STUDY OF EXPRESSION OF αvβ6 INTEGRIN IN POTENTIALLY MALIGNANT LESIONS – AN IMMUNOHISTOCHEMISTRY STUDY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfilment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH VI

ORAL PATHOLOGY AND MICROBIOLOGY

MAY 2018

Acknowledgement

ACKNOWLEDGEMENT

I would like to take this opportunity to express my gratitude to everyone who has helped me through this journey. I would like to start with my very respected and benevolent teacher, Dr.

Ranganathan K, Professor and Head, Department of Oral and Maxillofacial Pathology, Ragas

Dental College and Hospital. I consider myself blessed to have the opportunity to study under his guidance. He has always been a source of inspiration to excel in academics and career. I would offer my obeisance unto him for having taken interest in my study and instilling knowledge.

Thank you so much sir.

I owe enormous debt of gratitude to my professor Dr. Uma Devi K Rao for helping me in completing my thesis. She always has been a pillar of support and encouragement all throughout my post graduate life. She was approachable for any help and always made me feel at home by her caring nature. I want to take this opportunity to acknowledge and thank her for the help and support. Thank you mam.

I express my profound gratitude to my revered guide Dr. Elizabeth Joshua, Professor for her encouragement, advice and guidance which helped me complete my dissertation. She has been an integral part of my post graduation and I want to take this opportunity to acknowledge and thank her for her help and support. Your patience and perfection is one thing I want to learn. Thank you so much mam for all the support and guidance. I earnestly thank Professor, Dr. T. Rooban for his valuable support and concern in helping me to complete this study. I want to take this opportunity to acknowledge and thank him for the help and support throughout my post graduate life.Thank you sir.

I extend my sincere thanks to Professor Mr. Kanakaraj Chairman Jaya Educational trust &

Dr. N.S.Azhagarasan,, Principal, Ragas Dental College for providing me with an opportunity to utilize the facilities available in this institution in order to conduct this study.

I am immensely grateful to Mrs. Kavitha Wilson, Geneticist and Research assistant, for her valuable suggestions which enabled me to systematically proceed through the study.

I extend my special thanks to my readers Dr Lavanya N, Dr Lavanya C for their valuable support. I would like to thank them for their support during my post-graduate course.

I extend my sincere thanks to my senior lecturers Dr Sudarsan, Dr Kavitha and Dr

Joseph for their valuable support.

My sincere thanks Mr. Rajan, Lab Assistant, for his assistance in his valued support for the study and attender Mrs. Laila, Ragas Dental College and Hospital for their constant help in completion of my study.

I acknowledge gratefully the help of my batchmates Dr.Ishwarya, Dr. Janani, Dr. Sivashankari,

Dr.Bakiyalakshimi and Dr.Rajaseker for their support and encouragement.

I am dedicating this work to my mother Mrs. Pathipooranam Thangasamy and my

Brother Mr. Arul mani and my grandmother Mrs. Sivananjiammal and to my sister Mrs.

Muthu selvi and her husband Mr. Sugumaran. I thank them for all their sacrifices, understanding, support and constant encouragement.

CONTENTS

1. INTRODUCTION 1

2. AIM AND OBJECTIVES 3

3. MATERIALS AND METHODS 5

4. REVIEW OF LITERATURE 13

5. RESULTS 32

6. DISCUSSION 36

7. SUMMARY AND CONCLUSION 42

8. BIBLIOGRAPHY 44

9. ANNEXURES 53

I - Institutional Review Board approval form

II - Dissertation protocol

III - Primary antibody

IV - Secondary antibody

V - Department declaration form

VI - Plagiarism check form

VII – Abbreviations

Introduction

Introduction

INTRODUCTION

Integrins are the principal cell surface receptors that enable both normal and transformed cells to attach to and respond to their extra-cellular environment. Integrins mediate cell to cell or cell to extra-cellular matrix

(ECM) adhesion, providing adhesion for stationary cells. They mediate the traction during cell movement and importantly the promotion of many signalling pathways that regulated diverse processes including proliferation, migration, cell survival, differentiation, tumor invasion and metastasis1.

Oral Potentially Malignant Disorders are Erythroplakia, Leukoplakia,

Oral Submucous Fibrosis (OSF), and Erosive Lichen Planus. In erythroplakia the malignant transformation rate is 90%. Leukoplakia has a malignant transformation rate of 3.6-17.5%. Some authors say that OSF has a malignant transformation rate of about 0.5-6%. Malignant transformation rate of erosive lichen planus is reported to be less than 0.4-3.7% (Pei-Shan Ho et al) 63.

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx and oesophagus, with a malignant transformation rate of 7-13%5. The frequency of malignant change in lichen planus ranges from

0.4 to 3.3% (Scully et al, 1998) and 17% of oral leukoplakia may progress to malignany of the oral cavity (Gupta et al, 1980; Silverman et al, 1984) 2.

The integrin αvβ6 was highly expressed throughout the whole lesion in

90% of the squamous cell carcinoma, in 41% of the leukoplakia specimens,

1

Introduction

85% of the lichen planus samples and over 80% of oral submucous fibrosis related oral cancers, but is not expressed in tissues with inflammatory hyperplasia or chronic inflammation 2,5. The expression of αvβ6 integrin could be associated to the malignant transformation of oral leukoplakia and lichen planus and oral submucous fibrosis. Potentially malignant lesions are an immune inflammatory process and if persistent will result in activation of oncogenes and loss of tumor suppressor genes5. In carcinogenesis, epithelial cells breach the basement membrane and proliferate and migrate within the connective tissues3.

Although epithelial cell may express a variety of cell-adhesion molecules, integrins are the most important extracellular matrix (ECM) receptors and are known to play a major role in tumor invasion and progression3. Integrin αvβ6 is also expressed to a certain extent among cases of verrucous hyperplasia, verrucous carcinoma, carcinoma in situ and lichen planus and clinically diagnosed leukoplakia lesions. However epithelial cells of inflammatory, drug-induced or idiopathic hyperplasia or chronic inflammatory lesions do not express αvβ6 integrin26.

2

Aim and Objective

Aim and Objectives

AIM AND OBJECTIVES

The aim of this study is to investigate expression of αvβ6 integrin in potentially malignant lesions.

1. Detection of αvβ6 integrin in Mild dysplasia (n=10), Moderate and

Severe dysplasia (n=10) by immunohistochemistry (IHC).

2. Detection of αvβ6 integrin in Oral submucous fibrosis (n=10) by IHC.

3. Detection of αvβ6 integrin in normal healthy mucosa (n=10) by IHC.

HYPOTHESIS (Null)

αvβ6 integrin is not expressed in potentially malignant lesions.

STUDY SETTING

The study was conducted in the Department of Oral and Maxillofacial

Pathology, Ragas Dental College and Hospital, Chennai, using archival formalin-fixed-paraffin embedded blocks of mild, moderate, severe epithelial dysplasia, oral submucous fibrosis and normal mucosa using IHC. This study was approved by Institutional Review Board (IRB) of Ragas Dental College and Hospital, Chennai (Annexure I).

STUDY SUBJECTS:

The study material comprised of 40 formalin-fixed, paraffin embedded tissue specimens. The samples were divided into 4 groups namely: Group I,

Group II, Group III and Group IV.

Group I: 10 normal oral mucosal tissue specimens.

3

Aim and Objectives

Group II: 10 histopathologically confirmed mild epithelial dysplasia tissue specimens

Group III: 10 histopathologically confirmed moderate and severe epithelial dysplasia tissue specimens

Group IV: 15 histopathologically confirmed oral submucous fibrosis tissue specimens

4

Materials and Methods

Materials and Methods

MATERIALS AND METHODS

1. Tissue samples of normal oral mucosa (n=10), Mild dysplasia (n=10),

Moderate and Severe dysplasia (n=10), Oral submucous fibrosis

(n=10), were taken from archival blocks.

2. A detailed case history including patient’s age, gender, occupation,

past medical & dental history, history of drugs and trauma were

recorded.

3. Extra oral and intra oral examination was done.

4. Biopsy was performed from the lesional site. Normal oral mucosa was

taken when the patients were undergoing minor surgery for extraction

of impacted teeth.

5. The tissue biopsied was immediately transferred to 10% buffered

formalin.

6. After adequate fixation, tissue was embedded in paraffin.

7. From the paraffin embedded blocks 5 microns thick, sections were cut

and used for routine hematoxylin and eosin (H & E) staining and

immunohistochemical (IHC) staining.

8. Tissue sections of placenta were used as positive control for αvβ6

integrin positivity.

9. During embryogenesis αvβ6 is expressed at high levels in the

developing lung, skin and kidney and which is not expressed on

healthy oral epithelium but is upregulated during tissue remolding,

5

Materials and Methods

including wound healing, carcinogenesis and fibrosis 11, 4. αvβ6 binds

to its ligand via the tripeptide recognition sequence arginine – glycine

– aspartic acid (RGD). Known ligands of αvβ6 are extracellular

proteins such as fibronectin, tenascin-C, vitronectin, Latency-

associated peptide of TGF-β1 and TGF-β3.

HEMATOXYLIN AND EOSIN (H & E) STAINING

REAGENTS

 Harris’s hematoxylin

 1% acid alcohol

 Eosin

 Xylene

 Alcohol

PROCEDURE

The slides were dewaxed in xylene and hydrated through graded alcohol to water. The sections on the slides were flooded with Mayer’s hematoxylin for 5 minutes. The slides were washed in running tap water for 5 minutes. The slides were differentiated in 1% acid alcohol for 5 minutes. The slides were washed well in running tap water for 5 minutes. The tissue sections on the slides were then stained in eosin for 30 seconds. The slides were washed in running tap water for 1 minute. The slides were then dehydrated through alcohol, cleared, mounted and viewed under light microscope (LM).

6

Materials and Methods

IHC STAINING (αvβ6 INTEGRIN)

Armamentarium:-

 Microtome

 Autoclave

 Hot air oven

 Slide warmer

 Couplin jars

 Measuring jar

 Weighing machine

 APES coated slides (Amino propyl triethoxysilane)

 Slide carrier

 Aluminium foil

 Micro-pipettes

 Toothed forceps

 Electronic timer

 Beakers

 Rectangular steel tray with glass rods

 Sterile gauze

 Cover-slips

 Light microscope

REAGENTS USED

1. APES (3 amino propyl triethoxysilane)

7

Materials and Methods

2. 0.1 mol/L citrate buffer (50X)

3. 0.45% H2O2

4. DAB detection system

5. Distilled water

6. Mayer’s Hematoxylin

ANTIBODIES USED

1. Primary antibody – Anti αvβ6 integrin rabbit (bs-5791R) polyclonal antibody - Biotech desk,

2. Secondary antibody – Poly excel- HRP micro polymer IHC detection system – Pathnsitu.

PREPARATION OF PARAFFIN SECTION

After the slides were dried, tissue sections of 5 micron thickness were made in a rotary manual microtome. The ribbons of tissue section were transferred onto the APES coated slides from the tissue float bath such that two tissue bits come on to each slide with a gap in between. One of the tissue sections towards the frosted end of the slide was labeled negative to which negative serum, the secondary and the chromogen were added and the tissue section away from frosted side is the positive to which the primary antibody, secondary antibody and chromogen were added.

IMMUNOHISTOCHEMISTRY PROCEDURE

Frozen sections (6µm) were cut and fixed with -20°C acetone for 5 minutes and stored at -70°C until used. Antigen retrieval of primary antibody was microwaving for 30 min in 0.1 mol/L citrate buffer (Ph- 6). Endogenous

8

Materials and Methods

peroxidase was neutralized with 0.45% hydrogen peroxidase in methanol for

15 min and primary antibodies were applied in TBS (pH 7.6) for 1 hour. Poly excel HRP secondary antibody applied for 30 min. Peroxidase was visualized using DAB+ for 7 min and counter stained in Mayer’s hematoxylin and Slides were then washed in distilled water to remove excess chromogen. The tissue sections were mounted with DPX. The slides were then observed under the microscope. Throughout the procedure care was taken not to dry the tissues.

POSITIVE CONTROL

The Placenta contains Chorion and amnion layer. Placenta and fetal membranes from normal vaginal deliveries exhibited strong intensity of staining for αvβ6 integrin in the epithelial layer of the amnion. A case of placenta specimen known to express Alpha v beta 6 integrin positivity were fixed, processed, embedded, sectioned, stained in same manner and used as positive control. One positive control tissue slide was included for each batch of staining. The tissue section away from the frosted end of the slide was taken as the positive control.

IHC PROCEDURE

1. APES coated slides with 2 paraffin embedded tissue fixed with -20°C

acetone for 5 minutes and stored at -70°C until used.

2. Keep in citrate buffer (pH 6) for 30 minutes at 37ºC for antigen

retrieval.

3. Neutralized with 0.45% hydrogen peroxidase in methanol for 15

minutes.

9

Materials and Methods

4. Primary antibodies were applied in TBS (pH 7.6) for 1 hour.

5. Poly excel HRP secondary antibody - was applied for 30 minutes.

6. DAB detection system was applied.

7. Counter stained in Mayer’s hematoxylin for 7 minutes.

8. Slides were then washed in distilled water to remove excess

chromogen.

9. Slides to be mounted using dibutyl phthalate styrene (DPX).

10. Slides to be observed under the light microscope and graded.

CRITERIA FOR EVALUATION OF STAINING

Evaluation of H & E sections:

 The H & E stained sections were thoroughly examined.

 Oral dysplasia was graded as mild, moderate and severe dysplasia

and were examined.

 Oral submucous fibrosis cases were also examined.

Evaluation for IHC:

 Corresponding sections as examined by H & E were stained by IHC to

detect αvβ6

expression.

 The positive control used for IHC was normal membrane and placenta.

 Positive cells were counted in the epithelial cells of normal mucosa,

oral submucous fibrosis, mild dysplasia, moderate dysplasia, severe

dysplasia.

10

Materials and Methods

 The staining intensity of αvβ6 was scored on a scale of 1 to 3 (1-weak,

2-moderate, and 3-strong), and the proportion of cells staining

positively was scored on a scale of 1 to 4 (1- focal basal, 2-linear basal,

3-basal and Suprabasal, and 4-full epithelium thickness). The score for

intensity was added to the score for proportion to give a score in the

range of 0 to 7 and grouped as score = 0 (-), score = 1–4 (low), score =

5-7 (high).

TISSUE LOCALISATION:

αvβ6 stain is localized to the basal, suprabasal and full thickness of the epithelium.

CELLULAR LOCALIZATION OF STAIN

The normal localization of the staining includes both nucleus and cytoplasm. In our study the stain is localized to the cytoplasmic region. But the stain is not present in normal tissue. The stained slides were screened, examined systematically for αvβ6 integrin expression in the cytoplasmic portion.

INTENSITY OF STAINING

The staining intensity of αvβ6 was scored on a scale of 1 to 3 (1-weak,

2-moderate, and 3 strong), and the proportion of cells staining positively was scored on a scale of 1 to 4 (1- focal basal, 2-linear basal, 3-basal and

Suprabasal, and 4-full thickness of the epithelium thickness). The score for intensity was added to the score for proportion to give a score in the range of 0 to 7 and grouped as score = 0 (-), score = 1–4 (low), score = 5-7 (high)

11

Materials and Methods

intensively stained based on the intensity of staining taken up by the tissue as observed by two blinded observers independently with respect to positive control.

STATISTICAL ANALYSIS

Data were entered and analyzed using SPSSTM software (version 21.0).

Pearson’s Chi-square test done to compare intensity of staining between two groups. p value >0.05 was considered statistically significant. Kappa analysis was done to compare the intensity of αvβ6 integrin staining as observed between three observers.

12

Review of Literature

Review of Literature

STRUCTURE OF INTEGRIN

Representation of prototype αI-domain containing integrin heterodimer. αI(αA)-domain is part of the α-subunit and connected to the seven-bladed β-propeller domain. There are nine different types of integrins that contain αI domain. This domain contains Rossman fold and a MIDAS site for metal ion binding. β-propeller domain contains EF-hand motif for calcium binding and is connected with thigh domain followed by calf-1 and calf-2 domains. β-subunit contains a

β1(βA)- domain that process structural similarity with αI domain. This domain also contains

Rossman fold and MIDAS site for metal ion binding. The interface of βI domain of β-subunit

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Review of literature and β-propeller domain of α-subunit forms a ligand binding pocket. βI-domain is connected to hybrid domain followed by a plexin-somaphorin-integrin (PSI) domain, four EGF repeats and a

β-tail domain4.

STRUCTURE OF αvβ6 INTEGRINS

(1)Structurally, integrins are hetrodimers composed of two different, non-covalently associated,

α and β subunits. They appear by electron microscopy to have a large extra-cellular domain composed of a membrane distal, globular head that contains the ligand binding site, on two long stalks.

(2) The carboxy-(C) termini of the α and β subunits traverse the cell membrane and extend a short distance into the cytoplasm1.

LIGAND

EXTRA CELLULAR MATRIX

PLASMAMEMBRANE

CYTOPLASAM

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Review of literature

THE VARIOUS HETERODIMERIC COMBINATIONS OF α AND β SUBUNITS25.

INTEGRIN FAMILY OF PROTEINS AND THEIR LIGANDS

β1 α1 Colls, laminins

α 2 Colls, laminins, chondroadherin

α 3 Laminins (such as laminin-1, -5, -8, -10, and -11), Fn, thrombospondin, TIMP-2,

uPAR, collagen, epiligrin, entactin

α 4 Fn, VCAM

α 5 Fn, Fg, Upar

α 6 Laminins, merosin (laminin α2 chain), Kalinin

α 7 Laminins, merosin (laminin α2 chain),

α 8 Fn, vitronectin, Tn-C, osteopontin, and nephronectin

α 9 angiostatin, Tn-C, osteopontin, and ADAMs, VCAM-1, tTG,

α 10 Colls

α 11 Colls

α v Fn, vitronectin

β 2 α L ICAM-1, -2 and -3

α M Fg, ICAMs, iC3b, factor-Xa, denatured ovalbumin

α X Fg, iC3b

α D VCAM, ICAMs

β 3 α IIb Coll, Fn, vitronectin, Fg, vWF, thrombospondin

α v Fn, vitronectin, Fg, vWf, thrombospondin, FGF-2, MMP-2 and some ADAM

proteins

β 4 α 6 Laminins

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Review of literature

β 5 α v Vitronectin, Upar

β 6 α v Fn, Tn

β 7 α 4 Fn, VCAM, MAdCAM

α E E-cadherin

α v Colls, laminins, Fn

β N α v Fn, Colls

β 8 α v Vitronectin, Fn

Abbreviations used are

Colls: collagens; Fn: fibronectin; TIMP-2: tissue inhibitor of metalloproteinase; uPAR: urokinase-type plasminogen activator (uPA) receptor; VCAM: vascular cell adhesion molecule; Fg: fibrinogen; Tn-c: tenacin-C; ADAMs: a disintegrin and metalloproteinase proteins; tTG: tissue-type transglutaminase; iC3b:inactivated complement component 3b; ICAM: intercellular cell adhesion molecule; vWf: vonWillebrand factor; FGF-2: fibroblast growth factor

2; MMP: matrix metallo-proteinases and MAdCAM: mucosal addressin cell adhesion molecule.

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Review of literature

LIGAND OF αvβ6 INTEGRIN10

Ligand Type of protein

Fibronectin ECM protein

Tenascin-C ECM protein

Vitronectin ECM protein

LAP of TGFb-1 Cytokine

LAP of TGFb-3 Cytokine

Foot-and-mouth disease virus Viral capsid

(FMDV)

Coxsackievirus 9 (CAV-9) Viral capsid

Activation of different matrix metalloproteinases by αvβ6 in various cancers. Activation of

MMP9 has the highest prevalence15.

KERATINOCYTES INTEGRINS7

INTEGRINS MAJOR LIGAND EXPRESSION

α2β1 Collagen Constitutive (Basal layer)

α3β1 Laminin Constitutive (Basal layer)

α6β4 Laminin Constitutive (Basement membrane)

αvβ5 Vitronectin Weak (buccal mucosa)

α5β1 Fibronectin Induced in culture, on wounding, in pathological conditions

αvβ6 Fibronectin; tenascin Induced in culture, on wounding, in pathological conditions

α9β1 Tenascin Upregulated during wound healing

αvβ8 Vitronectin Suprabasal

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Review of literature

INTEGRINS SUPERFAMILY

(G. J. Thomas et al 2006) The integrin superfamily 18 α and 8 β subunits have been identified combining to form at least 24 hetrodimeric integrins which can be considered in several sub families based on ligand specificity. The different subunits show selectivity in their binding partners; for example the αv subunit can pair with multiple β subunits (β2, β3, β4, β5,

β6, β8) but the β6 subunit can only bind with αv1.

INTEGRIN RECEPTORS

(Sumit Goswami et al 2013) There are a total of 24 different types of integrins. The integrin family of receptors, depending on their ligand recognition pattern can be broadly classified into four different categories4.

I) RGD binding

II) Collagen binding

III) Laminin binding

IV) Leucocyte binding types of integrins

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Review of literature

RGD – Arginine – Glycine – Aspartic acid

RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+1.

INTEGRIN SIGNALING

(Caroline H Damsky et al 2002) Integrins are in the proper relationship with ECM in order to respond to signals from growth factors. Integrins recruit growth factors receptors (GFRs) as well as cytoskeletal components, adaptor molecules and non-receptor tyrosine kinase to sites of adhesion when they are clustered and activated by ECM ligands. They affect cell polarity, directed migration, cell growth and survival and differentiation10.

(Paul H. Weinreb et al 2004) Integrin signaling is unique among all the cell surface receptors because signaling can propagate in both directions (outside to inside and inside to outside). Most of the time, cell surface receptor-mediated signaling involves initial binding of the ligand on the extracellular domain, which then initiates changes at the cytoplasmic domain leading to further propagation of signaling events; this is called outside – in signaling35. Another kind of signaling process called inside-out signaling. In this process, binding of intracellular ligands to the cytoplasmic domain results in some conformational change in the extracellular domain of the receptor35.

EPIDERMAL HYPERPROLIFERATION AND INFLAMMATION

(Fiona M. Watt et al 2002) Suprabasal integrins can activate MAPK directly or via stimulating release of interleukin -1α (IL-1α) in cultured human keratinocytes results in increased proliferation. MARK is activated through stimulating keratinocytes to release IL-1α.

IL-1α production by keratinocytes induces a dermal mononuclear infiltrate, leading to release of further cytokines and growth factors7.

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Review of literature

FUNCTION OF INTEGRIN

(John Lamar et al 2011) Several integrins are expressed in normal, unwounded epidermis and oral squamous epithelium, including α3β1 and α6β4 (both laminin-332 receptors),

α2β1 (a collagen receptor), α9β1 (a fibronectin and tenascin receptor) and αvβ5 (a vitronectin receptor). Integrin α6β4 is an essential component of hemidesmosomes, which are adhesion structures on the basal surfaces of keratinocytes that anchor the epidermis to the dermis59.

Proliferating keratinocytes are normally restricted to the basal cell layer where they are adhered through integrins to the underlying basement membrane (BM), a specialized ECM that separates epithelial cell layers from adjacent connective tissue. Differentiating keratinocytes down-regulate integrin expression as they detach from the BM and are displaced into the suprabasal layers59.

(Michael Busk et al 1992) Integrins that regulate the activation of ECM- producing cells include cytokines, chemokines, growth factors, components of the renin-angiotensin system

(RAS), angiogenic factors, peroxisome proliferator-activated receptors (PPARs), mammalian target of Rapamycin (mTOR), and products of oxidative stress12.

(G. Latella et al 2013) Integrins regulate cell-cell and cell-extracellular matrix interactions, thus influencing growth, differentiation, and development, as well wound healing and development of fibrosis17. αvβ6 ligands include fibronectin, tenascin, vitronectin and latency-associated peptide (LAP). αvβ6 integrin inhibit to reduce the tissue levels of pro- fibrogenic transcripts, such as procollagen α1(I), TGFβ1, TGFβ2, connective tissue growth factor (CTGF), tissue inhibitory matrix metelloproteinaseas-117.

(G. J. Thomas et al 2006) αvβ6 promotes adhesion and migration on several different

ECM ligands, which promotes increased MMP secretion and can activate TGFβ1 and TGFβ3, which leads to promote the survival of OSCC cells1. αvβ6 expression in carcinomas of the lung,

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Review of literature breast, pancreas, stomach, colon, ovary, salivary gland as well as oral and skin squamous cell carcinoma1.

(Louise A. Koopman Van Aarsen et al 2008) αvβ6 binding to LAP1 or LAP3 leads to activation of the latent precursor form of TGF-β1 and TGF-β3. Thus, up-regulated expression of

αvβ6 can lead to local activation of TGF-β, Up-regulated αvβ6 expression has been shown to be associated with increased epithelial to mesenchymal transition, increased tumor cell invasion, and increased metastatic potential in vivo14. Local activation of TGF-β1 and the ability to promote tumor progression, invasion, and metastasis. The TGF- β1 cytokine is a pleiotropic growth factor that can regulate cell proliferation, differentiation, and immune responses. In vivo by a Transforming Growth Factor- β–Regulated Mechanism14.

(Karsta Luettich et al 2004) The integrin αVβ6 is expressed principally on epithelial cells. Where it has been shown to be a receptor for RGD and non-RGD sites in ligands. The mature form of TGFβ1 binds to and activates of αVβ6integrin. Among others, binding of mature

TGFβ1 to αVβ6 integrin resulted in an enhanced immobilization and phosphorylation of proteins, which are associated with focal adhesions. Stimulation with mature TGFβ1 leads to upregulation of c-Jun in TGFβ1 sensitive cells. Mature TGFβ1 activates c-Jun via MEKK1/p38 and if this activation may influence cytoskeletal reorganization53.

COMPARING THE EXPRESSION OF INTEGRINS

(Jens Schittenhelm1 et al 2013) αv integrin subfamily, which has five members αvβ1,

αvβ3, αvβ5, αvβ6 and αvβ8. The αvβ3 and αvβ5 integrins, which are frequently expressed in tumor endothelial cells, which may affect tumor initiation and progression, in lung cancer αvβ3 and αvβ6 can bind ligands such as osteopontin and fibronectin24. Tumor progression in colorectal cancer can be promoted through αvβ6-mediated activation of TGF-β. In pancreatic ductal ade-

21

Review of literature nocarcinoma αvβ6 is upregulated compared to normal ducts. Comparable levels of expression only for αvβ3 and αvβ8 were in brain metastasis, while αvβ6 and αvβ5 were higher in primaries24.

MATRIX METALLOPROTEINASES (MMPS) AND INTEGRINS

(Ylipalosaari et al 2005) MMPs have several roles in malignant transformation and tumour progression. MMPs release cell-membrane bound precursors of growth factors, modulate apoptosis mediators, produce angiogenesis inhibitors by cleavage or allow endothelial cell invasion into tumour tissue. They also degrade extra cellular membrane (ECM) and basement membrane(BM) components, allowing cancer cells to migrate and to invade, and modulate the immune response by suppressing lymphocytoproliferation. MMPs contribute to epithelial and carcinoma cell migration by cleaving laminin-5, which is a major component of hemidesmosomes in BM and promotes static adhesion between the epithelium and BM26.

Integrin αvβ6 overexpression induces production of MMP-3 and -9 and, to a lesser extent,MMP-

2 in both ligand-dependent and -independent manners. Ligation of β6 integrin with fibronectin achieves activation of the FYN/FAK complex, which in turn couples β6 signaling to the Raf-

ERK/MAPK pathway. This pathway activates MMP-3 transcription and promotes OSCC cell proliferation and metastasis26.

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Review of literature

(Hai-Xia Li1 et al 2013) αvβ6 regulates expression of MMP-1, MMP-2, MMP-9 and MMP-13 in OSCC cells55.

αvβ6 INTEGRIN EXPRESS IN WOUND HEALING

(Kirsi Haapasalmi et al 1996) Integrins are expressed by basal keratinocyte. lntegrins of the β1 family were present in normal keratinocytes of oral mucosa, while αv, αvβ5 , and αvβ6 integrins were absent. In those 3-day-old wounds in which epithelial sheets were fused, αvβ6 integrin was present in the basal cell layer in the wound area, but absent from the non-wounded site8. αv integrin and αvβ6 integrin complex reacted with basal keratinocytes in all 7-day-old wounds. The ligands of αvβ6 integrin are fibronectin and tenascin, in 7-day-old wounds that displayed high expression of this receptor. Fibronectin was present throughout the normal connective tissue and abundantly in the wound granulation tissue matrix. Tenascin was present strongly in subepithelial connective tissue but weakly in deeper areas of the non-affected tissue.

Specimen were collected from punch biopsies were taken from wounds on 3 and 7th day8.

POTENTIALLY MALIGNANT DISORDERS

Betel quid chewing was found to be the strongest risk factor for both dysplasia and OSF .

While the effect of alcohol drinking on the two oral premalignant diseases was not substantial,

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Review of literature the significantly elevated risk of cigarette smoking was detected among OL patients. Among these risk factors, betel quid chewing accounted for 73.2 and 85.4% of attributable risks of contracting dysplasia and OSF, respectively. Combined with cigarette smoking, the population attributable risk proportion of dysplasia increased to 84.4%. In the same way, 86.5% of the a etiologic fraction for patients having either dysplasia or OSF was detected 27.

CLASSIFICATION OF ORAL POTENTIALLY MALIGNANT DISORDERS

1. High Risk

Erythroplakia.

Leukoplakia.

Oral Submucous Fibrosis (OSF).

Erosive Lichen Planus.

2. Life-style Related

Smokeless Tobacco Keratosis.

Reverse Smoker’s Palate.

Actinic Cheilitis.

3. Infections

Hyperplastic Candidiasis.

Viral (HPV, HIV, EBV, HBV, HSV).

Tertiary Syphilis.

4. Immunodeficiency

Solid Organ Transplantation.

Graft Versus Host Disease.

Chronic Cutaneous Lupus Erythematous.

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Review of literature

5. Inherited Disorders

Xeroderma Pigmentosum.

Dyskeratosis Congenita.

Epidermolysis Bullosa.

Bloom Syndrome.

Fanconi’s Anemia.

No single factor has been identified as the causative factor for potentially malignant disorders.

But a number of high risk factors has been put forwarded which has greater than normal risk of malignancy at a future date 64.

A. Extrinsic Factors

1. Tobacco in any form (smoking or chewing) is the single most major extrinsic cause

(people who smoke more than 80 cigarettes per day have 17-23 times greater risk).

2. Alcohol regardless of beverage type and drinking pattern – synergistic action along with

tobacco (risk of smokers who are also heavy drinkers is 6-15 times than that of

abstainers).

3. Virus infection – HPV, EBV, HBV, HIV, HSV.

4. Bacterial infection – treponema pallidum.

5. Fungal infection – candidiasis.

6. Electro-galvanic reaction between unlike restorative metals.

7. Ultraviolet radiation from sunlight – associated with lip lesions.

8. Chronic inflammation or irritation from sharp teeth or chronic cheek-bite (tissue

modifiers rather than true carcinogens).

25

Review of literature

B. Intrinsic Factors

1. Genetic (5% are hereditary).

2. Immunosuppression – organ transplant, HIV.

3. Malnutrition – iron (anemia), vitamin A, B, C deficiency.

Most common sites for PMDs in India are buccal mucosa followed by tongue, palate and floor of the mouth. Location of PMDs differs from distribution of OSCC, for which the tongue, alveolar ridge and floor of mouth are the most common sites 64.

EXPRESSION OF αvβ6 INTEGRIN IN ORAL LEUKOPLAKIA

(S Hamidi et al 1999) Fibronectin and tenascin were both present in the connective tissue, especially in areas close to the basement membrane zone. Known ligands of αvβ6 integrin are extracellular proteins such as fibronectin, tenascin-C, vitronectin, latency-associated peptide of TGF-β1 and TGF-β3 via the tripeptide recognition sequence arginine-glycine-aspertic acid were expressed underneath the oral epithelium, basal and suprabasal, and at the area near the basement membrane zone in the epithelial dysplasia. Specimen was collected from oral biopsy specimens of 29 cases of leukoplakia2.

EXPRESSION OF αvβ6 INTEGRIN IN ORAL LICHEN PLANUS

(S Hamidi et al 1999) Known ligands of αvβ6 integrin are extra cellular proteins such as fibronectin, tenascin-C, vitronectin, latency-associated peptide of TGF-β1 and TGF-β3. αvβ6 integrin binds to its ligand via the tripeptide recognition sequence arginine-glycine-aspertic acid was very strongly present around keratinocytes in basal and suprabasal cell layers of all the lichen planus. Specimen was collected from oral biopsy specimens of eight of lichen planus2.

26

Review of literature

αvβ6 INTEGRIN EXPRESSION IN ORAL SUBMUCOUS FIBROSIS

(Karwan A Moutasim et al 2009) The stroma of OSF contained myofibroblasts and that

TGF-β1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts.

They also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvβ6 expression. Arecoline-dependent αvβ6 up-regulation promoted keratinocyte migration an induced invasion, raising the possibility that this mechanism may support malignant transformation. Specimens are collected from 41 OSF cases compared with 14 cases of fibroepithelial hyperplasia by immunohistochemistry44.

HYPOTHETICAL MODEL OF αvβ6 ROLE IN OSF PATHOGENESIS

Alkaloids implicated in OSF

Genetic predisposition

αvβ6 integrin

Activation of TGF-β1

Tansdifferentiation of myofibroblasts

Upregulation of collagen 1

Pathological fibrosis

27

Review of literature

The pathogenesis of OSMF involves the mechanical as well as chemical trauma to the oral mucosa. The dry arecanut pieces present in gutkha and pan masala causes microtrauma to the oral mucosa. The traumatized mucosa undergoes chronic inflammation due to repeated microtrauma and irritation. This causes the oxidative stress and cytokines production due to chronic inflammation. The hypothesis that dense fibrosis and less vascularity of the corium, in the presence of an altered cytokine activity creates a unique environment for carcinogens from both tobacco and arecanut to act on the epithelium is widely being accepted 11.

The alkaloid like arecoline, arrecadine, guaccine, tannins, catechins, leaches out in saliva from arecanut and acts on the chronically inflamed mucosa. Increased amount of cytokines produced in oral mucosa like fibroblast growth factor, transforming growth factor and platelet- derived growth factor increases the production of collagen in the submucosal region. Also the inhibitory cytokine in collagen production interferon-alpha is decreased in oral mucosa which leads to decreased degradation of collagen. Alkaloid leached out from areca nut acts on the fibroblast and induces the phenotypic changes, which leads to the decreased capacity of fibroblasts to degrade and remodel the collagen fibers in the submucosal region. This leads to the increased amount of collagen fibers in the submucosal region of oral cavity leading to

OSMF.

The arecanut has psychotropic and antihelminthic property due to presence of areca alkaloids. Four alkaloids have been identified in biochemical studies, arecoline, arecaidine, guvacine & guvacoline, of which arecoline is the main agent. These alkaloids have powerful parasympathetic properties which produce euphoria and counteract fatigue.

Nitrosation of arecoline leads to the formation of areca nut specific nitrosamine namely nitrosoguvacoline, nitrosoguvacine and 3-methyl nitrosominopropionitrile, which alkylate DNA.

28

Review of literature

Metabolism of these areca nut specific nitrosamine lead to formation of cyanoethyl, which binds with o’methyl guanine in DNA. Prolonged exposure to this irritant leads to malignant transformation 66.

αvβ6 INTEGRIN IN ORAL SQUAMOUS CELL CARCINOMA

(Maria L. Nystrom et al 2006) Cyclooxygenases (COX) catalyze the key step in prostanoid and thromboxane biosynthesis and are targets of non-steroidal anti-inflammatory drugs (NSAID). COX-1, expressed constitutively in most mammalian cells, generates prostaglandins necessary for normal physiologic function, whereas COX-2, normally undetectable, is induced rapidly by stimuli, including cytokines, oncogenes, and tumor promoters. Elevated COX-2 expression occurs in many carcinomas, including oral squamous carcinoma (OSCC), where it contributes to tumor progression9. NSAIDs have marked antitumor activity via COX-2 inhibition. COX-2 inhibition might modulate αvβ6 function by reducing cell surface expression, decreasing ligand binding affinity, and/or interfering with αvβ6 integrin signaling. Both αvβ6 and COX-2 are expressed in OSCC, both which promote invasion, and that

NSAIDs may inhibit integrin function. NSAIDs could inhibit αvβ6-mediated invasive activity9.

OSCC cells requires COX-2- dependent activation of Rac-1, via upregulation of PGE2; abrogating COX-2, thus, blocks αvβ6-dependent invasion. They are collected from biopsy twenty OSCC, 39 epithelial dysplasias (18 transforming to OSCC and 21 non-transforming; clinical follow-up range, 5-31 years), and 14 benign polyps showing fibroepithelial hyperplasia were chosen at random9.

αvβ6 INTEGRIN EXPRESSION IN SQUAMOUS CELL CARCINOMA

(Thomas et al, 2001). αvβ6 promoted oral SCC invasion by increasing migration, pro-

MMP9 production and invasion. αvβ6 dependent processes of oral SCC cells are likely to be

29

Review of literature modulated in vivo by ligation to a high-affinity, αvβ6 specific ligand for oral SCC cells, the

TGFb-associated pro-peptide, LAP56.

INTEGRIN αVβ6 IN COLON CANCER CELLS THROUGH MMPs

(Ylipalosaari et al 2005) Inhibition of integrin αvβ6 expression in colon cancer cells suppresses MMP-9 secretion. This integrin-mediated event is dependent upon direct binding between the β6 integrin subunit and extracellular signal-regulated kinase 228. They are collected from the human colon cancer cell lines28.

(Guang Yun Yang et al 2008) αvβ6 may mediate the potential for colon cancer cells to colonize in and metastasize to the liver. The mechanisms that αvβ6 may be involved in include the promotion of MMP-9 secretion, the enhancement of migration on fibronectin, and the survival of cancer cells in the liver. They are collected from the human colon cancer cell lines39.

αvβ6 INTEGRIN A MARKER FOR OVARIEN CANCER

(Nuzhat Ahmed et al 2002) αvβ6 is present in malignant epithelia but not in normal epithelia. Normal ovarian surface epithelium was negative for αvβ6 integrin expression.21 The

αvβ6 expression is associated with ovarian cancer and that the expression of the integrin increases with advancing grade in all epithelial ovarian cancer21. They are collected from biopsy from normal ovaries (N=8) or in benign ovarian serous cyst adenomas (N=8)21.

αvβ6 INTEGRIN EXPRESSION IN COLORECTAL CANCER

(Richard C. Bates et al 2005) In colon carcinoma model of Epithelial-Mesenchymal Transition

(EMT), it was increased expression of the integrin αvβ6 is elicited as a consequence of the transition. Importantly, the consequences of the elevated αvβ6 expression are directly linked to both tumor cell function and to the mechanism of the Epithelial-Mesenchymal Transition34.

30

Review of literature

(Seong Beom Ahn et al 2014) Integrin αvβ6 is more frequently expressed at the invasive front of rectal cancer. While it plays an important role in tumour progression47. Integrin αvβ6 directly interacts with the extracellular signal-regulated kinase (ERK2) activating the

ERK/MAPK pathway that is often highly overexpressed in colorectal cancer (CRC) metastasis.

They are collected in 362 Stage B or C rectal cancer tissue samples at the tumour central region, invasive tumour front and adjacent non-neoplastic mucosa using immunohistochemistry47.

αvβ6 INTEGRIN EXPRESSION IN BREAST CANCER

(D Katoh et al 2013) In a colon cancer in vitro model, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 were found to cause upregulation of the β6 subunit and consequent increase of αvβ6 heterodimers contributing to Epithelial-Mesenchymal Transition

(EMT). They are collected from in a colon cancer in vitro model40.

αvβ6 INTEGRIN EXPRESSION IN GASTRIC CANCER

(Pei-Long Lian et al 2016) The expression of αvβ6 and MMP-9 are closely correlated, and the combinational pattern of αvβ6 and MMP-9 can serve as a more effective prognostic index for gastric cancer. They are collected from 126 specimens from patients with primary gastric carcinoma61.

31

Results

Results

RESULTS

Sample characteristics:

The study population includes 40 cases taken from the archival blocks. They were categorized into four groups. Group I (n=10) comprised of normal mucosa samples. Group II

(n=10) comprised of mild epithelial dysplasia samples. Group III (n=10) comprised of both moderate and severe epithelial dysplasia samples. Group IV (n=10) comprised of oral submucous fibrosis samples. All the samples were analyzed for the immunoreactivity of αvβ6 integrin stain.

Distribution of age in the study groups: (Table 1 & Graph 1):

The age of the patients in all the study groups was divided into 3 groups: 10-30 years,

31-50 years and those above 51 years of age. Group I, consists of 3 (30%) cases in the age group

10-30 years and 5 (50%) cases in the age group 31-50 years and 2 (20%) cases above 51 years.

Group II, consisted of 1 (10%) cases in the age group 10 -30 years and 1 (10%) cases in the age group 31-50 years and 8 (80%) cases above 51 years. Group III, consisted of 3 (30%) cases in the age group 31-50 years and 7 (70%) cases above 51 years. Group IV, 2(20%) cases in the age group 10-30 years and 5 (50%) cases in the age group 31-50 years and 3 (30%) cases above 51 years.

Distribution of gender in the study groups: (Table 2 & Graph 2):

In group I, 6 (60%) were males and there were 4 (40%) female. In group II, 7 (70%) were males and there were 3 (30%) females. In group III, were 6 (60%) males and 4 (40%) females. In group IV, 7 (70%) were males and 3 (30%) were females.

32

Results

Distribution of habits in the study groups (Table 3 & Graph 3):

Based on the prevalence of habits in the study groups, they were categorized into five groups. They were those without any habits, those with habit of chewing tobacco and areca nut alone, smoking alone, chewing arecanut and smoking, chewing tobacco and arecanut and consuming alcoholic beverages,. In group I (control group) none of them had any habits. In group II, there were 3(30%) with habit of chewing tobacco and arecanut alone, 2(20%) had no habits, 4(40%) with habit of smoking beedi alone 1(10%) with habit of pan chewing and beedi smoking. In group III, 3(30%) with habit of arecanut chewing alone, 4(40%) with had no habits and 2(20%) who did not have history of habit recorded, 1(10%) with habit of ghutka chewing and consuming alcoholic beverages. In group IV, 9(90%) with habit of arecanut and tobacco chewing alone and 1(10%) with habit of smoking and tobacco chewing. (p= 0.001)

Distribution of site in the study groups (Table 4 & Graph 4):

In group I, 5 (50) biopsies were from retro-molar region and 5 (50%) biopsies were from gingiva. In group II, 5 (50%) biopsies were from buccal mucosa and 2 (20%) biopsies were from lateral border of tongue and 1 (10%) biopsies was from lower lip and 1 (10%) biopsies was from angle of mouth and 1 (10%) biopsies was from left buccal vestibule. In group III, 5 (50%) biopsies were from buccal mucosa and 4 (40%) biopsies were from tongue and 1 (10%) biopsies were from gingiva. In group IV, 10(100%) biopsies were from buccal mucosa. (p=0.000)

Distribution of intensity of αvβ6 integrin in the study groups: (Table 5 & Graph 5):

Of the total number of cases subjected to αvβ6 Integrin staining, in group I, showed

10(100%) negative. In group II, showed 1(10%) weak, 2(20%) strong and 7(70%) negative, in group III, 1(10%) weak, 3(30%) moderate and 6 (60%) negative, In group IV, showed 3(30%) weak, 2(20%) moderate, 3(30%) strong and 2 (20%) negative.(p=0.031)

33

Results

Tissue localization in αvβ6 integrin positive stained cells in the study groups (Table 6 &

Graph 6):

Of the total number of cases subjected to αvβ6 Integrin staining, in group I, showed 10

(100%) negative, in group II, showed 1 (10%) supra basal, 2 (20%) full thickness of epithelium and 7 (70%) negative, in group III, showed 4 (40%) full thickness of epithelium and 6 (60%) negative, in group IV, showed 1 (10%) focal basal, 1 (10%) linear basal, 3 (30%) supra basal, 3

(30%) full thickness of epithelium and 2 (20%) negative. (p=0.049)

Comparison of αvβ6 integrin the score of intensity between normal (group I) mild dysplasia (group II) and moderate & severe dysplasia (group III): (Tables 7 & Graph 7):

Of the total number of cases subjected to αvβ6 Integrin staining, In group II, showed 3

(30%) high and 7 (70%) negative, in group III, 4 (40%) high and 6 (60%) negative.(p=0.089)

Comparison of αvβ6 integrin the score of intensity between the normal (group I) and oral submucous fibrosis (group IV) : (Tables 8& Graph 8):

Of the total number of cases subjected to αvβ6 Integrin staining, in group I, 10 (100%) showed negative. In group IV, showed 4 (40%) low, 4(40%) high and 2 (20%) negative.

(p=0.001)

Distribution of intensity, localization of αvβ6 integrin expression in patients with and without arecanut chewing (Tables 9& Graph 9):

We also analyzed the distribution of intensity, localization of αvβ6 integrin in patients with and without arecanut chewing of 40 cases, there were 14 (35%) cases with the habits of arecanut and 26 (65%) cases without the habits of arecanut chewing. (p=0.019)

34

Results

In the group of arecanut chewers (n=14) there were 10 (72%) cases expressed αvβ6 integrin. The expression of αvβ6 integrin was strong in 3 (21%), moderate in 5 (35%), and weak in 2 (14%). The expression of αvβ6 integrin was full epithelium in 5 (35%), supra basal layer in

4 (28%) and focal basal layer in 1 (7%).

In the group of non arecanut chewers (n=26) there were 5 (19%) cases expressed αvβ6 integrin. The expression of αvβ6 integrin was strong in 1 (3%), moderate in 1 (3%), and weak in

3 (11%). The expression of αvβ6 integrin was full epithelium in 4 (15%), and focal basal layer in

1 (3%).

KAPPA VALUE:

Three observers analyzed the slides for scoring the intensity of staining. The overall Kappa value for the inter observer variation is 0.6.

35

Tables and Graphs

TABLE 1: DISTRIBUTION OF AGE IN THE STUDY GROUPS (N=40)

AGE IN YEARS 10-30 31-50 ABOVE 51 GROUP I n=10 3 (30%) 5 (50%) 2 (20%) GROUP II n=10 1 (10%) 1 (10%) 8 (80%) GROUP III n=10 0 3 (30%) 7 (70%) GROUP IV n=10 2 (20%) 5 (50%) 3 (30%)

GRAPH 1: DISTRIBUTION OF AGE IN THE STUDY GROUPS (N=40)

80% 80% 70% 70%

60% 50% 50% 50%

10-30 YEARS 40% 30% 30% 30% 31-50 YEARS 30% ABOVE 51 YEARS 20% 20% 20% 10% 10% 10%

DISTRIBUTION OF STUDY GROUPS (%) DISTRIBUTIONOFSTUDY 0 0% GROUP I GROUP II GROUP III GROUP IV AGE GROUP IN YEARS

GROUP I – NORMAL MUCOSA

GROUP II – MILD EPITHELIAL DYSPLASIA

GROUP III – MODERATE & SEVERE DYSPLASIA

GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 2: DISTRIBUTION OF GENDER IN THE STUDY GROUPS (N=40)

GENDER MALE FEMALE GROUP I n=10 6 (60%) 4 (40%) GROUP II n=10 7 (70%) 3 (30%) GROUP III n=10 6 (60%) 4 (40%) GROUP IV n=10 7 (70%) 3 (30%)

GRAPH 2: DISTRIBUTION OF GENDER IN THE STUDY GROUPS (N=40)

70% 70% 70%

60% 60% 60%

50% 40% 40% 40% 30% 30% MALE 30% FEMALE

20%

10% DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION

0% GROUP I GROUP II GROUP III GROUP IV GENDER

GROUP I – NORMAL MUCOSA

GROUP II – MILD EPITHELIAL DYSPLASIA

GROUP III – MODERATE & SEVERE DYSPLASIA

GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 3: DISTRIBUTION OF HABITS IN THE STUDY GROUPS (N=40)

HABIT NO ARECANUT SMOKING PAN ARECANUT HABIT & ALONE CHEWING CHEWING & p Value TOBACCO & ALCOHOLIC CHEWING SMOKING BEVERAGES GROUP I 10 0 0 0 0 (n=10) p=0.001* GROUP 2 (20%) 3 (20%) 4 (40%) 1 (10%) 0 II (n=10) GROUP 6 (60%) 3 (30%) 0 0 1 (10%) III (n=10) GROUP 0 9 (90%) 0 1 (10%) 0 IV (n=10)

*p Value < 0.05 is significant

GRAPH 3: DISTRIBUTION OF HABITS IN THE STUDY GROUPS (N=40)

100% NO HABIT 100% 90% 90%

80% ARECANUT & 70% TOBACCO 60% CHEWING 60%

50% SMOKING ALONE 40% 40% 30% 30% 30% 20% PAN CHEWING & 20% SMOKING 10% 10% 10% 10% 0 0 0 0 0 0 0 0 0 0 DISTRIBUTION OF STUDY GROUPS (%) GROUPS OFSTUDY DISTRIBUTION 0% ARECANUT GROUP I GROUP II GROUP III GROUP IV CHEWING & ALCOHOLIC HABITS BEVERAGES

GROUP I – NORMAL MUCOSA GROUP II – MILD EPITHELIAL DYSPLASIA GROUP III – MODERATE & SEVERE DYSPLASIA GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 4: DISTRIBUTION OF SITE IN THE STUDY GROUPS (N=40)

SITE BUCCAL TONGUE GINGIVA RETRO LIP VESTIB ANGLE p Value MUCOSA MOLAR ULE OF MOUTH GROUP I 0 0 5 (50%) 5 (50%) 0 0 0 (n=10) p=0.000* GROUP II 5 (50%) 2 (20%) 0 0 1 1 (10%) 1 (10%) (n=10) (10%) GROUP 5 (50%) 4 (40%) 1 (10%) 0 0 0 0 III (n=10) GROUP 10 (100%) 0 0 0 0 0 0 IV (n=10)

*p Value < 0.05 is significant

GRAPH 4: DISTRIBUTION OF SITE IN THE STUDY GROUPS (N=40)

100%

1

0.9

0.8

0.7 BUCCAL MUCOSA 0.6 TONGUE 50% 50% 50% 50% GINGIVA 0.5 40% RETRO MOLAR 0.4 LIP 0.3 VESTIBULE 20% 0.2 ANGLE OF MOUTH 10% 10% 10% 10% 0.1 0 0 0 0 0 0 0 0 0 0 0 0% 0 0 0 0 0 DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION 0 GROUP I GROUP II GROUP III GROUP IV SITE OF LESIONS

GROUP I – NORMAL MUCOSA GROUP II – MILD EPITHELIAL DYSPLASIA GROUP III – MODERATE & SEVERE DYSPLASIA GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 5 : αvβ6 INTEGRIN STAINING INTENSITY BETWEEN THE STUDY GROUPS (N=40)

STAINING PRESENT ABSENT pVALUE GROUP I (n=10) 80% 20% GROUP II, (n=10) 30% 70% p=0.003 GROUP III (n=10) 40% 60% GROUP IV (n=10) 0% 100%

* p value < 0.05 is significant

GRAPH 5: αvβ6 INTEGRIN STAINING INTENSITY BETWEEN THE STUDY GROUPS (N=40)

100% 100%

90% 80% 80% 70% 70% 60% 60%

50% 40% PRESENT 40% 30% ABSENT 30% 20% 20%

10% 0%

DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION 0% GROUP I (n=10) GROUP II, GROUP III GROUP IV (n=10) (n=10) (n=10) STAINING INTENSITY

GROUP I – NORMAL MUCOSA GROUP II – MILD EPITHELIAL DYSPLASIA GROUP III – MODERATE & SEVERE DYSPLASIA GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 6: COMPARISON OF αvβ6 INTEGRIN STAINING INTENSITY BETWEEN THE STUDY GROUPS (N=40)

αvβ6 INTEGRIN NEGATIVE WEAK MODERATE STRONG p Value INTENSITY GROUP I n=10 10 (100%) 0% 0 0 GROUP II n=10 7 (70%) 1(10%) 0% 2 (20%) p=0.031* GROUP III n=10 6 (60%) 1(10%) 3(30%) 0 GROUP IV n=10 2 (20%) 3(30%) 2(40%) 3(30%)

* p value < 0.05 is significant

GRAPH 6: COMPARISON OF αvβ6 INTEGRIN STAINING INTENSITY BETWEEN THE STUDY GROUPS (N=40)

100%

100%

90% WEAK 80% 70% MODERATE 70% 60% STRONG 60% NEGATIVE 50%

40% 30% 30% 30% 30% 20% 20% 20% 20% 10% 10% 10%

DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION 0% 0% 0% 0% 0% 0% GROUP I GROUP II GROUP III GROUP IV STAINING OF αvβ6 INTEGRIN INTENSITY

GROUP I – NORMAL MUCOSA

GROUP II – MILD EPITHELIAL DYSPLASIA

GROUP III – MODERATE & SEVERE DYSPLASIA

GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 7: TISSUE LOCALISATION IN αvβ6 INTEGRIN POSITIVE STAINED CELLS IN THE STUDY GROUPS (N=40)

TISSUE FOCAL LINEAR SUPRA FULL P Value LOCALIZATION NEGATIVE BASAL BASAL BASAL EPITHELIUM GROUP I n=10 10(100%) 0 0 0 0 p=0.049* GROUP II n=10 7(70%) 0 0 1(10%) 2(20%) GROUP III n=10 6(60%) 0 0 0 4(40%) GROUP IV n=10 2(20%) 1(10%) 1(10%) 3(30%) 3(30%) * p value < 0.05 is significant

GRAPH 7: TISSUE LOCALISATION IN αvβ6 INTEGRIN POSITIVE STAINED CELLS IN THE STUDY GROUPS (N=40)

100%

100% FULL EPITHELIUM

90% BASAL & SUPRA BASAL 80% 70% LINEAR BASAL 70% 60% FOCAL BASAL 60% NEGATIVE 50% 40% 40% 30% 30% 30% 20% 20% 20% 10% 10% 10% 10%

0% 0% 0% 0% 0 0% 0 0 0% DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION 0% GROUP I GROUP II GROUP III GROUP IV TISSUE LOCALISATION IN αvβ6 INTEGRIN

GROUP I – NORMAL MUCOSA

GROUP II – MILD EPITHELIAL DYSPLASIA

GROUP III – MODERATE & SEVERE DYSPLASIA

GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 8: COMPARISON OF αvβ6 INTEGRIN OF THE SCORE OF INTENSITY BETWEEN NORMAL AND GRADES OF EPITHELIAL DYSPLASIA (GROUP I , GROUP II & III) (N=30)

GROUPS NEGATIVE LOW HIGH p Value (0) (1-4) (5-7) GROUP I n=10 10 (100%) 0 0 p=0.089 GROUP II n=10 7 (70%) 0 3 (30%) GROUP III n=10 6 (60%) 0 4 (40%)

GRAPH 8: COMPARISON OF αvβ6 INTEGRIN OF THE SCORE OF INTENSITY BETWEEN NORMAL AND GRADES OF EPITHELIAL DYSPLASIA (GROUP I , GROUP II & III) N=30

100%

100%

90%

80% 70% 70% 60% 60% NEGATIVE 50% 40% HIGH 40% LOW 30% 30%

20%

10% 0% 0 0% 0

DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION 0% GROUP II GROUP III GROUP I αvβ6 INTEGRIN INTENSITY

GROUP I – NORMAL MUCOSA

GROUP II – MILD EPITHELIAL DYSPLASIA

GROUP III – MODERATE & SEVERE DYSPLASIA

Tables and Graphs

TABLE 9: COMPARISON OF αvβ6 INTEGRIN THE SCORE OF INTENSITY BETWEEN NORMAL AND ORAL SUBMUCOUS FIBROSIS (GROUP I & GROUP IV) N=20

GROUPS NEGATIVE LOW HIGH p Value (0) (1-4) (5-7) GROUP I n=10 10 0 0 GROUP IV n=10 2 (20%) 4 (40%) 4 (40%) p=0.001*

* p value < 0.05 is significant

GRAPH 9: COMPARISON OF αvβ6 INTEGRIN THE SCORE OF INTENSITY BETWEEN NORMAL AND ORAL SUBMUCOUS FIBROSIS (GROUP I & GROUP IV) N=20

120%

100% 100%

NEGATIVE 80% LOW

HIGH 60%

40% 40% 40%

20%

20% DISTRIBUTION OF STUDY GROUPS (%) GROUPS STUDY OF DISTRIBUTION 0 0 0% GROUP I GROUP IV αvβ6 INTEGRIN INTENSITY

GROUP I – NORMAL MUCOSA

GROUP IV – ORAL SUBMUCOUS FIBROSIS

Tables and Graphs

TABLE 10 : DISTRIBUTIONS OF INTENSITY, LOCALIZATION OF αvβ6 INTEGRIN EXPRESSION IN PATIENTS WITH AND WITHOUT ARECANUT CHEWING (N=40)

Tissue Localization Intensity Focal linear Supra Full HABIT Negative Weak Moderate Strong basal basal basal epithelium p VALUE WITH ARECANUT 4 2 5 3 1 4 5 p=0.019* (n=14) 35% (29%) (14%) (36%) (21%) (7%) 0.00% (29%) (36%) WITHOUT ARECANUT 21 3 1 1 1 4 (n=26) 65% (80%) (12%) (4%) (4%) 0 (4%) 0 (15%)

* p value < 0.05 is significant

GRAPH 10: DISTRIBUTIONS OF INTENSITY, LOCALIZATION OF αvβ6 INTEGRIN EXPRESSION IN PATIENTS WITH AND WITHOUT ARECANUT CHEWING (N=40)

80.00% 80.00%

70.00% WITH ARECANUT (n=14)

60.00% WITHOUT ARECANUT (n=26) 50.00%

40.00% 36.00% 36.00% 29.00% 29.00% 30.00% 21.00% 14.00% PERCENTAGE (%) PERCENTAGE 20.00% 15.00% 12.00% 7.00% 10.00% 4.00% 4.00% 4.00% 0 0.00% 0 0.00%

INTENSITY TISSUE LOCALIZATION

Figures

ANTIBODY KIT

PRIMARY ANTIBODY SECONDARY ANTIBODY (αvβ6 INTEGRIN)

ARMAMENTARIUM

Figures

POSITIVE CONTROL - PLACENTA (HUMAN) 10X (αvβ6 INTEGRIN) 40X (αvβ6 INTEGRIN)

NORMAL MUCOSA (HUMAN)

10X (αvβ6 INTEGRIN) 40X (αvβ6 INTEGRIN)

CYTOPLASMIC EXPRESSION Of αvβ6 INTEGRIN

Figures

EPITHELIAL DYSPLASIA (MILD) 10X (αvβ6 INTEGRIN) 40X (αvβ6 INTEGRIN)

EPITHELIAL DYSPLASIA (MODERATE)

10X (αvβ6 INTEGRIN) 40X (αvβ6 INTEGRIN)

CYTOPLASMIC EXPRESSION Of αvβ6 INTEGRIN

Figures

EPITHELIAL DYSPLASIA (SEVERE)

10X (αvβ6 INTEGRIN) 40X (αvβ6 INTEGRIN)

ORAL SUBMUCOUS FIBROSIS

10X (αvβ6 INTEGRIN) 40X (αvβ6 INTEGRIN)

CYTOPLASMIC EXPRESSION Of αvβ6 INTEGRIN

Discussion

Discussion

DISCUSSION

The purpose of the present study was to investigate the expression of αvβ6 integrin by epithelial cells in epithelial dysplasia and oral submucous fibrosis and its association with malignant transformation of these lesions. Integrins are a family of cell surface receptors that mediate cell–cell and cell–extracellular matrix adhesion in various cell types including epithelial keratinocytes. αvβ6 integrin is reported to have multiple roles in the malignant transformation, including cell adhesion and migration, regulation of cell growth and inflammation, and signalling of matrix degradation2.

Oral epithelial dysplasia is a potentially malignant lesion that has the potential to undergo malignant transformation (WHO, 1997). As high as 17% of the lesions may progress to malignant lesions of the oral cavity. In epithelial dysplasia,

αvβ6 integrin activates the TGF-β1 through its interaction with the αvβ6 ligands such as fibronectin, tenascin, vitronectin and latency-associated peptide (LAP) of the cytokine,which in turn activate the basal keratinocytes in the progression of dysplastic lesion to malignancy2.

In oral submucous fibrosis,the pathogenesis involves increased accumulation of collagen within the lamina propria of the oral mucosa, eventually leading to scarring of underlying muscle. Binding of αvβ6 integrin to the LAP of both TGF-β1 and TGF-β3 results in activation of the cytokines, which promotes pathogenic organ fibrosis. Increased αvβ6 integrin expression promotes keratinocyte migration and induced invasion, suggesting that the αvβ6 integrin may play a role in malignant transformation.Approximately 7-13% of OSF cases have been reported to progress to

Oral Squamous Cell Carcinoma5.

36

Discussion

Archival blocks were taken from the department for the present study. The study population includes 40 cases. They were categorized into four groups. Group I

(n=10) comprised of clinically normal mucosa samples which was obtained from retromolar and gingiva region of the patients who came for removal of teeth and crown lenghthening. Group II (n=10) comprised of mild epithelial dysplasia samples.

Group III(n=10) comprised of moderate and severe epithelial dysplasia as a single group. Group IV (n=10) comprised of oral submucous fibrosis samples.

According to Lee CH et al the average diagnostic age of subjects in their study who were suffering from epithelial dysplasia was 47.97 ±11.8 years. In the present study the average diagnostic age of epithelial dysplasia was 56±10.5 years. the average diagnostic age of subjects suffering from OSF was 39 ±12 years27. In the present study the average diagnostic age of oral submucous fibrosis was

44±11.5years. Our study was consistent with the Lee CH et al study and the pattern of age distribution between the groups was statistically significant.

In all the groups (II, III and IV), males were more affected than females. The overall male and female ratio was 2:1. According to Lee CH27et al study 94% of subjects with epithelial dysplasia were males. In our study the male to female ratio was 1:1.Our finding was consistent with that of Antony George64 et al who had a similar equal prevalence in both males and females. This could be due to the increased habitual use of tobacco and alcohol among women 64 .

According to Lee et al betel quid chewing was found to be the strongest risk factor associated with both dysplasia and OSF. Along with cigarette smoking this risk is significantly elevated. The attributable risk of dysplasia developing from betel quid chewing was 73.2% and 85.4% respectively. Combined with cigarette smoking the risk of developing dysplasia increased to 84.4%.27

37

Discussion

In the present study 40% of epithelial dysplasia had the habit of smoking alone and

50% of epithelial dysplasia patients had the habit of tobacco chewing and smoking and 30% of epithelial dysplasia patients had the habit of arecanut and tobacco chewing. None of the patients in the control group had habits

Integrins are the principal cell surface receptors the enable both normal and transformed cells to attach to and respond to their extracellular environment. They mediate cell to cell or cell to extracellular matrix adhesion providing adhesion for stationary cells. αvβ6 is a fibronectin binding protein. It has both a cytoplasmic and a transmembrane domain43.

In this study, in 30% of cases in group II (mild dysplasia) 40% in group III

(moderate and severe dysplasia) and 80% of group III (OSF) cases expressed αvβ6 integrin. In the control group, comprising of normal oral mucosa, αvβ6 was not expressed in all the cases. Thus there was a significant difference in expression of

αvβ6 between normal mucosa and dysplastic mucosa and OSF. This non expression in normal epithelium is in concurrence with the study done by Mirza et al 11who states that αvβ6 is an epithelial specific integrin that is not expressed on healthy oral epithelium but is upregulated during tissue remodeling, including wound healing and carcinogenesis11.

A percentage of dysplastic epithelial lesions are known to undergo malignant transformation. Expression of αvβ6 integrin has a role in the tumorigenesis and in epithelial repair. Its induction in tumorigenensis could be attributed to its capacity to bind to fibronectin and tenascin in the extracellular matrix.

Of the 30 % of cases, of mild epithelial dysplasia, 2 cases showed expression in the entire epithelium of which one case with the entire epithelium had strong

38

Discussion

intensity and one case showed staining of weak intensity. The staining index based on intensity and localization, was done both these two patients had high scoring and these two patients had the habit of smoking for over a period of 20 years

One case of mild epithelial dysplasia with strong expression of αvβ6 and high scoring. This patient had areca nut chewing habit along with smoking for only three years. Of the 40% of cases of moderate and severe dysplasia, which expressed αvβ6,

2 patients had staining of the entire epithelium of moderate intensity and high scoring index. These two cases had areca nut chewing habit along with tobacco chewing.

20% of the cases with moderate dysplasia were on the lateral border of the tongue and these two cases presented clinically as an ulcer and did not have habits. These two cases showed expression in the entire epithelium. Though one case showed moderate intense staining and one showed weak staining, their overall scoring index was high.

This could be attributed to the fact that these two cases presented as ulcers and the inflammatory component and the healing process of the ulcers could have induced the high intense staining of αvβ611

Of the 80% of cases of oral submucous fibrosis which expressed αvβ6, 70 % of the cases showed moderate to strong expression. The staining localization varied from basal to suprabasal to entire epithelium in these cases. In those cases where the expression was seen in the entire epithelium, high scoring index was observed and in those where basal and supra basal expression was seen, the scoring index was low.

The mean duration of the habit of chewing arecanut, in these patients was 10±7 and the mean age was 39±12. From the findings of this study, we state that irrespective of the age and the duration of the habit, those who had the habit of chewing arecanut expressed αvβ6 and in those cases where the expression was seen in the entire

39

Discussion

epithelium, high scoring index was seen compared to those cases where it was expressed only in basal and supra basal, who had low scoring scoring index.

On comparing the intensity between areca-nut users and non-users, it was observed that of the 14 areca nut users, 10 (72%) had expression of αVβ6 integrin expression of varying degrees while 21 (81%) of the 26 non-areca nut users exhibited no expression of αVβ6 integrin expression. This indicates that use of areca nut has a definitive role to play in the αVβ6 integrin expression. This phenomenon has been previously suggested by several others including Moutasim KA et al in 20115. They demonstrated that oral keratinocytes cell lines exposed to areca nut extracts upregulates the integrins and αVβ6 integrin expression as well as its functions. They deciphered the involvement and modulation of TGF-β1 by αVβ6 integrin expression and subsequent myofibroblast transdifferentiation. These modifications could cause matrix accumulation, both by increasing the deposition such as collagen-I, the most common collagen to be involved in OSMF, and also by decreasing the degradation of matrix proteins such as TIMP-1. The present study is in consistent with these reports.

Our study results show that areca- nut exposure is one of the crucial factor (p value =

0.019 ), which could explain the high expression of αVβ6 integrin in those cases who had this habit compared to those who did not have the habit and five of these cases, three showed weak expression. On comparing the tissue localization, αVβ6 integrin expression was observed in full epithelium in 5 cases (50%), 4 (40%) in supra-basal layers and focally in 1 (10%) of the areca nut users. In non-areca nut users, 66.66%

(n=4) involved full epithelium. This was statistically significant. (Table -9). This finding was consistent with the published literature. Our findings could be explained as follows, areca nut use causes repeated, chronic microtrauma to the buccal mucosa,

40

Discussion

possibly via the sharp, coarse fibers67. The attempt of the epithelium to repair following the injury should cause an increased expression of αVβ6 integrin or alternatively, only certain epithelial cell phenotypes that express αVβ6 integrin may be altered and possibly control this epithelium driven inflammation. Other cytokines such as TGFβ1 could also be implicated in this mechanism.2

The increased αVβ6 integrin expression in terms of intensity and localization in areca nut chewers, as compared to non-areca nut chewers indicate that αVβ6 integrin expression and areca nut chewing plays a pivotal role in determining the inflammation process via the TGFβ1 and myofibroblast transdifferentiation. This could be one of the pathway by which the abnormal fibrosis in OSF is caused. Also, the elevated αVβ6 integrin expression in areca nut chewers could be the earliest hall mark of deviation from normalcy.

Considering the other stratifications in the study like age, dysplasia etc: the difference oral dysplasia expression was significant for arecanut use emphasizing its role in epithelial alteration as one of the initial events which leads to fibrosis eventually through TGF beta and SMAD68

41

Summary and Conclusion

Summary and Conclusion

SUMMARY AND CONCLUSION

 A total of 40 biopsies were included in this study, comprising of 10

cases of normal mucosa, 10 cases of mild dysplasia, 10 cases of

moderate and severe dysplasia, 10 cases of oral submucous fibrosis.

 The pattern of αvβ6 integrin staining in all the groups was cytoplasmic.

 In our study, subjects with mild dysplasia and subjects with moderate

and severe dysplasia showed 30% and 40% αvβ6 integrin positivity

respectively and also 80% of oral submucous fibrosis showed αvβ6

integrin positivity

 None of the normal subjects expressed αvβ6 integrin. In mild dysplasia

cases, moderate and severe dysplasia cases and in subjects with oral

submucous fibrosis, αvβ6 integrin positivity was expressed in 30%,

40% and 80% of cases respectively. This pattern of staining between

the groups was statistically significant (p=0.031).

 The difference in localization between epithelial dysplasia and oral

submucous fibrosis was statistically significant (p=0.049). In subjects

with mild dysplasia, all the cases showed high scoring index (p=0.089)

and in oral submucous fibrosis, 40% of cases showed high intensity,

40% showed low intensity (p=0.001) compared to normal cases.

 72% of those who used areca nut had expression of αvβ6 integrin but

only 19% of the non-areca nut chewers expressed αvβ6 integrin

(p=0.019).

42

Summary and Conclusion

 Our findings suggest that areca nut influences αvβ6 integrin

expression. The increase in αvβ6 integrin expression could influence

myofibroblast transdifferentiation through TGF β activation.

43

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