CYTOLOGY AND HPV DNA TEST WITH BIOPSY AS GOLD STANDARD.

DIAGNOSTIC VALIDITY OF HUMAN PAPILLOMA VIRUS E6 mRNA ASSAY AS A TRIAGE TEST FOR CERVICAL

CYTOLOGY AND HPV DNA TEST WITH BIOPSY AS GOLD STANDARD.

Dissertation submitted in

partial fulfilment of the requirements for the degree of

M.D. (PATHOLOGY) BRANCH - III

GOSCHEN INSTITUTE OF PATHOLOGY AND ELECTRON MICROSCOPY

MADRAS MEDICAL COLLEGE CHENNAI – 600 003

THE TAMIL NADU

DR. M.G.R. MEDICAL UNIVERSITY CHENNAI

APRIL 2017

CERTIFICATE

This is to certify that this Dissertation entitled “DIAGNOSTIC VALIDITY OF HUMAN PAPILLOMA VIRUS E6 mRNA ASSAY AS A TRIAGE TEST FOR CERVICAL CYTOLOGY AND HPV DNA TEST WITH BIOPSY AS GOLD STANDARD.” is the bonafide original work of Dr.R.VALLABI, in partial fulfillment of the requirement for M.D., (Branch III) in Pathology examination of the Tamilnadu Dr.M.G.R Medical University to be held in April 2017.

Prof. Dr K. RAMA., M.D., Prof. Dr. R.PADMAVATHI., M.D., PROFESSOR OF PATHOLOGY, DIRECTOR I/C & PROFESSOR, Institute of social obstetrics and, Institute of Pathology,

Govt Kasturba Gandhi hospital, Madras Medical College,

Madras Medical College, Chennai – 600003.

Chennai – 600003.

Prof. Dr. M.K.MURALITHARAN, M.S., M.CH.

DEAN,

Madras Medical College and Government General Hospital,

Chennai - 600003

DECLARATION

I, Dr.R.VALLABI, solemnly declare that the dissertation titled

“DIAGNOSTIC VALIDITY OF HUMAN PAPILLOMA VIRUS E6 mRNA ASSAY AS A TRIAGE TEST FOR CERVICAL CYTOLOGY AND HPV DNA TEST WITH BIOPSY AS GOLD STANDARD” is the bonafide work done by me at the Institute of pathology, Madras Medical College under the expert guidance and supervision of Prof. Dr.K.RAMA, M.D., Professor of Pathology, Institute of social obstetrics and Govt Kasturba Gandhi hospital, Madras Medical College. The dissertation is submitted to the Tamilnadu Dr.M.G.R Medical University towards partial fulfillment of requirement for the award of M.D., Degree (Branch III) in Pathology.

Place: Chennai

Date: Dr. R.VALLABI

ACKNOWLEDGEMENT

I express my sincere thanks to Prof.

Dr.M.K.MURALITHARAN, M.S., M.CH Dean, Madras Medical College and Government General Hospital, for permitting me to utilize the facilities of the Institution.

I take the opportunity to express my thanks to Prof.

Dr.R.PADMAVATHI, M.D., Director I/C and Professor, Institute of Pathology, Madras Medical College, Chennai for her keen interest, constant encouragement and valuable suggestions throughout the study.

I am extremely thankful to Prof. Dr.K.RAMA, M.D., Professor of Pathology,Institute of social obstetrics and Govt Kasturba Gandhi Hospital , Madras Medical College, for her valuable suggestions, constant support, advice and encouragements throughout the study.

I am truly thankful to Prof.Dr.GeethaDevadas M.D., D.C.P., Prof.Dr.Padmavathi M.D., Prof.Dr. SudhaVenkatesh M.D., Prof. Dr. M.P Kanchana M.D., Prof.Dr.Rajavelu Indira Prof. Dr.

S.Pappathi M.D., D.C.H., Prof. Dr.Ramamurthy, for their valuable

suggestions and encouragement throughout the study.

I thank the Director of Institute of social obstetrics and Govt Kasturba Gandhi for permitting me to utilize the materials of the institution.

I express my heartfelt sincere thanks to all my Assistant Professors for their help and suggestions during the study.

I would like to thank the Institutional Ethics Committee for approving my study.

On a personal level, I extend my gratitude to my all the members of my family for their constant support

I am thankful to the statistician, for helping me in statistical analysis.

I thank my Friends, Colleagues, Senior Postgratuates, Junior

Postgraduates, Technicians and the Staffs for their continuing support and

helpful advice.

ABBREVIATIONS

WHO - World health organization HPV - human papilloma virus

ASCCP - American society for colposcopy and clinical pathology

OCP - oral contraceptive pills

HPE - Histopathological examination VIA - visual inspection with acetic acid VILI - visual inspection with lugol’s iodine LGL - Low grade lesion

HGL - High grade lesion

ASCUS - Atypical squamous cells of unknown significance LSIL - low grade squamous intraepithelial neoplasia HSIL - high grade squamous intraepithelial neoplasia CIN - cervical intraepithelial neoplasia

SCC - Squamous cell carcinoma

SCC-WD - Squamous cell carcinoma well differentiated SCC-MD - Squamous cell carcinoma moderately

differentiated

SCC-PD - Squamous cell carcinoma poorly differentiated

PCR - Polymerised chain reaction

CONTENTS

S. NO. TITLE PAGE NUMBER

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 24

5 OBSERVATION AND RESULTS 30

6 DISCUSSION 57

7 SUMMARY 72

8 CONCLUSION 76

BIBLIOGRAPHY

ANNEXURES

MASTER CHART

INTRODUCTION

1

INTRODUCTION

Cervical cancer ranks fourth among the leading causes of cancer death worldwide after carcinoma breast, colorectal and lung¹.Cervical cancer accounts for the second most common cancer in developing countries like India in women.

Every year 528000 new cases of carcinoma cervix are diagnosed. Of this one-fifth of cases occur in India¹.The most important etiological factor responsible for development of carcinoma cervix and precursor lesions is chronic Human Papilloma virus (HPV) infection^2. Invasive carcinoma of cervix usually progress from dysplasia over a period of several years. Reproductive age group women with mean age of 38 years are affected which adds to social and economic burden⁽³⁾. Squamous cell carcinoma of cervix accounts for 75-80%⁽⁴⁾ of all carcinomas of cervix.

The cervical squamous cell carcinoma is almost preceded by cervical intraepithelial neoplasia which is having a long latency period, so with screening procedures it is possible to diagnose cervical cancer in its pre-neoplastic stages.

Human papilloma virus (HPV) virus infection is closely associated with carcinoma cervix. Major cause of cervical cancer is identified as high risk human papilloma virus .At a given time about 11.4% of women in general population are predicted to harbor HPV infection and HPV 16 &18 attributes to 70.9% of invasive cervical cancer in the world⁽⁵⁾. Currently we use cytology and visual based methods for screening which are detecting only 15-20% of women who

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actually have abnormality significant enough to need treatment, other 80-85%

were probably false alarms.

HPVDNA testing has already been included in primary cervical screening programs and it significantly sensitive than cytology. But in majority of cases irrespective of age HPV infection is often transient and the cellular abnormalities often disappear and the neoplastic transformation is a rare complication. So screening with HPVDNA lacks specificity.

So it is essential to bring a test in primary screening program which will actually detect this neoplastic transformation. One such promising biomarker is expression of viral oncogene HPV E6 and E7.The viral oncogenes E6 and E7 will bind to the p53 and pRb genes respectively and will initiate the neoplastic transformation of cervical cells. Usually in transient infections these viral oncogenes are expressed at low levels, but they are over expressed when the viral genome integrates with host genome which is the essential step in carcinogenesis of cervical cancer.

Also in various studies it is reported that E6/E7 expression significantly correlated with the severity of cervical lesions. And so in case of potentially progressive high risk HPV infections it will be a promising biomarker. In cervical screening programs E6/E7 mRNA expression will have a great potential for predicting malignancy ⁽⁶⁾

AIMS AND OBJECTIVES

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AIMS AND OBJECTIVES

To assess the expression of HPV DNA in cervical biopsy samples

To assess the expression of HPV E6 mRNA in the cervical biopsy samples

To correlate expression of HPV E6 mRNA with conventional cytology, HPV DNA and cervical biopsy

To validate the expression of HPV E6 mRNA as a triage test for cervical cytology and HPV DNA test with Histopathology as gold standard.

REVIEW OF LITERATURE

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REVIEW OF LITERATURE

ANATOMY:

The cervix in Latin means “neck” which is the lowermost part of uterus, that protrudes in to the upper part of vagina. The cervix measures about 2.5 -3cm in length, in adult nulligravida and is positioned slightly downward and backward ^(7,8).

Cervix is divided in to two portions:

1. Portio vaginalis-the part that is protruding in to the vagina 2. Supravaginalportion: the part that is lying above vaginal vault

Ectocervix or exocervix is the outer part that is protruding into the vagina.

The part related to endocervical canal is the endocervix. The opening of endocervical canal to the ectocervix is the external os and upper limit of endocervical canal is the internal os. The cervix is innervated chiefly in the endocervix & peripheral deeper portions of ectocervix. So the inner two thirds of ectocervix is relatively insensitive to pain.

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HISTOLOGY OF CERVIX

Cervix is made of mixture of fibrous, elastic & muscular tissue which is lined by squamous epithelium & columnar epithelium. Ectocervix is lined by mature non –keratinizing stratified squamous epithelium which has three zones:

1. Germinal /Basal/Parabasal/cell layer-helps in continuous epithelial renewal 2. Mid zone /stratum spinosum-prominent part of cervical epithelium

3. Superficial zone-contains the most mature cells

A single layer of columnar epithelial cells line the endocervix, which also line the underlying glands. These cells have finely granular cytoplasm, filled with mucin and basally placed nuclei. Lymphocytes and Dendritic cells are also seen in the epithelium and stroma of ectocervix and endocervix.

The region between original squamocolumnar junction and the functional/

physiologic/new squamocolumnar junction is the transformation zone. This zone is characterized histologically by the presence of metaplastic epithelium. In epithelisation the mature squamous cells move below the endocervical epithelium and push the endocervical cells off the basement membrane and extension of these process to the clefts.

Squamous metaplasia is the proliferation and differentiation of the endocervical reserve cells to the squamous cells. The reserve cells initially look like parabasal and basal cells. When these cells acquire cytoplasm, they are called

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as immature squamous metaplasia. when these cells acquire glycogen, they are called as mature squamous metaplasia. The transformation zone is the site for all squamous intraepithelial neoplasia

INCIDENCE

The commonest cancer cause of death among women in developing countries is cervical cancer⁽⁹⁾. And it is the second most common cancer of women in reproductive age group⁽¹⁰⁾. Developing and in low, middle income countries 86% of all deaths are due to cervical cancer, hence it is taken as one of the indicator of health inequities⁽¹¹⁾.

In India, 122,844 women are diagnosed with cervical cancer every year and death due the cervical cancer is about 67,477⁽¹²⁾. Total population of women aged older than 15years in India is about 43.2million, who are at risk of developing cancer⁽¹²⁾. Highest age standardized incidence of cervical cancer in south Asia is in India⁽¹²⁾.

In Tamilnadu northeastern districts have a high incidence of cervical cancer which is attributed to infection with human papilloma virus⁽¹³⁾. These regions show high prevalence of HIV infection too ⁽¹⁴⁾. South and southeast Asian countries have high burden of cervical cancer which is due to the high prevalence of HPV infection and inadequate screening ⁽¹⁵⁾.

7 RISK FACTORS:

The risk factors associated with precancerous lesions and carcinoma cervix are as follows:

• Infection with high risk human papilloma virus ¹⁶

• Age at first sexual intercourse

• More than one sexual partner

• Multiparity

• Low socioeconomic status

• Nutritional deficiency

• Immunosuppression

• Sexually transmitted diseases

• Acquired immunodeficiency syndrome

• Smoking¹⁷

• Use of oral contraceptives

• Inadequate screening

The most important risk factors are HPV infection and inadequate screening. Cervical cancer risk is doubled with smoking, OCP use and immunosuppression¹⁷

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Nutritional deficiency especially of micronutrients like folate , vitamin E is associated with increased risk of cervical cancer¹⁸. Genetic susceptibility for cervical cancer is related to HLA class II,HLA B7 and DQB1.

Low socioeconomic status is associated with early sexual intercourse, poor hygienic status and increased comorbid conditions and hence increases the risk of HPV infection. Hence the incidence of cervical cancer is more in developing Countries than developed countries. Thus all the risk are interconnected and their combination further increases the cervical cancer risk but adequate screening at regular intervals and proper treatment at right time can prevent cervical carcinoma.

HPV AND CERVICAL CANCER

Carcinoma cervix is unique among human cancers because it is the first carcinoma found to be directly attributable to the effects of infectious agent. This association was first discovered by Dr.Heraldzurhausen. He was awarded Nobel prize in medicine in 2008 for the same ⁽¹⁹⁾. HPV belong to the family of Papillomaviridae ⁽¹⁶⁾, they are epitheliotropic viruses predominantly infecting skin and mucous membrane. HPV is a small non-enveloped DNA viruses,55nm in diameter. Based on DNA sequencing more than 200 types of HPV were recognized among which 85 are well characterized.⁽¹⁹⁾

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Epidemiologic classification of Human papillomavirus types

Established high risk-16,18,31,33,35,39,45,51,52,56,58 &59 Probably high risk- 26, 53,66,68,73 and 82

Established low risk-6,11,13,40,42,43,44,54,61,70,72,81,and CP6108 Each of the papilloma viruses has genomic organization which is almost similar and it is divided into three functional regions ⁽²⁵⁾. There are three regions in the viral genome such as

Early region – E1, E2, E5, E6, E7 Late region- L1, L2

Upstream regulatory gene or long control region

The transcription of codons in the early region is controlled by LCR which is a noncoding region. Early region codes for proteins required for viral replication and transforming genes like E5,E6 and E7⁽¹⁹⁾.The late region codes for the structural proteins like L1 and L2⁽²²⁾.

For initiation of viral replication E1, an ATP dependent helicase is required in association with E2. E2 is also acts as a transcriptional repressor of E6and E7 proteins.

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Binding of E6 proteins to P53 causes its proteolyticdegradation.P53 induces the cell cycle inhibitor p21 and causes cell cycle arrest at G1/S check point. Thus E6protein causing proteolytic degradation of P53 which inhibits apoptosis and leads to cell cycle progression.

E7 protein by binding with Rb gene product which is a tumour suppressor gene, lead to cell cycle progression in a uncontrolled manner. It also activates cyclin A and cyclin E and inhibits cyclin dependent kinase inhibitorsWAF-1 and which increase cell cycle progression further. So if E6 and E7 protein are over expressed it will lead to uncontrolled cellcycle progression and apoptosis blockage. Late coding region are produced late in the course of infection contains L1 and L2 which are viral capsid proteins^(20,21,22)

Mostly HPV infections begin in the squamo-columnar junction of the transformation zone. It involves the posterior lip than the anterior lip of thecervix.

The lesion involving the exocervical portio are of low grade and that of the endocervical canal are of high grade.

HPV produces two types of infections latent and productive infection

In Latent infection, HPV genome resides freely inside the nucleus in circular form called episome and there will be no replication. There is no morphological changes in the cervical epithelium. The viral Genome can only be detected by molecular methods.

The viral genome replicates independently that of the host genome in the productive infection and produces infectious virions and this occurs in the

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superficial and intermediate layers of the squamous epithelium, because HPVDNA replicates heavily in highly differentiated cells, that is the superficial layer. HPV associated effects in the cells can be seen both grossly and microscopically.

Acanthosis, nuclear atypia, multinucleation, kiolocytosis, cytoplasmic vacoulisation are some characteristic microscopic changes.

Precursor lesions of cancer cervix

Cervical pre-neoplastic lesions are initially classified as mild, moderate and severe Dysplasia. Later WHO classified them as CIN 1, CIN2 and CIN3.

Now the Bethesda system classifies them as low grade intraepithelial neoplasia (CIN1) and high grade intraepithelial neoplasia(CIN 2&3).

The term cervical intraepithelial neoplasia was introduced by Richart,who said that all precursor lesions are monoclonal proliferations ⁽²⁶⁾.Mild dysplasia corresponds to CIN 1,moderate and severe corresponds to CIN 2 and CIN 3 respectively.

According to ZurHausen et al infection with high risk types leads to precursor lesion and persistence of infection leads to invasive cancer^(27,28). thus HPV types are correlated with different types of CIN, with CIN 1 indicating a self-limited sexually transmitted infection and CIN 2 –CIN3 being the only true cervical cancer precursor⁽²⁹⁾.

CIN 1 or the productive viral infection have high viral load and exhibit cytological changes like koilocytic changes. They produce condylomas and flat

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lesions, have a common ploidy status^(30,31) which often self- limiting even without treatment.57% of CIN 1 cases regress spontaneously without treatment and have potential to progress to invasive 11% only progress to carcinoma.CIN 2 and CIN 3 lesions show dysplastic changes squamous cell carcinoma if not treated ⁽³²⁾. According to Sptizer et al mild to moderate dysplasia progression was 1% per year, moderate to severe dysplasia progression was 16% within 2years and 25%

within 5years. So we can prevent the progression of precancerous lesion by early detection and treatment of HPV ⁽³³⁾.

Arends et al conducted a study in which approximate CIN 1likelihood of regression is 60%, persistence is 30%, progression to CIN 3 is 10% and progression to invasive cancer is 1%. similarly for CIN 2 are 40%, 40%, 20% and 5%.and like hood of CIN 3 going for regression is 33% and progression to invasive cancer is 12%⁽³⁵⁾.

According to Sherman et al,93% of tumours expressed HPV DNA.HPV 16 was found in 50%,HPV 14 in 14%, HPV 45 in 8%,HPV 31 in 5% of specimens.

HPV 16 predominated in squamous cell carcinomas. HPV 18 predominated in adenocarcinomas and adenosquamous carcinomas ⁽³⁴⁾.

Pathology of precursor lesions CIN lesions are graded as follows

CIN 1-neoplastic cells will occupy lower one third of the epithelium

CIN 2-Neoplastic cells occupying lower one third to twothird of the epithelium

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CIN 3-Neoplastic cells occupying two third to full thickness of the epithelium Neoplastic cells will show the following features like

• Increased N/C ratio

• Increased nuclear membrane

• Nuclear pleomorphism

• Nuclear hyperchromatism

• Coarse chromatin

LOW GRADE SQUAMOUS INTRAEPITHELIAL LESION(LSIL) LSIL will have following features

• Nuclear atypia in the lower one third of epithelium

• Koilocytic changes like cytoplasmic vacuolation, bi and multinucleation

• Architectural changes like papillomatosis and acanthosis

Infection with trichomonas and candidiasis also show koilocytic changes but they are not associated with nuclear atypia

HIGH GRADE SQUAMOUS INTREPITHELIAL LESION (HSIL) HSIL features include the following

• Nuclear atypia in more than one third to full thickness of the epithelium

• Nuclear crowding

• Loss of polarity

• Increased nuclear pleomorphism

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• Anisonucleosis

• Indistinct cell borders

• Abnormal mitotic figures

Reparative and atrophic changes resemble HSIL HSIL can be managed colposcopically using cryosurgery, laser ablation and loop electrosurgical excision procedure, thus screening becomes essential for preventing progression of these to invasive carcinomas.

INVASIVE SQUAMOUS CELL CARCINOMA

Carcinoma cervix most commonly affects elderly women more than 40’s that is 20 years older than those affected by intraepithelial lesions⁽³⁶⁾ the most common risk factor is infection with HPV and the most common types are HPV 16 and 18. Initial HPV infection and development of carcinoma cervix takes about 10years^(37).

According to WHO classification, carcinoma cervix is divided into three categories as follows,

• Squamous cell carcinoma

• Adenocarcinoma

• Other epithelial tumours

Squamous cell carcinoma accounts for more than 70-80% of cases of all the types. Squamous cell carcinoma is further divided into following types,

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• Microinvasive squamous cell carcinoma

• Invasive squamous cell carcinoma

• Keratinizing

• Non keratinizing

• Verrucous

• Papillary

• Warty

• Basaloid

• Squamo-transitional

• Lymphoepithelioma like carcinoma

There is usually a very long latency period between initial HPV infection and development of full fledged cervical cancer. Hence adequate screening and appropriate treatment at the initial stages can prevent the progression of precursor lesions to cervical cancer

SCREENING:

Definition and principle

Identifying specific diseases or condition among asymptomatic individuals⁽³⁸⁾. So screening test have to be applied on large population and hence should be convenient, inexpensive, safe and painless⁽³⁹⁾. Higher margin of error

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than the diagnostic tests and less accuracy are the main disadvantage of screening test.

This table demonstrates the relationship of screening test parameters and screened disease and formula for calculating parameters of screening test^(40,41)

Sensitivity= proportions of persons with disease in whom the screening test is also positive (A/A+C)

Specificity=proportions of those without the disease in whom the screening test is also negative (D/D+C)

Positive predictive value=probability of disease in subjects with a positive test result (A/A+B)

Negative predictive value=probability of absence of the disease in subjects with a negative test result (C/C+D)

Efficacy of screening test means that the test should be able to diagnose a disease earlier that it would be without screening and with sufficient accuracy that the screening will produce less false positive and false negative results.

Effectiveness of early detection by screening test means that the person with disease who are diagnosed early should have a better clinical outcome than

Screening test Disease present Disease absent positive True positive(A) False positive(B) negative False negative(C) True negative(D)

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those persons who are diagnosed without screening ⁽⁴²⁾. Screening service offered should not do any harm such as wrong diagnosis. False positive screening test result, treatment will do more harm than good. It will also cause unnecessary anxiety among healthy people and these persons may be exposed to risks by exposing the screened persons for further examination ^(39,41). False negative screening test will give false assurance of not having the disease. The screening test is useful if only the death of patient due to disease is postponded after early diagnosis. The objective of screening test is to reduce the mortality and morbidity, and improving quality of persons in population ⁽⁴²⁾.

According to the article presented in the American journal of clinical pathology on the screening guidelines for cervical cancer the following recommendations were proposed according to evidence based systemic review⁽⁴³⁾ (ANNEXURE II).

Cervical cancer screening method

For cervical cancer to be controlled there are two modalities one is primary prevention and the other is secondary prevention. Primary prevention is the population based approach to reduce the risk of development of cervical cancer by behavioral intervention like sexual and health care seeking behavior or mass immunization against high risk HPV infection. Screening for precancerous lesion and treating them is the secondary prevention.

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Inadequate screening is most important contributor to cervical cancer in current scenario. Cancer screening must be made done to all women past 21 years to bring down its incidence especially in developing countries

The objectives of screening are as follows,

• To prevent morbidity and mortality of cervical cancer

• To prevent over management of precursor lesions that in most cases regress The screening programs for cervical cancer commonly used in developing countries includes

• Cytology based screening

• Visual approaches to screening

• HPV Screening

Cervical carcinogenesis involves three steps , the HPV infection, progression to high grade preinvasive lesion and invasion⁽⁴⁴⁾. Cytological abnormalities in most of the lesions are transient and will return to normal with HPV DNA negativity. For HSIL to develop in to invasive cancer it takes about 10 years.

Latent infection results from viral infection of mitotically active epithelial cell. Morphology of these cells appear normal and hence the cells appear normal in cytology and in visual techniques. So at this stage the only modality to detect the lesion is through HPV DNA testing and E6/E7 mRNA expression through molecular techniques ⁽⁴⁵⁾.

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Cytologic and histologic changes become to appear only at the stage of productive viral replication which is referred as LSIL. Accuminata/flat warts manifest in the replicative viral infection which are detected by colposcopy. Only at the stage of productive viral infection our cytology and visual methods becomes operative.

If the viral clearance does not take place and the infection is going to persist, which usually happens in high risk virus groups, there will be integration of viral genome with host genome, this stages can be picked up with HPV E6/E7 mRNA detection methods which will detect this viral integration and thus will help in predicting the progression to invasive cancers

Visual inspection with acetic acid is used as mass screening programs in low income countries like India in which 3-5% solution of acetic acid is applied to cervix and nuclear dense lesions can be seen as acetowhiteareas. According to sankarnarayan et al who conducted study on this test in India found that specificity was 82% (range 64-90%) and the sensitivity was 84%(66-96%)and it was having a high false positive rate⁽⁴⁶⁾.

For more than 60 years of cervical cancer screening, the papanicolaou smear(PAP), the cervical cytology, remained the main stay of screening because of its simplicity and low cost. However an abnormal cytology report requires confirmation by biopsy. Agreement is about 50% for cytology and histology diagnosis.

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Soost HJ et al in his study on validation of cytology results has reported that sensitivity was 78% (30-87%) and the specificity was 62%(61-94%)⁽⁴⁷⁾. There are several potential sources of error in pap smear screening, like we may not sample the lesion, sometimes the abnormal cells may not transfer from applicator to slide, inadequate preservation of cells or reading error may occur.

Also the cytology slides interpretation is subjected to inter observer variability. It is also less sensitive for glandular lesions than the squamous lesions. Cytology sensitivities varies between countries and the lab.

Originally HPV DNA testing was reflexly used to help the triage of atypical pap smears to colposcopy or follow up. ASCUS-LSIL triage study group 2003 recommended HPV DNA testing to resolve uncertain cytologies ^(48,49). But the American congress of obstetrician and gynecologists said not to do HPV testing if there is gross cervical lesion/severely abnormal cytology result ⁽⁵⁰⁾. Following cytology HPV testing was used in the screening of asymptomatic women with visually normal cervix to predict future risk of HPV.

HPV TESTING

Detection of HPV is a consistent feature of cervical cancer^(51).The sensitivity of HPV DNA testing is 68.2% and specificity is 91.6%⁽⁵¹⁾. In women under 30years, HPV infection is transient and regresses. According to ASCCP guidelines 2012, HPV testing is done only after 30 years.

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The molecular techniques currently used are as follows,

• Amplified methods Signal amplification

The hybrid capture 2 HPV DNA assay Target amplification

Polymerase chain reaction

• Non amplified methods

• The southern blot hybridisation

International HPV screening study group and Ronco et al in their summary of four European randomized trials they concluded that HPV DNA testing provided 60-70% greater protection against invasive cervical cancer over 6.5 years of follow up. Each of the four studies compared cytology with HPV testing with cytology alone. ^(52,53)

WHO has guidelines for cervical cancer screening ⁽⁵⁴⁾ mainly focuses on women aged 30-35 years. It recommends HPV testing as a primary screening.

WHO recommends immediate treatment with cryo/loop electrical excision if high risk HPV DNA is detected. According to American Cancer Society 2012 guidelines, screening should start at the age of 21 years with cytology alone which is performed every three years. If the women aged 30-65years, cytologic screening every three years or cytology and HPV co-testing every five years should be done⁽⁵⁵⁾. Screening discontinued after 65 years if the women had three negative pap and HPV tests in the preceding 10 years. Women treated for preinvasive

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lesion should continue screening annually for 20 years. Thus the negative test of HPV have a protective effect twice that of the negative cytology report. But specificity of HPV DNA test appears to be less.

HPV E6/E7 mRNA assays

Role of cervical cytology will become more limited in the near future as the HPV test may influence the subjective reading of cytology. The valuable tool will be the one which will indicate the productive phase of high risk HPV to the transformation phase, one such promising marker is detection of E6/E7 mRNA transcripts. Persistent high risk HPV infection and integration of the HPV genome into the host chromosomes lead to loss or disruption of E2 and subsequent up regulation of E6 and E7 expression is associated with progression to high grade intraepithelial lesion and invasive carcinomas^(20,21,22).The malignant transformation requires expression of viral oncogenes E6 and E7. They mediate degradation of the tumor suppressors p53 and pRb and thus cell cycle regulation is interfered .Low risk types E6 and E7 proteins are less competent in interfering with p53 and pRb functions than E6/E7 proteins from high risk types^(23,24). Therefore, low risk HPV infections are associated with benign proliferations, such as genital warts and lowgrade intraepithelial lesions prone to regress.

In high grade lesions parabasal cells highly express E6/E7 oncogenes, so detection of these E6/E7 mRNA transcript provides direct indication of integrated

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HPV /modified episomal HPV and high oncogenic activity. Different methods of detecting mRNA transcripts includes amplification technique which includes isothermal such as NASBA, transcription mediated and signal mediated and also isothermal solution phase amplification

Leone et al describes real time NASBA amplification using molecular becons probes is the most preferred technique, Nuclei acids research 1998,vol 26,2150-2155 .Some screening methods will preferably detect E6mRNA from cancer associated HPV TYPES 16,18,31, and 33 and preferably 45.These methods will specifically detect mRNA transcripts which contain all or part of the E6 open reading frame, including naturally occurring splice variants and transcripts which contain all or part of E7 open reading frame.

Different Cross sectional studies have shown that mRNA testing correlates better with the severity of lesion and seems to be more appropriate for risk evaluation than HPV DNA testing^{(57-70) .}Soltlar et al showed increasing prevalence of E6/E7 transcripts whenever lesions severity are higher (95% with CIN III)⁽⁶⁸⁾.

Krus et al showed overall prevalence of HPV 97%,mRNA test was positive in 92%⁽⁷¹⁾.Castle et al showed in 540 women selected for screening mRNA test correlated with severity of lesion, more than 90% of CIN III cases and all carcinomas tested positive⁽⁶⁹⁾

MATERIALS AND METHODS

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MATERIALS AND METHODS

This is a prospective study conducted at the Institute of social obstetrics and Kasturba Gandhi hospital for women and children, Madras medical college between November 2015 and July 2016

This study involved 41 women who have been diagnosed with positive VIA / VILI in colposcopy are subjected to conventional cytology and cervical punch biopsy. The part of the biopsy sample is collected in RNA Later solution for HPV DNA testing and HPV E6 mRNA expression. The results of above test are compared with histopathology as gold standard

INCLUSION CRITERIA:

1. Women in the age group of 21-65 2. Non pregnant women

3. VIA/VILI Positive

4. Women without prior treatment for cervical pre-neoplastic and neoplastic lesions

EXCLUSION CRITERIA:

1. Pregnant women 2. Menstruating women 3. Intra Uterine Device users

4. Women who have undergone prior treatment for cervical neoplastic and pre-neoplastic lesions

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After obtaining informed consent, detailed history was obtained (ANNEXURE III). After complete physical examination, specimens- cervical scrapings and cervical punch biopsy were collected.

Conventional PAP smear:

To collect samples for conventional PAP, Ayers spatula was inserted in to cervix and gently rotated at 360 degree and sample is smeared into a grease free slide and fixed in alcohol. After fixation, smear was stained with PAP stain (ANNEXURE IV). The PAP smears were examined and reported according to 2001 Bethesda system of classification (ANNEXURE V).

Cervical biopsy:

Cervical biopsy were taken using punch biopsy forceps and put in 10%

neutral buffered formalin and sent for Histopathological examination. After histopathologic processing, the tissue sections were stained with haematoxylin and eosin (ANNEXUREIV). The reports are given as

Normal

Chronic non-specific cervicitis CIN I

CIN II CIN III

26 Invasive squamous cell carcinoma

Well differentiated Moderately differentiated Poorly differentiated Adenocarcinoma

HPV DNA Typing and HPV E6 mRNA Expression:

The tissues were collected in RNA later solution and transported by cold storage to laboratory and kept at 4⁰c for overnight, the excess RNA later solution drained and tissues were frozen at -80⁰C.

DNA Isolation from tissues:

Tissues were washed thrice with Phosphate buffer saline (PBS) buffer.

Approximately 30 mg of tissue were minced and homogenized in tissue lysis buffer. Proteinase K was added to the homogenate and incubated at 37°C overnight. After incubation, equal volume of P:C:I (Phenol/ choloroform/

isoamylalcolhol) was added to the lysate and centrifuged at 10,000 rpm at 25°C for 25 min. The aqueous phase was transferred into a fresh tube and 2.5 volume of 100% ethanol was added to it to precipitate DNA. DNA pellet was transferred to Eppendorf tube, containing 70% Ethanol and centrifuged at 10,000 rpm for 15 min

27

at 4°C. DNA pellet was air dried and dissolved in appropriate volume of 1X Tris EDTA.

RNA isolation from tissue:

Total RNA was isolated from the tissue samples by RNeasy mini kit (Qiagen®) as per manufacturer’s protocol. The RNA samples were quantified using Nano drop (Thermo Scientific, USA) and the integrity of the RNA samples were checked by 1 % agarose gel electrophoresis and stored at -80°C until further use.

cDNA conversion:

A 20 µl volume containing 5 µg of total RNA, 1x buffer, 100 µM random primers, 0.1M DTT, 100mM dNTP and 200 Units of Superscript III (Invitrogen, USA) was used for cDNA synthesis, with the following thermal conditions: RNA was pre incubated with random hexamers for 20min at 65˚C followed by 90 min at 50˚C and 15 min at 72˚C. Further, a 1:25 dilution was prepared from the 20 µl stock cDNA and stored at -20°C.

28 Polymerase Chain Reaction (PCR):

Polymerase Chain reaction was performed to find out the presence of HPV 16 and 18 in cervical cancer tissue samples and β-Globin gene was used as positive control. PCR was carried out in a total volume of 10 µL using 1X PCR buffer, 1.5 mM MgCl₂, 100 µM dNTPs, 60 nM each of HPV16 and HPV18 specific forward and reverse primers, 0.5 Units of Taq polymerase and 100ng/Rxn tissue DNA. The thermal cycling conditions were 94°C for 10 min; 40 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec followed 72°C for 7 min and 4°C hold. The primers used for the analysis are HPV 16 E6 Fwd - 5’

CTGCAATGTTTCAGGACCCA 3’, HPV 16 E6 Rvs-5’

TCATGTATAGTTGTTTGC AGCTCTGT 3’; HPV 18 Fwd - 5’

AAACCGTTGAATCCAGCAGAA 3’, HPV 18 Rvs -5’

GTCGTTCCTGTCGTGCTCG 3’ and β-Globin Fwd - 5’

GGATCTGTCCACTCCTGATGCTG 3’, Rvs - 5’

TCACTCAGTGTGGCAAAGGTGC 3’.

Expression analysis:

Expression of HPV 16 E6 mRNA was quantified by Custom designed Taqman Assay using ABI Quantstudio6 with primers specifically amplifying HPV16 and 18 E6. PCR reaction was carried in a total volume of 10 µl containing 0.5 µl of 20X Primer-Probe mix (1X), 5 µl of 2X Universal Master Mix (1X), Applied Biosystem, USA and 1 µl of 1:25 cDNA with 3.5 µl H₂O. Thermal

29

protocol followed was: 50°C for 2 min, 95°C for 10min, 40 cycles of 95°C for 15 sec and 60°C for 60 sec.

The presence of HPV DNA 16 and 18 is reported as present or absent.The results of HPV E6 mRNA expression is given as amplification plot ,which is then computed to numerical expression with special software.

STASTISTICAL ANALYSIS

The information collected from various screening methods like cytology, HPVDNA and HPV E6 mRNA were recorded .Statistical analysis were done with SPSS 20 version. Comparative analysis were done with Chi-Square test and P values less than 0.05 were considered statistically significant.

The P value was calculated for conventional cytology, HPV DNA, HPV E6 mRNA in cervical non- neoplastic, preneoplastic and neoplastic lesions, showing their statistical significance in correlation with biopsy. The sensitivity, specificity, positive predictive value and negative predictive value were also calculated and they were compared with other similar methods.

The incidence and distribution of cervical non-neoplastic, preneoplastic and neoplastic lesions in relation to various clinicopathological factors likeage, menstrual status, quadrant of cervix involved were also studied.

OBSERVATIONS AND RESULTS

30

OBSERVATION AND RESULTS

This was a prospective study conducted in the Institute of Social Obstetrics and Govt. Kasturba Gandhi Hospital for Women and Children, Madras Medical College, between November 2015 and July 2016

Epidemiology of cervical non neoplastic, pre-neoplastic and neoplastic lesions in our institute (November 2015 to July 2016)

A total of 304 cervical biopsies were reported as cervical non neoplastic preneoplastic and neoplastic lesion by HPE. Of 304 cases, 208 (67.6%) were chronic cervicitis, 61(20.8%) were CIN I,3 (1%) was CIN II,5(1.5%) were CIN III and 27(8.9%) were malignancies (table 1 &chart 1).

TABLE 1

DISTRIBUTION OF CERVICAL NON-NEOPLASTIC, PRENEOPLASTIC AND NEOPLASTIC LESIONS

n=304

BIOPSY CERVICITIS CIN I CIN II CIN III MALIGNANCY

FREQUENCY 208 61 3 5 27

PERCENTAGE 67.6% 20.8% 1% 1.6% 8.9%

31

The distribution of subtypes of Squamous cell carcinoma and Adenocarcinoma among the malignant cases is given in table 2 .SCC-WD constitutes 14.8%, SCC-MD constitutes 59.2%, SCC-PD constitutes 14.8% and adenocarcinoma constitutes 11.1%

TABLE 2

DISTRIBUTION OF MALIGNANT CERVICAL LESIONS

n=27

BIOPSY SCC-WD SCC-MD SCC-PD ADENOCA

FREQUENCY 4 16 4 3

PERCENTAGE 14.8% 59.2% 14.8% 11.1%

Age wise distribution of cervical non neoplastic, preneoplasticand neoplastic lesions is given in table 3 and chart 2.All CIN lesions are common in the 31-40 year age group. All malignant lesions are equally common in the 41-50 and 51-60 year age group. Chronic cervicitis was common in 31-40 year age group.

32 TABLE 3

AGE WISE DISTRIBUTION OF CERVICAL NON-NEOPLASTIC, PRENEOPLASTIC AND NEOPLASTIC

n=304

BIOPSY

AGE(YEARS)

21-30 31-40 41-50 51-60 61-70

CERVICITIS- 208

37(17.8%) 76(36.5%) 59(28.4%) 26(12.5%) 10(4.8%)

CIN I-61 12(19.6%) 29(47.5%) 16(26.2%) 1(1.6%) 3(4.9%)

CIN II-3 0 3(100%) 0 0 0

CIN III-5 0 4(80%) 0 1(20%) 0

MALIGN-27 0 2(7.4%) 9(33.3%) 9(33.3%) 7(25.9%)

Chi-Square=117.180** P<0.001

The distribution of menstrual status in cervical non-neoplastic,preneoplastic and neoplastic lesions is given in table 4 and chart 3.More than 70% of lesion of cervicitis and CIN occur in the reproductive age group. Most of the malignant lesions occur in post-menopausal age group.

33 TABLE 4

DISTRIBUTION OF CERVICAL NON NEOPLASTIC, PRENEOPLASTIC AND NEOPLASTIC LESIONS-MENSTRUAL STATEWISE

n=304

BIOPSY

MENSTRUAL STATUS

REPRODUCTIVE POST MENOPAUSAL

CERVICITIS -208 165(79.3%) 43(20.7%)

CIN I-61 51(83.6%) 10(16.4%)

CIN II-3 3(100%) 0

CIN II-5 3(60.0%) 2(40%)

SCC-24 6(25%) 18(75%)

ADENO CA-3 1(33.3%) 2(66.7%)

Chi-Square=41.211* P<0.001

The distribution of lesions according to quadrant is given in table 5 and char 4, most of the lesions are seen around 3-6 ^o clock position. All invasive carcinomas were seen circumferentially abutting the os

34 TABLE 5

DISTRIBUTION OF LESIONS IN CERVICAL BIOPSIES-QUADRANT OF CERVIX INVOLVED WISE

LESIONS

QUADRANT

12-3 3-6 6-9 9-12 CIRCUM

FREQUENCY 37 90 70 78 29

PERCENTAGE 12.2% 29.6% 23% 25.7% 9.5%

CHARACTERISTIC OF CURRENT STUDY GROUP

41 VIA/VILI positive cases are randomly selected from the patients attending the colposcopy department for our study. Of these 25 were low grade lesion, 5 were high grade lesion, and 11 were invasive carcinomas (table 6 and chart 5)

TABLE-6

DISTRIBUTION OF VIA/VILI DIAGNOSIS IN THE STUDY GROUP

n=41

VIA/VILI LGL HGL INV CA

FREQUENCY 25 5 11

PERCENTAGE 61% 12.2% 26.8%

35

The 41 patients were subjected to conventional cytology, cervical biopsy HPV DNA typing and HPV E6 mRNA assay. The biopsy report was the confirmatory diagnosis. So it was taken as the gold standard. Of the 41 VIA/VILI positive cases biopsy turned out to be chronic cervicitis 10, CIN I-15,CINII-3,CIN III-2,SCC-11(table 7 and chart 6)

TABLE 7 BIOPSY RESULTS n=41

Frequency Percentage (%)

CHRONIC CERVICITIS 10 24.4

CIN I 15 36.6

CIN II 3 7.3

CIN III 2 4.9

SCC- 11 26.8

Total 41 100.0

In this study group, the age wise distribution of biopsy diagnosis is as follows, majority of patients with chronic cervicitis were equal in the age group 31-40 & 41-50years. CIN lesions were more in age group 31-40. SCC are predominantly above 40years (TABLE 8)

36 TABLE 8

AGE WISE DISTRIBUTION OF BIOPSY IN THE STUDY GROUP

n=41, % with in age group

BIOPSY

AGE IN YEARS

21-30 31-40 41-50 51-60 61-70

CERVICITIS 1(30%) 4(28.6%) 2(28.6%) 2(30%) 1(16.7%) CIN I 3(70%) 5(35.3%) 3(39.7%) 2(30%) 2(33.3%)

CIN II 0 2(11.1%) 1(10.3%) 0 0

CIN III 0 2(11.1%) 0 0 0

SCC 0 1(14.0%) 3(21.4%) 4(40%) 3(50%)

Majority of cervical nonneoplastic and preneoplastic lesions were commonly seen in reproductive age group women in the study .But all the squamous cell carcinomas were seen in the postmenopausal women (table 9

&chart 7)

37 TABLE 9

DISTRIBUTION OF BIOPSY RESULTS IN THE STUDY GROUP- MENSTRUAL STATUSWISE

n=41

BIOPSY

MENSTRUAL STATUS

REPRODUCTIVE POST-MENOPAUSAL

CERVICITIS 9(31%) 1(8.3%)

CIN I 14(48.3%) 1(8.3%)

CIN II 2(6.9 %) 1(8.3%)

CIN III 1(3.4%) 1(8.3%)

SCC 3(10.3%) 8(66.7%)

Pearson Chi-Square=15.969* P=0.003

The distribution of cervical nonneoplastic, preneoplastic and neoplastic lesions according to quadrant of cervix involved is given in table 10 and chart 8.Most of the lesions of chronic cervicitis were found in 9-12 o^, clock position.

CIN I lesions were found in 12-3 o^, clock position. In CIN II and CIN III in 9-12 and 3-6 positions respectively .and all SCC were seen abutting the os.

38 TABLE 10

DISTRIBUTION OF BIOPSY RESULTS IN THE STUDY GROUP- QUADRANT OF CERVIX INVOLVED WISE

n=41

BIOPSY

QUADRANT OF CERVIX INVOLVED (O^, Clockwise)

12-3 3-6 6-9 9-12 circumferencial

cervicitis 1(14.3%) 1(16.7%) 1(25.)% 6(54.5%) 1(7.7%)

CIN I 5(71.4%) 3(50%) 3(75%) 3(27.5%) 1(7.7%)

CIN II 1(14.3%) 0 0 2(18.2%) 0

CIN III 0 2(33.3%) 0 0 0

SCC 0 0 0 0 11(84.6%)

Pearson Chi-Square=52.767* P<0.001

Of the 25 low grade lesions of VIA/VILI ,8 turned out to be cervicitis,15 turned out to be CIN I,2 turned out to be CIN II. So8 were false negative by VIA VILI.5 high grade lesions 2 were cervicitis,1 was CIN II ,2 were CIN III.SO 2 false negative. All the invasive carcinomas were reported as squamous cell carcinoma in biopsy. This is given in table 11 and chart 9.Concordance with biopsy is good in case of high grade lesion and invasive carcinoma.

39 TABLE 11

COMPARISON OF VIA/VILI WITH BIOPSY DIAGNOSIS n=41

VIA/VILI

BIOPSY

CERVICITIS CIN I CIN II CIN III SCC

LGL=25 8(32.%) 15(60%) 2(8%) 0 0

HGL=5 2(40.0%) 0 1(20%) 2(40%) 0

INV CA= 0 0 0 0 11(100%)

Pearson Chi-Square=59.696* P<0.001

These patients were subjected to conventional cytology. The results were given as NIL-9,ASCUS-1,LSIL-18,HSIL-2,POSITIVE FOR MALIGNANCY- 11.(table 12&chart 10)

TABLE 12

CYTOLOGY RESULTS n=41

CYTOLOGY Frequency Percentage

(%)

ASCUS 1 2.4

HSIL 3 7.3

LSIL 18 43.9

NIL 8 19.51

POSITIVE FOR MALINGNANCY 11 26.8

Total 41 100.0

40

Of the 25 low grade lesion in VIA/VILI, 7 were reported as NIL,1 was reported as ASCUS, 17 reported as LSIL. OF the 5 high grade lesions 1were reported as NIL, 1reported as LSIL and 3 reported as HSIL. All the invasive carcinoma reported as SCC in cytology. Concordance of VIA/VILI and cytology was 100% in invasive carcinomas only. (table 13 and chart 11)

TABLE 13

COMPARISON OF CYTOLOGY WITH VIA/VILI n=41,

VIA/VILI

CYTOLOGY

NIL ASCUS LSIL HSIL SCC

LGL=25 7(77.8%) 1(100%) 17(94.4%) 0 0

HGL=5 1(22.2%) 0 1(5.6%) 3(100%) 0

INVASIVE CA=11 0 0 0 0 11(100%)

Pearson Chi-Square=57.400* P<0.001

The 8 NIL cases turned out to be cervicitis in biopy,18 LSIL cases 14 tuned out to be CIN I,1 to be cervicitis, 1 to be CIN III,2 to be CIN II.1 ASCUS turned out to be CIN I,3 of the HSIL turned out to be CIN II&CIN III . AND all the 11 POSITIVE FOR MALIGNANCY turned out to be SCC only. The concordance of cytology with biopsy 100% in SCC and HSIL, but in LSIL it was less. (table 14 & chart 12)

41 TABLE 14

COMPARISON OF CYTOLOGY WITH BIOPSY

n=41,

LBC

BIOPSY DIAGNOSIS

CERVICITIS CIN I CIN II CIN III SCC

NIL 8(90.%) 0 0 0 0

ASCUS 0 1(6.7%) 0 0 0

LSIL 1(10%) 14(93.3%) 2(66.7%) 1(25%) 0

HSIL 0 0 1(33.3%) 2(75%) 0

SCC 0 0 0 0 11(100%)

Pearson Chi-Square=90.883* P<0.001

Presence of HPV16 & HPV18 E6 DNA in Cervical cancer samples:

Presence of HPV DNA in cervical cancer tissue was confirmed by PCR reaction. Below shown are the representative gel pictures of HPV16 E6, HPV18 E6 and β-Globins amplification respectively.

a) Amplicon of HPV16 E6 DNA in Lane 2, 8,9,13.

Lane 6: 100 bp ladder

42

HPV DNA RESULT

Presence of HPV16 & HPV18 E6 DNA in Cervical cancer samples:

Presence of HPV DNA in cervical cancer tissue was confirmed by PCR reaction. Below shown are the representative gel pictures of HPV16 E6, HPV18

amplification respectively.

Amplicon of HPV16 E6 DNA in Lane 2, 8,9,13.

Lane 6: 100 bp ladder

Presence of HPV16 & HPV18 E6 DNA in Cervical cancer samples:

Presence of HPV DNA in cervical cancer tissue was confirmed by PCR reaction. Below shown are the representative gel pictures of HPV16 E6, HPV18

Amplicon of HPV16 E6 DNA in Lane 2, 8,9,13.

78bp

43

b) Amplicon of HPV18 E6 DNA in Lane 3.

Lane 1: 100 bp ladder

c) Amplicon of β-Globin in Lane 2-9.

Lane 1: 100 bp ladder

121bp

132bp

44

Among 41 cervical cancer samples, 18 samples were HPV 16 positive and 2 was HPV 18 positive. Percentage of samples positive for HPV DNA 16 and 18 was 48.78%.among the 20 cases which are positive HPV 16 was found to be positive in 80% Of cases, Only 20 % of cases showed positivity for HPV 18 .HPV 18 Positive cases were found to be CIN I in biopsy.HPV 16 was positive in I cervicitis,5 CIN I,2 cases of CIN II and CIN III,10 of SCC(table 15,chart 13 )

TABLE 15

DISTRIBUTION OF HPV DNA 16 &18 IN RELATION TO BIOPSY

HPV 16&18

BIOPSY DIAGNOSIS

CERVICITIS CIN I CIN II CIN III SCC

POSITIVE 1(5.6%) 5(16.6%) 2(11.1%) 2(11.1%) 10(55.6%) 20

NEGATIVE 9(39.9%) 10(51.5%) 1(4.3%) 0 1(4.3%) 21

TOTAL 10(24.4%) 15(36.6%) 3(7.3%) 2(4.9%) 11(26.8%) 41 Pearson Chi-Square = 21.203* p<0.001

HPV DNA 16 was totally positive in 43.9% of cases and HPV DNA 18 was positive in 4.9% 0f cases

45 HPV_DNA16

Frequency Percent

NEGATIVE 23 56.1

POSITIVE 18 43.9

Total 41 100.0

HPV_DNA18

Frequency Percent

NEGATIVE 39 95.1

POSITIVE 2 4.9

Total 41 100.0

Among 8 cases 0f NIL reported in cytology HPV DNA was Positive in 1case, 1 case of ASCUS was negative for HPV DNA, Of 17 LSIL cases HPV was Positive in 7 cases,3 of the HSIL cases were positive for HPV DNA,among 11 SCC only one was negative for HPV DNA(table 16,chart 14)

46 TABLE-16

COMPARISON OF CYTOLOGY WITH HPV DNA

CYTOLOGY

HPV DNA

NEGATIVE POSITIVE

NIL 8 (87.5%) 1(12.5%)

ASCUS 1 (100%) 0

LSIL 12 (70.5%) 6(29.4%)

HSIL 0 3(100.0%)

SCC 1(10%) 10(90.0%)

HPV E6 mRNA expression RESULTS:

Below are the amplification plots showing amplification curve of HPV16 E6 mRNA and GAPDH (glyceraldehydes 3 phosphate dehrogenase) (Reference gene).

AMPLIFICATION PLOT FOR E6 mRNA

47

AMPLIFICATION PLOT FOR GAPDH

These graphical values are converted to numerical values with special software.

Among 10,chronic cervicitis case HPV E6 mRNA was positive in 1,5 0f CIN I cases were positive,1 0f both CIN II and CIN III was positive, except one case of SCC all Were positive for HPV E6 mRNA.(TABLE17,CHART 15)

48 TABLE-17

DISTRIBUTION OF HPV E6 mRNA EXPRESSION IN RELATION WITH BIOPSY

n=41 HPV E6

mRNA

BIOPSY DIAGNOSIS TOTAL

CERVICITIS CIN I CIN II&III SCC

POSITIVE 1(10%) 5(33.3%) 2(40.0%) 10(90.9%) 18(43.9%) NEGATIVE 9(90%) 10(66.7%) 3(60.0%) 1(9.1%) 23(56.1%)

TOTAL 10 15 5 11 41

Among 9 NIL cases HPV E6 mRNA was positive in 1case,among 17 LSIL cases HPV E6 mRNA was positive in 5cases,one case of ASCUS was negative for HPV E6 mRNA, among 3 HSIL cases 2 was posiive,10 of the 11 SCC were positive for HPV E6 mRNA shown in (table 18 and chart 16)

49 TABLE-18

COMPARISON OF CYTOLOGY WITH HPV E6 mRNA

n=41

CYTOLOGY

Total POSITIVE FOR

MALINGNANCY ASCUS HSIL LSIL NIL

POSITIVE 10

(90.9%)

0 0.0%

2

(66.6)% 5 (29.4)% 1 (11.1%)

18 (43)%

NEGATIVE 1

(9.1%)

1 (100.0%)

1 (33.3%)

12 (70.6%)

7 (88.9%)

23 (56.0%)

Total 11

(100.0%)

1 (100.0%)

3 (100.0%)

17 (100.0%)

9 (100.0%)

41 (100.0%) Among 20 of the HPV DNA positive cases only 18 were positive for HPV E6 mRNA (TABLE 19).

TABLE-19

COMPARISON OF POSITIVITY AMONG HPV DNA AND HPV E6 mRNA

BIOPSY DIAGNOSIS TOTAL

CERVICITIS CIN I CIN

II & III SCC HPV DNA-

POSITIVE 1 5 4 10 20

E6mRNA-

POSITIVE 1 5 2 10 18

50

Table 21,22,23 NUMBER OF TRUE POSITIVE,TRUE NEGATIVE,FALSE POSITIVE, FALSE NEGATIVE FOR CIN I CASES IN CYTOLOGY,

HPV DNA, HPV E6 mRNA

TABLE 21 FOR CYTOLOGY IN CINI

CYTOLOGY

BIOPSY

POSITIVE NEGATIVE

POSITIVE 14 2

NEGATIVE 1 24

Sensitivity=93.3%

Specificity=92.31%

PPV=87.50%

NPV=96.00%

TABLE 22 FOR HPV DNA IN CIN I

HPV DNA

BIOPSY

POSITIVE NEGATIVE

POSITIVE 5 1

NEGATIVE 10 25

51 Sensitivity=33.33%

Specificity=96.15%

PPV=83.33%

NPV=71.43%

TABLE 23:FOR HPV E6 mRNA IN CINI

HPV E6mRNA

BIOPSY

POSITIVE NEGATIVE

POSITIVE 5 O

NEGATIVE 10 26

Sensitivity=33.33%

Specificity=100%

PPV=100%

NPV=72.22%

52

TABLE 24: COMPARISON OF SENSITIVITY, SPECIFICITY, PPV AND NPV IN CIN I CASES, FOR CYTOLOGY, HPV DNA, AND HPV E6 mRNA

CYTOLOGY IN % HPV DNA IN% HPV E6 mRNA IN%

SENSTIVITY 93.33 33.33 33.3

SPECIFICITY 92.31 96.15 100

PPV 87.50 83.33 100

NPV 96. 71.43 72.22

TABLE 25, 26, 27: TRUE POSITIVE, TRUE NEGATIVE, FALSE POSITIVE,FALSE NEGATIVEIN CIN II&III CASES FOR CYTOLOGY,

HPV DNA,HPV E6 mRNA

TABLE 25; FOR CYTOLOGY IN CIN II&III

CYTOLOGY

BIOPSY

POSITIVE NEGATIVE

POSITIVE 3 2

NEGATIVE 2 34

Sensitivity=60%

Specificity=94.44%

PPV=60%

NPV=94.44%

53

TABLE 26 FOR HPV DNA IN CINII&III

HPV DNA

BIOPSY

POSITIVE NEGATIVE

POSITIVE 4 1

NEGATIVE 1 35

Sensitivity=80%

Specificity=97.2%

PPV=80%

NPV=97.22%

TABLE 27: FOR HPV E6 mRNA IN CINII&III

HPV E6 mRNA

BIOPSY

POSITIVE NEGATIVE

POSITIVE 2 0

NEGATIVE 3 36

Sensitivity=40%

Specificity=100%

PPV=100%

NPV=92.31%

54

TABLE 28:COMPARISON OF SENSTIVITY,SPECIFICITY,PPV AND NPV FOR CIN II&III CASES FOR CYTOLOGY,HPV DNA,

HPV E6 mRNA

CYTOLOGYIN

%

HPV DNA IN% HPV E6 mRNA IN %

SENSTIVITY 60 80 40%

SPECIFICITY 94.44 97.2 100%

PPV 60 80 100%

NPV 94.44 94.59 92.31%

TABLE 29,30,31 TRUE POSITIVE,TRUE NEGATIVE,FALSE POSITIVE, FALSE NEGATIVEIN CASES OF SCC FOR CYTOLOGY, HPV DNA,

HPV E6 mRNA

TABLE 29. FOR CYTOLOGY IN SCC

CYTOLOGY

BIOPSY

POSITIVE NEGATIVE

POSITIVE 11 0

NEGATIVE 0 30

55 Sensitivity=100%

Specificity=100%

PPV=100%

NPV=100%

TABLE 30 FOR HPV DNA IN SCC

HPV DNA

BIOPSY

POSITIVE NEGATIVE

POSITIVE 10 0

NEGATIVE 1 30

Sensitivity=90.91%

Specificity=100%

PPV=100%

NPV=96.77%

56

TABLE 31: FOR HPV E6 mRNA In SCC

HPV E6 MRNA

BIOPSY

POSITIVE NEGATIVE

POSITIVE 10 0

NEGATIVE 1 30

Sensitivity=90.91%

Specificity=100%

PPV=100%

NPV=96.77%

TABLE 32: COMPARISON OF SENSITVITY, SPECIFICITY, PPVAND NPV IN CYTOLOGY, HPV DNA, HPV E6mRNA IN SCC CASES

CYTOLOGY IN

%

HPV DNA IN

%

HPV E6 MRNA IN %

SENSTIVITY 100 90.91 90.91

SPECIFICITY 100 100 100

PPV 100 100 100

NPV 100 96.77 96.77

CHART1 : DISTRIBUTION CERVICAL NONNEOPLASTIC, PRENEOPLASTIC AND NEOPLASTIC

CHART 2: AGE WISE DISTRIBUTION OF CERVICAL NON NEOPLASTIC, PRENEOPLASTIC AND NEOPLASTIC LESIONS

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

CHRONIC CERVICITIS

18%

37%

28%

13%

3%1%

CHARTS AND PICTURES

DISTRIBUTION CERVICAL NONNEOPLASTIC, PRENEOPLASTIC AND NEOPLASTIC LESIONS IN OUR INSTITUTE

CHART 2: AGE WISE DISTRIBUTION OF CERVICAL NON STIC, PRENEOPLASTIC AND NEOPLASTIC LESIONS

IN OUR INSTITUTE

SCC-WD SCC-MD SCC-PD CIN I CIN II CIN III ADENO CA

0% 0% 0%

20%

0% 0%

0% 6%

25%

48% 100%

80%

25%

50% 0%

26%

0%

0%

50%

31%

0%

2% 0%

20%

67%

25% 13%

75%

3% 0% 0%

0% 0% 0% 2% 0% 0%

33%

DISTRIBUTION CERVICAL NONNEOPLASTIC, LESIONS IN OUR INSTITUTE

CHART 2: AGE WISE DISTRIBUTION OF CERVICAL NON STIC, PRENEOPLASTIC AND NEOPLASTIC LESIONS

CHRONIC CERVICITIS SCC-WD

SCC-MD SCC-PD CIN I CIN II CIN III ADENO CA

ADENO CA

0%

0%

0%

67%

0%

33%

71-80 61-70 51-60 41-50 31-40 20-30