“CYCLIN D1 EXPRESSION IN ODONTOGENIC

KERATOCYSTS, DENTIGEROUS CYSTS AND

RADICULAR CYSTS A COMPARATIVE
IMMUNOHISTOCHEMICAL STUDY”

By

DR. YAMUNA . B. MACHA

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Dissertation submitted to the
Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka,
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In partial fulfillment of the requirements for the degree of

MASTER OF DENTAL SURGERY

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IN
ORAL PATHOLOGY AND MICROBIOLOGY

Under the guidance of

Dr. MAHARUDRAPPPA BASNAKER
M.D.S
Professor & HOD

DEPARTMENT OF ORAL PATHOLOGY AND

MICROBIOLOGY,
H.K.E’S S.N. INSTITUTE OF DENTAL SCIENCES AND
RESEARCH, KALABURAGI, KARNATAKA.

BATCH 2013-16
 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA

DECLARATION BY THE CANDIDATE

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I hereby declare that this dissertation entitled “CYCLIN D1
EXPRESSION IN ODONTOGENIC KERATOCYSTS,
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DENTIGEROUS CYSTS AND RADICULAR CYSTS” - A
COMPARATIVE IMMUNOHISTOCHEMICAL STUDY is a
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bonafide and genuine research work carried out by me under the

guidance of Dr. MAHARUDRAPPA BASNAKER, HOD, Professor &
Guide, Department of oral pathology and microbiology, H.K.E.S’s S.N.
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INSTITUTE OF DENTAL SCIENCES AND RESEARCH, Kalaburagi,

Karnataka.

Date: Signature of the Candidate

Place: Kalaburagi Dr. Yamuna B Macha

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 CERTIFICATE BY THE GUIDE

This is to certify that this dissertation entitled “CYCLIN D1

EXPRESSION IN ODONTOGENIC KERATOCYSTS,

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DENTIGEROUS CYSTS AND RADICULAR CYSTS” - A

COMPARATIVE IMMNOHISTOCHEMICAL STUDY is a bonafide

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research work done by Dr. YAMUNA B. MACHA in partial fulfillment
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of the requirement for the degree of MASTER OF DENTAL

SURGERY in ORAL PATHOLOGY AND MICROBIOLOGY.

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Date: Dr. MAHARUDRAPPA BASNAKER

Place: Kalaburagi Professor & HOD,
Department of Oral Pathology and
Microbiology,
H.K.E.S.’s S.N Institute of Dental
Sciences & Research,
Kalaburagi- 585105
Karnataka

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 ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE

INSTITUTION

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This is to certify that this dissertation entitled “CYCLIN D1
IE
EXPRESSION IN ODONTOGENIC KERATOCYSTS,
DENTIGEROUS CYSTS AND RADICULAR CYSTS A
EV

COMPARATIVE IMMUNOHISTOCHEMICAL STUDY is a

bonafide research work done by Dr. YAMUNA B MACHA under the
guidance of Dr. MAHARUDRAPPA BASNAKER, Guide, Professor
PR

& HOD, Department of oral pathology and microbiology, H.K.E.S’s .S.N.

Institute of Dental Sciences And Research, Kalaburagi.

Dr. MAHARUDRAPPA BASNAKER Dr. JAYASHREE MUDDA

Professor & Head, Principal,
Department of Oral Pathology Department of Periodontics
and Microbiology, H.KE.S’s. S.N Institute of
Dental Sciences & Research,
Kalaburagi

Date: Date:
Place: Kalaburagi Place: Kalaburagi

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 COPY RIGHT

DECLARATION BY THE CANDIDATE

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I hereby declare that the Rajiv Gandhi University of Health
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Sciences, Karnataka, shall have the rights to preserve, use and

disseminate this dissertation in print or electronic format for

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academic/research purposes.
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Date: Dr. YAMUNA B MACHA

Place: Kalaburagi

© Rajiv Gandhi University of Health Sciences, Karnataka

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 ACKNOWLEDGEMENT

I thank and praise the Almighty and my Parents for guiding me and helping

me throughout this endeavor and for all the goodness and mercy showered upon me

I take this opportunity to express my profound gratitude and deep regards to

my beloved guide Dr. MAHARUDRAPPA BASNAKER, Professor and HOD,

Department of oral pathology and microbiology, H.K.E.S’s S. N. Dental College,

Gulbarga, for his exemplary guidance, monitoring and constant encouragement

throughout the course of this thesis. The blessing, caring and excellent guidance given

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by his time to time shall carry me a long way in the journey of life on which I am

about to embark.
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I also take this opportunity to express a deep sense of gratitude to,

Dr. SATISH B.N.V.S., Professor, Department of oral pathology and microbiology

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for his cordial support, valuable information and guidance, which helped me in

completing this task through various stages.

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I am very much grateful to Dr. Prashant Kammar, Reader, Dr. Shrikala,

Dr. Niranjan Dr. Shashikant and Dr Allad for the valuable information provided

by them and for their continuous encouragement, and timely support.

I thank all my fellow PG friends, my batchmates Dr. Sunitha, Dr. Shruti; my

juniors, Dr.Sharan, Dr. Huma, Dr. Juveria , Dr. Keshav, Dr. Basavaprabhu,

Dr. Aishvarya for their constant encouragement and unending help. They have been

extremely helpful whenever I approached them. I am very much grateful to my

friends Dr Smita, Dr Savita and Harsha for their constant encouragement and

unending help.

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 I am grateful to Library Staff Mrs. Sharmila for her support in the library

and non teaching staff for their constant help and I am extremely thankful to

Mrs. Jyothi, Bio-Statistician, for her work and providing a final shape to my study.

I am very much indebted to this department and extend my appreciation to all the

Non-teaching staff of the Dept, Sangmesh Aralimar, Ramesh and Santosh who

have helped me directly or indirectly in making this study possible.

Words cannot express how much my husband has done for me. I thank him
whole heartedly for his love, understanding, support, blessings and sacrifices. I am
very grateful to my children Purvi and Purab , My husband Mr. Sushrut V.
Kareddy, my in-laws Dr Vishwanath Kareddy, Dr Vishalaxi Kareddy and my

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parents for their unwavering love and moral support at all times. Their words of
encouragement have been a constant source of inspiration.
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The list of people who have helped me directly or indirectly in preparing this
dissertation is endless and my apologies go with my thanks to all those I have omitted.
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Date: Dr. YUMUNA B. MACHA

Place: Kalaburagi

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 Dedicated

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to
My
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Beloved Parents
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&
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Teachers

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 TABLE OF CONTENTS

S. No. Title Page No.

1. Introduction 1-3

2. Aims and Objectives 4

3. Review of Literature 5-41

4. Materials and Methods 42-55

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5. Results 56-64

6. Discussion 65-84
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7. Summary and Conclusions 85-87
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9. Bibliography 88-98

10 Annexures 99-101
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 LIST OF TABLES

sr.no. Tables Page no.

1. Distribution of Cases According to Age 56

2. Distribution of Cases According to Gender 57

3. Distribution of cases according to site 58

Distribution of expression, staining pattern, staining 59

4. intensity and staining localization of cyclin d1 in
odontogenic cysts. Comparison by chi square test

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 LIST OF FIGURES

Sr. Page
Figures
No. No.

1. Instruments and armamentarium 50

2. H and E stained section of Odontogenic kertocyst 10X 71

3. H and E stained section of Odontogenic kertocyst 40X 71

4 Cyclin D1 expression in Odontogenic kertocyst mild expression 10X 72

5. Cyclin D1 expression in Odontogenic kertocyst mild expression 40X 72

6. Cyclin D1 expression in Odontogenic kertocyst moderate expression 10X 73

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7. Cyclin D1 expression in Odontogenic kertocyst moderate expression 40X 73

8. Cyclin D1 expression in Odontogenic kertocyst intense expression 10X 74

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9. Cyclin D1 expression in Odontogenic kertocyst intense expression 40X 74

10. H & E stained of daughter cyst in Odontogenic kertocyst 10X 75

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11. H & E stained of daughter cyst in Odontogenic kertocyst 40X 75

12. Cyclin D1 expression in daughter cyst of Odontogenic kertocyst 10X 76

13. H and E stained section of dentigerous cyst 10X 77

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14. H and E stained section of dentigerous cyst 40X 77

15. Cyclin D1 expression in dentigerous cyst mild expression 10X 78

16. Cyclin D1 expression in dentigerous cyst mild expression 40X 78

17. Cyclin D1 expression in dentigerous cyst moderate expression 10X 79

18. Cyclin D1 expression in dentigerous cyst moderate expression 40X 79

19. Cyclin D1 expression in dentigerous cyst intense expression 10X 80

20. Cyclin D1 expression in dentigerous cyst intense expression 40X 80

21. H and E stained section of radicular cyst 10X 81

22. H and E stained section of radicular cyst 40X 81

23. Cyclin D1 expression in radicular cyst mild expression 10X 82

24. Cyclin D1 expression in radicular cyst mild expression 40X 82

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25. Cyclin D1 expression in radicular cyst moderate expression 10X 83

26. Cyclin D1 expression in radicular cyst moderate expression 40X 83

27. Cyclin D1 expression in radicular cyst intense expression 10X 84

28. Cyclin D1 expression in radicular cyst intense expression 40X 84

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 LIST OF GRAPHS

Page
Sr.No. Graphs
No.

1. Distribution of cases according to age 56

2. Distribution of cases according to gender 57

3. Distribution of cases according to site 58

4 Distribution of cases according to pattern 60

5. Distribution of cases according to staining intensity 61

6. Distribution of cases according to staining localization 61

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 Introduction

INTRODUCTION

Definition of cyst: According to Kramer (1974) it's defined as a “pathological

cavity having fluid, semifluid or gaseous contents and which is not created by the

accumulation of pus”. Most cysts, but not all are lined by epithelium.1

Historical Perspective of Cyst

Cysts of the jaws and maxillofacial regions are not new lesions. There is

evidence of cystic lesions in the jaws of humans and other animals in the distant past.

Lesions of the jaws interpreted as cysts have been found in mummified specimens

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from the predynastic era.(c.4500 B.C) And from the fifth dynasty ( c.2800 B.C) in
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Egypt. Early description of cystic lesions of the jaws were written by Aulus Cornelius

Celsus (early part of first century), Pierre Fauchard (1690-1762) and John Hunter
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(1729-1793), among others.

From 1850, papers on the nature and treatment of jaw cysts became more
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frequent. Attempts to understand the relationship between various morphological

types of cysts led to classifications such as Paul Broca’s classification of odontomas

(1866) which included odontogenic tumors cysts and malformations 1.

Different types of odontogenic cysts and tumors arise from derivatives of

embryologic dental lamina. Three major categories of odontogenic cysts are usually

distinguished, radicular cysts, follicular or dentigerous cysts and odontogenic

keratocysts. Keratocysts account for 3% to 11% of odontogenic cysts and it is well

known that they show aggressive biological behaviour with higher rates of recurrence

than other types of odontogenic cysts and a tendency to invade adjacent tissue.

Chirapathomsakul D et al; (2006).2

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 1
 Introduction

Odontogenic cysts are further classified as developmental type or of

inflammatory origin with variable clinical and biological behaviour. Radicular cyst is

an inflammatory odontogenic cyst which is derived from the epithelial rest of

malassez, where as dentigerous cyst, odontogenic keratocyst and glandular

odontogenic cyst are developmental in origin.3

Rapid progress has been made in the last few years in elucidating the

molecular mechanisms underlying cell cycle regulation in mammalian cells. A four

phases of cell cycle is identified i,e G1, S, G2 and M. In G1 phase, the cells undergo a

period of growth. S phase is a period of DNA synthesis. In G2 phase preparations are

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made for cell division. In M phase, cell goes through prophase, metaphase, anaphase

and telophase and splits into two daughter cells, thus completing the cell cycle.
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The orderly progression of the cells through the cell cycle is precisely
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governed by a series of proteins called “cyclins”, whose influences affect the binding

and activation of the cyclin dependent kinase (CDK).This process is further regulated
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by phosphorylation, tumor suppressor genes and inhibited by cyclin dependent kinase

inhibitors.4

The cyclin D1 protein plays an important role in G1-S transition phase of

cycle. Cyclin D1 is one of the Rb pathway proteins with oncogenic properties which

controls G1-S transition.

Transition between different stages of cell cycle is regulated at checkpoints.

Several checkpoints are regulated by cyclin dependent kinases and their activating

partners , the cyclins. Cyclin D1 gene is located on chromosome 11q13, encodes a

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 2
 Introduction

critical cell cycle regulatory protein (Cyclin D1) that drives the cell cycle from G1

phase to S phase.

Amplification and expression of cyclin D1 gene has been reported in

various carcinoma, so the study of cyclin D1 expression may improve our

knowledge about the biological substrate of Odontogenic keratocyst behaviour

Robbins S et al , Pathologic basis of disease.5

Elevated levels of this protein might allow cells to escape from the cell

cycle checkpoint control and play an important role in tumorigenesis.

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Till date , only few studies related to cyclin D1 expression in odontogenic

cysts are conducted. The present study is undertaken to compare the staining
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pattern and intensity of expression of cyclin D1 in odontogenic keratocysts,
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dentigerous cysts and radicular cysts to ascertain biologic behaviour of these cyst

individually.
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H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 3
 Aims & Objectives

AIMS AND OBJECTIVES

1. To observe and evaluate the immunohistochemical staining pattern and

intensity of cyclin D1 expression in odontogenic keratocysts.

2. To observe and evaluate the immunohistochemical staining pattern and

intensity of cyclin D1 in expression in dentigerous cysts.

3. To observe and evaluate the immunohistochemical staining pattern and

intensity of cyclin D1 in the expression in radicular cysts.

4. To statistically compare and evaluate the expression of cyclin D1 among the

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three odontogenic cysts types namely keratocysts, dentigerous cysts and in

radicular cysts.
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H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 4
 Review of Literature

REVIEW OF LITERATURE

Odontogenic Cysts

The odontogenic cysts are derived from the epithelium associated with the

development of the dental apparatus. They represent an aberration at some stage of

odontogenesis.1

a. CLASSIFICATION OF JAW CYSTS

According to Shear 1

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Cysts of the jaws

● EPITHELIAL-LINED CYSTS
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Developmental origin
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1. Odontogenic

a. Gingival cyst of infants

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b. Odontogenic keratocyst

c. Dentigerous cyst

d. Eruption cyst

e. Gingival cyst of adults

f. Developmental lateral periodontal cyst

g. Botryoid odontogenic cyst

h. Glandular odontogenic cyst

i. Calcifying odontogenic cyst

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 5
 Review of Literature

2. Non-odontogenic

a. Midpalatal raphé cyst of infants

b. Nasopalatine duct cyst

c. Nasolabial cyst

Inflammatory origin

a. Radicular cyst, apical and lateral

b. Residual cyst

c. Paradental cyst and juvenile paradental cyst

d. Inflammatory collateral cyst

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● NON EPITHELIAL LINED CYSTS

1. Solitary bone cyst

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2. Aneurysmal bone cyst
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II Cysts associated with the maxillary antrum

1. Mucocele
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2. Retention cyst

3. Pseudocyst

4. Postoperative maxillary cyst

III Cysts of the soft tissues of the mouth, face and neck

1. Dermoid and Epidermoid cysts.

2. Lymphoepithelial (branchial) cyst

3. Thyroglossal duct cyst

4. Anterior median lingual cyst (intralingual cyst of Foregut origin)

5. Oral cysts with gastric or intestinal epithelium (oral alimentary tract cyst)

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 6
 Review of Literature

6. Cystic hygroma

7. Nasopharyngeal cyst

8. Thymic cyst

9. Cysts of the salivary glands: mucous extravasation cyst; mucus retention cyst;

ranula; polycystic (dysgenetic) disease of the parotid

10. Parasitic cysts: hydatid cyst; Cysticercus cellulosae; trichinosis

ACCORDING TO SHAFER 14

CLASSIFICATION BY ETIOLOGY

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1) DEVELOPMENTAL: Unknown origin but are not the result of an

inflammatory reaction
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1) Dentigerous cyst
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2) Eruption cyst

3) Odontogenic keratocyst

4) Gingival cyst of newborn

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5) Gingival cyst of adult

6) Lateral periodontal cyst

7) Glandular odontogenic cyst

2) INFLAMMATORY: Result of inflammation

1) Periapical cyst

2) Residual cyst

3) Paradental cyst

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 7
 Review of Literature

CLASSIFICATION BY TISSUE OF ORIGIN

A. DERIVED FROM RESTS OF MALASSEZ

a. Periapical cysts

b. Residual cyst

B. DERIVED FROM REDUCED ENAMEL EPITHELIUM

a) Dentigerous cyst

b) eruption cyst

C. DERIVED FROM DENTAL LAMINA (rests of serres)

a) odontogenic keratocysts

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b) gingival cyst of newborn

c) gingival cyst of adult

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d) lateral periodontal cyst

e) glandular odontogenic cyst

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UNCLASSIFIED

1. Paradental cyst

2. Calcifying odontogenic cyst

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 8
 Review of Literature

ODONTOGENIC KERATOCYST (Also called keratocystic odontogenic tumour

Keratocystic Odontogenic Tumor )

Definition :

A benign unicystic or multicystic , intraosseous tumour of odontogenic origin ,

with a characteristics lining of parakeratinized stratified squamous epithelium and

potential aggressive , infiltrative behaviour it may present solitary or multiple.

Multiple form is usually associated with nevoid basal cells carcinoma syndrome

(Nevoid Basal Cell Carcinoma Syndrome) 15.

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Epidemiology :

Keratocystic odontogenic tumors occur from the first to ninth decades of life
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with a peak incidence in the second and third decades respectively. The mean age of
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patients with multiple Keratocystic odontogenic tumors , with or without the Nevoid

Basal Cell Carcinoma Syndrome, is lower than those with single non recurrent

Keratocystic odontogenic tumors. Most series have shown a preponderance in

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males 15.

Etiology :

Recent studies have demonstrated the role of Protein patched homolog gene in

the etiology of Keratocystic odontogenic tumors.

Sites :

The mandible is involved more frequently than the maxilla , with figures

ranging from 65-83%. At about one half of them have been reported to originate at the

angle of the mandible , extending anteriorly and superiorly.

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 9
 Review of Literature

Clinical and radiological features :

The most important clinical feature of Keratocystic odontogenic tumors is its

potential for locally destructive behaviour , its higher recurrence rate , and its

tendency to multiply , particularly when associated with the Nevoid Basal Cell

Carcinoma Syndrome Patients. Patients reported usually complain of pain , swelling

or discharge. These tumours may reach a large size prior to discovery. Keratocystic

odontogenic tumor may penetrate cortical bone and involve adjacent structures 15.

Radiologically Keratocystic odontogenic tumors may appear as small , round

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or ovoid unilocular radiolucencies or may be larger with scalloped margins. A

mandibular radiolucency may involve body , angle and ascending ramus. The
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radiolucencies tend to be well demarcated with distinct sclerotic margins , but may be

diffuse sometimes. Maxillary lesions tend to be smaller , but more extensive

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involvement may occur. True multilocular mandibular lesions are not uncommon.

Adjacent teeth may be displaced but root resorption is a rare phenomenon 15.
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Macroscopy :

On gross examination the cyst linings are thin and fragile , and are usually

collapsed and folded 15.

Histopathology :

The Keratocystic odontogenic tumors are lined by a regular parakeratinized

stratified squamous epithelium , usually about 5-8 cell layers thick and without rete

ridges. There is a well defined , often palisaded , basal layer of columnar or cuboidal

cells. The nuclei of columnar basal cells tend to be oriented away from the basement

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 10
 Review of Literature

membrane and are often basophilic. The parakeratotic layers often have a corrugated

surface. Desquamated keratin is present in many of the cavities. Mitotic figures are

present frequently in the suprabasal layers. Linings are thin and fragile , and are

usually collapsed and folded 15.

Some linings may show features of epithelial dysplasia but malignant

transformation to squamous cell carcinoma is rare. In the presence of intense

inflammatory process , the epithelial lining loses its cellular architectural features.

Cystic jaw lesions that are lined by orthokeratinized epithelium do not form part of

the spectrum of a keratocystic odontogenic tumour 15

Histogenesis :

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It is usually agreed that Keratocystic odontogenic tumor arise from
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odontogenic epithelium. The available evidence points to two main sources of

epithelium , the dental lamina or its remnants and extensions of the basal cells from

the overlying basal cells.

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Genetics :

With regards to their genetic association it has been reported that Nevoid

Basal Cell Carcinoma Syndrome or Protein patched homolog gene has been mapped

to chromosomes 9q22.3 to q31 and which probably is thought to function as a tumour

suppressor. Studies on Nevoid Basal Cell Carcinoma Syndrome and sporadic

Keratocystic odontogenic oumor have provided molecular evidence of a two-hit

mechanism in the pathogenesis of the these tumours demonstrating allelic loss, at two

or more foci , of 9q22 leading to overexpression bc1-2 and Tp53 in the Nevoid Basal

Cell Carcinoma Syndrome. This supports the concept that Keratocystic odontogenic

H.K.E.S’s S.N Dental College, Kalaburagi, Dept. of Oral Pathology & Microbiology 11

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