I
“VALIDATION OF A NEW CLASSIFICATION SYSTEM
FOR CATEGORIZING ORAL EPITHELIAL DYSPLASIA”
Thesis Submitted to
THE KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH, BELGAUM
(KLE DEEMED UNIVERSITY)
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide Govt. of India Notification No.F.9-19/2000-U.3 (A)]
(Accredited ’A’ Grade by NAAC & Placed in ‘A’ Category of MHRD by GoI)
For the award of the Degree of
Doctor of PhilosophyDoctor of PhilosophyDoctor of PhilosophyDoctor of Philosophy
In In In In the Faculty ofthe Faculty ofthe Faculty ofthe Faculty of Dentistry Dentistry Dentistry Dentistry
((((Oral PathologyOral PathologyOral PathologyOral Pathology & Microbiology& Microbiology& Microbiology& Microbiology))))
by
Dr Alka Kale (Registration No: KLEU/Ph.D/07-08/ IZUN07001)
Under the Guidance of Prof. Dr Prakash V. Patil M.D, Ph.D. Dean, Mayo Institute of Medical Sciences
Gadia, Barabanki, UP.
2014
II
KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH,
(KLE DEEMED UNIVERSITY) BELGAUM
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide Govt. of India Notification No.F.9-19/2000-U.3 (A)]
(Accredited ’A’ Grade by NAAC & Placed in ‘A’ Category of MHRD by GoI)
Declaration Declaration Declaration Declaration bybybyby tttthe Candidatehe Candidatehe Candidatehe Candidate I hereby declare that the thesis entitled “Validation of A New
Classification System for Categorizing Oral Epithelial Dysplasia” is a bonafide
and original research carried out by me under the guidance of
Dr Prakash V. Patil, Ex-Professor & Head of Pathology, JNMC, Belgaum and
presently Professor of Pathology and Dean of Mayo Institute of Medical Sciences,
Barabanki. The thesis or any part thereof has not formed the basis for the award of
any degree/fellowship or similar title of any candidate of any University.
Place : Belgaum
Date : Dr Alka Kale M.D.S.
KLE VK Institute of Dental Sciences
Belgaum.
III
KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH
(KLE DEEMED UNIVERSITY) BELGAUM
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide Govt. of India Notification No.F.9-19/2000-U.3 (A)]
(Accredited ’A’ Grade by NAAC & Placed in ‘A’ Category of MHRD by GoI)
Certificate by the GuideCertificate by the GuideCertificate by the GuideCertificate by the Guide
This is to certify that the thesis titled “Validation of a New
Classification System for Categorizing Oral Epithelial Dysplasia” is a
bonafide research work done by Dr Alka Kale MDS
Place: Belgaum
Date :
Dr Prakash V. Patil MD, Ph.D. Professor of Pathology
Dean, Mayo Institute Medical Sciences
Gadia, Barabanki.
IV
KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH
(KLE DEEMED UNIVERSITY) BELGAUM
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide Govt. of India Notification No.F.9-19/2000-U.3 (A)]
(Accredited ’A’ Grade by NAAC & Placed in ‘A’ Category of MHRD by GoI)
CertificateCertificateCertificateCertificate bybybyby the Deanthe Deanthe Deanthe Dean
This is to certify that the thesis entitled “Validation of a New
Classification System for Categorizing Oral Epithelial Dysplasia” is a
bonafide and genuine research carried out by Dr Alka Kale M.D.S. under the
guidance of Dr Prakash V. Patil, Professor of Pathology & Dean, Mayo Institute
Medical Sciences, Barabanki, UP.
Dr S. G. Kardesai MD Director Academic Affairs KLE University
Belgaum 590010.
Prof. (Dr) V. D. Patil MD, DCH.
Registrar
KLE University
Belgaum 590010.
Date: Date:
V
KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH
(KLE DEEMED UNIVERSITY) BELGAUM
[Declared as Deemed-to-be-University u/s 3 of the UGC Act, 1956 vide Govt. of India Notification No.F.9-19/2000-U.3 (A)]
(Accredited ’A’ Grade by NAAC & Placed in ‘A’ Category of MHRD by GoI)
Copyright DeclarationCopyright DeclarationCopyright DeclarationCopyright Declaration
We hereby declare that KLE Academy of Higher Education and
Research, Belgaum (Karnataka) shall have the rights to preserve, use and
disseminate this thesis entitled “Validation of a New Classification System
for Categorizing Oral Epithelial Dysplasia” in print or electronic format
for academic and or research purpose.
Place: Belgaum
Date :
Guide :
Dr Prakash V. Patil MD, Ph.D. Professor of Pathology
Dean, Mayo Institute Medical Sciences
Gadia, Barabanki.
Research Scholar :
Dr Alka Kale MDS
KLE VK Institute of Dental
Sciences, Belgaum.
© KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH, BELGAUM
VI
ACKNOWLEDGEMENT
I express my sincere thanks to Dr Prabhakar Kore Hon. Chancellor, KLEUniversity, who with his great vision and undaunted effort created this empire ofEducation to enable plenty of scholars pursue their further studies and research work. Ihumbly thank Prof. (Dr) C. K. Kokate, Hon. Vice-Chancellor who has shaped KLEUniversity in a well acclaimed global university. He has always been an inspiration and arole model who has instilled in me the quest to improve by learning and research.
The work presented here is due to the encouragement and motivation given byProf. (Dr) Prakash V. Patil, (MD Pathology & Microbiology, Ph.D) Ex-Prof. & Head,Department of Pathology, J. N. Medical College, Belgaum and presently Professor ofPathology and Dean of Mayo Institute of Medical Sciences, Barabanki, for his constantguidance to me. Without his stewardship the completion of this research work andpresentation in the form of thesis would not have been possible. I am grateful to him.
I am thankful to Prof. Warnakulasuriya S, King’s College, London who had senthis valuable opinion and criticism on our earlier work, and to my teacher Prof. V K.Hazarey, Dean, Government Dental College, Nagpur for reviewing my ideas and givingvaluable inputs, suggestions and guidance in grading dysplasia. I am indeed obliged tohim.
I am indebted to the constant help and everlasting enthusiasm of my co ll ea gu esDr Seema Hallikerimath, Dr Punnya Angadi and Dr Deepa Mane throughout my study andalso to be the co-observers for the analysis of 100 cases. I thank them for constantlysu pp or ti ng me with their timely help & co ns tr uc ti ve su gg es ti on s . A special thanksto Dr Deepa Mane for helping with IHC procedures. I thank Dr Pushpak Shah for thephotomicrographs, Dr Chetan & all my department colleagues and past & presentpostgraduate students for their support and help. My thanks are also due to Dr MinalChoudhary, Prof. Oral Pathology, Datta Me gh e De nt al Co ll eg e , Wardha, Dr AlkaDive, Prof and Head, Or al Pa th ol og y & Dr Devendra from Lata Mangeshkar DentalCollege, Nagpur for the pilot study. I am es pe ci al ly ob li ga te d to the help rendered byDr Kunal Sah, Vinuth DP, Gaurav Sapra, Santosh Hiremath, Dipika Shukla & NiteshNaresh.
I am truly indebted to Prof. S. S. Mallapur, Faculty, Department of Statistics, KLEUniversity, Belgaum for his endurance to patiently evaluate my data and explain to me theimplication of the statistical analysis. I also extend my sincere thanks to Dr MamataHebbal and Dr Vaishali Keluskar for contributions made, Dr Anjana Bagewadi, Vice-Principal and Dr Anil Ankola, for sharing my administrative responsibilities. I amextremely thankful to Mr. Mahesh Desai for his constant help in preparing the entiredocument. Mr S. D. Pandit histopathology technician and all the non-teaching staff fortheir contribution.
Dr Alka Kale
VII
LIST OF ABBREVIATIONS
AgNOR : Argyrophilic Nuclear Organizing Region
APES : Aminopropylethoxysilane Solution
CK /K : Cytokeratin
CK-19 : Cytokeratin – 19
DFC : Dense Fibrillar Components
EGFR : Epidermal Growth Factor Receptor
FC : Fibrillar Centres
GC : Granular Components
H&E : Hematoxylin and Eosin
HOIN : High grade oral intraepithelial neoplasia
HUGO : Human Genome Organization
IHC : Immunohistochemistry
Ki-67 : Kiel, Germany 67th well on the plate
Ks : Simple Kappa
Kw : Weighted Kappa
LOIN : Low grade oral intraepithelial neoplasia
MMP-9 : Matrix metalloproteinase -9
NORs : Nucleolar Organising Regions
OED : Oral Epithelial Dysplasia
OIN : Oral Intraepithelial Neoplasia
OL : Oral Leukoplakia
OSCC : Oral Squamous Cell Carcinoma
PCNA : Proliferating Cell Nuclear Antigen
PMD : Potential Malignant Disorder
PVL : Proliferative Verrucous Leukoplakia
SCC : Squamous Cell Carcinoma
SIN : Squamous Intraepithelial Neoplasia
TAM : Tumor Associated Mucosa
WHO : World Health Organization
VIII
ABSTRACT
Introduction: The mortality of oral cancer has increased seven times in the last 50 years
in-spite of millions worth research going on globally. India has one of the highest rates in
oral cancer in the world partly attributed to high prevalence of tobacco chewing.
Alarmingly 194 million people aged 15 years and older have tobacco habit. In parallel to
the increase in Oral Cancer, border-line malignant lesions which range from epithelial
dysplasia to intra epithelial carcinoma have also increased in numbers. These lesions are
significant part of routine oral pathology service. Not all precancerous lesions and
conditions transform to cancer. Morphological alterations amongst some may have an
increased potential for malignant transformation.
The current histopathological grading of oral dysplasia lesions is notably unreliable
mainly due to lack of objective and validated grading features. Many studies have shown
wide variability in the diagnosis and grading of oral epithelial dysplasia (OED) with
results demonstrating only poor to moderate agreement. The customary grading system of
oral epithelial dysplasia into mild, moderate and severe does not allow accurate prediction
of which cases may eventually transform into malignancy. Many studies have remarked
that precancers with epithelial dysplasia have shown to develop into cancer more readily
than lesions without. Nevertheless all precancer or epithelial dysplasia do not develop into
cancer, whereas, some have even shown to regress with time.
Objectives: The present study was undertaken to assess the different grading systems to
determine the inter observer and intra observer variation and to study the individual
features defining atypia and general disturbance in the epithelium of oral epithelial lesions;
dysplasia. An attempt is also made to correlate the histopathological features with
Immunohistochemistry markers for cytokeratin (CK-19), basement membrane
enhancement protein (Ki-67) and extracellular matrix change (MMP-9) so as to
substantiate the observation of atypia observed in H&E sections. The study will also try to
validate a new binary classification which will objectively define each feature and classify
oral epithelial dysplasia as ‘risky’ and ‘non-risky’ using the H&E stains as gold standard.
Methodology: Pilot study to evaluate inter and intra observer variability was done on 25
precancerous cases. 100 cases of potentially malignant disorders were subjected to inter
and intra observer variability using two classifications: Smith and Pindborg (1969), Binary
Classification of Kujan (2006) and WHO (2005). Evaluation was doneto validatea new binary
IX
classification based on scores for individual features that was also substantiated by IHC.
The data was statistically evaluated by Kappa analysis and Fisher’s Exact Test.
Results: Almost perfect agreement was obtained by the new binary classification (Non-
risky & Risky) (k = 0.790) in comparison with the other classification where k value =
0.27 and kw =0.483. Amongst the 100 cases evaluated we found 8 cases to be of high risk
and were finally graded as ‘risky’. Immunoexpression of three markers was found to be
maximum in these 8 cases. The two features of epithelial dysplasia identified and found to
be statistically significantly present in the risky cases were abnormal and superficial
mitosis and marked cellular and nuclear pleomorphism and anisonucleosis.
Conclusions: Binary classification with scores for individual features of Epithelial
dysplasia was found to be the better method of grading which was statistically significant.
Abnormal and superficial mitosis and marked cellular and nuclear pleomorphism and
anisonucleosis were identified as features to be present in a given H&E stained
histological section of oral dysplasia to be graded in the ‘risky’ category suggesting their
chances of malignant transformation.
Keywords: Binary Grading, Potentially Malignant Disorders, Oral Epithelial dysplasia,
Tobacco, Leukoplakia, Erythroplakia.
X
CONTENTS
Sl. No. Sections Page No.
1. INTRODUCTION 1
2. AIM AND OBJECTIVES 6
3. REVIEW OF LITERATURE 7
i. General
a. The Epidemiology of Head and Neck Precancers
b. Epithelial Dysplasia
c. Clinical features of Potentially Malignant Disorders
d. Histopathology of Potentially Malignant Disorders
e. Grading Systems for Oral Epithelial Dysplasia
f. Subjectivity / Variability in Oral Epithelial Dysplasia
g. Various Methods of Evaluation of Oral Epithelial Dysplasia
h. Molecular Aspects of Potentially Malignant Disorders
i. Correlation of Dysplasia with Eventual MalignantTransformation
ii. Ki-67 Protein
iii. Matrixmetalloproteinase
iv. Cytokeratin
7
7
9
14
18
22
39
45
56
60
64
66
74
4. METHODOLOGY 79
5. RESULTS 89
6. DISCUSSION 105
7. CONCLUSIONS 127
8. BIBLOGRAPHY 130
9. ANNEXURES – 1 to 9
XI
LIST OF TABLES
Table No. Particulars Pages
1 Statistical analysis of 25 cases for Pilot Study 89
2 Statistical analysis of 100 study cases for Inter observervariability
90
3 Statistical analysis of 100 study cases for Intra observervariability
90
4 Scoring for atypical architectural and cytological features forthe proposed classification
91
5 Distribution of cases into ‘non-risky’ (total score ranged from1-15 ) and ‘risky’ (total score ranged from 16 to 84) groups
92
6a Calculation for risk categories using median and inter quartilefor 100 cases
92
6b Classification of risk categories for evaluation of 59 riskycases
92
7 Risk categorization of Risky and Non-risky cases 93
8 Ten Important Features with higher scores considered in‘Risky group’ are segregated with a total score of 61
94
9a Calculation for risk categories using median and inter quartilefor study group for 10 important features
95
9b Classification of risk categories for evaluation of cases for 10features
95
10 Risk categorization of risky and non risky groups for the 10features
96
11a CK-19 Area of expression in Epithelium of the study groups(Fisher Exact Test)
97
11b CK-19 Expression in Epithelium of the study groups (FisherExact Test)
97
12a Ki-67 Expression of area in the study groups (Fisher ExactTest)
98
12b Ki-67 Expression in study groups (Fisher Exact Test) 98
13a MMP-9 Expression in Epithelium of the study groups (FisherExact Test)
99
13b MMP-9 Expression in stroma of the study groups (FisherExact Test)
99
XII
LIST OF FIGURES
Figure No. Particulars Pages
1 ‘Non-risky’ dysplasia (a - 10x, b - 40x, H&E stain) 100
2 ‘Risky’ dysplasia (a - 10x, b & c - 40x, H&E stain) 100
3 ‘Non-risky’ dysplasia with minimal MMP-9 Expression (a- 10x,b- 40x, IHC stain)
101
4 ‘Risky’ dysplasia with enhanced MMP-9 Expression (a & b - 10x,c- 40x, IHC stain)
101
5 ‘Non-risky’ dysplasia. Restricted Ki-67 expression in basal celllayer. (a- 10x, b- 40x, IHC stain)
102
6 ‘Risky’ dysplasia. Enhanced Ki-67 expression in suprabasal layer.(a- 10x, b & c- 40x, IHC stain)
102
7 ‘Non-risky’ dysplasia. Restricted CK-19 expression in basal celllayer. (a -10x, b- 40x IHC stain)
103
8 ‘Risky’ dysplasia. Enhanced CK-19 expression in basal andsuprabasal layers. (a & b -10x, c- 40x, IHC stain)
103
9 Individual Features of Epithelial Atypia (H&E stain) 104
Introduction
1
INTRODUCTION
A disease will affect the seething mass of activity within a living cell and reflect
dysfunction. Corten et al1 stated that the cells tend to maintain a relatively normal range of
structure and function designated as normal, until the cellular stresses become serious. The
specialty of pathology begins with the examination of disease at cellular level which
modifies its structure and function in response to changing demands and advances.
Cancer evokes the strongest emotions in mankind. It connotes pain, protracted
suffering, hideous growth and death. An array of malignant neoplasms can arise from oral
tissues but squamous cell carcinomas of the oral mucosa constitute 90% of oral
malignancies worldwide. Being one of the most mutilating diseases, it is the sixth most
common malignant neoplasms in the world. However, in Indian sub-continent, it is the most
common cancer accounting for 1/3rd of all malignancies.
The significance of oral cancer involves its mortality and the disfigurement and
dysfunction associated with treatment. The rate of curability of cancer depends on the stage
and site. Small cancers for example on anterior tongue, floor of the mouth and buccal
mucosa have a local control rate upto 90%. Moderately advanced lesions without evidence
of spread to cervical lymph nodes have control rates upto 80-90%, whereas the intra oral
carcinomas with cervical lymph node metastasis, the survival rate is less than 50%.2
The mortality of oral cancer has increased seven times in the last 50 years in-spite of
millions worth research going on globally. India has one of the highest rates in oral cancer in
the world partly attributed to high prevalence of tobacco chewing. Forms of tobacco
chewing include pan (piper betel leaf with sliced areca nut, lime, catechu and other spices
chewed with or without tobacco), pan masala or gutkha (a chewable tobacco containing
areca nut) and mishri ( a powdered tobacco rubbed on the gums as toothpaste). According to
WHO 65% of men and 33% woman consume tobacco. Out of this 29.3% men and 2.3%
women smoke tobacco; 28.1% men and 12.0% women chew tobacco, whereas 46.5% men
and 13.8% women either smoked or chewed. Alarmingly 194 million people aged 15 years
and older have tobacco habit.79% of people who consumed some form of tobacco lived in
rural areas.
Introduction
2
In India, the prevalence of smoking and chewing varies. Chewing tobacco / pan
masala is common in Central, Eastern, Western (except Goa) & Northeastern states (except
Meghalaya) compared to Northern and Southern states, whereas, smoking is high in
Northern states except Punjab.3,4 Byakodi et al reported 1.12% (112 cases) prevalence rate
of oral cancer from 35,122 cases screened.5
R. Shankarnarayan in his research communication reported that out of 1151oral
biopsies received from 2003 – 2007, 344 were potentially malignant lesions and 422 were
oral cancers. He reports that though there is no clear cut trend in prevalence, a gradual
increase in number of cases was noted.6
In parallel to the increase in Oral Cancer, border-line malignant lesions which range
from epithelial dysplasia to intra epithelial carcinoma have also increased in numbers. These
lesions are significant part of routine oral pathology service. This is due to increased
awareness, improved consciousness towards health, availability of health care centers at
primary, secondary and tertiary levels, which results in early detection of oral borderline cases.
Over 90% of all oral cancers are squamous cell carcinomas (SCC) arising from the
lining mucosa. SCC may arise de novo in clinically normal mucosa, though a majority is
preceded by visible changes of the mucosa. There are several histologically distinct lesions
of oral mucosa which have malignant potential. Among these are leukoplakia, erythroplakia,
proliferative verrucous leukoplakia, oral submucous fibrosis etc. These vary in their
malignant potential and genetic background. The lesions may be found in association with
and / or preceding SCC.
Bouquot JE et al (2006) have rated the malignant transformation potential of
precancers, with proliferation verrucous leukoplakia and erythroplakia topping the list with
maximum potential whereas smokeless tobacco keratosis and lichen planus to have the least
malignant potential. 7
The term ‘Precancer’ has assumed newer terminologies as “Potentially malignant
lesions, oral precursor lesions, premalignant conditions etc”. These lesions present either as
white macule with excess surface keratin or as red, non-blanching macules with minimal
surface keratin. Histologically these precancerous lesions present with epithelial dysplasia,
which is described by Stedman's Medical Dictionary as a disorder of differentiation of
Introduction
3
epithelial cells which may regress, remain stable, or progress to invasive carcinoma. The
features of epithelial dysplasia include: Increased nuclear to cytoplasmic ratio, acanthosis,
atypical mitosis, pleomorphism, nuclear hyperchromatism, enlarged darkly stained nucleoli,
loss of cellular adhesion and abnormal keratinization. These features vary with individual
tissue specimens and assessment varies with pathologists viewing the same tissue specimen.
Prognostic significance of a lesion is difficult to determine because of the above factors.7,8
Leukoplakia is the most commonly encountered potentially malignant disorders. 2%
of leukoplakia may undergo malignant transformation per year. In lesions with epithelial
dysplasia this rate could vary between 1.1% -17.5%.9 Longitudinal studies of rural
populations in India revealed 80.6% of oral cancers being preceded by precancerous lesions
or conditions. 10,11
Though erythroplakia is uncommon, it often presents with dysplasia or intra
epithelial carcinoma when compared to leukoplakia which generally show no dysplasia or
may just present with mild to moderate dysplasia. Similarly the malignant transformation
potential for smokeless tobacco keratosis has been questionable. It is known that the tobacco
habit itself is said to carry a risk four times greater than normal mucosa. About 16% of
biopsied lesion showed mild dysplasia, and a few chewers showed severe dysplasia. Some
authorities have used clinical grading scale for smokeless tobacco keratosis based on
intensity of whiteness, erythema and fissuring. Unfortunately none of these grading systems
have successfully correlated severe or high grade lesions with an increased risk of
malignancy.
Not all precancerous lesions and conditions transform to cancer. Morphological
alterations amongst some may have an increased potential for malignant transformation.
These are also indicators of risk of likely future malignancies elsewhere in clinically normal
appearing oral mucosa and not only site specific predictors.12
There is always a challenge for oral pathologists to assess the degree of dysplasia in
potentially premalignant oral lesions with accuracy, so that the burden of malignant
transformation can be predicted and treatment modalities can ensure reduced morbidity.4
The current histopathological grading of oral dysplasia lesions is notably unreliable
mainly due to lack of objective and validated grading features. Many studies have shown
Introduction
4
wide variability in the diagnosis and grading of oral epithelial dysplasia (OED) with results
demonstrating only poor to moderate agreement.13
The customary grading system of oral epithelial dysplasia into mild, moderate and
severe does not allow accurate prediction of which cases may eventually transform into
malignancy. Many studies have remarked that precancers with epithelial dysplasia have
shown to develop into cancer more readily than lesions without.14 Nevertheless all precancer
or epithelial dysplasia do not develop into cancer, whereas, some have even shown to
regress with time.15
The limitations inherent in the histologic grading, suggest the necessity to improve
histologic assessment and the need to identify more reliable makers. In the last decade
approach to understanding of molecular carcinogensis has revealed newer directions. Many
molecular markers functioning as elements of cell cycle control have been recognized.
These have been tested individually and in combinations to develop diagnostic markers for
identification of dysplasia and their ability to predict malignant potential.16
Most of the authors have concluded that though the progress in molecular oncology
has significantly advanced the knowledge on tumorigenesis, yet the practical applications of
these genetic makers remain unresolved in detecting oral dysplasia. None of the markers, the
authors studied like EGF, TFG-α, EGFR, Ras, Myc, Cyclins, p53, p21, pRb including DNA
ploidy, loss of heterozygosity, apoptosis, Cox1& 2, cell adhesion molecules, angiongensis,
proliferation and differentiation markers and viruses, either singly or in combination appear
to be ready for use in routine clinical diagnostic practices. Not many studies have focused
correlating the findings at molecular level with the clinical attributes of terminal outcome.
From the observation of Gayani P et al it can be concluded that the molecular studies
are not only nonspecific but also require expertise, armamentarium and facilities which may
not be available for day to day diagnosis at clinics and primary or secondary health
centres.16
A variety of precancers are successfully evaluated and managed despite ongoing
controversies. Bouquot JE states that one of the major advances in this field has been the
simple recognition that a premalignancy is not guaranteed to eventually transform into
cancer. Thus, he defines premalignant lesion as an identifiable, benign tissue or cellular
Introduction
5
alteration associated with greater than normal risk of malignant transformation or
“degeneration”. Much of contemporary oral oncology research is focused on the
identification of clinical, histopathologic and molecular biological parameters of oral
precancers and early cancers which might aid in the recognition of those lesions with the
higher risk of transformation.17
The present study was undertaken to assess the different grading systems to
determine the inter observer and intra observer variation and to study the individual features
defining atypia and general disturbance in the epithelium of oral epithelial lesions; dysplasia.
An attempt is also made to correlate the histopathological features with
Immunohistochemistry markers for cytokeratin (CK-19) basement membrane enhancement
protein (Ki-67) and extracellular matrix change (MMP-9) so as to substantiate the
observation of atypia observed in H&E stained tissue sections. The study will also try to
validate a new binary classification which will objectively define each feature and classify
OED as ‘risky’ and ‘non-risky’ using the Hematoxylin and Eosin stains as gold standard.
Aim & Objectives
6
AIM AND OBJECTIVES
Primary Objective (Aim) :
To propose a new classification for dysplasia to categories dysplasia into ‘non risky’
and ‘risky’ groups
Secondary Objective :
1. To histopathologically evaluate 100 cases of potentially malignant disorders (PMD)
for degree of dysplasia based on the classification system given by (a) Smith and
Pindborg’s classification (1969) as mild, moderate and severe, using photographic
standardization and scores (b) WHO classification (2005) as mild, moderate &
severe dysplasia and (c) Binary classification as low risk and high risk cases given
by Kujan Omar (2006).
2. To assess the inter and intra observer variability and agreement for the above
mentioned 3 classification systems.
3. To histopathologically evaluate the degree of dysplasia for the same 100 cases of
potential malignant disorders (PMD) using the proposed classification.
4. To assess the inter and intra observer variability and agreement for the proposed
classification.
5. To identify individual histopathological features within the proposed classification
that would aid in categorization of risk behavior.
6. To correlate the proposed classification for degree of dysplasia with
immunohistochemical markers for : CK-19 for epithelial differentiation, Ki-67 as
proliferative marker and MMP-9 as marker for epithelial - connective tissue
interaction.
7. To correlate the observations of IHC with histopathological features analyzed with
the new proposed classification.
Review of Literature
7
REVIEW OF LITERATURE
Oral Cancer is one of the top three cancers in India. Out of which 30% is oral
squamous cell carcinoma (OSCC). The variation in incidence and pattern of oral cancer is
due to regional differences in the prevalence of risk factors. Literature review demonstrates
that nationwide incidence varied considerably based on study designs, sampling
methodology as well as by age, gender and location. Variations in age specific incidence
rates also increased with age, which drops at the age of 70 years and is consistent with
multiple studies. The diagnosis of oral cancer also depends on the clinical examination
conducted which can lead to variations.
a. THE EPIDEMIOLOGY OF HEAD AND NECK PRECANCERS
Prevalence and Incidence : Investigations in Western population have reported that
leukoplakia affects approximately 30-40 of every 1000 adults (3-4%) whereas prevalence
rates in India is about 170/1000 in some communities, though this may varies with other
population. Oral lichen planus, affects one of every 1000 adults in Western populations; in
certain subpopulations it is seen as 1/32 persons. No data are available for erythroplakia,
but incidence of oral/pharyngeal carcinoma in situ, which represents erythroplakias, is one
per million persons each year.17
Patient Age and Gender : Precancers are usually found more frequently in men than in
women. More than 60% and 80% of leukoplakia and erythroplakia patients are male and
the proportion of males increases with increasing age at diagnosis. More than 95% of
smokeless tobacco keratosis patients are men, except in populations wherein the chewing of
tobacco is popular among women. Only 30-50% of oral lichen planus patients are males.
Two-thirds of oral submucous fibrosis patients in India are females, but this proportion
varies as the men in some cultures use areca nut more than do the women.
Smokeless tobacco keratosis prevalence varies with the popularity of the smokeless
tobacco habit in a given population. In Western investigations it has been diagnosed in as
many as 9% of adult males. As a general rule, up to half of daily users will show at least a
mild degree of keratosis. In some Indian communities the prevalence is higher but is
confused with leukoplakia in reported epidemiologic investigations. Oral submucous
fibrosis is rare in Western cultures but occurs in as many as four per 1000 persons in rural
communities of the Indian subcontinent and 11/1000 among chewers of betel quid.17
Review of Literature
8
Smokeless tobacco keratosis is typically visible within a few years of the onset of the
smokeless tobacco habit and remains indefinitely unless the habit is discontinued. The
average age at diagnosis of smokeless tobacco keratosis is usually thought to be 45-60 years.
These lesions are initiated much earlier, however, and population-based prevalence studies
in young adults and teenagers have found keratotic changes in almost a third of young
smokeless tobacco users. Western investigation of smokeless tobacco keratosis to provide
age-specific prevalence rates identified a biphasic pattern with increased prevalence in
young adult males and in males older than 65 years of age.17
Coelho KR18 reported that 57% men and 11% women between 15-59 years use some
form of tobacco. Use of smokeless tobacco is more prevalent as it is accepted socially and
culturally in India. Gutkha, Zardha, Kharra, Mawa and Khani are all dry mixtures of lime,
areca nut flakes and condiments with or without powdered tobacco, custom mixed and are
commercially available and sold by vendors. Pan or guktha is kept in the cheek and chewed
or sucked for 10-15 min, sometimes kept overnight.
High incidence of oral cancer is attributed to the use of tobacco as quoted in several
studies : - Regression of leukoplakia with reduced tobacco consumption (Mehta et al);
Cessation of tobacco habits dropped the incidence of leukoplakia (Gupta et al) ; Tobacco
chewing (betel quid or Khaini) and smoking (Bidi & Cigarette) are common cause of oral
cancer – 48% cases stage III and IV (Khandekar et al) ; Significant association between oral
cancer incidence and daily frequency of tobacco chewing (p<0.001), which increased 9.2
fold among women chewing tobacco 10 times a day or more, with the highest risk during the
first twenty years of chewing (Jayalakmi et al). As quoted in the study by Coelho et al.18
Coelho K R.18 noted the oral cancer incidence from 1990 to 2005 and stated, an
increase in incidence of oral cancer among young adults especially tongue cancer. Age
related variability in the incidence of oral cancer in different region of India has increased
over the years.
500 students were selected and subjected to questionnaire and urine rapid nicotine
test in a study conducted by Saddichha S. et al19 55.6% confirmed tobacco use. Out of which
78% reported to be cigarette smoking, 20% used khaini and 2% of the college students
consumed gutkha. They further reported that 60% - 80% patients present with advanced
disease as compared to 40% in developed countries. In India 2.5 million people are living
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9
with disease; there are 0.8 million new cases and 0.55 million deaths due to oral cancer
every year.
Makwana NR et al20 reported from the questionnaire based study on consumption of
tobacco containing products that 28.41% of school children were of 10-13 years of age,
33.56% were 14-16 years and 36.26% were 17-19 years old. They stated that the reason for
addiction was due to peer pressure i.e. friends in 4.4% of children, 1.4% stated that movies
influence them and 94.2% did not respond.
Hazarey VK et al21 in a hospital-based cross-sectional study on 1000 OSF cases
reported the male-to-female ratio as 4.9:1. Occurrence of OSF was seen <30 years among
men when compared with women (OR = 4.62, 3.22–6.63, P = 0.0001). Reduced mouth
opening, altered salivation and taste sensation was noted. Exclusive areca nut chewing habit
was significantly more prevalent in women (OR = 44.5, 25.4–79.8, P = 0.0001). Whereas
significant increase for gutkha (Areca quid with tobacco) (OR = 2.33, 1.56–
3.54, P = 0.0001) and kharra / Mawa (crude combination of areca nut and tobacco)
(OR = 6.8, 4.36–11.06, P = 0.0001) chewing was found in men when compared with
women.
b. EPITHELIAL DYSPLASIA
Baillie and Royal Society Committee in Edinburgh first proposed the concept of
premalignancy in 1806. Sir James Paget in 1851 was first to apply this concept to pipe
smokers with “Smokers Patch”. Alterations in oral mucosa have been closely involved in the
advancement of our understanding of precancers or potentially malignant lesions. An
association between benign oral mucosal lesion and its subsequent malignant transformation
was clearly implicated by Sir James Paget in 1870. Oral “ichthyasis” (white keratotic
plaque) was a significant precursor to lingual carcinoma. In 1877 Schwimmer coined the
term “Leukoplakia” for white tongue changes seen prior to lingual cancer development in
tertiary syphilis. The term : “erythroplakia” was first used by Queyrat in 1911 to describe a
red macule of the genital organ in a syphilitic male patient. In 1924 Darier, a French
dermatologist had applied the term “erythroplakia of Queyrat” to a lesion of the oral
mucosa.17
The term dysplasia was introduced by Reagon in 1958 where he described the
features of dysplasia in relation to exfoliated cells from cervical lesion.22 In Medical
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10
terminology the word dysplasia means an abnormality in development while
histomorphologically dysplasia expresses the cellular and structural changes in the
epithelium.
Pindborg (1977) defined epithelial dysplasia as lesions in which part of the thickness
of the epithelium is replaced by cells showing varying degree of cellular atypia. The term
intra epithelial neoplasia and atypical epithelial hyperplasia were being synonymously used.
When the appearance of cancer is preceded by some other lesion in the epithelium
that lesion may show cellular changes from that point towards the possible subsequent
development of malignancy. The individual cellular changes are referred as ‘atypia’ and the
general disturbance in the epithelium is designated as ‘dysplasia’. Some features of
epithelial dysplasia are also represented in normal growth, proliferation, maturation and
organization of cells.23
Cellular responses which involve stimulation of cell growth such as hyperplasia or
change in cell type as in metaplasia do not by themselves predispose to malignancy.
Abnormal cell proliferation shows itself as visible changes as dysplasia in the microscope
which literally means “disordered growth”.24
Dysplastic cells are part of the multistep pathway to overt malignancy. Until these
cells get the ability to invade the connective tissue and metastasize, they are not defined as
‘malignant’. If these dysplastic cells are studied by tissue culture, they will show some
features of a “transformed cell”. The progressive changes of mild to severe dysplasia and
then the development of invasive activity are best seen in epithelial tissue. The dysplastic
cells invade the lower, middle and then the full thickness of the epithelium and eventually
invade the basement membrane.24
Oral epithelial dysplasia : a histopathologic marker of premalignancy, is a diagnostic
term used to describe the histopathological changes seen in a progressive chronic and
premalignant disorders of the oral mucosa. Clinically it may present as leukoplakia,
erythroplakia or leukoerythroplakia. It may also be seen in verrucous or papillary
leukoplakias or in the margins of chronic mucosal ulcers. It is also consistently seen in the
apparently normal mucosa adjacent to the squamous cell carcinoma.25
To develop a better understanding of the biology of oral precancer and to achieve
consistency in diagnosis an international working group comprising specialists in the fields
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of epidemiology, oral medicine and pathology and molecular biology met in London in May
2005 for a workshop co-ordinated by WHO collaborating center for oral cancer and
precancers. They suggested the following recommendations : -
Concept of Precancer : -
1. In longitudinal studies, area of tissue with certain alterations in clinical appearances
identified at the first assessment as “precancers” have undergone malignant change
during follow-up.
2. Some of these alterations, particularly red and white patches, are seen to co-exist at
the margins of overt oral squamous cell carcinomas.
3. A proportion of these may share morphological and cytological changes observed in
epithelial malignancies, but with frank invasion.
4. Some of the chromosomal, genomic and molecular alterations found in clearly
invasive oral cancers are detected in these presumptive “precancer” or “pre-
malignant” phases.
The terms “pre-cancer’, ‘precursor lesions’, ‘premalignant’, ‘intra epithelia
neoplasia’ and ‘potentially malignant’ have been internationally used broadly to describe
clinical presentations that may have potential to become malignant. The 2005 WHO
monograph on Head and Neck tumors, uses the term ‘Epithelial precursor lesions’.
A much earlier working group of WHO in 1978 classified the lesion into two broad
groups as (i) Lesions and (ii) Conditions with following definitions: -
A precancerous lesion is “a morphological altered tissue in which oral cancer is
more likely to occur than its apparently normal counterpart”.
A precancerous condition is “a generalized state associated with a significantly
increased risk of cancer”.
It is now known that even the clinically ‘normal’ appearing mucosa in a patient
harboring a precancerous lesion may have dysplasia on the contralateral anatomic site or
molecular aberrations in other oral mucosal sites suggestive of a pathway to malignant
transformation.
The consensus of the 2007 working group recommended the term “potentially
malignant disorders”. This describes all those lesions and conditions which may transform to
cancer. Potentially malignant disorders are indicators of risk of likely future malignancies
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elsewhere in clinically normal appearing oral mucosa and not only the site specific
predictors. This working group did not favour subdividing precancers to lesions and
conditions and had the consensus view to refer to all clinical presentations that carry the risk
of cancer under the term ‘potentially malignant disorders’ to reflect the wide spread
anatomical distribution.12
Three major problems are attached to the importance of epithelial dysplasia in
predicting malignant development. (i) The diagnosis is essentially subjective and (ii) not all
lesions exhibiting dysplasia will eventually become malignant and some may regress (iii)
carcinoma can develop from lesions in which epithelial dysplasia was not diagnosed in
previous biopsy. As epithelial dysplasia does not seem to be invariably associated with or
may not be a necessary pre-requisite for malignant development there is a need to develop
other methods for predicting the malignant potential of precancerous lesions.14
The features that are predominantly caused by alterations of cell kinetics are found in
the proliferative compartments of the epithelium. This relates to an increase growth or cell
division. Those features that relate to disturbed maturation manifest in the form of irregular
stratification and increased keratinization of individual cells beneath the keratin layer.26
Most pathologists grade oral epithelial dysplasia according to a combination of
cellular and tissue changes. Low grade lesions are considered to be less ‘severe’ than high
grade lesions but this assessment is subjective. It is not unusual for different pathologist to
place emphasis on different aspects of dysplastic alteration, thereby arriving at varying
diagnostic conclusions. Regardless of individual emphasis, certain oral precancer grading
criteria are in general use :
The cellular atypia or dysplasia is similar to that seen in squamous cell carcinoma.
There is no evidence of invasion into underlying stroma
The epithelium with the greatest proportion of atypical cells has the greatest risk of
being or becoming a carcinoma.
The epithelium with the most extreme atypia of cells has the greatest risk of being or
becoming a carcinoma.
The final grading or diagnosis should be based on the most severely involved area of
change, even if that area includes no more than a few rete processes.17
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Epidemiological data suggests that a proportion of many oral mucosal lesions
including idiopathic white patch (leukoplakia), speckled leukoplakia, erythroplakia, chronic
hyperplastic candidosis, oral submucous fibrosis, discoid lupus erythematosus and
dyskeratosis congenita undergo malignant transformation. At present, it is not possible to
predict which dysplastic oral lesion will proceed to malignancy and which will regress. A
molecular study found identical genetic changes in the dysplastic oral lesions and oral
cancers in the same patients, suggesting that the progeny of cells in a dysplastic oral lesion
may, with further modification, ultimately form a malignant lesion. This is the first evidence
that a clone of oral keratinocytes proceeds through stages of transformation from normal, to
dysplastic, to malignant. The findings suggests that oral keratinocytes suffer genetic damage
at each stage boundary until the accumulated genetic damage is sufficient for autonomous
proliferation and tumor formation.2
The New WHO classification (2005) applies the term dysplasia for “presence of
architectural disturbances accompanied by cytologic atypia.” The degree of dysplasia is
determined as a measure of tissue and cellular deviation from normal.
The features of epithelial dysplasia are summarized below as quoted by (Soames JV
and Southam JC 27:
1. Nuclear and cellular pleomorphism: Nuclei and cells show variation in size and
shape- anisocytosis and anisonucleosis.
2. Reversal of nuclear cytoplasmic ratio: Increase in size of nucleus with respect to
the cytoplasm.
3. Nuclear hyperchromatism: Abnormally intense nuclear staining.
4. Prominent nucleoli.
5. Increased and abnormal mitoses: Normal mitoses are increased in number and
found higher up in the epithelium than is usual i.e. away from basal layer :
suprabasal mitoses. These may have an abnormal form.
6. Disturbed polarity of the basal cells or of cellular orientation. The cells in the
basal layer have no definable long axis and the nuclei have no polarity.
7. Basal cell hyperplasia: The presence of several layers of cells having basaloid
appearance. It is often associated with drop shaped retepegs.
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14
8. Drop shaped retepegs: The retepegs are wider at their deeper part than they are
more superficially.
9. Irregular epithelial stratification or disturbed maturation: The cells no longer
show a proper sequence of morphological and maturational changes as they pass
from the basal layer to the surface.
10. Abnormal keratinization: Keratinization occurs below the normal keratin layer,
either as individual cell keratinization within the stratum spinosum or as disturbed
maturation of group of cells resulting in the formation of intraepithelial keratin
pearls.
11. Loss or reduction of intercellular adhesion (or cohesion) : Cells of the stratum
spinosum remain no longer adherent to each other.
It should be noted that not all these changes are necessarily seen in any one case.27
c. CLINICAL FEATURES OF POTENTIALLY MALIGNANT DISORDERS
The clinical features and prognosis of oral precancers (PMD) will depend on the
exact precancer involved. As a general rule they present as white lesions with excess surface
keratin or as red lesions with minimal surface keratin.
Leukoplakia : is defined as a chronic white mucosal macule which cannot be scraped off,
cannot be given another specific diagnostic name, and does not typically disappear with
removal of known etiologic factors, excepting tobacco. Oral leukoplakia occurs most
frequently on the lip vermilion, buccal mucosa and gingival mucosa, but many may be
misdiagnosed with frictional keratosis, though the tongue, lip vermilion and oral floor are
the sites most likely to present with leukoplakia containing dysplastic cells. Leukoplakia
("white patch") is the most common and well-researched oral precancer.
It should be emphasized that oral leukoplakia is a diagnosis of exclusion which
requires the clinician to be well acquainted with all other white oral lesions so as to be able
to rule them out prior to using the term leukoplakia for a particular keratosis in any patient.
It must be emphasized that the term leukoplakia is a clinical one. Even though biopsy and
microscopic evaluation is frequently required in order to identify dysplastic or malignant
cells, the presence of such cells does not alter the clinical diagnosis.
Case-control studies have strongly implicated smoked & smokeless tobacco and
alcohol abuse in the etiology of oral cancer, but have not further correlated white mucosal
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15
plaques with malignant transformation. It is not known whether oral cancers in the smokers,
identified in epidemiologic studies arise from leukoplakia or from clinically normal
mucosa. However various investigations have found leukoplakia adjacent to a third (range:
10-100%) of oral squamous cell carcinomas.17
Numerous follow-up studies have established malignant transformation rates for
leukoplakia, ranging from 1-28%, with an average of 4% overall. The few investigations
which have examined the latter feature have found that 7% (range: 0.1%-40%) of oral
leukoplakia will have at least one focus of squamous carcinoma in the initial biopsy. Cancer
arising from a leukoplakic lesion may be diagnosed two to four years after the leukoplakia is
diagnosed, but transformation may occur within months or may occur after decades.
Leukoplakia is associated with progression to cancer when its surface becomes
thickened and rough or granular and when its surface becomes verruciform, or when red
areas of minimal keratin production are interspersed amongst the background of thickened
keratin (erythroleukoplakia, speckled leukoplakia).
Erythroplakia : Is a clinical term for a chronic red mucosal macule which cannot be given
another specific diagnostic name and cannot be attributed to traumatic, vascular or
inflammatory causes. Such lesions are less common than white precancers but very careful
observation will reveal erythroplakia in association with an early invasive oral carcinomas.
Erythroplakia may also be associated with leukoplakia (erythroleukoplakia), and in mixed
red and white lesions it is the red portion that is more worrisome than the white. Mucosa of
the lateral and ventral tongue, the oral floor and the soft palate is the common site for
erythroplakia reported in most of the cases.17
This lesion has been referred as "the dangerous oral mucosa" because under the
microscope, it typically presents as carcinoma in situ, severe epithelial dysplasia or
superficially invasive carcinoma. In high risk clinical situation, such as oral floor lesions in
heavy smokers and alcohol abusers, 80% of these red patches may contain focal areas of
micro invasive cancer at the time of initial biopsy.
Though follow-up studies are not available for erythroplakia, its usual microscopic
counterpart, carcinoma in situ, has been shown to transform into invasive carcinoma in
approximately one of every four cases. This malignant transformation occurs despite the
fact that carcinoma in situ lesions are routinely treated by conservative surgical removal or
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16
laser ablation. Without therapy this disease transforms into invasive carcinoma in 60-90%
of the cases within 5-10 years after initial diagnosis.17
Smokeless Tobacco Keratosis: Also referred to as snuff pouch, snuff dipper's lesion, or
tobacco pouch is a chronic white or gray/translucent mucosal macule localized in areas of
direct contact with smokeless tobacco. This lesion cannot be scraped off, may disappear
with cessation of the tobacco habit, and is poorly demarcated from surrounding mucosa.
The altered mucosa is soft, velvety to feel and further on palpation, the tobacco chewer's
cheek will usually reveal a distinct "pouch". That results due to flaccidity in the chronically
stretched muscles in the area of tobacco placement. During a clinical examination, this
usually stretched mucosa appears folded or fissured. Induration, ulceration and pain are not
associated but occasional inflammatory erythema may be noted.17
It usually takes 5-10 years of tobacco habit for smokeless tobacco keratosis to
become apparent, but it may be present after less than a year. Once it occurs, it typically
remains unchanged indefinitely unless the daily smokeless tobacco contact time increases.
In such a case, it will gradually become thickened to the point of appearing as a distinctly
white, leathery or nodular plaque and become clinically indistinguishable from leukoplakia.
Development of smokeless tobacco keratosis in users is dependent on the type of
habit practiced in a society. For example Snuff is reported to produce more keratosis than
chewing tobacco, and people who keep the quid in a single selective site are more prone to
keratosis than those keeping in multiple sites. A specific brand of tobacco used, duration of
the habit, excessive daily contact-hours of tobacco on oral mucous membranes, an
increased amount of tobacco consumed daily, and a deficiency of beta-keratin or vitamin A
are other factors leading to high risk of keratosis.17
The malignant transformation potential of smokeless tobacco keratosis has not been
determined, but the tobacco habit itself is said to carry a risk four times greater than normal
mucosa, as observed in case-control studies of oral cancer patients. Few if any keratotic
lesions with serious dysplasia are reported in investigations using large numbers of tobacco
chewers, 16% of biopsied cases show at least mildly dysplastic cells in smaller
investigations.
Clinical grading scales for smokeless tobacco keratosis according to intensity of
whiteness, erythema and fissuring have been provided by some authorities. Unfortunately,
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none of these has successfully correlated severe or high-grade lesions with an increased risk
of malignancy.
Oral Submucous Fibrosis: Patients complain of burning sensation of the oral mucosa,
occasional mucosal ulceration, marble-like blanching of the mucosa, and palpable fibrous
bands of the buccal mucosa, soft palate and lips. Leukoplakic lesions are commonly seen
along with oral submucous fibrosis and oral carcinoma development has been shown to
occur in 5% of users during a 15 year period of follow-up. This irreversible precancerous
condition is strongly associated with the habit of chewing areca nuts. Whether cancers
develop from leukoplakia or from the non-leukoplakic mucosa is as yet not established, but
it is presumed that leukoplakia behave in the same precancerous fashion as in persons
without oral submucous fibrosis. The increased risk of oral cancer in betel quid chewers is
mostly relevant to those who include tobacco in the quid, although all other features of the
disease remain the same.
Proliferative Verrucous Leukoplakia (PVL) : First described in 1985, it is a white
mucosal plaque or discoloration which virtually always develops nodular, papillary or
verruciform surface projections and which gradually, sometimes rapidly, spreads laterally to
encompass a large mucosal area. It is a very special form of oral precancer. Four of every
five affected patients are female and the mean age at diagnosis is 62 years (range: 22-89
years). The usual site of female involvement is the buccal mucosa (63% of cases), while the
tongue is most frequently affected in men (82% of cases). Etiologic factors are elusive.
Two-thirds of patients do not have a tobacco habit, but there are especially strong
associations with human papillomavirus (89%) and candida albicans (50%).
Almost half of PVL samples will demonstrate epithelial dysplasia at initial diagnosis
and few cases are spared this change eventually. Oral carcinoma will develop in more than
70% of affected patients during the decade following this diagnosis. Aggressive and
frequent surgical interventions and very close follow-up has been identified thus far as the
best treatment option.
Lichen planus : Is an autoimmune disorder that presents on the bilateral buccal mucosa as
intertwining, thin strands or streaks of white keratosis known as Wickham's striae. Lesions
tend to vary in intensity and are known to be induced by a variety of medications and other
antigens, stress, overwork, including betel quid or tobacco habit. The excess keratin
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production may be the result of reduced proliferation of basal keratinocytes, with enhanced
maturation or differentiation of the epithelium. Skin involvement as purple to brownish,
pruritic papules are seen in some patients, but only the oral lesions are considered to be
precancerous. The more severe oral involvement demonstrates an erythematous background
(erosive lichen planus) and may present with bullae or ulcers (bullous lichen planus,
ulcerative lichen planus).
The erosive or atrophic forms of the disease, with its red mucosal background, are
considered to be more worrisome than the typical case of white striae on otherwise normal
mucosa. The malignant transformation rate for erosive lichen planus is probably less than
1%, according to follow-up studies. There are many who claim no malignant transformation
potential for oral lichen planus.
Malignant transformation in Lichenoid dysplasia too is debatable which presumes
that lichenoid microscopic changes associated with dysplastic cells are simply an immune-
mediated response to an otherwise unremarkable epithelial dysplasia. According to this
theory such lesions are not true lichen planus at all.
Recurring oral melanotic macule : Though oral melanoma is a rare cancer, it has been
estimated that one-third of these are preceded by an apparently innocuous mucosal
melanosis. A recent example of recurring oral melanotic macule has been reported to have
eventuated after seven years in melanoma. None of the six early biopsies demonstrated
histopathologic characteristics of melanoma; they were classic examples of oral melanotic
macule. DNA flow cytometry, however, showed obvious aneuploidy of cells in the early
biopsies, as well as in the final melanoma suggesting that histologically benign-appearing
oral melanosis may actually be the radial growth phase of oral melanoma in some cases. 17
d. HISTOPATHOLOGY OF POTENTIALLY MALIGNANT DISORDERS
Histopathologic features of oral precancers
Experience has taught us that certain cellular and tissue alterations are associated with
premalignancy and malignancy. Altered cells appear to be more primitive than normal and
so these changes are presumed to be examples of immature or inappropriate differentiation,
although pathologists typically refer to them as dysplasia or atypia. Animal models for oral
carcinogenesis have reliably demonstrated that normal epithelium passes through stages of
more and more severe dysplasia prior to the onset of invasive neoplasia. As a general rule,
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fewer than 20% of oral leukoplakia will demonstrate dysplasia where 8% will have severe
dysplasia. On the other hand, erythroplakia show severe changes more than 90% of the
time.14
Cellular changes in dysplasia : Specific alterations of individual epithelial cells are
important in the determination of epithelial dysplasia. Cells and nuclei take on a more
primitive appearance, similar to those of basal cells with enlarged nuclei (nuclear
hyperplasia), dark-staining nuclei (hyperchromatism), enlarged, often eosinophilic nucleoli
(prominent nucleoli), and with an increased nuclear-to-cytoplasmic ratio. These cells also
appear to be placed closely together than normal keratinocytes (increased cellular density).
Such changes are not exclusive to carcinogenesis, as they may be seen in reactive epithelium
or epithelium influenced by a variety of systemic alterations. Flow cytometry and image
cytometry add significantly to the pathologist's ability to assess nuclear changes associated
with eventual cancer development.
There is often an increase in mitotic activity in dysplastic epithelium, but this also is
seen in many reactive lesions. Enlarged, tripolar or star-shaped mitotic figures (abnormal
mitoses), however, are much more indicative of precancerous changes. Abnormal mitosis
may also be defined as mitotic figures found in unusual locations above the basal cell layer.
A key alteration of dysplastic epithelial cells is variation in the shape of the cells and
nuclei. This pleomorphism is unusual outside of cancers and precancers. Premature
production of keratin below the surface layer is another important alteration, but it is much
more commonly seen in oral carcinomas than in oral premalignancy. This dyskeratosis may
be represented by individually keratinized cells or by tight concentric rings of flattened
keratinocytes (epithelial pearls). The pathologist must be careful not to misinterpret a
keratin-filled surface indentation cut tangentially as true intraepithelial keratosis. Not all
dyskeratosis is related to malignancy or premalignancy. Individually keratinized cells, for
example, are also characteristic of hereditary benign intraepithelial dyskeratosis (Witkop
disease).
Cellular necrosis and loss of cellular cohesiveness (acantholysis) are major signs of
poorly differentiated carcinoma but are extremely rare in the epithelial dysplasia of oral
precancer. When present, these features must be distinguished from intercellular edema,
intraepithelial inflammatory cells and degenerating cells with pyknotic nuclei and
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vacuolated cytoplasm. Virus-induced koilocytes may be seen but are not necessary to the
diagnosis, nor does their appearance appear to be of prognostic significance.
When dysplasia is seen in epithelium which otherwise has the microscopic features
of lichen planus like liquifaction degeneration of basal cells, saw-toothed rete processes,
hyperkeratosis, sub-epithelial band of inflammatory/immune cells, etc, the lichen planus
features are essentially ignored and the lesion is graded according to the above mentioned
criteria for epithelial dysplasia, although the term lichenoid dysplasia may be applied to the
case.
Tissue (morphological) changes : Many oral precancers show excess surface keratin
(hyperkeratosis, hyperparakeratosis, hyperorthokeratosis) and most show hyperplasia of the
spindle cell layer (acanthosis), but both changes are common to a number of mucosal
lesions without any cancer transformation potential and neither is necessary for the
diagnosis of dysplasia.
Smokeless tobacco keratosis is characterized by a somewhat unique intracellular
vacuolization or "edema" of superficial layers, perhaps interspersed with streaks of
parakeratinized cells. This change most likely results from a low-grade chemical burn or
from the alkaline tobacco used. It has been referred to as surface etching and has no
implications to the risk of malignant transformation.12
Basal cell hyperplasia is of major importance to the diagnosis as well as to the
grading of dysplasia. Other tissue changes, however, are important features of oral
epithelial dysplasia, especially certain alterations of the rete processes.
Extremely elongated rete processes with minimal cellular atypia are of little
concern, as they are characteristic of a variety of hyperplastic conditions, including
papillomavirus infections, frictional keratosis, psoriasis and pseudoepitheliomatous
hyperplasia. Rete processes with a bulbous enlargement of the lowermost region (drop-
shaped rete processes), however, are worrisome regardless of their size, especially if
secondary projections or nodules are seen to arise from the basal layer and branch in
different angles into the lamina propria and connective tissue papillae. There is no
physiological explanation for secondary nodules extending laterally from a rete peg of the
oral mucosa. To many, these alterations are ominous enough to alter the histopathologic
grading of a lesion to a higher level.
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Dysplastic epithelium may be atrophic as well as acanthotic and some experts
believe that atrophic forms have a higher risk of malignant transformation. Atrophic
epithelium often lacks rete processes and may be ulcerated, thereby mimicking a traumatic
or inflammatory lesion with thin, regenerating epithelium creeping in from the margins.
Regenerating epithelium often has granulation tissue beneath it to distinguish it from
precancerous dysplasia in atrophic epithelium. The very nature of thin, atrophic epithelium
presents a diagnostic grading dilemma because basilar hyperplasia very quickly extends to
the surface. There are no standards for this situation but Speight et al (1996) have
recommended that these be regarded as severe dysplasia. Leukoplakia of the vermilion
border of the lower lip in actinic cheilitis (actinic cheilosis, farmer's lip, sailor's lip) are
especially prone to these difficulties.14
An alarming morphological alteration of dysplastic epithelium is loss of
stratification (loss of polarization) due to an apparent inability to properly differentiate and
mature from basal cells to prickle cells to flattened keratinocytes. Cells high in the
epithelium have the same immature appearance as those in the basal layers. This feature is
especially pronounced in severe epithelial dysplasia and carcinoma in situ.
The pathologist must pay particular attention to the appearance of the epithelial cells
at the lateral surgical margins, and the presence or absence of dysplasia should be
mentioned in the histopathologic description of the lesion. The presence of squamous
metaplasia of the excretory ducts of the minor salivary glands should also be mentioned,
especially when cellular atypia is evident. Many treatments for oral precancers remove or
destroy only the most superficial portions of the submucosal tissues, thus leaving behind the
salivary glands and their ducts.17
As stated by Odell EW28 cellular pleomorphism, loss of basal cell polarity and
irregular stratification reflect a loss of the structural organization that is found in normal or
hyperplastic epithelia with a well defined basal layer, a structured prickle cell compartment
and a uniform superficial zone which may or may not be keratinized. These features also
need to be considered along with the subtle changes by more obvious features such as
premature or individual cell keratinization or deep squamous pearl formation. Cellular
pleomorphism implies differences from the cell size and shape appropriate for a given layer
in the epithelium and the presence of new or bizarre forms. Altered polarity, sometimes with
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the appearance of spindle shaped cells and increased size unaccompanied by cell division
are all components of pleomorphism. Crowding of cells (‘hypercellularity’), usually basal or
in elongate or bulbous rete processes and either generalized or focal, is seen more frequently
than reduced inter cellular cohesion although these features may co-exist in the same
dysplastic lesion.
An increase in the proportion of the epithelium occupied by the proliferative
compartment is a common feature of dysplasia. Multiple layers of cells with basaloid
features, a high nuclear cytoplasmic ratio and mitoses high in the epithelium are
manifestations of this. Dysplasia in which these features predominate may have a
deceptively uniform appearance, as in atrophic epithelium such an expanded proliferation
compartment may occupy almost the full thickness. Bizarre mitoses and extremes of
anisonucleosis are rare in oral epithelia and should perhaps be reconsidered as a component
of dysplasia. Hyperchromatism of normal sized and shaped nuclei is common in
hyperplastic conditions.28
e. GRADING SYSTEMS FOR ORAL EPITHELIAL DYSPLASIA
Histopathological Grading of Oral Epithelial Dysplasia (OED)
Of all disciplines in clinical medicine, histopatholgy is often credited with being the
most scientific. There is no doubt that pathological examination has led to many of the
currently used disease classifications and that morphological observations and correlation of
the observations with clinical parameters has provided a sound basis for clinical medicine as
it is today. With the increasing demands for ‘evidence based medicine’, notoriously
subjective histopathological approaches do need to be redefined as concepts as well as
diagnostic criteria, scientifically validated. Grading, in fact, is an attempt to impose discrete
categories on what is in effect a continuous grey scale. By assigning a grade one artificially
creates discrete sub-entities in a biological continuum.29
There is a generally held view that assessment of dysplasia in premalignant lesions is
important because dysplastic lesions are more likely to undergo malignant change and
because it is felt that the chances of malignant transformation increases with increasing
severity of dysplasia. There is a broad measure of agreement among pathologists about the
altered epithelial features that comprise dysplasia, but there is great variation in interpreting
the severity of dysplasia and not all investigators describe the criteria they use.30
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23
Pindborg JJ et al13 in 1985 during the II meeting the International Association of
Oral Pathologists (Amsterdam) held an exercise to study subjectivity in evaluating epithelial
dysplasia. A surprisingly wide spectrum of diagnostic suggestions was found indicating the
need for an internationally accepted set of criteria for OED.
The diagnosis of epithelial dysplasia has caused considerable distress for
pathologists because of ambiguous diagnostic criteria and difference of opinion among
pathologists about what constitutes “epithelial dysplasia”. Therefore Abbey LM et al31
studied the intra examiner and inter examiner reliability in diagnosis of OED. The exact
agreement (interobserver) with sign-out diagnosis was 50.5%, whereas examiners agreed
with their own diagnosis (intraobserver) 50.8%, of the time. This lead to the conclusion that
accurate reproducible agreement among experienced board certified oral pathologists is
difficult to achieve.
Karabulut A et al32 investigated observer’s variability in grading epithelial dysplasia
between two general pathologists and two oral pathologists for 100 sections of oral
leukoplakia to grade from no dysplasia to carcinoma in situ. The inter observer agreement
rates were in the range of 49-69%. The calculated kappa values 27%-45% showing poor to
moderate agreement between the pathologists. These values were also similar when
comparing the two pairs of pathologists with the same education, indicating that the inter
observer variability was due to individual differences rather than to their educational
background.
The ideal situation would be to create a system for gauging epithelial dysplasia, then
to follow the patients with dysplasia and study how often and how soon the dysplasia turns
into carcinoma. However, ethical considerations do not allow the follow-up, for longer
periods of time for moderate and severe dysplasia and carcinoma in situ. “We are in fact
faced by an insoluble problem : although we may be able to produce objective system to
gauge epithelial dysplasia, we will not be able to test it” stated Pindborg JJ et al.13
The severity of dysplastic features is designated as grade of epithelial dysplasia.
Many dysplastic features in varying combinations have been used for grading. However,
difficulties have been encountered in assessing and standardizing the different degrees of
epithelial dysplasia. Many systems of grading oral epithelial dysplasia have been proposed
in order to standardize the severity of dysplastic features and the relationship of epithelial
Review of Literature
24
dysplasia in its varying grades to the subsequent development of cancers has still to be
worked out.33
Any grading system is said to be clinically useful if they are reproducible between
different observers. In addition, the parameters considered in the histological assessment
should be biologically meaningful, reflecting the malignant potential of the lesion.34
The various grading systems put forth by different authors are as follows:- 1. Smith
and Pindborgs ’ Photographic method (1969), 2. Banoczy and Csiba (1976), 3.WHO (1978),
4. Burkhardt and Maerkar (1981), 5. Lumermann H et al (1995), 6. Neville et al (1995), 7.
Speight P M et al (1996), 8. Bouquot JE (1997), 9.Soames JV (1998), 10.Kuffer and
Lombardi (2002), 11. Brothwell DJ (2003), 12. Takasi Saku Japanese society of Oral
Pathology 2004, 13. WHO (2005), 14. Binary System by Kujan O (2006).
1. Smith and Pindborg (1969)35 attempted to standardize the grading of dysplasia by
photographic method. They placed the diagnosis of epithelial dysplasia on an objective and
semi quantitative level by: (i) Concentrating the observer’s attention on one
photographically standardized microscopic feature at a time and (ii) Enabling the observer to
assess each feature individually and allocate a weighed score to each one.
The system was subjective involving the comparison of histological sections with a
series of standardized photographs. They used 13 histologic features which were
standardized by a set of photographs. Each feature was graded Absent, Slight and Marked as
follows:
Histologic features for Smith & Pindborg (1969) classification of oral epithelial dysplasia.35
Type of change Severity of dysplasia
1. Drop shaped retepegs None Slight Marked
2. Irregular epithelial stratification None Slight Marked
3. Keratinization of cells below keratinized layer None Slight Marked
4. Basal cell hyperplasia None Slight Marked
5. Loss of intercellular adherence None Slight Marked
6. Loss of polarity None Slight Marked
7. Hyperchromatic nuclei None Slight Marked
8. Increased nucleo-cytoplasmic ratio in basal andprickle cell layers
None SlightIncrease
MarkedIncrease
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9. Anisocytosis and anisonucleosis None Slight Marked
10 Pleomorphic cells and nuclei None Slight Marked
11 Mitotic activity Normal SlightIncrease
MarkedIncrease
12 Level of mitotic activity Normal Slight Marked
13 Presence of bizarre mitoses None Slight Marked
Scoring: A grading of ‘none’ was scored – 0 (zero). Grading of ‘slight’ or ‘marked’ was
scored – 1 to 10. The total score of all the features was taken as epithelial dysplasia index
(EDI) which was a semi objective score. It could vary from 0-75. The grading finally was
done as follows: Total score (EDI) 0-10 : Not dysplastic, 11-25 : Mild dysplasia, 26-45 :
Moderate dysplasia and 46-75 - Severe dysplasia.35
Fishman SL et al36 studied clinico-pathologic correlation of oral premalignancy.
They scored 153 cases of premalignant lesions for various histologic characteristics
following the criteria suggested by Smith and Pindborg (1969). According to them, certain
histologic changes were found to be more prominent in lesions felt to be premalignant.
These changes progressed as the severity of diagnosis increased. These changes were :-
Irregular epithelial stratification, Intraepithelial keratinization, Loss of polarity of basal cells,
Hyperchromatic nuclei, Increased nucleo-cytoplasmic ratio and Anisocytosis and
anisonucleosis.
Thus using the criteria suggested by Smith and Pindborg (1969), the authors scored
histologic changes and developed a meaningful index wherein the histologic changes were
described in a quantitative manner. They concluded that this photographic method can be
used as routine process.36
Katz HC et al37 evaluated 214 cases of epithelial dysplasia using Smith and Pindborg
(1969) method of standardization. They found the system to be of considerable value for
purposes of standardization and eliminated observer bias by the use of standardized
photographs. But they questioned the accuracy of the weightage given to each of the
histological characteristics. They suggested testing it further, as to which histological criteria
was of greatest value in predicting the potential for malignant change, though they found
that 98% of cases graded as severe dysplasia showed presence of superficial mitosis
suggesting that it could be an ominous feature.
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26
This system despite providing more objective data, could not find general favor
among pathologists and in routine diagnostic pathology as quantification was tedious.
Warnakulasuriya S (2001)17 commented on this system and noted that even inflammatory or
reactive lesions which are considered non-neoplastic may show some features of dysplasia.23
2. Banoczy and Sciba (1976)38 diagnosed epithelial dysplasia using the following criteria
suggested by Mehta et al (1971) : - Irregular epithelial stratification, Increased density of the
basal cell layer or prickle cell layer or both, Increased number of mitotic figures (a new
abnormal mitoses may be present), Dropped shaped retepegs, Increased nuclear cytoplasmic
ratio, Loss of intercellular adherence, Nuclear pleomorphism, Nuclear hyperchromatism,
Enlarged nucleoli, Keratinization of single cells or cell groups in the prickle cell layer, Loss
of intercellular adherence.
They graded epithelial dysplasia as:- Mild - When 2 of the above listed histological
changes were present. Moderate - When 2 to 4 changes were present. Severe - When 5 or
more of the changes were present. They found that out of 120 cases of epithelial dysplasia
analyzed, the most frequently appearing histological features were: a. Irregular epithelial
stratification. b. Drop shaped retepegs. c. Hyperplasia of the basal cell layer.
Based on their classification of dysplasia, they described the distribution of
leukoplakia lesions by age, sex, clinical types of leukoplakia, location and follow up results.
They correlated these factors with the grade of dysplasia. This system was based on
subjective interpretation of the features and didn’t take into account which factor could be
important in determining the malignant potential.38
3. WHO System (1978): In an attempt to standardize the criteria for oral precancer,
established a collaborating reference center in 1967. The center aimed to characterize and
define those lesions that should be considered as oral precancer and to determine, if possible
their relative risk of becoming malignant. In its report in 1978, WHO defined and listed the
12 histologic characteristics of epithelial dysplasia as follows :- 1) Loss of polarity of basal
cells. 2) The presence of more than one layer of cells having basaloid appearance-Basal cell
hyperplasia. 3) An increased nuclear-cytoplasmic ratio. 4) Drop shaped retepegs. 5)
Irregular epithelial stratification. 6) Increased number of mitotic figures. 7) The presence of
mitotic figures in the superficial half of the epithelium. 8) Cellular polymorphism. 9)
Nuclear hyperchromatism. 10) Enlarged nucleoli. 11) Reduction of cellular cohesion. 12)
Keratinization of single cells or cell groups in the prickle cell layer.37
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WHO 1978 graded epithelial dysplasia as: Mild, Moderate, Severe : -
Mild dysplasia: Slight nuclear abnormalities, most marked in the basal third of the
epithelial thickness and minimal in the upper layers, where the cells show maturation and
stratification. A few, but no abnormal mitosis may be present, usually accompanied by
keratosis and chronic inflammation.
Moderate dysplasia: More marked nuclear abnormalities and nucleoli tend to be present,
with changes most marked in the basal 2/3rd of the epithelium, nuclear abnormalities may
persists up to the surface, but cell maturation and stratification are evident in the upper
layers. Mitoses are present in the parabasal and intermediate layers, but none is abnormal.
Severe dysplasia: Marked nuclear abnormalities and loss of maturation involving more than
2/3rds of the epithelium, with some stratification of the most superficial layers. Abnormal
mitoses may be present in the upper layers.
Severe grades of dysplasia may merge into the lesion customarily designated as
carcinoma in situ, in which the whole or almost the whole thickness of epithelium is
involved. In the present knowledge, it was not possible to say whether the presence of severe
dysplasia carried a different degree of risk of subsequent development of invasive carcinoma
than the presence of carcinoma in situ.
It was generally believed that mild degrees of epithelial dysplasia did not indicate
any great danger for the patient, although, special reference had to be made to certain high
risk sites such as the floor of the mouth and the ventral surface of the tongue. Moderate
dysplasia, however called for a more cautious approach and severe dysplasia indicated that
there was a considerable risk of the development of cancer. They suggested that generally
the degree of dysplasia could be linked to the degree of probability of development of
malignancy.37
4. Burkhardt and Maerkar (1981)39 listed 6 relevant histological and cytological
parameters, based on which diagnosis and classification of epithelial dysplasia could be
made: 1) Basal cell hyperplasia. 2) Loss of basal cell polarity. 3) Cellular pleomorphism. 4)
An increase in mitotic figures. 5) Dyskeratosis. 6) Abnormal and absent epithelial
stratification. Additional indicators for dysplasia were i) An increase in sub epithelial
lymphocytes, plasma cells ii) intraepithelial cells (stroma reaction) and iii) Presence of
Candida organisms.
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They graded dysplastic criteria for classification according to degree of dysplasia and
characteristics of carcinoma in situ.
Degree Characteristics
Low Basal cell hyperplasia.
Basal cell polarity disrupted.
Medium Basal cell hyperplasia.
Loss of basal cell polarity.
Moderate degree of cellular polymorphism.
Slight increase in rate of mitosis.
Occasional dyskeratosis.
High Basal cell hyperplasia.
Basal cell polarity lost.
Marked cellular pleomorphism.
Increase in ratio of mitosis.
Numerous dyskeratosis
Abnormal epithelial stratification.
Ca-in
situ
Characteristics of high degree dysplasia more marked.
Epithelial stratification lost.
Stroma not yet invaded.
They classified leukoplakia lesions into following groups:
a) Leukoplakia without or with low degree dysplasia: These lesions showed no signs of
dysplasia. Those with minor dysplastic changes were included in this group as low degree
dysplasia could also represent a completely harmless reversible epithelial irritation. This
group of lesions was not considered as precancerous.
b) Leukoplakia with moderate degree of dysplasia: They held an intermediate position,
having more marked dysplastic criteria. They had to be considered as risk lesions when
planning treatment.
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c) Leukoplakia with a high degree of dysplasia and carcinoma in situ : These had to be
considered as precancerous and were identified by the fact that there was a marked degree of
dysplasia. Endophytic growth and downward extension of epithelial retepegs were common
findings.
d) Carcinoma-in situ: Was characterized by the additional feature of complete loss of
epithelial stratification. It was regarded as early form of cancer not showing invasive
growth.
e) Candidal leukoplakia: The incidence of candidiasis increased with the degree of
dysplasia. Fungal growth in the lesion had to be regarded both as risk factor and risk
indicator.
In their study, the degree of dysplasia correlated with the clinical parameters that
were known to relate to relatively poor prognosis like age, sex, location, the dental status,
and exogenous irritants. Patients exhibiting moderate to high degrees of dysplasia developed
malignancy. The authors proposed a treatment and follow up plan for different degrees of
epithelial dysplasia.
For no/low degree of dysplasia : i. Aim at total excision, ii. If excision total, no follow up
observation and iii. If excision incomplete, further observation at longer intervals (3-6
months).
For medium degree dysplasia : i. aim for total excision, ii. if removal incomplete initially,
total excision to follow, iii. with total excision, no follow up observation, iv. if excision
incomplete, further observation at shorter interval (4-8 weeks).
For marked dysplasia (carcinoma in situ) : i. total excision in every case, ii. follow up as
for cancer patients (every 4 weeks during the first year) and iii. In addition to this, Candida
infection requires antifungal therapy.39
Girod SC et al (1999)40 in their study classified the epithelial dysplastic lesions
according to Burkhardt and Maerkar (1981)39 and studied the expression of p53, PCNA and
Ki-67 in different degrees of epithelial dysplasia. The expression pattern correlated better
with the different degrees of dysplasia.
5. Lumermann H. et al (1995)25 considered: Basal cell hyperplasia, -Nuclear enlargement
and hyperchromaticity, drop shaped retepegs as ‘minimal’ criteria for the diagnosis of oral
epithelial dysplasia. The dysplastic changes were graded as:-
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30
1. Mild epithelial dysplasia: ‘Minimal’ dysplastic alterations confined to the lower third
of the epithelium.
2. Moderate epithelial dysplasia: Dysplastic changes seen in upto 2/3rds of the thickness
of the epithelium.
3. Severe epithelial dysplasia: Dysplastic cells fill more than 2/3rds but less than the
entire thickness of the epithelium.
4. Carcinoma in situ: The entire thickness of the epithelium contains less differentiated
basaloid or squamous epithelial cell with enlarged, hyperchromatic nuclei and a variable
number of typical and atypical mitotic figures with no invasion into the submucosa.
5. Verrucous hyperplasia with dysplasia: The epithelium exhibits considerable
thickening with surface papillations, hyperparakeratoses and parakeratin plugging and
occasional dysplastic cells confined to the lower 1/3 rd of the epithelium.
After the microscopic analysis and grading of the dysplasia, each case was correlated
with the clinical data. The authors described the distribution of the lesions according to age,
sex, site and follow up. 16% of patients with epithelial dysplasia showed invasive squamous
cell carcinoma within a mean period of 36.6 months. Interesting feature was, 2 of the 11
patients with histological verrucous hyperplasia with mild dysplasia developed squamous
cell carcinoma, indicating the serious nature of this variant of epithelial dysplasia.
Their study confirmed that the dysplastic changes are multicentric, with presence of
normal or hyperplastic epithelium in the skip areas of dysplastic epithelium. The grade of
dysplasia was different in various portions of the same biopsy specimen suggesting that the
incisional biopsy samples were not representative of the true nature of the lesion. This
implied that the examination of the entire lesion was necessary for accurate grading of
dysplasia.27
6. Neville BW et al (1995)41 graded dysplasia as:
Mild: Hyperchromatic and slightly pleomorphic nuclei are noted in the basal and suprabasal
cell layers of stratified squamous epithelium.
Moderate: Dysplastic changes extend from the basal layer to the mid portion of the spinous
layer and are characterized by nuclear hyperchromatism, pleomorphism, cellular crowding
and hyperkeratosis of the epithelial cell layer along with prominent granular cell layer.
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Severe: Cellular crowding and disordered arrangement throughout most of the epithelial
thickness, although slight maturation and flattening of the cells appears to be present at the
epithelial surface. Epithelial cells are less matured as they progress toward the
hyperparakeratotic surface.
Carcinoma in situ: When the entire thickness of the epithelium is involved, the term
carcinoma in situ is used. Dysplastic cells extend from the basal layer to the surface of the
mucosa (top to bottom change) with no invasion into the underlying connective tissue.28
7. Speight PM et al (1996)42 considered the thickness (height) to which the cellular and
tissue changes may extend, as important in grading dysplasia. According to them : a. Mild
forms of dysplasia represented recognizable changes limited to the parabasal layers (lower
third), b. Moderate dysplasia represented recognizable changes extending to middle third,
and c. Severe dysplasia represented as recognizable changes extending to the upper layers.
However, Warnakulasuriya S. 2001 commented that there was wide variation in the
thickness of the covering epithelium in the oral cavity, with much undulation it led to
practical difficulties in using this grading system.
8. Soames JV (1998)27 The features used for evaluating the grades of dysplasia have been
summarized as follows : Mild dysplasia : Epithelial dysplasia involving the lower third of
the epithelium showing pleomorphism, suprabasal mitosis and loss of ordered stratification.
Moderate dysplasia : Epithelial dysplasia involving about 2/3rd of the epithelium Severe
dysplasia : Showing marked cellular atypia and disturbed maturation of the epithelium.
9. Kuffer and Lombardi (2002)43 felt that the choice of clinical rather than histological
criteria in the diagnosis and terminology of precancer is the cause of a disorderly mixture of
dysplastic and non-dysplastic lesions. Therefore, they proposed to dismember the classical
“oral precancerous lesions” to classify all cases which histologically do not show dysplasia
into the category of “risk lesions”(ex. Simple tobacco keratosis) and to place lesions with
dysplasia (i.e. already engaged in the process of malignant transformation ) into the category
of “precursors” of squamous cell carcinoma(ex; tobacco keratosis with dysplasia). This
“precursor” term seems to be the most accurate to characterize the limited but already
malignant intraepithelial alterations of dysplasia and carcinoma in situ, which herald the
onset of an invasive squamous cell carcinoma.
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32
Drawbacks:
1. As there was considerable difference in potential for transformation between lesions
without dysplasia or with mild to moderate dysplasia and those with severe dysplasia, the
application of the term “risk lesion” to lesions without dysplasia which have a “zero risk” of
transformation (ex. Frictional keratosis) was inappropriate. It could lead dentist with less
expertise in this area to exaggerate the risks posed by the lesions with little or no possibility
of developing into cancer.
2. The use of the term “precursor of oral squamous cell carcinoma” to denominate dysplastic
lesions suggested that they were unequivocally associated with the future development of
cancer. This had no scientific evidence. On the contrary, as demonstrated by Mincer et al
(1972), 20 % of oral dysplasia regressed and 40% showed no modification in severity.
According to Gupta et al (1980), 13% of cases regressed and 40 % showed no modification
in severity.44
The concept of dysplasia had been criticized among gynecologists and pathologists
because of possibility of confusion between mild dysplasia and squamous metaplasia or
changes due to epithelial repairs and still worse because of the difficulty and lack of
consistency to differentiate between severe dysplasia and carcinoma in situ. The fact that
“dysplasia” considered as “premalignant” was usually treated conservatively and
“carcinoma in situ” considered as “malignant” was surgically treated, was criticized by
Richart RM and he demonstrated that dysplasia and carcinoma in situ were different aspects
of the same disease “Cervical intraepithelial neoplasia (CIN)” and treatment should be
same for both. This concept of CIN (one or more clones of transformed cells slowly
replacing normal keratinocytes starting from basal and parabasal layers to progressively
invade the whole epithelial height) has now replaced almost completely that of cervical
dysplasia. It has been extended with some modification to oral mucosa as “oral intra
epithelial neoplasia” (OIN) and in general as squamous intraepithelial neoplasia (SIN)
as described by Gnepp GM.45
As for CIN, there are 3 grades of OIN are as follows : - OIN 1: Mild dysplasia- less than
1/3rd involvement of the epithelium, OIN 2: Moderate dysplasia- 1/3rd to 2/3rd involvement
and OIN 3: Severe dysplasia – full thickness involvement or equivalent to carcinoma in
situ.
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This system uses OIN terminology to emphasize that all levels of dysplasia are
actually different levels of true neoplasia, albeit prior to invasion. This system is largely
based on subjective interpretation and lacks consistency in diagnosis among pathologists.
The “Bethesda classification” for cervical cytopathology, includes only 2 grades : - 1.
“Low grade squamous intraepithelial lesion (LSIL)” corresponding to CIN 1 and 2. “High
grade squamous intraepithelial lesion (HSIL)” corresponding to CIN2, CIN3.
Based on this “Bethesda classification” the former system with 3 grades for OIN
was replaced by a 2 grade system, which helped in better stratifying patients for clinical
protocols. Accordingly they chose to report the diagnosis of oral dysplastic lesions as :
1. Low grade oral intraepithelial neoplasia (LOIN) - including OIN 1 (mild dysplasia) or as
2. High grade oral intraepithelial neoplasia (HOIN) - including both OIN 2(moderate) and
OIN 3 (severe dysplasia).
According to the authors, the regression of oral dysplasia without any further
treatments, other than biopsy and cessation of smoking, were difficult to prove. A few cases
of low grade OIN regressed but not high grade OIN.
Drawback: It was inappropriate to consider OIN2 as high grade precursor lesions alongside
severe dysplasia and carcinoma in situ, as they had markedly lower potential for malignant
change.44
10. Brothwell DJ et al (2003): In an attempt to determine the extent of observer agreement
in diagnosis of oral epithelial dysplasia, they graded 64 sections of epithelial dysplastic
lesions according to 5 point scale routinely utilized at their institution (Faculty of dentistry,
University of Toronto). The criteria were: 0 = No dysplasia, 1 = Mild dysplasia: Increased
number of cells in the basal and parabasal epithelial regions showing nuclear
hyperchromatism and pleomorphism, 2 = Moderate dysplasia: Bulbous retepegs with
increased number of cells showing nuclear hyperchromatism and pleomorphism,
involving the basal, parabasal and prickle cell layer. 3 = Severe dysplasia: Bulbous retepegs
with increased numbers of cells showing nuclear hyperchromatism and pleomorphism
involving the entire thickness of epithelium. 4 = Carcinoma in situ: Markedly atypical
changes showing nuclear hyperchromatism and pleomorphism and encompassing the entire
thickness of the epithelium, with the suggestion of early superficial connective tissue
invasion, but without convincing evidence.
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34
Using this system, and with statistical analysis, the authors proved that intra and inter
observer agreement in grading the dysplastic lesions was substantial to an almost perfect
range. This study emphasized the use of appropriate statistical methods and calibration to
eliminate the bias in assessment of oral epithelial dysplasia.46
11. Takashi Saku et al47 proposed a new concept for histopathology of oral borderline
lesions. This group classified dysplasia as (i) Mild (ii) Moderate (true) dysplasia (iii)
Carcinoma in situ. (i) Mild epithelial dysplasia is applied to lesions with atypical features
whose potential for malignant transformation is not known. (ii) Moderate dysplasia is
regarded as true dysplasia which has carcinomatous potential. They also describe the term
epithelial hyperplasia as hyperplastic epithelial lesions with normal shape of rete processes
and normal cellular stratification. They suggested that basal cells are neither germinal cells
nor a source for squamous epithelial regeneration but are terminally differentiated cells.
Most of the cells with proliferative potential are present in the parabasal cell layer, which
may be regarded as the germinal center of the oral squamous epithelium.
When there is a focal proliferation of basaloid (parabasal) cells in the lower part of
rete processes, they replace not only the basal cells but also the lower prickle cells. Such
proliferating cell foci make a straight interface with parakeratinsed cells in the upper layer,
which shows distinct contrast between the upper and lower layers of the epithelium. This
sudden transition is caused by loss of prickle cells due to apoptosis. This phase is referred to
as “two phase” appearance. When any traces of the two phase appearance are recognized,
the lesion is diagnosed as moderate (true) dysplasia.
(iii) Carcinoma in situ is defined as true but not yet invasive neoplasm staying within an
epithelial layer : It is an advanced stage of moderate (true) dysplasia. In moderate dysplasia
the cells are only proliferating, whereas in carcinoma in situ the cells differentiated as
keratinocytes can be recognized. These are basal cell like cells referred to as ‘basal cell
minics’. Dyskeratotic cells in lower layers are important in diagnosing carcinoma in situ.
This working group classified carcinoma in situ into 4 histological types : basaloid,
verrucous, acantholtic and atrophic. In basaloid type most of the constituent cells are basloid
cells replacing the whole layers of epithelium. The verrucous type is characterized by round
shaped reteridges with gradual differentiation towards keratinization and enhanced
keratinsation in surface with keratin plugs. The acanthotic type is the most difficult subtype
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35
because it resembles simple epithelial hyperplasia. There will be distinctive keratinization
on the surface , the reteridges shape are slightly indented, individual cell dyskeratosis or
sudden keratinsation occurs in the lower layers of rete processes, which has no distinct
alignment of basal cell mimics. In the atrophic type the surface is flat and lacks the
hyperkeratotic layer. Rete processes are small and basal cell mimics are clearly aligned
along the basement membrane. Cell cohension appears loosened within the rete processes
with dense band of lymphocytic infiltration.47
12. WHO Classification (2005)48 A working group that convened for an editorial and
consensus conference in Lyon, France in July 16-19-2005 put forward criteria for grading
epithelial dysplasia which is published as WHO classification(2005).They have used a
combination of architectural & cytological changes with more explicit consideration of
levels of changes within the epithelium. Criteria used were in two groups
Architecture Criteria : 1. Irregular epithelial stratification, 2. Loss of polarity of basal
cells, 3. Drop-shaped rete ridges, 4. Increased number of mitotic figures, 5. Abnormal
superficial mitoses, 6. Premature keratinisation in single cells (dyskeratosis), 7. Keratin
pearls within rete ridges,
Cytology Criteria : 1. Abnormal variation in nuclear size (Anisonucleosis), 2. Abnormal
variation in nuclear shape (nuclear pleomorphism), 3. Abnormal variation in cell size
(anisocytosis), 4. Abnormal variation in cell shape (cellular pleomorphism), 5. Increased
nuclear cytoplasmic ratio, 6. Increased nuclear size, 7. Atypical mitotic figures, 8. Increased
number and size of nucleoli, 9. Hyperchromatism.
Grading of dysplasia
Mild dysplasia : In general architectural disturbance limited to the lower third of the
epithelium accompanied by cytological atypia define the minimum criteria of dysplasia.
Moderate dysplasia : Architectural disturbance extending into the middle third of the
epithelium is the initial criterion for recognizing this category. However, consideration of
the degree of cytologic atypia may require upgrading i. e. those lesions that show marked
cytological alteration should be elevated to a higher grade level regardless of how
extensively the atypical cells fill the epithelium.
Severe dysplasia : Recognition of severe dysplasia starts with greater than two thirds of the
epithelium showing architectural disturbance with associated cytologic atypia. However,
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36
architectural disturbance extending into the middle 3rd of the epithelium with sufficient
cytologic atypia may be up graded from moderate to sever dysplasia.
Carcinoma in situ : The theoretical concept of carcinoma in situ is that malignant
transformation has occurred but invasion is not present. It is not always possible to
recognize this morphologically. The following is recommended for the diagnosis of
carcinoma in situ: full thickness or almost full thickness architectural abnormalities in the
viable cellular layers accompanied by pronounced cytologic atypia. Atypical mitotic figures
and abnormal superficial mitoses are commonly seen in carcinoma in-situ.48
13. Binary System of Grading Dysplasia - Omar Kujan (2006)49 : Proposed a new binary
system of grading oral epithelial dysplasia for prediction of malignant transformation. They
used same morphological criteria used by the WHO classification (2005) and graded the
lesion into low- risk or high-risk based on scoring the features.
High-risk lesion (with potential susceptibility for malignant transformation) was based on
observing at least four architectural changes and five cytological changes.
Low-risk lesion (does not have the potential susceptibility for malignant transformation)
was based on observing less then four architectural changes or less than five cytological
changes.
They conducted a pilot study of 28 cases & they could predict the clinical out comes
with certainty in 84.9% of cases i.e. cases given as high risk progressed to carcinoma. The
negative value was also as high as 85% with only 5 cases of low risk category going for
malignancy. In comparison with WHO classification, where prognostic implications for
grading a case as moderate dysplasia were problematic, the binary system was more
objective by classifying them as low risk or high risk to predict the clinical outcome.
Thus they suggested that the new binary system complements the WHO 2005
classification and that it may help clinicians to make critical decisions especially regarding
cases of moderate dysplasia.
A study conducted by Omar Kujan et al (2007)49 showed low inter observer
variability in grading of oral epithelial dysplasia but good to substantial agreement was seen
when a cumulative scoring of these features was used to grade degree of dysplasia. The
efficacy of this system needs to be evaluated further.
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37
Malignant transformation rates for oral epithelial dysplasia as quoted in various
studies described in the article by Bouquot JE et al50
Sl.no Reference Country
No. ofpatients
Withdysplasia
Withmalignancy
Followup Malignancy
1 Gupta et al (1980) India 10.5 Yrs 7%2 Schepman et al 1998 Netherlands 166 166 20 8.5 Yrs 8%3 Silverman et al 1984) USA 257 22 8 7.2 Yrs 36%4 Banoczy & Csiba (1976) Hungary 68 68 9 6.3 Yrs 13.2%5 Amagasa et al (1985) Japan 12 - - 10 Yrs 50%6 Vedtofte (1987) Denmark 14 - - 3.9 Yrs 35.7%7 Mincer et al (1972) USA 45 45 5 3 Yrs 11%8 Pindborg et al (1995) India 61 61 4 7 Yrs 6.6%9 Lumerman etal (1995) USA 44 44 7 1.5 Yrs 16%10 Jaber et al (2003) England UK 359 630 20 3.3 Yrs 5.6%11 Cowan et al (2001) UK Ireland 745 165 24 ? 14.5%12 Burkhardt & Maerker (1981) Germany 200 200 21 5.5 Yrs 10.5%
The above table demonstrates that dysplastic lesions are more likely to progress to
oral squamous cell carcinoma. The cancer risk in moderate to severe dysplasia appears to be
at least 2-3 times more than that in mild dysplasia or hyperplasia. This suggests that
whatever may be the inter or intra observer variability in diagnosis of oral epithelial
dysplasia, it still remains the gold standard and is an important predictor of potential
malignant transformation.50
Interestingly, in 1988 the “Bethesda classification” for cervical cytopathology,
included only two grades.45 According to this, lesions would be classified as low-grade
squamous epithelial lesions, corresponding to former cervical intraepithelial neoplasia
grade 1, and high-grade squamous epithelial lesions, corresponding to grades 2 and 3. This
system has also been mentioned in some reports for oral lesions.40
After the publication of those papers on the binary system for grading epithelial
dysplasia in oral leukoplakia( OL), a study was performed with 218 patients with OL, from
which 39 (17.9%) developed into cancer.44 The authors reported that high-risk OL was
associated with a 4.57-fold increased risk for malignant transformation, compared with low-
risk OL. The authors suggested that high-risk dysplasia would be a significant indicator for
evaluating malignant transformation risk in OL. Subsequently, the same research group
published a study in which they identified significant risk factors for malignant
transformation in a long term follow-up cohort of patients with oral epithelial dysplasia. Of
the 138 patients in this study of Maria AVDC51 with histologically confirmed oral dysplasia,
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38
115 had OL and 23 had lichen planus. From these 138 lesions, 37 (26.8%) developed into
cancer and the “high-risk” degree of dysplasia was an independent risk factor for
transformation. Moreover, high-risk degree of dysplasia was associated with a 2.78-fold
increased risk of transformation compared with low-risk degree. The authors then suggest
the utilization of high-risk dysplasia as a significant indicator for evaluating malignant
transformation risk in patients with potentially malignant lesions. According to them, this
would also help guiding treatment in clinical practice. In spite of these great achievements, it
must be mentioned that malignancy also developed in some patients previously presenting
low-risk potentially malignant lesions.51
14. Grading of dysplasia Bouqout JE17 : Most pathologists grade oral epithelial dysplasia
according to a combination of cellular and tissue changes. Low-grade lesions are
considered to be less severe than high-grade lesions but this assessment is a subjective one
and it is not unusual for different pathologists to place emphasis on different aspects of
dysplastic alteration, thereby arriving at somewhat different diagnostic conclusions.
Regardless of individual emphasis, however, certain oral precancer grading criteria are in
general use:
a. the cellular atypia or dysplasia is similar to that seen in squamous cell carcinoma, b. there
is no evidence of invasion into underlying stroma (the diagnosis would then change to
carcinoma), c. the epithelium with the greatest proportion of atypical cells has the greatest
risk of being or becoming a carcinoma, d. the epithelium with the most extreme atypia of
cells has the greatest risk of being or becoming a carcinoma, e. the final grading or diagnosis
should be based on the most severely involved area of change, even if that area includes no
more than a few rete processes.
Oral epithelial dysplasia is subdivided into three prognostically significant
categories, mild, moderate and severe. Mild (grade I) dysplasia demonstrates proliferation
of atypical or immature basal cells above the parabasal region but not extending beyond the
lower third of the epithelium. Moderate (grade II) dysplasia demonstrates a similar
proliferation into the middle one-third of the epithelial cross-section. The term severe
(grade HI) dysplasia is reserved for abnormal proliferation from the basal layer into the
upper third of the epithelium. Carcinoma in situ, thought by some to be a premalignancy
and by others to be an actual pre-invasive malignancy, requires top-to-bottom change with
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undifferentiated, primitive cells from the basal layer to the topmost layer, although most
authorities accept a thin layer of surface keratin with abrupt transition into the underlying
atypical cells. The grading of dysplastic oral epithelium must also take into consideration
the degree of cellular atypia, as described above, and those lesions with marked alteration
are elevated into a higher grade level.
It is difficult to determine the amount of basal cell hyperplasia in oral precancers
because of transverse sectioning of tissue samples and because of the natural undulation of
the inferior margin of squamous epithelium. It may be helpful to make this determination
from the top of the connective tissue papillae because of the great variability of rete process
length in acanthomatous epithelium, but there is no established standard and in many cases
the basal hyperplasia is confined to the rete processes themselves.17
f. SUBJECTIVITY / VARIABILITY IN ORAL EPITHELIAL DYSPLASIA:
The topic of subjectivity in assessing & grading oral epithelial dysplasia has often
been raised and is a cause of considerable distress among pathologists. This is largely due to
lack of well defined criteria that can be used as guidelines for grading and differences in
opinion among pathologists about what actually constitutes epithelial dysplasia. In addition
there is still a cloud over what criteria could best describe the malignant potential of oral
epithelial dysplasia. The wide intra and inter observer variability encountered in grading oral
epithelial dysplasia was reported by several authors.
At the 2nd meeting of the International Association of Oral Pathologists in 1984, an
exercise was held to study subjectivity in evaluating oral epithelial dysplasia, carcinoma in-
situ and initial squamous cell carcinoma. A poster with a color photomicrographs was
presented to be evaluated anonymously by the 72 participants. The authors diagnoses were
based on the criteria described in the report of the WHO (1978)23 international collaborating
centre for oral precancerous lesions, which ranged from mild epithelial dysplasia to early
squamous cell carcinoma. In one case 37% of the participants agreed with the original
diagnosis, whereas the best rate of agreement was 78% in another photograph (Pindborg,
1985).13 The agreement thus ranged between 1-78%. Fischer D et al (2004)52 discern
regarding this study that the data did not consider the seriousness of diagnostic
disagreements nor were the chance agreements taken into account analytically and all the
participants were not experienced oral pathologists.
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Abbey LM et al (1995)31 evaluated inter examiner and intra examiner reliability to
objectively assess the degree of difficulty in diagnosing epithelial dysplasia. 6 oral
pathologists examined 120 oral biopsies exhibiting simple hyperkeratosis to severe
dysplasia. Exact agreement with the original sign-out diagnoses averaged 50.5%. The intra
examiner agreement was 50.8%. Agreement distinguishing dysplasia from no dysplasia
compared with original diagnosis was 80.3%. Reproducibility of the diagnosis by the same
pathologists was also a problem but was reported as 73.3%.
The results supported the view point that diagnosing epithelial dysplasia of oral
cavity is not an exact science. The authors opined that the pathologist’s diagnosis is a
necessary part of the process of treating a patient even though variation in opinion exists
among pathologists. They emphasized that the pathologist may make error in diagnosis of
epithelial dysplasia versus no dysplasia, one in 5 times when morphologic characteristics
alone are used. Therefore they mentioned that relevant clinical factors may play a role and
clinician should weigh all there factors & then arrive at a management strategy.
Karabult et al (1995)32 investigated the extent of agreement in grading epithelial
dysplasia between pathologists with the same or different educational background. 2 of the
examiners were oral and maxillofacial pathologists with dental background and the other 2
were general pathologists with medical background.
The degree of agreement ranged from 27% to 45 % (0.27-0.45) and was judged as
poor to moderate and it didn’t vary among pathologists of different backgrounds. The
authors concluded that inter examiner variability was due to individual disagreement and not
due to educational background. This variability may depend on which histologic and
cytologic characteristic are considered important for diagnosis, on the variability in the
observation of there characteristic and on the variability of grading there characters into
different categories of epithelial dysplasia, According to them, the problems arise due to
lack of sufficient knowledge of which criteria are important for predicting future
development of cancer, lack of objectivity in evaluating the established criteria, lack of
calibration of the method and arbitrary system of grading.
In a study by Abbey LM et al (1995)31, it was tested whether clinical information
could modify the diagnosis of epithelial dysplasia. But only one of the 6 pathologists came
close to reproducing the results he had achieved in the initial study. So the clinical
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41
information didn’t improve the accuracy and consistency among pathologists. In fact, there
was a decrease in accuracy.
Lumermann H et al (1995)25 used their histologic criteria to grade 308 cases in their
study to define the rate of development of invasive squamous cell carcinoma in dysplastic
lesions. They compared the independent grading of 2 experienced pathologists and found
that inter examiner agreement was 54% only.
Sudbo J et al (2001)53 assessed inter examiner variability by reporting 196 lesions
which were followed up for a period of 4 to 165 months (average 103 months). All
histological sections were reviewed by 4 separate pathologists working at 3 different
institutions. The sections were graded according to WHO criteria as mild, moderate, severe
and carcinoma in situ. The calculated kappa values were in the range of 0.17-0.33,
confirming poor agreement among the scorers. To improve the agreement, they considered
further only 2 diagnostic groups as: 1. Low grade (mild dysplasia and moderate dysplasia) 2.
High grade (severe dysplasia) constituting a presumptive favorable and poor prognostic
group, respectively. Even then the agreement among pathologists didn’t improve (Kappa
values ranged 0.21-0.34). The design and statistical analysis used by previous studies were
criticized by Brothwell DJ et al (2003).46 They described a different method of statistical
analysis in assessing observer agreement and proved that there was substantial intra-and
inter observer consistency and almost perfect conformity in the grading of oral epithelial
dysplasia. 64 histologic sections were examined by 3 oral pathologists and the original sign-
out diagnoses were used as the equivalent of a 4th examiner for comparison and analysis
purposes. Dysplasia was graded according to their institutional criteria on a 5 point scale.
Inter and intra examiner agreement was assessed by using % agreement, Ks and Kw using
quadratic weighting system. The Kw values were 0.68-0.83 and 0.82-0.96 suggesting
substantial to almost perfect range of inter observer agreement and intra observer agreement
respectively. There was slight inter observer bias and this could be due to different opinion
considering the important criteria in grading dysplasia. This suggested that variation in
assessment of dysplasia might decrease if appropriate statistical method is used.
Substantial variation has been reported in the grading of OED. The design and
statistical analysis used by these studies has not been adequate to allow determination of the
extent and source of the variation. The accuracy and precision of a measurement or
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42
diagnosis is normally evaluated by looking at the validity and reproducibility of examiner
observations, respectively. Validity refers to the degree to which examiner observations
represent the actual situation of interest. To assess validity, an observation is compared with
a ‘gold standard’, which is considered to best represent the truth. Observer accuracy is then
assessed by examining the degree of conformity with the gold standard.
Reproducibility refers to the degree to which observer measurement or diagnosis
remains the same on repeated independent observations of an unchanged characteristic and
considers the consistency obtained on two or more observations of the same subject. This
consistency can be assessed between different observers (inter observer) or within a single
observer (intra observer).
When studying the accuracy obtained in grading OED, the situation is slightly
different. There is no test available, which is thought to be better than the pathologist’s
observation; an accepted gold standard is not available for assessing the validity obtained
when grading OED. Therefore, reproducibility, normally used to assess precision, is used to
provide an indication of validity. When combined for this purpose, inter and intra observer
agreement levels give an estimate of the degree of bias and validity present in situations like
grading OED, where an appropriate gold standard is not available.46
A study by Fischer et al (2004)52 achieved a fair to good agreement with overall
Kappa values of 0.59 diagnosing just presence or absence of dysplasia which they believe is
higher than that obtained in other studies reported. When the histologic diagnosis was
simplified into three general categories of ‘no abnormality / hyperplasia’, ‘mild / moderate /
severe dysplasia’ & ‘carcinoma in situ / carcinoma’, the Kappa value improved and was
around 0.7. They emphasized that presence of inflammation may modify the reliability of
diagnosis as it may induce reactive atypical/dysplasia or may reduce the pathologists ability
to observe the dysplastic change leading to under reporting of lesions exhibiting
inflammation. The limitation of this study was the small sample size, differing educational
background of the pathologists, use of 2 sets of slides which could not always be identical.
They suggest combination of border line lesions into one category to improve the
diagnostic accuracy as has been suggested in the ‘Bethesda system’ for cervical
intraepithelial neoplasia.
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In a study by Kujan O et al (2006)49 three oral pathologists & one general pathologist
examined 68 oral epithelial dysplastic lesions & scored them according to WHO 2005
criteria. The features of each of these cases were then correlated with clinical outcome to
understand which were more commonly associated with malignant transformation. There
was poor to moderate agreement in grading the individual architectural & cytological
characteristics but a good to substantial agreement was seen when cumulative scoring of
features were used to agree on the degree of dysplasia.
In a study by Warnakulasuriya S et al (2008)54 they proposed to adapt a binary
system to reduce inter and intra observer variability. They suggested that the highest
agreement scores were found for : increased number of mitotic figures, drop shape rete
ridges, increased nuclear size and abnormal variation in cell shape. On the other hand,
irregular epithelial stratification, loss of polarity of basal cells, abnormal variation in nuclear
size, atypical mitotic figures and hyperchromatism had high disagreement scores.
Considering the problems in making reliable distinctions between the different
grades the Working Group considered collapsing the four grades to two when reporting the
presence or absence of epithelial dysplasia: ‘no/questionable / mild’ as low risk, ‘moderate
or severe’ implying high risk. If mild dysplasia is used as the cutoff point when deciding
whether or not to remove the lesion surgically, near misses of grading are not particularly
relevant in this context, and the variability could be significantly reduced by having two
grades. The working group considered the two class classification and was of the view that
reducing the number of choices from 3 to 2 may increase the likelihood of agreement
between pathologists. The utility of this was recently tested and has been shown to have
merit in that better agreement was reached between those experienced in examining oral
biopsies with improvement in kappa agreements.54
Several points to increase the objectivity were put forward and authors commented
on various factors that could be responsible for the wide inter & intra observer variability
encountered in oral epithelial dysplasia.
The points considered were-
To enforce a systematic way of evaluation for the participating pathologist; they could
start examining the slides by looking at each individual feature of architecture and
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44
cytological changes, and then they could build up their judgment on the degree of
dysplasia on the basis of evaluating these features.
The uncertainty on how to classify a particular lesion among pathologist could be
overcome by objective scoring for each criterion of dysplasia features.
Another source of variation might be related to the difference in the understanding of
these features in terms of recognition and impact on clinical outcomes. They felt that
even though only certain features were associated significantly with the clinical
outcomes, other features which showed no statistical significant association are also
important for the whole grading process.
Lack of standardized method for oral epithelial dysplasia; Although, the 2005 WHO
classification defined each category and pointed out how to grade the oral epithelial
dysplasia lesions, no definitive or exemplar photomicrographs that could illustrate the
individual architectural and cytological features were included .In the absence of such
information, assessment of these features depend on the educational process of grading
oral epithelial dysplasia.
It is the mission of the authoritative and regulatory bodies of the oral pathology
profession over the world to carry out more workshops, studies & meetings to improve
the educational process of grading oral epithelial dysplasia. A minimum data set similar
to that of Royal College of Pathologists (UK) for reporting head and neck cancer should
be set up.
To minimize the heterogeneity in reporting oral epithelial dysplasia; some pathologists
usually grade some oral epithelial dysplasia lesions as having features of mild dysplasia
with focal areas of moderate dysplasia. This essentially confuses the clinicians. The
advantage of minimum data set would minimize the heterogeneity of the future research
on epithelial dysplasia and helps to have homogenous data when meta-analysis or
systemic review is attempted.
Use of molecular markers: several papers exploring the prognostic and predictive values
of different molecular markers on oral epithelial dysplasia lesions are already published.
However it is not known how consistent the authors of these papers were in their
histological grading of the studied oral epithelial dysplasia lesions.
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45
Reduction in number of categories in grading dysplasia: Studies on grading dysplasia
from other sites of the body demonstrated that increase in inter observer agreement
between pathologists probably is best accomplished by having fewer categories of
readily separable entities.
To conclude, grading of oral epithelial dysplasia is not an exact science and
pathologists are doing their best to reach an optimal result. This has led to the researches to
search for alternative and advanced methods for objective assessment of dysplasia and
determine its malignant potential. 54
g. VARIOUS METHODS OF EVALUATION OF ORAL EPITHELIAL DYSPLASIA:
The assessment of epithelial dysplasia by light microscopic examination of
hematoxylin and eosin stained tissue sections so far is not totally satisfactory. The grading of
dysplasia is dependent on morphological experience of the pathologist, thus it is more or less
an expression of a nominalistic concept of disease. There have been a number of other
approaches to the problem of grading dysplasia with the aim to establish a more
fundamental molecular and biochemical basis for assessing dysplastic lesions. These studies
correlate the molecular and biochemical markers with the grading of dysplasia which was
used as “gold standard” to establish a more objective method of assessing epithelial
dysplasia. Quantifiability and reproducibility could be added virtues as suggested by
Burkhardt A (1985).55
The methods are as follows : Burkhardt (1985)55, Brennan (2007)56
A. Morphological methods: Exfoliative and aspiration cytology, Quantification of
histopathology : Computer aided analysis, Stereological technique and Use of image
analysis, Histochemistry, Immunohistochemistry, In situ hybridization, Electron
microscopy.
B. Functional methods: Cellular proliferation studies, Analysis of immune status,
DNA histograms, Analysis of cell products in circulating blood, Experimental
models, Microsattellite analysis / Loss of Heterozygosity, Microarray technology .56
Exfoliative cytology was seen to be of limited use, amongst the different methods
for cytological and histological evaluation of dysplasia, as it gave high false negative results
and little information was obtained regarding the changes in the deeper layers of oral
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46
mucosa.55 Aspiration cytology was seen to give better results than the smear, but it is a
micro invasive procedure.57 The micronucleus test done on the exfoliated cells as described
by Heddle JA (1973),58 was said to be a screening method for the assessment of
chromosomal damage and thus of mutagenic influences on the mucosa. An elevated
frequency of micronuclei, which were chromatin particles derived from acentric
chromosomal fragments was suggested to be indicative of the mutagenic influences.
Kramer (1970)59 said that by combining the ability of the pathologist to recognize
dysplastic features with the potential of the computer for pattern recognition, a diagnostic
hint can be obtained by processing the histological data by computer. But they opine that the
ultimate result depends on the recognition of the different dysplastic features by the
pathologist and hence prone for subjective alterations.
Franklin CD et al (1980)60 described stereological or two dimensional morphometry
technique by measuring dysplastic parameters on light or electron microscopic level. In an
experimental study on carcinogenesis, they found 3 histological features which were able to
differentiate benign and premalignant lesions, namely increase in nuclear cytoplasmic ratio,
nuclear chromatism, epithelial / mesenchymal interface. Since they found that many of the
parameters measured by these methods could be readily estimated by the human eye, the
quantification of the same was found to be unnecessary. Whether it plays any part in routine
diagnosis was not clear.55
Cahn LR et al (1962)61 noted that the chemical characteristics in the epithelium of
the premalignant lesions could be studied by application of different stains to the dysplastic
tissue sections, for example, they report that the PAS reaction indicated the presence or
absence of the glycogen. Burkhardt A (1985)55 concluded that the simple histochemistry
gave an insight into the understanding of the chemical nature in dysplastic lesions, but had
no diagnostic significance. Further he found the metabolic requirements of malignant and
premalignant cells to differ from those of the tissue of origin, by their enzyme
histochemical studies. Of the many different enzymes studied, the shift from predominantly
aerobic respiration to predominantly anaerobic glycolysis in many premalignant and
malignant cells was seen to be the fundamental metabolic change observed by Johnson et al
(1980).62 Besides the enzymes engaged in energy metabolism, Burkhardt A (1985)55 also
noted that the activities of hydrolytic enzymes may be altered in these lesions, and that an
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increase in the production of hydrolytic enzymes by tumour cells may be related to their
invasive capacity. Although the use of enzyme histochemistry for routine purposes has been
stressed by Heyden C et al (1974),63 Burkhardt A (1985)55 opines that the variety of factors
such as sensitivity of the reactions, duration and temperature during transport, marked
regional tissue variations, greater work load in the success of immunohistochemistry,
resulting in inaccurate, variable and unreliable results lead to the apparent loss of interest in
enzyme histochemistry.
Immunohistochemistry was a very promising field of research which had
contributed much to our understanding of tissue alterations in different premalignant and
malignant lesions.55 He reviewed and stated that, since its inception by Coons A. H et al in
1941 stated that, there had been enormous and continued development in this field, in
pursuit of better methods and thus in the better understanding of premalignant and malignant
lesions. He further stated that the development of this method had been made possible, that
such tests could be applied on paraffin sections of fixed tissues and also made highly
specific monoclonal antibodies available. The aim of most of the immunohistochemical
studies had been to determine tumour associated antigenic constituents, to study the
variation in the expression of some antigens in normal, premalignant, malignant and in other
lesions, in other words to find a specific "Tumour marker". The various investigations had
been focused on epithelial surface antigens, intracellular products, constituents of the
basement membrane zone and changes in the adjacent tissue including the so called ‘stromal
reaction’.
The following markers have been used to assess dysplasia as stated by Scully C 64 :
1. Cell surface antigens: a. Carbohydrates. b. ABH blood group antigens. c.Thompson-
Friedrich antigen. d. Growth factors and receptors (EGF, EGFR). e. Integrins f.
Granulocyte colony stimulating factor receptor.
2. Intracellular components: a. Cytokeratins. b. Monoclonal antibody 17.13
3. Oncogenes : a. Tumor suppressor and apoptosis related genes – p53, Bcl-2. b. Cell
cycle regulators. c. Proliferation markers: PCNA, Ki-67, BrdU
4. Tissue Markers : a. Heat Shock Proteins. b. Matrix Metalloproteinases. c.Vascular
Endothelial growth factor
5. AgNOR
6. DNA content (DNA ploidy)
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Silver binding nuclear organizer regions: AgNOR: Silver binding nuclear organizer
regions (AgNOR) are loops of ribosomal DNA that can transcribe for ribosomal RNA. In a
malignant cell, chromosome disarray with multiple nucleoli appears to result in an increase
in AgNOR counts suggesting poor prognosis for oral cancer. Mean count and area of
distribution of AgNOR within the nucleus was useful in diagnosing histological features
representing oral epithelial dysplasia.
In a study by Ray JG et al (2003)65, a mean Ag NOR count of 2.37 was used as a
potential cut point to distinguish between non dysplastic leukoplakia and dysplastic
leukoplakia. They compared epithelial dysplasia grading with mean AgNOR count as the
new diagnostic test due to its quantifiability and objective status and minimized
disagreements. Both the methods correlated in 65% of lesions. 17.3% of cases classified as
non-dysplastic by H & E method also had a high AgNOR count and were classified as
dysplastic by AgNOR. At the same time, 19.2% of cases classified as dysplastic by H&E
were classified as nondysplastic by the AgNOR count.
However, since mean AgNOR count of a lesion was an easily reproducible measure,
it would serve as a more objective criterion than the hematoxylin and eosin stain based
assessment for epithelial dysplasia. This proved AgNOR as a confirmatory adjunct to H&E
for diagnosing epithelial dysplasia. Additional attributes for use AgNOR are it is easy to
perform, is inexpensive & can be performed on paraffin embedded specimen. 65
Gross genomic alterations: (DNA ploidy status) (Scully C, 2003)66
DNA ploidy is a measurement of nuclear DNA content and forms a surrogate measure
of gross genetic damage. DNA ploidy status in the cells of the lesion suspected to be
premalignant had implications for the prognosis of the lesions. Subudo J et al (2001)34
conducted a study of the DNA content of 150 dysplastic oral leukoplakic lesions. They
graded the dysplasia according to WHO criteria. The genomic content was measured by
DNA image cytometry and the lesions were divided into: a) Aneuploid (high risk) lesions b)
Diploid (low risk) lesions and c) Tetraploid lesions (Intermediate risk).
They compared the genomic status with the histologic grade and the follow up clinical
outcome. It was found that no statistically significant correlation existed between histologic
grade and the DNA content of the dysplastic lesions. However, 3% of the low risk diploid
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49
lesions, 60% of the intermediate risk tetraploid lesions and 84% of high risk aneuploid
lesions transformed into malignancy.
In a study of 150 patients with dysplastic oral lesions followed up for a mean of 18.6
years, it was noted that three of 105 diploid cases as against 21of 25 aneuploidy cases
developed into oral squamous cell carcinoma giving a positive predictive value of 84%. It is
significantly noted that lesions regarded as mild dysplasia histologically were aneuploid &
progressed to squamous cell carcinoma thus enhancing its efficacy in determining genetic
change ahead of histology.
The DNA ploidy status thus gave promising results as an objective marker for risk
assessment as it gave high positive and negative predictive values. The advantages include
use of automated cytometry on nuclei obtained from routinely processed samples. Thus
DNA ploidy may form a more realistic option to measure gross genomic damage.
Immunohistochemistry could be the most promising & reliable method of study at a
cellular level, and its use in grading of epithelial dysplasia could help in understanding them
better and in arriving at a universal grading system. But results with immunohistochemistry
have to be regarded with great caution until the reliability is well established and with use of
adequate standards.66
The in situ hybridization technique was thought to be of limited use in studying
premalignant lesions, as false positive results could be obtained and it had to be corroborated
by molecular hybridization to label oral carcinomas. Electron microscopy gives an insight
into the ultra-structural details of cellular differentiation in epithelial tissues and certain of
these were thought to be indicative of dysplastic lesions. Thus by knowing the defined
criteria, dysplastic lesions could be differentiated from normal tissue .As the quantification
of each feature is prone for subjective evaluation, the grading of epithelial dysplasia
becomes difficult.55 Banoczy J et al (1980)67, in their SEM studies found changes of cell
surface in dysplasia in the form of atypical or irregular pattern of microvilli, pleomorphism
and disruption of microridges. Burkhardt A (1985)55 stated that the simple electron
microscopy and SEM added to the understanding of the biology of disturbances of epithelial
differentiation in malignancybut do not contribute to routine diagnostic procedure.67
Besides these morphological methods described, a more functional type of
approach to the problem in assessing dysplastic lesions are also available. Hume WJ and
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Potten CS (1979)68 extensively reviewed methods in the study of cellular proliferation.
Such studies included the counting of mitotic figures in conventional tissue slides (Mitotic
index) or after mitotic arrest in metaphase, and radioactive labeling with titrated thymidine.
The estimation of mitotic index was of limited use in epithelial dysplasia due to various
reasons such as - diurnal variations, difficulty in obtaining normal control values, the
increase in mitotic index not being specific of epithelial dysplasia, as it was also seen in
wound healing etc. Though radioactive labeling was described to be the most reliable
method their applications for obvious reasons was seen to be limited in humans. In vitro
methods of the same were of questionable biological significance.55, 68
The analysis of the changes in the immunostatus of an individual with premalignant
lesions did not lead to any affirmative test that could aid in diagnosing or understanding
them better. Bier J et al (1983)69 stated that the changes noted were all non-specific and
hence did not bear any prognostic implications. Kovesi G and Fekete B (1982)70 were of the
view that the assay of modified leukocyte adherence inhibition shows promising results as a
test for cancer. Tests for the analysis of the cell products such as enzymes involved in
metabolism of nucleic acids etc in serum, was done to find out if any significant changes in
these cell products helped in the prediction of malignant change. The reliability of these tests
however was not clear to be routinely used.
DNA histograms obtained by cytophotometric measurement for oral dysplastic
lesions, based on cells suspension/cytological smears following staining of nuclear DNA
have been applied. Saku T & Sato E (1983)71 put forward a dysplastic pattern of DNA
histograms suggestive of high diploid deviation of DNA ploidy pattern thought to have high
risk for malignant transformation. But this technique has high percentage of false negativity,
cannot be done on tissue section, it is time consuming, requires expensive hardware and
considerable experience therefore its value in conventional histology appears still to be
assessed.
Scully C. et al (1981)72 emphasized that analysis of cell products in circulating blood
could provide promising results. They reported an increase in concentration of β2
microglobulin, alkaline deoxyribonuclease and thymidine kinase in serum of patients with
oral dysplasia and cancer. High serum levels of deoxyribonuclease activity in dysplasia can
be a sign of poor prognosis but too little is known about the reliability and usefulness of
these tests.
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Evson JW (1981)73 stated that in experimental studies using animal model like
hamster, mice and rats can help in assessing the progress of epithelial dysplasia which could
contribute to prevention and elimination of factors enhancing the development of
malignancy.
Microsattellite markers using DNA sequences which are relatively short tandem
repeats of nucleotide sequences, and one to four base pair units are used to detect the
allelic imbalance or loss of heterozygocity (LOH).66 Lee JS (1993); Shin DM (2001)74, 75
High frequency of LOH at 3P,9q & 17P was seen in dysplastic lesions. LOH at 9P 21 also
commonly occurs in dysplasia. Dysplasia may show chromosomal polysomy which is
associated with genomic instability reported to be increasing during tumorigenesis.
The assessment of multiple marker is complex and the techniques are not routinely
available therefore simpler and less expensive methods are usually sought. Recent advances
has made it possible of identifying molecular changes in exfoliated cells such as in saliva.76
A study done by Spafford K et al (2001)77 revealed genetic changes in exfoliated cells
identical to those found in oral squamous cell carcinoma in 80% of saliva samples.
Microarray analysis : The advent of microarray technology has allowed for
screening of the entire genome. This contrasts sharply with prior molecular biology
techniques that identified individual genes alone and thus has the potential to disclose new
pathways in the pathophysiology of dysplasia. The genes found to be unregulated in
dysplastic tissue include inflammation-related genes, cyclo-oxygenease-2(COX-2PTGS2)
and several gene receptors (PTGER 3b1, a2). Further studies are required for the validation
of role of these genes in dysplasia.56
Burkhardt A (1985)55 concluded that the future development and progress in these
advanced methods were to be expected as there were many promising aspects in these
techniques. But they suggested that at present there are still many questions related to the
diagnosis and grading of dysplastic lesions. Hence, they said that the assessment of
biological potential of oral epithelial dysplasia still relies on the “light microscopic
histological examination" and there was no alternative to this old and well proven approach.
Further they added that any advanced method apt to replace light microscopy must be able
to predict the true biological potential more accurately than the conventional histology.
Additionally, the aim of any advanced method must be to unmask those cases with
seemingly harmless histological picture but progress to malignancy.
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Field cancerization and oral cancer
The concept and term ‘field cancerization’ was first put forward by Slaughter and
his co-workers in 1953. According to them, cancer develops multifocally as a result of
diffused and repeated carcinogenic assault and progresses at various rates within the entire
field. Slaughter stated that “Cancer does not arise as an isolated cellular phenomenon but
rather as an anaplastic tendency involving many cells at once. Subsequently the term,
“lateral cancerization” was used to indicate that the lateral spread of tumors was due to
progressive transformation of cells adjacent to a tumor, rather than the spread & destruction
of adjacent epithelium by preexisting cancer cells. The entire mucosa within a field due to
its high risk of malignant transformation has been referred to as “Condemned mucosa”. 78
This term encompassed the following issues: 1) Multiple cells form independent epithelial
tumors
2) Carcinogenic exposure affects multiple cells in a field.
Carcinogenesis is a multistep process and evidences indicate that presence of a field
lesion with genetically altered cells is a distinct biological stage in epithelial carcinogenesis
with important clinical implications.
Slaughter in 1953 defined field cancerization as “Increased risk of cancer
development in entire upper aerodigestive tract due to multiple genetic abnormalities in the
whole region after prolonged exposure to carcinogen”. Tobacco followed by alcohol has
been implied as the most common carcinogen. Boudewijin et al (2003)78 have proposed a
definition of field cancerization based on molecular findings as “the presence of one or more
areas consisting of epithelial cells that have genetic alterations. A field lesion (or shortly
field) has a monoclonal origin, and does not show invasive growth & metastatic behavior,
the hallmark criteria of cancer”. They state that a field lesion is preneoplastic and it may
have histological aberrations characteristic for dysplasia.
Gabriel DD et al (2007)79 described field cancerization as “the process where by
cells in a particular tissue or organ are transformed such that the genetically altered but
histologically normal appearing cells predate the development of neoplasia or coexist with
malignant cells, irrespective of clonality”.
Theories of field cancerization : (Monique G et al 2000)80
1. Multiple lesions develop independent of each other due to multiple genetic abnormalities
in the whole region following exposure to carcinogens. This theory is most accepted.
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53
2. Other theory states that any transforming event is rare & suggests that multiple lesions
arrive due to widespread migration of transformed cells through the whole aerodigestive
tract. Two types of migration may be involved: (a) Migration of tumor cells via saliva:
e.g. micrometastasis (b) Intraepithelial migration of the progeny of the initially
transformed cells.
To understand the concept of field cancerization studies have evaluated these two
theories. Tumor associated mucosa (TAM) of smoking head and neck squamous cell
carcinoma patients show alterations not found in non smoking patients. Suggesting that
altered cells are harbored in associated margins of tumor in smoking patients, which are
absent in non-smoking patients. If the migration theory is considered, then changes should
have been observed in normal tumor associated mucosa of non smoking patients.
Additionally, it was also found that field changes like increase in nuclear area, proliferation,
p53 mutation were even observed in healthy smokers without tumor being a source for these
cells. Therefore, these results support the idea that field changes in head and neck squamous
cell carcinoma patients are carcinogen induced independent events than being a result of
migration of transformed cells.
There is a co mm on cl on al or ig in in multiple primary tumors even if the lesions
were more than 7 cm. This was based on similarity of genetic changes. Three theories have
been proposed to explain the common clonal origin of multiple primary tumors : first single
cell or small clusters of cells migrate through the submucosa, secondly they are shed in
lumen of an organ (oral cavity) at one place & regrow at another. The most recent finding in
head and neck is that a large contiguous genetically altered field exists in the epithelium
from which multiple clonally related neoplastic lesions develop which is explained based on
studies by Jang SJ et al (2001) 81; Braakhuis BJ et al (2002)82; Simon R (2001). 83 This gives
impetus to lateral clonal spread of the cancer giving rise to new primaries. Based on this
Braakhuis BJ et al (2002) suggested the use of the term ‘second field tumors’ for those
lesions that are anatomically distinct but demonstrate genetical similarities & true second
primaries are those lesions that did not share genetic similarity and develop as independent
events. 82
Recently studies have shown presence of large altered field in the epithelium in
which multiple clonally related neoplastic lesions develop. This indicates that a large
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54
proportion of multiple primary tumors in the same or adjacent anatomical area have
developed within a single preneoplastic field. Based on this, a genetic progression model for
oral cancer was put forward where field change plays a central role as stated by Boudewijin
JM et al (2004).84
Patch is a clonal unit (2 mm in diameter) of mutated cells i.e. genetic change in stem
cell due to p53 mutation and has transformed this mutation to its daughter cells. This patch
appears to be first manifestation of oral cancer. Patch is cluster of cells showing p53
mutation.
Field of genetically altered cells -Tumor adjacent normal epithelium demonstrates genetic
alteration making it a field at risk. They are larger than patches, atleast 4mm in diameter but
may be over 7cm also. They show allelic loss at various chromosome arms 3p, 9p,17p &
may precede development of cancer.
Studies on Normal appearing mucosa adjacent to squamous cell carcinoma for
evaluation of field cancerization
Morphological changes : In tissue from adjacent normal margins of oral squamous cell
carcinoma (OSCC) may show epithelial hyperplasia, hyperkeratinization with
accompanying atrophy and fibrosis of the submucosa, round cell infiltration and capillary
telangectasia. Dyskaryosis with marked atypia, obvious in-situ or carcinoma is also reported
in studies. These findings are evidence of multi-centric origin and represent lateral spread of
oral squamous cell carcinoma.
Wright A et al (1985)85 studied histologically presence of dysplasia in epithelium
immediately adjacent to neoplasm in 80 cases of excised OSCC based on the concept that
most neoplasm’s are frequently preceded by precancerous lesions in an altered field. 80% of
cases showed presence of dysplasia and most common features seen were presence of basal
cell hyperplasia, nuclear hyperchromatism, loss of polarity, loss of adherence and disturbed
epithelial maturation. The occurrence of increased mitosis and abnormal mitosis was
relatively infrequent. One more important finding was that the dysplastic changes in the
adjacent epithelium were usually multi-centric.
Field change in oral cancer by quantitative assessment of cytomorphology of smears
taken from normal buccal mucosa in oral cancer & cancer free patients was reported by
Ogden GR et al(1990).86 They tried to detect changes arising in tissue peripheral to cancer
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55
and effects of potential etiological factors (smoking) on occurrence of field change. A
statistically significant reduction in cytoplasmic area in normal mucosa of cancer group was
seen which suggested that decrease in cytoplasmic area preceded an increase in nuclear area
within tissue that become malignant. The authors believe that this decrease in cytoplasmic
area may not be related to smoking as half the patients did not smoke. This finding is
supported by clinical observation of patients developing second primary tumors even after
cessation of smoking. They concluded that normal oral mucosa of cancer patients is
different from that of cancer free individuals and this appears not to be dependent of alcohol
or tobacco. Cancer free smokers though showed a slight increase in nuclear area as
compared to cancer free non smokers.
Kannan S. et al (1996)87 examined fifteen cases of adjacent oral epithelium to OSCC
were under light & electron microscopy. They performed the incisional biopsy in mucosa
immediately adjacent to the cancer. 67% of the lesions exhibited hyperplasia or mild
dysplasia while 33% showed moderate to severe dysplasia.
Ultra-structural observation of all the biopsies showed subcellular alterations deep in
the lower half of the epithelium similar to those seen in frank oral carcinomas. The
important changes observed were bizarre nuclei of basal and lower spinal cells, enlarged &
multiple nuclei, presence of inter chromatin and perichromatin granules, loss of desmosomes
and marked spongiosis as well as disturbed cellular maturation evidenced by abnormal and
irregular distribution of maturation markers like keratohyaline granules and tonofilaments.
In addition, basal lamina was very thin but was intact thus heralding its breakdown in future.
The authors believe that electron microscopy could be valuable in detecting subtle changes
due to field cancerization in adjacent epithelium of OSCC.
Thompson PJ et al(2002)88 undertook study to quantify the incidence of field change
in oral mucosa by taking ‘mirror image’ biopsies in 26 patients with unilateral OSCC from
clinically normal looking mucosa at corresponding anatomic sites. 58% of patient showed
histologically abnormal tissue on microscopic examination consisting of reactive change or
cellular atypia associated with chronic irritation, frank dysplasia, carcinoma in-situ or micro
invasive squamous cell carcinoma. The authors also found increased vulnerability for
dysplastic change in lesions present in the lateral / ventral part of tongue & floor of the
mouth though these observations were not statistically significant. They suggest use of
multiple biopsies, long term follow up & use of chemoprevention.
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h. MOLECULAR ASPECTS OF POTENTIALLY MALIGNANT DISORDERS:
Cytokeratins are intermediate filament proteins found in cytoplasm of all epithelial
cells. There are at least 20 different keratins polypeptides that are expressed in different
combinations, depending on type of epithelium and degree of differentiation. Presence of
cytokeratins 7, 8, 13, 16, 19 was observed at abnormal anatomical sites or at abnormal
intraepithelial levels in normal mucosa from head and neck OSCC patients as observed in
studies by Copper MP et al (1993)89; Ogden GR( 1993)90; Bongers V et al (1996)91; Bosch
FX et al (1989).92
Blood group antigens are cell surface carbohydrates. These show changes in
expression related to tissue type, differentiation state and cell motility capacity. Type 2 chain
ABH carbohydrate structures are distributed broadly in epithelial and endothelial cells
independent of patients ABO blood group. In normal oral epithelium, type 2 chain ABO
blood group antigens are expressed in parabasal cells. Bonger et al (1996)91 in their study
found a fourfold lower expression of type 2 chain ABH antigen in exfoliated cells from
normal mucosa from six different places distant from squamous cell carcinoma as compared
with healthy individuals. They believe that this may be a negative marker for field change
and risk indication.
Cyclins are cell cycle regulators and are functional when associated with their
concomitant Cyclin dependent kinases. Cyclin D1 regulates the G1-S transition and is
associated with CdK4 and 6. Clearly defined foci of Cyclin D1 expression in sections of
normal mucosa adjacent to squamous cell carcinoma was observed which was not observed
in the sections of normal mucosa of healthy individuals as reported by Bartkova J et al
(1995).93
King LE et al (1985)94 have shown increased expression of epidermal growth
factor receptor (EGFR) in tumor associate normal mucosa which could be due to elevated
mRNA or amplification of EGFR gene. Ligand binding to the extracellular domain of EGFR
causes receptor dimerization which activates tyronine kinase function. This leads to
autophospharylation, resulting in proliferation. EGFR expression was high in
nonsmoking/non drinking head and neck OSCC patients as well as in smoking/drinking
head and neck squamous cell carcinoma patients. It was also observed that the EGFR
expression was less elevated when the epithelium was located more distant to the tumor.
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These results suggest paracrine effect on EGFR expression due to factors related to tumor
and not due to carcinogen (smoking).
Oijen V et al(1998)95 demonstrated increased number of proliferating cells using the
marker Ki 67 in both adjacent tumor tissue and control oral epithelium in smokers and non
smoker in the absence of histologically visible epithelial alterations and suggested that high
proliferation increases the likelihood of malignant change.
Bcl2 is an anti-apoptotic markers. Its family plays an important role in regulation of
the apoptotic pathway. In normal tissue there is a balance between cell proliferation and
apoptosis (programmed cell death). Alterations in both pathways contribute to the clonal
expression of cancer cells. There was lack of bcl2 expression adjacent normal tumor
associated mucosa in OSCC as compared to healthy control mucosa. The authors suggest
that the expression of bcl2 has to be interpreted in the context of levels of other bcl2 and Bax
family members as stated in the study by Birchal MA et al (1997).96
The expression of all gluthione S-transferase isoenzyme was significantly higher in
suprabasal and superficial layers of normal oral mucosa of OSCC patients who subsequently
developed second primary tumors as compared to its expression in normal mucosa from
OSCC patients who were free of disease after 7 years follow-up reported Bonger V et
al(1995).97 The authors believe that this increase is intriguing as elevated levels of their
detoxification enzymes actually protect against carcinogenic attacks and probably this might
represent a futile effort for combating the carcinogenic metabolites in tobacco. They believe
that a high level of this enzyme has predictive value for development of second primary
tumors.
Loss of function of tumor suppressor gene p53 might result in uncontrolled cell
division and genomic instability. Mutant p53 has higher stability than wild type p53, which
allows accumulation to the levels detectable by immunohistochemistry. Oijen V et al
(1999)98 observed a uniformly spread focal over expression of p53 occurred more frequently
in normal epithelium of smoking OSCC patients as compared to nonsmoking OSCC patients
and from healthy controls. Increase p53 cell cluster were not detected in mucosa of healthy
smokers. Tobacco in addition to other genetic and environmental factors could contribute to
abundance of p53 cell cluster in tumor associated mucosa. All these findings suggest that
p53 plays a pivotal role in early carcinogenesis and development of field cancerization. In
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the recent patch field concept put forward by Boudewjan JM et al (2004)84 p53 positive cell
clusters are considered as the initial change (patch) in the progression of field cancerization.
Thus, to summarize the data on these molecular markers, it appears that normal
mucosa associated with carcinoma shows a range of alteration and genetic abnormalities.
Additional research is required to assess which field changes depicted by these molecular
markers have actual carcinogenetic influence & which are merely epiphenomenal.
Various clonal markers have been used to explain whether oral field cancerization
is by migration or independency theory. Patrick KH et al (2003)99 stated if multiple primary
tumors arise due to migration of malignant cells from a primary source, then all should show
identical genetic alterations. In case of independent origin, these alterations will be different.
To qualify as a clonal marker, such a genetic alteration should occur early in the
development of the primary lesion, be maintained during the progression of the lesion,
exhibit sufficient variability, be applicable in the majority of the lesions and should not
occur by chance & be present 100% of time.
Karyotype is the number and appearance of chromosome in the nucleus of
eukaryotic cell arrested in metaphase between two lesions and comparing their appearance
and search for ploidy and chromosomal breaks/rearrangements. Karyotypes of tumor have
been used as clonal markers. However, this is a rather insensitive method of detection and
requires tissue culture which in turn causes additional genetic alterations so it may not be
accurate reported by Worsham MJ et al (1995).100
Partridge M et al (2000)101 reported a case control study using microsatellite assay
with markers like 3p, 8p, 9p, 13q, Rb was done in two group of patients, one with dysplasia
who subsequently developed carcinoma on same side of mouth and another group with
similar lesions who did not develop lesions on a fixed time of follow up to identify high risk
individual for development of oral squamous cell carcinoma in a field of cancerization. They
found that dysplastic lesions harboring allelic imbalance at more polymorphic loci within
critical chromosomal regions developed tumor more frequently than matched controls with
allelic imbalance at fewer loci. They suggested that although dysplastic lesions may not be
precursor lesions per se but they developed in fields that harbored the genetic alterations
associated with tumorgenesis because microsatellite assay could identify the benign from the
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more troublesome true precursor lesions. Process of field cancerization is very extensive
and excision of lesion might leave back mucosa which harbors aberrations that cannot be
seen. Thus normal appearing mucosa may have source of cells for development of
subsequent tumors. This risk could be detected by use of allelic imbalance for prediction of
subsequent malignancy. Based on their findings, the authors advocate complete excision of
all dysplastic lesions showing allelic imbalance of two more key microsatellite markers
regardless of degree of dysplasia to produce margins that are atleast morphologically normal
followed by dietary advice and chemoprevention. They suggest the screening of clinically
normal mucosa as a routine work up in head and neck cancer patients by histology or
molecular tests if possible to establish evidence of field cancerization and for overall risk
assessment.
Tabor BRH et al (2001)102 quantitatively measured loss of heterozygosity in head
and neck squamous cell carcinoma. They reported 10 out of 28 of patients have tumor
associated genetic alterations in biopsy taken from macroscopically normal mucosa adjacent
to the tumor. This frequency was based on analysis of 4 biopsies that were taken in each
quadrant surrounding the tumors using 15 microsatellite markers in 6 chromosomes. In
majority of cases (7 of 10), there genetically altered cells were also found in margins of
specimens removed by the surgeon. These lesions were more than 4mm in diameter and
contained more than 50% of aberrant cells.
Analysis of mitochondria DNA markers by Ha PK et al (2002)103 in normal
adjacent mucosa of dysplastic lesions and identical mitochondrial DNA mutations were
found in 3 out of 8 lesions suggesting a monoclonal origin. They emphasized that these
changes could be detected in saliva and their levels decrease with treatment indicating its
use in early detection and monitoring of oral cancer.
Chromosomal Aberrations: Genomic instability increases the risk to develop head
and neck squamous cell carcinoma. Analysis of normal and malignant lesion adjacent to
head and neck tumors for chromosome instability using in situ hybridization has suggested
that molecular aberration can be detected before histological evidence of morphologic
changes.
A whole series of chromosomal changes were observed by Ai H et al (1999)104 using
fluorescence in-situ hybridization in cells from brushes of macroscopically normal cheek
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mucosa opposite the site of OSCC and correlated with smoking. A trend towards
aneusomies of chromosome 2, 6 and Y was observed in normal mucosa from smokers.
RNA expression analysis of oral keratinocytes can be used to detect early stages of
oral cancers was stated by Joel L et al (2008).105 RNA analysis was done using hamster
mucosa brush cytology samples measured with RTPCR. The authors suggested that brush
oral cytology may prove useful as a source of RNA for gene expression analysis during the
progression of diseases of oral epithelium such as OSCC.105
Taboor MP et al (2001)106 suggested the presence of genetic alterations in the mucosa
samples was found to be associated with histopathological changes. The majority of the
genetically altered fields were classified as dysplastic, however the cases without genetic
alterations were observed in the mild dysplasia group.
i. CORRELATION OF DYSPLASIA WITH EVENTUAL MALIGNANT TRANSFORMATION:
The development of a malignancy in stratified squamous epithelium occurs
spontaneously or as a gradual process in which multiple minor individual cellular and tissue
alterations eventually culminate in frank malignancy.14
There is a generally held view that assessment of dysplasia in pre-malignant lesions
is important because dysplastic lesions are more likely to undergo malignant change and
because it is felt that the chances of malignant transformation increase with increasing
severity of dysplasia. There is a broad measure of agreement among pathologists about the
altered epithelial features that comprise dysplasia, but there is great variation in interpreting
the severity of dysplasia and not all investigators describe the criteria they use.30
Banoczy J and Csiba A (1976)38 diagnosed a case as showing dysplasia when it had
two or more histological dysplastic features. This would tend to increase the proportion of
cases described as dysplastic by inclusion of those with minimal dysplasia that might not be
regarded as dysplastic by other pathologists. In their series of 500 cases of clinical
leukoplakia, 120 showed dysplasia. At follow-up, 13.2% of these dysplasia progressed to
malignancy. Dysplasia was graded into three categories of severity on the basis of the
numbers of histological dysplastic features evident. In their series, all of the instances of
malignant transformation arose from cases with dysplasia, as assessed by their criteria,
evident on biopsy.38
Pindborg JJ, Daftary DK & Mehta FS (1977)107 investigated carcinomas developing
from non-dysplastic lesions in a rural Indian population. They found malignant
i
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transformation after a shorter average time follow-up in dysplastic lesions, and this change
also occurred in a younger age group.
The data from Silverman S et al (1984)108, support the concept that dysplasia is an
important predictor of risk. However, 82% of the cases of malignant transformation were in
patients diagnosed as not showing dysplasia on biopsy. They studied 257 patients in
the USA with leukoplakia, of whom 45 progressed to carcinoma in the follow-up period of
eight years. Only 22 of these patients had dysplasia in the initial biopsy. They do not state
their criteria for recording a case as dysplastic. Biopsies that other pathologists would have
regarded as containing mild dysplasia might not have been designated as such. Of the 45
cases that progressed to malignancy, 8 were derived from the 22 cases with dysplasia,
suggesting a 36% incidence of malignant transformation as contrasts with the 15.7%
incidence in the cases not showing dysplasia on biopsy.
Such a follow-up studies suggest that dysplastic lesions are more likely to proceed to
carcinoma than non-dysplastic lesions, and they also provide some evidence in support of
the concept that more severe dysplasia is associated with increased risk. However, the grade
of dysplasia is not a reliable predictor of prognosis. It also appears that carcinoma can
develop from an epithelium showing only mild dysplasia or no dysplasia. This could be a
false conclusion as it might be due to the wrong site being biopsied by bad judgment or due
to bad luck. It is also important to stress that not all cases in which dysplastic epithelium is
present inevitably progress to carcinoma.30
The microscopic features of dysplasia are now relatively well established.
Lesions with specific dysplastic grades or changes have been followed in order to
determine their natural history. This is difficult because of the following facts: 1. the
biopsy procedure itself has removed the cells upon which the diagnosis is based, 2. the
more severe dysplasia are typically removed or destroyed prior to follow-up, 3. the
grading of dysplasia is an extremely subjective pursuit and there is often poor correlation
between experienced pathologists.49
Keeping the above facts in mind, it seems to be accepted that the biological
behavior of severe epithelial dysplasia and carcinoma in situ are identical. Investigations
have found that 20.35% of severely dysplastic lesions develop carcinoma, which is
similar to the figures for carcinoma in-situ. At the opposite end of the spectrum, mild
epithelial dysplasia so seldom eventuate in carcinoma, and are so similar to reactive
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epithelial changes, that few pathologist consider them a serious threat or recommend
complete removal of the associated white lesion.109
One of the ironies of oral pre-cancers is the apparent fact that the correlation
between the clinical appearance of leukoplakia and cancer transformation is relatively
good while any microscopic changes less than severe are not helpful in determining
prognosis. Recent investigators have even concluded that they were better able to
correlate the presence of papillomavirus with the clinical appearance of oral pre-cancers
than with the microscopic grade of dysplasia.109
There is a substantial need to improve the histologic assessment of epithelial
dysplasia, since epithelial dysplasia does not seem to be invariably associated with or even a
necessary pre-requisite for malignant development, it may be necessary to develop other
methods for predicting the malignant potential of pre-malignant lesions.30
Nevertheless, taking into account all the uncertainties and anomalies of behavior of
these lesions, it does seem justifiable in our present state of knowledge to consider in
general terms that the degree of dysplasia is linked to the degree of probability of the
development of malignancy. In any event, it is a safe rule to advise the complete removal,
wherever possible, of all lesions showing more than slight degree of dysplasia. Also, it
should again be emphasized that there are certain 'high-risk sites', especially the floor of the
mouth and ventral surface of the tongue, where greater attention should be given to even the
lesser degrees of dysplasia.23
Thus, the challenge is to predict which lesions will eventually develop into
carcinoma. Conventional clinical and histopathological aspects are not optimal for decisions
on management, which is influenced by the perceived risk of malignant development.
Resource-consuming management procedures might be spared if the risk of malignant
development could be predicted with reasonable certainty and management of risk patients
could be improved.30
Summerlin DJ (1996)110 states that cases of epithelial dysplasia, however, may
remain static or even regress, whereas examples of squamous cell carcinoma have been
shown to arise de novo. Epithelial dysplasia, though represents a pre-malignant condition
should be treated as if it will progress to carcinoma.
Progress in molecular oncology has significantly advanced our knowledge on
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63
tumorgenesis; yet the practical applications of these genetic markers remain unresolved in
detecting oral dysplasia. None of the molecular markers, single or in combination appear to
be ready for use in routine clinical diagnostic practices, although many molecules have
been studied as intermediate markers. Not many studies have focused correlating the
findings at molecular level with the clinical attributes of terminal outcome. Authors suggest
that studies should aim to develop these markers for routine application with improvement
in sensitivity, predictability, and reproducibility. 16
Recent advances in the use of quantitative high throughput methods has enabled the
profiling of gene expression (microarray), protein expression (proteomics), screening of
epigenetic changes, e.g. by pyro-sequencing and single nucleotide changes (SNP) of
hundreds of pre-selected genes simultaneously. These techniques which require minute
amounts of tissue when carried out in controlled way and using careful selected samples
with proper validation will help the potentially malignant and malignant tissue samples.
Combined with routine histopathology studies, these broad based studies of gene and their
products appear to have a great deal of potential for the identification of diagnostic and
prognostic markers of pre-cancer, and data from multicenter studies using microarray
technology may be engaged in the future for a better understating of aetiopathogenesis of
pre-cancer.16
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Ki-67 PROTEIN :
The Ki-67 protein was originally defined as the prototype monoclonal antibody Ki-
67, which was generated by immunizing mice with nuclei of the Hodgkin lymphoma cell
line L428. The name is derived from the city of origin (Kiel, Germany) and the number of
the original clone in the 96-well plate. Antigen Ki-67 also known as Ki-67 or MKi67 is
a protein that in humans is encoded by the MKi67 gene. The Ki-67 protein is a cellular
marker for proliferation. It is strictly associated with cell proliferation.
During interphase, the Ki-67 antigen can be exclusively detected within the cell
nucleus, whereas in mitosis most of the protein is relocated to the surface of
the chromosomes. Ki-67 protein is present during all active phases of the cell cycle (G1, S,
G2, and M) but is absent from resting cells (G0).111 The estimated half-life of ki-67 antigen is
60-90 minutes. It starts to be expressed in the S phase, progressively increasing through S
and G2 phases and reaching a plateau at mitosis. After cell division, the cells return to G1
with a stock of ki-67 antigen, whose level decreases rapidly during this phase. 112
Antigen Ki-67 (pKi-67) is associated with ribosomal RNA transcription. During
mitosis pKi-67 is present on all chromosomes, forming reticulate structure surrounding
metaphase chromosomes. Inactivation of antigen Ki-67 leads to inhibition of ribosomal
RNA synthesis. The expression of which is strictly associated with cell proliferation but is
absent in non-proliferating cells. This is widely used in pathology as a proliferation
marker to measure the growth fraction of cells in human tumors.113
Ki-67 antibodies have been used widely for the estimation of the growth fraction of
clinical samples of human neoplasms and of normal cells in culture. Molecular and
functional characterization of pKi-67 is necessary for a greater understanding of the role of
the antigen in the cell cycle of normal and of neoplastic cells. Western blotting has revealed
that Ki-67 reacts with two polypeptides of 345 and 395 kDa. A full length cDNA coding for
pKi-67 has been cloned by immunoscreening of human cells. Analysis using the cDNA has
identified two differentially spliced mRNAs encoding polypeptides of predicted molecular
masses of 359 kDa and 320 kDa. Both sequences contains 16 ‘Ki-67 repeat’ sequences, each
of which includes a conserved 66 bp ‘Ki-67 motif’. The deduced amino acid sequence of the
cDNA has revealed two potential nuclear localisation signals, a number of potential
sequences, implicated in proteolysis of the antigen and numerous potential sites for
phosphorylation, amidation, N-myristoylation and ATP/GTP binding. Despite recent
advances in the molecular characterisation of pKi-67, a detailed analysis of the function of
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the antigen is yet to be determined. Significantly, incubation of IM-9 cells with synthetic
antisense deoxyoligonucleotides complementary to the deduced translation start site of pKi-
67 prevents incorporation of thymidine, suggesting that pKi-67 is necessary for cell cycle
progression.
Indirect immunofluorescence has revealed that a large number of antigens are
located exclusively in the nucleolus of interphase cells. A number of antigens which are
located in the nucleolus during interphase associate with chromosomes during mitosis.
Furthermore, a small number of antigens are found in the nucleolus of proliferating cells
only. However, pKi-67 appears unique in that it associates with nucleoli of proliferating
cells only and with chromosomes during mitosis.
The nucleolus contains rRNA gene repeats and is the site of ribosome synthesis.
Nucleoli display a unique higher-order structure. During the cell cycle, nucleoli undergo
characteristic reorganisation. During nucleologenesis, prenucleolar bodies form around
Nucleolar organising regions (NORs) which subsequently fuse to form the interphase
nucleolus. Early in mitosis, nucleoli disperse, and nucleolar antigens become redistributed.
Some nucleolar antigens remain associated with nucleolar domains, whilst others are
dispersed throughout the mitotic cytoplasm or distributed around chromosomes. Ultra
structural analysis of mature nucleoli has defined three distinct nucleolar sub-structures,
each associated with distinct nucleolar functions.
The fibrillar centres (FC) are surrounded by the dense fibrillar components (DFC).
The fibrillar components of the nucleolus are embedded within the granular components
(GC). Many nucleolar proteins are compartmentalised within nucleolar domains. For
instance, fibrillarin is localised within the DFC, nucleophosmin/B23 is localised within the
GC whilst DNA topoisomerase I and RNA polymerase I are localised within the FC.
Compartmentalisation of proteins within the nucleolus reflects compartmentalisation of
specific nucleolar functions.
Since Ki-67 is located within nucleoli of proliferating cells only, it has been
suggested that the antigen may regulate nucleolar metabolism by, for example, increasing
rates of ribosomal synthesis required by rapidly dividing cells. However, it is not entirely
clear with which nucleolar compartment Ki-67 is associated. Immunoelectron microscopy
has revealed that Ki-67 antibodies stain regions surrounding the FC, probably localised
within the DFC. Ki-67 staining is reported to be absent from the FC, GC and nucleolar
interstices.114
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MATRIXMETALLOPROTEINASE (MMP) :
MMPs are a family of structurally related but genetically distinct enzymes that
degrade extracellular matrix and basement membrane components and regulate cell–matrix
composition. MMPs are calcium-dependent zinc-containing endopeptidases belonging to
metizincin superfamily. The interactions of cells with the ECM are critical for the normal
development and function of the organism. By regulating the integrity and composition of
the ECM structure, these enzyme systems play a pivotal role in the control of signals elicited
by matrix molecules, which regulate cell proliferation, differentiation, and cell death. The
turnover and remodeling of ECM must be highly regulated since uncontrolled proteolysis
contributes to abnormal development and to the generation of many pathological conditions
characterized by either excessive degradation or a lack of degradation of ECM
components.115
History : Woessner in 1962 first described MMP as protein enzyme in mammalian uterus
which could degrade collagen. Gross and Lapiere identified MMP, while attempting to
establish how a tadpole loses its tail during metamorphosis and discovered the first member
of this family (MMP-1). A higher molecular mass species (72 kDa) with gelatinolytic
activity in comparison with MMP- 1 was identified as MMP-2. Goldberg and colleagues
sequenced and purified MMP-2 from human rheumatoid synovial fibroblasts and isolated in
the 1970s and initially denoted as 72-kDa type IV collagenase / gelatinase A. MMP-3 was
identified as a lower molecular mass species (54 kDa) with proteoglycan and casein
degrading activity. In the late 1980s, Ed Harris and colleagues first proposed using the name
MMP for this family of enzymes. Subsequently, the International Union of Biochemistry
and Molecular Biology designated the family with the unique name MMPs and assigned
each family member with an enzyme number. By 1991, MMP-1, -2, -3, -7, -8, -9, and -10 as
well as tissue inhibitor of metalloproteinase (TIMP)-1 and -2 had been named and
characterized. Since then, the family has expanded gradually; the latest member discovered
is MMP-28, making a total of 23 human MMPs. MMPs activity is tightly regulated in
several ways, including their activation, the presence of specific endogenous inhibitors,
TIMP and by gene expression. MMP s may be secreted by tumour cells, fibroblasts,
endothelial cells and macrophages as well as mast cells and neutrophils .116
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Functions : MMP s are involved in physiological processes such as development,
morphogenesis, angiogenesis, bone remodeling, wound repair, apoptosis. These are also
involved in pathological processes such as in periodontal diseases, cancer invasion and
metastasis. 117
TYPES OF MMP s
MMPs are classified into subgroups as collagenases, gelatinases, stromelysins, and
matrilysins on the basis of their substrate specificity and molecular structure. The
membrane-type MMPs (MT-MMPs) are subclass of MMPs that additionally contain a
transmembrane and intracellular domain, a membrane linker domain, and are membrane
associated as described Verma RP et al (2007)118 and Vihinen P et al (2002).119
1. Collagenases includes MMP-1, MMP-8 and MMP-13
• Collagenases are proteinases capable of cleaving types I, II, III, V and IX collagens and
play a decisive role in remodeling and degradation of the Extra Cellular Matrix (ECM).
MMP-1 hydrolyzes type III collagen more rapidly than type I, while MMP-8 shows a
slight preference for type I collagen. Conversely, MMP-13 prefers type II collagen and
hydrolyzes this collagen much more rapidly than MMP-1 or MMP-8.118,119
• Gelatinases includes MMP-2 and MMP-9 : MMP-2 is widely distributed in skin
fibroblasts, keratinocytes, chondrocytes, endothelial cells, monocytes, osteoblasts and in
a number of other normal and transformed cells. MMP-9 is produced by keratinocytes,
monocytes and alveolar macrophages, polymorphonuclear leukocytes and in a number of
malignant or transformed cells. In OSCC, MMP-9 is expressed in carcinoma and
inflammatory cells around carcinoma islands. MMP-2 is mainly found in carcinoma-
associated fibroblasts (CAFs). Gelatinases has the ability to break type IV collagen,
found in the basement membrane. They play an important role in angiogenesis, tumor
invasion and metastasis, and have frequently been associated with poor prognosis. They
are also responsible for the final degradation of fibrillar collagens after initial cleavage
by collagenases. Gelatinases are secreted as inactive proforms that are activated
extracellularly. 118,119
2. Stromelysins : MMP 3, MMP 10, MMP 11, MMP 12 : Degrade various components of
the ECM and activate collagenases via the proteolytic removal of a propeptide.
Stromelysin 1 (MMP - 3) and stromelysin-2 (MMP - 10) are closely related to structure
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and substrate specificity. This subgroup also includes stromelysin-3 (MMP-11) and
metalloelastase (MMP - 12). 118,119
3. Matrilysin : MMP7 and MMP 26 : These are the smallest MMPs. They lack the C-
terminal hemopexin domain, common to other MMP family members and they have
markedly smaller molecular weights and are mainly expressed in epithelial cells in
different glandular structures. MMP-7 acts in intestinal mucosal defense by activating
antibacterial peptides, defensins. Matrilysin-2 (endometase, MMP-26) is expressed in
uterine endometrium and placenta and in cancers of lung, prostate and breast. 118,119
• Membrane type MMPs (MT- MMP) : MMP 14, MMP15, MMP 16, MMP 24, MMP
17 and MMP 25. These can activate proMMP-2 and most of them degrade ECM
components. In addition they contain a transmembrane and cytosolic domain. These are
anchored to cell membrane by a transmembrane domain in the C-terminus or attached to
cells by a C-terminal GPI anchor.
4. Additional MMPs include: MMP 19, MMP 20, MMP 21, MMP 22, MMP 23, MMP
28, MMP 29 : MMP-19 is expressed in mammary gland, placenta, lung, pancreas, ovary,
spleen, intestine, blood vessels of skin and uterine ligaments and in activated peripheral
blood mononuclear cells. MMP-20 (Enamelysin) has a restricted expression pattern in
dental tissues and is thought to play a role in tooth enamel formation. MMP-23 is
expressed in adult ovary, testis and prostate, suggesting its role in reproduction. It is also
expressed in adult heart, intestine, colon, placenta and lung. MMP-28 (Epilysin) is
expressed in testis and at lower levels in lungs, heart, colon, intestine and brain.
Expression of epilysin is detected in the basal epidermis of intact skin and in basal
keratinocytes in wounds.
REGULATION OF MMP s
Loffek S et al (2011)120 and Yan C et al (2007)121 suggested Cell-ECM interactions trigger
cellular signaling that promotes cell differentiation, migration, and mobilization which is
essential for normal cellular homeostasis. MMPs act on matrix and matrix substrates, such
as cytokines, chemokines, growth factors, cell surface receptors, and adhesion molecules.
MMPs can affect cell behavior in many ways. The cleavage products of MMPs signal in an
autocrine or paracrine manner. They cleave intercellular junctions or the BM regulating
epithelial tissue architecture and activate the action of latent and deactivate the action of
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active signaling molecules. Balanced and regulated degradation of ECM proteins by MMPs
are involved in many physiological processes. Conversely, excess MMP activity plays a role
in several pathological processes also. Therefore, understanding of the regulation of MMP
activity, under physiological and pathological conditions, could lead to novel therapeutic
intervention.
MMP expression has both constitutive and inducible components; MMP-2 and
MMP-1 are constitutively expressed in many cell types while MMP-9 is highly inducible.
The function of MMP s can be influenced by various effectors which include growth factors,
cytokines, chemical agents, reactive oxygen species etc. The enhanced MMP gene
expression may be down-regulated by suppressive factors like transforming growth factor
beta, retinoic acids, and glucocorticoids. Significantly, fragments of ECM cleaved by MMPs
can also potentiate the expression of other MMPs. Activity of MMP is also controlled in the
pericellular environment by modulation of activation and by the action of inhibitors.
Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein
and is expressed on the surface of tumor cells which stimulates adjacent fibroblasts and
tumor cells to produce several matrix metalloproteinases.
The catalytic activity of MMPs is strongly controlled at four different levels:
1. Gene expression with transcriptional and post-transcriptional regulation;
2. Extracellular localization and tissue or cell type of MMP release, termed
compartmentalization;
3. Pro-enzyme activation by removal of the pro-domain; and
4. Inhibition by specific inhibitors, i.e. tissue inhibitors of matrix metalloproteinases
(TIMPs), and by non-specific proteinase inhibitors, e.g. -2 macroglobulin.120,121
Role of MMPs in Oral Pathologies : MMPs has a marked role in tissue destructive oral
diseases. The expression and activity of MMPs in adult tissues is normally quite low, but
increases significantly in various pathological conditions that may lead into unwanted tissue
destruction, such as inflammatory diseases, tumour growth and metastasis. The best known
examples of unwanted tissue destruction related to over expression and activity of MMP
includes periodontitis, peri-implantitis, dental caries and OSCC.122
MMPs in Oral Premalignant and Malignant Lesions : OSCC is preceded by white or
red lesions can show varying degrees of epithelial dysplasia from mild to severe. The risk of
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progression is associated with histological grade, but it is currently impossible to predict
accurately which lesions will progress to malignancy. Degradation of the basement
membrane and invasion of the underlying connective tissue by neoplastic cells is recognized
as a fundamental step in the development of many epithelial cancers.123
Advances have been made in identifying the molecular determinants of
carcinogenesis. Alterations in specific oncogene and tumor suppressor genes have been
identified and shown to have causal roles in the initiation, maintenance and progression of
tumors. This has however, largely ignored the substantial contribution of the tumor
microenvironment to the malignant phenotype. The ‘seed and soil’ hypothesis of Paget
dates to 1889, where the molecular determinants of the ‘seed’ are much better delineated
than those of the ‘soil’ for either primary or metastatic lesions. An enzyme produced by the
normal cellular component of the tumor could be a significant player in carcinogenesis.124
Important events that determine the onset of dissemination of tumor cells are related mainly
to the invasion of surrounding tissue and the induction of angiogenesis. Local growth,
spread and metastasis are features that are shared among all malignant tumors, and require
the presence of the appropriate microenvironment of the host. For tumors to invade and
metastasize, neoplastic cells must be capable of degrading the extracellular matrix and
accessing blood vessels and lymphatics. Mounting evidence supports the view that
extracellular proteinases, such as the MMPs, mediate many of the changes in the
microenvironment during tumor progression.124
Tumour microenvironment regulation by MMP
Proteolysis of ECM: The principle components of the ECM are collagens and numerous
other proteins including laminins, entactin, and proteoglycans that make up the basement
membrane. Tumor cells overexpress proteases and/or induce expression of these enzymes in
neighboring stromal cells in order to degrade the basement membrane and invade the
surrounding tissue. Several MMPs have been implicated in the ECM degradation associated
with tumor growth and angiogenesis. This proteolytic activity is also required for a cancer
cell to invade a nearby blood vessel i.e. intravasation and then extravasate at a distant
location and invade the distant tissue in order to seed a new metastatic site.125
Modulation of Cell Adhesion, Migration and Epithelial to Mesenchymal Transition :
The loss of intercellular adhesion and the acquisition of degradative properties are
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prerequisites for tumor cells to become fully invasive. E-cadherin-mediated cell-cell
adhesion largely contributes to the maintenance of epithelial tissue integrity. The loss of E-
cadherin expression at the cell membrane is correlated with dedifferentiation,
aggressiveness, metastasis, and poor prognosis in different cancer types. MMPs modulate
the interactions between tumor cells by cleaving E-cadherin, and between tumor cells and
ECM by processing integrins, which also enhances the invasiveness of tumor cells. The
disorganization of E-cadherin/catenin complexes and expression of MMPs are frequently
involved in the capacity of epithelial cells to acquire an invasive phenotype. 126
MMPs have also been implicated in the epithelial to mesenchymal transition (EMT), a
hallmark of cancer progression to metastasis. During EMT, tumor cells acquire migratory
characteristics and more readily invade into surrounding tissues and metastasize to
secondary sites. Activation of growth factors and cleavage of adhesion molecules are some
of the proposed mechanisms underlying MMP induced EMT. Over expression of MMP-3, a
component of the tumor microenvironment, causes EMT and induces genomic instability in
cultured mammary epithelial cells leading to all stages of neoplastic progression, malignant
transformation, and mammary carcinomas in transgenic mice. 125, 126
MMPs Affect Growth Signals : Unregulated proliferation is a common feature of cancer
cells. There are two principal ways in which the tumor achieves this condition: by acquiring
self-sufficiency in growth-promoting signals or by becoming insensitive to antigrowth
signals. MMPs may be critically involved in disrupting the balance between growth and
antigrowth signals in the tumor microenvironment, as they potently influence the
bioavailability or functionality of multiple important factors that regulate growth.
One fundamental signaling pathway with essential roles in tissue homeostasis is the TGF-β
pathway. TGF - βnormally exerts tumor suppressive effects by enforcing cytostasis. Active
TGF-βis derived from an inactive pro-form by proteolytic conversion by furin or other
proteinases, such as MMP-9, which is usually expressed by inflammatory cells. Proteolytic
activation of TGF-βby MMPs has tumor-promoting effects by selectively driving stroma-
mediated invasion and metastasis of the tumor.
Ligands for the epidermal growth factor receptor (EGFR) are potent drivers of cell
proliferation and important regulators of tissue homeostasis. Malfunction of this system by
genetic mutations of the molecules involved is frequently observed in breast cancer and
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other malignant diseases. Activation of EGFR results in the upregulation of MMP-9, which
in turn degrades E-cadherin, a potent control element of many cellular functions including
cell-cell adhesion and differentiation. The cleavage of E-cadherin by MMPs or ADAM
proteinases has an impact on cancer cell proliferation. The association between EGFR,
MMP-9 and E- cadherin may play an important role in ovarian cancer and metastasis, as
activated EGFR and MMP-9 in these specimens co-localise with a region of reduced E-
cadherin.126
Wuertz B and F Ondrey (2010)127 from their study on role of MMP-9 in oral
keratinocytes concluded that TNF alpha and cigarette smoke may assist carcinogenesis in
vivo by inducing expression of MMP-9, potentiating invasion. They also stated that oral
leukoplakia cells are baseline cells which strongly produce functional MMP-9 that cannot be
stimulated further with TNF or CSC.
The Tumor Angiogenesis : The tumor vasculature is derived from sprouting of
local blood vessels (angiogenesis) and circulating (vasculogenic) progenitor cells derived
from the bone marrow i.e. vasculogenesis. The new vessels are often irregular and leaky due
to lack of the pericyte cover, which results in tumor cells to penetrate more easily. The
major MMPs involved in tumor angiogenesis are MMP-2, MMP -9, and MMP -14, and to a
lesser extent MMP-1 and MMP-7. Genetic MMP-9 deficiency was found to result in
retarded bone growth attributed to inhibition of angiogenesis.128
Lymphangiogenesis plays an important role in tumor biology, given that it is directly linked
with the formation of lymphatic metastases. MMPs certainly have a general impact on
lymphangiogenesis as supported by the use of broad-spectrum MMP inhibitors. However,
only a few reports directly link MMPs to the formation of new lymphatic vessels. The
modulation of VEGF bioavailability by MMPs, especially by MMP-9, may also affect
lymphangiogenesis and, in turn, promotes dissemination of metastases into the lymph.
MMPs Regulate Apoptosis : Evading programmed cell death, or apoptosis, is another
strategy that increases the cell number and the size of tumors. MMP function interferes with
the induction of apoptosis in malignant cells, which may involve the cleavage of ligands or
receptors that transduce proapoptotic signals. MMP-7 cleaves Fas ligand from the surface of
doxorubicin- treated cancer cells, lowering the impact of chemotherapy on the tumor by
abrogating apoptosis.129
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Adipocyte Regulation Affects Tumor Progression : Unquestionably, there are
consequences for the local paracrine crosstalk between the tumor cells and adipocytes.
Adipose secretory products collectively referred to as adipokines, have been identified as
contributors to the negative consequences of adipose tissue expansion in cancer,
cardiovascular disease, and diabetes. As adipocytes are a prominent part of the tumor stroma
and they do contribute to cancer progression. Moreover, adipokines such as leptin, regulate
the expression and activation of MMPs.129
To date, MMP-11 is induced by adipose tissue that directly affects cancer progression.
MMP-11 is expressed in adipose tissue as the tumor invades the surrounding environment
and negatively regulates adipogenesis by reducing preadipocyte differentiation and reversing
mature adipocyte differentiation. Adipocyte dedifferentiation leads to the accumulation of
non malignant peritumoral fibroblast-like cells, which favor cancer cell survival and tumor
progression.129
Tumor Invasion and Metastasis: The lethal outcome of the vast majority of all cancers is
due to the dissemination of metastatic tumor cells and the outgrowth of secondary tumors at
distant sites. Metastasis not only depends on features of the cancer cells disseminating from
the primary tumor but also requires the formation of a receptive environment, a metastatic
niche, which is specifically suited for the engraftment of tumor cells at the distant organ. It
is likely that MMPs and other proteinases are crucially involved in the formation of a
metastatic niche. MMP-9 turns out to be critical for the formation of the metastatic niche,
which is most likely linked with its ability to liberate VEGF and thereby support
angiogenesis. MMP-9 releases soluble factors to recruit stem and progenitor cells from the
bone marrow, which may be of particular significance in this context.129
Richard C et al (2004)130 in his retrospective study examined changes in MMP 1,2
and 9 using polymerase chain reaction in oral dysplasia and oral squamous cell carcinoma,
found higher levels of MMP-1 and 9 mRNA and are significantly associated with oral
dysplasia’s that progress to oral cancer compared with those that did not progress. Peschos
D et al (2006)131 checked the expression of MMP-9 in benign, premalignant and malignant
laryngeal lesions. In conclusion, their study indicates that the expression of MMP-9 is up-
regulated in a stepwise fashion, with two main steps, the first one, when a dysplastic lesion
evolves and the next one, when the dysplasia progresses to invasive carcinoma. MMP-9
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expression was related neither to survival nor to the other available clinicopathological
parameters patient age. Suvi TV et al (2013)132 reported that MMP 9 is detected in
malignant transformation of various cells and is associated with tumor metastasis and poor
prognosis. In the aggressive human tongue squamous cell carcinoma cell line, MMP-2 was
only found in its latent form, whereas MMP-9 was found in its active form. In an oral
squamous cell carcinoma cell line SCC-25, CAFs increase the expression of MMP-9, in
vitro, which is thought to occur via a fibronectin integrin v 6 pathway. MMP-9 may not be
the only, or even the most important, proteolytic enzyme in the OSCC invasion process, but
it may be important for indirect cell signaling by controlling the bioavailability and
bioactivity of molecules that target specific receptors, which regulate cell growth, migration,
inflammation, and angiogenesis.132
Gao J (2010)133 studied the expression of MMP-2/MMP-9 in normal oral mucosa,
lymph node-negative tongue cancers, lymph node-positive tongue cancers and their
metastasized tumours in cervical lymph nodes. They hardly found MMP-2/MMP-9
expression in normal epithelium. In lymph node-negative tongue cancer, 45% and 40% of
these primary tumors were positively stained for MMP-2/MMP-9. Importantly, in lymph
node-positive tongue cancer, 71% and 79% of these primary tumors were positive for MMP-
2 / MMP-9, respectively. Over expression of MMP-2/MMP-9 was present in the metastatic
lymph nodes. Their result implied the significance of MMP-2 and / or MMP-9 in predictive
value for the actual or potential presence of cervical metastases. Such activity has prognostic
value, and provides impetus for further development of biotherapies targeted at specific
inhibition of MMP activities.133
CYTOKERATIN
Cytokeratins (K) are proteins of intermediate filaments found in the intracytoplasmic
cytoskeleton and is responsible for the structural integrity of epithelial tissue. A major role
of these proteins is to act as a resilient yet adaptable arrangement that empowers epithelial
cells with the ability to sustain mechanical and non-mechanical stresses reported by
Coulumbe PA (2002).134
Jürgen S et al (2006) 135 reported that the term “CYTOKERATIN” was coined by
Franke et al in 1978. In 2006 a new systematic nomenclature for keratins was created and
now the proteins previously called "cytokeratins" are simply called keratins. This
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nomenclature is in accordance with nomenclature of Human Genome Organization (HUGO)
for both gene and protein names. These are made up of intracellular filamentous proteins,
which include microfilaments (6-8nm), intermediate filaments (10nm) and microtubules
(25nm). Intermediate filaments (IF) form the major part and are relatively stable component
of cytoskeleton. Approximately fifty different IF genes are expressed in different cells of the
human body.135
Cytokeratins consists of 20 biochemically and antigentically different polypeptides
with molecular weights ranging from 40 kDa – 67 kDa with an isoelectric pH of 4.9 – 7.8.
These proteins have been broadly divided into two subfamilies. These are recognised by
basic proteins. The low molecular weight proteins (52-67 kDa) are numbered as 1 to 8 and
the more acidic proteins with higher molecular weight (40-56 kDa) are numbered 9 to 20.
At least one member of each family is always co expressed in any given epithelial tissue.
Structure of Cytokeratin (keratin):
These have a homologous, central helical rod domain flanked by variably sized
amino and carboxyl terminal domains. DNAs of members of subfamily cross hybridize with
one another. The central rod domain of one keratin monomer binds with another keratin
monomer to form a coiled coil dimer. One dimer associates with another to form a tetramer.
Many tetramer join to form the final intermediate filament of size around 10 nm. The keratin
forms a hetrodimers, the filament being constructed from a protein pair which consists of a
type I and a type II keratin. Intermediate filament proteins are characterized by a highly
variable central alpha-helical rich rod-shaped domain and non-helical N & C terminal
globular domains at either end. Non--helical tail domain is typical for all keratins except for
K 19 report suggested by Chu PG et al 2002.136
Types and Pairing of Cytokeratins given by Hiroshi U et al (2005)137 :
Keratins (Ks) can be classified based on distribution, amino acid sequence and charge and
based on molecular weight.
Based on molecular weight: The molecular weight of cytokeratins ranges from 40 kDa to
67 kDa and based on which they are divided into low, intermediate and high molecular
weight keratins. Generally low molecular weight pairings are seen in simple epithelia, the
higher molecular weight parings are present in stratified non-keratinizing epithelia and the
highest molecular weight keratins are found in skin and corneal epithelium.
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Based on distribution: Soft and hard keratins. Soft keratins are epithelial cytokeratins, are
20 in number and are expressed in various types of epithelia, while hard keratins are
constituents of hard keratinizing structures such as hair and nails.137
Based on amino acid sequence: Type I and type II keratins. Type I or Acidic Keratins are
numbered 9 to 20 and Type II or basic keratin are numbered 1 to 8. It is based on the
isoelectric pH which is the point at which protein does not migrate in an electric field and
has got least migrating power.
Differences between Type I and Type II Keratins:
The major difference between type I and type II K is the molecular weight and isoelectric
pH. Cytokeratin 1 (K1) has the highest molecular weight and the highest isoelectric pH,
while K19 has the lowest molecular weight and a low isoelectric pH. Basic cytokeratins are
8 KDa larger than the acidic member. These cytokeratins are expressed in combinations
which characterize the type of epithelium i.e. each epithelial cell expresses specific pairs of
type I and type II keratins (with the exception of K19).
Thumb rules for Keratin pairing:
By Morgan PR et al (1987)138, Mei-Hua Lu et al (2000)139
1. Keratins are expressed as pairs containing one member from each subfamily
2. The two members of each pair are in the same size rank or order within their
respective family eg: Largest acidic keratin (K10) is expressed with largest basic
keratin (K1). i.e. K 1 & 10.
Each epithelial cell expresses specific pairs of type I and type II keratins (with the exception
of K19). Unpaired lowest molecular weight K19 is the type I keratin and is the smallest
keratin and is exceptional since it widely lacks the non--helical tail domain which is typical
for all other keratins.138,139
Molecular weight and chromosomal localization of Keratins: In the human
genome, the keratin genes are clustered at two different chromosomal sites, chromosome
17q21.2 (type I keratins, except K18) and chromosome 12q13.13 (type II keratins including
K18).
Keratin Expression in Adult Epithelia: The different epithelia in the human body
express cytokeratins that are characteristic of the type and degree of maturation or
differentiation within an epithelium. K5 & K14 are the major keratin expressed in the basal
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cell layer of both keratinised and non keratinised epithelia. In the undifferentiated basal cell
layer which contain stem cells, K5 & K14 are strongly expressed and are down-regulated in
the differentiating suprabasal cell layers, where as K19 is expressed in basal cell layer only
in non keratinised epithelium. In suprabasal cell layer K1 & K10, K4 & K13 is normally
expressed in keratinized and non keratinized epithelium respectively. K 8 & K18 is
expressed embryonically and in the simple epithelia. They are the first keratins to appear in
embryogenesis and also the oldest keratins found during evolution. K6 & K16 is a marker of
fast cell turn over and is usually expressed in keratinized epithelium as given by Tencate AR
(2003) 140 and Hisham IO (2006).141
Keratin Expression Profile in Head and Neck Pathology:
Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma: During the process of
malignant transformation of oral epithelium, oral leukoplakia represents one of the first
morphologically recognizable epithelial alterations. Hence the knowledge on alteration of
cytokeratin expression is important. According to Lindberg K et al (1989).142 CK 8/18 and
suprabasal expression of K19 is correlated with the premalignant transformation in oral
epithelium.
Mild dysplasia, arising from non keratinized epithelium - K1/10 synthesis is
enhanced and K4/13 is retained. This is explained by the presence of K1/10 mRNAs,
synthesis of which is increased in dysplastic state. Moderate dysplasia from non keratinized
epithelium - K1/10 filaments completely replace the K4/13 complex. Keratins K4/13 or
K1/10 that are characteristically present in suprabasal cell layers shows reduced expression
or loss in epithelial dysplasia. Severe dysplasia - both K4/13 and K1/10 are absent. K5 and
K14 are expressed in parabasal and spinous cell layers and may reflect basilar hyperplasia in
dysplastic epithelium, where as in normal epithelia these keratins are expressed only in basal
cells. Su L et al (1996)143 found that K19 expression in epithelial dysplasia varies from case
to case but abundant nRNA appears to be consistent and suggested that K19 expression is
diminished in the tissue with higher degree of keartinization. Differing levels of K19
expression may indicate instability in this keratin gene in the course of malignant
transformation. Perhaps in WDSCC K19 was negative and in PDSCC with K14 negative
found to have enhanced K19. Hence they suggested that synthesis of K19 was to
compensate for the loss of K14 so as to maintain the intermediate filament network.143
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The expression of K5 is a hallmark of squamous epithelium and is predominantly
seen in the basal/stem layers of stratified epithelium. Stem cells of stratified epithelium have
been repeatedly described as the major cellular targets for cancer causing mutations and
therefore might give in a long term rise to the development of SCC's. Stigbrand T et al.
showed positive correlation between increased expression K8 and K 18 and an increase in
histological grade of anaplasia. A relationship between K8 and K18 expression and location
of carcinoma cells at the tumor invasion front or in other areas of stroma interaction has
been reported. In normal fetal buccal mucosa and tongue epithelium until 27 weeks of
gestation K8 and K18 is expressed. Hence it could be explained that 8/18 expression in
tumor cells reincarnate towards embryonic expression. Study done by Thomas F et al.
showed that determination of the K 8/18 and 19 expression in OSCC can be correlated with
its prognostic potential. Furthermore in our previous study we observed that the expression
of K 8/18 was negative in all normal mucosa, and was enhanced in poorly differentiated
OSCC than well differentiated. 144
Tumors and Cysts of Odontogenic Origin: Few studies have described keratin and
vimentin in the epithelial component of odontogenic neoplasms and dental germ. The
research conducted by Crivelini MM et al (2003)145 have described the expression of K5, 7,
8, 13, 14, 17 and 19 in the human enamel organ. K19 is expressed in all odontogenic
epithelial tumors, enamel organ and as well as in dental lamina. From these observations it is
suggested that K19 may be marker for cells of odontogenic origin or ameloblast
differentiation marker.145
Tumors of salivary gland origin: Expression of K7, 8, 14 and 19 is seen in Luminal
cells of intercalated ducts of normal salivary gland and also seen in pleomorphic adenoma,
basal cell adenoma, adenoid cystic carcinoma and epithelial-myoepithelial carcinoma.146
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MATERIAL AND METHODS
After obtaining an institutional ethical clearance the present retrospective study is based
on grading of epithelial dysplasia to evaluate the inter and intra observer variability with most
commonly used classification system. The grading of epithelial dysplasia has also be done using
a proposed new binary classification system.
Pilot Study :
A pilot study was conducted to assess the inter and intra observer variability in grading
OED using a set of 25 archival cases collected from different oral pathology centers (KLE VK
IDS - Belgaum, DMDC - Wardha, GDC - Nagpur and LMDC - Nagpur). These were reviewed
independently by four oral pathologists trained under one centre. Three classification systems
were undertaken to determine the degree of dysplasia namely WHO 1978, Smith & Pindborg’s
1969 and WHO 2005. The Oral Pathologists were blinded to the clinical and histopathological
diagnosis. Findings were recorded on individual score sheets independently and a final
assessment for each case was recorded by the observer as “no dysplasia / mild / moderate /
severe dysplasia”. The results were tabulated on excel sheet for analysis of the inter and intra
observer variability. WHO 1978 classification showed poor to good agreement, maximum being
in the moderate group, whereas Smith and Pindborg’s was fair to moderate agreement, following
a moderate range. WHO 2005 demonstrated moderate agreement among all observers (Annex.
No. 1). Following this the set of same 25 cases were assessed by the binary system suggested by
Kujan Omar et al 2006, as ‘low risk and high risk, based on features described by WHO 2005 as
cytological changes and architectural features. Moderate to almost perfect agreement was
observed. Suggesting the degree of agreement was best in the binary system followed by
classification given by Smith and Pindborg.
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Estimation of Sample Size:
The sample size was calculated using the following formula :
Where: n=sample size
N= no. of subjects in the entire population.
n* = depends (r and pa-pe)
r = relative error
pa = overall agreement probability
pe = chance agreement probability
Taking into consideration, if raters will agree about 50% of times and relative error of
20%. i.e. pa-pe = 0.5 and r=20
Selection of cases (Samples) :
Based on the results obtained from the pilot study the present research protocol was
designed to further validate these grading systems and also to propose a new classification. The
validation of these classifications is also based on immunohisto chemical markers.
For the present study 100 cases of potentially malignant disorder (PMD) were retrieved
from the archives of the department which reported to KLE VK Institute of Dental Sciences
OPD from 1995 to 2012. From cases with signed out diagnosis of hyperplasia, mild, moderate,
severe dysplasia and carcinoma in-situ were selected. Samples with sufficient depth of tissue
available were included. Biopsy samples obtained from lesions on buccal mucosa were
considered. 100 slides from 100 different cases were finally included, which formulated the
study material.
In the series of selected 100 cases, the 25 cases included for pilot study were not
considered for the final assessment.
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Demographic data :
Clinical data was obtained from the biopsy requisition form for these selected 100 cases.
The demographic data like age, sex, type of habit, frequency and duration of tobacco habit was
noted. The clinical provisional diagnosis ranged as Leukoplakia, erythroplakia,
erythroleukoplakia and oral sub mucous fibrosis. Confidentiality and maintenance of all the
obtained records of all the cases included in this research protocol will be strictly maintained by
stating the waiver of consent (Annex. No. 2a).
Procedure :
Four sections of 4μeach were prepared by sectioning paraffin embedded tissue blocks.
One section was stained with H & E for histopathological evaluation and the one section each
was obtained on gel coated slide and stained immunohistochemically for MMP9, Ki-67 and with
CK-19 using HRP-IHC. The staining procedures and analysis is been stated in manual of
operation and standard operating procedures (Annex. No. 3).
Assessment Method for Epithelial Dysplasia:
For final assessment the grading classifications given by Smith and Pindborg’s grading
system (1969), WHO (2005) and the Binary grading system (2006) were used for evaluation of
H&E stained sections of 100 cases. No calibration exercise was attempted. Each slide was
labeled only with a serial order number. No clinical data or patient information was provided
with the slides to the evaluator. Four independent oral pathologists reviewed the slides. Sections
were observed under 4x; 10x; 100x magnification using Olympus BM2500 compound
microscope. The architectural characteristics of each feature was examined under the low
magnification (4x & 10x) and finished with cytological characteristics at high magnification
(40x).
A score sheet was designed for recording the individual features to aid systematic
analysis. Separate score sheets based on the selected three classifications were prepared. The
lesions were graded as ‘0 for no dysplasia ; 1 for Mild ; 2 for Moderate and 3 for Severe
dysplasia / carcinoma in-situ. For Binary grading system ‘1 for Low risk and 2 for High risk’ was
scored (Annex. No. 4).
Individual scores of all raters were obtained and in addition to the individual scores of the
raters a consensus score of 3 raters (SH, DM & PA) was obtained to compare with the
investigator’s (AK) score (Annex. No. 5). Based on the proposed classification of the present
Material & Methods
82
study the observers evaluated all the 100 cases as ‘non-risky’ and ‘risky’ for inter and intra
observer variability for all 18 dysplastic features (Annex. No. 6). For 10 important features
predicted for malignant transformation all the 100 cases were evaluated (Annex. No. 7a&b).
Classifications considered for the pilot study and the present study :
1. Histologic features for Smith & Pindborg 1969 classification of oral epithelial dysplasia.
Type of change Severity of dysplasia & scores
1. Drop shaped reteridges None-0 Slight-2 Marked-4
2. Irregular epithelial stratification None-0 Slight-2 Marked-5
3. Keratinization of cells below keratinized layer None-0 Slight-1 Marked-3
4. Basal cell hyperplasia None-0 Slight-1 Marked-4
5. Loss of intercellular adherence None-0 Slight-1 Marked-5
6. Loss of polarity None-0 Slight-2 Marked-6
7. Hyperchromatic nuclei None-0 Slight-2 Marked-5
8. Increased nucleo-cytoplasmic ratio in basal and
prickle cell layers
None-0 Slight
Increase-2
Marked
Increase-6
9. Anisocytosis and anisonucleosis None-0 Slight-2 Marked-6
10 Pleomorphic cells and nuclei None-0 Slight-2 Marked-6
11 Mitotic activity Normal-0 Slight
Increase-1
Marked
Increase-5
12 Level of mitotic activity Normal-0 Slight-3 Marked-10
13 Presence of bizarre mitoses None-0 Slight-6 Marked-10
2. WHO criteria for grading dysplasia (1978) : It graded epithelial dysplasia as: Mild,
Moderate, Severe.
Mild dysplasia: Slight nuclear abnormalities, most marked in the basal third of the epithelial
thickness and minimal in the upper layers, where the cells show maturation and stratification. A
few, but no abnormal mitosis may be present, usually accompanied by keratosis and chronic
inflammation.
Material & Methods
83
Moderate dysplasia: More marked nuclear abnormalities and nucleoli tend to be present, with
changes most marked in the basal 2/3rd of the epithelium, nuclear abnormalities may persists up
to the surface, but cell maturation and stratification are evident in the upper layers. Mitoses are
present in the parabasal and intermediate layers, but none is abnormal.
Severe dysplasia: Marked nuclear abnormalities and loss of maturation involving more than
2/3rds of the epithelium, with some stratification of the most superficial layers. Abnormal
mitoses may be present in the upper layers.
Severe grades of dysplasia may merge into the lesion customarily designated as
carcinoma in situ, in which the whole or almost the whole thickness of epithelium is involved.
In the present knowledge, it was not possible to say whether the presence of severe dysplasia
carried a different degree of risk of subsequent development of invasive carcinoma than the
presence of carcinoma in situ.
3. WHO criteria for grading dysplasia (2005) :
Architecture criteria Cytology criteria
1. Irregular epithelial stratification 1. Abnormal variation in nuclear size
(Anisonucleosis)
2. Loss of polarity of basal cells 2. Abnormal variation in nuclear shape
(Nuclear pleomorphism)
3. Drop-shaped rete ridges 3. Abnormal variation in cell size
(Anisocytosis)
4. Increased number of mitotic figures 4. Abnormal variation in cell shape
(Cellular pleomorphism)
5. Abnormal superficial mitoses 5. Increased nuclear cytoplasmic ratio
6. Premature keratinisation in single cells
(dyskeratosis)
6. Increased nuclear size
7. Keratin pearls within rete ridges 7. Atypical mitotic figures
8. Increased number and size of nucleoli
9. Hyperchromatism
Material & Methods
84
Grading of dysplasia:
Mild dysplasia: In general architectural disturbance limited to the lower third of the epithelium
accompanied by cytological atypia define the minimum criteria of dysplasia.
Moderate dysplasia: Architectural disturbance extending into the middle third of the
epithelium is the initial criterion for recognizing this category. However, consideration of the
degree of cytologic atypia may require upgrading i. e. those lesions that show marked cytological
alteration should be elevated to a higher grade level regardless of how extensively the atypical
cells fill the epithelium.
Severe dysplasia: Recognition of severe dysplasia starts with greater than two thirds of the
epithelium showing architectural disturbance with associated cytologic atypia. However,
architectural disturbance extending into the middle 3rd of the epithelium with sufficient cytologic
atypia may be up graded from moderate to severe dysplasia.
Carcinoma in situ: The theoretical concept of carcinoma in situ is that malignant transformation
has occurred but invasion is not present. It is not always possible to recognize this
morphologically. The following is recommended for the diagnosis of carcinoma in situ: full
thickness or almost full thickness architectural abnormalities in the viable cellular layers
accompanied by pronounced cytologic atypia. Atypical mitotic figures and abnormal superficial
mitoses are commonly seen in carcinoma in-situ.
4. Binary Classification (2006) :
High-risk lesion (with potential susceptibility for malignant transformation) was based on
observing at least four architectural changes and five cytological changes.
Low-risk lesion (does not have the potential susceptibility for malignant transformation) was
based on observing less than four architectural changes or less than five cytological changes. The
features to classify as High and Low Risk were based on the architectural and cytological
features suggested by WHO 2005 classification.
PROPOSED WORKING CLASSIFICATION
The proposed classification is based on Binary System (2006) and WHO (2005) criteria of
epithelial dysplasia. Additional architectural and cytological features are identified and included
in this classification. Each features of epithelial dysplasia is scored on a rating scale of 1 to 10.
The two groups identified are :
Material & Methods
85
• Non risky lesions : The total score ranged from 1 to 15
• Risky lesions : The total score ranged from 16 to 84
• ARCHITECTURAL FEATURES:
Sl. No. Particulars Scores
1. 1.1. Basal cell hyperplasia 4
2. 1.2. Drop shaped rete ridges 2
3. 1.3. Few normal mitotic figures 1
4. 1.4. Acanthosis 1
5. 1.5. Loss of polarity of basal cells 5
6. 1.6. Drop shaped rete ridges with nodules or forking 4
7. 1.7. Loss of cohesiveness with prominent intercellular bridges 5
8. 1.8. Abnormal and superficial mitoses 10
9. 1.9. Increased number of mitotic figures 5
10. 1.10. Dyskeratosis 2
11. 1.11. Loss of epithelial stratification 5
12. 1.12. Keratin pearls within rete ridges 5
• CYTOLOGICAL FEATURES:
Sl. No. Particulars Scores
1. 2.1. Mild to moderate degree of nuclear and cellular pleomorphism 5
2. 2.2. Marked nuclear & cellular pleomorphism &anisonucleosis 10
3. 2.3. Hyperchromasia of cells 5
4. 2.4. Increased nuclear-cytoplasmic ratio 4
5. 2.5. Atypical mitotic figures 6
6. 2.6. Increased number and size of nucleoli 4
INFLAMMATION :
Sl. No. Particulars Scores
1. 3.1 Mild 1
2. 3.2 Moderate 2
3. 3.3 Severe 3
Material & Methods
86
Procedure for H&E staining and Immunohistochemistry :
Four sections each of selected archival blocks were cut, one slide stained routinely with H&E
to confirm the diagnosis and the other slide stained with immnunohistochemical stain. The sections
required for immunoexpression is taken on a gel coated slides using 2%, 3-aminopropylethoxysilane
solution (APES) (Sigma Aldrich, St.Louis, MO, USA, Product code: A3648). The buffers required
for IHC are PBS as wash buffer and citrate buffer for antigen retrieval. The immunohistochemical
protocol was followed as per the standard recommendations of the company.
The IHC staining is performed using Super Sensitive one step Polymer HRP system
(QD-600, Biogenex, Bangalore) with pre-diluted primary antibodies using anti-Human antibody
for CK-19 (clone RCK 108, Biogenex, Bangalore), Anti MMP-9 (EP1-255Y, Biogenex,
Bangalore), Ki-67 (BGX-297, Biogenex, Bangalore). The storage and dilution of these
antibodies and IHC kit is done as per the manufacturers’ recommendation (Annex. No. 3).
Assessment of Immunohistochemical stained sections:
All the IHC stained slides were evaluated for immunoexpression of MMP 9, Ki
67, CK 19. The intensity of immunohistochemical staining was graded based on subjective
evaluation of color exhibited (brown color) by antigen, antibody and chromogen complex. For
Ki-67, the immunoexpression was noted for nuclear staining, CK-19 in cytoplasm and
MMP-9 for stromal reaction. Evaluation of immunoexpression was noted under x10
magnification under light microscope.
1. Analysis of immunoexpression of MMP-9
The immunoexpression of MMP-9 was based on intensity and area of expression
in the epithelium and stroma on a 4 point scale from 0 to 3. Intensity of expression was
graded as 0 when there was no evidence of immunoexpression. Intensity was considered
positive depending upon the colour intensity ranging from 1 : golden brown, 2 : light
brown, 3 : dark brown. Area of expression was based on a percentage of positivity in the
stroma and epithelium. The range was considered 1: when 1 to 25% of positive
expression was noted, 2 : 26 to 50% of positivity and 3 : more than 50% of positive
expression.
Material & Methods
87
Intensity Area in epithelium and stroma
0 = -ve 0 = -ve
1 = + , golden brown 1 = 1-25%
2 = + + , light brown 2 = 26-50%
3 = + + + , dark brown 3 = >50%
2. Analysis of immunoexpression of Ki-67
The immunoexpression of Ki-67 was mainly assessed in nuclei of epithelial cells.
This marker was evaluated in the epithelium based on the area of expression and
percentage of positive immunoexpression on a four point scale from 0 to 3. When
there was no evidence of immunoexpression it was graded as 0. In the epithelium this
proliferative marker was assessed based on the layers of the stratified squamous
epithelium involved. Whenever expression was noted only in the basal cell layer it
was graded as 1, suprabasal layer was graded as 2 and layers above this was graded as
3. Percentage of number of cells for positive immunoexpression was assessed. Area of
expression was graded as 1: when 1 to 25% positive epithelial cells were noted, 2: 26
to 50% positive cells and 3: more than 50% cells. The area with maximum number of
positive cells was considered in each section.
Area Percentage no. of cells
0 : No expression 0 = -ve
1 : Basal layer 1 = 1-25%
2 : Suprabasal layer 2 = 26-50%
3 : Above suprabasal layer 3 = >50%
3. Analysis of immunoexpression of Ck-19
The immunoexpression of Ck-19 was based on intensity and area of expression in
the epithelium on a 4 point scale from 0 to 3. Intensity of expression was graded as 0
when there was no evidence of immunoexpression. In te ns it y wa s co ns id er ed
po si ti ve depending upon the colour intensity ranging from 1 : golden brown, 2 : light
Material & Methods
88
brown, 3 : dark brown. Area of expression was based on a percentage of positivity in the
epithelium. It was considered as 1 for 1 to 25% of positive expression, 2 : 26 to 50% of
positivity and 3 : more than 50% of positive expression in the epithelium.
Intensity Area
0 = -ve 0 = -ve
1 = + golden brown 1 = 1-25%
2 = + + light brown 2 = 26-50%
3 = + + + dark brown 3 = >50%
STATISTICAL ANALYSIS:
Statistical analysis was done using :
1. Weighted Kappa and Non-Weighted Kappa tests for assessment of inter and intra observer
variability for all the classification system
Unweighted kappa takes into consideration agreement for individual cases
Weighted kappa considers the magnitude of disagreement (ordered categories) and
hence is a better measure for reliability
Kappa Agreement (Flesis and Cohen 1973)
< 0.40 Poor
0.40-0.75 Fair to Good
0.76 to 0.80 Excellent
0.81-0.99 Almost perfect
2. Fisher's Exact Test was done to evaluate the correlation of the immunohistochemical
markers with grades of epithelial dysplasia. p<0.001 was considered as statistically
significant.
Results
89
RESULTS
In the pilot study 25 cases of OED were evaluated for inter and intra observer variation
using four classifications :- a) Grading of dysplasia given by WHO (1978), b) Smith and
Pindborg Classification using photographic standards (1969) c) Classification of WHO (2005)
and d) Binary Classification by Kujan Omar et al (2006).
For the present study 100 cases of OED were subjected to evaluation for two selected
classifications: Smith and Pindborg (1969) and WHO (2005). A new classification is proposed
which included scoring system and has two categories for grading dysplasia as ‘non-risky’ and
‘risky’. IHC evaluation for Ki-67, CK-19 and MMP-9 is done to understand behavior at the
molecular level. The data obtained has been statistically analyzed.
Table No. 1 : Statistical analysis of 25 cases considered for Pilot Study
Classification/ Observers 1
st& 2
nd1
st& 3
rd1
st& 4
th2
nd& 3
rd2
nd& 4
th3
rd& 4
th
Smith &Pindborg
(1969)
k=0.307Fairkw =0.419Moderate
k=0.241Fairkw =0.417Moderate
k=0.415Moderatekw =0.577Moderate
k=0.304Fairkw =0.451Moderate
k=0.390Moderatekw =0.667Good
k=0.443Moderatekw =0.58Moderate
WHO(1978)
k=0.552Moderatekw=0.67Good
k=0.23Fairkw =0.444Moderate
k=0.114Poorkw =0.316Fair
k=0.430Moderatekw =0.515Moderate
k=0.458Moderatekw =0.531Moderate
k=0.288Fairkw =0.414Moderate
WHO(2005)
k=0.597Moderatekw =0.706Good
k=0.310Fairkw =0.419Moderate
k=0.212Fairkw =0.404Moderate
k=0.326Fairkw =0.451Moderate
k=0.405Moderatekw =0.490Moderate
k=0.460Moderatekw =0.438Moderate
Binaryclassification
(2006)
k=0.920AlmostPerfect
k=0.562Moderate
k=0.448Moderate
k=0.513Moderate
k=0.522Moderate
k=0.387Fair
Table no. 1: WHO (1978) showed good to poor agreement with maximum having moderate
agreement. Smith and Pindborg (1969) had fair to moderate agreement following a moderate
range. Whereas WHO (2005) demonstrated moderate agreement amongst all observers.
Moderate to almost perfect agreement was observed in binary classification by Kujan O. The
inference drawn from the pilot study suggests that the degree of agreement was best in the
binary system followed by classification given by Smith and Pindborg. (K=kappa, kw=weighted
kappa)
Results
90
Table No. 2: Statistical analysis of 100 study cases considered for INTER OBSERVER variability
Classification Weighted kappa (kw) Unweighted kappa (k) p value
Smith & Pindborg (1969) 0.483 (Moderate) 0.275 (Fair) < 0.001
WHO (2005) 0.419 (Moderate) 0.310 (Fair) < 0.001
Proposed classification - 0.790 (Good ) < 0.001
Table no. 2 : Smith and Pindborg’s classification showed fair to moderate agreement in both
weighted and unweighted kappa statistics (p<0.001). Similarly, WHO (2005) classification
also showed fair to moderate inter observer agreement. Good agreement was evident with
proposed classification
Table No. 3 : Statistical analysis of 100 study cases for INTRA OBSERVER variability
Classification Weighted kappa Unweighted kappa p value
Smith & Pindborg (1969) 0.742 (Good) 0.612 (Good) < 0.001
WHO (2005) 0.706 (Good) 0.597 (Moderate) < 0.001
Proposed classification - 0.810 (Almost perfect) < 0.001
Table No. 3 : The intra observer agreement was found to be almost perfect in the proposed
classification as compared to the other classification.
Results
91
Table No. 4 : Scores given for atypical architectural and cytological features for theProposed Classification
Sl. No. Particulars Scores
Architectural Features
1. 1.1. Basal cell hyperplasia 4
2. 1.2. Drop shaped rete ridges 2
3. 1.3. Few normal mitotic figures 1
4. 1.4. Acanthosis 1
5. 1.5. Loss of polarity of basal cells 5
6. 1.6. Drop shaped rete ridges with nodules or forking 4
7. 1.7. Loss of cohesiveness with prominent intercellular bridges 5
8. 1.8. Abnormal and superficial mitoses 10
9. 1.9. Increased number of mitotic figures 5
10. 1.10. Dyskeratosis 2
11. 1.11. Loss of epithelial stratification 5
12. 1.12. Keratin pearls within rete ridges 5
Cytological Features
1. 2.1. Mild to moderate degree of nuclear and cellular pleomorphism 5
2. 2.2. Marked nuclear & cellular pleomorphism & anisonucleosis 10
3. 2.3. Hyperchromasia of cells 5
4. 2.4. Increased nuclear-cytoplasmic ratio 4
5. 2.5. Atypical mitotic figures 6
6. 2.6. Increased number and size of nucleoli 4
Inflammation
1. 3.1 Mild 1
2. 3.2 Moderate 2
3. 3.3 Severe 3
Results
92
Table No. 5 : Distribution of cases into ‘non-risky’ (total score ranged from 1-15 ) and
‘risky’ (total score ranged from 16 to 84) groups.
Category No. of Cases Percentage Score
Non risky 41 41% 2-15
Risky 59 59% 20-84
Table No. 5 : 59 cases were identified as risky dysplasia as compared to 41 cases of ‘non-
risky’ dysplasia. Suggesting that 59 cases out of 100 cases are likely to develop into
malignancy.
Table No. 6a : Calculation for risk categorization using median and inter quartile for 100cases
X SD Median 1st Quartile 3rd Quartile
Non Risk Group Score 7.7 4.1 8 4 11.5
Risk Group Score 53.5 13.01 56 45 66
Table No. 6b : Classification of risk categories for evaluation of 59 ‘risky’ cases
Category Score Number of cases
Low risk 20-44 14(23.7%)
Medium risk 45-66 39(66.1%)
High risk 67-84 6(10.2%)
Table No. 6a & 6b : Entire score for the proposed classification comprising of 1 to 84 was
evaluated for mean and standard deviation. Median and the inter quartile range was used to
define cut off points and the cases were classified into low, medium and high risk based on the
scores above. Out of the 59 ‘risky’ cases 6(10.2%) cases had highest score (67-84) were
suggestive to be at higher risk for malignant transformation.
Results
93
Table No. 7 : Risk categorization of ‘Risky’ and ‘Non-risky’ 100 cases for each feature
Features Low Risk (14) Medium Risk (39) High Risk (6) Non Risk (41)
ArchitecturalNo. ofCases % No. of
Cases % No. ofCases % No. of
Cases %
1.1 11 78.5 34 87.2 6 100.0 0 0
1.2 2 14.3 14 35.9 1 16.7 3 7.3
1.3 7 50 21 53.8 1 16.7 23 56.1
1.4 10 71.4 37 94.9 5 83.3 30 73.2
1.5 7 50 32 82.1 6 100.0 1 2.4
1.6 5 35.7 32 82.1 5 83.3 0 0
1.7 10 71.4 34 87.2 6 100.0 5 12.2
1.8* 1 7.1 4 10.3 5 83.3 0 0
1.9 6 42.8 35 89.7 6 100.0 2 4.8
1.10 1 7.1 17 43.6 6 100.0 1 2.4
1.11 4 28.6 30 76.9 6 100.0 0 0
1.12 14 100 39 100.0 6 100.0 0 0
Cytological2.1 9 64.3 39 100.0 6 100.0 0 0.0
2.2* 1 7.1 3 7.7 4 66.7 0 0.0
2.3 9 64.3 38 97.4 6 100.0 19 46.3
2.4 11 78.6 39 100.0 6 100.0 6 14.6
2.5 11 78.6 39 100.0 6 100.0 4 9.7
2.6 5 35.7 34 87.2 6 100.0 2 4.8
Table No. 7 : Cases of both non-risky and risky categorized into three risk categories as per the
scores for 18 individual features. Feature 1.8* (Abnormal and superficial mitoses) and 2.2*
(Marked nuclear & cellular pleomorphism & anisonucleosis) were significantly associated with
the risk group as compared to the non-risky group. (Fisher Exact Test p<0.0010 & p<0.003)
Results
94
Table No. 8 : Ten Important Features with higher scores considered in ‘Risky group’ aresegregated with a total score of 61.
Feature no. Particulars Scores
1.5 Loss of polarity of basal cells 5
1.7 Loss of cohesiveness with prominent intercellular bridges 5
1.8 Abnormal and superficial mitoses 10
1.9 Increased number of mitotic figures 5
1.11 Loss of epithelial stratification 5
1.12 Keratin pearls within rete ridges 5
2.1 Mild to moderate degree of nuclear and cellular pleomorphism 5
2.2 Marked nuclear & cellular pleomorphism & anisonucleosis 10
2.3 Hyperchromasia of basal cells 5
2.5 Atypical mitotic figures 6
Total = 10 Total score 61
Results
95
Table No. 9a: Calculation for risk categorization using median and inter quartile forstudy group for 10 important features
Studygroups x SD Median 1st Quartile 3rd Quartile
Non RiskyGroup Score 5.4 3.68 6 1.5 7
RiskyGroup Score 37.5 10.72 39 32 44
Table No. 9b. : Classification of risk categories for evaluation of cases for 10 features
Category Score Number of Cases
Low risk 12-31 13(22%)
Medium risk 32-44 38(64.4%)
High risk > 45 8(13.6%)
Table No, 9a and 9b : The total score of the ten individual features selected comprising of 01
to 61 was evaluated for mean and standard deviation. The median and the inter quartile range
was used to define cut off points and the cases were again classified into low, medium and high
risk based on the ten individual features. Out of the 59 ‘risky’ cases 8(13.6%) cases had
highest score (> 45) were suggestive to be at higher risk for malignant transformation.
Results
96
Table No. 10 : Risk categorization of ‘risky’ and ‘non-risky’ groups for the 10 features
Risky group Non risky group
Low Risk (13) Medium Risk (38) High Risk (8) Non Risk (41)
No. ofCases % No. of
Cases % No. ofCases % No. of
Cases %
1.5 7 53.8 30 78.9 8 100.0 1 2.4
1.7 7 53.8 35 92.1 8 100.0 5 12.2
1.8* 0 0.0 4 10.5 6 75.0 0 0
1.9 7 53.8 32 84.2 8 100.0 2 4.8
1.11 2 15.4 31 81.6 7 87.5 0 0
1.12 1 7.7 17 44.7 7 87.5 0 0
2.1 8 61.5 37 97.4 7 87.5 0 0
2.2* 0 0.0 3 7.9 5 62.5 0 0
2.3 9 69.2 36 94.7 8 100.0 19 46.3
2.5 11 84.6 37 97.4 8 100.0 4 9.7
Table No. 10 : Cases of both non-risky and risky categorized into three risk categories as per
the scores for 10 selected individual features. Feature 1.8* (Abnormal and superficial mitoses)
and 2.2* (Marked nuclear & cellular pleomorphism & anisonucleosis) were significantly more
commonly associated with the ‘risky’ group as compared to the ‘non-risky’ group (Fisher Exact
Test p<0.001).
Results
97
Table No. 11a : Area of expression of CK-19 in Epithelium of the study groups (Fisher exactTest)
Area 0 1 2 3 p value
Risky 0 15 32 12p<0.001
Non risky 20 15 6 0
Table no. 11a shows that in ‘risky’ group the area of expression of Ck-19 in 32 cases the
expression was seen in 26 to 50% of epithelial cells. In a ‘non-risky’ group 20 cases were
negative for Ck-19 expression but 6 cases had 26 to 50% positive cells. The area of CK-19
expression in the epithelium was significantly high in the risky as compared to non-risky group.
Table No. 11b : CK-19 Expression of intensity in Epithelium of the study groups (Fisherexact Test)
Table No. 11 b : The intensity of expression was light brown in 30 cases and dark brown in 14
cases of ‘risky’ group. Whereas in the ‘non-risky’ 20 cases were negative and in 21 cases the
intensity ranged from light brown to dark brown. Intensity of CK-19 expression in the
epithelium was significantly high in the ‘risky’ as compared to ‘non-risky’ group. The eight
cases with scores 45 & above had more intensity (+++) and more area (>50%) of expression of
CK-19.
Intensity 0 1 2 3 p value
Risky 0 15 30 14p<0.001
Non risky 20 19 2 0
Results
98
Table No. 12a. : Ki-67 Expression in the study groups (Fisher exact Test)
Table No. 12 a shows that 39 ‘risky’ cases had 26 to 50% positive cells for ki-67 as compared
to the non-risky group where 24 cases were negative and 11 cases had1 to 25% positive
expression. The Ki-67 expression in the epithelium was significantly higher in the ‘risky’ as
compared to ‘non-risky’ group.
Table No. 12b. : Area of Ki-67 positivity in study groups (Fisher exact Test)
Table No. 12 b shows 42 cases in the risky group had ki-67 immnoexpression upto to the supra
basal layer, whereas 6 cases had expression above the suprabasal layer. In the ‘non-risky’ 24
cases were negative but 6 cases had ki-67 immunoexpression upto the suprabasal layer. The Ki-
67 expression in the epithelium was significantly higher in the ‘risky’ group as compared to
‘non- risky’ group. The eight cases with scores 45 & above had expression upto suprabasal
layer (+++) where >50% of cells had expression of Ki-67.
Expression 0 1 2 3 p value
Risky 0 11 39 9p=0.000
Non risky 24 11 6 0
Area 0 1 2 3 p value
Risky 0 11 42 6p=0.000
Non risky 24 11 6 0
Results
99
Table No. 13a : MMP-9 Expression in Epithelium of the study groups (Fisher Exact Test)
Groups Intensity Area
0 1 2 3 0 1 2 3
Risky 2 42 13 0 2 19 33 4
Non-Risky 9 26 2 1 9 19 11 1
p value p=0.001 (S) p=0.002 (S)
Table No. 13 a shows the intensity of MMP-9 expression was golden brown in 26 ‘non-risky’
cases and 42 ‘risky’ cases. In 13 risky cases area of MMP-9 expression in epithelial cell was
seen between 26-50% whereas in non-risky 26 cases had 1 to 25% positive cells. The MMP-9
epithelium was significantly higher in the risky as compared to ‘non-risky’ group.
Table No. 13b : MMP-9 Expression in stroma of the study groups (Fisher exact Test)
Groups Intensity Area
0 1 2 3 0 1 2 3
Risky 1 20 26 11 1 17 37 2
Non-Risky 2 15 18 4 2 19 15 2
p value p=0.551 (NS) p=0.700 (NS)
Table No. 13 b : shows the intensity of MMP-9 expression was light brown in 18 ‘non-risky’
cases and 26 ‘risky’ cases. In 37 risky cases area of MMP 9 expression in stromal cell was seen
between 26-50% whereas in non-risky 19 cases had 1 to 25% positive cells. No association was
observed in the stromal expression of MMP-9 in the ‘risky’ as compared to ‘non-risky’ group.
H&E Photographs
100
H&E Stained Sections of Dysplasia
Figure 1. Section showing ‘Non-risky’ dysplasia (a - 10x, b - 40x, H&E stain)
Figure 2. Section showing ‘risky’ dysplasia (a - 10x, b & c - 40x, H&E stain)
a b
a cb
MMP-9 Photographs
101
MMP-9 Immunoexpression in Dysplasia
Figure 3. ‘Non-risky’ dysplasia with minimal MMP-9 Expression (a- 10x, b- 40x, IHC stain)
b
Figure 4. ‘Risky’ dysplasia with enhanced MMP-9 Expression (a & b - 10x, c- 40x, IHC stain)
a c
a b
Ki-67 Photographs
102
Ki-67 Immunoexpression in Dysplasia
a b
b
Figure 6. ‘Risky’ dysplasia. Enhanced Ki-67 expression in suprabasal layer.(a- 10x, b & c- 40x, IHC stain)
c
a
Figure 5. ‘Non-risky’ dysplasia. Restricted Ki-67 expression in basal cell layer.(a- 10x, b- 40x, IHC stain)
CK-19 Photographs
103
CK-19 Immunoexpression in Dysplasia
Figure 7. ‘Non-risky’ dysplasia. Restricted CK-19 expression in basal cell layer.(a -10x, b- 40x IHC stain)
b
Figure 8. ‘Risky’ dysplasia. Enhanced CK-19 expression in basal and suprabasal layers.(a & b -10x, c- 40x, IHC stain)
c
a b
a
Epithelial Atypia Photographs
104
Fig. F : Loss ofCohesiveness (40x)
Fig. G : Loss ofstratification (40x)
Fig. A : Dysplastic OralEpithelium (10x)
Fig. B : Prominent IntercellularBridges (10x)
Fig. E : Increased Nucleoli (40x)
Fig. C : Loss of BasalCell Polarity (40x)
Fig. D : BasilarHyperplasia &
Hyperchromasia(40x)
Fig. I : Superficial &Abnormal Mitosis (40x)
Fig. H : Marked Pleomorphism (40x)
a
CBA
D E F
G H I
Figure 9(A-I) : Features of Epithelial Atypia (H&E Stain)
Discussion
105
DISCUSSION
Oral squamous cell carcinoma (OSCC) is one of the most commonly encountered
malignancies, which not only involves mortality but also disfigurement and dysfunction
associated with treatment. For intra oral cancers with cervical lymph node metastasis, the
five year survival rate is less than 50%.2 To improve the mortality and morbidity adequate
early diagnosis is urgently needed. It is not known if all oral cancers are preceded by
precursor lesions but a majority of OSCC do present as visible changes of the oral mucosa.
These precursor lesions are defined as an altered epithelium with an increased likelihood for
progression to OSCC. The altered epithelium shows a variety of cytological and architectural
changes that have traditionally been brought under the common denominator: Dysplasia.
In the oral cavity the most common lesions recognized as potentially malignant are
leukoplakia and erythroplakia. In the Indian subcontinent oral submucous fibrosis has been
reported to have malignant potential. It is known that 50% of OSCC arise from apparently
clinically normal mucosa. The prognostic significance of an individual lesion is difficult to
determine. None of the currently available molecular markers have proved to neither be
prognostically significant nor have yet been evaluated in large prospective studies. The gold
standard for the assessment of oral potentially malignant lesions is microscopic evaluation of
standard H&E stained sections for the presence of architectural and cytological changes,
generally referred as epithelial dysplasia.147
Assessment of dysplasia depends upon the microscopic diagnosis which is based on
grading the changes considering combination of architectural and cytological features.147
Grading of dysplasia is subjective and lacks intra and inter observer reproducibility due to
insufficiency of validated morphological criteria and the biological nature of dysplasia.54
Hence, due to the absence of a consensus, several systems are currently employed.
During the last decade many classifications of potentially malignant lesions
synonymously referred as epithelial dysplastic lesions, premalignant conditions, squamous
intra epithelial neoplasia etc. have been proposed. The majority of the classifications in the
current literature have followed criteria similar to those in common use for the grading of
epithelial lesions of the uterine cervix.148 In cervical epithelium there is clear distinction
between normal and abnormal layers of the epithelium and consequently the degree of
dysplasia can be assessed by determining the horizontal level of this border in the epithelium,
Discussion
106
resulting in substantial intra and inter observer consistency. Such a sharp distinction between
normal and abnormal layers in the epithelium of the upper aero-digestive tract is less obvious
and consequently the definitions for degree of dysplasia are much more susceptible for
discussion, thus resulting in substantial intra and inter observer variability.149
A pilot study was conducted to assess the inter and intra observer variability in
grading OED using a set of 25 archival cases with signed out diagnosis as hyperplasia in 2
cases, mild dysplasia in 3 cases, moderate in 7 cases, severe dysplasia in 8 cases and
carcinoma in situ in 4 cases. Four oral pathologists graded them independently using three
classification systems. The observers were blinded for clinical and histopathological
diagnosis. The classification systems followed were (1) WHO (1978) (ii) Smith and
Pindborg’s (1969) system (iii) Architectural & Cytological criteria given by WHO in 2005.
The results of the pilot study were evaluated statistically for assessment of inter and
intra observer variability:
1. Smith and Pindborg’s System: The inter and intra observer K values ranged from
0.241(fair) to 0.443 (moderate) whereas kw values ranged from 0.417(moderate) to
0.667(good) (Table No. 1). Agreement was better between the raters as each category is
assigned a score and the sum of the scores for the 13 categories provided an epithelial
dysplasia index for each slide. It produces a dysplasia assessment on an ordinal scale
which facilitates statistical analysis. The disadvantage was that the analysis was time
consuming as each feature had to be compared with the monograph pictures.
2. WHO Criteria (1978) : has summarized the features of epithelial dysplasia under two
heads namely (a) Epithelial organization & (b) Cytological features. The inter and intra
observer K values ranged from 0.114(poor) to 0.552 (moderate) whereas kw values
ranged from 0.316(fair) to 0.67(good) suggesting wide variation in observation ranging
from poor to good agreement, maximum observations being in the moderate group (Table
No. 1).When only a few features are present, more emphasis is placed on them but which
independent feature has to be given weightage to grade dysplasia will vary from case to
case. This lead to disparity in observations which was not only between histopathologist
(inter observer) but also when the same investigator viewed the case on a different
occasion (intra observer).
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3. WHO Criteria (2005) : WHO consensus group modified the features published in 1978
and broadly categorized as changes in the architecture (strata) of the epithelium and the
cytological features which manifest as cellular atypia and divided into grades of mild,
moderate and severe. The more numerous the features, the more severe is the grade of
dysplasia. However, dysplasia represents a spectrum of change rather than discrete
identifiable stage. In the pilot study the inter and intra observer K values ranged from
0.212(fair) to 0.597(moderate) whereas kw values ranged from 0.404(moderate) to
0.706(good) (Table No. 1). This system lacks guide lines in interpretation of the
presence, degree and significance of each feature and hence contributed to inconsistent
assessment.
Kujan O et al (2007)167 demonstrated a variation in the inter observer agreement on
grading OED using two different grading systems; the 2005 WHO classification and new
binary system. A further analysis of the inter-observer agreement on the architectural and
cytological features that were used in the grading process was undertaken to help to
understand where the inter-observer variation stems from. Their data showed wide inter-
observer variability in recognizing the individual features of OED. The observers have
shown poor to moderate agreement on grading these individual features, but a good to
substantial agreement was seen when a cumulative scoring was used to agree on the
degree of dysplasia either by using the new binary system of ‘‘low-risk’’ or ‘‘high-risk’’
or by using the 2005 WHO classification. This study states that the observers had
compensated the discrepancies in recognizing these features and this could be related to
the fact that most pathologists do initially screen the slides at low magnification and
make a provisional diagnosis. They then use a higher magnification to ascertain the final
diagnosis by looking for specific details in the slide.
4. Binary grading system (2006) : Thus the set of same 25 cases were once again
evaluated using the binary system of grading as ‘high / low risk’ as given by Kujan O et
al.49 The K values were calculated which ranged from k=0.387(fair) to k=0.920 (almost
perfect) (Table No. 1). Fair to almost perfect agreement among the observers was noted.
A statistically significant overall correlation was found between histopathological
grading of dysplasia using this low / high risk system for all observers (p<0.001). But
difficulties in assessment observed were the same as encountered with WHO criteria –
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108
2005. The disagreement was due to interpretation of individual features of dysplasia. By
reducing features to definable components increased the objectivity and insight but
evaluation and justification of specific features could not be achieved as the cut-off point
for a ‘high risk’ lesion was based on observing at least four architectural changes and five
cytological changes. The cut-off point for a ‘low risk’ lesion considered less than four
architectural changes or less than five cytological changes as suggested by the authors.
Based on the observations from pilot study and after considering an extensive
search of literature, the criteria under architectural features and morphological features
defined by WHO in 2005 were redefined to form the basis for the new proposal to grade
epithelial dysplasia for the present study. We have made an attempt to score the
individual features of dysplasia. The criteria for assigning the scores was based on the
severity and occurrence as seen in OSCC or in tissues adjacent to OSCC, features
considered for severe dysplasia by other authors and those suggested by Smith and
Pindborg. This was suggested with the objective of reducing the inter and intra observer
variability and for making assessment objectively reproducible. (Table No. 4)
The two basic requirement of any grading system should be : reproducibility
and prognostic value. Reproducibility refers to the degree to which observer
measurement or diagnosis remains the same on repeated independent observations of an
unchanged characteristic. This consistency can be assessed between different observers
(inter observer) or within a single observer (intra observer). Prognostic value is the
natural evolution of a disease without treatment and predictive value is the natural
evolution of the disease in response to treatment.150 For studying accuracy in grading
dysplasia of upper aero-digestive tract, there is no test available, which is thought to be
better than the pathologist’s observation when an accepted gold standard is not available
for assessing the validity obtained for grading these lesions. Therefore, reproducibility is
needed to provide indication of validity. The inter and intra observer agreement levels
give an estimate of the degree of bias and validity, where an appropriate gold standard is
not available.46
Hence, we tried to assess the inter and intra observer agreement levels to give an
estimate of the degree of bias and validity for assessing epithelial dysplasia. To make our
grading system reproducible two important aspects were identified (i) to obtain highest
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109
agreement for individual features of dysplasia / atypia and (ii) to assign scoring system
for individual features to reduce subjectivity and make assessment easy.
From the analysis of pilot study and from data obtained from the studies of Smith and
Pindborg, Kujan O, RJ Oliver, WM Tilakarane, the highest agreement scores were seen for
(1) atypical and abnormal mitosis (ii) Marked cellular and nuclear pleomorphism (iii)
irregular epithelial stratification (iv) loss of cohesiveness (v) Keratin pearls within the
epithelium (vi) loss of polarity of basal cells. The interpretation of the features required to be
present for a diagnosis varies from observer to observer as does their actual recognition and
the assessment of their degree of importance. There is a relatively high degree of
inconsistency in evaluation due to the overwhelming dependence upon subjective factors.
Therefore it was decided that an attempt should be made to place the diagnosis of epithelial
dysplasia on an objective level. A total of 18 features were identified to score for grading
dysplasia in the present study which was scored on a numerical scale of 1 to 10.
Rationale for assigning scores to the observations made in the present study:
Each individual characteristic dysplastic feature to be evaluated is listed along with
the scores as shown in Table No. 4. Many of the clinically diagnosed potentially malignant
disorders histopathologically show excess surface keratin, a prominent basal cell layer ,
hyperplasia of spinous cell layer (acanthosis) and a few normal mitotic figures in the
proliferative layers. These changes may be common to a number of mucosal lesions without
any cancer transformation potential. Warnakulasuriya S. et al54 suggested that the magnitude
of surface keratinization (keratosis or hyperkeratosis) is of no importance in the assessment
of dysplasia. As these features are of common occurrence and less significant for malignant
transformation, they were assigned lower scores of 1 and 2. Cells appear primitive and show
nuclear hyperplasia (enlarged nuclei), hyperchromatism of nucleus of basal cells, enlarged,
often eosinophilic nuclei (prominent nucleoli) and increased nuclear to cytoplasmic ratio.
These features are not exclusive to carcinogenesis as they are seen in reactive epithelium or
epithelium influenced by a variety of systemic alterations. Similarly not all dyskeratosis is
related to dysplasia, for example individual cell keratinization can be seen in Witkop disease
and in smokeless tobacco keratosis. Dyskeratosis is rare in carcinoma in situ, perhaps
because the cells are too primitive to produce keratin in quantities capable of being seen with
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110
light microscopy. It is also rare in moderate dysplasia and never seen in mild dysplasia.42
These cellular changes in dysplasia were assigned lower scores in the present assessment.
Elongated rete-processes lined by well polarized basal cells with normal stratification
and minimal or no cellular atypia were given lower score. These changes are seen in a variety
of inflammatory hyperplastic conditions like infections, frictional keratosis, psoriasis and
pseudoepitheliomatous hyperplasia, hence considered for lower scores in the present study.
On the other side of the spectrum some architectural and cytological features were
considered for higher score. The lower most region of the epithelial rete ridges with bulbous
enlargement results due to excessive basal cell proliferation. Especially if secondary
projections or nodules and forking are seen to arise from the basal layer that branch at
different angles into the subepithelial lamina propria or irregular ‘drop-shaped’ rete ridges
running in different directions. Budding of the rete-processes is a sinister feature. Small
clusters of basaloid cells may bulge or project from the basal layer into the stroma. These
features are considered for higher score. There is no physiological explanation for secondary
nodules and forking and hence were considered as ominous enough to alter the
histopathologic grading of a lesion to a higher level.
Dysplastic epithelium may also be atrophic which lacks retepegs and may be
ulcerated, thereby mimicking a traumatic or inflammatory lesion with thin, regenerating
epithelium creeping-in from the margins. Regenerating epithelium has granulation tissue
beneath it to distinguish it from dysplasia. Speight PM et al42 suggest that, in atrophic
dysplastic epithelium the basal cell hyperplasia appears to extend to the surface and they
recommend such lesions as severe dysplasia. This feature was considered for higher scores.
Loss of cellular cohesiveness not accompanied by severe inflammatory changes,
referred to as increased intercellular space or prominent intercellular bridges or loss of
intercellular adherence: is a feature of invasive carcinoma and is rarely seen otherwise. Intra
epithelial deposits of extra cellular matrix molecules can be regarded as intra epithelial
stroma. This result in the loss of intercellular adherence.47 Whereas Bouquet JE suggests that
loss of cohesiveness must be distinguished from intercellular edema, intra epithelial
inflammatory cells or degenerating cells with pyknotic nuclei and vacuolated cytoplasm,
which may also cause loss of cellular cohesiveness. These mild expansions of intercellular
spaces in few areas or along the length of epithelium are marked by the presence of
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111
inflammation in the underlying connective tissue.50 The loss of cohesiveness of cells in
pathologies of buccal mucosa could be due to the fact that there are fewer desmosomes in the
keratinocytes of the non-keratinized stratified squamous epithelium (SSE) as compared to
those of cornified SSE. The smaller number of desmosomes in non-cornified epithelia
reflects the lesser amount of keratin filament bundling (tonofilament formation) observed in
those cell types as noted by Garrant PR.151 Hence when otherwise loss of cohesiveness is
present it should be considered as a definite feature of dysplasia and thus assigned higher
score in our study.
Loss of polarity due to irregular orientation of basal cells has been scored high.
Polarity is lost when the normal alignment of the basal cells with their long axis
perpendicular to the basement membrane becomes disturbed.35 Due to hyperchromasia the
cells have basaloid appearance which indicates active proliferation. The oral mucosal
epithelium has germinal center in the parabasal area, from which cells with proliferative
potential are differentiated into prickle cells as well as basal cells. “The loss of polarity of
basal cells” (WHO classification) can be interpreted as loss of basal cell alignment. This loss
of alignment results from focal proliferation of basaloid (parabasal) cells. Therefore it is
important to pay careful attention to this feature to consider for accurately diagnosing
dysplasia.47 The loss of basal cell alignment is more clearly observed in dysplasia with a two
phase appearance. This is generated by a focal proliferation of basaloid cells in the lower part
of rete processes which replace not only basal cells but also lower prickle cells. Such
proliferating cell foci make a straight interface with parakeratinized cells in the upper layer,
which shows distinct contrast between the upper and lower layer of the epithelium. This
sudden transition is caused by loss of prickle cells due to their apoptosis. This feature is
referred to as the ‘two phase appearance’. There is a stepwise development of two phase
dysplasia, from small buddings of basaloid cells, their fusion, to fully developed ones. The
working committee for new histopathological criteria for borderline malignancy of oral
mucosa47 suggested that such lesions are diagnosed as true epithelial dysplasia.
Loss of epithelial stratification which is a more comprehensive loss of the normal
layered effect throughout the entire thickness and length of the epithelium is considered a
high risk feature and has been given higher score. Loss of differentiation of epithelial cells in
oral mucosa is the most fundamental morphological change of dysplasia. Bouquot JE et al
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(2006)50 describe this as an alarming morphological alteration of dysplastic epithelium which
is due to an apparent inability to properly differentiate and mature. Cells high in the
epithelium have the same immature appearance as those in the basal layers. Dysplastic
epithelium may be atrophic or acanthotic and some authors believe that atrophic forms have a
higher risk of malignant transformation.
Mitosis : The proliferative compartment contains two pools of dividing cells : Stem
cells and Transit amplifying cells. Stem cells are located in the basal cell layer and are slow
cycling cells; divide one to five times while migrating laterally and upward towards the
epithelial surface, producing a clone of differentiating cells.151 These different types of
proliferating cells cannot be distinguished by histologic methods. Regardless of whether the
cells are of the stem or amplifying type, cell division is a cyclic activity. The only phase
distinguished histologically is mitosis, which can be further subdivided into the recognizable
stages of prophase, metaphase, anaphase and telophase.
The mitotic activity can be affected by a number of factors such as time of the day,
stress and inflammation. Though slight inflammatory cell infiltrate stimulates mitosis, severe
inflammation causes a marked reduction in proliferative activity.152 In healing tissues,
inflammatory or reactive proliferations, metaphase of the dividing cell in the cell cycle is
most commonly seen and is considered as normal mitotic activity. Abnormal or atypical
mitosis appear as bizarre shaped mitotic cells and are easily identified in routine H&E stained
sections. In potentially malignant disorders and cancers different phases of mitosis are seen.
In the present study identification of abnormal and atypical mitotic cells, their presence in the
different layers of epithelium and their numbers were considered for highest score and hence
considered as high risk feature.
Cellular pleomorphism implies differences or bizarre forms in the cell size and cell
shape inappropriate for a given layer in the epithelium. Altered polarity sometimes with the
appearance of spindle shaped cells, and increased size unaccompanied by cell division are
components of pleomorphism. Bizarre mitoses and extreme of anisonucleosis are quite rare
in oral epithelium and hence should be considered as component of dysplasia.28
Abnormal mitosis, disturbed polarity, hyperchromatism, enlarged nucleoli,
hyperchromatism and intra epithelial keratinization were considered as features of
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113
significance for malignant transformation by Tilakaratne WM et al153 and the same is
considered for the present study.
Increased number and size of nucleoli : The nucleus of a fixed cell appears to have a
complex net like framework along with granules of varying size irregularly arranged. These
granules represent chromatin, a name given by Flemming because they stain brilliantly with
basic coal tar dye. The vast majority of nucleus has within them small, round, sharply
demarcated bodies called nucleoli. These are of two types, “true nucleoli” or plasmosomes
which take cytoplastic stains and “false nucleoli” or karyosomes which are reserve store
house of basic chromatin. They are known to contribute their substance to the chromosomes
at the time of cell division. The plasmosomes are related to metabolic activity in certain cells,
especially in the production of secretary products.154
Koss LG155 suggested that in normal cell, nucleoli, show cells with active RNA and
protein production. Size and configuration of nucleoli depends on two factors (i) Level of
production of RNA in nucleolus (ii) Cytoplasmic demand of the RNA. Therefore, large
nucleoli and increase of numbers reflect uninhibited formation of RNA in the presence of
limited cytoplasmic demand. These regions are called nucleolar organizer regions (NOR).156
While still within the nucleolus, these rRNA molecules are assembled together with
many protein molecules into two subunits of future ribosomes. The subunits leave the
nucleus through pores in the nuclear membrane and enter the cytoplasm. Here the two
ribosome subunits are brought together to form a functional ribosome at an early stage in
translation and separated again when the ribosome has fully translated a molecule of
messenger RNA (mRNA). Synthesis of ribosomes happens in the nucleolus. Ribosomes are
actually sites of protein synthesis.157
Recent studies showed that the nucleolus has emerged as a highly complex and
multifunctional regulatory compartment. The nucleolus is an extremely sensitive structure to
monitor ; it responds to cellular apoptotic stresses such as radiation, or exposure to cytotoxic
agents. Nucleolar proteins such as nucleophosmin, nucleostemin, have important roles in
these non-ribosomal functions, including regulation of cell growth and death, stress
responses, mitosis and the cell cycle.158,159
In the present study, increase in number and size of nucleoli, a feature also suggested
by WHO 2005 as a feature of dysplastic has been given higher score. Abnormalities in the
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nucleolar morphology of cancer cells attracted the attention of tumor pathologists as early as
the late 19th century, when Pianese G et al160 reported that hypertrophic and irregularly
shaped nucleoli were characteristic of malignant cells. The morphological and functional
changes of the nucleolus, widely observed in cancer tissues, are the consequence of the
increased metabolic necessities that characterize proliferating cells. 160
Study by Rajput DV et al161 suggested that NOR’s are the sites of rRNA synthesis
that reflect protein synthesis. AgNOR’s that stain for NOR associated proteins is known to
increase with increase in cell ploidy, increased transcriptional activity and in state of active
cell proliferation. Malati N. et al (2008) 162 from their study concluded that AgNOR quantity
is strictly proportional to the proliferative of AgNORs per nucleus suggests a marker of
proliferative activity. Quantitative assessment of size, shape and pattern of distribution acts
as a marker of premalignant / malignant change. Studies have shown that Nucleoli are
usually inconspicuous in normal cells. Nucleoli are prominent, more in number, enlarged and
irregular with occasional sharp pointed projections in malignant cells. This is due to
increased protein synthesis. It is reported that number and irregularity of nucleoli increased
with increasing grades of carcinoma.162
Keratisation of cells below the keratinized layer and Keratin pearls within reteridges :
Keratin formation is a feature of normal tissue maturation. As the basal cells of oral
epithelium move from one layer to the other and as the cells increase in age with progressive
maturation, they result in formation of keratin in the oral epithelium. This pattern of
maturation differs in different type of epithelium. Keratin is the protein formed by ribosomes,
secreted through the golgi apparatus and consist of aggregation of tonofilaments, involucrin
and filaggrin. As the epithelial cells contain these keratin filaments, they are referred to as
keratinocytes.
In dysplastic epithelium the irregular maturation and proliferation of cells lead to
abnormal formation of keratin and lower layers of epithelium. These are recognized as
individual cell keratinization and as keratin pearls. The presence of this is similar to the
keratin pearls formed in squamous cell carcinoma. Hence this feature in grading dysplasia is
scored high.
The changes seen in lamina propria should also be considered in grading dysplasia.
Lamina propria plays an important role in determining the phenotype of the overlying
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115
epithelium and directs the pattern of epithelial morphogenesis. Specificity is the result of
epithelial mesenchymal interaction. Though regional specificity exits, which may be consider
as being the result of extrinsic inductive stimuli from underlying lamina propria or an
intrinsic property of the basal layer of the epithelium.163 Inflammation in the lamina propria
was considered for grading dysplasia as it influences epithelial proliferation, keratinization
and cytological changes like cellular edema and altered nuclear cytoplasmic ratio in the
present study.
Appraisal of comparison of Epithelial Dysplasia classifications:
Grading is hampered by the arbitrary division into distinct categories of a continually
progressing process without any naturally and sharply defined borders. It is in fact, an
attempt to impose discrete categories on what is in effect a continuous grey scale and
therefore any grading scheme is by definition artificial.29 The ultimate diagnosis depends on
the emphasis put upon each of the characteristics in grading by the observer. Several studies
have shown inter and intra observer variability in the assessment of presence or absence of
individual characteristic features and thus the grading of dysplasia using different
classification system.
In the present study 100 cases of PMD with epithelial dysplasia were assessed using
Smith and Pindborg’s classification given in 1969, WHO in 2005 and the proposed new
classification for assessing the inter and intra observer variability. Kappa agreement was used
to evaluate the degree of variation in grading dysplasia for each cytological and architectural
feature. Both unweighted kappa and weighted kappa analysis were considered.
Unweighted or simple kappa (k) is a measurement of inter-rater agreement which
provides quantitative estimate of reliability and represents agreement greater than that
expected by chance alone. As stated by Cohen the kappa values fall between ‘-1’ : complete
disagreement, ‘0’ : agreement accepted by chance and ‘1’ : perfect agreement. Thus kappa
gives credit only for complete agreement.
Whereas weighted kappa (kw) considers the magnitude of disagreement by giving
credit to complete and partial agreement and hence a better measurement for reliability.
Weighted kappa results in a better inter and intra observer agreement values.164, 165 In the
present study the interpretation of kappa values suggested by Flesis and Cohen (1973)166
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116
were considered, where the values < 0.40 was rated as poor, 0.40-0.75 as fair to good, 0.76-
0.80 as excellent and above 0.81 as almost perfect.
The overall inter observer variability in our study for Smith and Pindborg and WHO
2005 classification showed moderate to fair agreement kw=0.48 & 0.149 and k=0.275 &
0.310 respectively (Table No. 2). Whereas the proposed classification had 90% agreement
(p=0.001) k=0.79. The intra observer variation for the 100 cases of dysplasia showed similar
observation when compared between the different classifications. Grading was reproducible
with good agreement for Smith and Pindborg and almost perfect agreement (91%, k=0.81)
for the proposed classification (Table No. 3).
Great variability in inter and intra observer agreement has been reported in several
studies. They ranged from poor to substantial agreement by use of different statistical
methods. Pindborg et al reported a wide range of agreement (1-78%) for 9 photomicrographs
evaluated by 72 pathologists. Abbey LM et al (1995)3l examined 120 oral biopsies of a range
of pathologies from hyperkeratosis to severe dysplasia. The agreement ranged from 35.8% to
55.8%. The kappa values improved to 0.70 – 0.88 for inter examiner comparisons when the
grading was expanded to “within one histological grade”.
Sudbo J et al (2001)53 s ho we d po or inter observer agreement using 3 point ordinal
scale (k values: 0.17 – 0.33). A slight improvement was observed when 2 categories of
“favorable” (Mild and moderate dysplasia) and “poor” (severe dysplasia) were used. (K
value ranged 0.21 – 0.34). Brothwell DJ et al (2003)46 showed a substantial agreement using
a 5 point ordinal scale with a group weighed Kappa (Kw) of 0.74 (94%, Cl=0.64 – 0.85).
However similar to other studies, in their study the group unweighted Kappa showed a fair to
moderate agreement (ks) of 0.37 with 95% Cl = 0.32 – 0.42.
Fischer D et al (2004)52 reported an overall kappa weighted value of 0.59 (95%
cl=0.45 – 0.72) An improvement in the kappa agreement was observed when the histological
diagnosis was simplified into three general categories of “no abnormality / hyperplasia”,
“mild / moderate / severe dysplasia” and “Carcinoma in situ” (Kw=0.70 (95% Cl : 0.56-
0.84). Subjective judgment and individual histopathological experience of the participating
pathologists was the core for the histopathological assessment.
Kujan O et al (2006)49 observed in their study that for a substantial group weighted
agreement was reported when four observers graded oral biopsy specimens into categories
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117
varying from hyperplasia (no dysplasia) to carcinoma in-situ (Kw=0.63, (95% CI: 0.42-
0.78)). A fair to moderate agreement was found using unweighted kappa test (ks=0.22,
(95CI:0.11 : 0.35). These variations were due to the fact that the unweighted kappa
considered all disagreements between raters. Whereas weighted kappa yields a higher
reliability when disagreements are small compared to, when they are large. An improvement
in the pairwise and overall agreement was reported in their study when a binary grading
system was used. They observed a better agreement between those experienced in examining
oral mucosal biopsies as compared with the general pathologist. Bias could also be due to
established departmental practice in oral pathology as stated by Kujan O et al.
All previously published reports on the inter observer agreement on grading OED
lesions showed considerable variations. Warnakulasuriya S et al26 suggested that this might
be due to the lack of objectivity in the evaluation of established criteria, arbitrary division of
the grading, lack of calibration of criteria and grading, and lack of sufficient knowledge
about which criteria are important for the prediction of malignant potential.
To overcome some of these limitations, we in our proposed classification have given
score to each of the 18 features identified for dysplasia and are predicted to have potential for
malignant transformation. The sum total of the scores ranged from 1 to 84. When the scores
ranged from 1 to 15 for a PMD it was categorized as ‘non-risky’ and when the scores ranged
between 16 to 84 the lesion was graded as ‘risky’. Accordingly 59% of the cases were
grouped under ‘risky’ (Table No. 5) suggesting that these are likely to develop into
malignancy. Whereas literature suggests, 2% to 17% chance of malignant transformation in
PMD. The scores assigned to individual dysplastic characters were in agreement with that
given by Smith and Pindborg for evaluating the dysplastic features more objectively. The
total score in our study was probably over estimated for the group categorized as ‘risky’. The
features considered under the group of ‘risky’ were further evaluated for risk categorization.
Entire score of the proposed classification comprising of 1 to 84 was evaluated for mean and
standard deviation (Table no. 6a, 6b). Median and inter quartile range was used to define cut
off point for scoring dysplasia and the cases were regrouped into low risk (score between 22 -
44) which consisted of 23.7% (14) cases, medium risk with scores between 45-66 were
66.1% (39) cases and in the high risk group we found 10.2% (6) cases with a scored between
67-84. This suggested that out of 59 cases initially grouped as ‘risky’, 6 cases could be
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118
considered for having potential to undergo malignant transformation as they were on the
higher side of the cut-off values.
To evaluate the 18 individual dysplastic features the ‘non-risk’ and ‘risky’ cases were
categorized for each feature of dysplasia (Table no. 7). Feature assigned number 1.8 i.e.
abnormal and superficial mitosis and 2.2 i.e. marked nuclear and cellular pleomorphism and
anisonucleosis were significantly associated with ‘risky’ group as compared to ‘non-risky’
where the Fisher’s Exact Test value were p<0.001, p<0.003 respectively. Fisher’s Exact test
of statistical significance is useful for categorical data that result from classifying objects into
different ways: it is used to examine the significance of association (contingency) between
two kinds of classification of samples.
To further evaluate the significance of individual features, those dysplastic features
that had score of 5 and above were segregated. A total of 10 such important features
predicted for malignant transformation were identified (Table No. 8). These are loss of
polarity, loss of cohesiveness with prominent intercellular bridges, abnormal and superficial
mitoses, increased number of mitotic figures, loss of epithelial stratification, keratin pearls
within rete ridges, mild to moderate degree of nuclear and cellular pleomorphism, marked
nuclear & cellular pleomorphism & anisonucleosis, hyperchromasia of basal cells and
atypical mitotic figures. The total score of these 10 features was calculated as 61.
All the 100 cases were once again categorized into the three risk categories as per the
scores of ten above mentioned selected features. Calculations using median and interquartile
range to define the cut-off point based on which of the ‘risky’ cases were regrouped as low,
medium and high risk (Table No. 9a & 9b). 13.6% (8) cases were identified with the total
score of more than 45, suggesting these cases which had score more than cut-off value could
be at a higher risk of undergoing malignant transformation.
Cases of both ‘non-risky’ (41 cases) and ‘risky’ (59 cases) were categorized into risk
categories as per the scores of 10 above mentioned selected individual dysplastic features.
Abnormal and superficial mitosis (feature no.1.8) and marked nuclear & cellular
pleomorphism & and anisonucleosis (feature no. 2.2) were significantly and more commonly
associated with ‘risky’ group as compared to the ‘non-risky’ group (Fischer’s Exact Test
p<0.001) (Table No. 10). These observations suggest that these two features observed in the
biopsy of any potentially malignant disorder would be important criteria for consideration for
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119
malignant transformation. Another important feature observed in the ‘risky’ category was
loss of cohesiveness which histologically presents as prominent intercellular bridges. Our
results are in agreement with the data obtained from the studies of Smith and Pindborg,
Kujan Omar, Oliver RJ, Tilakaranate WM.
Variation could result due to the difference in the understanding of these features in
terms of recognition and impact on clinical outcomes. Though some features are associated
significantly with the clinical outcomes, the other features which showed no statistical
significant association are as suggested by Patridge M. et al (1997)167 considered to be
important for the whole grading process.
One of the main objectives of the study by Kujan O et al (2006)49 was to assess the
prognostic value of the binary system using qualitative morphological characteristics. They
found the new binary grading system to be a very good predictor for the malignant changes
in oral epithelial dysplasia with reasonable values of sensitivity and specificity (85% and
80%, respectively). They predicted the clinical outcomes of dysplastic lesions with certainty
(28/33;84.9%), while the negative predictive value of system was 85%.
They also suggested that cases with either hyperplasia or mild dysplasia could be
considered as ‘watch and wait’, whereas the cases with severe dysplasia or CIS in situ need
intervention. The cases with moderate dysplasia belong to the category of ‘watch and wait’.
Their data showed that moderate dysplasia cases had significant potential for malignant
changes (14/30 ; 46.7%). 14 out of 16 (87.5%) cases, in their study with moderate dysplasia
had been assigned as high risk and progressed to OSCC. Their results further demonstrated
that patients whose OED lesions had been assigned as “high risk” tended to have
significantly greater transformation rates than those with lesions assigned as “low risk”
(p=0.004, by log-rank test). In contrast to other studies, the results of the Kujan O et al
(2006) suggest that the new binary grading system has the potential to help the clinicians to
make more appropriate treatment decisions when dealing with OED.
Appraisal of IHC in Epithelial Dysplasia:
Epithelial alterations are the first morphological changes seen during the progress of
malignant transformation of the oral epithelium. These potential malignant disorders are
important lesions for clinical preventive investigations. For better understanding not only are
the clinicopathological evaluation essential but also the molecular-biological changes during
Discussion
120
transformation process should be known. A variety of cell biological markers mainly
involved in cell proliferation and apoptosis are studied and some association is described in
the literature.
Identical genetic changes in dysplastic oral lesions and oral cancers in some patients
suggest that the progeny of the cells in dysplastic lesions may further modify into a
malignant lesion. A clone of oral keratinocytes may proceed through stages of transformation
from normal, to dysplastic, to malignant. The major event in oral malignancy is excessive
proliferation of oral keratinocytes, which break through the basal membrane and spread in
the stroma as suggested by Upasani OS et al (2004).168
Though progress in molecular oncology has significantly advanced our knowledge on
tumorogenesis; yet the practical applications of these genetic markers remain unresolved in
detecting oral dysplasia. Although many molecules have been studied as intermediate
markers, not many studies have focused correlating the findings with the clinical attributes of
terminal outcome. Combined with routine histopathology studies, the broad based studies of
molecular markers could have a great potential for the determination of prognosis of PMD.
The aim of the present study was to correlate the molecular changes in the epithelium
and basement membrane zone of lamina propria of PMD. An attempt is made in the present
study to correlate the histopathological features with IHC markers for CK-19, Ki-67 and
MMP-9 so as to substantiate the observation of dysplasia observed in H&E stained tissue
sections.
Cytokeratins (CK) are epithelial specific intermediate filament proteins. They are 20
different types and exhibit distinct patterns of expression in specific epithelial tissues. The
CK polypeptides have common epitopes against which antibodies are developed and these
tend to cross react. A large number of monoclonal and polyclonal antibodies have been
developed that are being used for diagnosis using IHC. CK are connected to the nuclear
envelope on one side, while they interact with plasma membrane proteins. These in turn
interact with ECM proteins. Thus CK are involved in transduction of signals and transport of
nutrients from inside to outside of cells and vice a versa. CK-19 is expressed in simple
epithelium and basal cells of non-keratinizing squamous epithelium reported by Takeda TK
et al (2006).169
Discussion
121
Basal cells situated along the base of the epithelial ridge have a flattened cell surface
in contact with connective tissue. These non-serrated cells contain only a few cytoplasmic
organelles and appear to be least differentiated cells. These represent the stem cells and
transit amplifying cells. They are characterized by a high nucleus to cytoplasmic ratio,
expression of K-19, a relative lack of keratin filament bundle and high levels of β1
integrins.151
In the present study for all 100 cases the intensity of CK-19 immunoexpression in the
epithelial cells and in the different cell layers were observed (Table no. 11a & 11b). The
immunoexpression of Ck-19 was found to be statistically significantly higher (p<0.001) in
the ‘risky’ cases as compared to ‘non-risky’. In the ‘risky’ group all the cases showed CK-19
expression whereas 20 ‘non risky’ cases had no CK-19 expression. In the ‘risky’ group, 44
cases out of 59 had more than 26% of cells with immunoexpression and the intensity of
expression ranged from light brown to dark brown suggesting increase in the number of
proliferating cells. 21 ‘non-risky’ cases had immune expression of CK-19 with maximum
having less than 26% of immunopositive cells with intensity of expression being light brown
suggesting the presence of proliferating cells in this group. The intensity of expression of
CK-19 was significantly higher in the ‘risky’ group as compared to the ‘non-risky’ (p <
0.001). We also found that 6 out of the 8 cases (75%) which were categorized as high risk
with scores more than 45 in the ‘risky’ group had high expression (+++, >50%) of CK-19.
These findings suggest disturbance in the distribution of proliferating cells and the stem cells
during proliferation in the epithelium.
Lindberg K.(1989)142 in their study suggested CK-19 as a marker of premalignancy
when the supra basal cells of dysplastic epithelium were immunopositive. This is in
accordance with our observation. Takeda TK et al (2006)169 reported CK-19 and p63 over
expression in their series of cases and suggested that CK-19 positive cells are transient
applying cells located in the parabasal layers. Loss of asymmetric cell division leads to
increase in number of stem cells in oral dysplasia. This disturbance of stem cells and
asymmetry in cell division can be recognized as dispolarity of basal cells, an essential
histological feature of dysplasia. Thus an increase in suprabasal cells could be an indicator of
higher grade of dysplasia. They also suggested that the decrease in CK-19 expression could
be due to alteration of stem cell function and that these cells could be replaced by
Discussion
122
proliferating cells in the basal layer of dysplastic epithelium. Hence this feature could be a
useful index in grading.
The expression of terminal differentiation products differ from keratinized to non-
keratinized SSE in which K4 and K13 keratins are expressed in the suprabasal cells, whereas
the suprabasal cells of cornified SSE express high molecular weight keratins (K1, K2, K10 &
K11). This normal pattern of keratin expression is altered in hyperplastic and cancerous
lesions of the oral mucosa. The appearance of K8, K18 and K-19, typical of simple highly
proliferative epithelial cells, becomes prominent in oral leukoplakia and stratified squamous
cell carcinoma was reported by Lan Su et al (1996).143
Ki-67 is a nuclear antigen present in the proliferating cells. It is present in all phases
of cell cycle, where its expression is low in G1 phase and increase to peak levels in the G2
and mitotic phase. In our study ki-67 expression and the area involved in the epithelial cell
layer showed a significantly marked increase of immunoexpression in the ‘risky’ group as
compared to the ‘non-risky’ (p<0.001). In the ‘risky’ group 48 cases had immunoexpression
in more than 26% of the epithelial cells and in 6 cases these cells were extending above the
suprabasal layer. Whereas in the ‘non-risky’ group 17 cases were immune positive for Ki-67,
maximum of which had less than 25% of immune positive cells and were limited to the basal
layer. The 8 cases with scores of more than 45 (high risk in the risky group) in the proposed
classification had more intensely stained and more than 50% immunopositive cells in the
upper layers of dysplastic epithelium (Table 12a & 12b). As Ki-67 is located within nucleoli
of proliferating cells only, it may be suggested that the antigen may regulate nucleolar
metabolism by increasing rates of ribosomal synthesis required by rapidly dividing cells
which can be histologically interpreted as basilar hyperplasia. Our findings are in accordance
to observations of Takeda TK et al169, they found increase in ki-67 expression in the basal
and parabasal cells of dysplasia group as compared to normal oral epithelium. They did not
notice any significant difference between the mild and moderate dysplasia cases. Takashi
Saku47 suggested that differentiation of keratinocytes happens in two directions : 1) towards
the surface – keratinization and 2) towards basal cells. Thus basal cells are neither the
germinal cells nor are they a source for squamous epithelial regeneration, but are terminally
differentiated cells. The cells with proliferating potential are located in the parabasal cell
layer which may be regarded germinal center of oral squamous epithelium.
Discussion
123
When epithelial cell nuclei are Ki-67 positive they are regarded as cells in
proliferation. In normal epithelium these are located in parabasal cells. In ‘risky’ dysplasia
group in our study, ki-67 positive cells were seen up to the middle third of epithelium. With
the presence of dyskeratosis or sudden keratinization in the lower layer of the epithelium the
ki-67 positive cells could indicate that the keratinocyte differentiation is not well regulated
that could lead to lack of basal cell alignment and loss of stratification.
Oral cancer may result from mutation of ras oncogene and a simultaneous
deactivating mutation of p53, a tumors suppresser gene in oral keratinocyte. Oral cancer may
result from mutation in other oncogenes and in EGFr / ras / kinase cascade / c-myc signaling
pathway. Authors correlated this with the stages of carcinogenesis from normal mucosa, to
squamous hyperplasia (9p), to dysplasia (3p, 17p), to carcinoma in situ (11q, 13q, 14q), to
invasive carcinoma (6p, 8q, 4q).2
In dysplasia the oral keratinocytes proliferation is limited within the epithelial
compartment. Eventually the proliferating keratinocytes may break through the epithelial
basement membrane and expand into the underlying connective tissue resulting in carcinoma.
Frank invasion into the stroma by cancer cells are obvious histologically. However in some
cases the invasiveness is not clearly determined. This so called micro invasion cannot be
ruled out in carcinoma in-situ cases. Takashi Saku47 suggests that invasion of cancer cells is
accompanied by a neoplastic stromal change around the cell nests. On H&E stained sections,
the newly formed neoplastic stroma is more diffusely eosinophilic than the lose connective
tissue of the lamina propria but more basophilically myxoid than surrounding fibrous
granulation tissue. Carcinoma in-situ does not induce such diffuse neoplastic stroma. To
evaluate the basement membrane and stromal changes MMP-9 was considered in the present
study.
MMPs are a family of structurally related but genetically distinct enzymes that
degrade extracellular matrix and basement membrane components and regulate cell–matrix
composition. MMPs may be secreted by tumour cells, fibroblasts, endothelial cells and
macrophages as well as mast cells and neutrophils.116
In our study MMP-9 immunoexpression was studied in the epithelium and connective
tissue in all 100 cases of PMD for grading dysplasia. The intensity and area of expression in
the epithelium was found to be statistically significant when compared between the ‘risky’
Discussion
124
and ‘non-risky’ cases (Fisher Exact Test, Intensity: p<0.001, Area: p<0.002). (Table No. 13a
& 13b). The intensity of MMP-9 expression in the epithelium was golden brown in majority
of the ‘risky’ and ‘non-risky’ cases. Where in more than 26% of epithelial cells had enhanced
MMP-9 immunoexpression in 37 cases of ‘risky’ dysplasia. The immunoexpression in
stromal cells did not show any significant correlation / association in the intensity and area
between the two study groups (Fisher Exact Test, Intensity: p<0.551 NS, Area: p<0.700 NS).
The expression of MMP-9 in ‘risky’ group was in accordance with the study on
OSCC where it was noted that MMP-9 is expressed in carcinoma and inflammatory cells
around carcinoma islands.118,119 For tumors to invade and metastasize, neoplastic cells must
be capable of degrading the extracellular matrix and accessing blood vessels and lymphatics.
Mounting evidence supports the view that extracellular proteinases, such as the MMPs,
mediate many of the changes in the microenvironment during tumor progression.124
Richard et al130 in his retrospective study examined changes in MMP 1, 2 and 9 using
polymerase chain reaction in oral dysplasia and oral squamous cell carcinoma and found
higher levels of MMP-1 and 9 mRNA that was significantly associated with dysplasia that
progressed to oral cancer as compared with those that did not. Peschos et al131 checked
expression of MMP-9 in benign, premalignant and malignant laryngeal lesions. They
suggested a two-step model of up-regulation of MMP-9 expression, first when a dysplastic
lesion evolves and the next when the dysplasia progresses to invasive carcinoma. MMP-9
expression was related neither to survival nor to the other available clinicopathological
parameters.
In the present study the stromal reaction of MMP-9 did not show any significant
difference between the cases. Though the stromal reaction in the high risk category cases
from the ‘risky’ dysplasia group were more pronounced as compared to the other cases.
Recent studies have shown that MMPs contribute to tumor progression, through its effect on
cell proliferation, survival and angiogenesis.170
In addition to carcinoma cells, cancers consist of tumor-associated stromal cells,
which include fibroblasts, endothelial cells, leukocytes, macrophages, nerve cells, and
adipocytes. The cancer cells crosstalk with stromal components during cancer progression
and they are mediated by transmembrane receptors, which are expressed on cancer cells and
stromal cells.132 Tumor-associated cells promote angiogenesis, inflammation, invasion, and
Discussion
125
ECM modeling through cell-cell contact and the production of growth factors, hormones,
cytokines, and proteinases such as MMPs.170
MMP 9 is detected in malignant transformation of various cells and is associated with
tumor metastasis and poor prognosis. In the aggressive human tongue squamous cell
carcinoma cell line, MMP-2 was only found in its latent form, whereas MMP-9 was found in
its active form. In an oral squamous cell carcinoma cell line SCC-25, increase expression of
MMP-9, in vitro, this was thought to occur via fibronectin integrin v 6 pathway. MMP-9
may not be the only factor for OSCC invasion, but it may be important for indirect cell
signaling by controlling the bioavailability and bioactivity of molecules that target specific
receptors, which regulate cell growth, migration, inflammation, and angiogenesis.132
Gao J (2010)133 studied the expression of MMP-2/MMP-9 in normal oral mucosa,
lymph node-negative tongue cancers, lymph node-positive tongue cancers and their
metastasized tumors in cervical lymph nodes. They hardly found MMP-2/MMP-9 expression
in normal epithelium. In lymph node-negative tongue cancer, 45% and 40% of these primary
tumors were positively stained for MMP-2/MMP-9. Importantly, in lymph node-positive
tongue cancer, 71% and 79% of these primary tumors were positive for MMP-2/MMP-9,
respectively. Over expression of MMP-2/MMP-9 was present in the metastatic lymph nodes.
Their result implied the significance of MMP-2 and / or MMP-9 in predictive value for the
actual or potential presence of cervical metastases. Such activity has prognostic value, and
provides impetus for further development of biotherapies targeted at specific inhibition of
MMP activities.
Progress in molecular oncology has significantly advanced our knowledge on
tumorgenesis; yet the practical applications of these genetic markers remain unresolved in
detecting oral dysplasia. None of the molecular markers single or in combination appear to
be ready for use in routine clinical diagnostic practices, although many molecules have been
studied as intermediate markers.16
Furthermore, as suggested by Gayani P. et al (2009)16 recent advances in the use of
quantitative methods has enabled the profiling of gene expression (microarray), protein
expression (proteomics), screening of epigenetic changes, e.g. by pyro-sequencing and single
nucleotide changes (SNP) of hundreds of pre-selected genes simultaneously. These
techniques which require minute amount of tissue when carried out in controlled way and
Discussion
126
using carefully selected samples with proper validation will help the potentially malignant
and malignant tissue samples. Combined with routine histopathology studies, these broad
based studies of gene and their products appear to have a great deal of potential for the
identification of diagnostic and prognostic markers of pre-cancer, and data from multicenter
studies using microarray technology may be engaged in the future for a better understating of
aetiopathogenesis of pre-cancer.
CONCLUSION
Conclusions
127
CONCLUSIONS
Oral epithelial dysplasia : a histopathologic marker of premalignancy, is a diagnostic
term used to describe the histopathological changes seen in progressive chronic and
premalignant disorders of the oral mucosa.. It may also be seen in verrucous or papillary
leukoplakia or in the margins of chronic mucosal ulcers. It is also consistently seen in the
apparently normal mucosa adjacent to the squamous cell carcinoma. Clinically it may present
as leukoplakia, erythroplakia or leukoerythroplakia 25
A histological dysplasia classification system should ideally meet two basic
requirements. First it should be easily applicable in daily routine practice with low inter and
intra observer variability. Secondly, it should allow a clear separation and give clear
suggestions to the clinician to distinguish between patients who need treatment to prevent
progression and those whom no treatment is needed and should be kept on routine
observation.
We propose a new binary classification system consisting of two categories where the
individual architectural and cytological features are given a score. This new classification has
been validated and has shown good to almost perfect agreement for both inter and intra
observer variation. This classification has been designed with the following objectives : a) to
reduce the inter and intra observer variability, b) to make it objectively reproducible,
c) transmit maximum information possible from interpretation of routine H&E stained
sections, d) give a clear diagnosis for clinicians to make critical treatment decision.
From the observations of the present study, ten important morphological and
architectural features are identified to be considered for categorizing potentially malignant
disorder histologically as ‘risky’. Abnormal superficial mitosis and marked cellular and
nuclear pleomorphism were two important features that could predict malignant
transformation are found to be statistically significant and that should be identified and
considered for risk categorization and malignant transformation.
The IHC markers using Ki-67 for observing the irregular epithelial cell proliferation,
Ck-19 for loss of polarity and basal cell hyperplasia in the suprabasal layers and MMP-9 for
stromal changes, micro-invasion and changes in the basement membrane and lamina propria
have substantiated the H&E observation.
Conclusions
128
Our proposed system of grading dysplasia is designed for histopathological
evaluation using H&E stained sections. It was designed to extract a larger set of data with
detailed observations of individual features of dysplasia based on architectural and
cytological changes. Our aim was to generate objective data to correlate with defined patterns
of dysplastic features.
Grading dysplasia is not an exact science and pathologists are doing their best to
reach an optimal result. In this study we have tried to weigh individual features by defining
their importance and scoring them for OED grading process obtaining a common consensus
on the features. However, data reveals that the proposed classification system can be used to
improve or develop simpler and easy diagnostic methods.
The outcome of ‘risky’ dysplasia for malignant transformation could not be assessed
in the study due to lack of follow up data. Within the limitations of the analysis we have
shown that many individual features of dysplasia can be assessed reproducibly as they were
categorized under high risk with a definite score. Reducing features to definable components
increases objectivity and insight, though it makes it difficult for comparison with the
published data.
Future studies should compare lesional responses related to patient population and
their geographic areas of origin. As well as specific carcinogenic factors, diagnostic
usefulness of aids like Toluidine blue application, oral brush biopsy techniques and
spectroscopy and the natural history of the potentially malignant disorders should be
correlated.
Relevance of the observations and future scope of this study
• The proposed classification is objective and is easy to understand. As the inter and
intra observer agreement is statistically significant and hence is reproducible.
• The proposed classification has been assigned with scores for specific features that
has helped to minimizes the inter and intra observer variability.
• Reporting and grading of oral epithelial dysplasia in potentially malignant disorders
has been made easy and valid with the use of routine H&E stain section from any
histopathology diagnostic service, center or laboratory.
Conclusions
129
• Larger sample size studies, at various oral pathology centers, in different
geographical locations could help in developing this proposed classification
universally acceptable.
• Our classification of ‘risky’ and ‘non-risky’ will be helpful for clinical application in
imparting proper treatment, patient education, ‘wait & watch’ policy and follow-up.
Hence, the need is for:
• Application of the proposed binary classification as ‘risky’ and ‘non-risky’ for
routine use.
• Multicentric focused group assessment using the proposed binary classification.
• Longitudinal follow-up study for evaluating the malignant potential of ‘risky’ and
‘non-risky’ cases of potentially malignant disorders.
Bibliography
130
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CONTENT
Sl. No. Contents Page No.
1 Background of the proposed research 1 – 3
2 Aim and Objectives 3
3 Materials and Methods 4 – 12
4 Results 12 – 15
5 Discussion 15 – 27
6 Conclusion 27 – 29
7 Bibliography 29 – 32
1
BACKGROUND OF THE PROPOSED RESEARCH
The significance of oral cancer involves its mortality and the disfigurement and
dysfunction associated with treatment. The rate of curability of cancer depends on the stage and
site. Small cancers for example on anterior tongue, floor of the mouth and buccal mucosa have a
local control rate upto 90%. Moderately advanced lesions without evidence of spread to cervical
lymph nodes have control rates upto 80-90%, whereas the intra oral carcinomas with cervical
lymph node metastasis, the survival rate is less than 50%.1
The mortality of oral cancer has increased seven times in the last 50 years in-spite of
millions worth research going on globally. India has one of the highest rates in oral cancer in the
world partly attributed to high prevalence of tobacco chewing.
In parallel to the increase in Oral Cancer, border-line malignant lesions which range from
epithelial dysplasia to intra epithelial carcinoma have also increased in numbers. These lesions are
significant part of routine oral pathology service. This is due to increased awareness, improved
consciousness towards health, availability of health care centers at primary, secondary and tertiary
levels, which results in early detection of oral borderline cases.
Over 90% of all oral cancers are squamous cell carcinomas (SCC) arising from the lining
mucosa. SCC may arise de novo in clinically normal mucosa, though a majority is preceded by
visible changes of the mucosa. There are several histologically distinct lesions of oral mucosa
which have malignant potential. Among these are leukoplakia, erythroplakia, proliferative
verrucous leukoplakia, oral submucous fibrosis etc. These vary in their malignant potential and
genetic background. The lesions may be found in association with and / or preceding SCC.
The term ‘Precancer’ has assumed newer terminologies as “Potentially malignant lesions,
oral precursor lesions, premalignant conditions etc”. These lesions present either as white macule
with excess surface keratin or as red, non-blanching macules with minimal surface keratin.
Histologically these precancerous lesions present with epithelial dysplasia, which is described by
Stedman's Medical Dictionary as a disorder of differentiation of epithelial cells which may
regress, remain stable, or progress to invasive carcinoma. The features of epithelial dysplasia
include: Increased nuclear to cytoplasmic ratio, acanthosis, atypical mitosis, pleomorphism,
nuclear hyperchromatism, enlarged darkly stained nucleoli, loss of cellular adhesion and
abnormal keratinization. These features vary with individual tissue specimens and assessment
2
varies with pathologists viewing the same tissue specimen. Prognostic significance of a lesion is
difficult to determine because of the above factors.2,3
Not all precancerous lesions and conditions transform to cancer. Morphological
alterations amongst some may have an increased potential for malignant transformation. These
are also indicators of risk of likely future malignancies elsewhere in clinically normal appearing
oral mucosa and not only site specific predictors.4
There is always a challenge for oral pathologists to assess the degree of dysplasia in
potentially premalignant oral lesions with accuracy, so that the burden of malignant
transformation can be predicted and treatment modalities can ensure reduced morbidity.5
The current histopathological grading of oral dysplasia lesions is notably unreliable
mainly due to lack of objective and validated grading features. Many studies have shown wide
variability in the diagnosis and grading of oral epithelial dysplasia (OED) with results
demonstrating only poor to moderate agreement.6
The customary grading system of oral epithelial dysplasia into mild, moderate and severe
does not allow accurate prediction of which cases may eventually transform into malignancy.
Many studies have remarked that precancers with epithelial dysplasia have shown to develop
into cancer more readily than lesions without.7 Nevertheless all precancer or epithelial dysplasia
do not develop into cancer, whereas, some have even shown to regress with time.8
The limitations inherent in the histologic grading, suggest the necessity to improve
histologic assessment and the need to identify more reliable makers. In the last decade approach
to understanding of molecular carcinogensis has revealed newer directions. Many molecular
markers functioning as elements of cell cycle control have been recognized. These have been
tested individually and in combinations to develop diagnostic markers for identification of
dysplasia and their ability to predict malignant potential.9
Most of the authors have concluded that though the progress in molecular oncology has
significantly advanced the knowledge on tumorigenesis, yet the practical applications of these
genetic makers remain unresolved in detecting oral dysplasia. Not many studies have focused
correlating the findings at molecular level with the clinical attributes of terminal outcome.
The present study was undertaken to assess the different grading systems to determine the
inter observer and intra observer variation and to study the individual features defining atypia
and general disturbance in the epithelium of oral epithelial lesions; dysplasia. An attempt is also
3
made to correlate the histopathological features with Immunohistochemistry markers for
cytokeratin (CK-19) basement membrane enhancement protein (Ki-67) and extracellular matrix
change (MMP-9) so as to substantiate the observation of atypia observed in H&E stained tissue
sections. The study will also try to validate a new binary classification which will objectively
define each feature and classify OED as ‘risky’ and ‘non-risky’ using the Hematoxylin and Eosin
stains as gold standard.
AIM AND OBJECTIVES
Primary Objective (Aim) :
To propose a new classification for dysplasia to categories dysplasia into ‘non risky’ and
‘risky’ groups
Secondary Objective :
1. To histopathologically evaluate 100 cases of potentially malignant disorders (PMD) for
degree of dysplasia based on the classification system given by (a) Smith and Pindborg’s
classification (1969) as mild, moderate and severe, using photographic standardization
and scores (b) WHO classification (2005) as mild, moderate & severe dysplasia and (c)
Binary classification as low risk and high risk cases given by Kujan Omar (2006).
2. To assess the inter and intra observer variability and agreement for the above mentioned
3 classification systems.
3. To histopathologically evaluate the degree of dysplasia for the same 100 cases of
potential malignant disorders (PMD) using the proposed classification.
4. To assess the inter and intra observer variability and agreement for the proposed
classification.
5. To identify individual histopathological features within the proposed classification that
would aid in categorization of risk behavior.
6. To correlate the proposed classification for degree of dysplasia with
immunohistochemical markers for : CK-19 for epithelial differentiation, Ki-67 as
proliferative marker and MMP-9 as marker for epithelial - connective tissue interaction.
7. To correlate the observations of IHC with histopathological features analyzed with the
new proposed classification.
MATERIAL AND METHODS
After obtaining an institutional ethical clearance the present retrospective study is based
on grading of epithelial dysplasia to evaluate the inter and intra observer variability with most
commonly used classification
a proposed new binary classification system.
Pilot Study :
A pilot study was conducted to assess the inter and intra observer variability in grading
OED using a set of 25 archival cases
IDS - Belgaum, DMDC - Wardha, GDC
independently by four oral pathologists trained under one centre. Three classification systems
were undertaken to determine the degree of dysplasia namely WHO 1978, Smith & Pindborg’s
1969 and WHO 2005. The Oral Pathologists were blinded to the clinical and histopathological
diagnosis. Findings were recorded on individual score sheets independently and a final
assessment for each case was recorded by the observer as “no dysplasia / mild / moderate /
severe dysplasia”. The results were tabulated on excel sheet
observer variability. WHO 1978
in the moderate group, whereas
a moderate range. WHO 2005
No. 1). Following this the set of same 25 cases were assessed
Kujan Omar et al 2006, as ‘low risk and high risk, based on features described by WHO 2005 as
cytological changes and architectural features.
observed. Suggesting the degree of
classification given by Smith and Pindborg
Estimation of Sample Size:
The sample size was calculated using the following formula :
4
MATERIAL AND METHODS
obtaining an institutional ethical clearance the present retrospective study is based
on grading of epithelial dysplasia to evaluate the inter and intra observer variability with most
commonly used classification system. The grading of epithelial dysplasia has also be done using
a proposed new binary classification system.
A pilot study was conducted to assess the inter and intra observer variability in grading
OED using a set of 25 archival cases collected from different oral pathology centers (KLE VK
Wardha, GDC - Nagpur and LMDC - Nagpur). These were reviewed
independently by four oral pathologists trained under one centre. Three classification systems
ermine the degree of dysplasia namely WHO 1978, Smith & Pindborg’s
1969 and WHO 2005. The Oral Pathologists were blinded to the clinical and histopathological
diagnosis. Findings were recorded on individual score sheets independently and a final
for each case was recorded by the observer as “no dysplasia / mild / moderate /
The results were tabulated on excel sheet for analysis of
WHO 1978 classification showed poor to good agreement, m
, whereas Smith and Pindborg’s was fair to moderate agreement, following
a moderate range. WHO 2005 demonstrated moderate agreement among all observers
Following this the set of same 25 cases were assessed by the binary system suggested by
Kujan Omar et al 2006, as ‘low risk and high risk, based on features described by WHO 2005 as
cytological changes and architectural features. Moderate to almost perfect agreement
Suggesting the degree of agreement was best in the binary system followed by
classification given by Smith and Pindborg.
The sample size was calculated using the following formula :
obtaining an institutional ethical clearance the present retrospective study is based
on grading of epithelial dysplasia to evaluate the inter and intra observer variability with most
system. The grading of epithelial dysplasia has also be done using
A pilot study was conducted to assess the inter and intra observer variability in grading
collected from different oral pathology centers (KLE VK
Nagpur). These were reviewed
independently by four oral pathologists trained under one centre. Three classification systems
ermine the degree of dysplasia namely WHO 1978, Smith & Pindborg’s
1969 and WHO 2005. The Oral Pathologists were blinded to the clinical and histopathological
diagnosis. Findings were recorded on individual score sheets independently and a final
for each case was recorded by the observer as “no dysplasia / mild / moderate /
for analysis of the inter and intra
ood agreement, maximum being
air to moderate agreement, following
emonstrated moderate agreement among all observers (Annex.
by the binary system suggested by
Kujan Omar et al 2006, as ‘low risk and high risk, based on features described by WHO 2005 as
Moderate to almost perfect agreement was
agreement was best in the binary system followed by
Where: n=sample size
N= no. of subjects in the entire population.
n* = depends (r
r = relative error
pa = overall agreement probability
pe = chance agreement probability
Taking into consideration, if raters will agree about 50% of times and relative error of
20%. i.e. pa-pe = 0.5 and r=20
Selection of cases (Samples) :
Based on the results obtained from the pilot study the present research protocol was
designed to further validate these grading systems and also to propose a new classification. The
validation of these classifications is also based on immunohisto chemical markers.
For the present study 100 cases of potentially malignant disorder (PMD) were retrieved
from the archives of the department which reported to KLE VK Institute of Dental Sciences
OPD from 1995 to 2012. From cases
severe dysplasia and carcinoma in
available were included. Biopsy samples obtained from lesions on buccal mucosa were
considered. 100 slides from 100 different cases
study material.
In the series of selected 100 cases, the
considered for the final assessment.
Demographic data :
Clinical data was obtained from the biopsy requisition form f
The demographic data like age, sex, type of habit, frequency and duration of tobacco habit w
5
n=sample size
of subjects in the entire population.
depends (r and pa-pe)
r = relative error
overall agreement probability
chance agreement probability
Taking into consideration, if raters will agree about 50% of times and relative error of
pe = 0.5 and r=20
Selection of cases (Samples) :
Based on the results obtained from the pilot study the present research protocol was
designed to further validate these grading systems and also to propose a new classification. The
tion of these classifications is also based on immunohisto chemical markers.
For the present study 100 cases of potentially malignant disorder (PMD) were retrieved
department which reported to KLE VK Institute of Dental Sciences
From cases with signed out diagnosis of hyperplasia, mild, moderate,
severe dysplasia and carcinoma in-situ were selected. Samples with sufficient depth of tissue
lable were included. Biopsy samples obtained from lesions on buccal mucosa were
from 100 different cases were finally included,
In the series of selected 100 cases, the 25 cases included for pilot
considered for the final assessment.
Clinical data was obtained from the biopsy requisition form f or these selected 100 cases.
The demographic data like age, sex, type of habit, frequency and duration of tobacco habit w
Taking into consideration, if raters will agree about 50% of times and relative error of
Based on the results obtained from the pilot study the present research protocol was
designed to further validate these grading systems and also to propose a new classification. The
tion of these classifications is also based on immunohisto chemical markers.
For the present study 100 cases of potentially malignant disorder (PMD) were retrieved
department which reported to KLE VK Institute of Dental Sciences
with signed out diagnosis of hyperplasia, mild, moderate,
Samples with sufficient depth of tissue
lable were included. Biopsy samples obtained from lesions on buccal mucosa were
finally included, which formulated the
25 cases included for pilot study were not
r these selected 100 cases.
The demographic data like age, sex, type of habit, frequency and duration of tobacco habit w as
6
noted. The clinical provisional diagnosis ranged as Leukoplakia, erythroplakia,
erythroleukoplakia and oral sub mucous fibrosis. Confidentiality and maintenance of all the
obtained records of all the cases included in this research protocol will be strictly maintained by
stating the waiver of consent.
Procedure :
Four sections of 4μeach were prepared by sectioning paraffin embedded tissue blocks.
One section was stained with H & E for histopathological evaluation and the one section each
was obtained on gel coated slide and stained immunohistochemically for MMP9, Ki-67 and with
CK-19 using HRP-IHC. The staining procedures and analysis is been stated in manual of
operation and standard operating procedures.
Assessment Method for Epithelial Dysplasia:
For final assessment the grading classifications given by Smith and Pindborg’s grading
system (1969), WHO (2005) and the Binary grading system (2006) were used for evaluation of
H&E stained sections of 100 cases. No calibration exercise was attempted. Each slide was
labeled only with a serial order number. No clinical data or patient information was provided
with the slides to the evaluator. Four independent oral pathologists reviewed the slides. Sections
were observed under 4x; 10x; 100x magnification using Olympus BM2500 compound
microscope. The architectural characteristics of each feature was examined under the low
magnification (4x & 10x) and finished with cytological characteristics at high magnification
(40x).
A score sheet was designed for recording the individual features to aid systematic
analysis. Separate score sheets based on the selected three classifications were prepared. The
lesions were graded as ‘0 for no dysplasia ; 1 for Mild ; 2 for Moderate and 3 for Severe
dysplasia / carcinoma in-situ. For Binary grading system ‘1 for Low risk and 2 for High risk’ was
scored.
Individual scores of all raters were obtained and in addition to the individual scores of the
raters a consensus score of 3 raters (SH, DM & PA) was obtained to compare with the
investigator’s (AK) score. Based on the proposed classification of the present study the observers
evaluated all the 100 cases as ‘non-risky’ and ‘risky’ for inter and intra observer variability for
all 18 dysplastic features. For 10 important features predicted for malignant transformation all
the 100 cases were evaluated.
7
Classifications considered for the pilot study and the present study :1. Histologic features for Smith & Pindborg 1969 classification of oral epithelial dysplasia.
Type of change Severity of dysplasia & scores1. Drop shaped reteridges None-0 Slight-2 Marked-42. Irregular epithelial stratification None-0 Slight-2 Marked-53. Keratinization of cells below keratinized layer None-0 Slight-1 Marked-34. Basal cell hyperplasia None-0 Slight-1 Marked-45. Loss of intercellular adherence None-0 Slight-1 Marked-56. Loss of polarity None-0 Slight-2 Marked-67. Hyperchromatic nuclei None-0 Slight-2 Marked-58. Increased nucleo-cytoplasmic ratio in basal and
prickle cell layersNone-0 Slight
Increase-2MarkedIncrease-6
9. Anisocytosis and anisonucleosis None-0 Slight-2 Marked-610 Pleomorphic cells and nuclei None-0 Slight-2 Marked-611 Mitotic activity Normal-0 Slight
Increase-1MarkedIncrease-5
12 Level of mitotic activity Normal-0 Slight-3 Marked-1013 Presence of bizarre mitoses None-0 Slight-6 Marked-10
2. WHO criteria for grading dysplasia (1978) : It graded epithelial dysplasia as: Mild,
Moderate, Severe.
Mild dysplasia: Slight nuclear abnormalities, most marked in the basal third of the epithelial
thickness and minimal in the upper layers, where the cells show maturation and stratification. A
few, but no abnormal mitosis may be present, usually accompanied by keratosis and chronic
inflammation.
Moderate dysplasia: More marked nuclear abnormalities and nucleoli tend to be present, with
changes most marked in the basal 2/3rd of the epithelium, nuclear abnormalities may persists up
to the surface, but cell maturation and stratification are evident in the upper layers. Mitoses are
present in the parabasal and intermediate layers, but none is abnormal.
Severe dysplasia: Marked nuclear abnormalities and loss of maturation involving more than
2/3rds of the epithelium, with some stratification of the most superficial layers. Abnormal
mitoses may be present in the upper layers.
Severe grades of dysplasia may merge into the lesion customarily designated as
carcinoma in situ, in which the whole or almost the whole thickness of epithelium is involved.
In the present knowledge, it was not possible to say whether the presence of severe dysplasia
carried a different degree of risk of subsequent development of invasive carcinoma than the
presence of carcinoma in situ.
8
3. WHO criteria for grading dysplasia (2005) :
Architecture criteria Cytology criteria1. Irregular epithelial stratification 1. Abnormal variation in nuclear size
(Anisonucleosis)2. Loss of polarity of basal cells 2. Abnormal variation in nuclear shape
(Nuclear pleomorphism)3. Drop-shaped rete ridges 3. Abnormal variation in cell size
(Anisocytosis)4. Increased number of mitotic figures 4. Abnormal variation in cell shape
(Cellular pleomorphism)5. Abnormal superficial mitoses 5. Increased nuclear cytoplasmic ratio6. Premature keratinisation in single cells(dyskeratosis)
6. Increased nuclear size
7. Keratin pearls within rete ridges 7. Atypical mitotic figures8. Increased number and size of nucleoli9. Hyperchromatism
Grading of dysplasia:
Mild dysplasia: In general architectural disturbance limited to the lower third of the epithelium
accompanied by cytological atypia define the minimum criteria of dysplasia.
Moderate dysplasia: Architectural disturbance extending into the middle third of the
epithelium is the initial criterion for recognizing this category. However, consideration of the
degree of cytologic atypia may require upgrading i. e. those lesions that show marked cytological
alteration should be elevated to a higher grade level regardless of how extensively the atypical
cells fill the epithelium.
Severe dysplasia: Recognition of severe dysplasia starts with greater than two thirds of the
epithelium showing architectural disturbance with associated cytologic atypia. However,
architectural disturbance extending into the middle 3rd of the epithelium with sufficient cytologic
atypia may be up graded from moderate to severe dysplasia.
Carcinoma in situ: The theoretical concept of carcinoma in situ is that malignant transformation
has occurred but invasion is not present. It is not always possible to recognize this
morphologically. The following is recommended for the diagnosis of carcinoma in situ: full
thickness or almost full thickness architectural abnormalities in the viable cellular layers
accompanied by pronounced cytologic atypia. Atypical mitotic figures and abnormal superficial
mitoses are commonly seen in carcinoma in-situ.
9
4. Binary Classification (2006) :
High-risk lesion (with potential susceptibility for malignant transformation) was based on
observing at least four architectural changes and five cytological changes.
Low-risk lesion (does not have the potential susceptibility for malignant transformation) was
based on observing less than four architectural changes or less than five cytological changes. The
features to classify as High and Low Risk were based on the architectural and cytological
features suggested by WHO 2005 classification.
PROPOSED WORKING CLASSIFICATION
The proposed classification is based on Binary System (2006) and WHO (2005) criteria of
epithelial dysplasia. Additional architectural and cytological features are identified and included
in this classification. Each features of epithelial dysplasia is scored on a rating scale of 1 to 10.
The two groups identified are :
• Non risky lesions : The total score ranged from 1 to 15
• Risky lesions : The total score ranged from 16 to 84
• ARCHITECTURAL FEATURES:
Sl. No. Particulars Scores1. 1.1. Basal cell hyperplasia 42. 1.2. Drop shaped rete ridges 23. 1.3. Few normal mitotic figures 14. 1.4. Acanthosis 15. 1.5. Loss of polarity of basal cells 56. 1.6. Drop shaped rete ridges with nodules or forking 47. 1.7. Loss of cohesiveness with prominent intercellular bridges 58. 1.8. Abnormal and superficial mitoses 109. 1.9. Increased number of mitotic figures 510. 1.10. Dyskeratosis 211. 1.11. Loss of epithelial stratification 512. 1.12. Keratin pearls within rete ridges 5
• CYTOLOGICAL FEATURES:
Sl. No. Particulars Scores1. 2.1. Mild to moderate degree of nuclear and cellular pleomorphism 52. 2.2. Marked nuclear & cellular pleomorphism &anisonucleosis 103. 2.3. Hyperchromasia of cells 54. 2.4. Increased nuclear-cytoplasmic ratio 45. 2.5. Atypical mitotic figures 66. 2.6. Increased number and size of nucleoli 4
10
INFLAMMATION :
Sl. No. Particulars Scores1. 3.1 Mild 12. 3.2 Moderate 23. 3.3 Severe 3
Procedure for H&E staining and Immunohistochemistry :
Four sections each of selected archival blocks were cut, one slide stained routinely with H&E
to confirm the diagnosis and the other slide stained with immnunohistochemical stain. The sections
required for immunoexpression is taken on a gel coated slides using 2%, 3-aminopropylethoxysilane
solution (APES) (Sigma Aldrich, St.Louis, MO, USA, Product code: A3648). The buffers required
for IHC are PBS as wash buffer and citrate buffer for antigen retrieval. The immunohistochemical
protocol was followed as per the standard recommendations of the company.
The IHC staining is performed using Super Sensitive one step Polymer HRP system
(QD-600, Biogenex, Bangalore) with pre-diluted primary antibodies using anti-Human antibody
for CK-19 (clone RCK 108, Biogenex, Bangalore), Anti MMP-9 (EP1-255Y, Biogenex,
Bangalore), Ki-67 (BGX-297, Biogenex, Bangalore). The storage and dilution of these
antibodies and IHC kit is done as per the manufacturers’ recommendation (Annex. No. 3).
Assessment of Immunohistochemical stained sections:
All the IHC stained slides were evaluated for immunoexpression of MMP 9, Ki 67, CK
19. The intensity of immunohistochemical staining was graded based on subjective evaluation ofcolor exhibited (brown color) by antigen, antibody and chromogen complex. For Ki-67, the
immunoexpression was noted for nuclear staining, CK-19 in cytoplasm and MMP-9 for stromalreaction. Evaluation of immunoexpression was noted under x10 magnification under light
microscope.
1. Analysis of immunoexpression of MMP-9
The immunoexpression of MMP-9 was based on intensity and area of expression in the
epithelium and stroma on a 4 point scale from 0 to 3. Intensity of expression was graded as 0
when there was no evidence of immunoexpression. Intensity was considered positive depending
upon the colour intensity ranging from 1 : golden brown, 2 : light brown, 3 : dark brown. Area
of expression was based on a percentage of positivity in the stroma and epithelium. The range
was considered 1: when 1 to 25% of positive expression was noted, 2 : 26 to 50% of positivity
and 3 : more than 50% of positive expression.
11
Intensity Area in epithelium and stroma0 = -ve 0 = -ve1 = + , golden brown 1 = 1-25%2 = + + , light brown 2 = 26-50%3 = + + + , dark brown 3 = >50%
2. Analysis of immunoexpression of Ki-67
The immunoexpression of Ki-67 was mainly assessed in nuclei of epithelial cells. This
marker was evaluated in the epithelium based on the area of expression and percentage ofpositive immunoexpression on a four point scale from 0 to 3. When there was no evidence of
immunoexpression it was graded as 0. In the epithelium this proliferative marker wasassessed based on the layers of the stratified squamous epithelium involved. Whenever
expression was noted only in the basal cell layer it was graded as 1, suprabasal layer wasgraded as 2 and layers above this was graded as 3. Percentage of number of cells for positive
immunoexpression was assessed. Area of expression was graded as 1: when 1 to 25%positive epithelial cells were noted, 2: 26 to 50% positive cells and 3: more than 50% cells.
The area with maximum number of positive cells was considered in each section.
Area Percentage no. of cells0 : No expression 0 = -ve1 : Basal layer 1 = 1-25%2 : Suprabasal layer 2 = 26-50%3 : Above suprabasal layer 3 = >50%
3. Analysis of immunoexpression of Ck-19
The immunoexpression of Ck-19 was based on intensity and area of expression in the
epithelium on a 4 point scale from 0 to 3. Intensity of expression was graded as 0 when there was
no evidence of immunoexpression. In te ns it y wa s co ns id er ed po si ti ve depending upon the
colour intensity ranging from 1 : golden brown, 2 : light brown, 3 : dark brown. Area of
expression was based on a percentage of positivity in the epithelium. It was considered as 1 for 1
to 25% of positive expression, 2 : 26 to 50% of positivity and 3 : more than 50% of positive
expression in the epithelium.
Intensity Area0 = -ve 0 = -ve1 = + golden brown 1 = 1-25%2 = + + light brown 2 = 26-50%3 = + + + dark brown 3 = >50%
12
STATISTICAL ANALYSIS:
Statistical analysis was done using :1. Weighted Kappa and Non-Weighted Kappa tests for assessment of inter and intra observer
variability for all the classification system
Unweighted kappa takes into consideration agreement for individual cases
Weighted kappa considers the magnitude of disagreement (ordered categories) and
hence is a better measure for reliability
Kappa Agreement (Flesis and Cohen 1973)
< 0.40 Poor
0.40-0.75 Fair to Good
0.76 to 0.80 Excellent
0.81-0.99 Almost perfect
2. Fisher's Exact Test was done to evaluate the correlation of the immunohistochemical
markers with grades of epithelial dysplasia. p<0.001 was considered as statistically
significant.
RESULTS
In the pilot study 25 cases of OED were evaluated for inter and intra observer variation
using four classifications :- a) Grading of dysplasia given by WHO (1978), b) Smith and
Pindborg Classification using photographic standards (1969) c) Classification of WHO (2005)
and d) Binary Classification by Kujan Omar et al (2006).
For the present study 100 cases of OED were subjected to evaluation for two selected
classifications: Smith and Pindborg (1969) and WHO (2005). A new classification is proposed
which included scoring system and has two categories for grading dysplasia as ‘non-risky’ and
‘risky’. IHC evaluation for Ki-67, CK-19 and MMP-9 is done to understand behavior at the
molecular level. The data obtained has been statistically analyzed.
Statistical analysis of 25 cases considered for Pilot Study : WHO (1978) showed good to poor
agreement with maximum having moderate agreement. Smith and Pindborg (1969) had fair to
moderate agreement following a moderate range. Whereas WHO (2005) demonstrated moderate
agreement amongst all observers. Moderate to almost perfect agreement was observed in binary
classification by Kujan O. The inference drawn from the pilot study suggests that the degree of
13
agreement was best in the binary system followed by classification given by Smith and Pindborg.
(K=kappa, kw=weighted kappa)
Statistical analysis of 100 study cases considered for INTER OBSERVER variability : Smith and
Pindborg’s classification showed fair to moderate agreement in both weighted and unweighted
kappa statistics (p<0.001). Similarly, WHO (2005) classification also showed fair to moderate
inter observer agreement. Good agreement was evident with proposed classification.
Statistical analysis of 100 study cases for INTRA OBSERVER variability : The intra observer
agreement was found to be almost perfect in the proposed classification as compared to the other
classification.
Distribution of cases into ‘non-risky’ (total score ranged from 1-15 ) and ‘risky’ (total
score ranged from 16 to 84) groups : 59 cases were identified as risky dysplasia as compared
to 41 cases of ‘non-risky’ dysplasia. Suggesting that 59 cases out of 100 cases are likely to
develop into malignancy.
Calculation for risk categorization using median and inter quartile for 100 cases.
Classification of risk categories for evaluation of 59 ‘risky’ cases : Entire score for the
proposed classification comprising of 1 to 84 was evaluated for mean and standard deviation.
Median and the inter quartile range was used to define cut off points and the cases were classified
into low, medium and high risk based on the scores above. Out of the 59 ‘risky’ cases 6(10.2%)
cases had highest score (67-84) were suggestive to be at higher risk for malignant transformation.
Risk categorization of ‘Risky’ and ‘Non-risky’ 100 cases for each feature : Cases of both
non-risky and risky categorized into three risk categories as per the scores for 18 individual
features. Feature 1.8* (Abnormal and superficial mitoses) and 2.2* (Marked nuclear & cellular
pleomorphism & anisonucleosis) were significantly associated with the risk group as compared
to the non-risky group. (Fisher Exact Test p<0.0010 & p<0.003).
Ten Important Features with higher scores considered in ‘Risky group’ are segregated
with a total score of 61 : Loss of polarity of basal cells, Loss of cohesiveness with prominent
intercellular bridges, Abnormal and superficial mitoses, Increased number of mitotic figures,
Loss of epithelial stratification, Keratin pearls within rete ridges, Mild to moderate degree of
nuclear and cellular pleomorphism, Marked nuclear & cellular pleomorphism & anisonucleosis,
Hyperchromasia of basal cells, Atypical mitotic figures.
14
Calculation for risk categorization using median and inter quartile for study group for 10
important features. Classification of risk categories for evaluation of cases for 10 features :
The total score of the ten individual features selected comprising of 01 to 61 was evaluated for
mean and standard deviation. The median and the inter quartile range was used to define cut off
points and the cases were again classified into low, medium and high risk based on the ten
individual features. Out of the 59 ‘risky’ cases 8(13.6%) cases had highest score (> 45) were
suggestive to be at higher risk for malignant transformation.
Risk categorization of ‘risky’ and ‘non-risky’ groups for the 10 features : Cases of both non-
risky and risky categorized into three risk categories as per the scores for 10 selected individual
features. Feature 1.8* (Abnormal and superficial mitoses) and 2.2* (Marked nuclear & cellular
pleomorphism & anisonucleosis) were significantly more commonly associated with the ‘risky’
group as compared to the ‘non-risky’ group (Fisher Exact Test p<0.001).
Area of expression of CK-19 in Epithelium of the study groups (Fisher exact Test) : ‘Risky’
group the area of expression of Ck-19 in 32 cases the expression was seen in 26 to 50% of
epithelial cells. In a ‘non-risky’ group 20 cases were negative for Ck-19 expression but 6 cases
had 26 to 50% positive cells. The area of CK-19 expression in the epithelium was significantly
high in the risky as compared to non-risky group.
CK-19 Expression of intensity in Epithelium of the study groups (Fisher exact Test) : The
intensity of expression was light brown in 30 cases and dark brown in 14 cases of ‘risky’ group.
Whereas in the ‘non-risky’ 20 cases were negative and in 21 cases the intensity ranged from light
brown to dark brown. Intensity of CK-19 expression in the epithelium was significantly high in
the ‘risky’ as compared to ‘non-risky’ group. The eight cases with scores 45 & above had more
intensity (+++) and more area (>50%) of expression of CK-19.
Ki-67 Expression in the study groups (Fisher Exact Test) : 39 ‘risky’ cases had 26 to 50%
positive cells for ki-67 as compared to the non-risky group where 24 cases were negative and 11
cases had1 to 25% positive expression. The Ki-67 expression in the epithelium was significantly
higher in the ‘risky’ as compared to ‘non-risky’ group.
Area of Ki-67 positivity in study groups (Fisher Exact Test) : 42 cases in the risky group had
ki-67 immnoexpression upto to the supra basal layer, whereas 6 cases had expression above the
suprabasal layer. In the ‘non-risky’ 24 cases were negative but 6 cases had ki-67
immunoexpression upto the suprabasal layer. The Ki-67 expression in the epithelium was
15
significantly higher in the ‘risky’ group as compared to ‘non- risky’ group. The eight cases with
scores 45 & above had expression upto suprabasal layer (+++) where >50% of cells had
expression of Ki-67.
MMP-9 Expression in Epithelium of the study groups (Fisher Exact Test) : The intensity of
MMP-9 expression was golden brown in 26 ‘non-risky’ cases and 42 ‘risky’ cases. In 13 risky
cases area of MMP-9 expression in epithelial cell was seen between 26-50% whereas in non-
risky 26 cases had 1 to 25% positive cells. The MMP-9 epithelium was significantly higher in
the risky as compared to ‘non-risky’ group.
MMP-9 Expression in stroma of the study groups (Fisher Exact Test) : The intensity of
MMP-9 expression was light brown in 18 ‘non-risky’ cases and 26 ‘risky’ cases. In 37 risky
cases area of MMP 9 expression in stromal cell was seen between 26-50% whereas in non-risky
19 cases had 1 to 25% positive cells. No association was observed in the stromal expression of
MMP-9 in the ‘risky’ as compared to ‘non-risky’ group.
DISCUSSION
Assessment of dysplasia depends upon the microscopic diagnosis which is based on
grading the changes considering combination of architectural and cytological features.10 Grading
of dysplasia is subjective and lacks intra and inter observer reproducibility due to insufficiency
of validated morphological criteria and the biological nature of dysplasia.11 Hence, due to the
absence of a consensus, several systems are currently employed.
During the last decade many classifications of potentially malignant lesions
synonymously referred as epithelial dysplastic lesions, premalignant conditions, squamous intra
epithelial neoplasia etc. have been proposed. The majority of the classifications in the current
literature have followed criteria similar to those in common use for the grading of epithelial
lesions of the uterine cervix.12 In cervical epithelium there is clear distinction between normal
and abnormal layers of the epithelium and consequently the degree of dysplasia can be assessed
by determining the horizontal level of this border in the epithelium, resulting in substantial intra
and inter observer consistency. Such a sharp distinction between normal and abnormal layers in
the epithelium of the upper aero-digestive tract is less obvious and consequently the definitions
for degree of dysplasia are much more susceptible for discussion, thus resulting in substantial
intra and inter observer variability.13
16
Based on the observations from pilot study and after considering an extensive search of
literature, the criteria under architectural features and morphological features defined by WHO in
2005 were redefined to form the basis for the new proposal to grade epithelial dysplasia for the
present study. We have made an attempt to score the individual features of dysplasia. The criteria
for assigning the scores was based on the severity and occurrence as seen in OSCC or in tissues
adjacent to OSCC, features considered for severe dysplasia by other authors and those suggested
by Smith and Pindborg. This was suggested with the objective of reducing the inter and intra
observer variability and for making assessment objectively reproducible.
The two basic requirement of any grading system should be : reproducibility and
prognostic value. Reproducibility refers to the degree to which observer measurement or
diagnosis remains the same on repeated independent observations of an unchanged characteristic.
This consistency can be assessed between different observers (inter observer) or within a single
observer (intra observer). Prognostic value is the natural evolution of a disease without treatment
and predictive value is the natural evolution of the disease in response to treatment.14 For
studying accuracy in grading dysplasia of upper aero-digestive tract, there is no test available,
which is thought to be better than the pathologist’s observation when an accepted gold standard
is not available for assessing the validity obtained for grading these lesions. Therefore,
reproducibility is needed to provide indication of validity. The inter and intra observer agreement
levels give an estimate of the degree of bias and validity, where an appropriate gold standard is
not available.15
Appraisal of comparison of Epithelial Dysplasia classifications:
Grading is hampered by the arbitrary division into distinct categories of a continually
progressing process without any naturally and sharply defined borders. It is in fact, an attempt to
impose discrete categories on what is in effect a continuous grey scale and therefore any grading
scheme is by definition artificial.16 The ultimate diagnosis depends on the emphasis put upon
each of the characteristics in grading by the observer. Several studies have shown inter and intra
observer variability in the assessment of presence or absence of individual characteristic features
and thus the grading of dysplasia using different classification system.
In the present study 100 cases of PMD with epithelial dysplasia were assessed using
Smith and Pindborg’s classification given in 1969, WHO in 2005 and the proposed new
classification for assessing the inter and intra observer variability. Kappa agreement was used to
17
evaluate the degree of variation in grading dysplasia for each cytological and architectural
feature. Both unweighted kappa and weighted kappa analysis were considered.
Unweighted or simple kappa (k) is a measurement of inter-rater agreement which
provides quantitative estimate of reliability and represents agreement greater than that expected
by chance alone. As stated by Cohen the kappa values fall between ‘-1’ : complete disagreement,
‘0’ : agreement accepted by chance and ‘1’ : perfect agreement. Thus kappa gives credit only for
complete agreement.
Whereas weighted kappa (kw) considers the magnitude of disagreement by giving credit
to complete and partial agreement and hence a better measurement for reliability. Weighted
kappa results in a better inter and intra observer agreement values.17, 18 In the present study the
interpretation of kappa values suggested by Flesis and Cohen (1973)19 were considered, where
the values < 0.40 was rated as poor, 0.40-0.75 as fair to good, 0.76-0.80 as excellent and above
0.81 as almost perfect.
The overall inter observer variability in our study for Smith and Pindborg and WHO 2005
classification showed moderate to fair agreement kw=0.48 & 0.149 and k=0.275 & 0.310
respectively. Whereas the proposed classification had 90% agreement (p=0.001) k=0.79. The
intra observer variation for the 100 cases of dysplasia showed similar observation when
compared between the different classifications. Grading was reproducible with good agreement
for Smith and Pindborg and almost perfect agreement (91%, k=0.81) for the proposed
classification.
Great variability in inter and intra observer agreement has been reported in several
studies. They ranged from poor to substantial agreement by use of different statistical methods.
Pindborg et al reported a wide range of agreement (1-78%) for 9 photomicrographs evaluated by
72 pathologists. Abbey LM et al (1995)20 examined 120 oral biopsies of a range of pathologies
from hyperkeratosis to severe dysplasia. The agreement ranged from 35.8% to 55.8%. The kappa
values improved to 0.70 – 0.88 for inter examiner comparisons when the grading was expanded
to “within one histological grade”.
Sudbo J et al (2001)21 sh ow ed po or inter observer agreement using 3 point ordinal
scale (k values: 0.17 – 0.33). A slight improvement was observed when 2 categories of
“favorable” (Mild and moderate dysplasia) and “poor” (severe dysplasia) were used. (K value
ranged 0.21 – 0.34). Brothwell DJ et al (2003)15 showed a substantial agreement using a 5 point
18
ordinal scale with a group weighed Kappa (Kw) of 0.74 (94%, Cl=0.64 – 0.85). However similar
to other studies, in their study the group unweighted Kappa showed a fair to moderate agreement
(ks) of 0.37 with 95% Cl = 0.32 – 0.42.
Fischer D et al (2004)22 reported an overall kappa weighted value of 0.59 (95% cl=0.45 –
0.72) An improvement in the kappa agreement was observed when the histological diagnosis was
simplified into three general categories of “no abnormality / hyperplasia”, “mild / moderate /
severe dysplasia” and “Carcinoma in situ” (Kw=0.70 (95% Cl : 0.56-0.84). Subjective judgment
and individual histopathological experience of the participating pathologists was the core for the
histopathological assessment.
Kujan O et al (2006)23 observed in their study that for a substantial group weighted
agreement was reported when four observers graded oral biopsy specimens into categories
varying from hyperplasia (no dysplasia) to carcinoma in-situ (Kw=0.63, (95% CI: 0.42- 0.78)). A
fair to moderate agreement was found using unweighted kappa test (ks=0.22, (95CI:0.11 : 0.35).
These variations were due to the fact that the unweighted kappa considered all disagreements
between raters. Whereas weighted kappa yields a higher reliability when disagreements are small
compared to, when they are large. An improvement in the pairwise and overall agreement was
reported in their study when a binary grading system was used. They observed a better
agreement between those experienced in examining oral mucosal biopsies as compared with the
general pathologist. Bias could also be due to established departmental practice in oral pathology
as stated by Kujan O et al.
All previously published reports on the inter observer agreement on grading OED lesions
showed considerable variations. Warnakulasuriya S et al24 suggested that this might be due to the
lack of objectivity in the evaluation of established criteria, arbitrary division of the grading, lack
of calibration of criteria and grading, and lack of sufficient knowledge about which criteria are
important for the prediction of malignant potential.
To overcome some of these limitations, we in our proposed classification have given
score to each of the 18 features identified for dysplasia and are predicted to have potential for
malignant transformation. The sum total of the scores ranged from 1 to 84. When the scores
ranged from 1 to 15 for a PMD it was categorized as ‘non-risky’ and when the scores ranged
between 16 to 84 the lesion was graded as ‘risky’. Accordingly 59% of the cases were grouped
under ‘risky’ suggesting that these are likely to develop into malignancy. Whereas literature
19
suggests, 2% to 17% chance of malignant transformation in PMD. The scores assigned to
individual dysplastic characters were in agreement with that given by Smith and Pindborg for
evaluating the dysplastic features more objectively. The total score in our study was probably
over estimated for the group categorized as ‘risky’. The features considered under the group of
‘risky’ were further evaluated for risk categorization. Entire score of the proposed classification
comprising of 1 to 84 was evaluated for mean and standard deviation. Median and inter quartile
range was used to define cut off point for scoring dysplasia and the cases were regrouped into
low risk (score between 22 - 44) which consisted of 23.7% (14) cases, medium risk with scores
between 45-66 were 66.1% (39) cases and in the high risk group we found 10.2% (6) cases with
a scored between 67-84. This suggested that out of 59 cases initially grouped as ‘risky’, 6 cases
could be considered for having potential to undergo malignant transformation as they were on
the higher side of the cut-off values.
To evaluate the 18 individual dysplastic features the ‘non-risk’ and ‘risky’ cases were
categorized for each feature of dysplasia. Feature assigned number 1.8 i.e. abnormal and
superficial mitosis and 2.2 i.e. marked nuclear and cellular pleomorphism and anisonucleosis
were significantly associated with ‘risky’ group as compared to ‘non-risky’ where the Fisher’s
Exact Test value were p<0.001, p<0.003 respectively. Fisher’s Exact test of statistical
significance is useful for categorical data that result from classifying objects into different ways:
it is used to examine the significance of association (contingency) between two kinds of
classification of samples.
To further evaluate the significance of individual features, those dysplastic features that
had score of 5 and above were segregated. A total of 10 such important features predicted for
malignant transformation were identified. These are loss of polarity, loss of cohesiveness with
prominent intercellular bridges, abnormal and superficial mitoses, increased number of mitotic
figures, loss of epithelial stratification, keratin pearls within rete ridges, mild to moderate degree
of nuclear and cellular pleomorphism, marked nuclear & cellular pleomorphism &
anisonucleosis, hyperchromasia of basal cells and atypical mitotic figures. The total score of
these 10 features was calculated as 61.
All the 100 cases were once again categorized into the three risk categories as per the
scores of ten above mentioned selected features. Calculations using median and interquartile
range to define the cut-off point based on which of the ‘risky’ cases were regrouped as low,
20
medium and high risk. 13.6% (8) cases were identified with the total score of more than 45,
suggesting these cases which had score more than cut-off value could be at a higher risk of
undergoing malignant transformation.
Cases of both ‘non-risky’ (41 cases) and ‘risky’ (59 cases) were categorized into risk
categories as per the scores of 10 above mentioned selected individual dysplastic features.
Abnormal and superficial mitosis (feature no.1.8) and marked nuclear & cellular pleomorphism
& and anisonucleosis (feature no. 2.2) were significantly and more commonly associated with
‘risky’ group as compared to the ‘non-risky’ group (Fischer’s Exact Test p<0.001). These
observations suggest that these two features observed in the biopsy of any potentially malignant
disorder would be important criteria for consideration for malignant transformation. Another
important feature observed in the ‘risky’ category was loss of cohesiveness which histologically
presents as prominent intercellular bridges. Our results are in agreement with the data obtained
from the studies of Smith and Pindborg, Kujan Omar, Oliver RJ, Tilakaranate WM.
Variation could result due to the difference in the understanding of these features in terms
of recognition and impact on clinical outcomes. Though some features are associated
significantly with the clinical outcomes, the other features which showed no statistical significant
association are as suggested by Patridge M. et al (1997)25 considered to be important for the
whole grading process.
One of the main objectives of the study by Kujan O et al (2006)23 was to assess the
prognostic value of the binary system using qualitative morphological characteristics. They
found the new binary grading system to be a very good predictor for the malignant changes in
oral epithelial dysplasia with reasonable values of sensitivity and specificity (85% and 80%,
respectively). They predicted the clinical outcomes of dysplastic lesions with certainty
(28/33;84.9%), while the negative predictive value of system was 85%.
They also suggested that cases with either hyperplasia or mild dysplasia could be
considered as ‘watch and wait’, whereas the cases with severe dysplasia or CIS in situ need
intervention. The cases with moderate dysplasia belong to the category of ‘watch and wait’.
Their data showed that moderate dysplasia cases had significant potential for malignant changes
(14/30 ; 46.7%). 14 out of 16 (87.5%) cases, in their study with moderate dysplasia had been
assigned as high risk and progressed to OSCC. Their results further demonstrated that patients
whose OED lesions had been assigned as “high risk” tended to have significantly greater
21
transformation rates than those with lesions assigned as “low risk” (p=0.004, by log-rank test). In
contrast to other studies, the results of the Kujan O et al (2006) suggest that the new binary
grading system has the potential to help the clinicians to make more appropriate treatment
decisions when dealing with OED.
Appraisal of IHC in Epithelial Dysplasia:
Epithelial alterations are the first morphological changes seen during the progress of
malignant transformation of the oral epithelium. These potential malignant disorders are
important lesions for clinical preventive investigations. For better understanding not only are the
clinicopathological evaluation essential but also the molecular-biological changes during
transformation process should be known. A variety of cell biological markers mainly involved in
cell proliferation and apoptosis are studied and some association is described in the literature.
Identical genetic changes in dysplastic oral lesions and oral cancers in some patients
suggest that the progeny of the cells in dysplastic lesions may further modify into a malignant
lesion. A clone of oral keratinocytes may proceed through stages of transformation from normal,
to dysplastic, to malignant. The major event in oral malignancy is excessive proliferation of oral
keratinocytes, which break through the basal membrane and spread in the stroma as suggested by
Upasani OS et al (2004).26
Though progress in molecular oncology has significantly advanced our knowledge on
tumorogenesis; yet the practical applications of these genetic markers remain unresolved in
detecting oral dysplasia. Although many molecules have been studied as intermediate markers,
not many studies have focused correlating the findings with the clinical attributes of terminal
outcome. Combined with routine histopathology studies, the broad based studies of molecular
markers could have a great potential for the determination of prognosis of PMD.
The aim of the present study was to correlate the molecular changes in the epithelium and
basement membrane zone of lamina propria of PMD. An attempt is made in the present study to
correlate the histopathological features with IHC markers for CK-19, Ki-67 and MMP-9 so as to
substantiate the observation of dysplasia observed in H&E stained tissue sections.
Cytokeratins (CK) are epithelial specific intermediate filament proteins. They are 20
different types and exhibit distinct patterns of expression in specific epithelial tissues. The CK
polypeptides have common epitopes against which antibodies are developed and these tend to
cross react. A large number of monoclonal and polyclonal antibodies have been developed that
22
are being used for diagnosis using IHC. CK are connected to the nuclear envelope on one side,
while they interact with plasma membrane proteins. These in turn interact with ECM proteins.
Thus CK are involved in transduction of signals and transport of nutrients from inside to outside
of cells and vice a versa. CK-19 is expressed in simple epithelium and basal cells of non-
keratinizing squamous epithelium reported by Takeda TK et al (2006).27
Basal cells situated along the base of the epithelial ridge have a flattened cell surface in
contact with connective tissue. These non-serrated cells contain only a few cytoplasmic
organelles and appear to be least differentiated cells. These represent the stem cells and transit
amplifying cells. They are characterized by a high nucleus to cytoplasmic ratio, expression of K-
19, a relative lack of keratin filament bundle and high levels of β1 integrins.28
In the present study for all 100 cases the intensity of CK-19 immunoexpression in the
epithelial cells and in the different cell layers were observed. The immunoexpression of Ck-19
was found to be statistically significantly higher (p<0.001) in the ‘risky’ cases as compared to
‘non-risky’. In the ‘risky’ group all the cases showed CK-19 expression whereas 20 ‘non risky’
cases had no CK-19 expression. In the ‘risky’ group, 44 cases out of 59 had more than 26% of
cells with immunoexpression and the intensity of expression ranged from light brown to dark
brown suggesting increase in the number of proliferating cells. 21 ‘non-risky’ cases had immune
expression of CK-19 with maximum having less than 26% of immunopositive cells with
intensity of expression being light brown suggesting the presence of proliferating cells in this
group. The intensity of expression of CK-19 was significantly higher in the ‘risky’ group as
compared to the ‘non-risky’ (p < 0.001). We also found that 6 out of the 8 cases (75%) which
were categorized as high risk with scores more than 45 in the ‘risky’ group had high expression
(+++, >50%) of CK-19. These findings suggest disturbance in the distribution of proliferating
cells and the stem cells during proliferation in the epithelium.
Lindberg K.(1989) 29 in their study suggested CK-19 as a marker of premalignancy when
the supra basal cells of dysplastic epithelium were immunopositive. This is in accordance with
our observation. Takeda TK et al (2006)27 reported CK-19 and p63 over expression in their series
of cases and suggested that CK-19 positive cells are transient applying cells located in the
parabasal layers. Loss of asymmetric cell division leads to increase in number of stem cells in
oral dysplasia. This disturbance of stem cells and asymmetry in cell division can be recognized
as dispolarity of basal cells, an essential histological feature of dysplasia. Thus an increase in
23
suprabasal cells could be an indicator of higher grade of dysplasia. They also suggested that the
decrease in CK-19 expression could be due to alteration of stem cell function and that these cells
could be replaced by proliferating cells in the basal layer of dysplastic epithelium. Hence this
feature could be a useful index in grading.
The expression of terminal differentiation products differ from keratinized to non-
keratinized SSE in which K4 and K13 keratins are expressed in the suprabasal cells, whereas the
suprabasal cells of cornified SSE express high molecular weight keratins (K1, K2, K10 & K11).
This normal pattern of keratin expression is altered in hyperplastic and cancerous lesions of the
oral mucosa. The appearance of K8, K18 and K-19, typical of simple highly proliferative
epithelial cells, becomes prominent in oral leukoplakia and stratified squamous cell carcinoma
was reported by Lan Su et al (1996).30
Ki-67 is a nuclear antigen present in the proliferating cells. It is present in all phases of
cell cycle, where its expression is low in G1 phase and increase to peak levels in the G2 and
mitotic phase. In our study ki-67 expression and the area involved in the epithelial cell layer
showed a significantly marked increase of immunoexpression in the ‘risky’ group as compared
to the ‘non-risky’ (p<0.001). In the ‘risky’ group 48 cases had immunoexpression in more than
26% of the epithelial cells and in 6 cases these cells were extending above the suprabasal layer.
Whereas in the ‘non-risky’ group 17 cases were immune positive for Ki-67, maximum of which
had less than 25% of immune positive cells and were limited to the basal layer. The 8 cases with
scores of more than 45 (high risk in the risky group) in the proposed classification had more
intensely stained and more than 50% immunopositive cells in the upper layers of dysplastic
epithelium. As Ki-67 is located within nucleoli of proliferating cells only, it may be suggested
that the antigen may regulate nucleolar metabolism by increasing rates of ribosomal synthesis
required by rapidly dividing cells which can be histologically interpreted as basilar hyperplasia.
Our findings are in accordance to observations of Takeda TK et al27, they found increase in ki-67
expression in the basal and parabasal cells of dysplasia group as compared to normal oral
epithelium. They did not notice any significant difference between the mild and moderate
dysplasia cases. Takashi Saku31 suggested that differentiation of keratinocytes happens in two
directions : 1) towards the surface – keratinization and 2) towards basal cells. Thus basal cells
are neither the germinal cells nor are they a source for squamous epithelial regeneration, but are
24
terminally differentiated cells. The cells with proliferating potential are located in the parabasal
cell layer which may be regarded germinal center of oral squamous epithelium.
When epithelial cell nuclei are Ki-67 positive they are regarded as cells in proliferation.
In normal epithelium these are located in parabasal cells. In ‘risky’ dysplasia group in our study,
ki-67 positive cells were seen up to the middle third of epithelium. With the presence of
dyskeratosis or sudden keratinization in the lower layer of the epithelium the ki-67 positive cells
could indicate that the keratinocyte differentiation is not well regulated that could lead to lack of
basal cell alignment and loss of stratification.
Oral cancer may result from mutation of ras oncogene and a simultaneous deactivating
mutation of p53, a tumors suppresser gene in oral keratinocyte. Oral cancer may result from
mutation in other oncogenes and in EGFr / ras / kinase cascade / c-myc signaling pathway.
Authors correlated this with the stages of carcinogenesis from normal mucosa, to squamous
hyperplasia (9p), to dysplasia (3p, 17p), to carcinoma in situ (11q, 13q, 14q), to invasive
carcinoma (6p, 8q, 4q).1
In dysplasia the oral keratinocytes proliferation is limited within the epithelial
compartment. Eventually the proliferating keratinocytes may break through the epithelial
basement membrane and expand into the underlying connective tissue resulting in carcinoma.
Frank invasion into the stroma by cancer cells are obvious histologically. However in some cases
the invasiveness is not clearly determined. This so called micro invasion cannot be ruled out in
carcinoma in-situ cases. Takashi Saku31 suggests that invasion of cancer cells is accompanied by
a neoplastic stromal change around the cell nests. On H&E stained sections, the newly formed
neoplastic stroma is more diffusely eosinophilic than the lose connective tissue of the lamina
propria but more basophilically myxoid than surrounding fibrous granulation tissue. Carcinoma
in-situ does not induce such diffuse neoplastic stroma. To evaluate the basement membrane and
stromal changes MMP-9 was considered in the present study.
MMPs are a family of structurally related but genetically distinct enzymes that degrade
extracellular matrix and basement membrane components and regulate cell–matrix composition.
MMPs may be secreted by tumour cells, fibroblasts, endothelial cells and macrophages as well as
mast cells and neutrophils.32
In our study MMP-9 immunoexpression was studied in the epithelium and connective
tissue in all 100 cases of PMD for grading dysplasia. The intensity and area of expression in the
25
epithelium was found to be statistically significant when compared between the ‘risky’ and ‘non-
risky’ cases (Fisher Exact Test, Intensity: p<0.001, Area: p<0.002). The intensity of MMP-9
expression in the epithelium was golden brown in majority of the ‘risky’ and ‘non-risky’ cases.
Where in more than 26% of epithelial cells had enhanced MMP-9 immunoexpression in 37 cases
of ‘risky’ dysplasia. The immunoexpression in stromal cells did not show any significant
correlation / association in the intensity and area between the two study groups (Fisher Exact
Test, Intensity: p<0.551 NS, Area: p<0.700 NS).
The expression of MMP-9 in ‘risky’ group was in accordance with the study on OSCC
where it was noted that MMP-9 is expressed in carcinoma and inflammatory cells around
carcinoma islands.33,34 For tumors to invade and metastasize, neoplastic cells must be capable of
degrading the extracellular matrix and accessing blood vessels and lymphatics. Mounting
evidence supports the view that extracellular proteinases, such as the MMPs, mediate many of
the changes in the microenvironment during tumor progression.35
Richard et al36 in his retrospective study examined changes in MMP 1, 2 and 9 using
polymerase chain reaction in oral dysplasia and oral squamous cell carcinoma and found higher
levels of MMP-1 and 9 mRNA that was significantly associated with dysplasia that progressed to
oral cancer as compared with those that did not. Peschos et al37 checked expression of MMP-9 in
benign, premalignant and malignant laryngeal lesions. They suggested a two-step model of up-
regulation of MMP-9 expression, first when a dysplastic lesion evolves and the next when the
dysplasia progresses to invasive carcinoma. MMP-9 expression was related neither to survival
nor to the other available clinicopathological parameters.
In the present study the stromal reaction of MMP-9 did not show any significant
difference between the cases. Though the stromal reaction in the high risk category cases from
the ‘risky’ dysplasia group were more pronounced as compared to the other cases. Recent studies
have shown that MMPs contribute to tumor progression, through its effect on cell proliferation,
survival and angiogenesis.38
In addition to carcinoma cells, cancers consist of tumor-associated stromal cells, which
include fibroblasts, endothelial cells, leukocytes, macrophages, nerve cells, and adipocytes. The
cancer cells crosstalk with stromal components during cancer progression and they are mediated
by transmembrane receptors, which are expressed on cancer cells and stromal cells.39 Tumor-
associated cells promote angiogenesis, inflammation, invasion, and ECM modeling through cell-
26
cell contact and the production of growth factors, hormones, cytokines, and proteinases such as
MMPs.38
MMP 9 is detected in malignant transformation of various cells and is associated with
tumor metastasis and poor prognosis. In the aggressive human tongue squamous cell carcinoma
cell line, MMP-2 was only found in its latent form, whereas MMP-9 was found in its active
form. In an oral squamous cell carcinoma cell line SCC-25, increase expression of MMP-9, in
vitro, this was thought to occur via fibronectin integrin v 6 pathway. MMP-9 may not be the
only factor for OSCC invasion, but it may be important for indirect cell signaling by controlling
the bioavailability and bioactivity of molecules that target specific receptors, which regulate cell
growth, migration, inflammation, and angiogenesis.39
Gao J (2010)40 studied the expression of MMP-2/MMP-9 in normal oral mucosa, lymph
node-negative tongue cancers, lymph node-positive tongue cancers and their metastasized
tumors in cervical lymph nodes. They hardly found MMP-2/MMP-9 expression in normal
epithelium. In lymph node-negative tongue cancer, 45% and 40% of these primary tumors were
positively stained for MMP-2/MMP-9. Importantly, in lymph node-positive tongue cancer, 71%
and 79% of these primary tumors were positive for MMP-2/MMP-9, respectively. Over
expression of MMP-2/MMP-9 was present in the metastatic lymph nodes. Their result implied
the significance of MMP-2 and / or MMP-9 in predictive value for the actual or potential
presence of cervical metastases. Such activity has prognostic value, and provides impetus for
further development of biotherapies targeted at specific inhibition of MMP activities.
Progress in molecular oncology has significantly advanced our knowledge on
tumorgenesis; yet the practical applications of these genetic markers remain unresolved in
detecting oral dysplasia. None of the molecular markers single or in combination appear to be
ready for use in routine clinical diagnostic practices, although many molecules have been studied
as intermediate markers.9
Furthermore, as suggested by Gayani P. et al (2009)9 recent advances in the use of
quantitative methods has enabled the profiling of gene expression (microarray), protein
expression (proteomics), screening of epigenetic changes, e.g. by pyro-sequencing and single
nucleotide changes (SNP) of hundreds of pre-selected genes simultaneously. These techniques
which require minute amount of tissue when carried out in controlled way and using carefully
selected samples with proper validation will help the potentially malignant and malignant tissue
27
samples. Combined with routine histopathology studies, these broad based studies of gene and
their products appear to have a great deal of potential for the identification of diagnostic and
prognostic markers of pre-cancer, and data from multicenter studies using microarray technology
may be engaged in the future for a better understating of aetiopathogenesis of pre-cancer.
CONCLUSION
Oral epithelial dysplasia : a histopathologic marker of premalignancy, is a diagnostic term used
to describe the histopathological changes seen in progressive chronic and premalignant disorders
of the oral mucosa.. It may also be seen in verrucous or papillary leukoplakia or in the margins of
chronic mucosal ulcers. It is also consistently seen in the apparently normal mucosa adjacent to
the squamous cell carcinoma. Clinically it may present as leukoplakia, erythroplakia or
leukoerythroplakia 41
A histological dysplasia classification system should ideally meet two basic
requirements. First it should be easily applicable in daily routine practice with low inter and intra
observer variability. Secondly, it should allow a clear separation and give clear suggestions to the
clinician to distinguish between patients who need treatment to prevent progression and those
whom no treatment is needed and should be kept on routine observation.
We propose a new binary classification system consisting of two categories where the
individual architectural and cytological features are given a score. This new classification has
been validated and has shown good to almost perfect agreement for both inter and intra observer
variation. This classification has been designed with the following objectives : a) to reduce the
inter and intra observer variability, b) to make it objectively reproducible, c) transmit
maximum information possible from interpretation of routine H&E stained sections, d) give a
clear diagnosis for clinicians to make critical treatment decision.
From the observations of the present study, ten important morphological and architectural
features are identified to be considered for categorizing potentially malignant disorder
histologically as ‘risky’. Abnormal superficial mitosis and marked cellular and nuclear
pleomorphism were two important features that could predict malignant transformation are found
to be statistically significant and that should be identified and considered for risk categorization
and malignant transformation.
The IHC markers using Ki-67 for observing the irregular epithelial cell proliferation, Ck-
19 for loss of polarity and basal cell hyperplasia in the suprabasal layers and MMP-9 for stromal
28
changes, micro-invasion and changes in the basement membrane and lamina propria have
substantiated the H&E observation.
Our proposed system of grading dysplasia is designed for histopathological evaluation
using H&E stained sections. It was designed to extract a larger set of data with detailed
observations of individual features of dysplasia based on architectural and cytological changes.
Our aim was to generate objective data to correlate with defined patterns of dysplastic features.
Grading dysplasia is not an exact science and pathologists are doing their best to reach an
optimal result. In this study we have tried to weigh individual features by defining their
importance and scoring them for OED grading process obtaining a common consensus on the
features. However, data reveals that the proposed classification system can be used to improve or
develop simpler and easy diagnostic methods.
The outcome of ‘risky’ dysplasia for malignant transformation could not be assessed in
the study due to lack of follow up data. Within the limitations of the analysis we have shown that
many individual features of dysplasia can be assessed reproducibly as they were categorized
under high risk with a definite score. Reducing features to definable components increases
objectivity and insight, though it makes it difficult for comparison with the published data.
Future studies should compare lesional responses related to patient population and their
geographic areas of origin. As well as specific carcinogenic factors, diagnostic usefulness of aids
like Toluidine blue application, oral brush biopsy techniques and spectroscopy and the natural
history of the potentially malignant disorders should be correlated.
Relevance of the observations and future scope of this study
• The proposed classification is objective and is easy to understand. As the inter and intra
observer agreement is statistically significant and hence is reproducible.
• The proposed classification has been assigned with scores for specific features that has
helped to minimizes the inter and intra observer variability.
• Reporting and grading of oral epithelial dysplasia in potentially malignant disorders has
been made easy and valid with the use of routine H&E stain section from any
histopathology diagnostic service, center or laboratory.
• Larger sample size studies, at various oral pathology centers, in different geographical
locations could help in developing this proposed classification universally acceptable.
29
• Our classification of ‘risky’ and ‘non-risky’ will be helpful for clinical application in
imparting proper treatment, patient education, ‘wait & watch’ policy and follow-up.
Hence, the need is for:
• Application of the proposed binary classification as ‘risky’ and ‘non-risky’ for routine
use.
• Multicentric focused group assessment using the proposed binary classification.
• Longitudinal follow-up study for evaluating the malignant potential of ‘risky’ and
‘non-risky’ cases of potentially malignant disorders.
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