DENTAL SCIENCE, MATERIALS AND TECHNOLOGY

PATHOPHYSIOLOGY OF ORAL DISEASES

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services. DENTAL SCIENCE, MATERIALS AND TECHNOLOGY

Additional books in this series can be found on Nova’s website under the Series tab.

Additional e-books in this series can be found on Nova’s website under the e-books tab.

DENTAL SCIENCE, MATERIALS AND TECHNOLOGY

PATHOPHYSIOLOGY OF ORAL DISEASES

P. C. ANILA NAMBOODIRIPAD AND E. ANURADHA SUNIL

New York

Copyright © 2016 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher.

We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN.

For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected].

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication.

This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data

Names: Namboodiripad, P. C. Anila. | Anuradha Sunil, E. Title: Pathophysiology of oral diseases / editors, P.C. Anila Namboodiripad and E. Anuradha Sunil (Palissery House, Arimpur, Thrissur District, Kerala State, India). Description: Hauppauge, New York : Nova Science Publisher's, Inc., [2016] | Series: Dental science, materials and technology | Includes index. | Description based on print version record and CIP data provided by publisher; resource not viewed. Identifiers: LCCN 2016017787 (print) | LCCN 2016017080 (ebook) | ISBN 9781634852364 () | ISBN 9781634852203 (hardcover) | ISBN 9781634852364 (ebook) Subjects: LCSH: Mouth--Diseases. | Teeth--Diseases. | Physiology, Pathological. Classification: LCC RC815 (print) | LCC RC815 .P37 2016 (ebook) | DDC 616.3/1--dc23 LC record available at https://lccn.loc.gov/2016017787

Published by Nova Science Publishers, Inc. † New York

Contents

Preface vii Section 1: Developmental Anomalies of Oral Tissues 1 Chapter 1 Developmental Anomalies Affecting Number of Teeth 3 Chapter 2 Developmental Anomalies Affecting Shape of Teeth 7 Chapter 3 Developmental Anomalies Affecting Structure of Teeth 17 Chapter 4 Developmental Anomalies Affecting Tongue 25 Section 2: Benign and Malignant Disorders 29 Chapter 5 Benign and Malignant Epithelial Tissue Tumors 31 Chapter 6 Benign and Malignant Connective Tissue Tumors 41 Chapter 7 Benign and Malignant Tumors of Salivary Glands 57 Chapter 8 Odontogenic Cysts 69 Chapter 9 Odontogenic Tumors 95 vi Contents

Chapter 10 Potentially Malignant Disorders 105 Section 3: Systemic Disorders 109 Chapter 11 Bone Disorders 111 Chapter 12 Disorders of the Skin 129 Section 4: Bacterial, Viral and Fungal Lesions 147 Chapter 13 Bacterial Lesions of Oral Cavity 149 Chapter 14 Viral Infections of the Oral Cavity 157 Chapter 15 Fungal Infections of Oral Cavity 169 Chapter 16 Regressive Alterations of Teeth 175 About the Authors 179 Index 181

Preface

Teaching a large number of ever-inspiring students for the past few years was the motivation for the authors to compile this book. This book would not have seen the light of the day if it were not for them. Knowledge of the pathogenesis of diseases affecting the oral cavity is of great help in understanding its clinical features, radiographic features, histological features as well as the finalization of treatment for those diseases. This knowledge became the reason to compile this book that explains the various pathophysiological causes of the diseases. Some of the diseases are still being explained by their old pathogenesis; however, some others are being attributed to some new causes, following the advent of new and innovative technologies. This book is a sincere attempt to simplify the subject of Oral Pathology by elaborating upon the underlying causes of the various diseases. Apart from students, the author owes her gratitude to Dr. HM Dholakia, who made the subject of Oral Pathology sound so simple; Dr. Alka Kale, whose boundless energy and dynamic personality is an inspiration; Dr. Ramakant Nayak, who harps on the importance of constant updation of knowledge that is required for one to better the subject of Oral Pathology and to the management, principals, and staffs of a large number of dental colleges in India where the idea of the book was concieved. The constant encouragement and support received is the motivation for the author to compose this book.

viii

Finally, the author says a million thanks to her family—especially her parents and daughters—who ungrudgingly bore with her lack of time for them and finally a special thanks to her ever-encouraging husband, without whose help this book would not have seen the light of the day.

The author would also like to thank the God Almighty who gave her the strength to achieve her dream of authoring this book.

Dr PC Anila Namboodiripad, BDS, MDS, Professor, Oral and Maxillofacial Pathology

Section 1: Developmental Anomalies of Oral Tissues

Chapter 1

Developmental Anomalies Affecting Number of Teeth

Introduction

Development of Teeth

Parallel to the lip edge in the late embryonic period (44 days), there is the formation of the epithelial lamella or the primary epithelial band which plays a role in the development of the teeth. This epithelial lamella divides into two parts, one to form the vestibule and the other to give rise to the teeth. The oral vestibule forms on the labial side, by furrowing of the labio-dental lamina, a ridge formed from the epithelial lamella. The furrow is called as the labio- dental sulcus which later forms the oral vestibule. The labio-dental lamina oriented towards the oral cavity gives rise to the dental lamina, which is a U- shaped or horseshoe shaped band from which the teeth arises. The neural crest cells in the form of the ectomesenchyme, and the oral ectoderm interact with each other to give rise to the 10 roundish teeth buds in the upper and lower arches and they represent the primordium of the deciduous teeth. Lingual to these roundish swellings, other swellings form, and they are the anlagen of the permanent teeth. The roundish buds give rise to the bud stage of tooth formation. The buds go through the cap, early bell and advanced bell stages to finally form the enamel organ that forms enamel, the dental papilla, that is enclosed within the invaginated portion of the cap, forming dentin and the pulp, and the dental sac 4 P. C. Anila Namboodiripad and E. Anuradha Sunil which is made up of ectomesenchymal cells derived from the neural crest cells, surrounding the enamel organ, giving rise to the alveolar bone, cementum and the periodontal ligament. Downward extension of the enamel organ determines the morphology of the root. The epithelium that forms the root is the Epithelial root sheath of Hertwig which fragments, and disintegrates, to help in the formation of the outer covering of the root called as the cementum and the formation of root pulp called as the ‘ root canal ‘and the root dentin.

Initiation Proliferation Morpho Apposition Eruption Attrition differen- tiation

Figure 1. Developmental stages in tooth and root formation.

Anodontia

Introduction

When the follicles that needed to grow teeth are completely or partially absent, it results in a condition called as anodontia or oligodontia. This is a congenital abnormality affecting the number of the teeth (normal number of adult teeth- 32, deciduous teeth-20). When all teeth have been extracted either due to caries or pathology, the condition is called as pseudoanodotia.

Pathogenesis

The absence or the mutation in the homeobox genes are said to play a role in anodontia. Homeobox genes are basically genes that play a role in the early embryonic development of various parts of the body including the teeth. They are a set of a large family of related genes that control the formation of many Developmental Anomalies Affecting Number of Teeth 5 structures of the body. They play this role by acting as transcription factors. They consist of 235 functional genes and about 65 pseudo genes i.e., genes that are similar in structure, but that which do not provide instructions for preparation of the proteins. Homeobox genes are normally seen occurring in clusters and are present on every human chromosome. The genes that play a role in anodontia include the PAX, MSX, HOX, etc. Any absence or defect in these genes results in the anodontia. Absence of teeth can also occur in correlation with other developmental anomalies like the oromandibular limb dysgenesis, or in case of the hereditary anhidrotic ectodermal dysplasia etc. Hereditary ectodermal dysplasia has a defective EDA gene and hence is said to be genetic in nature.

Supernumerary Teeth

Introduction

When more than the normal complement of teeth is present in the oral cavity, the condition is called as the Supernumerary teeth or hyperdontia. The extra teeth can be seen at any site in the oral cavity.

Pathogenesis

The etiological role in the formation of supernumerary teeth can be both genetic and environmental. The various theories put forth include:

1) ‘Atavism theory’: This theory suggests that reversion in the phylogenetics of extinct primates where there were three incisors in each quadrant, as against the present day two per quadrant, may be one of the factors in the favor of occurrence of supernumerary teeth. This theory has found no takers. 2) Another theory put forth in favor of supernumerary teeth includes the ‘Dichotomy theory.’ This theory states that supernumerary teeth forms when one tooth bud splits into two, resulting in the formation of two teeth, which are either of equal or of unequal sizes one of which

6 P. C. Anila Namboodiripad and E. Anuradha Sunil

may be dysmorphic or of abnormal shape. Excess teeth of equal size are called as supplementary teeth and if unequal, it is called as supernumerary teeth. 3) The ‘dental lamina hyperactivity theory’ elaborates that the supernumerary teeth may arise from the epithelial remnants of the dental lamina and the supplementary teeth from the lingual extension of the accessory tooth bud. This is the most accepted theory of supernumerary tooth formation 4) Genetic theory: According to this theory the occurrence of the supernumerary teeth is said to run in families. A sex linked inheritance has been suggested. The condition is found to be more common in males than in females.

Supernumerary Roots

Introduction and Pathogenesis

Supernumerary roots are excess roots found in teeth and are more than the number of roots expected. The most common teeth affected are mandibular canines, premolars, and molars, especially the third molars. Canines and most premolars, except for maxillary first premolars, usually have one root. Maxillary first premolars and mandibular molars usually have two roots. Maxillary molars usually have three roots. When an extra root is found on any of these teeth, the root is described as a supernumerary root. These supernumerary roots may occur due to the disturbances in the Hertwig's epithelial root sheath forming the root. There will be an increased number of divisions of the epithelial diaphragm (a horizontal extension of the epithelial root sheath of Hertwig) to form multiple roots instead of the stipulated number. Cases have been reported wherein both the deciduous and permanent dentition exhibit supernumerary roots.

Chapter 2

Developmental Anomalies Affecting Shape of Teeth

Figure 2. Developmental stages in tooth formation.

Histophysiological Stages of Tooth Development

The histophysiological stages of tooth development include

 initiation,  proliferation,  histodifferentiation,  morphodifferentiation,  apposition

8 P. C. Anila Namboodiripad and E. Anuradha Sunil

Microdontia

Introduction

Microdontia is a condition of having disproportionately small teeth. Classified into:

 True generalized microdontia  Relative generalized microdontia  Microdontia affecting a single tooth.

Pathogenesis

Development of tooth is a continuous process in which a number of physiologic growth processes and various morphologic stages interplay to achieve the tooth's final form and structure. True generalized microdontia show genetically normal small teeth. All the teeth in the oral cavity are affected uniformly. Relative generalized microdontia is a condition wherein a child inherits the large jaws of one parent and normal sized teeth from the other parent making the teeth appear relatively small. Microdontia affecting a single tooth. It is an autosomal dominant condition with incomplete penetrance. Prevalence varies from 0.8 to 8.4% of population. When a peg shaped tooth is present, the remaining permanent teeth often exhibit a slightly smaller mesiodistal length. The proliferation and the morphodifferentiation stage of tooth development are affected in case of the microdontia affecting the maxillary lateral incisor, and the tooth fails to resemble the normal tooth. Dentin is the structure that determines the shape of the tooth. Though the odontoblasts and the dentin histological structure are normal, the morphology of the tooth is affected and ‘peg laterals’ may be formed.

Developmental Anomalies Affecting Shape of Teeth 9

Macrodontia

Introduction

Macrodontia is a condition of having disproportionately large teeth. Classified into

 True generalized macrodontia  Relative generalized macrodontia  Macrodontia affecting a single tooth.

Pathogenesis

The proliferation and the morphodifferentiation stage of tooth development are affected in case of macrodontia affecting a single tooth. It is considered more of a ‘text book’ term than an actual occurrence. It is both a poorly researched and documented characteristic because very less data is available for analysis. The published incidence of macrodontia ranges from 0.03% to 1.9% on the one end, while it can be as high as 3.6% on the other end. Most of the macrodontic tooth is a case of either a germination or fusion. Although true generalized macrodontia related with hyperpituitarism and suprarenal hyperfunction is rare, relative generalized macrodontia is more common but poorly defined.

Fusion and Gemination

Introduction

Gemination is a developmental anomaly in which a developing tooth splits into two separate teeth. Fusion is a condition where two developing teeth merge to become a single tooth. Gemination is derived from the latin word geminus meaning twin. These teeth have disfigured enamel, separate pulpal space; one pulp chamber and two canals or they may appear like a large crown with a large pulpal space. Evolution, trauma, heredity and environmental factors such as 10 P. C. Anila Namboodiripad and E. Anuradha Sunil thalidomide embryopathy, hypervitaminosis A and the fetal alcohol exposure of the pregnant mother are said to play a role in the development of this condition. Nutritional deficiency, endocrinal disturbances and ionizing radiation are also said to play a role in the causation of this condition together with hyperdontia. The condition is also seen to occur in chondroectodermal dysplasia and achondroplasia. Gemination is seen in both the deciduous and permanent dentition.

Pathogenesis

When a tooth bud splits into two, the two teeth are said to be ‘twins.’ The condition is also called as germination. Thus twinning is defined as an effort to make two teeth from one enamel organ. The tooth that is formed will have two completely or incompletely separated crowns having a single root and a root canal. Sometimes the twinning may be complete, that means, inclusive of both the crown and the root. The main cause for this condition is said to be trauma but familial tendency has also been suggested as a factor. Fusion normally occurs due to joining of the two developing tooth buds which may be positioned between two simultaneously developing neighboring teeth whose interdental bone has been resorbed probably due to trauma, or it may occur due to the joining together of a tooth and its supernumerary counterpart.

Talon’s Cusp

Introduction

The talon’s cusp is an additional cusp that is composed of enamel, dentin and the pulp tissue mainly on the lingual surface of the primary or permanent, anterior teeth. It is called as dens evaginatus when it is seen in relation with the posterior teeth. It shows a predilection for the maxilla over the mandible and the maxillary lateral incisors may be the most commonly affected teeth.

Developmental Anomalies Affecting Shape of Teeth 11

Pathogenesis

Both heredity and environmental conditions are said to be responsible for the formation of the talon’s cusp. It is commonly seen to occur during the morphodifferentiation stage of the tooth development. There is an outward folding of the inner enamel epithelial cells and a hyperactivity of the dental lamina cells early in odontogenesis together with a temporary hyperactivity of the dental papilla. Endocrinal disturbances may be responsible for initiating this proliferation. The shape and function of the ameloblasts and odontoblasts remain unaffected. The condition may coexist with other developmental anomalies such as mesiodens, odontomes, and unerupted or impacted teeth. It may also occur in syndromes such as the Mohr syndrome, Sturge Weber syndrome, Rubinstein Taybi syndrome, Ellis van Crevald syndrome etc.

Taurodontism

Introduction

Taurodontism is a developmental condition found in the molar teeth of humans whereby the body of the tooth and its pulp chamber is enlarged at the expense of the roots.

Pathogenesis

A number of pathogenetic factors have been considered in the etiology of taurodontism. The various theories include

 ‘a primitive pattern’ theory,  a mutation, and a  retrograde character,  an atavistic feature,  X linked trait or an autosomal dominant trait.

Other factors playing a role include a delay in the aging of the pulp chamber, odontoblastic deficiency and an alteration in the epithelial root sheath of Hertwig. 12 P. C. Anila Namboodiripad and E. Anuradha Sunil

Taurodontism is said to occur in individuals who have heavy masticatory habits, for example, the Neanderthals and the Inuit or the Eskimos. It has also been found to be predominant in individuals who use teeth as tools such as for sharpening, holding etc. Osteomyelitis, developmental homeostasis which is disturbed, and people who have undergone high dose of chemotherapy or bone marrow transplantation are also said to manifest this developmental anomaly. Taurodontism appears most frequently as an isolated anomaly, but it has also been associated with several developmental syndromes and anomalies including Down syndrome, amelogenesis imperfecta, ectodermal dysplasia, Klinefelter syndrome, Mohr syndrome, tricho-dento-osseous syndrome, Wolf- Hirschhorn syndrome and Lowe syndrome. Other rare syndromes which show the presence of taurodontism include Smith-Magenis syndrome, Mc Cune Albright syndrome, William’s syndrome, and Van der Woude syndrome.

Dilaceration

Introduction

Tooth dilacerations is a developmental disturbance which involves the disfigurement of tooth either at level of the crown or root portion resulting in the altered morphology or shape of the tooth, commonly seen as extreme bends or curves in an otherwise straight tooth.

Pathogenesis

The normal growth and remodeling of the maxilla and the mandible causes a simultaneous fluctuation in the tooth positions relative to the alveolar and basal bone during the root formation. Therefore a decreased bone remodeling rate is equal to decreased bone plasticity which is suggested to increase the resistance to movement of the root growth site relative to the mineralized root which further results in the stretching of the root sheath and dilaceration or flexion. The earlier theory of dilacerations, which considered trauma as an etiology, could not explain the angulations at the mid root position or the apical position of the teeth. A new theory stating that these changes took place due to the plasticity of alveolar bone was introduced. It was also stated that as Developmental Anomalies Affecting Shape of Teeth 13 the permanent teeth replaced the deciduous teeth some amount of shifting of the dental follicle would occur, together with the bending of the root of the tooth.

Figure 3. Showing the process of dilacerations.

Another theory which was proposed in favor of dilaceration was with the shift of the enamel epithelium and its developing mineralized portion, away from the dental papilla and the cervical loop. This shift was found to be away from the central axis of the tooth. No individual structure of the teeth, like the enamel, dentin, pulp or cementum was affected in this case and the only sign visible was the bending of the root. The other factors that play a predisposing role in dilaceration may be

 the rate of eruption of the tooth,  length of the root,  length of the eruption path,  transverse and upward eruption of the teeth especially in the case of the molars,  jaw rotation and the  root resorption. 14 P. C. Anila Namboodiripad and E. Anuradha Sunil

Damage to the permanent teeth in the form of dilacerations may also occur following trauma to its deciduous counterpart. This may result in the displacement of the permanent teeth causing the direction of the root growth to be altered. Different parts of the alveolar bone, the apical, the middle, and the coronal portions all have different turnover time. Thus the movement of the root from one plane to the other may cause root dilacerations. There are other relative dento-skeletal alterations occurring during root formation, such as the mesial drift of the dentition and transverse growth of the maxilla. So the doubt arises as to why dilacerations do not occur following the normal mesial drifting of the socket as the growth of the jaws takes place, and the plasticity gradient of the same after the root apexification is completed. The question has been answered thus that “dilacerations does not occur in such conditions due to the presence of the occlusal forces, craniofacial growth type or due to genetics.”

Dens Invaginatus

Introduction

Dens invaginatus is a rare developmental disturbance of teeth, showing a set of varied morphological variations. The teeth affected on the radiograph shows an infolding of enamel and dentine which extends deep into the pulp cavity and into the body of the root and may sometimes even reach upto the root apex.

Pathogenesis

Etiology of Dens Invaginatus The etiology of dens invaginatus malformation is controversial and remains unclear. Over the last decade several theories have been proposed. They include:

 The buckling of the enamel organ which may occur due to the growth pressure may be one of the etiological factors for dens invaginatus. Developmental Anomalies Affecting Shape of Teeth 15

 Another factor for the occurrence of the dens invaginatus may be in the case of the inner enamel epithelium failing to grow normally or remaining stationery, and the remainder of the enamel organ growing at a normal pace. This may result in the enamel organ engulfing the inner enamel epithelium resulting in the formation of the dens invaginatus.  Or as per Rushton (1937), dens invaginatus arises due to the rapidly proliferating inner enamel epithelium invaginating into the adjacent dental papilla. He named this condition as a “benign neoplasma of limited growth.”  Ohlers on the other hand made an observation similar to Rushton and proposed that there was an invagination of the hyperproliferative inner enamel epithelium but the structure into which it invaginated was not mentioned by him. That is, there was no mention of the dental papilla.  Bruszt on the other hand suggested a fusion of two tooth germs- one inside the other.  Fischer and Sprawson said that the dens invaginatus occurred due to infection in the developing tooth germ.  Trauma was suggested as a cause for occurrence of dens invaginatus by Gustafson & Sundberg (1950) but why lateral incisors were commonly affected and not any other teeth, was the big question.  Foramen caecum was suggested as one of landmark of the enamel organ and this structure was found infolded into the tooth resulting in the formation of the dens invaginatus. Hence according to the authors the invagination may originate starting right at the incisal edge of the tooth.  Grahnen, et al. stated the importance of genetic factors in etiology of dens invaginatus.

Dens Evaginatus

Introduction

The Dens evaginatus (DE) is a developmental anomaly affecting the premolars and it is seen to commonly occur bilaterally. It may be associated with any teeth in the arch with a slight predilection for females. The arch prevalence for the dens evaginatus may vary in relation to the populations 16 P. C. Anila Namboodiripad and E. Anuradha Sunil reviewed. Though the DE occurs predominantly in the permanent dentition it may occur in either the deciduous or the permanent teeth. When associated with the anterior teeth it is predominantly seen on its lingual surface and is referred to as the talon’s cusp. The tubercle on the premolars may be seen to occur either between the two cusps or on the inclined plane of the buccal cusp of the premolar.

Pathogenesis

Dens evaginatus is said to occur following the invagination of the hyperproliferative inner enamel epithelium into the stellate reticulum in the bell stage of tooth development, as opposed to the invagination of the inner enamel epithelium into the dental papilla that occurs in case of the dens invaginatus. The result is in the formation of a tubercle or a solid elevation on the occlusal surface of the posterior teeth. To understand the mechanism behind this developmental occurrence in the tooth, the molecular biology of its development has to be understood. The enamel organ has transient signaling centers located right at its center and these determine the process of formation of the future cusps. The cells lodging these signaling centers are known as the enamel knot. The enamel knot is surrounded by highly proliferative epithelium and an underlying mesenchyme. This enamel knot is also said to play a role in the induction of the dental papilla. The signaling molecules in the enamel knot includes FGF-4and 9, BMP- 2, 4 and 7, and the TGF. These cells of the enamel knot disappear by the early bell stage of tooth formation that is after the initiation of the hard tissue formation of the teeth. Secondary enamel knots arise in the early bell stage of tooth formation, but this time at the sites of the future cusp tips as opposed to primary enamel knots located in the center of the enamel organ. The main cusps are formed by the secondary enamel knot, in the bell stage and any additional cusps are formed later. This is controlled by signaling molecules in the dental papilla together with the formation of the Hertwig’s root sheath at the cervical loop.

Chapter 3

Developmental Anomalies Affecting Structure of Teeth

Figure 1. Anomalies affecting enamel.

18 P. C. Anila Namboodiripad and E. Anuradha Sunil

Amelogenesis Imperfecta

Introduction

Amelogenesis imperfecta (AI) is a disorder which is genetic in origin. It affects both the structure and morphology of enamel of nearly all the teeth of the jaws in a similar manner, and may be coexist with the morphological or biochemical changes that may affect the other parts of the body. The prevalence rate ranges from 1:700 to 1:14000. The enamel in case of amelogenesis imperfect may be hypoplastic, hypomineralised or both and may result in teeth that may be discoloured, sensitive or prone to disintegration. AI exists in isolation or in association with other abnormalities in case of numerous syndromes. It may show autosomal dominant, autosomal recessive, sex-linked and sporadic inheritance patterns.

Pathogenesis

After the initial laying down of the organic matrix of enamel that comprises of proteins such as amelogenin, and non-amelogenin proteins such as amelin, tufltin, and enamelin, the matrix is reabsorbed, to make space for the expanding hydroxyapatite crystals, both in terms of size and volume. This ameloblast mediated proteolytic destruction, during the maturation stage of enamel formation, is brought about by a protease called as KLK4. This degradation continues until almost the entire tissue volume is occluded by the hydroxyapatite crystals containing minerals such as calcium and magnesium. Towards the end of the maturation stage the newly formed enamel is rich in its mineral content by about 95%. AI may show autosomal dominant, autosomal recessive, sex-linked and sporadic inheritance patterns. In families with an X-linked form it has been shown that the disorder may result from mutations in the amelogenin gene, AMELX. The enamelin gene, ENAM, is implicated in the pathogenesis of the dominant forms of AI. Autosomal recessive AI has been reported in families with known consanguinity. The proposed theory of pathogenesis of amelogenesis imperfecta could be

1) The reduction of the prism density resulting in the hypomineralisation of enamel. The individual crystals in the enamel fail to grow to its full extent. Developmental Anomalies Affecting Structure of Teeth 19

The TGF-β is the main gene playing a role in prism formation and is expressed in the secretory stage of amelogenesis. This TGF-β promotes the MMP20 through the Runx2. This MMP20 plays a role in the development of the extracellular matrix of enamel comprising of amelogenin, enamelin, tuftelin and amelin. Thus failure in processing of the enamel matrix leads to retention of the enamel matrix proteins within ameloblasts and they fail to lie out completely. The protease KLK4 gene is said to play a role in the matrix removal. Prism formation is thus disorganized and it is seen irregularly, to result in pit formation on the tooth crown, the cervical area is normally spared as the amount of matrix to be deposited here is lesser. The mineralization goes on normally both in terms of density and appearance.

A) After the normal enamel matrix deposition takes place the ameloblasts move away from the dentin surface and therefore exposes it for further action. B) The ameloblasts improve the cell adhesion linkage to bear the ever mounting deposition of enamel. As the cuspal volume of enamel grows, the cells of the enamel namely the ameloblasts, adjusts the cell to cell contact to bear with the force required to ward off the increasing monolayer of enamel. Suggestion has been given that most likely the ITGB6 up regulates MMP20 expression by means of the TGFβ activation. This on the other hand cleaves through the cadherin, allowing the ameloblasts to reposition them in relation to one another and thereby resulting in the sinusoidal pattern of the enamel rods and rod sheaths. MMP20 is also said to play a role in reabsorbsion of the enamel matrix proteins, predominantly the amelogenin and also the amelin, tuftlin, enamelin which is a necessity for their final degradation prior to the completion of mineralization. Hence it has been suggested that whenever the enamel is affected, the cadherin cleavage and matrix processing are affected due to the ITGB6 mutation hence resulting in deep cracks in the ameloblast monolayer in the cuspal portion leading to pitting, distorted prism architecture and residual matrix proteins that slows down the final enamel mineralization.

2) MMP20 is also essential for the ameloblast-ameloblast contact or cell to cell contact. It thus plays a role in cadherin processing. Thus any compromise in the cell to cell contact would result in an abnormal placement of the enamel prisms resulting in the disruption in the Hunter Schreger bands pattern.

20 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 2. Cartoon summarizing the mechanism of the AI subtype.

Hypoplastic Type of Amelogenesis Imperfecta

In case of the hypoplastic type of AI, the enamel organ degenerates too soon and in some cases there may be a total absence of the differentiation of the inner enamel epithelium. The above mentioned genes may play a role in the causation of these alterations.

Figure 3. Depicting the formation of enamel matrix and its degradation to accommodate the hydroxyapatite crystals. Developmental Anomalies Affecting Structure of Teeth 21

Hypocalcification Type of Amelogenesis Imperfecta

If the protein matrix does not degrade at the right time there may be a disturbance in the crystal formation due to either calcium or a phosphorus deficiency and hence it results in defective calcification, further resulting in the hypocalcified type of AI. It thus shows a large pooling of amelogenenin and also the albumin, as the degradation of both these products are extremely delayed.

Hypomaturation Type of Amelogenesis Imperfecta

Delayed degradation of the matrix and hence delayed growth of enamel crystals results in the hypomaturation of the enamel. It results in the hypomaturation type of AI

Developmental Anomalies Affecting Dentin

Dentinogenesis Imperfecta and Dentin Dysplasia 1 and 2

Introduction Type 1 Dentinogenesis Imperfecta is a hereditary condition that occurs in people who also have osteogenesis imperfecta and in those who have brittle bones which easily break. The Type 2 and Type 3 type of DI do not have associated inherited disorders. It may show varying signs and may affect both the primary and permanent dentitions.

Pathogenesis Dentin formation involves numeorus genes that produce a complex extracellular matrix that is highly organized, and that eventually mineralizes in a highly controlled fashion. Type I collagen (product of COL1A1 andCOL1A2 genes) is the most abundant dentin protein. This complex molecule has the structure of a heterotrimer and forms the foundation for several mineralized tissues including bone and dentin. The collagen molecules interact with a variety of non-collagenous proteins such as the bone sialophosphoprotein, 22 P. C. Anila Namboodiripad and E. Anuradha Sunil osteopontin, osteonectin, dentin sialoprotein to name a few, to help initiate and regulate the mineralization process in these tissues. Interestingly, mutations in either type I collagen or proteins that interact with it can cause the DI. Dentinogenesis type 1 is normally said to be caused by mutations in the COL1A1 and the COL1A2 genes. A deficiency of dentin sialophosphoprotein (DSPP) has also been suggested as a causative factor in dentinogenesis imperfecta. Mutations in the DSPP (dentin sialophosphoprotein) gene mapped to chromosome 4q21 and responsible for the formation of the non-collagenous proteins is said to play a role in the etiology of the Dentinogenesis imperfecta 2 and 3 and Dentin Dysplasia type2. The mutations in the genes may cause either a defective or improper mineralization of dentin. This defective gene would be lodged in the forming odontoblasts and hence it may affect the odontoblastic cell, or influence protein processing or transporting of the proteins inside the cell, during rapid dentin formation. This may result in defective odontoblastic differentiation and mineralization of the dentin matrix. The defective gene forms a matrix which is deficient in phosphorus ion, which is an important mineral during dentin formation. Mutations in the DSPP gene results in the formation of a very soft dentin, especially the mantle dentin. This dentin is found to be discolored, weak and more likely to crack. Probably the hearing loss in the individuals who have Dentinogenesis imperfecta may also be due to the defective DSPP genes. These patients have absent enamel prisms in the developing teeth, since the nidus for initiation of enamel formation is in the dentin and may also be the reason for the defective mantle dentin. Abundant insoluble mutant DSPP and its degradation products (dentin phosphoprotein, bone phosphoprotein, osteopontin, osteocalcin, osteonectin and dentin sialoprotein, DSP) expressed by odontoblast cells could possibly be hypothesized to deactivate or indirectly interfere with the metabolism of other proteins in dentin and result in the phenotypes of DI.

Dentin Dysplasia Type 1

Introduction

Dentin dysplasia (DD) type I is characterized by short or total absence of roots, obliterated pulp chambers, and peri-apical radiolucencies. It affects both Developmental Anomalies Affecting Structure of Teeth 23 the primary and secondary dentitions. It is found to be even rarer than DI.DD's incidence is 1:100,000.

Pathogenesis

 Wesley et al. suggested that the condition is caused by an atypical contact of odontoblasts with ameloblasts leading to uncharacteristic differentiation and/or function of these odontoblasts.  Logan et al. stated that the dental papilla is responsible for the abnormalities in root development. They proposed that innumerable degenerative foci within the papilla become calcified, leading to reduced growth and final obliteration of the pulp space.

Regional Odontodysplasia

The condition is usually identified on routine radiographic examination of the patient who has visited the dental OP with complaints of failure of eruption or pulpitis or a necrosis in absence of caries. The radiograph would show features such as “ghost like appearance of teeth” and a similar radiodensity of both the enamel and the dentin.

Pathogenesis

The condition is not hereditary and the most common pathogenesis is the absence of the blood supply which is required to mineralize the teeth. Both the enamel and dentin matrix will fail to calcify completely. Somatic mutations affecting the dental lamina, vascular disorders, local or systemic viral infections, differentiation failures of neural crest cells, local trauma, nutritional deficiency, and radiation may also result in the occurrence of the regional odontodysplasia.

Chapter 4

Developmental Anomalies Affecting Tongue

Development of Tongue

The tongue begins to form at almost time as the palate. It is found to arise from various swellings on the pharyngeal floor. There is the appearance of the tuberculum impar (median lingual swelling) on the lower edge of the mandibular arch at the same time of the midline fusion of the first (mandibular) and the second (hyoid) pharyngeal arches. On either side of it, two more protuberances are seen called as the lateral lingual prominences. These lateral lingual swellings grow over the median lingual swelling to fuse to form the anterior 2/3 of the tongue. It therefore consists of both the ectodermal and endodermal parts. The primordium of the thyroid arises at the caudal end of the tuberuculum impar. (Stage 10, ca. 28 days) as an unpaired ventral endodermic bud. The thyroid then migrates in front of the larynx leaving behind a foramen caecum on the dorsal surface of the tongue. The 2nd, 3rd pharyngeal arches, the copula and a small portion of the 4th pharyngeal arch gives rise to the posterior third of the tongue. Hence this part is made up of the endoderm only. Between the anterior 2/3rd and the posterior 1/3rd the terminal sulcus forms. The most caudal end of the tongue forms the hypobranchial eminence that indicates the entrance to the trachea.

26 P. C. Anila Namboodiripad and E. Anuradha Sunil

Median Rhomboid Glossitis

Introduction

Median rhomboid glossitis occurs on the dorsum of the tongue. It is an enigmatic inflammatory or infectious condition.

Pathogenesis

This condition is thought to occur due to failure of retraction of the tuberculum impar during development. In the case of normal development this tuberculum impar after it descends into thyroid area, is closed over by the two fused lingual swellings from either side. These lingual swellings are ideally located on either side of the tuberculum impar prior to the fusion. But since this condition is rare in childhood, when it must first manifest, this theory has been rejected. The present day theory states that median rhomboid glossitis is a type of distinct type of candidiasis which when occurring on the midline of the tongue; the name given to it is the ‘median rhomboid glossitis.’ Actinomyces has also been implicated in the etiology of the median rhomboid glossitis. The tongue is a favorable site for the candida to lodge since it is a warm and protected space provided by the lingual papillae. This site is also not a self cleansing area and this area cannot easily be cleaned by the brush too. The median rhomboid glossitis may also show the presence of surface nodules. Excessive pressure on the palate when applied by the involved tongue, while producing certain sounds such as the ‘g’, ‘k’ and ‘j’, accounts for the nodularity on the tongue surface. The presence of the median rhomboid glossitis is also an indication of the immunocompromised state of the person. It could also be an indication for the Human immunodeficiency virus infection.

Developmental Anomalies Affecting Tongue 27

Geographic Tongue

Introduction

Geographic tongue is a form of tongue inflammation where red, smooth, migratory patches covered over by a white border, form on the surface of the tongue.

Pathogenesis

 Geographic tongue is considered as a psoriasiform tongue. That is, it is said to occur coincidently with the psoriasis of the skin.  It is not an infectious condition like HIV. It does not spread between partners.  It is not linked to usage of tobacco.  It is said to be a side effect of consumption of the non metal like lithium.  Dental materials may contribute to the initiation of the migratory glossitis.  It may occur due to vitamin B deficiencies.  It may also be seen in relation to allergic conditions such as atopy and asthma, eczema and contact allergy  Said to also be associated with hormones since it is predominantly seen in females on oral contraceptives  It has even been considered to be a hereditary condition with a polygenic mode of inheritance. Defective genes are said to be the HLA-DR5, HLA-DRW6 and HLA-DRCw6.

Section 2: Benign and Malignant Disorders

Chapter 5

Benign and Malignant Epithelial Tissue Tumors

Benign Tumors of Epithelial Tissue Origin

Squamous Papilloma

Introduction Neoplasia caused by Human papilloma virus (HPV) causes squamous cell papilloma. Skin papillomas are called as ‘warts’ or ‘verrucas’ and those on the genital tract are called as the ‘genital warts.’

Pathogenesis The cause of the squamous papilloma was found to be the virus called as HPV. There are various known subtypes of HPV, but subtypes 6 and 11 have been found positive in a large number of squamous papillomas and HPV 16 and 18 positive in another section of the papillomas. Some cases showed a total absence of HPV in the human papillomas. These viruses have been found to interfere with the proper functioning of the ‘gate keeper protein,’ the p53. They play a role in the inactivation of the gene that acts as regulators of the cell proliferation namely the p53. E6 protein of the HPV-16 is said to be capable of binding to protein p53, encouraging its degradation and altering the control of cell growth. Six early proteins of the HPV play a role in gene regulation and cell transformation and they include E1,E2,E3,E4,E5,E6 and E7 (E stands for 32 P. C. Anila Namboodiripad and E. Anuradha Sunil

‘early’ that is before DNA replication starts and are encoded by the HPV genome) and the 2 late proteins, L1 and L2(‘late’ indicates ‘after DNA replication’) which forms the shell of the virus. It is the genotypic variations in the E6 and E7 base proteins that decide the oncogenic potential of the HPV genes. For example; the E7 protein of HPV 16 is more oncogenic than the E7 protein of HPV 6. The mode of transmission of oral HPV is unclear. What exactly happens when an HPV enters a cell is that the circular viral genome breaks at the level of the E1 and E2 regions but never in the E6 and E7 region. E2 to E5 are lost when the transcription takes place and this causes the loss of control over the E6 and E7 regions. These then get directly involved in the cell cycle by inhibiting the normal functions of p53 and pRb (human retinoblastoma gene). The role of the p53 then, is to arrest the cell cycle by allowing its repair. If it fails to repair the cell then it induces the programmed cell death to occur and prevents the error from getting into the next stage. If there is a p53 mutation, E6 suppresses the properties of p53 gene product, and acts as a second hit in the ‘two hit hypothesis’ theory of neoplasia. It plays a role in the degradation by the ubiquitin pathways. The E7 protein on the other hand acts with retinoblastoma protein (pRb), which is the crucial factor for the cellular cycle control and causes release of the transcription factor E2F which will cause the stimulation of the cellular division. E7 also binds and inactivates the protein kinase inhibitors p21 and p27.

Figure 1. Shows the diagrammatic representation of HPV-induced oral carcinogenesis.

A model for the interaction of HPV E6 and E7 with the DNA damage pathway. Benign and Malignant Epithelial Tissue Tumors 33

Keratoacanthoma (KA)

Introduction Keratoacanthoma is a benign tumor that resembles the squamous cell carcinoma both clinically and histologically. It is also called as a ‘self healing carcinoma.’ It is found to arise in relation to hair follicle and the associated sebaceous glands.

Pathogenesis Various etiological factors have been considered in the formation of keratoacanthoma:

1) Said to be genetic in nature: genes have been said to play a role in formation of the keratoacanthoma. 2) Human Papilloma Virus is said to be another etiological factor for the causation of this lesion 3) Immune factors: since the lesion may be seen in individuals with malignant disorders or in immunosuppressed individuals’ immunology is said to an etiological factor for the keratoacanthoma. 4) Envoirnmental factors like sunlight, chemicals carcinogen, and trauma have also been implicated in the etiology of the keratoacanthoma. 5) It is also said to occur in persons who are on medications that target the Ras/Raf/MAP kinase pathways. 6) Keratoacanthoma is commonly seen to occur in cigarette smokers and hence is said to be another etiological factor for KA. 7) This lesion is also seen in case of patients with psoriasis being treated with psoralen 8) Said to occur at sites of burns.

No details about the pathogenesis for any of the above etiological factors are available.

34 P. C. Anila Namboodiripad and E. Anuradha Sunil

Nevus

Introduction

Nevus cells are melanocytes that do not have cytoplasmic extensions or processes called as dendrites. They do have an abundant cytoplasm and coarse granules and they are commonly seen at the dermoepidermal junction or in the dermis of the skin. These nevus cells are epitheloid in shape when located at the dermoepidermal junction, transform into the lymphocytoid cells as they reach the dermis and further into the neuroid morphology when they enter the dermis. They are hence classified into the Type A, Type B and the Type C cells respectively. Nevus cells occur in the form of clusters and the melanocytes may also be evenly dispersed as single units. Nevus cells are the primary components of a melanocytic nevus.

Pathogenesis

The main pathogenesis for the nevus is the point mutation in the BRAF gene (a part of the Ras-Raf-Mek-Erk-MAPK (mitogen-activated protein kinase) signaling cascade): a serine/threonine protein kinase gene or the Neuroblastoma ras viral oncogene. No details are elaborated in case of the role of the above genes in the pathogenesis of nevus.

Malignant Epithelial Tissue Tumors

Basal Cell Carcinoma

Introduction Basal cell carcinomas are malignant neoplasias arising from the basal layer of the skin. They appear in the form of open sores, red patches, pink growths or scars. They usually occur due to intense UV exposure, and may result in a disfiguring mass if allowed to grow, but never seems to metastasize.

Pathogenesis Mutation in the gene MC1R or the melonocortin gene is said to cause basal cell carcinoma and the malignant melanoma. The UV radiation causes Benign and Malignant Epithelial Tissue Tumors 35 damage to a lot of proteins of the skin when it is exposed to it. The MC1R is said to provide protection against this damage. There are also genes that produce proteins which help in removing the damaged proteins such as the lipid peroxide and the DNA hydro peroxides. These genes if defective may also cause the occurrence of the basal cell carcinoma as these proteins are toxic to the body. The genes that play a role in damaged protein removal include the CYP2D6 and the GST genes. HPV is also said to play a role in the basal cell carcinoma but HPV plays a major role in Squamous Cell Carcinoma than Basal cell carcinoma. The cell membrane receptor of the epidermal cells form a complex with the sonic hedgehog protein(SHH) which is a component of the hedgehog protein, which is an extracellular protein of the hedgehog pathway (HH). The combined protein complex initiates a cascade of cellular events that leads to cell proliferation. The cell receptor complex comprises of the

1) Patched (PTCH) 1 protein that is the ligand binding component of the Horse Hedgehog (HH) receptor complex in the cell membrane 2) Smoothened (SMO) protein which plays a role in transducing the hedgehog signaling to the downstream genes.

When active:

SHH binds to PTCH1 ↓ PTCH1 inhibition of SHH is released ↓ HH pathway is activated ↓ Activation of transcription factors ↓ Expression of cell cycle regulator genes

So improper HH pathway and any of the genes playing a role in the pathway like the PTCH1, PTCH2, and SMO genes may cause human malignancies including the basal cell carcinoma. 36 P. C. Anila Namboodiripad and E. Anuradha Sunil

PTCH 1 may also prevent the binding of the protein to the SMO. This unbound SMO permits uncontrolled proliferation of the cells through activation of the following:

a. GLI1 (glioma-associated oncogene 1; also called zinc finger protein) and GLI2, the transcription factor genes. b. Genes helping in cell proliferation like the cyclin D, cyclin E, myc, and c. Proteins playing a role in the angiogenesis. d. Numerous other genes that may play a role in Basal Cell Carcinoma and they include: 1. XRCC 3, a DNA repair gene 2. CDKN2A and CDKN2B both cyclin-dependent kinase inhibitor genes 3. K5 gene a basal keratinocyte gene and the 4. BRM gene

Mutations in the p53 genes have been found to play a role in formation of Basal cell carcinoma. Genes such as the CD95, BCL-2, PDGFRα, or cFLIP, are presently under study as also the FOX gene. Hence it was proposed that these genes may be the ‘drivers’ and not ‘passengers’ in the cause for BCC. Gremilin 1 has been identified in the stroma of patients with BCC and is said to be a gene that antagonizes the genes that play a role in tumor growth such as the BMP2 and the BMP 4. Hence this gene sustains the growth of the tumor.

Verrucous Carcinoma

Introduction

Verrucous carcinoma (VC) was first described by Ackerman in 1948 and is described as a low-grade, well-differentiated variant of squamous cell carcinoma (SCC).

Benign and Malignant Epithelial Tissue Tumors 37

Pathogenesis

Surprisingly no pathogenesis of the verrucous carcinoma has been described in literature. Etiological factors for the carcinoma include the tobacco chewing, HPV 6 and 11. Verrucous carcinoma has been associated with smoking but not drinking.

Malignant Melanoma

Introduction

It is a type of cancer arising from the melanocytic cells of the skin.

Pathogenesis

The role of melanin is not only producing pigmentation but also protecting the skin against the UV rays. This protection is brought about by genes that break down the protein formed, following UV exposure. So melanin is an antioxidant protecting the skin against various malignancies. Melanin takes on the property of a chelator by converting from an antioxidant to a prooxidant. It also produces a large amount of superoxide anion. Melanin is converted from an antioxidant to a pro-oxidant, takes on properties of a metal chelator, becomes a redox generator, and produces large amounts of superoxide anion. Thus the oxidative stress that is increased, produces transcriptional factor activation namely AP-1and Ref-1 and this whole process lead to drug resistance. This activity if defective would result in malignancy of melanocytes and is called as the melanoma. The uptake of metals into the cells is controlled by an enzyme the metallothioneins. Non metals would also bind melanin. The non metals include pesticides, dyes, organic amines, polychlorinated biphenyls and herbicides.

38 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 2. A model for chemoprevention of early melanoma progression. Oxidation of melanin leads progressively to generation of a redox-active tautomer (quinone-imine), intracellular redox cycling (enhanced by metals or other substances bound by melanin) with melanosomal and DNA damage, transcription factor activation and enhancement, and activation of the natural antiapoptotic (drug-resistant) phenotype of the melanocyte. Antioxidants include a number of cellular antioxidants (ascorbic acid, α- tocopherol, and glutathione) whereas upstream inhibitors of oxidation might include such drugs as inhibitors of cholesterol synthesis or inhibitors of mitochondrial activity. Metal uptake into cells is regulated by metallothioneins, and polymorphisms should contribute to differential uptake and risk. ROS, reactive oxygen species and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia.

Activation of the human telomerase reverse transcriptase (Htrt) and a deficiency in the p16INK4a-Rb pathway results in the occurrence of the immortalization of the melanocyte. This thus results in the Radial Growth Phase (RGP) of melanoma and it also indicates the role of keratinocytes in the survival of the melanocytes and its growth, only near or in the epidermis. In the RGP melanoma there is a high apoptotic rate of these melanocytes. Once the melanoma enters the Vertical Growth Phase, there is the repressed apoptosis which allows the cells to survive in the absence of the keratinocytes. This is brought about by the PTEN loss, which inhibits apoptosis mainly through the AKT activation and also the over-expression of a number of protein kinases or RAS activation, and β-catenin activation. There is also a loss of the E cadherin and an altered expression of the N-cadherin and αVβ3 integrin as the RGP melanoma transforms into the VGP melanoma. For the tumor growth, migration and the angiogenesis, a close relationship must also exist between the melanocytes and the fibroblasts in the connective tissue. For the melanocyte development, survival, migration and differentiation in the KIT-SCF signaling is essential. This KIT expression is highly expressed in Benign and Malignant Epithelial Tissue Tumors 39 all the benign variants of the melanoma and also it is highly suppressed in the invasive and metastatic melanomas.

Figure 3. The disruption of the p16ink and p16ARF by the inactivation of the CDKN2A gene results in the melanocytic nevus transforming into a dysplastic nevus. The retinoblastoma pathway may be affected resulting in the occurrence of the malignant melanoma.

The role of CCND1 gene is also found to be important in the occurrence of the melanoma. It has already been implicated in previous tumors like the centrocytic lymphomas and parathyroid cancers. CRAF is the primary mediator that initiates cell proliferation and it is not certain whether it is the BRAF or the CRAF that transmits signals from mutated Neuropeptide RAS to MEK. The activated RAS triggers the MAPK pathway through interactions with the RAF oncoproteins (BRAF and CRAF). The MEK phosphorylation leads to the activating phosphorylation of the MEK (MAPK/ERK kinase) which in turn activates the phosphorylation of the ERK. Cell proliferation is the primary step in the initiation of malignancies. Mutations in this BRAF contribute to the melanoma formation. Increased MAPK activity prevents the apoptosis and initiates the cell proliferation through increased cell cycle progression. PTEN deletion results in Akt activation which further results in phosphorylation and inactivation of Raf.

The Altered State of Melanin during Melanogenesis Melanin is normally in a reduced antioxidant state. Exposure to ROS leads to an oxidized condition which facilitates binding of transition metals (and other compounds such as polyphenol chlorinated biphenyls). Metal uptake into the cell is closely regulated by a family of metallothioneins. This situation sets 40 P. C. Anila Namboodiripad and E. Anuradha Sunil up the oxidized melanin as a redox generator, which leads to increasing intracellular oxidative stress, a widespread adaptive response including TGF up regulation, cell death, a high rate of cellular turnover and secondary intrinsic drug resistance. Studies have also shown that the melanoma cells have an augmented ROS as compared to the normal melanocytes which may be shown by the increased superoxide anion.

Oral Squamous Cell Carcinoma

Introduction

Squamous cell carcinoma (SCC) is an uncontrolled growth of abnormal cells arising within the spinous cells of the squamous epithelium. SCCs often look like scaly red patches, open sores, elevated growths with a central depression, or warts; they may crust or bleed.

Pathogenesis

Oncogenes and tumor suppressor genes are said to play a role in the pathogenesis of oral squamous cell carcinoma. The primary among them being the K-ras and the p53. Cytogenetic alterations can occur due to defect or the mutation in the genes namely the bcl-1, int-2, n-ras and hst-1 have been mapped to chromosome 11q13 and also the check point genes namely the cyclins and the CDKs. Cyclin D1, Cyclin A, Cyclin B, Cyclin B1are all said to play a role in oral carcinogenesis. The action takes place at various levels of the cell cycle G1, S, G2 and M phase. TGF-α and TGF-β mutations are said to occur early in occurrence of squamous cell carcinoma. The p53 protein activates the production of the p21 protein. There is also a mutation in the Rb gene and the APC gene. The p16ink4 inhibits phosphorylation of Rb gene when apoptosis is induced. P-cadherin, E- cadherin, β1 integrins and the α6 β4 are commonly affected in the poorly differentiated squamous cell carcinoma. HPV types 2, 6, 11, 16, 18, 31, 33 and 35 have also been implicated in the initiation of the squamous cell carcinoma. Apart from the various pathogenesis of oral squamous cell carcinoma mentioned above there is large number of other pathogenesis mentioned in literature and to elaborate on all of them would be beyond the scope of this book.

Chapter 6

Benign and Malignant Connective Tissue Tumors

Benign Tumors of Connective Tissue Origin

Giant Cell Granuloma

Introduction Reparative giant cell granuloma (RGCG) is a non odontogenic lesion which is not a true neoplasm but may occur in reaction to trauma or inflammation. It is a rare lesion in the head and neck region.

Pathogenesis The etiopathogenesis of the giant cells in the giant cell granuloma may be:

1) Derived from the multinucleated osteoclasts, or 2) It may be the result of fusion of the endothelial cells or 3) The fusion of the background spindle shaped cells. 4) The nuclei of these giant cells are found positive for Cyclin D1 and the surrounding macrophages are found positive for Cyclin B1 and the MI B-1. But their roles in the giant cell granuloma remain unknown.

42 P. C. Anila Namboodiripad and E. Anuradha Sunil

Oral Lipoma

Introduction Lipoma is a benign, rubbery, soft, encapsulated tumor of adipose tissue usually made up of mature fat cells.

Pathogenesis There were the following theories proposed for the development of the lipoma 1) ‘Hypertrophy theory.’ In this theory the adipose tissue was found to be increased in size in some areas of the body. But this theory did not have many takers as the lipoma was found in areas where fat tissue was negligent; and secondly during starvation the fat was found lost from the body but not from the fat deposits of the lipoma lesions. 2) Another theory to explain the lipoma included the ‘metaplasia theory.’ According to this theory it was thought that the mesenchymal cells underwent metaplasia into lipoblasts which laid down the adipose tissue. It also supported the theory of lipoma occurring anywhere in the body. These mesenchymal cells were the multipotential embryonic cells that differentiated into the fat cells under hormonal influence especially during adolescence. 3) The third theory stated that lipoma was formed due to the trauma and the chronic irritation of the soft tissues.

Since the theories 1 and 3 did not explain many features of the theory of the lipoma formation the metaplastic theory was found to be most acceptable.

Neurofibroma, Schwannoma and Malignant Peripheral Nerve Sheath Tumor

Introduction Neurofibromas are the benign peripheral nerve sheath tumors. A neurilemmoma is the other benign peripheral nerve sheath tumor apart from the neurofibroma. It is benign and usually encapsulated neoplasm made up of Schwann cells.

Benign and Malignant Connective Tissue Tumors 43

Pathogenesis Somatostatin receptors (SST) have been said to play a role in the formation of the schwannoma, neurofibroma and malignant peripheral nerve sheath tumor. Among the SST receptors, the SST2A was found positive in schwannomas but not in the neurofibromas and the malignant peripheral nerve sheath tumors. These receptors have been found predominantly within the Schwann cells. But the exact role they play in the formation of these neoplasias is unknown. VEGF an endothelial cell marker was found lesser in the neurofibroma than the schwannoma and the bFGF (β Fibroblastic growth factor) was found increased in the schwannoma.

Malignant Connective Tissue Tumors

Osteosarcoma

Introduction Malignant tumor of bone comprising of malignant osteoblasts play a role in formation of the osteosarcomas. Intramedullary and surface osteosarcomas are the two types of osteosarcomas. In case of the intramedullary type it has been further subdivided into the three; namely the typical type, the telangiectatic type and the highly differentiated variant. Surface osteosarcomas have been divided into periosteal, paraosteal and a high grade type. Histologically the osteosarcoma has been divided into the osteoblastic, chondroblastic and the fibroblastic types. Other types include giant cell, small cell and the mixed types of osteosarcomas.

Pathogenesis Osteosarcoma is seen predominantly in the growing bones especially during puberty. It is also seen in patients affected by the Paget’s disease, fibrous dysplasia and other bone disorders. People constantly exposed to radiation, for e.g., women who fix the radium dials in watches to make them luminescent, may also suffer from osteosarcoma. People exposed to the 44 P. C. Anila Namboodiripad and E. Anuradha Sunil chemical agents, like methlycholanthrene, chromium salts; beryllium oxide etc. may also succumb to osteosarcoma.

Figure 1. W. Michael Kuehl,P. Leif Bergsagel. Molecular pathogenesis of multiple myeloma and its premalignant precursor. Clin Invest. 2012; 122(10):3456-3463.

A simian virus SV40 has been stated as another one of the etiological factors for osteosarcoma. Exaggerations of the chromosome 6p21, 8q24 and 12q14 and the loss of heterozygosity of chromosome 10q21.1have been implicated as the etiological factors for osteosarcomas. Also the loss of the chromosome 9, 10, 13 and 17 and gain of chromosome 1 are other chromosomes suggested as a causative factor for the osteosarcoma. P53 mutations seen commonly in other malignancies may be seen in some osteosarcomas The Rb tumor gene which is important in the cell cycle control is also said to play a role in the osteosarcoma. Myc in the bone marrow stromal cells may be increased in the osteosarcoma. Another transcription factor involved in cell proliferation is c-Myc, whose amplification has been implicated in osteosarcoma pathogenesis and resistance to chemotherapeutic drugs. Overexpression of Myc in bone marrow stromal cells leads to osteosarcoma development and loss of adipogenesis (Shimizu et al., 2010). Overexpression of MET (Ferracini et al., 1995; Rong et al., 1993) and c-Fos (Wu et al., 1990) has been reported in more than 50% of Benign and Malignant Connective Tissue Tumors 45 osteosarcoma cases, whereas c-Myc is overexpressed in less than 15% of cases (Barrios et al., 1993; Ladanyi et al., 1993). c-Myc, c-Fos, and cathepsin L have been shown to be overexpressed in a high proportion of relapsed tumors and metastases (Gamberi et al., 1998; Park et al., 1996). Transforming growth factor beta (TGF-β) family proteins are also implicated in osteosarcomagenesis through impairment of osteoblast proliferation, differentiation and cell death. High-grade osteosarcomas are found to express (TGF-β) in significantly higher amounts than low-grade osteosarcomas (Franchi et al., 1998). IGF (insulin-like growth factor)-I and IGF-II are growth factors that are often overexpressed by osteosarcomas (Rikkof et al., 2009). Runx2 has a very low expression in osteosarcoma cell lines. Runx2 also associates with BMPs, Rb, and p27KIP1 playing a crucial role in cell cycle and bone differentiation regulatory pathways. Bone morphogenetic proteins (BMPs) such as bone morphogenetic protein-6 and bone morphogenetic protein receptor 2 are expressed in more than 50% of osteosarcomas and related to poor prognosis (Gobbi et al., 2002; Guo et al., 1999; Yoshikawa et al., 2004).

Rhabdomyosarcoma

Introduction Based on histopathologic features, two subtypes, embryonal (ERMS) and alveolar (ARMS) were identified. They are found to be associated with distinct clinical characteristics and genetic alterations.

Pathogenesis The tumor is said to arise from primitive mesenchyme with a propensity for muscle formation rather than a malignancy arising from the skeletal muscle cells. Hence the tumor may be located even in bones even though the bone is not a site for occurrence of muscle cells.

1) The condensation of the mesenchyme is said to form a muscle tumor. The loose primitive mesenchyme condenses to form an embryonic muscle. 2) Embryonal tissue that was trapped during the early phase of tissue formation may also be the cause for this tumor. It may also arise from embryonal muscle tissue that was trapped during the early phases of tissue development. 46 P. C. Anila Namboodiripad and E. Anuradha Sunil

3) Though the exact pathogenesis for the RMS is unknown, studies have shown that the cause for the occurrence for this tumor may be the mutations that overturn the balance between the differentiation and the proliferation of the primitive mesenchymal tissue during myogenesis and hence the development of the rhabdomyosarcoma 4) The chromosomal translocations in the t(2;13)(q35;q14) or t(1;13)(p36;q14) or 1;13ARMS is said to result in the generation of the PAX3-FKHR and the PAX7-FKHR fusion products. This contributes to the oncogenic behavior of the tumor by altering the growth, differentiation and apoptosis pathways.

On the other hand the ERMS have allelic loss in chromosome 11p15.5. This allele on the other hand is said to suppress the tumor suppressor gene. It causes the loss of maternal genetic information and the duplication of the paternal genetic information. The gene matched to this chromosome is the IGF II gene and in case of RMS (rhabdomyosarcoma) it stimulates tumor growth. Both of them are found to target the p53 and the Rb pathways through amplification of the genes MDM2 and the CDK4. The amplifications are frequent in ARMS but rare in the ERMS. Embryonal RMS (eRMS) is characterized by loss of heterozygosity on the short arm of chromosome 11 (11p15.5), suggesting inactivation of a tumour-suppressor gene. In contrast, the majority (80-85%) of the alveolar RMS (aRMS) have the reciprocal chromosomal translocations. The cell motility and invasion in epithelial cells is brought about by the aberrant expression of the MET oncogenes in both the ERMS and the ARMS. The MET is found to code the receptor for the HGF/scatter factor which is said to play a role in the cell motility and invasion. The overexpression of MET is said to create the same role in the myoblasts like embryonal myoblasts where they migrate into the surrounding connective tissue. PAX is another gene which is said to play an inhibitory role in the myogenic differentiation of the cultured myoblasts. PAX3/7-FOXO1 can suppress MyoD expression and also induce the NFκB signaling and this inhibits the myogenesis through the Cyclin D1/CDK4 complexes. They may sequester the MyoD which could hinder the cell cycle and the differentiation of the myoblasts. The GLI1 is said to play a role when primitive myoblasts are present. But the association of the GLI1 is found to be associated with an undifferentiated muscle tumor rather than with the ERMS or ARMS. Benign and Malignant Connective Tissue Tumors 47

Lymphomas

Non Hodgkin’s Lymphoma

Introduction Non Hodgkin’s lymphomas are malignancies of the lymphocytes that are diverse in nature and include all tumors of the lymphocytic lineage excepting the Hodgkin’s lymphoma.

Pathogenesis Based on the classification of lymphomas numerous types have been stated.

B-Cell Lymphoma Abnormal lymphocyte production, maturation or action may cause a malignant phenotype of the lymphocytes and this is seen due to mutation of the gene responsible for the lymphocyte production. These mutations are responsible for the expansion of the monoclonal population of the malignant lymphocytes. The type of lymphomas depends on the stage of lymphocyte production or maturation of the lymphocytes resulting in tumors related to the immature B cells, naïve B cells, activated B cells and hence they produce tumors such as Burkett’s lymphoma, diffuse large B cell lymphoma and mantle cell lymphoma.

T-Cell Lymphoma T cells arise from the CD34 stem cell and give rise to both T and B cells. The T cells migrate early into the thymus where they transform into mature T cells. The T cells form a complex with its receptor α, forming a mature TCR complex and then decide to develop into either a CD4+ or the CD8+ cell. They identify suitable MHC and subsequently leave the thymus. In the thymus, the process starts from thymic lymphoid cells developing into T-cell receptor (TCR) alpha, forming the mature TCR complex, and then developing into CD4+/CD8+ cells. Interaction between TCR and peptide/MHC leads to a commitment to be either CD4+ or CD8+ cells. Subsequently, T cells undergo a positive selection (to recognize host MHC, self-MHC restriction) and negative selection (to not bind MHC too strongly, which would lead to autoreactive clone, self-MHC tolerance) and are ready to leave the thymus. Those that were unable to perform its function of CD4+ or CD8+ production, 48 P. C. Anila Namboodiripad and E. Anuradha Sunil die in the thymus through apoptosis. Mutations can occur at any of the above mentioned stages and result in the formation of the lymphomas.

Figure 2.

Source: Courtesy: Front. Pediatr.24 August 2015. MicroRNAs and potentialtargets in osteosarcoma: review. Valerie B. Sampson, Soonmoon Yoo,Asmita Kumar, Nancy S. Vetter and E. Anders Kolb

Figure 3. Network of miRNAs and target genes in OS. The figure depicts altered miRNA genes that play a critical role in the development and progression of OS. The majority of miRNAs are downregulated (tumor suppressors) and target genes are overexpressed (oncogenes). Upregulated miRNAs (oncogenes) are depicted by upward arrows and target genes are repressed (tumor suppressors). Abbreviations: MDR1, multi-drug resistance 1; FasL, Fas ligand;IGF-1R, insulin-like growth factor 1receptor; EGFR, epidermal growth factor receptor; ROCK1, Rho-associated coiled- coilkinase 1; Bcl2, B-celllymphoma-2;Bcl-xL, B-cell lymphoma-extralarge;Mcl-1, myeloid leukemia cell differentiation protein;PTEN, phosphatase and tensin homolog;MMP-13, matrix metalloprotease-13;N-Cad, N-Cadherin; SATB2, special AT-rich sequence-binding protein 2; RUNX2, runt-related transcription factor2; DTL, denticleless protein homolog). Solid gray arrows represent activated signaling pathway;solid blunt lines represent inhibition of signals; dotted gray lines represent indirect signaling pathways. Benign and Malignant Connective Tissue Tumors 49

Information about the activation of the proto-oncogenes, e.g., bcl-1, 2, 6 and c-myc or the disruption of the p53 tumor suppressor gene is an important knowledge for the clinician so that the proper treatment can be implemented. The NHL is divided into the mantle cell, follicular, diffuse large cell and Burkett’s lymphoma. The ‘first hit’ leading to the Follicular Lymphoma is probably the translocation of the t (14; 18). This is based on the genetic aberration. Naïve B cells carrying the translocation, exit the bone marrow and lodge in secondary lymphoid tissues where they undergo the germinal center reaction, but survives it due to the presence of the bcl-2 which occurs in the germinal centers during such malignancies, unless it is otherwise absent. These cells may also be rescued from apoptosis due to its weak B cell affinity. But it has been found that there must be another mechanism playing a role together with t (14; 18) which may help in the occurrence of the HL.

Hodgkins Lymphoma

Introduction Hodgkin’s lymphoma is another type of lymphoma which does not fit into the category of the Non Hodgkin’s lymphoma. Mixed cellular infiltrate is a feature of the Hodgkin lymphoma (HL) together with the presence of the Hodgkin’s and the Reed Sternberg cells (HRS) which comprises a minor component.

Pathogenesis

EBV infected B cells express latent membrane protein that ↓ Upregulates NF-kB (Transcription factor for activating lymphocytes) Mutation in ↓ Negative regulators of NF-Kb Leads to excessive B cell proliferation/protection of B cell from proapoptotic signals ↓ Increased number of B cells ↓ Mutation in Immunoglobulin gene of B cell ↓ Production of RSCs EBV, NF, RSCs

Figure 4. 50 P. C. Anila Namboodiripad and E. Anuradha Sunil

Evidence states that HRS cells are from the B cell clone that has lost their B cell characteristics. In normal circumstances these B cells undergo apoptosis. But there is said to be some mechanism that prevents the B cells from undergoing the apoptosis and hence they survive, and the mechanism is said to be a ‘virus.’ The virus said to play a role in inhibition of apoptosis has been identified as the EBV. The EBV alters the cell to such an extent that it replaces the survival signals that are normally provided by the B-cell receptor (BCR) during germinal center reaction that the B cell receptors on the cell surface are also altered and they now appear as the HRS cells. The survival of the B cells from apoptosis is brought about by the LMP2A expression. Constitutive activation of Notch1(that plays a role in development) by LMP2A, and subsequent inhibition of E2A and downregulation of EBF, two transcription factors that regulate B-cell development, appears to be involved in both survival signaling and transcriptional regulation. Thus, LMP2A is likely to play a key role in both the survival and reprogramming of EBV-positive HRS cells. The cells are modified to resemble an activated B cell receptor due to this expression. The cells showing absence of the EBV may have had the latent infection during childhood resulting in the appearance of episomes i.e., showing the absence of disintegration of the viral particles. There is compelling evidence that HRS cells are clonal B cells that have lost their B cell phenotype. EBV is said to play a role in reprogramming and survival through dysregulation of several signalling networks and transcription factors, including nuclear factor (NF)-κB. Activation of NF-κB, AP-1, members of the JAK/STAT signaling and the NOTCH-1 signalling pathway, are features of all HRS cells and the gene mutations affecting this pathway appear common in EBV-negative HL. The HRS also attracts cells supportive to its microenvironment like the immune and the stromal cells. Also there is a coincident production of the cytokines and the chemokines by the HRS, thereby circumventing the normal apoptotic pathway and allowing the survival of BCR (B cell receptor)-deficient B cells. LMP1 mimics a constitutively active CD40 molecule, which is a receptor for activating the B cells of the germinal center, although providing a more potent and sustained signal. Activation of the NF-kB pathway, which is a feature of HRS cells, leads to upregulation of antiapoptotic genes, including c- FLIP and XIAP, which are likely to contribute to HRS cell survival.

Benign and Malignant Connective Tissue Tumors 51

Figure 5. EBV produces a gene rearrangement and gene mutation in the B cell and hence it escapes apoptosis and shows the presence of receptors that are different from other B cells. LMP2A is the factor that protects Bcells from undergoing apoptosis. The concept of the same cells in negative EBV cases suggest that EBV is negative at present but may have been EBV positive earlier and the cell has lost its viral episome’ hit and run hypothesis’

Burkitt’s Lymphoma

Introduction

This is a very rare and fast growing form of a Non Hodgkin’s lymphoma. It is a high grade B cell lymphoma that is very aggressive. It is commonly seen in the sub Saharan Africa and is said to be caused by EBV.

52 P. C. Anila Namboodiripad and E. Anuradha Sunil

Pathogenesis

Figure 6. Depicting the pathogenesis of Burkitt’s lymphoma.

The BL cells show a characteristic germinal center derivation. It comprises the CD10+ CD77+ Bcl2. High levels of the bcl-6, c-myc are seen in the germinal centers due to the translocation of the chromosome t (8:14), t (2:8), or t(8:22) is considered the hallmark for the development of the BL. It places the c-myc allele into the heavy or light chain locus of the Ig. The presence of c-myc not only allows cells to enter cell cycle but also plays a role in apoptosis in the absence of the inhibiting signals such as the ras, bcl-2 or the EBNA 1. Though they exhibit high levels of HLA class II it fails to stimulate the CD4+T cells. The EBV has the property to convert the normal B lymphocytes into both the BL and the B- lymphoblastoid cells. Even though translocation of MYC is generally considered a hallmark for BL and this characteristic has been uniformly observed across the different BL subtypes, c-myc does not act alone. Further, in order for c-myc to initiate its transcriptional activities, it must first form a heterodimer with the constitutively expressed max. If the formation of c- myc/max heterodimers is prevented, cells do not undergo c-myc-induced transformation and alternately may even prevent its deregulation. The mistakes in the repair of the mutated c-myc results in translocation between chromosomes t (8; 14). But deregulated oncogene is unable to transform normal cells into cancer cells. This role is performed by the p13 k, which is a key element in the Burkitt lymphoma development. Benign and Malignant Connective Tissue Tumors 53

Another enzyme the AID (Activation-induced cytidine deaminase) has been implicated in the development of the BL and the deregulated expression of the AID causes a c-myc translocation in a cell latently infected with the EBV. A transcription factor found active in the BL is the TCF-3. Not much detail is known about this agent. It has been observed that malaria occurs together with the BL. The reason is the inverse proliferation of the B and the T cells: ↑ of the B cells and the ↓ of the T cells cause a reactivation of the EBV in the infected T cells. HIV represents another infection that may aggravate the pathogenesis of BL. Reduced CD4+ count may play a role in an EBV infection of the B cells. There are 5 viral glycoproteins that help the EBV to enter the B cell namely the viral gp 350 and the viral gp 42. The other viral proteins include gB, gH, and gL.

Multiple Myeloma

Introduction Multiple myeloma, also called as plasma cell myeloma is an uncommon cancer of the plasma cells.

Pathogenesis The underlying pathophysiologic phenomena of the clinical features include suppression of humoral- and cell-mediated immunity, elevation of IL- 6, abnormalities of the bone marrow microenvironment, and increased osteoclastic activity. Translocations in the chromosome 14q32 and other chromosomes such as 11, 4, 6, 16 results in the occurrence of the multiple myeloma. Deletion of whole or parts of chromosome 13 are also said to play a role in the malignant melanoma. The overproduction of the plasma cells results in secretion of the IL-6 which is also called as the osteoclast activating factor (OAF) and this is said to play a role in bone destruction. It causes the lysis of the long bones resulting in fractures. Light chains of the plasma cells may be excessively deposited in the kidneys (light chain deposition disease) resulting in the renal failure, in the nerves (neuropathy) and also leading to immunologic deficiencies that result in infections. 54 P. C. Anila Namboodiripad and E. Anuradha Sunil

It also results in production of the single clone of antibody or the monoclonal antibody (M protein) which is detected in the urine and is known as the Bence Jones proteins.

Hemangioendothelioma

Introduction Hemangioendothelioma are those vascular neoplasms that exhibit a borderline biological behavior that is intermediate between totally benign hemangiomas and highly malignant angiosarcomas.

Pathogenesis The pathogenesis is said to occur following the reciprocal translocation between the t (1; 3) (p36; q25). The genes involved in the translocation are the WWTR1 or the TAZ gene to the CAMTA1 gene. Gene fusion between two other genes YAP with TFE3 is also said to play a role in the formation of the hemangioendotheliomas. The above mentioned WWTR1 (WW domain-containing transcription regulator 1) and the CAMTA1 are both transcription factors. An evolutionary conserved signal transduction pathway playing a role in the regulation of the organ size and anoikis (a type of programmed cell death) was said to play a role in this tumor formation and they form the end effectors of the Hippo pathway. So basically the fusion occurs between an oncogene and a tumor suppressor gene. The CAMTA1 in mammals helps in formation of the cardiac muscle and also plays a role in the neural development and acts as a suppressor in formation of the neuroblastoma, glioblastoma multiforme and glial tumors.

Congenital Epulis

Introduction Congenital epulis, also known as granular cell tumor of the newborn or Neumann's tumor is a rare tumor of the newborn. It commonly arises from the anterior part of the maxillary gingiva and is seen as a mass protruding from a newborn’s mouth. It may hinder the child’s respiration or the feeding.

Benign and Malignant Connective Tissue Tumors 55

Pathogenesis The occurrence of the tumor in neonates and in females suggested that this tumor may be hormonally affected. However neither high estrogen nor progesterone has been detected in these individuals. The absence of markers for the primary tumors indicates an origin from an uncommitted mesenchymal cell. The similarity of this tumor with the Schwann cell granular cell tumor indicates that the lesion could arise from the Schwann cells. But the Schwann cell marker, S100 was found negative in the CGCE.

Granular Cell Tumor

Introduction Granular cell tumor is an uncommon poorly understood tumor.

Pathogenesis  Earlier thought to arise from the myoblasts but the markers for muscle cells was found negative.  The theory that it arises from the Schwann cells has been proved correct since it is found to be S100 positive.  Said to arise from fibroblasts or mesenchymal cells but authors have proven that it is not a neoplastic condition and it probably arose due to trauma.

Chapter 7

Benign and Malignant Tumors of Salivary Glands

Benign Salivary Gland Tumors

Pleomorphic Adenoma

Introduction This tumor is also called as the benign mixed tumor. It is one of the most common tumors of the salivary glands. The tumor is painless, slow growing, and composed of biphasic population of epithelial and mesenchymal cells.

Pathogenesis A large number of theories have been put forth to describe the origin of the Pleomorphic Adenoma (PA). It is proposed that the tumor is of monoclonal origin i.e., single source giving rise to both the epithelial and mesenchymal components for e.g., pluripotent intercalated duct cells. The tumor could be derived from the epithelial basal duct cells which undergo neoplastic differentiation to form

1) An epithelial component and the other 2) A mesenchymal component.

A gene PLAG1 mapped to chromosome 8q12.1 may play a role in Pleomorphic Adenoma pathogenesis. This gene probably induces the growth 58 P. C. Anila Namboodiripad and E. Anuradha Sunil factor production, mainly the IGF 2 and cytokine like growth factor 1, and which therefore results in cell proliferation. This has been studied by both the Western blot and immunohistochemical analysis. It was found that the PLAG1 expression has been identified in the epithelial, myoepithelial, and also the mesenchymal components of the Pleomorphic Adenoma. PLAG1 and HMGA2 genes are said to play a role in cell cycle regulation and growth factor signaling. Fusion of the PLAG1 gene and the FGFR1 gene was stated as another mechanism for the development of the Pleomorphic Adenoma. The PLAG1 gene was mapped to chromosome 8q12.1 and FGFR1 mapped to chromosome 8q12. The breakpoints for the fusion occurred in the 5' non coding regions of both genes, leading to activation and the promoter substitution of PLAG1 expression. Another partner gene identified in Pleomorphic Adenoma is the WIF1 (wnt inhibitory factor 1). Wnt proteins are extracellular signaling molecules that play a role in the control of embryonic development. Wnt proteins play a role in stem cell biology, in angiogenesis and even in carcinogenesis.

Figure 1. Normal cell has a multi looping transmembrane receptor, one end of which is in the cytosol and the other end attached to the wnt ligand. Through cytosolic factor like disheveled (dsl) they are attached to the beta catenin, a cadherin which plays a role in cell to cell adhesion and cytosol to nucleus signaling. Benign and Malignant Tumors of Salivary Glands 59

WIF1is expressed at a high level in normal salivary glands but on the other hand HMGA2 is not expressed. These 2 genes are fusion partners and the WIF1 is the suppressor of this wnt molecule. And a rearrangement in the WIF1 gene is said to play a role in development of the pleomorphic adenoma. A combination effect between upregulation of HMGA2 and a downregulation of WIF1 gene is the main cause for pleomorphic adenoma. Translocations involving chromosome 8q12 and 12q14-15 are also observed in the pleomorphic adenoma of salivary glands. Another translocation observed is the t (3; 8) (p21; q21) translocation seen in nearly half of pleomorphic adenomas. This results in promoter swapping between the gene (CTNNB1) for Il-catenin or β catenin and PLAG i, a novel zinc finger gene at 8q12 (1). This finding indicates that PLAGi activation due to promoter swapping is very likely to be a crucial event in salivary gland tumorigenesis. High amplification of the HMGA2 gene, either in its intact or disrupted form or in the form of fusion with the WIF1 gene indicates a malignant transformation of the tumor. Other authors try to play down its role and suggest that the real role of these genes is to provide elements for proper translation of the fusion transcripts. The highest expression levels of HMGA2 gene are detected in fetal tissues whereas gene expression is undetectable in normal adult tissues. The HMGA2 genes are also said to partner 2 other genes to play a role in occurrence of pleomorphic adenoma and they include the NFIB (nuclear factor I B gene) and HMGA2-FHIT (fragile histidine triad gene). These have been identified in pleomorphic adenomas with translocation t (9; 12) (p23; q12-15) and t(3;12) (p14.2;q14) respectively. There is a rearrangement of the HMGA2 gene and such changes are said to result in an increased expression of that gene. Thus, HMGA2 gene rearrangements may promote tumorigenesis in pleomorphic adenoma. Histologically, the pleomorphic adenoma is said to consist of features that indicate its origin from the myoepithelial cells. Those features include perinuclear tonofilaments, actin microfilaments, and remnants of basement membrane. Epithelial markers showed that benign pleomorphic adenomas of the major salivary glands are pure epithelial cell tumors. Pleomorphic adenoma was also said to arise from neoplastically transformed intercalated ducts cells. Viruses have been implicated in the pathogenesis of pleomorphic adenoma and predominant among them was the SV40 virus, as SV40 DNA sequences and associated T antigen were said to be identified in these tumors.

60 P. C. Anila Namboodiripad and E. Anuradha Sunil

Basal Cell Adenoma

Introduction Basal cell adenomas are rare salivary gland tumors comprising of basaloid cells arranged in a solid and trabecular pattern and nuclei of peripheral cells are said to show palisading. They lack the chondromyxoid matrix material characteristic of pleomorphic adenomas.

Pathogenesis Trisomy 8(three copies of the same chromosome) (genes on chromosome 8 are involved in brain development and function, and about 16% are involved in cancer) has been identified in the basal cell adenoma. In some cases alterations in chromosome 13 and 16 q has also been found. On the chromosome 16q12-13 there is alteration in the CYLD1gene. Translocations in the chromosome 8 and 16 results in acute myeloid leukemia. The CYLD1 is a deubiquinating enzyme and the loss of its activity shows an opposite and exaggerated activity of the NF-kappa B indicating tumorigenesis.

Canalicular Adenoma

Introduction It is a rare tumor that comprises less than 1% of all the salivary gland tumors. If present, it is commonly observed in the minor salivary glands of the upper lip in nearly 80% cases. The tumor is multifocal in nature and may be mistaken for metastasis.

Pathogenesis The multifocality suggests a field effect. It is thought to arise from the terminal ducts or the striated ducts of the salivary gland.

Oncocytoma

Introduction It is a benign tumor that is composed of cells that contain granular eosinophillic cytoplasm, called as oncocytes. It has also been called as an oxyphillic adenoma. Benign and Malignant Tumors of Salivary Glands 61

Pathogenesis There have been various theories that have been proposed for the oncocytic change in the acinar cells

1) Oncocytic change seen in the acinar cells due to metaplasia is said to be one of the theories put forth with regard to oncocytic change. 2) The second theory states that oncocytoma may be composed of acinar cells that have aged and those that have lost their capacity to perform their specific tasks. They are considered as burnt out. This theory was proposed by Hamperl 3) Another theory states the importance of mitochondria and this being the reason for transformation of the acinar cells. According to these authors, as the acinar cells age, the number of mitochondria in them increases in number due to the damage or the burning out of the mitochondrial enzymes. 4) Mitochondrial DNA damage was said to be another factor responsible for the oncocytic change and this was said to result in the disturbance of the respiration of the cells together with it a multi organ failure. This theory was proposed by Linnane et al. in 1989. 5) Sunmunn et al. stated that a previously hyperplastic or hypertrophic duct that was undergoing regressive alteration resulted in mitochondriopathy which could be a reason for the oncocytic change.

Oncocytoma in its early stages shows the presence of oncocytes having an increased number of mitochondria which undergoes pleomorphism hence resulting in biochemical changes and altered cell metabolism.

Warthin’s Tumor

Introduction Warthin’s tumor of the salivary glands is a benign tumor which is considered more of a hamartoma or a reactive proliferation of ductal salivary gland cells and lymphoid elements, than a neoplasia. Hamartoma is the abnormal proliferation of tissue native to its part.

Pathogenesis Authors have identified three main observations that are unique to the pathogenesis of Warthin’s tumor and they have been described as: 62 P. C. Anila Namboodiripad and E. Anuradha Sunil

1. One set of patients whose Karyotype is normal and the other where the karyotype was affected. 2. The next set of them wherein the changes occurred only in number of the chromosome for e.g., loss of Y chromosome or trisomy or monosomy 5, and 3. The third type of cases in whom the entire chromosome had undergone structural changes with one or two reciprocal translocations at (11; 19) (q21; p13). This probably suggests a link to mucoepidermoid carcinoma. This karyotyping was an individual event or may be a part of a complex karyotyping. 1. The complex karyotype carried a MECT1-MAML2 fusion transcript. But this transcript cannot be considered as a hallmark in the diagnosis of the Warthin’s tumor. 2. A theory put forth by another group of authors with regard to the pathogenesis of Warthin’s tumor stated that the heterotropic intercalated and striated duct cells got entrapped in the intraparotid or paraparotid lymph nodes. According to this theory the parotid gland was among the first salivary glands to develop during embryogenesis and the last one to get encapsulated. Lymph nodes get entrapped within them and they are found to lie on the inside of the capsule. Heterotropic salivary gland ducts may get entrapped in this lymphoid tissue to become neoplastic and result in the formation of the Warthin’s tumor. 3. The lymphoid tissue was considered by some authors, as a defensive reaction to the metaplastic epithelial change or Warthin’s tumor. These authors do not consider the Warthin’s tumor to be a neoplasia. 4. Because of its coexistence with autoimmune diseases like Hashimoto’s thyroiditis etc., and the presence of lymphoid element, this tumor is considered to be an autoimmune lesion. 5. So what is the factor that causes the epithelial portion of the Warthin’s tumor to become metaplastic? The authors who have propositioned this theory believe that  EBV could be an inciting factor for Warthin’s tumor and  Type 2 Nitrous Oxide is said to another stimulating factor for the tumor. Another gene suggested as the cause for the tumor is the WAMTPI gene (Warthin and mucoepidermoid tumour Benign and Malignant Tumors of Salivary Glands 63

translocation partner gene) and this gene has been implicated in the histogenesis of the Warthin’s tumor. A theory for pathogenesis of Warthin’s tumor states that the striated duct cells undergo oxyphilic metaplasia and produce an apocrine secretory activity. The duct epithelium proliferated due to the metaplasia which then incited a stromal reaction with proliferation of the immune cells together with the epithelial cells. The causative factor(s) that caused this metaplasia was unknown. Cigarette smoke could be one of the predisposing factors causing the metaplastic change. This has been speculated, because of the close relationship between smoking and Warthin’s. 6. Some authors have identified the presence of the class II HLA on the epithelial cells in the Warthin’s tumor. Similar HLA has also been identified on the epithelial cells of the some autoimmune salivary gland conditions like for e.g., The Sjogren’s syndrome involving the salivary and lacrimal glands. Thus according to these authors the epithelial cells acted as an antigen presenting cell (APC) that could stimulate the lymphoid stroma. Thus, these authors suggested that the expression of HLA-DR antigens and interleukin-1 production by the epithelial component of WT could especially excite the lymphoid cells within the salivary gland.

Malignant Salivary Gland Tumors

Mucoepidermoid Tumor

Introduction Mucoepidermoid carcinomas were first described by Masson and Berger in 1924. It represents a distinct type of tumor. It comprises of three cellular elements in varying proportions: mucus-secreting cells, the squamous cells, and the “intermediate” cells.

Pathogenesis Mucoepidermoid carcinoma may originate from excretory duct reserve cells, but the issue remains non-explanatory. Cytogenetics: Even though MEC is the most common type of malignant salivary gland tumors’ its pathogenesis and the key molecular events leading 64 P. C. Anila Namboodiripad and E. Anuradha Sunil to its development are yet to unravel. At least two partially overlapping cytogenetic subgroups have been identified, i.e., MECs with t (11; 19) (q21; p13) or variants thereof, and MECs with single or multiple trisomies, either observed as the sole abnormality or in combination with structural rearrangements. A recurrent t (11; 19) (q21; p13) translocation has been identified in Mucoepidermoid carcinomas of both the salivary glands and of the bronchopulmonary tissues. Such a translocation leads to the fusion of exon 1 from a gene of unknown function at 19p13, termed mucoepidermoid carcinoma translocated 1 (MECT1, also known as CTRC1, TORC1, or WAMTP1), with exons 2-5 of a member of mastermind-like gene family, MAML2, at 11q21. It has been demonstrated that the resultant fusion transcript MECT1-MAML2 activated the transcription of the Notch target gene HES1, independent of ligand stimulation. The translocation t (11; 19) (q21; p13) and the MECT1-MAML2 fusion transcript have been detected in 38-81% of mucoepidermoid carcinoma cases. This same translocation is also seen in case of acute leukemia, and an apparently identical rearrangement has been identified in Warthin’s tumor. Apart from Warthin’s tumor, it has not been demonstrated in any other salivary gland tumor. Immunohistochemistry using an MECT1-MAML2 antibody in fusion-positive mucoepidermoid carcinomas resulted in nuclear staining of all three major cell types, i.e., mucus-producing, epidermoid, and intermediate cells. However, stromal cells did not express the fusion protein. Expression of the hybrid gene in all cell types suggests that it may play an early role in tumor initiation. Such a distinct translocation and resulting fusion transcript may be a useful tool in diagnosing morphologically ambiguous MEC. In addition, there is an association between transcript expression and tumor stage, with fusion-positive tumors behaving in a less aggressive fashion. Fusion-positive patients had a significantly lowered risk of local recurrence, metastases, or tumor-related death compared to fusion-negative ones (median survival of more than 10 years compared to 1.6 years). In addition, there was a preponderance of highly differentiated low-grade tumors in fusion-positive patients compared to the fusion-negative group. Not all fusion-positive tumors carried the translocation t (11; 19), meaning that other cryptic translocations may contribute to the disease in such cases. Fusions may thus be found in MECs with complex 11; 19 rearrangements and also with variant translocations such as t (11; 17) and t (11; 13), as well as in tumors with apparently normal karyotypes and trisomies. The other common chromosomal abnormality was single or multiple trisomies, observed in 7 of 21 MECs in one series. Trisomies were mostly Benign and Malignant Tumors of Salivary Glands 65 observed in cases not harboring a t (11; 19). The most frequently encountered trisomies were +7, +8, and +X. Other recurrent abnormalities found were deletions of the terminal part of 6q. Apart from these abnormalities, the t (11; 19)-negative MECs showed a heterogeneous pattern of rearrangements with no obvious recurrent aberrations. Very recently, deletions of CDKN2A gene have been shown to be associated with poor prognosis in MECT1-MAML2 fusion-positive MECs. In the same study, neither activating EGFR mutations nor copy number gains at the EGFR locus was detected in fusion-positive and fusion-negative cases. Finally, the detection of HER-2 overexpression by immunohistochemistry has been correlated to adverse clinicopathologic features in the Mucoepidermoid carcinoma.

Acinic Cell Carcinoma

Introduction Acinous cell carcinoma makes up approximately 6-10% of the salivary gland cancers. It is one among the rare salivary gland cancers. Acinic cell carcinomas commonly arise in the parotid gland (86.3% in largest study) but submandibular and sublingual glands have also been seen to be affected.

Pathogenesis Histogenesis: Acinic cell carcinomas may arise from neoplastic transformation of the terminal duct cells (intercalated duct cells) with differentiation towards serous acinar cells. Another theory put forth suggests that the tumor could arise from transformation of terminally differentiated serous acinar cells. Cytogenetics: Multiple structural and numerical aberrations have been described in acinic cell carcinoma but no specific alteration has been identified. Loss of Y and trisomy 7, 8, and 21 has been reported. In one of the largest study to date, acinic cell carcinomas showed LOH (loss of heterozygosity) in at least one of the 20 loci tested on chromosomes 1, 4, 5, 6, and 17. Chromosomal arms 4p, 5q, 6p, and 17p were the most frequently altered, with 4p15-16, 6p25-q, and 17p11 regions showing the highest rate of abnormalities. In another study, analysis of different samples from a single case found evidence of polyclonality.

66 P. C. Anila Namboodiripad and E. Anuradha Sunil

Adenoid Cystic Carcinoma

Introduction Adenoid cystic carcinoma (ACC) is a rare form of malignant neoplasm that arises within secretory glands, especially the major and minor salivary glands of the head and neck. Glands of the trachea, lacrimal glands, breast, skin, and vulva may also be affected by the adenoid cystic carcinoma. This malignancy has a distinctive histologic appearance.

Pathogenesis Adenoid cystic carcinoma is characterized by a t (6; 9) (q22-23; p23-24) translocation. The translocation fuses exon 14 of MYB gene, on chromosome 6q22-23, to the last coding exons of NFIB gene, on chromosome 9p23-24. Most breakpoints occur in intron 14 of MYB and intron 8 of NFIB. The minimal common part of MYB that is deleted is exon 15 including the 3'-UTR which contains several highly conserved target sites for miR15a/16 and miR- 150 micro RNAs. These micro RNAs are known to negatively regulate MYB expression. Deletion of these target sites may lead to overexpression of MYB- NFIB transcript and activation of MYB targets, including genes associated with apoptosis, cell cycle control, cell growth/angiogenesis, and cell adhesion. Deregulation of expression of MYB and its target genes may be a key oncogenic event in the pathogenesis of adenoid cystic carcinoma. Mutations in the c-kit gene have recently been described in adenoid cystic carcinoma, but their occurrence is rare, and they most probably do not represent ‘driver’ mutations in this entity. The tumors were composed of four major cell types; intercalated duct, myoepithelial, secretory, and pluripotential reserve/stem cells. The cellular composition of adenoid cystic carcinoma is similar to that in the “terminal tubule” complex stage of a developing salivary gland except that in the tumor the pluripotential reserve/stem cells differentiate predominantly along the intercalated duct cell line rather than secretory cells as in the acinic cell carcinoma. Furthermore, adenoid cystic carcinoma appears to contain a far greater number of myoepithelial cells than acinic cell carcinomas.

Pathogenesis Regarding the Perineural Invasion The exact mechanism behind the perineural invasion is still a mystery yet a number of theories have been put forth; a recent theory states that reciprocal signaling interactions between the tumor and the peripheral nerve plays a role Benign and Malignant Tumors of Salivary Glands 67 in the perineural invasion. The tumor cells acquire the capacity to respond to signals in the peripheral nerves and hence promote the invasion. Neutotropic agents (cytokines within nerves) have been identified in other malignancies such as the prostrate and the pancreas and they include the NGF and other factors, such as neural cell adhesion molecule (NCAM), have been shown to have increased expression in Head and Neck squamous cell carcinoma. Older theory included the spread of the tumor along any path which showed the least resistance and this happens to be the peripheral nerves, and another theory stating the presence of lymphatic channels within nerves has been discounted. Lymphatic channels have been found unable to penetrate the epineurium, around a single nerve, hence this theory has been discarded

Carcinoma Ex Pleomorphic Adenoma

Introduction Carcinoma ex pleomorphic adenoma (CXPA) is a poorly understood malignancy that are rare and that develops from either a long-standing primary or a recurrent pleomorphic adenoma (PA).

Pathogenesis Genomic alterations in carcinoma ex- pleomorphic adenoma are identical to those found in pleomorphic adenomas. Alterations at 12q13-15 with amplification of HMGA2 and MDM2 genes have been reported. MDM2 (at 12q14-15) is one of the most frequently co-amplified genes together with HMGA2, suggesting a pathogenetic role for MDM2 in carcinoma ex- pleomorphic adenoma. The genes were co-amplified in the same homogeneously staining regions and double minute chromosomes (MDM2) in a case of carcinoma ex- pleomorphic adenoma with a del (5)(q22-23q32-33) and t(10;12)(p15;q15). However, there was little MDM2 protein expression, when assessed by immunohistochemistry, compared to high HMGA2 expression levels in the carcinomatous parts of the tumor. Cerb-B2 expression has been detected in one third of carcinoma ex- pleomorphic adenomas and could help distinguish it from atypical pleomorphic adenoma. Mutation and overexpression of TP53 are also frequent events in carcinoma ex pleomorphic adenoma.

Chapter 8

Odontogenic Cysts

Odontogenic Keratocyst or Keratocystic Odontogenic Tumor

Introduction

Odontogenic keratocyst is an uncommon, benign cystic tumor derived from the odontogenic apparatus.

Pathogenesis

The cyst is said to arise from the odontogenic apparatus namely the

 Dental lamina or its remnants  Basal cells extension from the overlying epithelium.  In case of the Nevoid Basal cell carcinoma syndrome the cyst is more likely to arise from the dental lamina and the satellite cysts have been considered to be the  Primordial cyst on the other hand was said to arise in place of a tooth, from the enamel organ, more specifically from the degeneration of the stellate reticulum within the enamel organ.

70 P. C. Anila Namboodiripad and E. Anuradha Sunil

The proliferative activity of the OKC was studied by various authors due to its highly aggressive nature. It was found that there was an increased proliferation of the markers such as the Ki 67 and PCNA in OKC compared to other odontogenic lesions. But the proliferative markers are not an indication for the neoplastic nature of this lesion. In some cases though the lesion is neoplastic there is no much exaggeration of the markers seen. Another theory in favor of the neoplasticity of the lesion is its heterozygosity. Allelic loss in tumour suppressor genes such as p16, p53, PCTH, MCC, TSLC1, LTAS2 and FHIT, have been found in case of the OKC indicating their aggressiveness as these marker have already been used to identify some of the common human neoplasias. Also the lesion showing the presence of larger number of daughter cysts show a more tendency for allelic loss. P53 has been found low expressed in OKC. Growth characteristics of the OKC have been co-related to the overexpression and underexpression as well as the deletions in the 3q13.1, 5p14.3, 7q31.3 genes and also the overexpression and amplification of the 12q13, EGFR3, glioma associated oncogene (GLI1). Faults in the cell adhesion molecule is said to play a role in detachment of the epithelium from the connective tissue. The most important genetic alteration is said to be in the Drosophilia Patched gene (PTCH1). Germline mutation in this gene is said to play a role in the NBCCS. The Ki-67 showed a higher labeling index in the cases that showed a PTCH1 mutation. The ‘two hit’ or the haploinsufficiency theory is another factor in favor of the development of the OKC. The first hit is a mutation in one allele, which, although it can be dominantly inherited, has no phenotypic effect. The second hit refers to loss of the other allele and is known as “loss of heterozygosity.” Precursor cells to be hit by the loss of an allele namely 9q22.3-q31, which leads to loss of gene dosage, have already been affected by the ‘first hit’ in case of the NBCCS, whereas the sporadic OKC occur in cells in which there is already ‘two somatic hits.’ The 9q22.3-q31 is already a targeted site in case of carcinomas such as basal cell carcinoma, squamous cell carcinoma etc. Another important event in tumorigenesis where there is no exact change in the gene sequence is the epigenetic alterations which occur in case of benign and malignant tumors of the head and neck. DNA methylation is one such event where there is an addition of the methyl group in cytosines and in case of the OKC the PTCH1 methylation may occur in place of a true mutation. Odontogenic Cysts 71

Methylation is the process rather than mutation, seen to occur in the P21gene and the absence of methylation in the Rb1 gene in OKC samples as opposed to normal dental follicles.

Figure 1. Possible molecular mechanisms related to PTCH1 inactivation. One or more of these genetic or epigenetic alterations may act in cooperation causing PTCH inactivation, leading to Sonic Hedgehog pathway activation.

Y. Kubota et al. showed that Interleukin-1α (IL-1α) is strongly expressed in odontogenic keratocysts. IL-1α may up-regulate matrix metalloproteinase-2 activation by increasing the expression of membrane-type 1 matrix metalloproteinase in the fibroblasts isolated from odontogenic keratocysts synergistically with type I collagen. Ogata et al. showed that in keratocystic odontogenic tumour fibroblasts, IL-1α may stimulate COX-2 expression. Andric et. al. investigated the expression of survivin, an inhibitor of apoptosis, in odontogenic keratocysts and compared it to the findings in non-neoplastic jaw cysts - periapical cysts they also studied a possible relationship between survivin expression and human cytomegalovirus presence within these cysts. It was found that survivin may contribute to the aggressive behavior of odontogenic keratocysts, and thus support the emerging opinion of their neoplastic nature. Gli2-mediated reactivation of quiescent epithelial rests to form keratocysts indicates that these cells retain the capacity to function 72 P. C. Anila Namboodiripad and E. Anuradha Sunil as progenitor cells on activation by an appropriate developmental signal. Furthermore, markers known to be rapidly induced in response to growth factors, tumor promoters, cytokines, bacterial endotoxins, oncogenes, hormones and shear stress, such as COX-2, may also shed a new light on biological mechanisms involved in the development of these benign but sometimes aggressive neoplasms of the jaws.

Dentigerous Cyst

Introduction Dentigerous cysts are benign non-inflammatory odontogenic cysts thought to be developmental in nature.

Pathogenesis The dentigerous cyst may be either extrafollicular or intrafollicular. The extrafollicular dentigerous cyst arises mostly as an envelopmental OKC. Intrafollicular dentigerous cyst is said to arise due to accumulation of fluid between the tooth crown and the follicle. The tooth in this case would not be hypoplastic. But if the cyst has arisen due to degeneration of stellate reticulum of an enamel organ the tooth is most likely to be hypoplastic. The fluid that accumulates, most probably arises from the capsular vessels either between the layers of the reduced enamel epithelium or between the crown of the tooth and the reduced enamel epithelium. Capsular ground substance based glycosaminosglycans (GAG) causes the expansion of the cyst either in response to normal metabolic turnover of the tissue or in response to inflammation. The GAGs further enter the lumen through channels in the epithelial lining. This may also result in the further cyst expansion. A permanent tooth counterpart entering into the radicular cyst of a deciduous tooth may be another reason for the formation of the dentigerous cyst. But is has been noticed that the radicular cysts in association with the deciduous teeth are rare. And the permanent teeth are more likely to invaginate into the radicular cyst of the deciduous tooth rather than cover over it, is the explanation given by other authors. But in the histology of these cysts it has been found that there is a presence of inflammatory cells apart from the cyst lining. It may be mistaken for the radicular cyst. A new theory states the role of a structure of primary cilia that are said to be involved in the pathogenesis of dentigerous cyst and the OKC. These cilias have been identified in the lining of both the aforementioned cysts. The cilias Odontogenic Cysts 73 have already been proved to be involved in the pathogenesis of cysts in the kidneys. They have been found to be normal inhabitant of the oral epithelium so it is not unusual for them to play a role in the pathogenesis of the cysts. The study is still underway for confirmation of this theory.

Figure 2. Diagram of ciliary structure.

The motile cilia have a central core of microtubules and an inner and outer dynerin structures required for motility. The organelle is covered over by a membrane which contains the multiple microtubules.

From ‘The Nature Publishing group.’ These primary cilia have been presenting a variety of receptor functions and hence it is not unusual for them to play a role in signaling. Apart from simple signal transduction function the cilia is also said to play a role in a large number of developmental activities and also plays a role in memory. 74 P. C. Anila Namboodiripad and E. Anuradha Sunil

Lateral Periodontal Cyst

Introduction Lateral periodontal cyst (LPC) is an uncommon but well recognized type of a developmental odontogenic cyst.

Pathogenesis There is no clear information about the pathogenesis of the lateral periodontal cysts but they have been postulated to arise from these three sources:

 Reduced enamel epithelium, of a developing adjacent tooth.  Remnants of dental lamina were suggested due to the presence of the glycogen rich islands of the lateral periodontal cysts. It is said that cyst LPC differs from that of the Dentigerous Cyst in that the LPC arises from the postfunctional (quiescent) cells of the dental lamina and the DC from the active cells of the dental lamina.  Cell rests of Malassez: But the cyst normally occurs on the facial aspect of the jaws and not the lingual aspect hence the probability of the cyst arising from the dental lamina and not the rests of Malassez is considered.  Also a possible origin from a primordial cyst of supernumerary tooth germs has also been considered. This theory has been suggested, due to the presence of a large number of supernumerary teeth in this region.  A pulpal infection opening on the lateral aspect of the root or a periodontal ligament infection, stimulating the rests of Malassez is also considered another possibility.  Clear cells’ presence in the cyst lining points to their resemblance to the clear cells in the dental lamina rests and not to the rests of Malassez or reduced enamel epithelium and neither can they arise from the radicular or the dentigerous cysts as both these cysts arise from the above two mentioned sources.  The final and the most recent theory states that the origin of the LPC is based on genetics. The reduced enamel epithelium covering the crown of the developing adjacent teeth was said to be the cause for the formation of this cyst. The replantation of the epithelial plaques which may give rise to a cyst, by canalisation within it shows the active Odontogenic Cysts 75

nature of the epithelial lining. But why does the cyst take so many years for formation is the next question, since LPC is predominant in the middle ages and the implantation must occur in childhood. Also if the lining was said to be so active how it can it not erupt into the oral cavity and how come it does resorb the bone and not the roots, are all questions for which there are no concrete answers. Also why the LPC is seen in the specific premolar areas and not in others cannot be explained by this theory.

Figure 3. The above sketches show the hypothetical pathogenesis of the lateral periodontal cyst. The eruption of a tooth through the lateral dentigerous cyst may result in the formation of the lateral periodontal cyst.

Figure 4. Figures illustrate the different origins of the lateral periodontal (left) and gingival cysts (right). Both cysts are said to originate from the epithelia comprising a part of the dental follicle. 76 P. C. Anila Namboodiripad and E. Anuradha Sunil

In view of this varied etiology, the authors suggested that the term should be qualified to indicate whether the cyst’s origin was pulp infection, infection through the gingival crevice or idiopathic stimulation of cell rests. It is now widely accepted that the term ‘lateral periodontal cyst’ should be confined to cysts in the lateral periodontal position in which an inflammatory etiology and a diagnosis of gingival cyst of the adult and collateral keratocyst have been excluded on clinical and histological grounds.

Gingival Cyst of the Adult and New Born

Introduction Gingival cyst of the adult and new born arises on the gingiva of the new born and the adult.

Pathogenesis Derived from the reduced enamel epithelium and the remnants of the dental lamina i.e., the cell rests of Serre after 4th month in-utero. It is also said to arise from the invagination of the rete peg of the overlying epithelium, or the traumatic implantation of the surface epithelium and from the degeneration from the aberrant mucous glands. It was postulated that the gingival cyst of adults and the lateral periodontal cyst have a common histogenesis and represent the intra-osseous and extra- osseous manifestations of the same lesion. Buchner and Hanssen considered that they probably were of the same epithelial origin.

Glandular Odontogenic Cyst

Introduction Glandular Odontogenic Cyst (GOC) is a rare odontogenic cyst that is unique with respect to its histopathologic features and its unclear histogenesis.

Pathogenesis Origin from dental lamina rests: Disintegration of the Dental Lamina: Shortly after the laying down of the hard tissues of the teeth, such as enamel and dentin, the complex pattern of dental laminae begins to section or disintegrate due to ectomesenchymal invasion and/or programmed cell death. Odontogenic Cysts 77

From the zone where the dental lamina joins with the oral epithelium, disorganization or fragmentation of the From the zone where the dental lamina joins with the oral epithelium, disorganization or fragmentation of the dental lamina progresses towards the developing enamel organ. Some cells of the laminae persist and tend to aggregate through proliferation into nests, known traditionally as epithelial pearls (Serres pearls or glands of Serres). The successional laminae (for formation of permanent teeth enamel organ) as well as the accessional laminae (for formation of supernumerary teeth or salivary gland) also disintegrate and give rise to epithelial cell remnants. Cells deactivate epithelial markers (E-cadherin, cytokeratin), up-regulate Slug and MMP2 (matrix metalloproteinase-2), and activate mesenchymal markers (vimentin), while residual lamina cells are removed by apoptosis. 3 Sometimes the dental lamina cells persist as rests (rests of Serre) which may later become active to form odontogenic cysts and tumors. The histological features of GOC strongly suggest an origin from the remnants of dental lamina because of the below mentioned features.

A. Rests of dental lamina:

1. Because of its histological features comprising of:

a. Nonkeratinised epithelial lining b. Mucus metaplasia of rest cells c. Aggressive nature of the cyst may indicate that it arises from the dental lamina rests.

2. High recurrence rate. 3. Cytokeratin (CK), 7,13,14,19 is positive on immunological testing and negative for CK 8 and 18 indicating an origin from odontogenic epithelium, rather than the salivary gland. 4. Wysocki et al. (1980) have pointed out that the characteristic glycogen- rich clear cells so commonly seen in dental lamina- derived cysts and remnants are never found in cysts derived from reduced enamel epithelium or rests of Malassez. Similarly, the epithelial linings of radicular and dentigerous cysts which are derived from the latter do not contain such clear cells.

B. Salivary gland origin: It was thought by the earlier authors that GOC was derived from salivary gland tissue because of the following factors. 78 P. C. Anila Namboodiripad and E. Anuradha Sunil

a) Histological features similar to low grade central mucoepidermoid carcinoma (LGMEC). b) First named as sialo-odontogenic cyst. c) Contains mucus cells. d) Is aggressive in nature like a malignant neoplastic salivary gland lesion. e) The distinguishing feature in GOC is found to be the typical thin epithelial lining without any solid epithelial proliferation as normally seen in MEC. f) Swirling spherical/epithelial plaque that is often seen in GOC is not observed in MEC. g) In cases where diagnosis of GOC or LGMEC cannot be made based on the morphological features alone, especially in small incisional biopsy samples, mammary serine protease inhibitor (maspin) immunolocalization can be used to distinguish these two lesions. The high levels of maspin in the epithelial-mucous cells (in both cytoplasm and nuclei) in LGMEC may serve as a tool to differentiate it from GOC. It has been suggested that many cases formerly diagnosed as central MEC can be the examples of GOC, and also some LGMECs would have originated from GOCs. h) CK expression has been demonstrated in GOC and central MEC. They found differences in CK18 and 19. CK18 is expressed by all MECs (mucoepidermoid carcinoma), but it is only expressed by 30% of the GOCs, whereas CK19 is expressed by all GOCs and only 50% of the central MECs. They suggested that GOC and central MEC are distinct entities that and expression of CKs 18 and 19 can be useful adjunctive tools in differentiating these two lesions. Immunohistochemical studies using cytokeratin 7, 13, 14, and 19 and their positivity strongly support the odontogenic nature. i) The detection of osteodentin and negative reaction for EMA (epithelial membrane antigen) in the glandular structure show that these features are not of glandular origin and support the concept of odontogenic differentiation in GOC. j) Platelet Derived Growth Factor (PDGF) signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme in development of salivary glands. These results suggest that the FGF/FGFR, ShhPtc, and Eda/Edar signaling cascades are critical for salivary gland organogenesis namely the submandibular gland. Similarly multiple signaling molecules, Odontogenic Cysts 79

including BMPs, FGFs, Shh, and Wnt proteins, have been implicated in tooth development.

Features distinguishing GOC from other odontogenic cysts:

a) The immunohistochemical expression of the apoptosis- inhibiting protein bcl-2, the cell cycle-related antigen ki-67 and the P53 which is involved both in cell cycle and apoptosis regulation was lower in GOC as compared to dentigerous cyst. They suggested that the biological behavior of GOCs may be associated with deregulation of cell death in the lining epithelium, indicated by increased expression of bcl2, while cell proliferation and P53 do not play a significant role. b) The aggressive biologic behavior of the GOC and its propensity for recurrence might be associated with, cell kinetics in the lining epithelium, as has been demonstrated in Odontogenic Keratocyst. It has been also demonstrated that the rate of recurrence increases with the radiographic complexity of the cyst. c) Botyroid odontogenic cysts, lateral periodontal cyst are slow growing as compared to GOC though they show histological features similar to the GOC.

Origin from epithelial rests of malassez (ERM): The formation of the ERM occurs during the development of the root, which begins before eruption. They break up to remain lodged in the surrounding dental follicle or may disintegrate. These rests are called the rests of Malassez. It was first identified by Serres as ‘restes de l’organe de l’email’ (Rests from the enamel organ). However, Malassez, in 1885, presented the first description of the cells and their distribution. Proliferation of the ERM has been implicated in developmental cyst formation, such as the gingival or lateral periodontal cyst. Inflammatory cysts, such as the paradental and peri- apical cysts, also arise commonly from the ERM (epithelial cell rests of Malassez). A number of odontogenic tumors may also arise from the ERM. Growth factors are important mediators of intercellular communication between connective tissues and epithelium; several growth factors have stimulatory effects on epithelial rests of Malassez, proliferation and differentiation. The recently described keratinocyte growth factor (KGF) appears to be of particular interest. It is possible that quiescence of the rests of Malassez is normally associated with low local levels of KGF or related 80 P. C. Anila Namboodiripad and E. Anuradha Sunil growth factors in the PDL but that inflammation enhances the expression of growth factors necessary for their activation. KGF production is markedly up- regulated during epithelial wound healing. Absence of inflammation and hence reduced expression of KGF indicates that GOC does not arise from the rests of Malassez.

Outer Enamel Epithelium Eriguchi (1959) has produced evidence for the existence of yet another source of odontogenic epithelial remnants not previously recognized. In embryos of 230-260 mm CRL (Crown of the foetus Rump length) the author demonstrated thin epithelial strands of polyhedric cells radiating from the outer enamel epithelium. The strands tend to proliferate and may reach 1.5-1.8 mm in length and thus, almost contacting the ridges of the overlying oral epithelium. They are often accompanied by or intermingle with minute blood vessels. The strands later show fragmentation with the formation of several spherical epithelial pearls that will blend with remnants from the dental laminae. The outer enamel epithelium-derived residues often show keratinisation similar to that found in the dental lamina-induced micro- keratocysts mentioned above. The residues are thought to act as source for the development of odontogenic lesions later in life, similar to those originating from dental lamina remnants. These findings have never been confirmed and convincing evidence is thus lacking.

Radicular Cyst

Introduction The radicular cyst or periapical cyst or apical periodontal cyst is among the most common cystic lesions in the oral cavity. It is an odontogenic cyst of inflammatory origin. Since the cyst is the sequelae of dental caries and the periodontal infection, which are the most predominant diseases of the oral cavity, radicular cysts are the commonest among all the cysts affecting the oral cavity.

Pathogenesis Pathogenesis of Radicular Cyst is considered in 3 Phases, which are as follows

 Phase of Initiation, Odontogenic Cysts 81

 Phase of Cyst Formation,  Phase of Cyst Enlargement

Phase of Initiation: The lining of the radicular cyst is said to arise from the proliferation of the cell rests of Malassez, and on the other hand in some cases, epithelial lining is also said to stem from:

a) Maxillary sinus wall lined respiratory epithelium, where the periapical lesion is in connection with the sinus wall. b) Oral epithelium from fistulous tract. c) Oral epithelium that proliferates apically from the periodontal pocket.

The mechanism responsible for stimulation of epithelial cells is not clear. It may be due to inflammation in periapical granuloma or some products of the dead necrotic pulp that may initiate the process & at the same time evoke an inflammatory reaction. Local changes in the connective tissue may also activate these cells. The cells of central portion of mass after proliferation of the epithelial cells become distanced further & further from nutrition in comparison with basal layer due to which they fail to obtain sufficient nutrition, they eventually degenerate, become necrotic & liquefy. This creates an epithelium lined cavity filled with fluid. There is another theory which suggests that the cyst may form through proliferation of epithelium which lines a pre-existing cavity formed through focal necrosis & degeneration of connective tissue in periapical granuloma. But such an area of necrosis near the epithelium or epithelial proliferation is not common.

Epithelial Proliferation The endotoxin from the necrotic tooth is the most important cause for the epithelial cells to proliferate. This endotoxin has mitogenic effect on epithelial cells. They were reported to stimulate keratinocyte proliferation directly, and the infection is also said to release a large number of inflammatory mediators, proinflammatory cytokines, and growth factors through innate and adaptive immune responses. Inflammatory mediators and proinflammatory cytokines produced by host cells that can be identified in radicular cysts include the prostaglandins (PGs), interleukin-1 (IL-1), Il-3, IL-4, IL-6, interferon (IFN), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-alpha (TGF-α). These mediators in turn stimulates the EGF(epidermal growth factor) 82 P. C. Anila Namboodiripad and E. Anuradha Sunil and causes it to act on the epithelial cell rests. Epithelial rests are also stimulated by the action of the keratinocyte growth factor (KGF) in the stromal fibroblasts. The chemokine and cytokines are found to be in much larger proportion than that in the granuloma. The epithelial cell rests of Malassez are quiescent or stable cells, which are generally in standby in the G0 phase of the cell cycle. For the cells to divide they need to pass through the G1, S and M phases. Proliferation markers are found in large quantities in the cyst, like the PCNA and the Ki-67 indicating an increased mitotic activity. Growth factors which are also found in large quantities in the cyst linings induce the proliferation of the epithelial cells which were earlier resting and quiescent cells.

Causes for Cyst Enlargement Include 1) Directly releasing of heparin into the luminal fluid, 2) Release of hydrolytic enzymes which could degrade capsular extracellular matrix components, and hence facilitating their passage into the fluid, and 3) The action of histamine on vascular permeability, thus leading to the transudation of serum proteins.

Mechanism of bone resorption in radicular cyst may also result in cyst expansion. The bone resorption is brought about by the osteoclasts whose presence is indicated by the presence of the RANKL and the OPG proteins in the radicular cyst wall. The mucinous or viscid consistency of the cyst fluid is mainly due to the mucous secretions from the goblet cells. At the sites of inflammation in the wall of the cyst, there are repeated small hemorrhages. Foamy macrophages play an important role here. They engulf extravasated RBCs. The cholesterol esters from the cell membranes of the RBC are engulfed by the macrophages. The acid lipase in the lysosomes breaks down this cholesterol into the cholesterol and fatty acids. Off-loading of cholesterol occurs when suitable acceptors are found in the external environment.

 Toller showed that radicular cyst exhibited a 5-11g/100 ml (mean 7.1g/100 ml) of protein due to various inflammatory factors playing a role.

Odontogenic Cysts 83

Types of Radicular Cysts Based on Its Pathogenesis

Simon (1980) pointed out that there are two distinct types of radicular cysts namely,

 Those containing cavities completely enclosed in epithelial lining, and  Those containing epithelium-lined cavities that is open to the root canals.

Simon (1980) designated the latter as ‘bay cysts.’ The presence of necrotic tissue in the cyst lumen attracts neutrophilic granulocytes, which extravasate and transmigrate through the epithelial lining into the cyst cavity where they perish. The lytic products of the dying cells in the cyst-lumen release a greater number of molecules. As a result the osmotic pressure of the cyst fluid rises to a level higher than that of the tissue fluid. The latter diffuses into the cyst cavity so as to raise the intraluminal hydrostatic pressure well above the capillary pressure. The increased intracystic pressure may lead to bone resorption and expansion of the cyst. However, the fact that an apical pocket cyst with lumen opening to the necrotic root canal, that becomes larger, would eliminate osmotic pressure was a factor that worked in favor of development of radicular cysts by the above mechanism.

Periapical Pocket Cyst The periapical pocket cyst is a radicular cyst containing an epithelium- lined pathological cavity which is open to the root canal of the affected tooth apex. As has been mentioned previously such lesions were originally described as ‘bay cysts’ and has been recently investigated in detail and renamed as the ‘periapical pocket cysts.’ It is postulated that a pocket cyst is initiated by a small bubble-like extension of the infected root canal space into the periapex. The microluminal space is enclosed in a stratified squamous epithelium which grows and forms an epithelial collar around the root tip. The epithelial collar forms an ‘epithelial attachment’ to the root surface so as to seal off the infected root canal and the microcystic lumen from the periapical setting. The presence of micro-organisms in the apical root canal attracts neutrophilic granulocytes by chemotaxis into the microlumen. But, the pouch- like lumen – biologically outside the body environment– acts as a ‘death trap’ and ‘garbage bag’ to the externalized and dying neutrophils. As the necrotic tissue and microbial products accumulate, the sac-like lumen enlarges to 84 P. C. Anila Namboodiripad and E. Anuradha Sunil accommodate the debris to form a huge bag of the root canal space into the periapical area. Thus these extensions of the root canal space of such lesions is found to be similar to the marginal periodontal pockets and hence the name ‘periapical pocket cyst’ as against a pointless nomenclature of ‘bay cyst.’

Calcifying Odontogenic Cyst

Introduction Calcifying cystic odontogenic tumour (CCOT) is a benign cystic neoplasm of odontogenic origin, characterized by an ameloblastoma-like epithelium with ghost cells that may calcify. COC is a controversial odontogenic cyst/tumor. It is a developmental cyst said to arise from the stratum intermedium part of the tooth germ. Due to the controversy regarding its histology a new terminology was coined for it namely “Calcifying cystic odontogenic tumor.” And another variant of the same was the COC or the calcifying odontogenic cyst.

 Peripheral COC: in gingival tissues, ie: extra-osseous  Central COC: within bone of jaws, ie: intra-osseous.

The following discussion pertains only to central COC

 a huge range of histopathologic patterns exists, ranging from a benign lesion that is primarily cystic, a benign lesion with a solid pattern of growth to a rare tumor with features of carcinoma.  Simple cystic, non-proliferative: single-chambered cyst lined by squamous or stellate reticulum–like cells with a varying degree of palisading of the basal cells; ghost cells present, eosinophilic dentinoid is mostly present and often juxtaepithelial in position, but no other hard tissues seen  Cystic, proliferative/ameloblastomatous: apart from simple cystic features, there is proliferation of ameloblastoma-like cords, islands, and sheets of epithelium with palisading of basal cell nuclei with reverse polarization within the wall; odontogenic epithelial proliferations that can superficially resemble ameloblastoma extend into the lumen as well as the connective tissue wall of the lesion Odontogenic Cysts 85

 Odontoma-associated: Odontoma-like tissues are seen in the wall of the lesion; when associated with an odontoma, patient’s age tends to be younger, with mean age 17 years  Solid lesions may be more aptly designated “Dentinogenic Ghost cell tumors”

Dentinogenic Ghost Cell Tumor

 Also called epithelial odontogenic ghost cell tumor, odontogenic ghost cell  Similar components to COC, but without a cystic structure, and with a neoplastic growth pattern.  It is controversial whether this is a developmental cyst or a true neoplasm  In 1992, WHO classified this lesion as odontogenic tumor but continued to use the term calcifying odontogenic cyst  In 2005, WHO redesignated the lesion as calcifying cystic odontogenic tumor  Although the condition is often described as being cystic in majority of cases the calcifying odontogenic cysts grow as more solid, seemingly neoplastic proliferations, and the term dentinogenic ghost cell tumor has been used to describe these lesions  Even the cystic lesion has an apparent neoplastic potential but most of them appear to be non-neoplastic

Pathogenesis Controversy exists as to whether tumor arises from the wall of the cyst or whether the cyst arose from the transformation of the solid tumor mass. The cyst was said to arise from the reduced enamel epithelium.

Ghost Cells The ghost cells were found in the thickened part of the lining epithelium. The ghost cells represent abnormal keratinisation that undergoes calcification in the future. The spinous cells in such situations may be widely separated by intercellular edema and the epithelium around the ghost cells is often convoluted. 86 P. C. Anila Namboodiripad and E. Anuradha Sunil

Atubular dentinoid was found in relation to the ghost cells. Foreign body giant cells were also found in relation to these ghost cells. Ultrastructurally the keratin was absent in these cells. There were a large number of theories regarding pathogenesis of the Ghost cells:

1) The ghost cells were thought to be normal or abnormal keratinisation. 2) The ghost cells were thought to be a squamous metaplasia undergoing calcification due to ischaemia. Praterois thought that the ghost cells indicated different stages of normal and altered keratinisation and were derived from metaplastic transformation of odontogenic epithelium. 3) Ghost cells were considered by some authors to be abortive enamel matrix in odontogenic epithelium. 4) Dentinoid was considered by Gorlin et al. as an inflammatory response to ghost cells. Some authors stated that it was not dentinoid but the osteoid which is laid down as a response of granulation tissue to ghost cells. 5) Sauk et al. on the other hand stated that the osteiod and dentinoid were seen in areas devoid of granulation tissue and that it was just an inductive phenomenon. 6) Sam Pyo Hong, et al. stated that ghost cells were probably the result of coagulative necrosis occurring at the same time when CCOT is undergoing liquefaction necrosis. This was suggested because of their negative reaction to cytokeratin antibody as opposed to the adjacent odontogenic epithelium indicating an altered keratin antigen. CK19 was positive in CCOT lining and all odontogenic epithelium and odontogenic tumors but ghost cells were negative for the CK19 staining and this proved the antigenic alterations. CK13 immunoreactivity was weak in ghost cells as compared to its positivity in CP and CCOT further indicating the degenerative nature of the ghost cells. Moreover, ghost cells do not express reactivity for cytokeratins[1,3,5,7] but express for AE1/AE3 and 34bE12. All these reactivities indicate a coagulative necrosis pathogenecity in the odontogenic epithelium of the CCOT. 7) Yamamoto et al. found high molecular weight keratins and lowered involucrin staining as compared to the oral epithelium. Thus, they deciphered that these cells go through an abnormal terminal differentiation by making altered homogenous acellular materials and Odontogenic Cysts 87

ghost cells which may be formed into probably different subclasses of keratins which has strong propensity to degenerate. Till date the pathogenesis of ghost cells is not completely known.

Fissural or Non Odontogenic Cysts

Nasopalatine Duct Cyst

Introduction These are the most common of the epithelial and non odontogenic cyst of the maxilla. It has also been called as the incisive canal cyst.

Pathogenesis The nasopalatine duct cyst is said to arise from the nasopalatine duct in the incisive canal. In the lower animals these glands of the nasopalatine duct played a role in perception of smell. In man vestigial remnants of the cord, canal or even ducts are found as epithelial islands that proliferate and undergo cystification resulting in the formation of the nasopalatine duct cyst. The epithelial lining was varied depending upon the site within the duct where the epithelial islands were found either towards its oral end or towards its nasal end. If near the nasal end the epithelial islands were ciliated columnar, if in the center the cells were pseudostratified and if towards the oral end it was stratified squamous in nature. Accordingly the epithelial lining of the cyst also varied. The vomeronasal organ of Jacobsson, a vestigial olfactory organ in man, placed just above the nasal end of the nasopalatine duct, was said to be another source for the formation of the nasopalatine duct cyst. Trauma or infection of these islands would cause the epithelial cells of this organ to proliferate and then degenerate resulting in the formation of the cyst. In such a case there must be the presence of inflammatory cells either in the stroma or the epithelial lining of the cyst. But was no inflammatory infiltrate within the cyst lining in any of the specimen. Hence this theory was disapproved. Mucous gland cells make up the third theory in the formation of the nasopalatine duct cyst. But the content of the cystic fluid was found to be more transudate like than exudates and was found to contain no mucin. Hence this cyst theory was also rejected. 88 P. C. Anila Namboodiripad and E. Anuradha Sunil

The final mechanism that is responsible for playing a role in the etiopathogenesis is thus still under speculation as none of the theories hold credence.

Globulomaxillary Cyst

Introduction Globulomaxillary cyst was named as such as it was considered as a non odontogenic cyst thought to arise at the line of fusion between the globular process and the maxillary process.

Pathogenesis Fissural cysts were said to occur at the sites of fusion of the developmental processes. In case of the globulomaxillary cyst, it was said to originate at the junction of the fusion between the globular, medial nasal and maxillary process. A slight variation was proposed in the above theory and it was considered that these cysts formed at the junction of the premaxilla and maxilla rather than the above mentioned sites, and that they should be called premaxillary- maxillary cysts. Embryologists have disproved this theory and stated that, in fact, at these junctions there is no actual ectodermal to ectodermal contact but contact takes place between the mesoderms which are covered over by the folded epithelium, and when fusion occurs there is a mesoderm to mesoderm fusion and the ectoderm grows over the underlying fused mesoderm. The theory of there being no globulomaxillary cyst but that these are actually just OKC or lateral periodontal cysts or radicular cysts present adjacent to the teeth has found favor with the embryologists.

Nasolabial Cyst

Introduction The nasolabial cyst occurs outside the bone in the nasolabial folds below the alae nasi. It is traditionally regarded as a jaw cyst although strictly speaking it should be classified as a soft tissue cyst.

Odontogenic Cysts 89

Pathogenesis This neoplasm is said to arise from the nasolacrimal duct which forms at the line of fusion between the lateral nasal and the maxillary process, ensconced in the nasolacrimal ridge extending between the inner canthus of the eye, where its cranial end is situated and the lateral wall of the nose where its caudal end is seen. The duct is lined by pseudostratified lining and the same cells are found to line the nasolabial cyst. The proliferation and the cystic degeneration of the epithelial islands is said to be the reason for the formation of the cyst.

Cysts of the Salivary Gland

Mucous Retention Cyst and Ranulas

Introduction A mucus cyst is a benign, common, mucus-containing cystic lesion of the minor salivary glands in the oral cavity. Mucoceles in the floor of the mouth are called as ranulas. They usually involve the major salivary glands. More precisely the ranula begins in the body of the sublingual gland.

Pathogenesis Mucus retention cysts results from the obstruction of salivary flow within a salivary gland duct, because of a sialolith, periductal scar, or impinging tumor. The retained mucin is surrounded by ductal epithelium, giving the lesion a cyst-like appearance microscopically. A sialolith represents the precipitation of calcium salts (predominantly calcium carbonate and calcium phosphate) around a central nidus of cellular debris or scattered mucin. The trauma causes destruction of large amount of the glandular acini and continuous secretion of mucin from the remainder of the acini. The above factors cause pooling of saliva distal to the obstruction resulting in a swelling, which histologically has an epithelium lined cavity filled with mucin, because the majority of salivary glands affected by the mucoceles or the salivary duct cyst are the minor mucous salivary glands. Ranula is said to have a similar pathogenesis as that of a mucus retention cyst but in this case the obstruction is in the duct (ducts of Rivinus) of the small major salivary gland namely the sublingual gland.

90 P. C. Anila Namboodiripad and E. Anuradha Sunil

Mucous Extravasation Cyst

Introduction Another cyst of the salivary gland is the mucous extravasation cyst and this is not a true cyst in the true sense; i.e., it is not lined by an epithelium.

Pathogenesis The mucus from the disintegrated cells, following trauma to the salivary gland, pools up and is then surrounded by the surrounding connective tissue that undergoes walling off to protect the spillage of the mucin to the neighboring areas. The epithelial structure, whether they are the duct or the acini, may persist after trauma, finally distintegrates resulting in the mucus extravasation cyst. Mucous extravasation type of cyst is basically a false cyst. It occurs due to extravasation of mucus from a severed duct into the surrounding connective tissue. The severance of the duct mostly follows a crush type injury. This type of cyst is not normally lined by an epithelium. The spreading of the mucus in the connective tissue stroma may be due to the assistance obtained by the additional invasive proteins namely the MMPs, TNF-α and the plasminogen activators.

False Cysts or Pseudocysts

Aneurysmal Bone Cyst

Introduction Aneurysmal bone cyst (ABC) is a solitary, expansile and erosive lesion of bone. The cause of this non-neoplastic lesion is unknown.

Pathogenesis Aneurysmal bone cyst is said to be located commonly at two predominant sites in the body as stated by Jaffe.

 The juxtacortical and  subperiosteal.  And the etiology for the former was considered as trauma and for the latter as occurring secondarily within a primary lesion. Another theory Odontogenic Cysts 91

for its occurrence may be due to a sudden vascular disturbance or following the development of an arteriovenous shunt.

The theory about the cyst arising in a primary lesion has received support. The primary lesion in question is said to be a benign giant cell tumor that has regained communication, during its course of formation, with a large blood vessel which has been damaged by the tumor. Fibrous dysplasia was another lesion in which the ABC was found to arise. Chondromyxoid fibroma may show aneurysmal cystic changes. Non-ossifying fibroma, chondroblastoma, giant cell tumor of bone, osteoblastoma, giant cell granuloma, fibrous dysplasia, myxofibroma and solitary bone cyst was also seen to undergo cystic changes. One case of Paget’s disease of bone showed the presence of large blood-filled spaces, as did several of the malignant lesions. These ABC occurs following the fusion of a large number of microcysts within the primary lesion. In the case of the giant cell lesions, the multinucleate giant cells may form a part of their margins. The central giant cell granuloma has a propensity to form microcysts because of its loose, oedematous, fibrillar connective tissue stroma in which lie many thin-walled blood vessels and extravasated erythrocytes. Microcyst formation is facilitated by localised areas of necrosis in the stroma brought about by stagnation and ischemia. The resulting microcysts are lined by stromal connective tissue. They enlarge by further stromal breakdown and coalesce with each other. Loss of stromal support leads to dilatation and rupture of the thin-walled vessels resulting in the haemorrhage into the stroma and the further formation of the microcysts. An association between the dilated blood vessels and microcysts has frequently been observed. Once a vascular connection is established between a larger vessel and a microcyst, haemodynamic pressure participates in its enlargement and little supportive resistance is offered if the surrounding stroma is loose and edematous. The spaces now assumes the dimensions of macrocysts which are surrounded by a layer of compressed stroma and these multiple expanding blood-filled cysts produce the pressure resorption of bone. Endosteal resorption of the cortical plates occurs ultimately and once these are breached, a ‘blow out’ of the lesion covered with periosteum, is seen. A layer of periosteal new bone may be deposited to form a thin shell covering the aneurysmal bone cyst. The rare development of aneurysmal bone cyst that occurs in malignant lesions probably explains the so-called malignant form of the cyst, occasionally reported in the literature. Some authors believe that the latter 92 P. C. Anila Namboodiripad and E. Anuradha Sunil represented the telangiectatic form of osteosarcoma which several authors have described, as resembling the aneurysmal bone cyst. To conclude no concrete evidence exists till date to explain the validity of the above claims.

Solitary Bone Cyst

Introduction A solitary bone cyst of the mandible or the traumatic bone cyst of jaw, (extravasation cyst, haemorrhagic cyst of the mandible, unicameral bone cyst, extravasation cyst and progressive bone cavity) is said to be an uncommon nonepithelial lined, radiolucent, mandibular lesion. The main etiological factor for the same, is said to be trauma together with other theories described later.

Pathogenesis The pathogenesis is based upon a traumatic etiology. Olech et al. introduced their hypothesis which stated that following the trauma to the bone there may be an intramedullary haemarrohage. There is a failure in the organization of the clot and its subsequent liquefaction. This is similar mechanism as seen in case of the “dry socket” following extraction of teeth. That bone in which the spongy bone is enclosed within the compact cortical bone has more propensities for undergoing the cystic changes. This would explain the most frequent sites of occurrence of this cyst, such as in the mandible and the metaphysis of long bones. It would also explain the fact that most solitary bone cysts develop in young individuals. The failure in the organization of the clot is especially seen in cases where the bone together with the endosteum has undergone necrosis, following trauma. The trabaculae of the bone is removed by osteoclastic activity, till viable connective tissue gains contact with the hematoma, but since the wound is large, the time taken for this action would be too long and subsequently, by that time, the clot liquefies. On surgically venturing into the cystic lesion it was either found to be empty or containing air, or containing blood and serosanguinous fluid. The latter two discoveries support the theory of the breakdown of the hematoma. This breakdown causes a rise in the osmotic pressure which results in further pressure resorption of the bone, or sometimes the occurrence of a swelling due to constructive periosteal deposition. Fluid gets diluted as the process repeats Odontogenic Cysts 93 and finally the osmotic pressure drops. As transudartion into the cyst occurs, the fluid is diluted so that intracystic pressure drops, but further bleeds may be responsible for progression of the lesion. After the bleeding stops there will the absorption of this fluid and the cyst becomes empty. Hence this explains the various types of cystic fluids encountered by the surgeons. Surgical intervention results in fresh bleeding but since the pathological area is now in contact with a healthy flap, the cyst finally heals though the mechanism responsible for the initiation of the cyst and the treatment mentioned above appears to be the same. This theory though it has many flaws is now considered as the pathogenesis of the solitary bone cyst. No other theories regarding the pathogenesis of the solitary bone cyst other than the one described above has been proposed till date.

Chapter 9

Odontogenic Tumors

Ameloblastoma

Introduction

Ameloblastomas are benign and locally aggressive tumors affecting predominantly the posterior aspect of the jaws especially the molar ramus area of the mandible and sometimes the maxilla.

Pathogenesis

The enamel organ plays a role in the formation of the only ectodermal structure of the tooth called as the ‘enamel.’ The neural crest derived dental papilla and the dental sac play a role in the formation of the other remaining structures of the teeth. The enamel organ is made up of four layers namely the outer enamel epithelium, the stellate reticulum, the stratum intermedium and the inner enamel epithelium. The absence of the stratum intermedium in the tumor indicates that probably the absence of this structure may be the reason for the lack of formation of the enamel and hence resulting in an ameloblastoma. Parathyroid hormone-related proteins, the fibroblast growth factor, and the matrix metalloproteinases (MMP-2, TIMP-2, and MMP- 14.) have been found in many tumors including the ameloblastoma, and they have been linked, to the bone or tissue destruction and tumor growth. These proteins probably play 96 P. C. Anila Namboodiripad and E. Anuradha Sunil a role in the aggressiveness of the ameloblastoma. These patients are also found to have hypercalcemia which is found to revert back to normal after the removal of the tumor. The tumor is thought to originate from sources that include residual epithelium of the tooth germ; and the cell rests of the enamel organ (cell rests of Malassez and cell rests of Serre), epithelial odontogenic cysts namely the dentigerous cysts), basal cells of the surface epithelium of the jaws and heterotrophic epithelium in other parts of the body. The peripheral ameloblast like cells are found to be rich in the Ki-67 in both the plexiform and follicular ameloblastomas. In the case of the unicystic ameloblastoma Ki-67 was found predominantly in the basal cells. The stellate reticulum cells are found to be negative for the Ki-67. In case of the desmoplastic ameloblastoma, though it is said to be an aggressive tumor there was a lowered proliferation of the cells than the peripheral ameloblastoma. Ki-67 is said to be a marker for increased cell proliferation. Caspase3, an indicator for apoptosis was positive in the central stellate reticulum cells. Bcl-2 an antiapoptotic protein was found positive in the peripheral ameloblast like cells of the ameloblastomas and negative in the stellate reticulum like central cells. But there was no difference in values of mediators like cytochrome oxidase c, AFAP-1, caspase 9 and AIF between normal enamel organ and ameloblastoma and between the primary and secondary ameloblastomas; hence they could not be used as markers for the identification of these tumors. Genes such as ameloblastin were found absent in ameloblastoma: amelin, tuftelin and enamelin were found positive. Hence it is said that the ameloblastin gene may be playing a role in the protection against the development of the ameloblastoma. The development of ameloblastoma is also dependent on the heparin binding domain deficient ameloblastin, p27 and the p21. Mutations of the ameloblastin gene is also said to play a role in the formation of other odontogenic tumors hence cannot be used as a tumor marker for ameloblastoma. The epithelial lining of cysts, especially dentigerous cysts, is another source for the formation of the ameloblastoma. Ameloblastoma may also undergo malignant change. But the cause or stimulus for the malignant change of these remnants is unknown. P53, ‘the gate keeper genes’ is found to play no role in the formation or the enlargement of the ameloblastoma. Osteoclastogenesis is said to be caused by genes such as RANKL and MMP-9. They cause a rapid destruction of the bone and bone marrow and Odontogenic Tumors 97 hence when present in tumors they help in its enlargement. Ameloblastoma is positive for these markers. IL-1α and IL-6 plays a role in the tumor size of the ameloblastoma and they may also play a role in bone resorption and causing the expansion of the ameloblastoma. A growth factor seen commonly in human malignancies is positive in both ameloblastoma and ameloblastic carcinoma. The protein involved is the Midkine growth factor which is a heparin-binding growth factor. The midkine is said to play a role in the tumor development and its progression. The Transforming growth factor protein β/SMAD, a transcription factor, is found to be reduced in ameloblastomas indicating a more aggressive behavior of this tumor. This protein plays a role in cell proliferation, differentiation and reduced apoptosis. WNT proteins, a set of signaling proteins, excepting the WNT-10 b are found present in ameloblastomas and are found heterogeneously expressed in different types of ameloblastomas, thus indicating a role in the invasion and differentiation of the tumor cells. The level of the E-cadherin or β-catenin remains the same in case of the normal tooth germ and the unicystic ameloblastomas. They play a role in cell differentiation. Rho GTPases are said to play a role in the phenotypic expression of the ameloblastoma that is solid or cystic, peripheral or central. And these Rho GTPases play a role in the cell cycle regulation, its polarization, invasion etc. The presence of the apoptosis inducing ligand (TRAIL) and its receptor and FAS (CD95), FAS ligand are all responsible for the slow growth and the failure of the tumor to metastasize in case of the ameloblastoma. Peripheral ameloblastomas, not overlying bone are said to arise from the pluripotent basal layer cells of the surface epithelium. Some authors suggest that such tumors may be representing just a variant of the salivary gland tumor or the adamantoid pattern of the squamous cell carcinoma.

AOT

Introduction

This tumor is aggressive since it has a short life span i.e., seen in the 2nd and 3rd decades of life and quite localized in its appearance in the canine premolar area.

98 P. C. Anila Namboodiripad and E. Anuradha Sunil

Pathogenesis

Though there is a controversy to the exact cell playing a role in AOT, the stratum intermedium, the reduced enamel epithelium, dental lamina and its remnants have been said to play a role. Hemidesmosomes have been identified by electron microscope in the luminal membrane of the duct-like and the adenomatoid structures that is connected to the basal lamina like structures on the surface. Enamelysin, amelogenin, sheathilin were observed in the luminal surface of the columnar cells. Thus all features point to a histogenesis from the reduced enamel epithelium because of the similarity of this basal lamina like structure which is found adjacent to the reduced enamel epithelium in normal enamel development of the human tooth. There was variable positivity in the epithelial cells of the AOT. Laminin was positive in luminal surface of AOT indicating its origin from the reduced enamel epithelium, seen during the protective stage of reduced enamel epithelium. Type IV collagen was seen at the interface between tumor epithelium and the stroma. Some epithelial cells resemble the dental lamina while others were found to be mature odontogenic cells due to α4 (IV) chain expression that had occurred on the cribriform areas and hyaline materials. The cells in the spindled areas showed high PCNA content indicating the sites for tumoral growth. Other authors suggest a pathogenesis arising from the gubernacular dentis. The gubernacular cord running along the gubercular canal is the gubernacular dentis. This gubernacular cord was also found to contain the dental lamina which played a significant role in the formation of the ameloblastoma and the OKC. The cord was found to be directly connected with the dental follicle. Hence the occurrence of AOT was seen more in the permanent dentition than the deciduous dentition. But the existence in the permanent molar could not be explained by this hypothesis since it has no deciduous predecessor. It was later found that permanent molar also has the gubernacular cord that has been found in teeth which have no predecessors. The AOT is said to arise from the enamel organ, dental lamina, cell rests of dental lamina and Hertwig’s epithelial root sheath but none of the factors have found favors with the various authors. Earlier theory stated the origin of the tumor from both the epithelial and salivary gland tissue. The duct like structures showed presence of amelogenin and the non ductal structures showed absence of this protein. The spindle shaped cells between the epithelial islands resembled the stellate reticulum and the area adjacent to the islands Odontogenic Tumors 99 resembled the stratum intermedium. The eosinophillic, luminar material resembled basement membrane material, laminin. Though the AOT is found in relation to an impacted tooth it does not envelope the crown like in case of a dentigerous cyst. It has a fibrous tissue separating the crown and the tumor, which may be a fibrous capsule of the tumor too. Since the tumor forms at 6 months of age it probably shows the presence of a tooth erupting through a lesion. It is normally found attached to the tooth at different levels that is the neck, the root and finally may enclose the entire crown.

Calcifying Epithelial Odontogenic Tumor

Introduction

Calcifying epithelial odontogenic tumor is a rare tumor first described by Pindborg in 1955. It comprises less than 1% of all odontogenic neoplasms. The tumor is histologically made of polygonal epithelial cells, eosinophilic deposits resembling amyloid and calcifications.

Pathogenesis

This tumor is said to arise from the dental lamina residue. Another theory states its origin from the stratum intermedium cells of the enamel organ. The polyhedral cells of the CEOT express laminins 1 and 5, fibronectin, vimentin and are negative for amyloid. Tumor epithelium is slightly positive for vimentin but negative for desmin. The epithelial cells are also strongly positive for alkaline phosphatase and shows ATPase localization in their cell membranes. The amyloid like material is found to arise from the lamina densa material degradation. A large number of dendritic cells may also be seen between epithelial cells and they are found to be similar to the Langerhans cells. Ameloblastin gene is found in the CEOT and also shows Amelogenesis Imperfecta like features. The epithelial cells slowly transforms from an odontogenic to squamous epithelium in the case of this tumor. Thus it is positive for keratin -14, keratin 10/13. The proteins that affect the progression and invasion of the tumor include the NF-KB, Ki-67 and MMP-9. The marker for the lymphatic 100 P. C. Anila Namboodiripad and E. Anuradha Sunil endothelial cells is seen in both the CCOT basal and the polyhedral cells and these cells are said to be associated with the extracellular matrix signaling and the cell proliferation in the CCOT.

Squamous Odontogenic Tumor

Introduction

Squamous odontogenic tumor is a rare benign odontogenic tumor.

Pathogenesis

The histogenesis is said to be from the epithelial rests of Malassez but there is no substantiating evidence available, to support this theory. Another source of origin of this tumor has been identified and that is from the basal layer of the oral mucosa. Some other authors state that the origin of the tumor may be from more than one source i.e., surface epithelium, Malassez rests, and dental lamina rests. Hence the variation in the behavior of the tumor is seen when it arises at different locations.

Clear Cell Odontogenic Carcinoma

Introduction

The clear cell odontogenic tumor is a benign but locally invasive odontogenic tumor and it has been included in the revised World Health Organization classification for odontogenic tumors. Clear cell odontogenic tumour (CCOT) was classified as a benign tumor in the earlier classification, but has been found to be a more menacing lesion and currently it has been designated as a carcinoma. These tumors show an aggressive growth, recurrences, and metastasize to distant sites.

Odontogenic Tumors 101

Pathogenesis

Although the origin is unknown, the tumor cells has been found to resemble clear cell rests of primitive dental lamina that are frequently observed in the same locations. The clear cells stain positively with PAS stain and undergo diastase digestion. The positivity is due to reduced number of organelles rather than due to the presence of glycogen granules. These granules are also mucicaramine negative. They show positivity to cytokeratins and EMA. The tumor cells may rarely act positive to antibodies against vimentin, S100 and actin.

Primary Intraosseous Carcinoma

Introduction

Type 1 PIOC ex odontogenic cyst 1. Malignancies in ameloblastoma Type II 1. Malignant ameloblastoma 2. Ameloblastic carcinoma Type III Clear cell odontogenic carcinoma Type IV Primary de novo Intraosseous squamous cell carcinoma Type V Intraosseous Mucoepidermoid Carcinoma

Pathogenesis The PIOC is said to arise from within the jaws and found to have no connection with the surface epithelium. It is said to form from the entrapped odontogenic epithelium which may be the rests of the dental lamina, or from the linings of odontogenic cysts or previously present odontogenic tumor. The squamous cells of the tumor may also arise from other sources and to diagnose it as the PIOC, the exact source of origin of the tumor has to be identified.

102 P. C. Anila Namboodiripad and E. Anuradha Sunil

Ameloblastic Fibroma/Fibrosarcoma

Introduction Ameloblastic fibroma (AF) was first described by Krause in 1891. It is an extremely rare, mixed benign tumor that may occur predominantly in the posterior region of either the mandible or maxilla and often associated with an unerupted tooth. It has a slight female predilection and is commonly seen in the first two decades of life, altering the tooth eruption sequence and causing a delay in tooth eruption pattern.

Pathogenesis Immunomarkers for dental lamina namely the CK 7, 13, 14 were found positive in case of the ameloblastic fibroma. The connective tissue determines the etiology for the ameloblastic fibroma. The proportion of the proliferation of the epithelium and connective tissue was under question. It has been found that the Ki-67 was found in higher proportion in the epithelial tissue part of the tumor as compared to the mesenchmal tissue part, indicating a higher proliferative index. This Ki-67 may also be used as a marker for indication of transformation of the ameloblastic fibroma to the ameloblastic fibrosarcoma. The apoptotic activity is also said to be an important factor in regulation of the tumor growth. An inhibitor of apoptosis calrectin was not immunodetected in AF. This calrectin was also negative in the mesenchymal portion of the normal tooth germs. Bcl-2 was found negative in the mesenchymal portion of the AF while it was positive in the epithelial portion. The latter marker was positive in the sarcomatous portion of the Ameloblastic fibrosarcoma and epithelial strands were negative for the Bcl-2. Thus these markers could be used to distinguish the benign tumor from its malignant counterpart. Vimentin was positive in the immature dental papilla cells and also in case of the AF. Nestin is another marker found in both the developing mesenchyme and the mesenchyme of the AF. The role of this protein is found to be unclear. Among the ameloblastic proteins only amelogenin was found positive in the AF. All the other proteins like amelotin, ameloblastin and enamelin were negative. This positivity shows that the tumor arises from the dental lamina cells.

Odontogenic Tumors 103

Odontoma

Introduction Odontoma is a developmental anomaly of odontogenic origin, resembling a hard tumor and composed of enamel, dentin, cementum, and pulp tissue.

Pathogenesis Trauma during the deciduous tooth formation and the inflammatory and infectious process, heredity, odontoblastic hyperactivity are said to be the etiologic factors for odontomas. A mutant gene which causes the odontogenic cells to intermix, would result in the formation of the odontome. There may also be a defect in the morphodifferentiation or histodifferentiation stages of tooth development resulting in the formation of this hamartoma. Hard keratin is present in the ghost cells of the odontoma and the CCOT and the Wnt signaling pathway plays a role in the pathogenesis of the ghost cells during metaplasia.

Cementoma

Introduction Cementomas have been classified by the WHO in 1971 into 4 groups:

1) Cementifying fibroma. 2) Benign cementoblastoma (true cementoma). 3) Periapical cemental dysplasia. 4) Gigantiform cementoma.

Pathogenesis No available material is available with regard to the pathogenesis of this tumor. The tumor was thought to arise from cells of Malassez that has transformed into the cementoblasts which later form the cementoma.

104 P. C. Anila Namboodiripad and E. Anuradha Sunil

Dentinoma

Introduction Dentinoma is a rare tumor of odontogenic origin, comprising of immature connective tissue, odontogenic epithelium and dentin which is dysplastic. Pathogenesis Not much data is available regarding the pathogenesis of the dentinoma. The origin of such a growth is discussed, and the possibility of its development as a result of proliferation of connective tissue and of cells of Hertwig's epithelial root sheath is taken into consideration. The epithelial remnants induce the undifferentiated cells of the connective tissue to transform into odontoblasts to produce dentine.

Chapter 10

Potentially Malignant Disorders

Leucoplakia

Introduction

Leukoplakia is a clinical term used to describe a white patch on the oral mucosa which cannot be given another diagnostic name, i.e., it is a diagnosis of exclusion. Leukoplakia, is a clinical diagnosis which has the unusual feature of being reliant not so much on definable appearances as on the exclusion of other lesions which present as oral white plaques such as lichen planus, chronic cheek bite, frictional keratosis, tobacco pouch keratosis, nicotine palatinus, leukoedema, white sponge nevus, etc. These lesions must be ruled out before a diagnosis of leukoplakia can be made.

Pathogenesis

Smoking and Leucoplakia The white patch mistaken as leucoplakia may more likely be a reactive lesion to local tissue irritation rather than a lesion induced by carcinogens in cigarette smoke, and hence smoker’s keratosis could be a better term than leucoplakia. The mechanism of the white appearance in actual leucoplakia is said to be due to the thickening of the keratin layer, called hyperkeratosis. This atypical keratin appears white when it becomes hydrated by saliva, and it reflects light off the surface evenly. Therefore it hides the normal pink-red 106 P. C. Anila Namboodiripad and E. Anuradha Sunil color of mucosae (the result of underlying vasculature showing through the epithelium).A similar state is seen on areas of thick skin such as the soles of the feet or the fingers after extended dipping in water. Another possible mechanism is increased thickening of the stratum spinosum, called acanthosis. The proliferative activity of the oral mucosa by the tobacco smoke, causes a tendency in the leucoplakia to undergo malignant transformation, and this it does through the stimulation of epidermal growth factor receptor (EGFR) that later activates the cyclin D1. But apart from the above pathogenesis the prolonged exposure to the tobacco smoke may also cause localized genomic instability and lack of resolution of the lesion even after the etiology has been withdrawn. HPV 16 infection that causes leucoplakia has a high propensity of converting to malignancy and this occurs even in the case of non smokers and non alcoholics. Other studies state that the HPV infected patients have the HPV infection as a ‘second hit’ and the ‘first hit’ being the genetic probability of malignancy in such patients. The E6 and E7 proteins of low-risk HPV types established in oral leukoplakia may kindle the suprabasal post mitotic infected basal cells to reenter the S-phase of the cell cycle, further resulting in epithelial proliferation and altered maturation, without causing a genomic instability associated with consequent cell transformation. This mechanism may be a co-factor for the development of the leukoplakia, but there is no tangible evidence to support this. p53 plays a major role in DNA repair and cell-cycle regulation after carcinogen-induced damage (tobacco, alcohol, etc) to the DNA of the oral epithelium. The carcinogen induces a stress which results in the protein production by activation of the phosphorylation of its serine residues of the p53 such that the level of the active p53 protein is determined based on this carcinogen induced damage. If the damage is negligent the cell cycle progress, would be detained to allow DNA repair. If on the other hand the level of p53 is high the cells enter apoptosis. So when there is a mutation of the p53 protein this protective pathway is lost and the epithelium undergoes malignant transformation. The p53 mutation is also found to occur in case of the UV-B irradiation. But if the p53 has already undergone 2-3 mutations there is a direct transformation to malignancy. Only the mutant p53 can be detected immunohistochemically not the normal p53. The overexpression of the telomerase in leucoplakic patients is an indication for a malignancy.

Potentially Malignant Disorders 107

Oral Submucous Fibrosis

Introduction

Oral submucous fibrosis (OSF) was first described in the early 1950s, as a potentially malignant disease largely seen in people of Asian descent. It is a slow chronic progressive disorder and its clinical manifestation depends on the stage of the disease at the time of detection. The majority of patients will have intolerance to spicy food, stiffness of lip, tongue and palate leading to limitation of mouth opening and tongue movement.

Pathogenesis

The possible etiological factors to date are capsaicin in chilies, areca nut, micronutrient deficiencies of zinc, iron, and essential vitamins. Apart from these factors there is a possible demonstration of various auto-antibodies and an association with specific HLA antigens has been suggested. It is hypothesized that there is either an increased collagen synthesis or reduced collagen degradation following areca nut usage. There is a high content of copper in the areca nut and the probable role of copper as an arbitrator of fibrosis is supported by the demonstration of the up regulation of lysyl oxidase in OSMF biopsies. Lysyl oxidase is required for stabilizing the collagen fibres. Due to the effect of the slaked lime (Ca (OH) 2); the arecoline gets hydrolyzed to arecadine, which has an immense effect on fibroblasts. As per the study by Harvey et al., exposures to 0.1-10 μg/ml arecoline will stimulate fibroblasts and concentrations greater than 25 μg/ml, slows the fibroblast growth and collagen synthesis. According to Jeng et al. the depletion of cellular glutathione (GTH) levels by arecoline, predisposes the fibroblasts of the oral mucosa to various genotoxic and cytotoxic stimulation.

Stablization of Collagen Structure by Tanins and Catechins Flavonoids in areca like the tannins and catechins, causes an increased fibrosis by forming a more stable and non soluble collagen structure by inhibiting the collagenase enzyme activity. As the disease progresses the type III collagen is completely replaced by type 1 collagen which is more likely to resist degradation. There is also an excess of alpha 1 (1) chains as compared to 108 P. C. Anila Namboodiripad and E. Anuradha Sunil alpha 2(1) chains, that suggests an alteration of collagen molecule during the disease progression.

Inhibition of Collagen Phagocytosis In fibrotic connective tissue lesion such as OSMF, that do not exhibit gross inflammation, the main route of collagen removal is by phagocytosis and not by the process of extracellular digestion and the phagocytic activity in OSMF is inversely dose dependent to levels of arecoline, nicotine and safrole in saliva.

Copper and OSMF Copper is a component of the enzyme lysyl oxidase which, as mentioned earlier, plays a major role in cross linking of collagen and elastin molecules. Copper also affects specific growth and development of fibroblasts. The cell doubling time of OSMF fibroblast was increased to 3.2 days as compared to 3.6 days in case of the normal fibroblasts. Lack of iron is said to cause improper vascular channel formation and associated decrease in vascularity. This makes diffusion of esters of arecoline easier. Also the iron also plays a role in normal epithelium maturation as it is a component of the enzyme cytochrome oxidase. In iron deficiency, there is a lowered level of this enzyme and subsequent atrophy of epithelium results, which may lead to the burning sensation and ulcerations of oral cavity in the areca chewers. This causes a decreased appetite in these patients leading to further anemia and results in a never ending vicious cycle.

Lichen Planus

Discussed in Chapter 15 -Skin disorders.

Section 3: Systemic Disorders

Chapter 11

Bone Disorders

Osteogenesis Imperfecta

Introduction

Osteogenesis imperfecta (OI) exhibits varied clinical features ranging from the bone problems, blue sclerae, short stature, hearing loss, respiratory problems, and a disorder of tooth development called dentinogenesis imperfecta. OI Type1 is characterized clinically by increased bone fragility often leading to fractures, ranging from few to 100, without secondary deformities; in combination with blue sclera, conductive or mixed hearing loss in late adolescence, short or sometimes normal height, and dentinogenesis imperfecta. Radio logically, in OI type I, bone fragility in combination with generalized demineralization, slender shafts of tubular bones with thin cortex and poorly trabeculated spongiosa are evident. Also the ossification of the cranial vault is often retarded, leading to a mosaic pattern of wormian bones. Several kinds of mutations in the COL1A1 gene cause the more severe forms of osteogenesis imperfecta, including types II, III, and IV.

Pathogenesis

Collagen is a protein that strengthens and supports many tissues in the body, including cartilage, bone, tendon, skin and the sclera.

112 P. C. Anila Namboodiripad and E. Anuradha Sunil

Role of Type I Collagen in Osteogenesis

Type I collagen creates layers of fibrils in the extracellular matrix of bone, giving bone it’s tensile strength and forming a template for the deposition of inorganic minerals. Type I collagen owes its ability to form fibrils to its very specific amino acid sequence. Collagen begins as procollagen molecules, which must be processed by enzymes outside the cell, to remove extra protein segments from their ends. Each rope-like procollagen molecule is made up of three chains: two pro-α1 (I) chains, which are produced from the COL1A1 gene, and one pro-α2 (I) chain, which is produced from the COL1A2 gene. Both are built from 338 uninterrupted repeats of the amino acid sequence. Glycine-X-Y, where X is often proline and Y is often hydroxyproline. Due to the regular pattern of prolines and hydroxyprolines, the alpha chains assume a left-handed helical conformation. Two alpha1 helices and one alpha2 helix then wind around each other to form a right-handed triple helix. Presence of a glycine in every third position is critical for triple helix formation, since only glycine, the smallest of all amino acids, fits into the confined space in the center of the triple helix. Triple helix formation starts at the C-termini of the alpha chains and proceeds toward the N-termini in a zipper-like fashion. Prior to and simultaneously with triple helix formation, prolines and lysines in the alpha chains are chemically modified by addition of hydroxyl or sugar groups. These modifications are believed to play a role in triple helix stabilization. Completed triple helices are secreted from the cells and spontaneously assemble into collagen fibrils. Fibril-forming collagens such as type I collagen are synthesized into larger precursors, known as procollagens, which contain globular N-terminal and C-terminal propeptides. After procollagens are processed, the resulting mature collagen molecules arrange themselves into long, thin fibrils. Individual collagen molecules are cross-linked to one another within these fibrils. The formation of cross-links results in very strong type I collagen fibrils, which are found in the spaces around cells. The association is then stabilized by the formation of interchain disulphide bonds. From the carboxyl end, helix formation proceeds towards the amino terminal end in a zipper-like fashion. Simultaneously, proline and lysine residues are hydroxylated by specific hydroxylases and hydroxylysine residues are glycosylated by sugar transferases. These modifications occur only in chains that are in nonhelical configuration and cease as soon as the protein folds into a triple helix. In the extracellular space, specific N- and C-peptidases cleave the terminal extensions. The mature helical molecules self-aggregate into highly ordered fibrils stabilized by intermolecular cross-links formed by Bone Disorders 113 oxidative deamination of lysine and hydroxylysine residues. Mechanical strength of connective tissue is due mainly to fibrils which form a template for matrix deposition. In bone, the fibrils are the template for mineralization, i.e., in this case, for incorporation of hydroxyapatite crystals. The mechanical properties of bone are dependent on an intimate association between the collagen fibrils and crystals of hydroxyapatite. Most cases of osteogenesis imperfecta have an autosomal dominant pattern of inheritance, which means one copy of the altered gene in each cell is sufficient to cause the condition. Many people with type I or type IV osteogenesis imperfecta inherit a mutation from a parent who has the disorder. Most infants with more severe forms of osteogenesis imperfecta (such as type II and type III) have no history of the condition in their family. In these infants, the condition is caused by new (sporadic) mutations in the COL1A1 or COL1A2 gene. Osteogenesis imperfecta is the most common disorder caused by mutations in the COL1A1 gene. More than 400 COL1A1 gene mutations that cause osteogenesis imperfecta have been identified. Most of the mutations that are responsible for osteogenesis imperfecta type I, the mildest form of this disorder, reduce the production of pro-α1 (I) chains. With fewer pro-α1 (I) chains available, cells can make only half the normal amount of type I collagen. A shortage of this critical protein underlies the bone fragility and other characteristic features of osteogenesis imperfecta type I. Some of the mutations that cause severe forms of osteogenesis imperfecta delete segments of DNA from the COL1A1 gene, resulting in an abnormally shortened, often nonfunctional pro-α1 (I) chain. Other genetic changes alter the sequence of amino acids in the pro-α1 (I) chain, usually replacing the amino acid glycine with a different amino acid. In some cases, amino acid substitutions alter one end of the protein chain (called the C-terminus), which interferes with the assembly of collagen molecules. These COL1A1gene mutations lead to the production of abnormal versions of type I collagen. When this abnormal collagen is incorporated into developing bones and other connective tissues, it causes the serious health problems associated with severe forms of osteogenesis imperfecta. The COL1A1 gene is located on the long (q) arm of chromosome 17 at position 21.33. More precisely, the COL1A1 gene is located from base pair 50,184,095 to base pair 50,201,641 on chromosome 17. Less commonly, osteogenesis imperfecta has an autosomal recessive pattern of inheritance. Autosomal recessive inheritance means two copies of the gene in each cell are altered. 114 P. C. Anila Namboodiripad and E. Anuradha Sunil

The parents of a child with an autosomal recessive disorder typically are not affected, but each carry one copy of the altered gene. Some cases of osteogenesis imperfecta type III are autosomal recessive; these cases usually result from mutations in genes other than COL1A1 and COL1A2. When osteogenesis imperfecta is caused by mutations in the CRTAP or LEPRE1 gene, this condition also has an autosomal recessive pattern of inheritance. The COL1A1 gene provides instructions for making part of a large molecule called type I collagen. Many people with type I or type IV osteogenesis imperfecta inherit a mutation from a parent who has the disorder. Most infants with more severe forms of osteogenesis imperfecta (such as type II and type III) have no history of the condition in their family. In these infants, the condition is caused by new (sporadic) mutations in the COL1A1 or COL1A2 gene. Mutations in the COL1A1, COL1A2, CRTAP, and LEPRE1 genes cause osteogenesis imperfect in 90 percent of all cases Most of the mutations that cause osteogenesis imperfecta type I occur in the COL1A1 gene. These genetic changes reduce the amount of type I collagen produced in the body, which causes bones to be brittle and to fracture easily. The mutations responsible for most cases of osteogenesis imperfecta types II, III, and IV occur in either the COL1A1 or COL1A2 gene. These mutations typically alter the structure of type I collagen molecules. A defect in the structure of type I collagen weakens connective tissues, particularly bone, resulting in the characteristic features of osteogenesis imperfecta. Mutations in the CRTAP and LEPRE1 genes are responsible for rare, often severe cases of osteogenesis imperfecta. In cases of osteogenesis imperfecta without identified mutations in the COL1A1, COL1A2, CRTAP, or LEPRE1 gene, the cause of the disorder is unknown. These cases include osteogenesis imperfecta types V and VI. Researchers are working to identify additional genes that may be responsible for these conditions. Most collagen mutations arise de novo, that is, they are new mutations that occur in one sperm or one egg as a random event and the couple has no increase in their risk of having a second child with OI. However, a small proportion of couples who have had one child with OI are at increased risk of having a second child with OI because one partner is a mosaic carrier. Collagen mutation in some cells of the body but not in others is called as mosaic carriers and hence has been called as ‘mosaic.’ The collagen mutation occurs during the fetal development of the mosaic carrier and all cells that arise from the first cell with a mutation will also carry the mutation. This includes germ cells (eggs or sperm) and some body cells. The mosaic carrier does not have symptoms of OI or may have Bone Disorders 115 very mild symptoms (blue sclerae, shorter stature, dentinogenesis imperfecta). OI is dominantly inherited and caused by a heterozygous mutation in either of the two genes, COL1A1 and COL1A2, encoding the chains of type I collagen.

Fibrous Dysplasia

Introduction

Fibrous dysplasia is an uncommon bone disorder in which scar-like (fibrous) tissue develops in place of normal bone. This can weaken the affected bone and cause it to deform or fracture. Fibrous dysplasia is a developmental abnormality characterized by a disorganized mixture of fibrous and osseous elements in the medullary region of affected bones. Fibrous dysplasia can affect any bone, and can divide into four sub types:

 monoostotic - single bone  polyostotic - multiple bones  craniofacial fibrous dysplasia - skull and facial bones alone  cherubism - mandible and maxilla alone (not true fibrous dysplasia)

Pathogenesis

Fibrous dysplasia is stated to occur as a result of a developmental failure in the transformation of primitive bone into mature lamellar bone and its inability to respond to mechanical stress. This failure of maturation results in a mass of immature secluded trabeculae intertwined in dysplastic fibrous tissue that are remodeling constantly but never or very nearly never completing the process of bone maturation and mineralization. This lack of remodeling and combination of deficient mineralization results in great loss of mechanical strength, leading to the pain, pathological fractures and deformity. The mutation in the GNAS gene, located at chromosome 20q13.2-13.3, which occurs after fertilization in somatic cells is said to be the cause of this condition which encodes the alpha subunit of the stimulatory G protein, G s α.

116 P. C. Anila Namboodiripad and E. Anuradha Sunil

The general function of Gs ↓ is to activate adenylate cyclase, which, in turn, ↓ produces cAMP, which, in turn

↓ activates cAMP-dependent protein kinase.

Further effects of Gs are therefore found in function of cAMP-dependent protein kinase. It hence provides a step in signal transduction. Amplification of the signal occurs because the receptor activates multiple Gs. But, each Gs activates only one adenylate cyclase. As a consequence of this mutation, amino acid arginine, in position 201 of the genomic DNA in the osteoblastic cells, is substituted by cysteine or histidine. The abnormal G1 protein stimulates cyclic adenosine monophosphate (AMP), and the osteoblastic cells expressing this mutation have a higher rate of DNA synthesis than normal cells. The dysplastic features are seen in all cells derived from these mutated cells and this may also result in varied clinical presentation. The Gnas mutation was first acknowledged in patients with McCune-Albright syndrome (MAS), a rare disorder presenting features such as polyostotic fibrous dysplasia, skin pigmentation, and one of several endocrinopathies. The G-proteins begin a cascade that ultimately leads to activation of the enzyme adenylyl cyclase that produces cAMP. Normally, there is an almost immediate deactivation of adenylyl cyclase and a breakdown of the cAMP. In MAS, that does not occur. Overproduction of cAMP leads to increased amounts of activity that affects each tissue differently, based on its designated function. Café-au-lait spots occur due to overproduction of the enzyme tyrosinase, which plays a role in melanin production. In Fibrous dysplasia, this mutation causes hyperproliferation and incomplete differentiation of marrow stromal cells to abnormal osteoblasts. cAMP also activates Fos, which inhibits osteoblastic-specific genes as well as stimulates cytokines that promote bone resorption by osteoclasts. Hypophosphatemia/ phosphaturia, sometimes found in FD and MAS, is caused by excess secretion of a phosphatonin fibroblast growth factor. Similarly, most of the endocrine problems associated with MAS Bone Disorders 117 can be related to Gs-activation. In addition, activating Gs-α mutation have been shown to potentiate WNT/β-catenin signaling, and removal of Gs-α leads to reduced WNT/β-catenin signaling and decreased bone formation. Activation of WNT/β-catenin signaling in osteoblast progenitors causes an FD-like phenotype and reduction of β-catenin levels, rescued differentiation defects of stromal cells derived from patients with FD. This suggests that activated G proteins have significant roles during both skeletal development and disease by modulating the WNT/β-catenin signaling strength. This abnormal growth leads to the formation of a disorganized fibrotic bone matrix with primitive bone formation, and lack of maturation to lamellar bone. Mineralization is also abnormal. There is a failure of the bone to align in response to mechanical stress. This defect is seen in the monostotic as well as the polyostotic forms of fibrous dysplasia. The extent of disease is related to the stage at which the postzygotic mutation in Gs α has occurred, whether during embryonic development or postnatally.

Osteoblastic Abnormalities in Fibrous Dysplasia

The histological analysis of fibrous dysplasia suggests abnormalities in the formation of osteoprogenitor cells. The number of immature osteoblasts was found to be low. The cells located along the bone surface were not inclined and elongated, and were different from the normal cuboidal mature osteoblasts. The marrow cavity was filled with elongated, immature, alkaline phosphatase-positive cells, and this cellular invasion does not allow the normal development of the resident blood cells and the fat cells. These histological features indicate that the preosteoblastic population is increased in these patients, whereas the count of mature cells was decreased. Osteoblast differentiation was also found to be defective and it is visible in the abnormal collagen organization in the immature bone. The collagen fibers show features of immature bone in that they are not parallelly oriented and they sometimes appear perpendicular to the bone surface, showing a comb-like structure. These abnormalities found are seen in both monostotic and polyostotic lesions reflecting a disorganization of collagen fibers synthesized by dysplastic cells. Together with the abnormal formation and direction of collagen, the non- collagenous protein formation is also affected. Osteopontin and the bone sialoprotein are decreased and the osteonectin level is increased, projecting the immature composition of the matrix of the dysplastic bone. These dysplastic osteoblasts give rise to more than the normal number of osteocytes which are 118 P. C. Anila Namboodiripad and E. Anuradha Sunil also lodged in larger than normal lacunaes.’ Their intercellular canaliculis which form a network are also found to be disorganized in the bone matrix. In this dysplastic bone, the osteoclasts, which are multiple, are also found to be dysfunctional and they show local lytic areas. These dysplastic cells exhibited an elevated intracellular cAMP associated with an increased proliferation rate. The less differentiated osteoblast phenotype was found to be associated with an increased cell proliferation in these dysplastic lesions and this was evidenced by the alteration of osteocalcin mRNA expression and protein synthesis. Osteocalcin expression was enhanced in osteoblasts of dysplastic bone when they were made to respond to 1, 25, dihydroxyvitamin D. The concomittant decrease in cell differentiation and increased proliferation of pre- osteoblastic cells in dysplastic lesions lead to a rapid deposition of an immature, poorly organized woven bone characteristic of the bone lesion at the histological level. Other important factors regulating bone formation include the members of the AP-1 family. It seems therefore possible that the increased osteoblastic cell proliferation and the abnormal osteoblast differentiation observed in fibrous dysplasia results in part from the increased expression of c-fos and other members of the in vitro, AP-1 control genes that are involved in osteoblast differentiation, and the c-fos expression that modulates the expression of osteoblast genes such as osteocalcin and osteopontin whose promoter contains AP-1 sites. The increased c-fos protein by cAMP may induce cells to enter into the G1 phase of the cellular cycle. This is consistent with the finding that c-fos is expressed by immature osteoblasts and controls osteoblast proliferation and differentiation. There may be other mechanisms playing a role in the cellular alterations observed in fibrous dysplasia. Studies have reported an increased level for estrogen expression in osteoblasts in both monostotic fibrous dysplasia and McCune-Albright syndrome. Endocrine receptors were found to be defective here. Estrogens have been said to play a role in proliferation of osteoblastic cells by increasing the expression of local growth factors such as TGF-β and IGF-1 which play an important role in controlling osteogenesis. McCune Albright syndrome patients have also reported a rise in the expression of the parathyroid hormone related peptide (PTHrP) in their osteoblastic cells.

Bone Disorders 119

Cleidocranial Dysostosis

Introduction

Cleidocranial dysplasia (CCD) is a skeletal dysplasia characterized by delayed closure of the cranial sutures, hypoplastic or aplastic clavicles, and multiple dental abnormalities. CCD was initially thought to engage only the bones of membranous origin but more current and comprehensive clinical investigations have shown that CCD is a widespread skeletal dysplasia affecting not only the clavicles and the skull but the complete skeleton. CCD was as a result considered to be a dysplasia instead of a dysostosis.

Pathogenesis

Mutations in the CBFA1 gene that presumably lead to synthesis of an inactive gene product were identified in patients with CCD. The function of CBFA1 during skeletal development was further elucidated by the generation of mutated mice in which the Cbfa1 gene locus was targeted. Main cause of CCD is the mutation in the RunX2 gene on Chromosome 6p21 and whose main role is to give instructions for making a protein that is involved in the development and maintenance of cartilage and bone. The formed protein is a transcription factor, and hence it binds to specific regions of DNA that helps to control the activity of a particular gene. According to various researchers the RUNX2 is a protein that acts as a “master switch,” controlling a number of other genes implicated in the development of osteoblasts. Mutations in this gene may include either a change in one amino acid in the RUNX2 protein or the introduction of a premature stop signal which may result in an abnormally short protein or sometimes the entire gene is missing. These mutations result in shortage of functional RUNX2 protein which may result in abnormal bone and cartilage development hence giving rise to signs and symptoms of CCD. Loss of genes in the neighborhood of RUNX2 may result in other disturbances in these individuals. In a third of CCD individuals, no mutation in the RUNX2 gene is seen. The etiology in such individuals is unknown. Mutations may be missense and they gather at arginine 225 (Arg225) of the RUNX2 protein. Nuclear accumulation of RUNX2 protein is affected. 120 P. C. Anila Namboodiripad and E. Anuradha Sunil

The different steps of endochondral bone formation are listed. Patterning genes regulate the shape and number of the individual skeletal elements. Precursor cells aggregate and subsequently differentiate into chondrocytes. Chondrocytes proliferate, hypertrophy, and calcify before they are replaced by bone. Some of the factors that are known to regulate this process are shown on the right. Cbfa1 controls the differentiation of precursor cells into osteoblasts, regulates chondrocyte differentiation towards hypertrophy, and is necessary for the invasion of calcified cartilage. Cbfa1 controls differentiation of aggregated precursor cells of the clavicular anlage.

Figure 1. Role of Cbfa1 in bone formation.

Cherubism

Introduction

Cherubism is a rare, self-limiting, fibro-osseous, genetic disease of childhood and adolescence characterized by varying degrees of progressive bilateral enlargement of the mandible and/or maxilla, with clinical repercussions in severe cases.

Pathogenesis

The main etiological factor for Cherubism is said to be the mutations in the SH3BP2 gene linked to chromosome 4p16.3and has been recognized in about 80%of patients. In the remaining 20% the genetic cause is unidentified. Bone Disorders 121

This gene is important because it is found to provide instructions for manufacturing a protein whose exact function is unknown. This protein is thought to play a role in the transmission of the chemical signals within cells of those predominantly playing a role in bone remodeling and certain immune system cells. So large scale active version of this protein is produced following mutations in the SH3BP2 gene. The mutations of SH3BP2 are still under study. The hyperactive protein may cause jaw bone inflammation and stimulates the production of osteoclasts that causes destruction of bone in the upper and lower jaws. Bone loss and inflammation may be the likely cause for the cyst-like growths which is a trait of cherubism. The second theory in pathogenesis as stated by Caballero and Vinals includes mesenchymal changes during jaw development, an odontogenic origin or even hormonal and traumatic factors. The third theory as stated by Silva et al. that includes the breaking down of Msx-1 gene, involved in the regulation of mesenchymal interaction during craniofacial development. Hyckel et al. proposed the molecular model of cherubism pathogenesis which is based on interaction between a disturbed parathyroid hormone related proteins (PTHrP) receptor with the Hox gene Msx-1 activity occurring following mutation in SH3BP2. This causes interference with jaw morphogenesis which further leads to the dysregulation of mesenchymal bone formation, and to the presence of osteoclasts in the developing giant cell granulomas. Cherubism is caused by mutations in SH3BP2. Studies of cherubism mice showed that tumor necrosis factor α (TNF-α)-dependent autoinflammation is a major cause of the disorder but failed to explain why human cherubism lesions are restricted to jaws and regress after puberty. It has been demonstrated that the inflammation in cherubism mice is MYD88 dependent and is rescued in the absence of TLR2 and TLR4. However, germ-free cherubism mice also develop inflammation. Mutant macrophages are hyper responsive to PAMPs (pathogen-associated molecular patterns) and DAMPs (damage-associated molecular patterns) that activate Toll-like receptors (TLRs), resulting in TNF- α overproduction. Phosphorylation of SH3BP2 at Y183 is critical for the TNF- α production. Finally, SYK depletion in macrophages prevents the inflammation. These data suggest that the presence of a large amount of TLR ligands, presumably oral bacteria and DAMPs during jawbone remodeling, may cause the jaw-specific development of human cherubism lesions. Reduced levels of DAMPs after stabilization of jaw remodeling may contribute to the age-dependent regression. 122 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 2. Showing the mutation of SH3BP2 gene and its action on bone to cause cherubism.

Paget’s Disease

Introduction

Paget’s disease occurs when there is a disturbance in the bone remodeling that characteristically begins with unwarranted bone resorption followed by an increase in bone formation. This osteoclastic hyper activity followed by substituted osteoblastic activity leads to the formation of a structurally disordered mosaic of bone which is still a woven bone, and which is mechanically larger, weaker, more vascular, less compact, and more prone to fracture than normal adult lamellar bone.

Pathogenesis

1. Viruses. Slow virus infection (intra-nuclear nucleocapsid-like structure) in [osteoclasts and osteoclast precursors].

a. Paramyxovirus

Human bone marrow cells shows increased numbers of large, highly nucleated osteoclasts and they express measles nucleocapsid protein, due to which IL-6 levels are increased. But these nuclear and Bone Disorders 123

cytoplasmic inclusions, which are a feature of pagetic osteoclasts, are not specific to this disease. Osteopetrosis shows the presence of similar inclusions in osteoclasts or macrophages and hence might represent a nonspecific stress response in osteoclasts. Similar nuclear inclusions are also seen in brain cells of patients with subacute sclerosing panencephalitis which is a fatal condition caused by a long- term measles virus infection of the brain. These inclusions are similar in size to those in Paget's disease, but their arrangement may appear different. Recent work has suggested that the inclusion bodies contain SQSTM1, VCP (valocin containing protein) and ubiquitin. These facts suggest that, rather than being viral particles, they may be protein degradation products, which may be exaggerated by aging.

b. Infection of human osteoclast precursors with canine distemper virus stimulates expression of NF-κB. c. Respiratory syncytial virus.

But no virus has been identified till date.

2. SQSTM1 gene is said to play a role in Paget’s disease of bone and is said to be caused by more than 20 mutations in the gene. The mutations in the SQSTM1gene are brought about by replacement of the amino acid proline with the amino acid leucine at protein position 392. Mutations result in hyperactivation of the chemical signaling pathway which encourages osteoclast formation. Paget’s disease of bone results when affected bone is broken down and replaced much sooner than normal and the new bone is weaker and less regular than normal bone. This altered bone remodeling causes certain bones to become unusually large, malformed, and easily fractured. It is unclear why the disease affects some bones but not others.

a. Mutations in an unknown gene results in sporadic and familial Paget’s disease of bone. This gene binds to a multifunctional protein that binds ubiquitin and controls activation of the nuclear factor kappa-B (NF-kB) signaling pathway. The protein functions as a scaffolding/adaptor protein with TNF receptor- 124 P. C. Anila Namboodiripad and E. Anuradha Sunil

associated factor 6 to arbitrate activation of NF-kB in response to upstream signals. On the other hand spliced transcript types programming either the same or different isoforms have been identified for this gene. b. The p62 is encoded by the SQSTM1 gene (sequestome 1); which may do the function of a scaffold protein in the RANK signaling pathway. This mutated protein can cause accelerated osteoclast activity but how it influences it, is unknown. The significance of the p62 in osteoclast function was identified during the inactivation of SQSTM1 which caused defects in RANKL-induced osteoclastogenesis. Overall, Paget’s disease is characterized by localized increases in osteoclast formation that lead to bone resorption and associated osteoblastic activity. c. p62 is also said to perform other functions in addition to bone remodeling, such as recycling worn-out cell parts, autophagy, apoptosis, and the body's immune responses and inflammatory reactions

The role of SQSTM1 as a scaffold in NF-κB signaling is certainly relevant to osteoclastogenesis and bone biology. SQSTM1 forms a complex with TRAF6 or receptor interacting protein and an atypical protein kinase C to facilitate NF-κB signaling stimulated by factors such as RANKL, TNF-α and IL-1. This complex allows optimal and sustained activation of NF-κB, but the effects of SQSTM1 on NF-κB signaling are complex.

Apert’s Syndrome

Introduction

Apert’s syndrome is an unusual autosomal dominant disorder featured by craniofacial anomalies, craniosynostosis, and severe symmetrical syndactyly (affecting the skin and the bone) of the hands and feet.

Bone Disorders 125

Pathogenesis

Apert’s syndrome is an autosomal dominant disorder, and a single copy of the altered gene in the cell is enough to cause the disorder. A mutation in the FGFR2 gene located on chromosome 10q causes Apert’s syndrome. The function of this gene is to signal the undifferentiated mesenchymal cells to become bone cells during embryonic development. Mutation in a specific part of FGFR2 gene which alters the protein that plays a role in signaling, results in prolonged signaling and the consequence of this would be the premature fusion of bones of hands and feet and the skull.

Down’s Syndrome

Introduction

Down syndrome is a lifelong condition in which a person is born with distinct physical features and some degree of cognitive disability.

Pathogenesis

The chromosomal karyotype for standard trisomy 21 is indicated as 47, XY, +21 in males and 47, XX, +21 in females. A large number of errors can happen during cell division. Disjunction occurs when the pairs of chromosomes in a cell undergoing division, are supposed to split up and go to different points during meiosis. But in rare cases this doesn’t occur and that defective pair completely goes to only one point without splitting. So the finally formed cell will have 24 chromosomes and the other cell will have 22 chromosomes only. This error is called “nondisjunction.” This may affect either the sperm or the egg and when one of them fuses with a normal set of chromosome the offspring chromosome would have 47 chromosomes instead of the normal 46 i.e., an additional 21st chromosome. Therefore the technical name, trisomy 21. In most of the cases the extra chromosome is from the egg and the reason may be due to increased maternal age and research is on to identify the exact cause for this disjunction. Molecular and cytogenetic studies indicated that duplicate21 (q22.1-22.2) is sufficient to cause Down syndrome. It contains genes that code for enzymes, 126 P. C. Anila Namboodiripad and E. Anuradha Sunil such as cystathionine beta-synthase (CBS), superoxide dismutase 1 (SOD1), glycinamide ribonucleotide synthase-aminoimidazole ribonucleotide synthase- glycinamide formyl transferase. GARS-AIRS-GART. Down’s syndrome is also seen to occur when part of chromosome 21q22.3 becomes translocated to another chromosome during embryogenesis and the affected individuals with this genetic change are said to have translocation Down syndrome. Another scenario is when a patient with Down’s syndrome has an extra copy of chromosome 21 in only some of the body's cells and the condition is called mosaic Down syndrome. Having these extra copies of genes on chromosome 21 causes disturbance in embryogenesis and causing the distinguishing features of Down syndrome and the amplified risk of health problems associated with this condition. This mosaic Down’s syndrome is not inherited similar to the Trisomy 21.

Achondroplasia

Introduction

One of the most common types of short-limb uneven dwarfism is called as Achondroplasia. Parrot in 1878 was the first to coin the term achondroplasia, suggesting absent cartilage formation. Histologically the name seems inappropriate but it has been used universally and has also been accepted by the International Working Group on Constitutional Diseases of the Bone.

Pathogenesis

Achondroplasia is a bone disorder commonly affecting the long bones and the base of the skull and is characterized by large head, short extremities, and trident-shaped hands. It is an autosomal dominant disorder that primarily affects the FGFR3 due to mutations affecting it. About 80% of parents are unaffected. Research suggests that the disease occurs due to increased signal transduction from the mutant receptors in the FGFR-3 genes and it has been proved by introducing drugs that reduce signal transduction such as the kinase inhibitors and neutralizing antibodies. It has hence been proved that functional Bone Disorders 127 impact of the mutations of the FGFR3 gene may be the key negative regulator of endochondral ossification and may play a role in targeting the chondrocyte growth plate. However, it was mapped to the p axis of chromosome 4. It has been seen that all patients with the typical features of achondroplasia have the same glycine to arginine substitution at position 380, which maps to the transmembrane domain of the FGFR. Signaling pathways seen downstream of FGFR-3, include the mitogen activated protein kinase (MAPK), signal transducer and activator of transcription (STAT), and phospholipase Cg pathways. The STAT signals are thought to inhibit the proliferation of the chondrocytes, but MAPK signals not only influence proliferation negatively, but also disrupt terminal differentiation and postmitotic matrix synthesis. Multiple pathways of the FGFR3 signal downregulate growth-promoting molecules, resulting in the inhibitory proliferation and differentiation of chondrocytes in growing bones. The transmembrane domain of the FGFR3 is affected in the achondroplasia.

Marfan Syndrome

Introduction

Marfan syndrome is an autosomal dominant inherited disorder that affects the body's connective tissues, which provides the strength, support, and elasticity to tendons, cartilage, heart valves, blood vessels, and other vital parts of the body.

Pathogenesis

Mutations in the fibrillin-1 (FBN1) gene on chromosome 15, which encodes for the glycoprotein fibrillin is said to be the cause of Marfan syndrome. Recent works have stressed the role of proteases and disturbances in tissue homeostasis as other causes for occurrence of MFS. Fibrillin, a 350 kDa glycoprotein, was discovered in 1986 and characterized as the main component of the extracellular microfibrils. The major element of microfibrils is fibrillin, which makes up the structural components of the suspensory ligament of the lens and serves as a matrix for elastin in the aorta and other connective tissues. Hence a deficient fibrillin 128 P. C. Anila Namboodiripad and E. Anuradha Sunil deposition leads to reduced structural integrity of the aorta of the heart, spinal dura, ligaments, lens zonules, and lung airways. TGF-β signaling pathway which may ultimately lead to incorporation of fibrillin into the connective tissue matrix may also be playing a role in the pathogenesis of Marfan syndrome. Fibrillin-1 is translated from an mRNA encoded by the FBN1 gene on chromosome 15. The protein is processed and secreted and then forms microfibrils in the matrix in association with other extracellular molecules. Microfibrils may function alone or form the basis of elastic fibers following deposition of elastin. Among molecules that are associated with fibrillin-1 is LTBP-1, a binding protein for the latent form of TGF-β. TGF-β is expressed as a precursor, which is cleaved to make the active form, but the two peptides remain associated and are then bound by a protein, LTBP-1.

Elastic Fibers, Micro Fibrils, and Fibrillin The elastic fiber which is laid down over the fibrillin matrix in the sites mentioned above is a complex structure, which contains lysyl oxidase, elastin, 10-12 nm microfibrils, and perhaps proteoglycans. By electron microscopy, elastic fibres can be seen to be composed of two morphologically distinct components: an amorphous fraction lacking any regular structure and constituting about 90% of the mature fiber, which consists exclusively of elastin, and a microfibrillar component consisting of 10-12 nm microfibrils. Specific and effective treatment can be brought about by understanding the molecular basis of human genetic disease and that is the mantra of human molecular genetics. Till date this optimism has often been more of a buildup than realization. The genetic story of the Marfan syndrome has only recently been unraveled and this pathway to treatment may be both multifaceted and full of discoveries into unexpected areas of biology. It is quite likely that in a short time we may observe the usage of TGF-β blockade in place of β- (adrenergic) blockade as a more effectual treatment of many aspects of Marfan syndrome — and this treatment is the penultimate goal of understanding this intricate phenotype.

Chapter 12

Disorders of the Skin

Pemphigus Vulgaris

Introduction

Pemphigus vulgaris is an uncommon, grave autoimmune disease in which vesicles and bullae erupt on the skin, the lining of the oral cavity, the genitals, and other mucous membranes.

Pathogenesis

Theories of the Pathogenesis of Pemphigus Vulgaris

1. The Desmoglein Compensation Theory Based on the position of Dsg 1 and Dsg in the skin and mucosa, two authors Amagai and Stanley projected the desmoglein reparation theory. This landmark theory stated that the survival of any one Dsg type is adequate to preserve the integrity of the epidermis and mucosa.

2. ‘Multiple Hits’ Hypotheses New advances in the study of pathogenesis of Pemphigus vulgaris states that patients develop antibodies against desmocollins, and plakins and nondesmosomal proteins, other than Dsg 1 and 3which function as cell- 130 P. C. Anila Namboodiripad and E. Anuradha Sunil membrane receptors and they include pemphaxin, thyroperoxidase, nicotinic acetylcholine receptor and some annexins.. The desmocollin 3 is detected throughout the basal, spinous and lower granular layer and the blocking of Dsg3 function with a monoclonal antibody results in the formation of vesicles and bullae. Apart from that, some patients also developed antimitochondrial antibodies, which could pierce keratinocytes and react with mitochondrial proteins. Hence at least 3 autoantibodies are said to play a role for e.g., autoantibodies directed against keratinocyte, desmosomal autoantigens and mitochondrial autoantigens. The ‘multiple hits’ theory which was recently published, explained the occurrence of acantholysis. But the Dsgs’ were said to play a major role. Autoantibodies against other antigens are also found but were nonpathogenic and their contributions remain disputable.

Figure 1. Showing the pathogenesis of pemphigus. Acantholysis of the spinous cell layer due to the autoantibody acting against the Dsg1 and Dsg. Disorders of the Skin 131

3. The Antibody-Induced Apoptosis Theory Researchers are considering the mechanism of keratinocyte detachment, as the underlying mechanism for acantholysis in pemphigus vulgaris. Apoptosis signaling is said to be induced by pemphighus IgG and the anti-Fas receptors under experimental conditions and the pathway involved include an increase in intracellular Fas R, Fas L, Bax and p53 and also a reduction in the level of Bcl-2; increased caspase 8, activation of caspase 1 and 3. It was found that the inhibitors of caspases 1 or 3 were effective in suppressing apoptosis and stopping acantholysis, and hence favoring the concept that apoptosis contributes to cell separation.

4. The Basal-Cell Shrinkage Hypothesis and the Apoptolysis Theory A new hypothesis of pemphigus pathogenesis was introduced in 2006, suggesting that after the binding of the keratinocyte receptor by the pathogenic pemphigus vulgaris autoantibody there was an involvement of a chain of signal transduction pathways that initiated the rupture of the cytoskeleton, resulting in the collapse and shrinkage of the keratinocytes. Desmosomal cadherin disruption caused by the autoantibodies may be the reason for the acantholysis. The cadherin may be a part of the signaling pathway involved in the PV pathogenesis and they include the p38 MAPK, RhoA, PKC, c-myc, and plakoglobin. The internalization and reduction of Dsg 3 in PV has been linked to the p38 MAPK activity. However, the p38 MAPK may also regulate desmosomal adhesion independent of intermediate filaments. For example, the alteration in the organization of the actin cytoskeleton and the actin filament dynamics via the heat shock protein 27, especially within the marginal junction-associated actin belt were shown to influence the autoantibody- induced loss of cell adhesion. A novel term, ‘apoptolysis’ was introduced in 2009 which stated that suprabasal acantholysis and cell death pathways occurred, following the basal cell shrinkage. The predominant difference between apoptosis and apoptolysis in PV was that of basal cell shrinkage and that no actual death of keratinocytes occurred. Experiments conducted have shown that the acantholysis and apoptosis can be prevented by the anti- Fas L antibody. This new concept of apoptolysis is critical for the description of PV development. However, whether apoptosis occurs prior to acantholysis or whether it is just an intermediate step in acantholysis is still under debate.

132 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 2. Model for PV pathogenicity.

The mitochondrial antigens have been shown to be attacked by the autoantibodies that have been identified in the sera of PV patients and this theory can be used to explain the acantholysis in patients lacking desmoglein antibodies. Pemphigus vulgaris IgGs go through keratinocytes (KCs) and exclusively bind to mitochondrial proteins, which is linked to the mitochondrial injury stimulating apoptosis and acantholysis. The mitochondrial proteins most commonly attacked in pemphigus vulgaris include those involved in O2 respiration, oxidative phosphorylation, tricarboxylic acid cycle and the assembly/inactivation of reactive oxygen species (ROS). The increased ROS Disorders of the Skin 133 production by the PV IgGs, following proton leakage from KCs, PV sera reduced the ability of KCs to respond to stress. An increase in lipid peroxidation shown by increased production of ROS is an indicator of the mitochondrial dysfunction in PV and the oxidative stress indicated by altered peroxidant-antioxidant balance and the intrinsic apoptotic pathway dependant on the mitochondria indicate an important role for mitochondria in keratinocytes to indirectly play a role in acantholysis. But how the PV anibodies enter the mitochondria is controversial and it has been suggested that a cell surface protein showing features of a mitochondria may play a role. Mitochondrial antibodies enter the keratinocytes which present with annexins, and the annexins can reposition to the cytosol to reach the mitochondria. Or an antibody in a complex with the Fc receptor present on the keratinocytes, may show the path for it to enter the mitochondria. Another theory for pathogenesis of PV as suggested by Lanza et al. includes the role of CDK2. Exposure of PV serum to synchronized human keratinocytes increased the quantity of cells in S phase, which resulted in the rounding of the cell, cell-cell disconnection, increased CDK2 expression in a dose-dependent manner, and also changed the expression of over 500 genes. The most significantly enriched genes were those involved in cell communication and cell- cell junction formation. Small interfering RNA against CDK2 reversed PV serum-induced CDK2 overexpression and reduced cell-cell detachment. Lanza et al. (2008) concluded that activation of CDK2- mediated signaling is a crucial event in the development of PV lesions.

Pemphigus Vegetans, Pemphigus Foliaceous, Paraneoplastic Pemphigus

Introduction

Pemphigus Vegetans was (PV) initially considered a variant of pemphigus vulgaris in 1876, by Neumann and in 1889, Hallopeau described a patient with pustules and vegetating plaques, suggestive of a variant of PV of Neumann. Both types of pemphigus vegetans show the expansion of vegetating plaques, especially in skin folds (perianal, axilla and inguinal). They majorly affect younger patients. 134 P. C. Anila Namboodiripad and E. Anuradha Sunil

Pemphigus foliaceus (PF) is a persistant autoimmune blistering disease mainly involving the skin of the face and upper part of the body, such as chest regions, rather than the lower part or the scalp. Paraneoplastic pemphigus (PNP) is a predominantly fatal paraneoplastic mucocutaneous blistering disease most likely caused by lymphoproliferative disorders.

Pathogenesis

Pemphigus is an autoimmune disease with the IgG found directed against desmoglein 1 in pemphigus vegetans and pemphigus foliaceous and desmoglein 3 in case of paraneoplastic pemphigus.

Cicatricial Pemphigoid

Introduction

Cicatricial pemphigoid is a rare, chronic, blistering and scarring disease that affects the oral and ocular mucosa.

Pathogenesis

The parts of the basement membrane zone i.e., the dermal-epidermal junction is made up of four compartments: sublamina densa, the basal cell plasma membrane, the lamina lucida, and the lamina densa. Hemidesmososmes are located in the dermal surface of the plasma membrane of the keratinocytes. They anchor the epidermis, through the lamina lucida and connecting (anchoring) filaments to the lamina densa and eventually to the underlying dermis. The basal set of intermediate filaments namely the keratins 5 and 14, also form a part of the hemidesmosome apart from the other four protein components, namely the Bullous pemphigoid antigen 1 (230 kD) and plectin that are the intracellular components and the Bullous pemphigoid antigen 2 (180 kD, type XVII collagen) and alpha 6 beta 4 integrin which are the transmembrane components. Disorders of the Skin 135

The lamina lucida contains the anchoring filaments, laminins 5, 6, and 7 which originate in the hemidesmosomes of the plasma membrane and insert into the lamina densa. The lamina densa is composed predominantly of type IV collagen. However, laminin, nidogen/entactin, and heparin sulfate proteoglycans are also found in this region. The sublamina densa zone which is located beneath the lamina densa contains anchoring fibrils (collagen VII), anchoring plaques (type 4 collagen and laminin), collagen fibers, and elastic fibers. Anchoring fibrils in addition to connecting anchoring plaques, bind to laminin 5 in the lamina lucida and to anchoring plaques and type IV collagen in the lamina densa. So it is the anchoring fibrils that connect all of these structures, in due course anchoring the epidermis and its basic basement membrane zone to the papillary dermis. In the cicatricial pemphigoid affecting the oral cavity, IgG and C3 may be confined to a small area to the lower lamina lucida and lamina densa or else to the recognized overlying the hemidesmosome. The sublamina densa remains uninvolved because of the involvement of the β4 integrin, BP180 or laminin 332. This anti-laminin Ab interferes with the adhesion of laminin-5 causing separation of the keratinocytes from the underlying basement membrane. BP180 forms a complex with laminin-5 and it spans the lamina lucida and interacts with the lamina densa-which may explain the scarring in this disease.

Bullous Pemphigoid

Introduction

Bullous pemphigoid (BP) is a chronic blistering of the skin. It ranges from mildly itchy welts to severe blisters and infection, and may affect a small area of the body or be widespread. The vast majority of those affected are elderly, but it has been seen at all ages.

Pathogenesis

Cell Junctions The hemidesmosomal junction comprises of a plaque which may be electron dense, disc shaped structure. Keratin intermediate filaments may be attached to the α6β4 integrins and BP180 which are the hemidesmosomal 136 P. C. Anila Namboodiripad and E. Anuradha Sunil proteins. Hemidesmosomal plaque components BP 230 and plectin are attached to the intracellular portions of α6β4 and BP180 whereas their extracellular domains contact the anchoring fibrils via filamentous proteins such as laminin 5 after they lengthen into the basement membrane zone. Anchoring fibrils originate in the lamina densa of the basement membrane, and they widen into the adjacent connective tissue and end in a structure known as anchoring plaques. Type VII collagen forms the major component of anchoring fibrils. BP230 gene is found to be mapped to 6p11–12, and the BP180 gene to the 10q24.3. BP230 is a plakin protein family member that associates the hemidesmosomes with keratin intermediate filaments. This intracellular protein has significant similarity in origin with plectin and desmoplakins I and II. BP230 is said to contain a central coiled domain region supported by two globular end domains whereas BP180 is a transmembrane protein with a type II orientation. It has an amino-terminal region located in the intracellular hemidesmosomal plaque, and a carboxyl terminal portion projecting into the extracellular portion of the BMZ. This sequence pattern is prognostic of a protein domain that forms into a collagen triple helix. The exact role of bullous pemphigoid antigens in the etiopathogenesis of bullous pemphigoid is not completely clear. BPAg1 (BP230) is an intracellular component of the hemidesmosome; BPAg2 (BP180, type XVII collagen) is a transmembranous protein with a collagenous extracellular domain. The binding of antibodies at the basement membrane activates complement and inflammatory mediators. Activation of the complement system is thought to play a critical role in attracting inflammatory cells to the basement membrane. These inflammatory cells are postulated to release proteases, which degrade hemidesmosomal proteins and lead to blister formation. Eosinophils are characteristically present in human patients' blisters as demonstrated by histopathologic analysis, although their presence is not an absolute diagnostic criterion. Interleukin 16, a major chemotactic factor responsible for recruiting CD4+ helper T cells to the skin and for inducing functional interleukin 2 receptors for cellular activation and proliferation, was found to be expressed strongly by epidermal cells and infiltrating CD4+ T cells in lesional bullous pemphigoid skin. The binding of autoantibodies to antigens initiates a series of immune inflammatory events, including neutrophillic infiltration; complement activation, macrophage activation, and mast cell degranulation, which lead to the development of clinical blisters or bullae. Disorders of the Skin 137

Figure 3. The diagrammatic representation of the dermal-epidermal anchoring complex.

1. Mast Cells The mast cell released during inflammation degranulates and plays an essential role in getting neutrophils to the target tissue and once the complement is activated it plays a key role in the inflammatory cascade leading to the blister formation.

2. Neutrophils Inflammation at the site of Bullous pemphigoid further aggravates due to recruitment of the neutrophils. Neutrophils release proteases which results in the local tissue damage of BM. Thus; the disease severity is directly correlated to the number of infiltrating neutrophils.

3. Eosinophils Eosinophils are also found in the BP lesions and are often found scattered throughout the upper dermis or collected at the edge of the dermal–epidermal junction. Eosinophil count in the circulating blood is elevated too. Blister fluid is found to contain a large amount of IL-5, eotaxin, and eosinophilic cationic protein (ECP). IL-5 encourages expansion and activation of eosinophils. 138 P. C. Anila Namboodiripad and E. Anuradha Sunil

Eotaxin is eosinophil-specific chemokine, regulating eosinophil migration produced by fibroblasts and probably keratinocytes. CCR3, the specific eotaxin receptor, has been stated to be strongly seen on eosinophils, basophils, and Th2 cells in BP. IL-5 and eotaxin probably increases the inflammatory reaction and adds to the invasion of granulocytes, which, by release of proteinases or cytotoxic agents such as eosinophil major basic protein (MBP) and ECP, finally causes the separation of epidermis and dermis at the position of the lamina lucida in the basement membrane zone.

4. Role of Proteolytic Enzymes Elevated levels of proteolytic enzymes, such as collagenase, neutrophil elastase (NE), plasminogen activators, cathepsin G, plasmin, matrix metalloproteinase-2 (MMP-2, gelatinase A), MMP-9 (gelatinase B), and MMP-13 have been isolated from blister fluid and lesional biopsies of BP patients. These enzymes are released into the ECM upon cell activation, by neutrophils and eosinophils, and are also believed to proteolytically degrade various extracellular matrix proteins including the extracellular domain of BP180. Other than plasmin, the MC-specific serine protease MCP-4 (chymase) is also able to activate MMP-9. The physiological inhibitor of NE namely α1-proteinase inhibitor is inactivated when the MMP-9 is proteolytically activated and this allows unrestrained activity of NE. These observations show that proteolytic enzymes released from inflammatory cells harm the BMZ directly, causing the separation of the dermoepidermal junction.

Mechanisms of Tissue Injury and Blister Formation

The mechanisms by which BP autoantibodies are thought to be pathogenic, include complement activation, recruitment of inflammatory cells, liberation of proteolytic enzymes and direct interference with the adhesion function of the autoantigens.

1. Role of Direct Mechanisms The blister formation in the BP usually requires cellular and humoral immunity as compared to that in pemphigus and anti-laminin 5 mucous membrane pemphigoid. There is news that apart from the mechanisms mentioned above, the disruption of the junction may also occur due to direct binding of the autoantibody independent of their Fc portions. Disorders of the Skin 139

There is competition with the natural ligand, in blocking the key binding sites of the antibody to the BP180antigen which may result in the impairing the functions of these molecules. The autoantibodies from BP patients largely belong to the non-complement fixing IgG4 subclass; lower concentrations of the IG1 subclass are also present. Hemidesmosomal disassembly or the inducing of the proinflammatory cytokines may also be caused by the activation of the intracellular signaling pathways which results in formation of blisters by autoantibodies. IL-6 and IL-8 is modulated due to autoantibody action but its pathogenic relevance is not known.

2. Role of Complement System Several early studies have already shown that BP autoantibodies fix complement in vitro and that C3 is detected by direct immunofluorescence along the basement membrane zone of perilesional skin in almost all cases of BP. Both BP autoantibodies and C3 are detected by immunoelectron microscopy at the site of immunological injury, i.e., the lamina lucida of the skin. Lesional skin of patients with BP shows components of both the alternative and the classical pathways of complement (including C4, C5, C1q and C5-9), factor B, properidin and B1H globulin. T cells giving rise to matrix metalloproteinase (MMP)–2, MMP-9, and MMP-13 were significantly increased in lesional bullous pemphigoid skin compared with that of healthy skin. Hence a role for MMP is suggested in the blistering of bullous pemphigoid. A cytokine named BAFF (B-cell activating factor –From the TNF family) that plays a role in survival and regulation of B-cell was found to be considerably higher in sera of bullous pemphigoid patients compared with healthy subjects, even though no major association was detected between serum BAFF levels and titers of anti-BPAg2 antibodies. In 2008, a responsibility for IgE class of autoantibodies, in relation to BP180, was established and a higher level of the IgE correlated with an even more severe clinical state. The coagulation cascade was found to be involved in the bullous pemphigoid patients, and hence is said to play a role in its severity. There was also an increase in eosinophils which was also said to play a role in the coagulation, hence increasing the potential thrombotic risk, blister formation, tissue damage, as well as inflammation. Reports in 2009, of bullous pemphigoid developing after adalimumab treatment for psoriasis brought forward the idea whether drugs can play a role 140 P. C. Anila Namboodiripad and E. Anuradha Sunil in inducing the disease or it may simply be the coincident association of bullous pemphigoid with psoriasis. The initiating factor for BP includes the binding of the pathogenic autoantibody to a specific antigen but the cause and the method of autoantibody production remains uncertain. These facts mentioned above may enable further studies to be conducted, to determine the source of the autoantibody concerned and the pathogenesis, as well as more useful therapy of BP.

Lichen Planus

Introduction

Lichen planus (OLP) is a pruritic, papular eruption characterized by its violaceous color and polygonal shape, sometimes with a fine scale. It is most often found on the flexor surfaces of the upper extremities, genitalia and on the mucous membranes.

Pathogenesis

Recent data has suggested that both antigen-specific and non-specific mechanisms are involved in the pathogenesis of OLP and it has been suggested that both factors play a role in lesion development. The primary event in OLP lesion formation and the factors that establish OLP vulnerability are unknown. Unspecified agents like altered protein, autoreactive peptide, contact allergen, hapten, drug reaction, and viral or infectious agents on a class I major histocompatibility complex molecule on the keratinocyte is believed to activate a CTL-inciting disease (chronic T lymphocytic lesion). Precipitating factors for the same may include trauma, stress, and infectious agents (i.e., hepatitis C virus (HCV). Most T cells in the epithelium and adjacent to the damaged basal keratinocytes are activated CD8+ lymphocytes. This could be in combination with increased Th1 cytokine production (IL-1, IL-8, IL-10, IL-12, TNF-a) and an increase in the expression of intercellular adhesion molecule-1 on Langerhans cells and macrophages, and major histocompatibility complex antigens by keratinocytes. Disorders of the Skin 141

The perivascular localization of CD4+ T cells is a pointer to the possible support offered to the cytotoxic CD8+ cells by secretion of Th1 cytokines. Keratinocyte damage by CD8+ T cells which releases cytokines would lead to basement membrane disturbance such as apoptosis through the perforin/granzyme or Fas/Fas ligand pathways, making way for the entry of more CD8+ T cells and further basal cell damage which causes a vicious cycle leading to chronicity of the disease. Both the cutaneous lichen planus and oral LP are found to have similar pathogenesis. The preponderance of T cells in the inflammatory infiltrate, also suggests a major role for humoral immune mechanism in the pathogenesis of cutaneous LP. The CD8+ T lymphocytes are attracted to the antigen presentation by the basal keratinocytes which presents with the major histocompatibility complex (MHC) class I on it. The lichen planus antigen is unknown, though it may be a self-peptide or a protein similar to the self-peptide, in which case lichen planus would be a true autoimmune disease. Keratinocyte apoptosis results in Civatte bodies. And this keratinocyte apoptosis caused by the immune system occurs through many mechanisms namely:

a. The caspase cascade, that requires the tie up between the ligand/protein found on the surface CD95L (FasL) of the cytotoxic T lymphocytes (CTL) to the keratinocyte CD95 (Fas) to trigger apoptosis; b. CTL and natural killer cell secretion of granzyme B, that forays into keratinocytes via the cell membrane pores from perforin, resulting in apoptosis; and c. The lymphocyte secreting the cytokines such as tumor necrosis factor thereby inducing matrix metalloproteinase 9, which can interfere with the basement membrane zone integrity and promote keratinocyte apoptosis through deprivation of basement membrane zone–derived cell survival signals.

Other authors have suggested the vacuolar degeneration of basal keratinocyte during apoptosis. But this phenomenon would cause severe and extensive oral erosions. Erosive LP is rare and the reticular variant is predominant, so it can be assumed that there is a reciprocal action by the oral mucosa that controls these erosions and it maybe by the increased cellular proliferation. Hence a mixed pattern of both apoptosis and increased cell turnover is seen occurring in LP. Another author also suggested that the cells 142 P. C. Anila Namboodiripad and E. Anuradha Sunil may undergo senescence rather than apoptosis as shown by the presence of positive p21waf1 expression in OLP, which indicates cell cycle arrest and senescence. This cell cycle arrest maintains the tissue structure and helps in the DNA repair mechanisms but senescence would point towards malignant change. The level of p21WAFK was found to vary in different research studies. As p53 expression has been identified as a response to DNA damage, the identification of p53 in OLP tissue is interpreted as an indication of precancerous potential by some researchers. The expression of NF-kappa B has been found higher in OLP than in cutaneous lichen planus (CLP), a fact that is considered consistent with the more persistent inflammation observed in OLP in comparison to CLP. IL-6 expression in serum and saliva of OLP patients is considered indicative of a Th2 cellular involvement in OLP, a fact that was underestimated initially in the pathogenesis of OLP. Other mechanisms which may be non-specific like the MMP activation and the mast cell degranulation are also said to play a role in the OLP pathogenesis. The expression or unmasking of the lichen planus antigen may be induced by drugs (lichenoid drug reaction), contact allergens in dental restorative materials or toothpastes (contact hypersensitivity reaction), mechanical trauma (Koebner phenomenon), viral infection, or other unidentified agents. There is also found to be a correlation between LP and LP variants to hepatitis C virus, syphilis, HIV, human herpesvirus 2, chronic bladder infections, human papilloma virus or the Helicobacter pylori. However, screening for hepatitis C virus infection is advisable, as case-control studies have shown an association of hepatitis C virus and LP. Carcinogenesis in OLP may be dictated by the incorporated signal from various tumor promoters and inhibitors such as MIF, MMP-9 and TNF-alpha, TGF-beta 1, IFN-gamma and IFN C gamma-12.

Lupus Erythematosus

Introduction

A collection of autoimmune diseases which shows a hyperactive human immune system and which attacks the normal healthy tissues is named as Disorders of the Skin 143

Lupus erythematosus. The different body systems affected by this disease includes the skin, joints, kidneys, blood cells, heart, and lungs.

Pathogenesis

Systemic lupus erythematosus (SLE, lupus) shows the presence of a uniform loss of the self tolerance with the stimulation of the autoreactive T and B cells leading to pathogenic autoantibodies and tissue injury. The etiopathogenesis is said to be multifactorial with various genetic and envoirnmental factors playing a role. Sex hormones and the hypothalomo- pituitary adrenal axis are also found to be contributory factors to the pathogenesis of SLE. Defective immune regulatory mechanisms, such as the clearance of apoptotic cells and immune complexes, are important contributors to the development of SLE. The loss of immune tolerance, increased antigenic load, excess T cell help, defective B cell suppression, and the shifting of T helper 1 (Th1) to Th2 immune responses leads to B cell hyperactivity and the production of pathogenic autoantibodies. Finally, certain environmental factors are probably required to trigger the disease. The predominant disturbance in the patients with the SLE is the autoantibody production. These antibodies are found to attach to the nucleus, cytoplasm and the cell surface. This antinuclear antibody is characteristic for the pathogenesis for the SLE. Also the anti-double stranded DNA (ds-DNA) and anti-Sm antibodies are exclusive to patients with SLE. The Sm antigen is a small nuclear ribonucleoprotein (snRNP) composed of a distinctive set of uridine rich RNA molecules attached to a common group of proteins associated with the RNA molecules. Anti-DNA antibody titers frequently vary over time and disease activity but anti-Sm antibody titers are usually constant. Their association with the glomerulonephritis is the most remarkable feature of anti-DNA antibodies. Anti-DNA antibodies can be identified from patients with active lupus nephritis and anti-DNA antibodies can induce nephritis. Immune mediated cells like the B cells, T cells, monocytic cells resulting in the polyclonal B cell activation also play a role in the pathogenesis of the SLE.

144 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 4. Depicting the causative factors for Lupus Erythematosus.

Figure 5. Showing the etiological factors responsible for formation of systemic lupus erythematosus. Disorders of the Skin 145

Irritating chemicals such as cell wall phospholipids, viral antigens and bacterial DNA can induce anti-DNA antibodies in mice. Self antigens and the environmental antigens are taken up by professional antigen presenting cells (APCs) to induce antibodies on the surface of B cells. They then present them to the T cells after processing into peptides through surface HLA molecules. The activated T cells on the other hand stimulate the B cells to produce pathogenic autoantibodies. Several cytokines may play a role together with the B and T cells and it includes the IL-10. Accessory molecules such as those of the CD40/CD40L and B7/CD28/CTLA-4 systems help the IL-10 cytokines. The B cells produced in these patients are abnormal. They are found to exhibit increased intracytoplasmic calcium responses than controls. They are more prone to the polyclonal activation by various agents. Abnormalities in T cell and its reduced number is also observed in patients with SLE probably because of the effects of antilymphocytic antibodies. Apoptosis is also found defective in these individuals and this may lead to a prolonged existence of the diseased lymphocytes in SLE. Infectious agents, diet, toxins/drugs may affect the production of inflammatory mediators and enhance the immunogenicity of the self antigens. Agents, such as ultraviolet (UV) light, causing photosensitivity, may also play a role in SLE. Protection against this UV radiation is brought by the Nitrous oxide which is found increased in these individuals. The consumption of meat and milk products of livestock fed with synthetic estrogens and synthetic estrogens used by postmenopausal women for contraception may also initiate apoptosis. Drugs such as procainamide and hydralazine, which are aromatic amines or hydrazines, can induce a lupus-like syndrome, predominantly in persons who are genetically slow acetylators. These chemicals and their derivatives are commonly used in the agriculture, industry, and in the various commercial applications.

Section 4: Bacterial, Viral and Fungal Lesions

Chapter 13

Bacterial Lesions of Oral Cavity

Tuberculosis

Introduction

Tuberculosis (TB) is an infectious disease, primarily affecting the lungs (a pneumonia), and is caused by the bacteria called as Mycobacterium tuberculosis. It normally spreads from person to person through aerosol or by droplet infection.

Pathogenesis

Primary Tuberculosis The immediate reaction following the entry of the organism through the routes such as the respiratory tract, oral mucosa etc. is the onslaught by the neutrophils. The glycolipid capped mannose on the organism acts as an antigen and initiates the procedure to engulf it. But the organism resists this digestion by interfering with the fusion between the lysosome and phagolysosome, multiplies in the phagosome, and causes macrophage necrosis. It thus makes the macrophage ineffective though the lysosome has initiated the maturation arrest of the organism. This macrophage would be engulfed by another macrophage. This is brought about under the control of genes called as the Natural Resistance-Associated Macrophage protein 1(NRAMP-1). On the other hand some macrophages are able to complete the 150 P. C. Anila Namboodiripad and E. Anuradha Sunil digestive action and destroy the microorganism. The macrophage that has engulfed the processed antigen will then present the MHC on its surface and it now becomes an antigen presenting cell (APC). This APC then releases the IL-12 which would attract the T cells, namely the helper T cell. In two weeks time the delayed hypersensitivity reaction would be initiated causing antibody production against the mycobacteria. Mantoux test is positive at this stage. The next in the defense line, against the microorganism would be, in the manufacture of the interferon gamma by the Th 1 cells which transforms the macrophages into the epitheloid cells. These epitheloid cells produce TNF which would recruit more monocytes from the circulation. The monocytes will again coalesce to form larger phagocytic cells which are then called as the ‘Langerhans giant cells.’ These cells are unique in that they have the cell which is large and they exhibit multiple nuclei that are arranged in a ‘horse shoe shaped’ manner adjacent to the cell membrane of the giant cell. Finally there is the collection of the inflammatory cells and the fibroblasts with the collagen fibers which walls of the above mentioned cells, to be now known as a ‘granuloma.’ This granuloma prevents the spread of the organism and its toxins into the neighboring areas. In the lung this granuloma is known as the ‘Ghon focus’ and if it accumulates in the lymph nodes it is called as the ‘Ghon complex.’ Upon fibrosis calcification and healing this complex is known by the name ‘Ranke’s complex.’ The Mycobacterium tuberculosis and the Mycobacterium bovis strains, but not the vaccine strain BCG Mycobacterium bovis was found to have a region of difference 1(RD 1) that was important site for indication of the virulence of the organism.

Secondary Tuberculosis Secondary tuberculosis occurs some time after exposure to the organism as compared to the primary which occurs immediately on being infected with the organism. When organism has remained latent in the individual without causing any symptoms, and when it has become active due to a lowered immunity it is called as secondary tuberculosis. The presentation is then similar to that of the primary tuberculosis except that it can become extensive as compared to the primary TB which is more localized.

Miliary Tuberculosis When the organism spreads through the blood stream or hematogenous spread of the organism to the different parts of the body or becomes disseminated it is called as the miliary tuberculosis. The name has come about Bacterial Lesions of Oral Cavity 151 because of the ‘millet seed’ like appearance on a chest radiograph of a patient with miliary tuberculosis. The clinical manifestation of this condition may be acute or is most commonly sub-acute or chronic.

Syphilis

Introduction

It is a highly contagious disease that mainly spreads through sexual contact. It is said to be caused by a spirochetal organism called as the Treponema pallidum.

Pathogenesis

The entry of the organism into the body is followed by the invasion of the epithelial cell and the fibroblast like and endothelial cells. Endothelial cells are traversed by the corkscrew type motility of the organism. This is followed by the production of the matrix metalloproteinase-1 in the connective tissue thus breaking down collagen which causes organism to enter the lymphatics and the bloodstream to disseminate following nutrient gradients (chemotaxis) and causing its varied clinical manifestations.

Actinomycosis

Introduction

It is rare type of bacterial infection caused by a dormant bacterial organism called as actinomyces. Most of the infections caused by bacteria are localized as they are unable to penetrate the body’s tissues. But in the case of the actinomycosis the infection is able to move slowly and steadily through the body tissues. The actinomycosis is not a contagious disease since the organism needs an anaerobic environment to thrive, which is available within the body, but absent once t is exposed to the environment.

152 P. C. Anila Namboodiripad and E. Anuradha Sunil

Pathogenesis

The disease is caused by the actinomyces organism which is a gram +ve bacilli and not a fungus as the name suggests. The organism is neither a parasite nor a saprophytic organism. In most cases, the bacteria live without causing any harm mainly on the throat, lining of the mouth, vagina and the digestive system. The virulence of the actinomyces organism is low and it normally produces infection in the humans following a breach in the tissue or through a necrotic tissue. It also has the characteristics of producing infection that has varying manifestations and varying clinical features. But once the infection is established it mounts an intense inflammatory response by producing pus and a granuloma formation. The inflammatory response is found to conclude by fibrosis. Prior to that, the infection spreads and invades surrounding tissues or organs. Draining sinuses from these swellings that spout ‘sulfur granules’ are seen, or called so because of it’s ‘sulfur yellow’ color, which is the hallmark of the infection. The sulphur granules are found teeming with the actinomyces organisms when viewed under the microscope.

Sarcoidosis

Introduction

Sarcoidosis is an inflammatory disease that mainly affects numerous organs in the body, but most commonly affected includes the lungs and lymph glands.

Pathogenesis

The main pathogenesis in sarcoidosis is the formation of the non caseating granuloma. The centre of this granuloma is said to show the presence of the antigen which is surrounded by the epitheloid cells and the multinucleated giant cells. Both CD4+and CD8+T cells, B cells and fibroblasts are found lining the periphery of the granuloma.

Bacterial Lesions of Oral Cavity 153

Figure 1. Immunopathogenesis of sarcoidosis (a proposed model).

The so called sarcoid antigen is engulfed by circulating dendritic cells. Dendritic cells stimulate the T cells. Activated T cells are highly polarized and they produce IL-2 which causes monoclonal proliferation of T cells. Also, upon TCR activation, T-bet production increases. T-bet upregulates and enhances the production of IFNγ which enables the granuloma formation. The future antigen clearance and increased IL-10 levels bring about disease clearance. Disease chronicity results in a predominance of TH2 cytokines which leads to lung remodelling by fibrosis.

Recurrent Aphthous Stomatitis

Introduction

Recurrent aphthous stomatitis is a disorder characterized by recurring ulcers in the oral mucosa in patients with no other signs of the disease. 154 P. C. Anila Namboodiripad and E. Anuradha Sunil

Pathogenesis

A large number of etiological factors have been suggested as the cause for the recurrent aphthous stomatitis. The etiological factors other than ones mentioned below cannot be pathogenetically explained as etiopathogenetic factors, since no substantiating evidence has been found between the etiology and the disease.

 Stress has been emphasized as a causative factor in RAU.  Stress as an etiological factor in RAS patients induces parafunctional habits in them such as lip or cheek biting that may result in trauma to the site and the patient acquiring ulceration. But correlating stress with RAU in experimental studies the relationship has been found negative, thus implying that stress may only be a contributory factor in RAS and plays no role in its etiology.  Oral organisms have been found at the site of the ulcer, suggesting that probably the bacteria such as oral streptococci, helicobacter pylori etc may play a role in the causation of RAS and though the role of virus has been suggested, it is still said to be under speculation. Viruses that have been suggested include the viruses from the herpes family like the EBV, CMV, HSV, HSV6, etc. These organisms may act as an antigenic stimuli initiating an immunologic reaction and damaging the oral mucosa in the bargain.  TNF-alpha is said to play a major role in formation of the RAU. The inciting factor may be any of the above mentioned antigens that stimulate the production of IL-2 and TNF-alpha, following its attack on the oral mucosal keratinocytes. This TNF-alpha causes expression of MHC 1 which further attracts the CD 8+ cells resulting in the ulcer formation of the oral mucosa.  Both types of immune responses humoral and acquired is said to play a role in RAS. It is said to result in neutrophil reactivation, complement addition, role of NK cells, B cells altered CD4+/CD 8+ ratio and T cell receptors in the peripheral blood. The Th1 is said to play a major role in RAS formation.  There was found to be an increase in the production of the IL-2, IFN-c and the TNF- alpha in the acute cases of RAS and also a simultaneous reduced secretion of the TGF- beta and the IL-10. This imbalance in Bacterial Lesions of Oral Cavity 155

the pro and the anti-inflammatory cytokines may be the reason for the ulcer formation in the RAS.  Also the increased Th1 as compared to the Th2 may also be the cause for the RAS. The heat shock proteins are a component of the anti- inflammatory cytokine and this is said to inhibit the differentiation of the monocytes into the dendritic cells. This may further aggravate the inflammatory process. The heat shock proteins are found at elevated levels in smokers thus explaining the reduced occurrence of aphthae in the smokers.  Autoimmunity is suggested as another etiological factor for RAS. Here the structures of the epithelium such as the desmosomes, hemidesmosomes get disrupted due to an auto-antibody antigen reaction at that site.

Chapter 14

Viral Infections of the Oral Cavity

Herpes Simplex Infection

Introduction

Herpes simplex is a common viral infection that presents with localized blistering and it affects mostly all people on one or more occasions during their lives. There are two main types of herpes simplex virus (HSV), although there may be an overlap.

 Type 1, which is mainly affects the face (cold sores or fever blisters)  Type 2, mainly affecting the genitalia (genital herpes)

158 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 1. Showing the structure of HSV-1.

Pathogenesis

The virus enters the host through disrupted skin or mucous membrane and the source of spread of the herpes simplex infection is normally from the bodily fluids such as the saliva, semen or the vaginal fluid or from herpes sore. After the organism enters the host cell, it multiplies without causing any symptoms. But if the immunity of the host is low, the virus destroys the cell. This results in the formation of a vesicle or bullae, due to the seeping in of the transudate from the surrounding blood vessels into the empty space left behind by the destroyed cells. The blister lasts for duration of only about 7 to 14 days and then disappears without scarring. The virus would have reached the site of manifestation from the skin along the nerve fibers. They cluster at the dorsal root ganglias where they remain latent and do not multiply. Whenever the host Viral Infections of the Oral Cavity 159 immunity is lowered they travel again to the surface skin to produce the manifestations of the disease. The virus may also pass through the bodily fluids and infect other persons. The main pathogenesis occurs in the epithelial cells of the oral mucosa. It enters the nucleus of the cell to alter its structure. The nucleus is enlarged, and is called a ‘giant cell,’ and this giant cell is a diagnostic feature for this disease. The involved cell undergoes apoptosis and releases the liquid material into the intraepithelial space and these results in the formation of the vesicles and the bullas which is later released into the surrounding tissues after a period of 7 to 14 days. The reactivation of this virus is normally triggered by stress, UV light, trauma or immunosuppression. The virus now moves along the nerve gradient to the peripheral epithelium to manifest the disease. In what way does HSV interact with and enter Dendritic Cell (DC)? The virus entry into the host cell passes through various barriers. The viral envelope is made up of 11membrane glycoproteins such as gD, gH, gL and gB. The first target is the heparin sulfate, a surface glycosaminoglycans to which the glycoprotein C (gC) and/or gB binds to. For the virus to penetrate the cell, the fusion needs to take place between the cell membrane and the glycoproteins B, D, H, and L. HSV represents a flexible and clever pathogen, which has developed a high number of immune evasion strategies to defend itself effectively against the attacks of the host cells. HSV takes advantage of the crucial position of DC in antigen presentation and T cell stimulation processes. The knowledge about the sophisticated interplay of HSV with DC enables us to introduce effective prevention strategies such as the development of effective HSV vaccines or new methods using the wide variety of HSV functions, as promising targets for immunization strategies. The defense strategy of the host consists of the dendrite mediated T helper cell response and antibody production. But in counteraction to this there is a down regulation of the CD4+ response which interrupts the MHC 2 antigen processing of the host. It thus prevents it from responding to the HLA DR and HLA DM polypeptides. Another defense strategy by the virus includes a biphasic mechanism where apoptosis of the host dendritic cell is inhibited. This is the early phase. In the late phase the pro-apoptotic mechanisms are said to play a role. The early inhibition of apoptosis enables sufficient time for the virus to multiply within the host dendritic cell and the delayed phase helps in efficient incapacitation and elimination of dendritic cell by HSV.

160 P. C. Anila Namboodiripad and E. Anuradha Sunil

Herpes Zoster

Introduction

Shingles, also known as zoster or herpes zoster, is a painful skin rash and is caused by the varicella zoster virus (VZV), which is the same virus that causes chickenpox.

Figure 2. HSV remains latent along a peripheral nerve fibre.

Pathogenesis

The virus spreads by airborne viral particles discarded from the skin of an infected person. The organism spreads without its envelope from cell to cell producing a characteristic vesicle on an erythematous base on the epithelium, replicates in the epithelium and then acquires a period of latency after traveling to the dorsal root ganglia and ascending the sensory nerves. During the period of reactivation following lowered immunity, the virus spreads distally from the ganglion to start new cutaneous and/or mucosal lesions. The Viral Infections of the Oral Cavity 161 virus invades T-cells of the blood and those T-cells carry the virus to the skin. There, the virus can regenerate itself as the glycoproteins of the virus which is required to form its envelope is unaffected. The superficial layers of the epithelium or the skin lack the endosomal pathway that removes the glycoprotein from the envelope. The mannose 6 phosphate in its glycoprotein envelope helps it to get attracted to the sensory nerves which have the suitable receptors where it would travel through, and then remain latent. It causes no damage to the sensory nerves but reactivates whenever the immunity of the individual is lowered. Whenever there is a lowered immunity the virus travels through the nerve pathway to reach the skin and rekindle the infection. As the enveloped virus builds up, it creates the characteristic chickenpox rash. Hence once the virus enters the aerosol form it can become infective again. The multiplication of the genes of the virus during its replication phase occurs in installments.

1) Initially the immediate early genes are produced. These genes encode regulatory proteins. 2) The next are the early genes which encodes enzymes necessary for replicating the viral DNA and 3) The late genes which encode the structural proteins 4) The envelope and other structures are developed as the virus buds out from the nuclear membrane or may arise from the endoplasmic reticulum. They are then transported to the cell membrane via the Golgi complex thus putting an end to the host cells.

How the virion gets reactivated and the genetic mechanism of the same is unknown.

HIV

Introduction

It is the disease or an infection caused by the human immunodeficiency virus. It alters and weakens the immune system and makes the person immune to other infections and diseases. 162 P. C. Anila Namboodiripad and E. Anuradha Sunil

Pathogenesis

Figure 3. Multiplication of the HIV has been studied by numerous scientists. And there is now a consensus in the method of the action of the virus. It is a known fact that viruses require a host to multiply. The host in this case is said to be the lymphocyte especially the T lymphocyte, and also sometimes the macrophage.

Viral Attachment

Once HIV comes into contact with a cell, the T-cell, it must at first connect itself to the cell so that it can fuse and inject its genetic material into it. Attachment is a specific binding that occurs between the surface proteins of the cell and proteins that serve as receptors. Usually, these receptors help the cell communicate with other cells. Two receptors, namely the CD4 and a beta- chemokine receptor (either CCR5 or CXCR4), are used by HIV to attach onto the cell. On the surface of the viral envelope, two sets of proteins, gp120 and gp41 attaches to CD4 and CCR5/CXCR4.

Viral Penetration/Fusion After the completion of the attachment, viral penetration occurs. Penetration allows the nucleocapsid or the genetic core of the virus to be inserted directly into the cell's cytoplasm. gp120 majorly contains three sugar- coated glycoproteins and, once gp120 attaches itself to CD4, these three proteins spread apart. The gp41 which is normally hidden is then exposed and this then binds to the chemokine receptor. Once this occurs, the viral envelope and the cell membrane are brought into close contact and melt into each other. Viral Infections of the Oral Cavity 163

Drugs called fusion inhibitors prevent the binding of gp41 and the chemokine receptor.

Figure 4.

Uncoating

Once HIV has penetrated the cell membrane, it is ready to release its RNA into the cell. The viral RNA is completely protected in the nucleocapsid. This nucleocapsid needs to melt to allow the conversion of the viral RNA into DNA, which is required if the virus has to enter a human T cell’s genetic core.

164 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 5.

Figure 6. Viral Infections of the Oral Cavity 165

Reverse Transcription

The process whereby the single stranded viral RNA is converted to double stranded DNA is called as reverse transcription and the enzyme necessary to perform this action is called as reverse transcriptase. This action is necessary for the virus to seize a T-cell's genetic machinery in order to reproduce itself. The transcription is a normal action in all human cells, but here the DNA converts into RNA. Reverse transcriptase uses nucleotides – which are the building blocks of DNA -- from the cell cytoplasm to make this process possible. Drugs called reverse transcriptase inhibitors block HIV's reverse transcriptase from using these nucleotides.

Integration

Figure 7. 166 P. C. Anila Namboodiripad and E. Anuradha Sunil

HIV must then put in its DNA (the preintegration complex) into the host cell's DNA, if HIV was successful in translating its instructions from RNA to DNA. This process is called integration and in order for integration to occur, the newly translated DNA must be transported across the nuclear membrane into the nucleus. HIV may carry this preintegration complex with the help of a viral protein, to the nucleus. Once the viral RNA has successfully crossed the nuclear membrane and been helped to the nucleus, HIV uses an enzyme integrase to insert HIV's double-stranded DNA into the T cell's existing DNA.

Figure 8.

Drugs that inhibit the HIV preintegration complex from traveling to the nucleus -- integrase inhibitors -- are currently in early clinical trials.

Viral Latency and Protein Synthesis

The host cell is now latently infected with HIV after the successful integration of the viral DNA. This viral DNA is addressed as provirus. This HIV provirus now awaits activation. When the immune cell becomes activated, this latent provirus awakens and instructs the T cell’s cellular machinery to produce the necessary components of HIV. From the viral DNA, Viral Infections of the Oral Cavity 167 two strands of RNA are constructed and transported out of the nucleus. One strand becomes the HIV protease, reverse transcriptase, integrase, and structural proteins and the other strand becomes the genetic material for the new viruses. Compounds that inhibit or alter the viral RNA have been identified as potential antiviral agents.

Cleavage and Viral Assembly

The viral protease enzyme cleaves the various viral subunits after they have been produced and processed, as they are now ready to convert into new viruses. Drugs called protease inhibitors bind to the protease enzyme and prevent it from separating, or cleaving, the subunits. The new HIV virions are created, if the cleavage is successfully completed. To form the membrane of the virus the cell’s membrane begins to undergo deformation. The viral RNA is wound tightly within the nucleocaspid formed from this membrane. Researchers are looking at drugs called zinc finger inhibitors, which interfere with the packaging of the viral RNA into the nucleocapsid.

Figure 9. 168 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 10.

Budding

The conclusive step of the viral life cycle is called budding. Here, the genetic material enclosed in the nucleocapsid fuses with the distorted cell membrane to form the new viral envelope. Once the virus is completely protected from the host environment, it pinches off from the cell and enters into the circulation and it is ready to start the whole multiplication process again. The host T cell is killed but how exactly it dies is not known and scientists have come up with many theories. First, is the process of apoptosis where the host cell tries to protect itself and tries to destroy the virus within it. The next theory is about the damage to the cell membrane which occurs due to the HIV particles budding off from the cell. The third theory is that of the killer cell, that recognizes that the cell is infected and injects it with chemicals and destroys it. Due to all these mechanisms the T cells begin to decline in number and there are not enough T cells to defend the body. At this stage, a person is said to have AIDS, and becomes prey to infections that a healthy immune system could have very well dealt with. There is still much that is not yet known about HIV's life cycle. More research will help the scientists to persuade HIV into giving up more secrets of how it survives and spreads in the body. In turn, this will initiate the design and development of new drugs and vaccines that could be produced to help stop the infection.

Chapter 15

Fungal Infections of Oral Cavity

Candidiasis

Introduction

C. albicans resides normally as a lifelong, harmless commensal. C albicans is one of the many eukaryotic species which is said to comprise about 8.7 million organisms in the world and of which only 7% of it is the fungi. Of the 7% fungi only 600 species are said to be pathogenic to human. Among the pathogenic fungi life threatening infections are said to be caused by Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Candida albicans. Candida organism is responsible for most of the hospital related infections. Superficial candidiasis and the life threatening systemic candidiasis are the two manifestations of the candidial infections. HIV is one of the major risk factors for developing oral candidiasis. Further risk factors for developing oral candidiasis include the wearing of dentures and the extremes of age. The various predisposing factors for candidiasis are less well defined than for oral candidiasis and include the use of antibiotics, oral contraceptives, diabetes mellitus, pregnancy and hormone therapy. Though superficial candidiasis is predominant and despite their frequency and associated morbidity, they are non-lethal. But in stark contrast, in case of the systemic candidiasis there is an associated high crude mortality rate, even with first line antifungal therapy. 170 P. C. Anila Namboodiripad and E. Anuradha Sunil

Other risk factors may play role too, namely venous catheters, trauma, and any GI surgeries that penetrate the mucosal barrier.

Pathogenesis

A large number of factors called as virulence factors, are found to play a role in the pathogenecity of the yeast organisms, namely the morphological transition from the yeast to the hyphael forms, the adhesins and invasins on the cell surface, thigmotropism, secretion of the hydrolytic enzymes and the formation of biofilms. There is a great interplay between the candidial organism and the host defense in the candida infections. Like any other infections that affect the human body, the lymphocytes and the other nonspecific inhibitors play a role. The organism undergoes multiple steps, like an induced endocytosis and an active penetration for it to reach the internal surfaces of the body. In case of the induced endocytosis, the host ligands such as the E cadherin on the epithelial cells and the N-cadherin on the endothelial cells act as the receptors, to the specialized proteins on the cell surface of the organism, and these have been called as the ‘invasins.’ Several factors and activities have been identified which contribute to the pathogenic potential of this fungus. Among them are molecules which mediate adhesion to and invasion into host cells, the secretion of hydrolases, the yeast- to-hypha transition, contact sensing and thigmotropism, biofilm formation, phenotypic switching and a range of fitness attributes. The candida organism is found to have three surface adhesion molecules and an aspartyl proteinase enzyme to enable the initial penetration into the keratinized surfaces. Fungal adhesion and physical forces are believed to be crucial for the penetration. The deeper penetration is facilitated by hypha and this is guided for by a mechanism of contact sensing, called the ‘thigmotropism.’ Once contact sensing occurs the organism switches to its hyphael form and that may be enabled by the aspartic protease (Saps) which is another factor that has been proposed to contribute to active penetration. Whereas on the other hand lipases and phospholipases, have not been shown to contribute majorly to this process. The invasins have been found to be two in number:

i. Als3 (agglutinin like sequence) (an adhesin) and Fungal Infections of Oral Cavity 171

ii. Ssa1. They bind to the host receptors by a clathrin dependent mechanism and results in the endocytosis. Macropinocytosis is found to be another mechanism that helps in the endocytosis.

The Als family gene is made up of 8 members, Als1-7to Als9. It encodes the glycosylphosphatidylinositol (GPI)-linked cell surface glycoproteins. The Als 3 is important for the adhesion. It is upregulated during the candidial infections, whether they are the mucosal or vaginal. Ssa1 is a cell-surface expressed member of the heat shock protein 70 (Hsp70) family. Another important adhesin of C. albicans is the Hwp1, which is a hypha- associated GPI-linked protein. The link between Hwp1 and C albicans is said to be by covalent bonds. Once the fungus enters the various phagocytic cells either the neutrophils or the macrophages, it switches conveniently from one form to the other. Like for e.g., in the PML, the cells produce the reactive oxygen species, antimicrobial peptides etc. The organism is now nutritionally starved. The organism then switches to the hyphae form and resists the unfavorable environment. In case of the macrophages the organisms switches from glycolysis to gluconeogenesis and later it makes use of the lipids and the amino acids as the food source. Schematic diagram illustrating the contribution of the various virulence factors contributes to C. albicans pathogenicity. C. albicans commonly colonizes the epithelial surface (stage 1) and causes superficial infections (stage 2), but under conditions when the host is compromised, the fungus establishes deep-seated infections (stage 3) by penetrating further into the epithelial tissue. Occasionally, C. albicans causes disseminated infections (stage 4), which allow the fungus to colonize and infect other host tissues and can be fatal. This infective process involves numerous virulence attributes including adhesins, hydrolytic enzyme production (Sap proteins, phospholipases, and lipases), hypha formation, and phenotypic switching. Sap2 (and possibly other Sap proteins) is known to degrade many human proteins, including mucin, extracellular matrix proteins, numerous immune system molecules, endothelial cell proteins, and coagulation and clotting factors. Therefore, the action of Sap proteins could be involved in all four stages of infection and probably greatly enhance the pathogenic ability of C. albicans.

172 P. C. Anila Namboodiripad and E. Anuradha Sunil

Figure 1. An overview of selected C. albicans pathogenicity mechanisms. Yeast cells adhere to host cell surfaces by the expression of adhesins. Contact to host cells triggers the yeast-to-hypha transition and directed growth via thigmotropism.

Figure 2. Fungal Infections of Oral Cavity 173

Mucormycosis

Introduction

Mucormycosis (also called zygomycosis) is a rare infection caused by organisms that belong to a group of fungi called Mucoromycotina in the order Mucorales. These fungi are classically found in the soil and in involvement with decaying organic matter, such as compost piles, leaves, or rotten wood.

Pathogenesis

The mucorales are common organisms to which a human is constantly exposed. They predominantly play a role in the patients with diabetes due to the ketoacidosis occurring in them, which has a property of removing iron from sequestering proteins in serum and this serves as a nutritional source for the fungus and in patients having defects in the phagocytic functions. The infection is also commonly seen in persons having increased free iron, which is a nutritional bed for the fungi. The presence of a glucose regulated protein GRP 78 is said to protect the patients from being infected by mucormycosis. But it provides no immunity against the other fungi, candida and aspergillus fumigatus.

Chapter 16

Regressive Alterations of Teeth

Internal Resorption

Introduction

The classification for internal resorption includes internal inflammatory resorption and internal replacement resorption. External resorption is classified into external replacement resorption, external inflammatory resorption, external surface resorption, external cervical resorption and the transient apical breakdown.

Pathogenesis

Internal resorption occurs following caries, pulpotomy, following the heat generated during cutting of dentin, trauma to the teeth or a cracked tooth and also sometimes in relation to the orthodontic treatment. In the root canal replacement type the dentin is severely damaged and the tissue is replaced by a hard tissue that resembles bone or cementum. Herpes zoster has been said to play a role in the degeneration of the odontoblasts during its systemic infectious state. It has been speculated that such a state occurs due to the attraction of the virus to the nerve endings in the pulp and is commonly seen during the involvement of the maxillary branch of the trigeminal nerve. 176 P. C. Anila Namboodiripad and E. Anuradha Sunil

Genes such as the IL-1 gene polymorphism has been suggested to play a role in the development of the internal resorption. The inflammation in the pulp attracts a large number of the inflammatory cells, predominantly the macrophages and together with it the odontoclasts. These clast cells initiate the resorption from the inside. The intensity of the inflammation primarily determines the internal resorption. The resorption is followed by formation of granulation tissue and this, walls of the infection and prevents its further spread into the surrounding tissue. The collateral blood supply from periodontal tissue helps in continuing with the resorptive process. In the case of the internal resorption, the inflammatory type, there is no associated hard tissue formation and the resorption ends with the formation of the granulation tissue. In the replacement type the resorbed area is replaced by tissue resembling bone or cementum. This may result in enlargement of pulp space with partially or fully obliterated pulp chamber.

Figure 1. Depicting the etiopathogenesis of internal resorption.

Regressive Alterations of Teeth 177

Pulp stones

Introduction

Pulp stones (denticles) are nodular, calcified masses of substance appearing in either or both in coronal and radicular portion pulp organ in teeth.

Pathogenesis

The focus or the loci for formation of the pulp stones are the degenerating neural sheath and blood vessels. Hence this may result in reduction in both these structures in the pulp chamber. The pulp also becomes fibrotic, together with the appearance of the pulp stones. True pulp stones show the presence of odontoblasts lining a focal area of pulp tissue. Epithelial cell nests, considered as the epithelial cell rests of Malassez, which had become entrapped during development, become surrounded by concentrically arranged fibers – an organic phase that is impregnated with mineral salts. True pulp stones were formed following an inductive interaction between the epithelium and the pulp tissue. The false pulp stones formed around a nidus. It has been suggested that the fibroblasts themselves have the property of inducing the formation of the apatite crystals. Unidentified mesenchymal stem cells and pericytes are cell lines that differentiate into odontoblast like cells when suitable conditions are prevalent. Experiments have shown that the mineral formed may be bone like or dentin like. Minor circulatory disturbances in the pulp vessels could result in the initiation of the said mineralization in the mitochondrial matrix when viewed through an electron microscope. Different forms of calcium phosphate may play a role in formation of these stones. Small stones may show just a single layer of the collagen fibers that has undergone mineralization. But the larger stones are formed by multiple layers of these collagen fibers wound one around the other and then undergoing mineralization. It has been found that the chemical constituent of the pulp stones are similar to that of the hydroxyapatite crystals, in terms of the presence of zinc, calcium and phosphorus. Immunohistochemical antibodies to the pulp stones such as against Type 1 collagen and non collagenous proteins like osteopontin, osteonectin and 178 P. C. Anila Namboodiripad and E. Anuradha Sunil osteocalcin, showed that type 1 collagen was evenly distributed in the stone, osteonectin and osteocalcin were not detected and osteopontin was found positive in the peripheral area of the pulp stone and played a major role in the calcification and it has come from the less differentiated pulp cells. The osteopontin presence in the pulp stone indicated its similarity to the atherosclerotic plaques in the heart tissue and the urinary stones in the kidneys. In the deciduous dentition the pulp stones were smaller in size and very low in proportion indicating that pulp stones increased in size and number as the tooth aged. Also the diffuse calcifications were more predominant in adult teeth as opposed to the deciduous teeth. A possibility of dental procedures conducted on the pathological tooth prior to the stone formation suggested that this factor may play a role in stone formation. But the correlation was said to be very weak. Another factor playing a role in initiating stone formation was the occurrence of caries in the involved teeth. Though the property of the pulp stone was found similar to the kidney stone but they never occurred simultaneously. But on the other hand patients having atherosclerotic plaques did show an increased occurrence of the pulp stones. Occurrence of pulp stones was also found to be higher in persons with developmental anomalies of teeth such as dilacerations, impactions, taurodontism and enamel pearls. Further studies have stated that the stones are formed due to the nanobacteria which produce the apatite crystals.

About the Authors

Dr PC Anila Namboodiripad, is an alumini of the prestigious KLE Society’s Dental College, Belgaum, Karnataka, India. She is currently working as a Professor in the Department of Oral and Maxillofacial Pathology at the Royal Dental College, Kerala. She has about 23years of teaching experience, guiding both the undergraduate and post graduate dental students. She is a life member of the Indian Association of Oral and Maxillofacial Pathologists. She has attended numerous National Conferences and has also had the privilege to chair scientific sessions. She has a number of Pubmed indexed articles in International journals to her credit. She is currently the Associate Editor of the prestigious Oral and Maxillofacial Journal, a Journal of the Kairali Society of Oral and Maxillofacial Pathologists. E-mail: [email protected]

Dr. E. Anuradha Sunil did her graduation and Post graduation in Oral Pathology from Govt. Dental College, Patna, India. Currently she is the Principal and also heads the Department of Oral Pathology in Royal Dental College, Kerala, India. She is a guide of Post- graduate students and also the editor of the Oral and Maxillofacial Pathology Journal of the local Kairali Society of Oral and Maxillofacial Pathologists. She is a member of the Indian Association of Oral and Maxillofacial Pathologists. She has attended National Level Conferences and also chaired scientific sessions.

Index

alkaline phosphatase, 99, 117 # allele, 46, 52, 70 allelic loss, 46, 70 10q24, 136 allergens, 142 allergy, 27 A alters, 50, 125, 161 Ameloblastoma, 95, 96, 97 acetylcholine, 130 Amelogenesis imperfecta, 18 achondroplasia, 10, 126 amelogenin, 18, 19, 98, 102 acid, 82, 112, 113, 123 amines, 37, 145 Acinic cell carcinoma, 65 amino acid, 112, 113, 116, 119, 123, 171 Actinomycosis, 151 anchoring, 134, 135, 136, 137 acute leukemia, 64 anemia, 108 acute myeloid leukemia, 60 Aneurysmal bone cyst, 90 adalimumab, 139 angiogenesis, 36, 38, 58, 66 adaptive immune responses, 81 anhidrotic ectodermal dysplasia, 5 Adenoid cystic carcinoma, 66 anodontia, 4 adenoma, 59, 60, 67 antibody, 54, 64, 86, 131, 133, 139, 143, adenosine, 116 150, 155, 159 adhesion, 19, 58, 66, 67, 70, 131, 135, 138, antigen, 59, 63, 78, 79, 86, 134, 140, 141, 140, 170, 171 142, 143, 145, 149, 152, 153, 155, 159 adipose tissue, 42 antioxidant, 37, 39, 133 Africa, 51 antiviral agents, 167 aggressive behavior, 70, 71, 96, 97 aorta, 127 agriculture, 145 APC, 40, 63, 145, 150 AIDS, 168 apex, 14, 83 airways, 127 apoptosis, 38, 39, 40, 46, 48, 49, 50, 51, 52, albumin, 21 66, 71, 77, 79, 96, 97, 102, 106, 124, alcoholics, 106 131, 132, 141, 145, 159, 168 182 Index apoptosis pathways, 46 blood, 23, 80, 91, 92, 117, 127, 137, 143, apoptotic mechanisms, 159 150, 158, 161, 176, 177 appetite, 108 blood stream, 150 arginine, 116, 119, 126 blood supply, 23, 176 arteriovenous shunt, 91 blood vessels, 80, 91, 127, 158, 177 ascorbic acid, 38 bloodstream, 151 asthma, 27 BMP, 16, 36 atherosclerotic plaque, 178 bonds, 112 atopy, 27 bone, 4, 10, 12, 14, 21, 22, 43, 44, 45, 49, atrophy, 108 53, 75, 82, 83, 84, 88, 90, 91, 92, 93, 95, attachment, 83, 162 96, 97, 111, 112, 113, 114, 115, 116, autoantibodies, 130, 131, 132, 136, 138, 117, 118, 119, 120, 121, 122, 123, 124, 139, 143, 145 125, 126, 175, 176, 177 autoantigens, 130, 138 bone biology, 124 autoimmune disease, 62, 129, 134, 141, 142 bone cells, 125 autosomal dominant, 8, 11, 18, 113, 124, bone form, 117, 118, 120, 121, 122 126, 127 bone marrow, 12, 44, 49, 53, 96, 123 autosomal recessive, 18, 113 bone resorption, 82, 83, 97, 116, 122, 124 axilla, 133 BRAF gene, 34 brain, 60, 123 breakdown, 91, 92, 116, 175 B BRM gene, 36 budding, 168 bacteria, 121, 149, 151, 152, 154 building blocks, 165 bacterial infection, 151 Bullous Pemphigoid, 135, 136, 139 barriers, 159 Basal cell adenoma, 60 Basal cell carcinoma, 34, 35, 36, 69, 70 C basal lamina, 98 basal layer, 34, 81, 97, 100 caecum, 15, 25 base, 32, 113, 126, 160 calcification, 21, 85, 86, 99, 150, 178 basement membrane, 59, 99, 134, 135, 136, Calcifying epithelial odontogenic tumor, 99 138, 139, 141 calcium, 18, 21, 89, 145, 177 basophils, 138 CAMTA1, 54 BCL-2, 36 canals, 9, 83 bending, 13 cancer, 37, 52, 53, 60 benign, 15, 33, 39, 42, 44, 54, 57, 59, 60, candida, 26, 169, 170, 173 61, 69, 70, 72, 84, 89, 91, 95, 100, 102 capillary, 83 beryllium, 44 capsule, 62 bilateral, 120 carboxyl, 112, 136 biological behavior, 54, 79 carcinogenesis, 32, 33, 40, 58, 106 biopsy, 78 carcinoma, 33, 35, 36, 37, 40, 62, 63, 64, bleeding, 93 65, 66, 67, 69, 78, 84, 97, 100, 101 cardiac muscle, 54 Index 183 caries, 4, 23, 175, 178 circulation, 150, 168 cartilage, 111, 119, 120, 126, 127 classification, 47, 100, 175 cascades, 78 Clear cell odontogenic carcinoma, 101 caspases, 131 cleavage, 19, 167 cathepsin G, 138 clinical diagnosis, 105 causation, 10, 20, 33, 154 clinical presentation, 116 CBS, 125 clinical trials, 166 CCND1, 39 clone, 48, 50, 54 CD8+, 47, 140, 141, 152 closure, 119 CD95, 36, 97, 141 clusters, 5, 34 CDKN2A, 36, 39, 65 coding, 58, 66 CDKN2A and CDKN2B both cyclin- cold sore, 157 dependent kinase inhibitor genes, 36 collagen, 21, 71, 98, 107, 108, 112, 113, cell biology, 58 114, 117, 134, 135, 136, 150, 151, 177 cell cycle, 32, 35, 39, 40, 44, 45, 46, 52, 58, collateral, 76, 176 66, 79, 82, 97, 106, 142 color, 106, 140, 152 cell death, 32, 40, 45, 54, 76, 79, 131 commercial, 145 cell differentiation, 97, 118 communication, 79, 133 cell division, 125 competition, 139 cell line, 45, 66, 177 complement, 5, 136, 137, 138, 139, 154 cell membranes, 82, 99 complexity, 79 cell metabolism, 61 composition, 66, 117 cell surface, 50, 133, 143, 170, 171, 172 compost, 173 Cementoma, 103 compounds, 39 Cerb-B2, 67 condensation, 45 chemical, 33, 44, 121, 123, 145, 168, 177 configuration, 112 chemokine receptor, 50, 162, 163 Congenital epulis, 54 chemoprevention, 38 connective tissue, 38, 46, 70, 79, 81, 84, 90, chemotaxis, 83, 151 91, 92, 102, 104, 108, 113, 114, 127, chemotherapy, 12 136, 151 Cherubism, 120, 121 consanguinity, 18 childhood, 26, 50, 75, 120 consensus, 162 cholesterol, 38, 82 consumption, 27, 145 chondroblastoma, 91 contraceptives, 27, 169 chondrocyte, 120, 126 controversial, 14, 84, 85, 133 chromium, 44 cooperation, 71 chromosome, 5, 22, 40, 44, 46, 52, 53, 57, copper, 107 58, 59, 60, 62, 66, 113, 115, 120, 124, correlation, 5, 142, 178 125, 126, 127, 128 cortex, 111 chronic irritation, 42 cortical bone, 92 Cicatricial Pemphigoid, 134 covalent bond, 171 cigarette smoke, 33, 105 covering, 4, 74, 91 cilia, 72, 73 cracks, 19 184 Index crown, 9, 10, 12, 19, 72, 74, 99 deprivation, 141 crust, 40 deregulation, 52, 79 crystals, 18, 20, 21, 113, 177, 178 derivatives, 145 Cyclin, 40, 41, 46 dermis, 34, 134, 135, 137 cycling, 38 destruction, 18, 53, 89, 95, 96, 121 cyclins, 40 detachment, 70, 131, 133 CYP2D6, 35 detection, 65, 78, 107 cyst, 69, 72, 74, 75, 76, 77, 78, 79, 80, 81, developmental process, 88 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, diabetes, 169, 173 99, 101, 121 diaphragm, 6 cystathionine, 125 diet, 145 cysteine, 116 diffusion, 108 cytochrome, 96, 108 digestion, 101, 108, 149 cytokines, 50, 67, 72, 81, 116, 139, 141, dilacerations, 12, 13, 14, 178 145, 153, 155 disability, 125 cytomegalovirus, 71 disease activity, 143 cytoplasm, 34, 60, 78, 143, 162, 165 disease progression, 108 cytoskeleton, 131 diseases, 80, 161 cytotoxic agents, 138 disorder, 18, 107, 111, 113, 114, 115, 116, 121, 124, 126, 153 displacement, 14 D distribution, 79 DNA, 32, 35, 36, 38, 59, 61, 70, 106, 113, defects, 117, 124, 173 116, 119, 142, 143, 145, 161, 163, 165, deficiency, 10, 11, 21, 22, 23, 27, 38, 54, 166 107, 108 dosage, 70 deformation, 167 Down syndrome, 12, 125, 126 degenerate, 81, 87 drug resistance, 37, 40 degradation, 18, 19, 20, 21, 22, 31, 32, 107, drugs, 38, 44, 126, 139, 142, 145, 167, 168 123 dwarfism, 126 dendrites, 34 dyes, 37 dendritic cell, 99, 153, 155, 159 dysplasia, 10, 22, 91, 103, 115, 116, 118, dens invaginatus, 14, 15, 16 119 dental caries, 80 Dentigerous cyst, 72 dentin, 3, 4, 8, 10, 13, 19, 21, 22, 23, 76, E 103, 104, 175, 177 dentin formation, 22 EBNA, 52 Dentinogenesis Imperfecta, 21, 22, 111, 115 EBV infection, 53 Dentinoma, 104 E-cadherin, 40, 77, 97 dentures, 169 ECM, 138 deposition, 19, 42, 53, 92, 99, 112, 113, ectoderm, 3, 88 118, 127, 128 ectodermal dysplasia, 5, 12 depression, 40 ectomesenchymal cells, 4 Index 185 eczema, 27 external environment, 82 edema, 85 extracellular matrix, 19, 21, 82, 100, 112, egg, 114, 125 138, 171 elastin, 108, 127, 128 extraction, 92 electron microscopy, 128 extravasation, 90, 92 embryogenesis, 62, 125, 126 ENAM, 18 F enamel, 3, 4, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 69, 72, 74, 76, 77, families, 6, 18 79, 80, 85, 86, 95, 96, 98, 99, 103, 178 FAS, 97 encoding, 115 fat, 42, 117 endocrine, 116 fatty acids, 82 endoderm, 25 fertilization, 115 endothelial cells, 41, 100, 151, 170 fetal development, 114 endotoxins, 72 fever blisters, 157 enlargement, 91, 96, 97, 120, 176 fibers, 117, 128, 135, 150, 177 environmental conditions, 11 fibroblast growth factor, 95, 116 environmental factors, 9, 143 fibroblasts, 38, 55, 71, 82, 107, 108, 138, enzyme, 37, 53, 60, 61, 82, 107, 108, 112, 150, 152, 177 116, 125, 138, 161, 165, 166, 167, 170, fibrosarcoma, 102 171 fibrosis, 107, 150, 152, 153 eosinophils, 137, 138, 139 fibrous cap, 99 epidermis, 38, 129, 134, 135, 138 Fibrous dysplasia, 43, 91, 115, 116, 117, epigenetic alterations, 70, 71 118 epithelial cells, 11, 46, 63, 81, 82, 87, 98, fibrous tissue, 99, 115 99, 159, 170 filament, 131 epithelium, 4, 13, 15, 16, 20, 40, 63, 69, 70, fitness, 170 72, 73, 74, 75, 76, 77, 79, 80, 81, 83, 84, flaws, 93 85, 86, 88, 89, 90, 95, 96, 97, 98, 99, flexor, 140 100, 101, 102, 104, 106, 108, 140, 155, fluid, 72, 81, 82, 83, 87, 92, 137, 138, 158 159, 160, 177 follicle, 13, 72, 75, 79, 98 epulis, 54 follicles, 4, 71 ERMS, 45, 46, 47 food, 107, 171 erythrocytes, 91 foramen, 25 estrogen, 55, 118 force, 19 etiology, 11, 12, 14, 15, 22, 26, 33, 76, 90, formation, 3, 4, 5, 6, 7, 11, 12, 14, 15, 16, 92, 102, 106, 119, 154 18, 19, 20, 21, 22, 33, 36, 39, 42, 43, 45, eukaryotic, 169 46, 48, 52, 54, 62, 72, 74, 75, 77, 79, 80, evidence, 50, 65, 80, 92, 100, 106, 154 87, 89, 91, 95, 96, 98, 103, 108, 112, exaggeration, 70 117, 122, 123, 124, 126, 130, 133, 136, exclusion, 105 137, 138, 139, 140, 144, 152, 153, 154, exons, 64, 66 155, 158, 159, 170, 171, 176, 177, 178 exposure, 10, 34, 37, 106, 150 fractures, 53, 111, 115 186 Index fragility, 111, 113 glycosaminoglycans, 159 fragments, 4 goblet cells, 82 fungus, 152, 169, 170, 171, 173 Granular cell tumor, 55 fusion, 9, 15, 25, 26, 41, 46, 54, 58, 59, 62, granules, 34, 101, 152 64, 65, 88, 89, 91, 125, 149, 159, 163 granulomas, 121 Gremilin, 36 growth factor, 43, 45, 58, 72, 79, 81, 97, G 106, 118 growth pressure, 14 ganglion, 160 GST, 35 gelatinase A, 138 GTPases, 97 gene expression, 59 gene regulation, 31 genes, 4, 14, 20, 21, 22, 27, 32, 33, 34, 35, H 36, 37, 40, 46, 50, 54, 58, 59, 60, 66, 67, 70, 74, 96, 114, 116, 118, 119, 120, 125, hard tissues, 76, 84 126, 128, 133, 149, 161 healing, 33, 150 genetic alteration, 45, 70 health problems, 113, 126 genetic disease, 120, 128 hearing loss, 22, 111 genetic factors, 15 heart valves, 127 genetic information, 46 heat shock protein, 131, 155, 171 genital herpes, 157 height, 111 genital warts, 31 helical conformation, 112 genomic instability, 106 Helicobacter pylori, 142, 154 Geographic tongue, 27 Hemangioendothelioma, 54 germ cells, 114 hematoma, 92 germination, 9, 10 hemidesmosome, 134, 135, 136 Giant cell granuloma, 41 hepatitis, 140, 142 gingival, 75, 76, 79, 84 HER-2, 65 gland, 59, 61, 62, 63, 65, 77, 78, 87, 89 heredity, 9, 11, 103 GLI1, 36, 47, 70 herpes, 154, 157, 158, 160 glioblastoma, 54 histamine, 82 glioblastoma multiforme, 54 histidine, 59, 116 glioma, 36, 70 histogenesis, 63, 76, 98, 100 Globulomaxillary cyst, 88 histological features, 77, 79, 117 glomerulonephritis, 143 histology, 72, 84 glossitis, 26, 27 history, 113, 114 gluconeogenesis, 171 HLA, 27, 52, 63, 107, 145, 159 glucose, 173 HLA antigens, 107 glutathione, 38, 107 HMGA2, 58, 59, 67 glycine, 112, 113, 126 homeostasis, 12 glycogen, 74, 77, 101 hormones, 27, 72, 95, 143, 169 glycolysis, 171 host, 48, 81, 158, 159, 161, 162, 166, 168, glycoproteins, 53, 159, 161, 162, 171 170, 171, 172 Index 187

HPV, 31, 32, 35, 37, 40, 106 individuals, 12, 22, 33, 55, 92, 119, 126, human, 5, 31, 32, 35, 38, 70, 71, 97, 98, 145 121, 123, 128, 133, 136, 142, 161, 163, induction, 16 165, 169, 170, 171, 173 industry, 145 human body, 170 infants, 113, 114 human immunodeficiency virus (HIV), 27, infection, 15, 50, 53, 74, 76, 80, 81, 87, 106, 53, 142, 161, 162, 163, 165, 166, 167, 135, 149, 151, 152, 158, 161, 168, 171, 168, 169 173, 176 human papilloma virus, 142 infectious agents, 140 humoral immunity, 138 inflammation, 27, 41, 72, 80, 81, 82, 108, Hunter, 19 121, 137, 139, 142, 176 hyaline, 98 inflammatory cells, 72, 87, 136, 138, 150, hybrid, 64 176 hydroxyapatite, 18, 20, 113, 177 inflammatory disease, 152 hydroxyl, 112 inflammatory mediators, 81, 136, 145 hyoid, 25 inguinal, 133 hyperactivity, 6, 11, 103, 143 inherited disorder, 21, 127 hypercalcemia, 96 inhibition, 35, 36, 50, 71, 78, 102, 138, 159 hyperpituitarism, 9 initiation, 7, 16, 22, 27, 39, 40, 64, 93, 177 hypersensitivity, 142, 150 injury, 90, 132, 139, 143 hypertrophy, 120 insulin, 45 hypothesis, 32, 51, 92, 98, 131 integration, 166 integrin, 38, 40, 134, 135 integrity, 127, 129, 141 I intercellular adhesion molecule, 140 interface, 98 identification, 96, 142 interference, 121, 138 idiopathic, 76 interferon, 81, 150 IL-8, 139, 140 interferon (IFN), 81 142, 154 immune response, 124, 143, 154 interferon γ (IFNγ), 153 immune system, 121, 141, 142, 161, 168, interferon gamma, 150 171 Internal resorption, 175 immunity, 53, 150, 158, 159, 160, 161, 173 internalization, 131 immunodeficiency, 26 intervention, 93 immunofluorescence, 139 intron, 66 immunogenicity, 145 invaginate, 72 immunohistochemistry, 65, 67 ionizing radiation, 10 immunolocalization, 78 iron, 107, 108, 173 immunoreactivity, 86 irradiation, 106 immunosuppression, 159 ischemia, 91 impacted teeth, 11, 99 islands, 74, 84, 87, 89, 98 in vitro, 118, 139 isolation, 18 incidence, 9, 23 incisors, 5, 8, 10, 15 188 Index

lipid peroxidation, 133 J lipids, 171 lipoma, 42 joints, 143 lithium, 27 LMP1, 50 K localization, 99, 141 locus, 52, 65, 119, 177 K5 gene a basal keratinocyte gene and the, lumen, 72, 83, 84 36 lupus erythematosus, 143, 145 karyotype, 62, 125 lymph gland, 152 keratin, 86, 99, 103, 105, 136 lymph node, 62, 150 keratinocytes, 36, 38, 79, 81, 130, 131, 132, lymphocytes, 47, 49, 52, 140, 145, 170 133, 134, 135, 138, 140, 141, 154 lymphoid, 47, 49, 61, 62, 63 Keratoacanthoma, 33 lymphoma, 47, 49, 51, 52 keratosis, 105 lysine, 112 ketoacidosis, 173 lysis, 53 kidneys, 53, 73, 143, 178 kinetics, 79 KIT-SCF, 38 M KLK4 gene, 19 macrodontia, 9 macrophages, 41, 82, 121, 123, 140, 149, L 150, 171, 176 magnesium, 18 labio-dental lamina, 3 major histocompatibility complex (MHC), labio-dental sulcus, 3 47, 140, 141, 150, 154, 159 lamella, 3 malaria, 53 laminin-5, 135 malignant melanoma, 34, 39, 53 Langerhans cells, 99, 140 malignant tumors, 70 larynx, 25 mandible, 10, 12, 92, 95, 102, 115, 120 latency, 160 mantle, 22, 47, 49 Lateral periodontal cyst, 74 manufacturing, 121 leakage, 133 MAPK/ERK, 39 lens, 127 Marfan syndrome, 127, 128 lesions, 42, 44, 70, 78, 80, 83, 85, 91, 105, marrow, 116, 117 117, 118, 121, 133, 137, 160 MAS, 116 leucine, 123 material degradation, 99 leukoplakia, 105, 106 materials, 27, 86, 98 liberation, 138 matrix metalloproteinase, 71, 77, 95, 138, Lichen Planus, 105, 108, 140, 141, 142 139, 141, 151 life cycle, 168 maxilla, 10, 12, 14, 87, 88, 95, 102, 115, ligament, 4, 74, 127 120 ligand, 35, 58, 64, 97, 139, 141 MBP, 138 lipases, 170, 171 MC1R, 34 Index 189

MCP, 138 morbidity, 169 MDM2, 46, 67 morphogenesis, 121 measles, 123 morphology, 4, 8, 12, 14, 18, 34 mechanical properties, 113 mortality, 169 mechanical stress, 115, 117 mortality rate, 169 MECT1-MAML2, 62, 64, 65 mosaic, 111, 114, 122, 126 median, 25, 26, 64 motivation, vii Median rhomboid glossitis, 26 mRNA, 118, 127 meiosis, 125 mucin, 87, 89, 90, 171 MEK, 39 mucormycosis, 173 melanin, 37, 38, 40, 116 Mucormycosis, 173 Melanogenesis, 39 mucosa, 100, 105, 106, 107, 129, 134, 141, melanoma, 37, 38, 39, 40 149, 153, 154, 159 mellitus, 169 mucous membrane, 129, 138, 140, 158 melt, 162, 163 mucus, 63, 64, 78, 89, 90 mesenchymal stem cells, 177 multiple myeloma, 53 mesenchyme, 16, 45, 78, 102 mutation, 4, 11, 18, 19, 22, 23, 32, 38, 40, mesoderm, 88 44, 46, 47, 50, 51, 65, 66, 70, 71, 103, MET, 45, 46 106, 111, 113, 114, 115, 116, 119, 121, metabolism, 22 120, 122, 123, 124, 126 metalloproteinase, 71 mycobacteria, 150 metastasis, 60 myoblasts, 46, 47, 55 methyl group, 70 myogenesis, 46 methylation, 70, 71 microdontia, 8 N microorganism, 150 microscope, 98, 139, 152, 177 natural killer cell, 141 migration, 38, 138 necrosis, 23, 81, 86, 91, 92, 149 miliary tuberculosis, 150 neonates, 55 mineralization, 19, 22, 113, 115, 177 neoplasm, 41, 42, 66, 84, 85, 89 mitochondria, 61, 133 nephritis, 143 mitogen, 34, 127 nerve, 42, 43, 66, 67, 158, 159, 160, 161, MMP, 90, 95, 96, 99, 138, 139, 142 175 MMP-2, 95, 138 nerve fibers, 158 MMP-9, 96, 99, 138, 139, 142 neural crest cells, 3, 4, 23 modifications, 112 neural development, 54 molecular biology, 16 neuroblastoma, 54 molecular weight, 86 neurofibroma, 42, 43 molecules, 16, 21, 58, 79, 83, 108, 112, 113, neuropathy, 54 114, 127, 128, 139, 143, 145, 170, 171 neutrophils, 83, 137, 138, 149, 171 monoclonal antibody, 54, 130 nevus, 34, 39, 105 monolayer, 19 nicotine, 105, 108 monosomy, 62 NK cells, 154 190 Index nodes, 62 P nodules, 26 non-amelogenin, 18 translocations at (11; 19) (q21), 62 nondisjunction, 125 p53, 31, 32, 36, 40, 46, 49, 70, 106, 131, normal development, 26, 117 142 NOTCH-1, 50 palate, 25, 26, 107 nuclear membrane, 161, 166 pancreas, 67 nuclei, 41, 58, 60, 78, 84, 143, 150, 159, parasite, 152 166, 167 parathyroid, 39, 118, 121 nucleotides, 165 parents, 114, 126 nutrient, 151 parotid, 62, 65 nutrition, 81 pathogenesis, 18, 23, 33, 34, 37, 40, 44, 46, 52, 53, 54, 57, 59, 61, 62, 63, 66, 72, 74, O 75, 83, 86, 87, 89, 92, 93, 98, 103, 104, 106, 121, 127, 129, 130, 131, 133, 140, obstruction, 89 141, 142, 143, 152, 159 odontoblastic cell, 22 pathology, 4 odontodysplasia, 23 pathway, 32, 35, 38, 39, 45, 46, 50, 54, 71, Odontoma, 85, 103 103, 106, 127, 128, 131, 133, 139, 141, oligodontia, 4 161 Oncocytoma, 60, 61 PDGF, 78 oncogenes, 46, 72 PDL, 80 oncoproteins, 39 pemphigus, 130, 131, 132, 133, 134, 138 oral cavity, 3, 5, 8, 75, 80, 89, 108, 129, 135 penetrance, 8 Oral submucous fibrosis, 107 peptide, 47, 118, 128, 140, 141, 145, 171 oral vestibule, 3 pericytes, 177 organ, 3, 4, 10, 14, 15, 16, 20, 54, 61, 69, periodontal, 4, 74, 75, 76, 79, 80, 81, 84, 88, 72, 77, 79, 87, 95, 96, 98, 99, 152, 177 176 organelles, 73, 101 periosteum, 91 organic matter, 173 peripheral blood, 154 organism, 149, 150, 151, 152, 158, 160, permeability, 82 169, 170, 171 peroxide, 35 orthodontic treatment, 175 phagocytic cells, 150, 171 osmotic pressure, 83, 92 phagocytosis, 108 ossification, 111, 126 phenotypes, 22, 38, 47, 50, 117, 118, 128 osteoclastogenesis, 124 phosphate, 89, 161, 177 Osteogenesis, 111, 113 phospholipids, 145 osteogenesis imperfecta, 21, 111, 113, 114 phosphorus, 21, 22, 177 osteonectin, 22, 117, 177 phosphorylation, 39, 40, 106, 132 Osteosarcoma, 43 photosensitivity, 145 overproduction, 53, 116, 121 physical features, 125 oxidation, 38 pigmentation, 37, 116 oxidative stress, 37, 40, 133 plaque, 78, 135 Index 191 plasma cells, 53 proteins, 5, 18, 19, 21, 22, 31, 35, 45, 53, plasma membrane, 134, 135 54, 58, 79, 82, 90, 95, 97, 99, 102, 106, plasminogen, 90, 138 116, 121, 129, 130, 132, 136, 138, 143, plasticity, 12, 14 155, 161, 162, 170, 171, 173, 177 playing, 11, 19, 35, 36, 45, 49, 54, 82, 88, proteoglycans, 128, 135 96, 98, 118, 121, 127, 143, 178 proteolytic enzyme, 138 Pleomorphic adenoma, 59 proto-oncogene, 49 pneumonia, 149 Pseudocysts, 90 point mutation, 34 psoriasis, 27, 33, 139 polarization, 84, 97 PTEN, 38, 39 polychlorinated biphenyl, 37 puberty, 43, 121 polymorphisms, 38, 176 pulp, 3, 4, 9, 10, 11, 13, 14, 22, 23, 76, 81, polyostotic fibrous dysplasia, 116 103, 175, 176, 177, 178 polypeptides, 159 pus, 152 population, 8, 47, 57, 117 precipitation, 89 Q precursor cells, 120 pregnancy, 169 quinone, 38 premolars, 6, 15, 16, 75, 97 preparation, 5 prevalence rate, 18 R prevention, 159 primary epithelial band, 3 radiation, 23, 43 primary tumor, 55 radiodensity, 23 probability, 74, 106 radium, 43 progenitor cells, 72 RAS, 38, 39, 154, 155 progesterone, 55 rash, 160, 161 prognosis, 45, 65 Rb, 38, 40, 44, 45, 46 programming, 124 RBC, 82 proliferation, 7, 8, 9, 11, 31, 35, 36, 39, 44, reactions, 124 46, 49, 53, 58, 61, 63, 70, 77, 78, 79, 81, reactive oxygen, 38, 132, 171 82, 84, 89, 96, 97, 100, 102, 104, 106, reactivity, 86 118, 127, 136, 141, 153 receptor, 35, 43, 45, 46, 47, 50, 51, 58, 73, proline, 112, 123 97, 106, 116, 118, 121, 123, 124, 126, promoter, 58, 59, 118 130, 131, 133, 136, 138, 161, 162, 170, prostaglandins, 81 171 protease inhibitors, 167 reciprocal translocation, 54, 62 protection, 35, 37, 49, 96 recruiting, 136 protein components, 134 recurrence, 64, 77, 79 protein family, 136 Recurrent aphthous stomatitis, 153 protein kinase C, 124 regenerate, 161 protein synthesis, 118 regression, 121 proteinase, 138, 170 regulator gene, 35 remodelling, 153 192 Index renal failure, 53 sexual contact, 151 repair, 32, 52, 106 shape, 6, 8, 11, 12, 34, 120, 140 reparation, 129 shear, 72 replication, 32, 161 shock, 155 researchers, 119, 142 shortage, 113, 119 residual matrix, 19 showing, 14, 50, 65, 70, 106, 117, 133 residues, 80, 99, 106, 112 sialophosphoprotein, 21, 22 resistance, 12, 44, 67, 91 signal transduction, 54, 73, 116, 126, 131 resolution, 106 signaling pathway, 123, 124, 127, 131, 139 respiration, 55, 61, 132 signals, 39, 49, 50, 52, 67, 121, 123, 127, respiratory problems, 111 141 response, 40, 72, 86, 117, 123, 142, 152, sinuses, 152 159 skeletal muscle, 45 restorative materials, 142 skin, 27, 34, 35, 37, 66, 106, 111, 116, 124, reticulum, 16, 69, 72, 84, 95, 96, 98, 161 129, 133, 134, 135, 136, 139, 143, 158, retinoblastoma, 32, 39 160, 161 reverse transcriptase, 38, 165, 167 SLE, 143, 145 Rhabdomyosarcoma, 45 smoking, 37, 63 risk, 38, 64, 106, 114, 126, 139, 169, 170 somatic cell, 115 RMS, 46 Somatostatin, 43 RNAs, 66, 133, 143, 163, 165, 166, 167 species, 38, 132, 169, 171 root, 4, 6, 10, 11, 12, 13, 14, 16, 22, 23, 74, speculation, 88, 154 75, 79, 83, 98, 99, 104, 158, 160, 175 spindle, 41, 98 root growth, 12, 14 spongy bone, 92 squamous cell carcinoma, 33, 36, 40, 67, 70, 97, 101 S stabilization, 112, 121 starvation, 42 S100, 55, 101 stem cells, 66 saliva, 89, 105, 108, 142, 158 stimulation, 32, 64, 76, 81, 106, 107, 143, salivary gland, 57, 59, 60, 61, 62, 63, 64, 159 65, 66, 77, 78, 89, 90, 97, 98 stimulus, 96 salts, 44, 89, 177 stomatitis, 153, 154 sarcoidosis, 152, 153 streptococci, 154 scatter, 46 stress, 72, 106, 123, 133, 140, 154, 159 Schwannoma, 42 stretching, 12 sclera, 111 stromal cells, 44, 45, 50, 64, 116 scope, 40 structural changes, 62 secretion, 53, 89, 116, 141, 154, 170 structural protein, 161, 167 senescence, 142 structure, 5, 8, 13, 15, 18, 21, 72, 73, 78, 85, sensation, 108 90, 95, 98, 107, 114, 117, 122, 128, 135, sensing, 170 142, 158, 159 serine, 34, 78, 106, 138 subacute, 123 serum, 82, 133, 139, 142, 173 Index 193 subgroups, 64 TP53, 67 substitution, 58, 113, 126 trachea, 25, 66 sulfate, 135, 159 transcription, 5, 32, 35, 36, 38, 44, 50, 53, sulfur, 152 54, 64, 97, 119, 127, 165 supernumerary teeth, 5, 6, 74, 77 transducer, 127 suppression, 53, 143 transduction, 126 survival, 38, 50, 64, 129, 139, 141 transformation, 31, 52, 59, 61, 65, 85, 86, SV40, 44, 59 102, 106, 115 swelling, 25, 89, 92 transforming growth factor (TGF), 16, 19, symptoms, 114, 119, 150, 158 40, 45, 81, 118, 127, 128, 142, 154 syndrome, 11, 12, 63, 69, 116, 118, 124, transition metal, 39 125, 126, 128, 145 translation, 59 synthesis, 38, 107, 116, 119, 127 translocation, 49, 52, 53, 54, 59, 63, 64, 66, syphilis, 142, 151 126 systemic lupus erythematosus, 144 transmission, 32, 121 trauma, 9, 10, 12, 14, 23, 33, 41, 42, 55, 89, 90, 92, 140, 142, 154, 159, 170, 175 T treatment, 49, 93, 128, 139 tricarboxylic acid cycle, 132 T cell, 47, 52, 53, 136, 139, 140, 141, 143, trigeminal nerve, 175 145, 150, 152, 153, 154, 159, 163, 166, trisomy 21, 125 168 tuberculosis, 149, 150, 151 tannins, 107 tumor cells, 67, 97, 101 taurodontism, 11, 12, 178 tumor development, 97 tendon, 111, 127 tumor growth, 36, 38, 46, 95, 102 tensile strength, 112 tumor necrosis factor, 81, 121, 141 therapy, 140, 169 tumorigenesis, 59, 60, 70 thigmotropism, 170, 172 tumour suppressor genes, 70 third molar, 6 turnover, 14, 40, 72, 141 threonine, 34 type 1 collagen, 107, 178 thymus, 47 thyroid, 25, 26 thyroiditis, 62 U TIMP-2, 95 tissue homeostasis, 127 ubiquitin, 32, 123 TLR, 121 ulcer, 154, 155 TLR2, 121 undifferentiated mesenchymal cells, 125 TLR4, 121 unmasking, 142 TNF-α, 81, 90, 121, 124, 142, 154 urine, 54 tobacco, 27, 37, 105, 106 UV light, 159 tooth, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, UV radiation, 34, 145 15, 16, 18, 19, 22, 23, 69, 72, 74, 75, 76, 79, 81, 83, 84, 88, 92, 95, 96, 97, 98, 99, 102, 103, 111, 175, 177, 178 194 Index

Wnt signaling, 103 V World Health Organization (WHO), 85, 100, 103 vaccine, 150 wound healing, 80 vagina, 152 WWTR1, 54 variations, 32 vasculature, 106 VEGF, 43 X Verrucous carcinoma, 36, 37 vesicle, 158, 160 XRCC 3, a DNA repair gene, 36 vessels, 72, 91, 177 viral infection, 23, 26, 122, 123, 142, 157 Y viruses, 31, 154, 162, 167 vitamins, 107 Y chromosome, 62 vulnerability, 140 yeast, 170, 172 vulva, 66

Z W zinc, 36, 59, 107, 167, 177 warts, 31, 40 zygomycosis, 173 Western blot, 58