of the patients. This will provide an opportunity for development of p53

targeted therapy for carcinoma cervix.

Few cases of carcinoma cervix were p53 negative suggesting that other factors

may be involved in the carcinogenesis. Therefore, further HPV studies and other

markers of carcinoma cervix are indicated.

97
 SUMMARY

1. In the present study, 30 of cases carcinoma cervix (29 cervical biopsies and 1

hysterectomy specimen) were studied from January 2012 to December 2012.

The gross and microscopic features with special emphasis on various

clinicopathological prognostic parameters were studied. Thirty cases of

hysterectomy specimens for other gynaecologic causes were used to study the

normal cervical epithelium.

2. All these cases were subjected to IHC for p53, the results of which were

correlated with various clinicopathological prognostic parameters of carcinoma

cervix.

3. Majority of the patients of carcinoma cervix were in 4th decade (36.7%).

4. Majority of the patients of carcinoma cervix were of parity 3 to 5 (53.3%).

5. Majority of the patients of carcinoma cervix presented with exophytic growth

(70%).

6. Majority of the patients of carcinoma cervix were of clinical stage 2b (26.7%).

7. Majority of the cases of carcinoma cervix were of squamous cell carcinoma

(86.7%), followed by 6.7% adenosquamous carcinoma, 3.3% adenocarcinoma

and 3.3% of CIN III. Of the 26 cases of SCC, 2 were microinvasive SCC (7.8%)

and others were invasive SCC (92.2%).

8. Most of the cases of carcinoma cervix were moderately differentiated (86.2%)

followed by 6.9% each of well differentiated and poorly differentiated

carcinoma.

9. Most of the SCC were large cell non keratinising type (61.5%) followed by

38.5% large cell keratinising type.

98
10. Other histologic feature like multifocal invasion was present in 90%, unifocal

invasion in 6.7%. Associated in-situ component was present in 23.1% cases of

carcinoma cervix.

11. The p53 expression was present in 86.7% of cases of carcinoma cervix.

However, no significant statistically association was observed (p value=0.4).

12. A statistically significant association of p53 positivity was seen with

microscopic type of carcinoma cervix (p value=0.044).

13. A statistically significant association of p53 score was seen with microscopic

type of carcinoma cervix (p value=0.041).

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

seen in the present study (p value=0.337).

16. The p53 positivity increased in later stages of the disease (stage 3a-100% p53

positivity). However, no statistically significant association of p53 positivity

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

seen in the present study (p value=0.315).

18. In later stages of the disease a high p53 score was observed (3b- 50% cases had

score of 6-8), however no statistically significant association of p53 score with

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

III-100% p53 positivity), no statistically significant association of p53 positivity

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

positivity (grade III-100%). However, no statistically significant association of

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

(Grade III – 100% cases showed score of 6-8), no statistically significant

association of p53 score with Broder’s grade of carcinoma cervix was seen in

the present study (p value=0.301).

22. The keratinising subtype of SCC showed a 100% positivity for p53, but no

statistically significant association of p53 score with histologic subtype of SCC

was seen in the present study (p value=0.884).

100
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Kankaya D, Sertçelik A. The role of p53, Bcl-2 and Ki-67 in premalignant

cervical lesions and cervical cancer. Eur J Gynaecol Oncol. 2007; 28(4): 290-3.

56. Singh M, Srivastava S, Singh U, Mathur N, Shukla Y. Coexpression of p53 and

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30(5-6): 276-85.

107
 PROFORMA

NAME : AGE /SEX :

IP/OPNO. BIOPSY/HYSTRECTOMY NO:

DATE : OCCUPATION:

ADDRESS:

PRESENTING COMPLAINTS

White discharge per vaginum

Duration of symptom

Foul smelling/non foul smelling

Colour

Appearance

Associated with itching

Blood stained or not

Lower abdominal pain

Loss of weight in last 6 months

FAMILY HISTORY OF CANCER: cervical/ uterine

MENSTRUAL HISTORY

Age of menarche

Age of menopause

Age at birth of 1st child

No. of children

108
H/o irregular menses

Intermittent bleeding

Contact bleeding

Postmenopausal bleeding

Treatment taken – if yes specify (progesterone/ surgical –D &C)

Hormone replacement: no /yes – estrogen only /e+p

Marital history : age at marriage

H/O OTHER DISEASES

Diabetes /Hypertension / Liver/ Kidney/ Thyroid Diseases

Diet : Intake of nicotine

Bowel and bladder movements

OTHER INVESTIGATIONS DONE– pap smear: if yes specify the report findings

H/O OTHER PREVIOUS SURGERIES: -/+ REASON:

CLINICAL FINDINGS (per speculum/per vaginum):

CLINICAL STAGING (FIGO):

TYPE OF SPECIMEN RECEIVED: cervical biopsy/ Hystrectomy specimen

PATHOLOGICAL FACTORS

109
Section 1: Reporting proforma for cervical cancer in excisional cervical biopsies
Description of specimen and core macroscopic items

cm x ……..cm and ............... cm

thick/deep

Number of fragments received, measurement of each and block designation:

..........................................................................................................................................
..........................................................................................................................................

Core microscopic 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

Distribution of invasive component

Unifocal
Multifocal

110
Other features

CIN (cervical intra-epithelial neoplasia):

Present
Absent

Grade

CIN 1
CIN 2
CIN 3
CGIN
Present
Absent
Grade:
Low
High

Lymphovascular space invasion

Present
Absent

p53 +/-

Intensity
Grade
p53 score

TYPE OF THERAPY: Surgery/Radiotherapy/Chemotherapy.

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

Maximum dimensions of tumour: …………mm x ..................... mm

Position of cervical tumour:

Macroscopic involvement of paracervical tissue

Core microscopic items

Type
Squamous carcinoma
Adenosquamous carcinoma
Adenocarcinoma Neuroendocrine
carcinoma Other
(Specify .......................................................... )

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:………………..................................................................................

Distance from distal vaginal epithelial margin: .......... cm

Position of this:……………………................................................................................

Pelvic nodes: (pelvic group includes obturator, internal, external and common iliac nodes)
Right
Left
Total number
Number involved

Para-

Other tissues and organs: Normal / Abnormal (describe)

Endometrium
Myometrium
Right adnexum
Left adnexum
p53 +/-
Intensity
Grade
p53 score

TYPE OF THERAPY: Surgery/Radiotherapy/Chemotherapy.

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

WDPV White discharge per vagina

LAP Lower abdominal pain

MH Menstrual history

IMB Intermenstrual bleeding

PMB Post menstrual bleeding

Cb Contact bleeding

DUB Dysfunctional uterine bleeding

Mn Mennorhagia

CB Cervical biopsy

H Hystrectomy

Y Presence of feature

N Absence of feature

Sp Specimen type

BG Broder’s grade

HTSCC Histologic subtype of SCC

UF Unifocal invasion

MF Multifocal invasion

p53 Int p53 intensity