CDDODIA00035

Nucleolar Organiser Regions (AgNORs), a quantative criteria in the
diagnosis of Human Oral Squamous Cell Carcinoma and Oral

Leukoplakia
By
Dr.Sushma Rao
Dissertation Submitted to the
Rajiv Gandhi University Of Health Sciences, Karnataka, Bangalore

In partial fulfillment
Of the requirements for the degree of
Master of Dental Surgery
In
Oral Medicine and Radiology
Under the guidance of
Dr.Asha Iyengar
Department Of Oral Medicine and Radiology
R.V.Dental College and Hospital,
Bangalore
September 2006

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ii
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.

Declaration by the Candidate

I hereby declare that this Dissertation entitled Nucleolar Organiser
Regions (AgNORs), a quantative criteria in the diagnosis of Human
Oral Squamous Cell Carcinoma and Oral Leukoplakia is a bonafide
and genuine research work carried out by me under the guidance of
Dr. Asha R Iyengar, Professor, Department Of Oral Medicine and
Radiology, R.V.Dental College and Hospital, Bangalore.

Date: Signature of the Candidate
Place: Bangalore Name: Dr.Sushma Rao

iii

Certificate by the Guide

This is to certify that the dissertation entitled Nucleolar Organiser
research work done by Dr.Sushma Rao in partial fulfillment of the
requirement for the degree of MASTER OF DENTAL SURGERY
(M.D.S.) IN ORAL MEDICINE AND RADIOLOGY.

Date: Signature of the Guide
Place: Bangalore Name: Dr.Asha R Iyengar
Designation and Department: Professor,
Department of Oral Medicine and
Radiology, R.V.Dental College and Hospital,
Bangalore.
iv

Endorsement by the HOD, Principal/Head of the
Institution

This is to certify that the dissertation entitled Nucleolar Organiser
research work done by Dr.Sushma Rao under the guidance of
Dr.Asha R Iyengar, Professor, Department Of Oral Medicine and
Radiology, R.V.Dental College and Hospital, Bangalore.

Seal and Signature Seal and Signature
of the HOD of the Principal
Name: Dr.K.S.Nagesh Name: Dr.K.S.Nagesh
Date: Date:
Place: Bangalore Place: Bangalore

v
COPYRIGHT

Declaration by the Candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences,
Karnataka shall have the rights to preserve, use and disseminate this
dissertation in print or electronic format for academic/research
purpose.

Place: Bangalore Name: Dr.Sushma Rao

RAJ IV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA,
BANGALORE.

vi
ACKNOWLEDGEMENT

It is my pleasure to express my deep sense of gratitude to my Professors
Dr.K.S.Nagesh and Dr. Asha R Iyengar for their concern, constant guidance,
support and encouragement.
I sincerely thank Dr.Bharathi, Professor, Department of Pathology for her
guidance, constant help and valuable time she spent during the course of my
dissertation work.
I also thank Dr.Veerendra Kumar, Professor, Department of Oral Pathology and
Dr.Jyothi Gupta, Assistant Professor, Department of Oral Medicine and
Radiology, for their guidance and their valuable suggestions.

Place: Name: Dr.Sushma Rao

vii
ABSTRACT
Background and Objectives
Nucleolar Organiser regions are loops of DNA that encode ribosomal RNA. They
have recently attracted much attention because of the claims that their frequency
within the nuclei is significantly higher in malignant cells and may reflect the
state of activation and the proliferation activity of the cells.
The present study was carried out to analyze the distribution of the AgNOR in
Oral Leukoplakia and Oral Squamous Cell Carcinoma, and in their various
histological grades, and to assess if the AgNOR distribution could give
information on the malignant potentiality in premalignant lesions and
aggressiveness of the malignant lesions.
Methodology
The study specimens comprised of 35 archival cases, of which 15 cases were of
Oral Leukoplakia and 20 cases of Oral Squamous Cell Carcinoma. The specimens
were stained by Hematoxylin and Eosin and Modified Silver staining method of
Ploton et al for the Nucleolar Organiser Regions. The specimens were analyzed
independently by the two observers and was further statistically analysed.
Results
The Mean AgNOR count in Oral Leukoplakia was 2.80 0.50 and in cases of Oral
Squamous Cell Carcinoma was 5.71 1.08. The Mean AgNOR count in cases of
mild dysplasia was 2.59 0.66, in moderate dysplasia was 2.92 0.43 and in
severe dysplasia was 2.79. The Mean AgNOR count in cases of well
viii
differentiated Oral Squamous Cell Carcinoma was 5.73 1.62 and in cases of
moderately differentiated Oral Squamous Cell Carcinoma was 5.671.19.
Interpretation and Conclusion
The Mean AgNOR count was higher in cases of Oral Squamous Cell Carcinoma
as compared to cases of Oral Leukoplakia, and the AgNOR counts increased with
the increase in the grades of dysplasia. The difference in the total AgNOR count
between the two observers in Oral Leukoplakia and Oral Squamous Cell
Carcinoma was highly statistically significant. Therefore the AgNOR method can
be used to provide information on the malignant potentiality in premalignant
lesions and aggressiveness of the malignant lesions.

KEY WORDS
Nucleolar Organiser Regions (NOR); Argyrophilic Nucleolar Organiser Regions
(AgNOR); Silver nitrate staining; Oral Leukoplakia; Oral Squamous Cell
Carcinoma.

ix
TABLE OF CONTENTS

Sl.No. Contents Page No.
1. Introduction 1-3
2. Objectives 4
3. Review Of Literature 5-70
4. Methodology 71-77
5. Results 86-91
6. Disscussion 97-109
7. Conclusion 110
8. Summary 111-112
9. Bibliography 113-124
10. Annexures 125-126

x
LIST OF TABLES

Tables Content Page No.
Table 1 Interobserver variation 92
Table 2 Groups and comparison 92
Table 3 Histological grades of Oral Leukoplakia 92
Table 4 Histological grades of Oral Squamous
Cell Carcinoma
93
Table 5 Cutoff value (95
th
percentile of Precancer) 93

xi
LIST OF PHOTOGRAPHS
Fig Contents Page No.
Fig 1 LEICA RM 2025 Microtome 78
Fig 2 Thermostatic bath and Hot plate

78
Fig 3 Chemicals used in Silver staining 79
Fig 4 LYNX N-200M TrinocularMicroscope with Nikon
CoolPix 4500 Digital Camera

79
Fig 5 Photomicrographs of H & E stained sections of
Oral epithelial hyperplasia
80
Fig 6 Photomicrographs of Silver stained sections of
Oral epithelial hyperplasia
80
Oral Leukoplakia- Mild Dysplasia
81
Oral Leukoplakia- Mild Dysplasia
81
Oral Leukoplakia- Moderate Dysplasia
82
Oral Leukoplakia- Moderate Dysplasia
82
Oral Leukoplakia- Severe Dysplasia
83
xii
Oral Leukoplakia- Severe Dysplasia
83
Oral Squamous Cell Carcinoma- Well
Differentiated
84
Oral Squamous Cell Carcinoma- Well
Differentiated
84
Oral Squamous Cell Carcinoma-Moderately
Differentiated
85
Oral Squamous Cell Carcinoma-Moderately
Differentiated
85

xiii
LIST OF GRAPHS

Graphs Contents Page No.
Graph 1 Number of cases in the groups
included in the study
94
Graph 2 Range of AgNOR counts in cases
of Oral Leukoplakia
94
Graph 3 Range of AgNOR counts in cases
of Oral Squamous Cell Carcinoma
95
Graph 4 Mean AgNOR counts in cases of
Oral Leukoplakia and Oral
Squamous Cell Carcinoma
95
Graph 5 Mean AgNOR counts in different
grades of dysplasia in Oral
Leukoplakia
96
Graph 6 Mean AgNOR counts in different
histological grades of Oral
96


1
Introduction
Oral cancer is one of the most prevalent cancers, and is one of the ten most
common causes of death worldwide
1
. It is a major health problem in India,
forming about 10% of the estimated 644,600 new cancers that occur in all parts of
the body each year
2
.

Amongst oral cancer, Squamous cell carcinoma accounts for over 90% of the
cases
2
. It has a wide range of etiological and risk factors. Therefore the patient
may remain asymptomatic for a long period of time or may present with mild
discomfort, dysphagia, limited mouth opening etc. Patient may also present with
a wide variety of precancerous lesions like Leukoplakia, Erythroplakia, Lichen
planus and Submucous fibrosis
1
.

As most Oral cancers arise from precancerous lesions or conditions, the effect of
primary prevention implies a reduction in the risk for oral cancer. Even though
only a small portion of precancers actually progresses to Oral cancer, this
development forms a source for over 70% of oral cancer in India
2
.

Despite improved surgical approaches, reconstruction techniques and advances in
radiation and medical oncology, the single most effective route of improving the
long term out come of oral cancer is early diagnosis, coupled with appropriate
treatment
3
. Therefore for early diagnosis, assessment of etiologic and risk factors
like tobacco, alcohol and precancerous lesions are important. A routine biopsy

2
Introduction
and histopathological examination should be made for all precancerous lesions to
eliminate any dysplastic changes
3
.

In the past few years several methods have been used for the identification of
proliferating cells in tissue sections and these have been applied especially to pre
malignant lesions with the aim of using them as a marker for an impending
malignancy, such as assessment of mitosis. DNA ploidy status, autoradiographic
methods, DNA and RNA insitu hybridization, monoclonal antibodies to detect
proliferation related antigen etc. Major disadvantages of these techniques are that,
they are time consuming, expensive and require sophisticated equipment and
technical expertise
4, 5
.

The silver binding Nucleolar Organiser Regions (AgNOR) is one such technique
and has been used in assessing the precancerous and cancerous lesions of breast
lumps parathyroid gland disorders and recently has been extended to oral lesions.
The silver binding Nucleolar Organiser Regions (AgNOR) have been attracting
much attention because of the claims that their frequency within the nucleus has
been significantly higher in malignant cells than in normal, reactive or benign
neoplastic cells
6
.

Studies have shown that there is a definite correlation between histopathological
grading system and AgNOR count, but a few significant co-relations between

3
Introduction
clinical features, histological features and AgNOR has not yet been substantially
documented in literature.The cost of silver staining is lower than that of most
immunohistochemical reactions generally used in pathology
7, 8
.

In continuation with the previous studies, the aim of this study is to assess the
usefulness of AgNOR as a quantitative criteria in the diagnosis of human Oral
Leukoplakia and Oral Squamous Cell Carcinoma.


4
Objectives of the study
Objectives of the study:

1. To analyze Nucleolar organizer Regions related parameter in Oral
Leukoplakia and its various histological grading.

2. To analyze Nucleolar organizer Regions related parameter in Oral Squamous
cell carcinoma and its various histological grading.

3. To assess the correlation in the difference between Nucleolar organizer related
parameters in Oral Leukoplakia and Oral Squamous Cell Carcinoma.


5
Review Of Literature
Cell is the structural and functional unit of living matter. The basic unit of the
body is the cell, and each organ is an aggregate of many different cells
9
.
In 1665 Hooke R, introduced the word cell for the smallest organized unit of
living material capable of existing independently
10
.
Leewenhook (1674) discovered free cells as opposed to the walled in cells of
Hooke and Grew and observed some organization within the cells particularly the
nucleus in some erythrocytes
11
.
Leydig (19
th
century) defined cells as a mass of protoplasm containing nucleus
12
.
Most mammalian cells lie within the range of 5-50 m diameter
13
. The cells of the
body are divided into three groups on the basis of their proliferation capacity
14

Continuously dividing cells (labile cells)
Quiescent cells (Stable cells)
Non dividing cells (permanent)
The cell is proportioned into two major components
9, 10

Nucleus
Cytoplasm

Cytoplasm
Cytoplasm is the protoplasm, which surrounds the nucleus and is bounded by cell
membrane. Protoplasm has been described as the physical basis of life, it exists in
colloidal state
18
.


6
Cytoplasm is mainly composed of water (70% or more by volume) with dissolved
inorganic cations (ions of hydrogen, potassium, sodium, calcium, magnesium,
iron etc.) and anions (chloride, bicarbonate, hydroxyl, phosphate, sulphate etc.),
but is also permeated with assemblies of large organic molecules which compose
the cellular structure and the system of enzymes and energy carriers providing the
basis of living processes within cells
10
.

Cell membrane
Cell is bounded by a cell membrane (plasma membrane or plasma lemma). It
consists of a bimolecular layer of mixed phospholipids with their hydrophilic
portions at the outer and inner surfaces of the membrane and their hydrophobic
chains projecting towards the middle of the bilayer
10
.
Cell membrane comprises of:
Cytoplasmic inclusions (stored food, secretary granules, pigments)
Cytoplasmic organelles
Membrane organelles
Plasma membrane
Endoplasmic reticulum
Golgi apparatus
Mitochondria
Lysosomes
Cytoplasmic RNA Ribisomes
Centrosomes
Various fibrils, filaments and tubules

7
Nucleus
The nucleus is usually spherical or ellipsoidal, often indented, 4-10 m in
diameter, and separated from the cytoplasm by a membranous nuclear envelope,
measures about 3-14 m diameter
10
.
The Latin word nucleus or the Greek word Karyon signifies the Kernel of a
nut
16
.
Leewenhook (1700) first observed cells nucleus in the red blood corpuscles of the
salmon
17
.
Brown (1803) recognized a conspicuous spherical body, nucleus in the interior of
plant cell and subsequently it was found in all animal cells. He coined the term
nucleus
9.

Structure of nucleus:
1. Nuclear membrane
10
is a flattened sac formed by the membranes of the
innermost two layers of the cytoplasmic membrane system separated by a
distance of about 20 nm.
The outer membrane is of endoplasmic reticulum proper and often bears
ribosomes; products of protein synthesis may accumulate between the two
membranes.
The inner membrane is quite distinct from other cell membranes, being the
attachment site for the ends of chromosomes, which often adhere to the surface as
a dense coating of chromatin during interphase. This membrane is also reinforced

8
with a network of 10nm filament, which, with other proteinaceous material,
forms dense nuclear lamina lining the inner surface of the nuclear envelope. It
controls the movement of macromolecules between the nucleoplasm and the
surrounding cytoplasm.

2. Ground substance (Karyolymph/karyoplasm)
18
is a feebly staining, clear
matrix, more viscous than the ground cytoplasm. The electron microscope shows
that it is finely granular containing dispersed chromatin. It is a colloidal
solution, holding the uncoiled, invisible portion of chromosome, serves as a
medium for diffusion of metabolites and larger molecules.

3. Chromatin
18
consists of strands of DNA and its associated protein. These
strands are about 20nm in diameter and occupy the entire nucleolus.

4. Nucleolus
18
: Each interphase nucleus contains one to four nucleoli, although in
a nucleus of condensed type, the nucleolus may be obscured. The number and
size (up to 1m or more) of the nucleolus is constant for any particular cell type.
Nucleoli are prominent and usually multiply in cells actively engaged in protein
synthesis. They disperse and disappear during cell division.
Nucleoli are discrete, darkly staining spherical or ovoid bodies, not delineated
by a membrane, and lie free within the karyoplasm or attached to the inner aspect
of nuclear envelope.

9
They are larger, more denser, and more regular in outline than masses of
heterochromatin. They contain 5-10% RNA with the rest protein and a small
amount of DNA and often are surrounded by a rim of condensed chromatin
termed the nucleus-associated chromatin.
With silver staining techniques, the nucleolus may show a coiled, thread like
structure termed the nucleolenema.

Electron microscopy shows four basic components
18
:
1. Granules of 12-15 nm diameter composed of RNA and proteins
2. Fibrils of 5 nm diameter also composed of ribonucleoprotein and often
closely packed.
3. Chromatin both as peripheral, nucleolar-associated chromatin and as fine
loops of chromatin passing from the peripheral rim into the interior.
4. A proteinaceous, amorphous material usually occurring in aggregation.
With electron microscopy nucleoli are seen to have
19
:
o A central filamentous zone (pars filamentosa)
o Outer granular zone (pars granulose)
o Both of which are embedded in an amorphous material (pars amorphosa)

Nucleoli lie at certain specific sites on certain chromosomes (the nucleolar
organizing sites) where there are gene sequences or cistrons that encode genetic
information for the synthesis of ribosomes
18
.

10
At these sites the ribosomal RNA is transcribed basically as subunits. Ribosomal
RNA is at first in the form of long fibres that constitute the fibrous zone of the
nucleoli. It is then broken up into smaller pieces (ribosomal subunits) that
constitute the granular zone
18
.

Nucleolar Organiser Regions (NORs)
Nucleolar Organiser Regions are loops of DNA that encode ribosomal RNA and
are considered important in the synthesis of protein
20
.
They are located on the short arms of acrocentric chromosomes (13, 14, 15, 21
and 22)
21
.
Nucleolar Organiser Regions at the ultra structural level i.e. fibrillar center should
be regarded as the true interphase counter part of NORs present on the five
human acrocentric chromosome
22
.
In prophase, the components of the fibrillar center disperse and in the telophase
tiny granules associate with NOR bearing chromosomes which are ultimately
rearranged into nucleolar structures
22
.

The relationship between interphase of AgNOR and cell proliferation indicates
that the quantity of interphase of AgNOR can be considered as a parameter of cell
kinetics. The amount of AgNOR is related to the cell cycle, increasingly
progressive from G
o
to S-phase and is proportional to the proliferative activity of
neoplastic cells
23
.

11
A rapidly dividing tumor population is more likely to have a greater proportion of
cells in the early stages of G
1
before individual NORs have associated and are
therefore more likely to be observed in greater numbers. Conversely tumors with
low rate of cell proliferation are more likely to display a single NOR
23
.
Thus the number of AgNORs may increase due to the activation of formally
inactive NORs or decrease as a result of an association of active NOR. The
tumor cells have large number of interphase AgNOR. The interphase NOR
distribution in tumor cells is a consequence of the increased demand for ribosomal
biogenesis which characterizes dividing cells
24
.
The number of AgNORs in interphase nuclei may reflect the state of activation
and /or degree of malignant transformation of certain tissues
25
.

The ribosomal genes are transcribed by the RNA polymerase I and ultimately
direct ribose formation and protein synthesis. Transcriptionally active NORs
associate to certain specific, acidic, non histone proteins which are an essential
component of the pre-RNA synthesis machinery, and are selectively stained by a
silver colloid technique and can be visualized as black dots (AgNOR)
23
.
The abundance and intensity of AgNORs are an indication of not only their
absolute number and dispersion but also their transcriptional activities
6
.


12
NORs chromosomal segments in ribosomal RNA (rRNA) are encoded and they
are thus responsible for the development of RNA containing nucleolus or nucleoli
into which the NORs project on large loops of DNA
6
.

The silver staining technique neither identifies rRNA nor rDNA but the acidic
proteins associated with these sites of rRNA transcription, these proteins are
designated as B
23,
C
23,
AgNOR proteins and RNA polymerase I
6
.
Theoretically, 20 AgNOR sites should be demonstrable in a human diploid
nucleus after DNA synthesis (i.e. in G
2
of the cell cycle). In practice, however, the
AgNORs in a normal cell are usually tightly aggregated with in the one or two
nucleoli evident in histological or cytological preparations; the individual
AgNORs are not discernable
6
.


13
The term Leukoplakia was first coined in 1877 by Schwimmer that was used to
indicate a white patch or a plaque occurring on the surface of a mucous
membrane
26
.
The word leukoplakia is derived from the Greek word Leukos meaning White
and Plakia meaning a Patch
26
. According to Williams (1963) presence of
hyperkeratosis is an essential requirement for the usage of term.
Silverman (1968) defined leukoplakia as a white patch or a plaque on the oral
mucosa, which cannot be scrapped off, cannot be reversed by removing the
irritant and is separate from any other recognizable disease entity
27.

WHO (1978) defined leukoplakia as a white patch or plaque that cannot be
characterized clinically or pathologically as any other disease
28

At an International seminar at Malmo in 1984
29
leukoplakia was defined as
A white patch or plaque that cannot be characterized clinically or pathologically
as any other disease and which is not associated with any physical or chemical
causative agent except the use of tobacco.
It was also recommended that the complete description of leukoplakia should
include the etiological, clinical, topographical and histological characteristics of
the lesion.


14
Etiology:
Certain local and systemic factors were proposed as the causative agents for
Leukoplakia
1,

30
that are as follows:
Local Factors
Tobacco smoking
Smokeless form of tobacco
Betel nut consumption with or without slaked lime
Alcohol consumption
Candidiasis
Chronic irritation due to malocclusion producing chronic cheek biting, ill-
fitting dentures, sharp and broken down teeth.
Electro galvanism
Viruses
Tertiary syphilis
Vitamin B12 and folic acid deficiency
Sideropenic dysphagia

At the international seminar held at Malmo (1984) the etiology of
leukoplakia was classified as:
Tobacco related
Idiopathic

15
Clinical features
1
:
The patient with leukoplakia is usually asymptomatic and rarely notices the
presence of a white patch of a plaque on the oral mucosal. It is usually the dentist
who sees it during routine examination of the oral cavity. It appears
predominantly as a white patch or a plaque on the oral mucosa that is not
scrappable. There is always the presence of an associated history of tobacco
usage.
Site of occurrence
1
:
The sites of occurrence vary according to the habit. It may be on the Labial
mucosa, Commissure, Buccal mucosa, Tongue, Palate or Gingiva.
Clinical appearance
1
:
Homogenous leukoplakia:
It is predominantly a white lesion, with uniform, flat, thin appearance having a
consistent texture throughout. It may have the following appearances:
Patch/plaque like, smooth surface
Wrinkled surface, described as cracked mud appearance
Corrugated surface, described as ebbing tide appearance
Fine white lines.


16
Non Homogeneous leukoplakia:
Here the surface structure is not consistent and presents as follows
o Exophytic / verrucous leukoplakia - It has irregular, blunt / sharp
projection.
o Nodular / speckled leukoplakia - It has white small specks or nodules on
an erythematous base. Nodules may be pinhead size or larger.
o Ulcerative / erosive leukoplakia - It has red areas of erosion or ulcer
associated with white patches. At times the ulcerated area exhibits
yellowish areas of fibrin. Commonly it may be associated with
pigmentation of varying intensity.
Proliferative Verrucous is a type of exophytic, non-homogeneous leukoplakia,
which starts as a simple, hyperkeratosis, but has tendency to become multifocal
and may develop into squamous cell carcinoma. It has increased recurrence rate
and is redundant to all types of treatment.
LCP classification and staging for oral leukoplakia
29
:
This was proposed in 1994 at an international conference in Uppsala, Sweden.
L. Provisional - A provisional diagnosis of oral examination had a
predominantly white appearance and cannot be diagnosed as any other
disease.


17
L. Definitive - A definite diagnosis of leukoplakia is made as a result of
identification if possible elimination of suspected etiological factor and in
persistent cases by histopathological examination.
L. Provisional
L 1
st
symbol represents the size of the lesion.
1 - Less than 2 cm
2 - Greater than 2 cm and less than 4 cm
3 - Greater than 4 cm
X - Not specified.
C - 2
nd
symbol represents clinical aspects of the lesion
1 - Homogenous leukoplakia
2 - Non homogenous leukoplakia
X - Not specified
L Definitive
P - 3rd symbol represents Histopathology of the lesion
1 - No dysplasia
2 - Mild dysplasia
3 Moderate dysplasia
4 - Severe dysplasia


18
Staging according to LCP classification
Stage I - L (any) C1, P1 P2
Stage II - L (any) C2, P1P2
Stage III - L (any), C (any) P3 P4
Criteria of staging
1. If there is a doubt concerning the LCP categories to which a particular
case should be allotted, then the lower category should be chosen.
2. If there are multiple, simultaneous leukoplakia, then the leukoplakia with
the highest L category should be used and the multiplicity must be
indicated in parenthesis e.g.1.2 (m)
3. When different clinical types of leukoplakia are present, the highest score
should be used.
4. When multiple biopsies are taken from multiple sites, the highest
pathological score of various biopsies should be used.
5. For reporting purposes, the oral sub site must be recorded.

Histopathologic features
31
:
Hyperkeratosis of the surface epithelium with or without acanthosis associated
with variable numbers of chronic inflammatory cells are noted in the subjacent
connective tissue.

19
The keratin layer may consist of Parakeratin or Orthokeratin or a combination of
both. With Parakeratin there is no granular cell layer and the epithelial nuclei are
retained in the keratin layer. With Orthokeratin, the epithelium demonstrates the
granular cell layer and the nuclei are lost in the granular cell layer.

Histopathologic alterations of the Dysplastic epithelial cells:
Enlarged nuclei and cells
Large and prominent nucleoli
Increased nuclear-to-cytoplasmic ratio
Hyperchromatic nuclei
Pleomorphic (abnormally shaped) nuclei and cells
Dyskeratosis (premature keratinization of the individual cells)
Increased mitotic activity
Abnormal mitotic figures

Histomorphologic alterations of the dysplastic epithelium evident at low power
magnification:
Bulbous or tear drop shaped rete ridges
Loss of polarity
Keratin or epithelial pearls
Loss of typical epithelial cell cohesiveness


20
Grades of epithelial dysplasia
1
:
Mild epithelial dysplasia alterations limited to the basal and parabasal
layers
Moderate epithelial dysplasia involvement from basal layer to the mid
portion of the spinous layer
Severe epithelial dysplasia alterations from the basal layer to a level
above the midpoint of the epithelium
Carcinoma-in-situ when the entire thickness of the epithelium is
involved

Carcinoma-in-situ is defined as dysplastic epithelial cells that extend from the
basal layer to the surface of the mucosa (top - to - bottom change). There is no
invasion of the atypical epithelial cells.

Malignant Transformation of Leukoplakia:
In the absence of any intervention, leukoplakia may persist, regress spontaneously
or progress to cancer.
Studies conducted by various researchers showed that at the time of identification
of leukoplakia, the biopsies revealed dysplasia in 12-25% of the cases and cancer
in 5-10% of the cases
27, 32, 33
.


21
Some authors reported 0-12.5% rate of malignant transformation
33, 35
, while in
another study rate of malignant transformation in snuff users was 6.2% and 19.5%
in patients not associated with snuff
34
.
In one study it was observed that more than 75% of oral carcinomas developed
from preexisting leukoplakia
36
.
Leukoplakia with dysplasia had increased risk of malignant transformation
1
.
According to some investigators, the malignant potential of leukoplakia also
depends on the site of occurrence. Increased risk of associated with leukoplakia
involving the dorsum of the tongue and the floor of the mouth.
The presence and severity of dysplasia is thought to have an impact on the
malignant risk of premalignant lesions. The current gold standard for predicting
the malignant potential of premalignant lesions is the presence and degree of
dysplasia
1
.
The risk of progression of leukoplakia varies from 0.13 to 24%, depending on the
patients and the follow-up period. The majority of leukoplakias (epithelial
hyperplasia, mild dysplasia) do not progress to malignancy. Candida colonization
has been associated with an increased risk of progression
1
.


22
Oral cancer is one of the 10 most frequent cancers world wide, with about three
quarters of all cancers occurring in developing countries
1
. It represents 5
th
most
common cancer in the world
1
. In Central and South East Africa, it accounts for
upto 40% of all cancers, where as in most industrialized countries, it is relatively
uncommon accounting for less than 4%.

There is a striking difference in the incidence and the mortality rates across the
world, with highest rates generally registered in a few developing countries
including India, Pakistan and Bangladesh, where this is the most common form of
cancer. On the basis of cancer registry data, it is estimated that annually about 1,
20, 000 new oral cancer cases develop in India.

Etiology
1,30
:
Tobacco - Smoking and Smokeless
Arecanut
Alcohol
Food habits - chillies
Nutritional deficiency -Iron deficiency anemia and Plummer Vision
syndrome
Chronic trauma from sharp teeth
Sunlight
Syphilis

23
Viruses - HPV 16, EBV, HHV 6, CMV, HSV 1
Genes
1
- Oncogenes can be produced by point mutations and
translocations of gene amplification. Aberrant expression of protoneogenic
epidermal growth factor receptor, members of rare gene family, C-myc,
int-2, hast-1, PRAD-1, bcl-1 are believed to contribute to cancer
development. The human epidermal growth factor receptor gene (EGFR)
maps to 7p, 13q
22
and encodes for transmembrane tyrosine specific
phospokinase, which is expressed primarily in cells of epithelial origin.
Binding of activating legends to the extracellular domain induces homo or
hetero dimidation and subsequent phosphorylation of the receptor
resulting in activation of the down stream signaling pathways. These
events lead eventually to cell proliferation, inhibition of apoptosis and
stimulation of invasion and metastasis.

Premalignant lesions and conditions
- Leukoplakia
- Erythroplakia
- Lichen Planus
- Oral Submucous Fibrosis
- Plummer Vinson syndrome
- Xeroderma pigmentosum


24
Carcinogenesis
37
:
Carcinogenesis is a multistep process involving interaction of various
carcinogens and several genetic factors, which play a role in cancer progression.
Multistage carcinogenesis: Environmental carcinogens and certain other
endogenous factor (genetic alteration and mutation) interacting in a complex
manner can give rise to development of cancer. Three steps are described:
Initiation - alteration in the cellular DNA
Promotion there is no DNA damage initial phases are reversible
Malignant conversion
Oral squamous cell cancer is the result of a multistage process from normal to
dysplastic lesions and ultimately to cancer.

Molecular Changes in Oral Premalignancy and malignancy
1

Carcinogenesis is a genetic process that leads to a change in morphology and in
cellular behavior. Major genes involved in head and neck squamous cell
carcinoma include protooncogenes and tumor suppressor genes (TSGs). Other
factors that play a role in the progression of disease may include allelic loss at
other chromosome regions, mutations to protooncogenes and TSGs, or epigenetic
changes such as deoxyribonucleic acid (DNA) methylation or histone
deacetylation. Cytokine growth factors, angiogenesis, cell adhesion molecules,
immune function, and homeostatic regulation of surrounding normal cells could
also play a role.

25
While proto-oncogenes increase cell growth and differentiation and are to be
likely involved in carcinogenesis, few have been reported in head and neck
squamous cell carcinoma. Proto-oncogenes associated with head and neck
squamous cell carcinoma include ras (rat sarcoma), cyclin-D1, myc, erb-b
(erythroblastosis), bcl-1, bcl-2 (B-cell lymphoma), int-2, CK8, and CK19.
TSGs negatively regulate cell growth and differentiation. Functional loss of TSGs
is common in carcinogenesis These TSGs have been associated with sites of
chromosome abnormalities where LOH has been reported to commonly involve
chromosome arms 3p, 4q, 8p, 9p, 11q, 13q, and 17p.
Loss on 3p and 9p are early and common events in oral carcinogenesis.
Chromosome arm 3p may code for FHIT and is involved in epithelial cancers
LOH on 13q correlates with lymph node metastases and recurrence in head and
neck squamous cell carcinoma.
Molecular staging may provide more precise predictions of the malignant
potential than conventional clinicopathologic features do as it represents the
fundamental biologic characteristics of each tumor.
The current model of carcinogenesis is a multistage process, with loss occurring
on chromosome arms 3p and 9p early in the lesions progress from benign to
dysplastic, with additional losses later in the disease, often involving 8p, 13q, and
17p. Putative TSGs at these sites of loss are p16 loss at 9p and p53 gene loss at
17p. Molecular markers are likely to become important clinical markers in
diagnosis and staging and in treatment planning.

26
Clinical features
1, 30
:
Carcinoma of oral mucosa may appear as on of the three general morphological
patterns in the early stages
Papillary or Verrucous type of lesion is an exophytic growth, and is seen
as a papillary mass of varying size with a broad base or a relatively narrow
pedicle. The surface structure may be pebbled, verrucous or relatively
smooth.
The ulcerative type of lesion appears as a discrete ulcer with raised
indurated margin or as a relatively large area of ulceration with firm
indurated tissue at the periphery.
White raised plague, velvety red or erythroblastic lesion or mixed red and
white lesion.

Invasive tumor front
38
:
Invasive tumor front is the tumor-host interface. One of the most promising recent
findings in recognition of the biological aggressiveness of oral cancer is the
recognition of the significance of structural and functional features of the most
advanced parts of a carcinoma, namely the invasive tumor front (ITF). It has been
hypothesized that disturbances of the mechanisms that control cell differentiation,
migration and renewal or death (apoptosis), as well as perturbations in normal
epithelial-mesenchymal interactions at the tumor-host interface, underlie the
malignant behavior of carcinomas.

27
Tumor cells at the most invasive parts of a malignant tumor differ substantially
from central and/or superficial ones. It has been postulated that the invasive tumor
front of the tumor consists of the most aggressive cells, which have the ability to
invade the surrounding structures, including the vessels, and thereby metastasize.
An increasing body of evidence now supports the idea that characteristics of the
invasive tumor front are of major significance for the prognosis of oral cancer.
A histopathological malignancy grading in oral squamous cell carcinoma was
proposed examining exclusively the most advanced invasive tumor cell layers at
the tumor-host interface. This approach yielded highly significant and
independent prognostic information within the same TNM stages.

Invasive tumor front grading is based on the assessment of qualitative histological
parameters:
Degree of keratinization
Nuclear polymorphism
Pattern of invasion
Lymphocytic infiltration
Therefore is subject to inter-observer variation.
Computer-aided diagnostic pathology (CADP) is useful for standardization to
avoid inter-observer variation


28
Histopathologic features
31
:
Oral squamous Cell Carcinoma arises from the dysplastic surface epithelium and
is characterised by invasive islands and cords of malignant squamous epithelial
cells.
The cells show abundant Eosinophilic cytoplasm with large Hyperchromatic
nuclei and increased nuclear-to-cytoplasmic ratio. Varying degrees of cellular and
nuclear pleomorphism are seen. Keratin pearls may be seen.
Oral Squamous Cell carcinoma can be classified according to Broders
30
as
Grade I 75% of cells well differentiated Well differentiated
Grade II 50% of cells well differentiated Moderately Differentiated
Grade III 25% of cells well differentiated Poorly differentiated

Well differentiated carcinoma may retain the ability to produce keratin, where as
the poorly differentiated carcinoma loses the anatomic pattern and function of
epithelium.
The tumors may be associated with mixed inflammatory infiltrate. Recognition of
the tumor invasion may be assisted by a study of type IV collagen (basement
membrane collagen) by immunocytochemistry. Invasion can occur into the
lymphatics, blood vessels and perineural spaces.


29
Historical Note
39
:
The use of silver solutions for staining cells and chromosomes is not new. It has
been used in histological studies of normal and degenerating nerve tissue as well
as in cancerous tissues.
The use of silver for staining was first used for staining polytene chromosomes in
Drosophila and later was used to stain somatic metaphase chromosomes of chick
embryos.
Various structural aspects of the human chromosomes (bands, centromeres,
satellites) were revealed by further modifications of the existing technique.
Thus by varying the silver solutions and conditions of staining it is possible to
show
The equivalent of G- and C- banding
Coiling of the chromonemata
Specific satellite DNA regions
Sites of ribosomal DNA
This differentiation of the chromosomal regions is thought to occur as a result of
preferential binding of silver to Histone and / or to non-histone proteins.

Silver staining technique for the demonstration of the NORs is an emperic
technique which appears to stain NOR proteins involved in the regulation of
transcription or post-transcriptional modification of rRNA transcripts
51
.


30
The silver stain was originally used to demonstrate the NORs of chromosomes, to
evaluate their function, and to identify chromosomes in cytogenetic preparations.
The technique was simplified and was first applied to histological sections by
Ploton et al. It was subsequently popularized, especially by Crocker and
associates, and the staining protocol that Crocker described has been used by most
workers in the field.
Crocker and others have suggested that the technique has practical utility in
diagnostic pathology for demonstration of neoplastic potential and for evaluating
the prognosis and aggressiveness of malignant neoplasms
51

A sizeable body of literature has developed evaluating this technique in various
diagnostic applications, which generally indicates successful use of the technique;
it also contains comments about problems. These caused difficulty in
interpretation of the results and limited its routine use
51
.
Among the problems are
51
:
Limited reliability and reproducibility
General background staining of tissues
Making weakly stained NORs difficult to identify and making NORs harder to
resolve
Staining of granules in the cytoplasm that can be confused with NORs
Black precipitates scattered over the slide that can be confused with
NORs


31
Fading of the sections, often within days
Poor staining with some tissue fixatives
Variation in the staining intensity and configuration among NORs, such that
short staining times that allow clear separation and quantitation of the
more strongly reacting NORs but are insufficient to demonstrate weakly
reactive NORs; but longer staining times that demonstrate weakly reactive
NORs, also produce over staining and confluence of strongly reactive NORs,
and with some tissues produce a continuous stained rim on the nucleolus
rather than individual NORs.

Several authors have proposed variations in the original procedure to reduce
these problems, including
51
:
Destaining of slides be-fore silver staining
Pre- or post-incubation of sections with alcoholic acetic acid other acids or
glycineto reduce background staining
Variations in solution concentration
Increase in pH
Variations in temperature
Staining slides while inverted or coating the sections with collodion to
reduce precipitates; conversion of the silver precipitate to a dye complex
Combination with other staining procedures


32
Replacement of the gelatin component with other colloids
Use of sodium thiosulfate and gold toning to improve permanence.
None of these modifications has entirely controlled the problems.

20
. They are located on the
short arms of acrocentric chromosomes (13, 14, 15, 21 and 22)
21
. The AgNOR
are argyrophilic non histone proteins whose precise biochemical nature is not well
understood. However they are easily demonstrated by the simple, specific,
one-step staining technique used in this study
40
.

6
.There is
now some evidence that the morphology, intensity, and spatial relationships of
AgNOR on chromosomes vary during cell cycle phases. Since they are reported
to be markers of rRNA transcriptional activities, an analysis of their number and
form is of great importance
40
.
The silver staining technique identifies neither rRNA not rDNA but the acidic
designated as B23, C23,AgNOR protein and RNA polymerase I
6
.


33
Theoretically, 20 AgNOR sites should be demonstrable in a human diploid
nucleus after DNA synthesis [i.e. in G
2
phase of the cell cycle], although the ease
with which they can be demonstrated depends to some extent on their
transcriptional activity
6
.
In practice, however, the AgNORs in a normal cell are usually tightly aggregated
within the one or two nucleoli evident in histological or cytological preparations.
The individual AgNORs are often not discernable
6
. .
The number of detectable NORs and AgNORs depends on several factors
6
.
The level of transcriptional activity
The number of NOR bearing chromosomes in the karyotype
The stage of cell cycle in which they are sought.
Three of the NOR related proteins studied in some detail are the
40

Silver binding proteins (AgNOR)
The 100 KD nucleolar protein
The 80 KD nucleolar protein.
The nucleolus is not a constant structure, for it disperses before mitotic cell
division and reorganizes afterwards
6
. The abundance and intensity of AgNORs
are an indication of not only their absolute number and dispersion but also their
transcriptional activity; indeed it is possible that the residual AgNOR proteins
persist even after transcriptional activity has ceased
6
.


34
A quantifiable apparent increase in the mean AgNOR count of a cell population in
tissue sections could result if either
6

a) Cell proliferation is so active that nucleolar disassociation is present in
many cells and the AgNORs are therefore dispersed through the nucleus
b) There is a defect of nucleolar association resulting in AgNOR
dispersion
c) Cell ploidy increases, resulting in a real increase of AgNOR bearing
chromosomes
d) Transcriptional activity increases resulting in prominence of otherwise
inconspicious AgNORs.

There is a striking discrepancy between AgNOR counts in chromosome spreads
and those reportedly observed in histological sections of similar cell populations.
Cytogenic studies reveal upto 20 NOR sites in the full chromosome population
from a normal human nucleus, where as histopathological studies record not more
than one or two AgNORs per nucleus of benign cells in sections
6
.
The difference may be due to difficulty in perceiving the individual AgNORs
when they are congregating within a relatively small nucleolus
6
.
In malignancy AgNORs become dispersed through the nucleus to a varying
extent, enabling the histologist to count them more readily
6
.


35
The quantification of AgNORs is therefore partly dependent on the degree of
dispersion or disaggregation of the relatively large number of AgNORs in the
nucleus
6
. Thus, the AgNOR count denotes not the absolute number of AgNORs
but rather a numerical index of dispersion
6
.


36
AgNOR Staining Technique:
20
. They are located on the
short arms of acrocentric chromosomes (13, 14, 15, 21 and 22)
21
.
The ribosomal genes are transcribed by the RNA polymerase I and ultimately
direct ribose formation and protein synthesis. Transcriptionally active NORs
associate to certain specific acidic, non histone proteins which are an essential
component of the pre-RNA synthesis machinery, and are selectively stained by a
silver colloid technique and can be visualized as black dots (AgNOR)
23
.
6
.

The number of AgNORs in interphase nuclei may reflect the state of activation
and /or degree of malignant transformation of certain tissues
25

A reliable technique for staining human chromosomal nucleolar organizers with
silver solutions was proposed by modifying the procedures described earlier by
(1) Omitting the use of the photo flood light, (2) retaining the heat treatment for
the silver impregnation, (3) controlling the development process by manipulating
pH, temperature and concentration of the Ammonical Silver-Formalin
developer
41
. The AgNOR appeared after incubation in 50% silver nitrate with no
developer introduced. This technique resulted in a highly selective staining of the

37
NORs with no visible staining or distortion of the chromosome arms. This
modification was referred to as Ag-I. The authors concluded that
41

By increasing the concentration of the Formalin from 3% to 10% with or
without pH changes resulted in faster AgNOR development.
Increase in temperature resulted in faster development
Increasing the ratio of AS, AS: F ratio from 1:1 produced better AgNORs
A one-step procedure for AgNOR staining was proposed which reduced the
careful controlled requirements of the earlier methods. This method needed
warming upto 70
0
C and gave good results for staining AgNORs on chromosome
preparations
42
.
A new method was developed which allowed the fine identification of nucleolar
components, particularly those which were stained by silver. In order to determine
the cytochemical nature of the components associated with AgNOR proteins, the
EDTA regressive preferential staining procedure for ribonucleoproteins was to
applied sections. By this means the precise localization of the AgNOR proteins
were studied simultaneously with that of ribonucleoprotein within interphasic
nucleoli and mitotic chromosomes. In interphasic nucleoli, stainable AgNOR
proteins were localized in fibrillar centres and part of the dense fibrillar
component. No silver deposits were seen on perichromatin or interchromatin
fibrils and granules. In interphasic and mitotic nuclei, AgNOR proteins were
never found within condensed chromatin but always in association with
ribonucleoprotein components. They concluded that the new method proposed

38
above appears to be a useful tool for the simultaneous study of the localization of
ribonucleoprotein and Ag-NOR proteins during the cell cycle
43
.

Pretreating deparaffinized sections with Schiff's reagent improved the specificity
of Goodpasture and Bloom's AgNOR staining after aldehyde fixation.

This
reduced the selective staining of acidic nucleolar proteins
44
.

A modified one-step silver technique, performed at 20
0
C, aimed at localizing the
argyrophilic proteins of the nucleolar organizer region, to various materials
including cells in smears, chromosomes, semi-thin sections of plastic embedded
cells and sections of paraffin embedded human pathological tissues was proposed.
They tested various modes of imaging, including bright-field, Nomarski contrast,
reflected light and combined Nomarski contrast with reflected light, and found
that the use of reflected light, based on the ability of silver to reflect incident light
specifically, gave images with an improved resolution compared to bright field.
They further concluded that the one-step staining method at room temperature
improved the specificity of the staining and optimized the conditions of
observation
45
.

The effect of pH on silver staining of the nucleolar organizer regions (NORs) of
human chromosomes was investigated between pH 6.5 and 12.0. Nonvolatile
mixtures of ethanolamine and ethanolammonium nitrate were used in place of

39
ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5
by the silver staining procedure of Howell and Black was used as the standard;
this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was
found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of
mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR
staining was incomplete, and between pH 8.5 and 6.5 there was no NOR
staining
46
.
The degree of staining was dependent on the fixation regime employed and results
varied greatly form one fixative to another. Alcohol-based fixatives generally
gave optimal results, Carnoys fluid was especially recommended. Mercurial and
dichromate-containing fixatives were found to have highly detrimental effects on
NOR staining. Routine 10 percent formol saline fixation also gave adequate
results where as 10 percent neutral buffered formalin gave optimal staining,
similar to alcohol-based fixation
47
.

The one step silver-staining was improved by post fixation of sections in acetic
acid-ethanol during rehydration, covering the sections with celloidin before
staining and reducing the concentration of the silver nitrate solution from 50% to
20%. The technique was economical because of the low silver content and small
volume of the incubation solution used
48
.


40
A simple modification to the silver staining of nucleolar organizer regions was
proposed by performing incubation with the slide inverted, this resulted in
minimal undesirable background staining. The method being straight forward and
fast, maintained a high degree of contrast between the background and the
AgNORs
49
.

The silver colloid technique was further modified for staining nucleolar organizer
regions in paraffin embedded tissues by the application of a gold toning step with
subsequent gold reduction if necessary, following incubation of sections in the
standard sliver colloid solution
50
.

An attempt was made for the improvement in silver staining technique for
Nucleolar organizer regions (AgNORs) as it suffered from limited reliability,
back ground staining, precipitates and fading of sections. An improved procedure
which incorporated pre-reduction of the sections was proposed with selection of
an optimal gelatin and post treatment of the section to produce a permanent
preparation
51
.

A further improvement in the visualization of proteins associated with nucleolar
organizer regions proteins (AgNORs) on formalin-fixed and paraffin-embedded
archival tissues was obtained after application of wet autoclave pretreatment
52
.


41
The lack of a standardized staining protocol results in frequent misinterpretation
of actual structures evaluated by the various authors during AgNOR enumeartion,
the AgNOR area obtained was closely related to the whole stained nucleolar area
obtained by prolonging the silver staining reaction beyond the optimal time for
selective NOR visualization. In order to compare data from different laboratories,
the authors proposed to stain the whole nucleolus by silver and to measure the
dimensions of the silver-stained nucleoli by image analysis
53
.

A sensitive staining method for nucleolar organizer regions using blue toning of
AgNORs was proposed. The staining (blue staining), performed as single step,
provided a higher amplification and better resolution than silver staining alone
54
.

A further modification in the AgNOR staining procedure was proposed which
provided highly contrasting AgNORs with minimal unspecific silver
precipitation, thus facilitating both manual and computerized counting. This
technique involved the use of microwave irradiation in order to shorten the
processing time, the use of gelatin as a protective colloid, and a Farmers solution
to optimize the specificity of the technique
55
.

The technique of silver staining of the nucleolar organiser (AgNOR) improved the
contrast, selectivity and speed when performed with microwave irradiation
56, 57
.


42
A new staining method for dual demonstration of Estrogen receptors and
argyrophilc Nucleolar Organizer Regions was proposed. The slides were analysed
with the image analysis system AMBA. The dual staining of both markers
resulted in a reproducible and specific staining result. They concluded that it was
justified to measure AgNORs in immunohistochemically stained cells
58
.

Three improvements to the original technique to overcome the difficulties was
proposed as follows (1) Pretreatment with 7% nitric acid produced very distinct
dark brown images of AgNORs on a yellow background. The gradient of
background colours allowed easy discrimination of nucleolar, nuclear and
cytoplasmic structures (2) A second improvement was achieved by coating the
slides with 7% cellodin solution in ethyl alcohol-ether prior to AgNOR staining
and acid pretreatment. (3) Placing sections face down on the staining solution
prevented the formation of nonspecific silver precipitates
59
.

Lindner
51
made an attempt for the improvement in silver staining technique for
Nucleolar organizer regions. He analyzed several variables that could affect the
staining procedure, which were as follows:
Effects of Oxidation and Reduction- Be fore Silver Staining
Oxidation of sections before silver staining produced a marked increase in the
background staining and the staining of other non-NOR elements. The reduction


43
of sections decreased the background staining and staining of non-NOR structures
without having any effect on the specific staining of the NORs.
Effects of Variation in Silver and Formic Acid Concentration
Concentrations originally reported by Ploton et al. were about optimum.
Effects of Temperature
The variations in room temperature produced large differences in NOR staining
density. They suggested that for a base staining time of 30 min, a time reduction
of 2 min for each degree C of temperature increase, and vice versa.
Reduction of Precipitates
Staining the slides inverted reduced the precipitates but did not eliminate them.
The gelatin in the staining solution reduced nonspecific staining reactions by a
protective effect.
Improvement of Stain Permanence
Sections receiving no treatment after the silver stain can fade in a few days as a
result of residual chemicals left in the sections and exposure to oxidants in the
mounting medium. The air treatment of the sections with sodium thiosulfate after
silver staining improved permanence. Gold toning with a gold chloride-sodium
thiocyanate complex was also used; the gold protective solution replaces the
silver with inert gold.
Effect of Specimen fixation
Alcohol or Formalin fixation resulted in better results than other fixatives.


44
Comterstaining
A counterstain with 0.01% safranin for 1 min produced a weak transparent
nuclear stain that does not interfere with quantitation of NORs. Other
counterstains could be used.
Interaction with Other Straining Procedures
Most stains based on organic dyes, notably H&E and Papanicolaou stains, were
completely removed during the strongly acidic silver- staining step without prior
destaining. The presence of these dyes did not affect the NOR staining. Sections
stained for NORs could later be stained with other procedures, such as H&E or
the Papanicolaou stain; some degrading of the staining was noted, but it was
acceptable for many purposes. With strong nuclear stains the NORs were no
longer visible.

45
AgNOR counting
Four classes of AgNOR patterns, were enumerated in cultured lymphocytes
60

Cells with a single (or rarely two) AgNOR clusters (several silver dots in a
matrix inside the nucleolus)
Cells with a single compact nucleus
Cells with two compact nucleoli
Cells with several silver dots scattered throughout the nucleus

A standardized means for the enumeration of NORs in the histological sections
was proposed by Crocker et al
22
. They suggested that the total AgNOR dots, both
intra and extra-nucleolar, should be enumerated. The individual AgNOR dots
were much easily discerned in cell imprint than in sections.
They observed three main types of AgNOR configuration
22
:
Type I: The NORs are fully aggregated to form a solitary rounded argyrophilic
structure, often called an AgNOR, these correspond to the nucleolus.Even with
shorter incubations in the AgNOR reaction, NORs cannot be resolved within the
nucleolus. Eg: Resting lymphocytes and quiescent cells
Type II: NORs can be visualized inside the nucleolus, commonly seen in
proliferating cells.
Type III: Distribution of small true AgNORs through out the nucleoplasm as
frequently seen in malignant cells.


46

Type I
Nuclear Membrane

Nucleoli

Type II
Nuclear Membrane

Nucleoli

Type III:

Nuclear Membrane


47
They proposed that all silver stained structure should be counted, but when seen
lying in clusters (aggregated) each cluster should be counted as one structure.
When AgNORs are seen separately within the nucleolus, each AgNOR should be
counted as one unit, together with smaller AgNORs seen outside the nucleolus.
They further suggested that total AgNOR dots both intra and extra Nucleolar
should be counted
22
.
Two counting strategies can be used if AgNORs represents nucleolar
disaggregation, which inturn may reflect cellular activity
22
:
Counting only single nucleolus and extranucleolar AgNORs
Counting all AgNORs
AgNORs in clusters are counted as a single structure
Intranuclear NORs Extranuclear NORs

N=4

N=4

NOR in clusters
N=2


48

AgNOR counts in Spitz Nevi and malignant melanomas were assessed in one
study, and it was noted that there was some focal accentuation of staining
intensity
40
.
They recorded the number of individually discernable and separable block dots in
each nucleus and computed the average for each case; where two or more dots
were so closely aggregated within a nucleolus that the precise number within the
aggregate could not be counted, the aggregate was recorded as one.
They proposed that:
A nucleus shown below would be accessed as having one AgNOR

Nucleus

1 AgNOR

A nucleus shown below would be accessed as having three AgNOR

Nucleus
3 AgNORs

49
The Subjective AgNOR pattern assessment (SAPA) Score
62
was designed to
assess the number of AgNORs per cell and variations in their size and shape

Parameter Score
1. Estimated number per cell
Less than 2
1
2-5 2
Greater than 5 3
2. Variation of satellite size and shape
Uniform
1
Moderate variation 2
Marked variation 3
3. Variation in cluster size and shape
Uniform
1
Moderate variation 2
Marked variation 3

50
A modified counting protocol was used in a study to assess the Nuclear Organiser
regions (AgNORs) in Odontogenic cysts and amelobalstomas
63

A (N=6) B (N=4)
A. The number of separate black dots within the nucleolus was recorded
B. Where the dots were not separated by a Halo of nucleoplasm, the
aggregated dots were recorded as one
C. Where the closely aggregated dots were sepatated by a Halo of
nucleoplasm, the aggregated dots were recorded as one
D. Nucleoli that contained separate discernable dots were recorded as one,
since there was no surrounding Halo of nucleoplasm

C (N=7) D (N=6)
Overlapping nuclei were excluded, as those were showing no discernable
AgNORs.

51
Various optical means of increasing AgNOR definition were investigated by
several researchers, including Nomarske and dark field technique. According to
one study it was sufficient to use a colour filter (such as green), since this reduced
chromatic aberration and increased the clarity of AgNOR perimeters
22
.
Automated image analysis was used to develop a procedure for standardizing the
measurement of silver stained protein in cancer cells. They indicated that with the
possibility of standardizing the AgNOR image analysis method, it should be the
preferred counting method
64
. Automated image analysis helps to reduce inter and
intra examiner variability and also allows both enumeration and quantification of
AgNOR size and type
65
.
Another modification was the use of a simple eyepiece graticule, which reduced
the eye fatigue and prevented recounting and was less time consuming
61
.


52
Errors in enumeration:
In one study the inter and intra observer errors were assessed and they were in
the range of 2-7%
22
. This may have been a result of two factors:
Most tumors are heterogenous with regard to type of cell population. To
overcome this, the counting should be determined by standard cumulative
means of techniques.
Section thickness: Attempts to standardize it has been cumbersome. Thick
sections contain all NOR profiles, where as in thin sections, NORs are
easily separated and lost. So they proposed that 3m section was ideal.


53
AgNOR Staining Applications:
The silver colloid technique was applied to identify nucleolar organiser region

associated protein (AgNOR) to 16 fibrous proliferations of

childhood and six low
grade fibrosarcomas. TheAgNORs were visualised as dots within the nuclei of
the cells, and on thebasis of their relative mean numbers of AgNORs, fibrous
proliferations of

childhood could be easily differentiated from low grade infantile

fibrosarcoma. They concluded that this technique, previously the province of the

cytogeneticist, may be of use to the pathologist in differentiating

infantile fibrous
proliferations
66

The silver colloid technique was applied to identify nucleolar organizer region
associated protein (AgNOR) to 68 cutaneous tumours. Basal cell carcinomas,
eccrine tumours, apocrine tumours, and hair follicle tumours had differences in
their numbers of AgNORs; these appeared as small black dots in their nuclei.
Dermatofibromas and squamous cell carcinomas showed a degree of variability in
the number of AgNORs depending on the cellularity of the former and
differentiation of the latter. Basal cell carcinomas possessed significantly many
more AgNORs per nucleus than the other neoplasms
67
.
A study was conducted on 29 cases of Spitz nevi (SN) and 39 cases of invasive
Malignant Melanomas (MM) by silver method. SN showed between 1.0 and 1.6
AgNOR per cell with a mean of 1.2; MM counts ranged from 1.2 to 4.2 with a

54
mean of 2.0. They concluded that the AgNOR method cannot reliably
differentiate SN fron MM; however a count of more than 2.0 AgNOR per cell
would favor the diagnosis of MM rater than SN
40
.
A study on 214 benign and malignant breast lesions by silver staining method
was conducted.The study comprised of 39 cases of fibroadenomas, 28 cases of
papillomas, 23 cases of sclerosing adenosis, 38 cases of epitheliosis, 9 cases of
lobular carcinoma in situ and 37 cases of intraduct carcinomas. Counts in 25-33%
of epitheliosis and intraduct carcinomas overlapped. The AgNOR counts in
carcinomas were also compared with Ploidy and growth phase fractions by flow
cytometry. 33 of 46 cancers with counts over 3 AgNOR dots per nuclear profile
contained aneuploid cells, where as the 8 of the 12 with counts below 3 comprised
of diploid cells only. Similar trends were noted with regard to growth phase
fractions also. They concluded that the above method alone cannot offer reliable
histological discrimination for malignancy in the breast. However the AgNOR
counting may provide information on breast cancer prognosis supplementary to
that obtained from DNA flow cytometry analysis
21
.
A modified silver stain technique was applied for visualizing nucleolar organizer
regions (AgNOR counting) on 24 benign and 23 malignant prostatic biopsies.
Marked inter-observer variation was found, particularly in sections with high
AgNOR counts. After averaging the AgNOR counts of both observers, there was


55
no significant difference in counts between the benign and the malignant
biopsies
68
.
A study was conducted on Silver-binding nucleolar organizer regions in sections
from formalin-fixed, paraffin-embedded tissue blocks of oral squamous cell
carcinoma. Thirty-nine cases, that comprised poor prognostic group and good
prognostic group, were examined with respect to the relation between AgNOR
counts and histologic grading, and correlation between AgNOR counts and
prognosis. The pooled mean AgNOR count in poor prognostic group was higher
than that in good prognostic group. Five-year survival rate of the cases with high
AgNOR counts (greater than or equal to 6.5) was significantly lower than that
with low AgNOR counts (less than 6.5). High AgNOR counts were highly
suggestive of poor prognosis in oral squamous cell carcinoma
15
.
A study on Silver-binding nucleolar organizer region proteins (AgNORs) was
counted in sections from formalin-fixed, paraffin-embedded tissue blocks of oral
submucous fibrosis (OSMF) and oral squamous cell carcinoma (SCC).
Moderately advanced and advanced cases of OSMF, and well-differentiated and
poorly differentiated cases of SCC, were studied with respect to the relation
between AgNOR counts and histologic grading. Normal oral mucosa collected
from age- and sex-matched subjects constituted the control group. The pooled
mean AgNOR counts in advanced OSMF were higher than in moderately
advanced cases and those in poorly differentiated SCC were higher than those of

56
well-differentiated SCC, although the comparison was not significant in the latter.
They concluded that AgNOR counts could hold promise for predicting the
biologic behavior of OSMF because the study demonstrated a correlation with
clinical and histologic grading
16
.
A study was conducted using AgNORs stain on 66 paraffin-embedded head and
neck squamous cell carcinomas and on 12 samples of normal tonsillar squamous
epithelium. Carcinomas had a significantly higher mean AgNOR count than the
benign epithelium. Among carcinomas, mean AgNOR count increased with stage
of the disease, but there was no significant correlation with histologic grade or
DNA ploidy as determined by flow cytometry. The data suggested that AgNOR
count could be used as a possible aid in differentiating benign from malignant
squamous epithelial proliferations in the head and neck, and also possibly as a
prognostic marker in these carcinomas
69
.
A morphometric study of nucleolar organizer regions (NOR) to analyze their
distribution, volume, number and shape in the different strata of human normal
oral mucosa epithelium and papilloma and in squamous cell carcinoma employing
microphotographs of silver-stained paraffin sections was conducted. The different
NOR-related parameters evidenced significant differences between normal
mucosa, papilloma and squamous cell carcinoma. The functional polarity of
normal mucosa epithelium and of papilloma was also evidenced in terms of NOR-
related parameters. The NOR parameters showed a greater change in squamous

57
cell carcinoma cases as compared to normal oral mucosa epithelium and
papilloma. AgNOR counts were higher in cases of squamous cell carcinoma
(8.042.44) as compared to papilloma (4.532.37) and normal oral mucosa
(2.951.42)
23
.
A study quantified Nucleolar organiser regions from a range of oral mucosal
biopsies consisting of benign, reactive, dysplastic and carcinomatous lesions,
using silver staining, to see if AgNOR counts were helpful in distinguishing them.
Mean counts were greater in carcinomas compared to epithelial dysplasias or
benign keratoses. Although these differences were significant, counts in each
diagnostic group overlapped so much that they were of no practical value in
distinguishing between individual lesions. However, the higher counts found in
many carcinomas were due to dispersion of AgNORs within the nucleoplasm, so
that the AgNOR type was helpful in making such a distinction. Whether those
dysplastic lesions with higher and more dispersed counts represent those at
greater risk of malignant transformation awaits longitudinal study
65
.
A study was conducted to evaluate the mean numbers of argyrophil nucleolar
organizer regions and the Ki-67 antigen expression as proposed cell kinetic
parameters on 80 biopsies of suspected oral dysplasias and 40 probes of oral
squamous cell carcinomas. The mean numbers of AgNORs per nucleus and the
percentages of cells in S-phase as determined by flow cytometry showed a


58
correlation. Related to the histological degree of dysplasia, both AgNOR counts
and percentages of cells in S-phase revealed a similar pattern of distribution.
Therefore, both the AgNOR counts and the Ki-67 antigen were considered to be
valuable tools for cell kinetic reference and can be considered supplementary
methods to flow cytometry in diagnosis and therapy of oral cancer
70
.

A study was conducted on Nucleolar Organiser Regions (NORs) and Proliferating
Cell Nuclear Antigen (PCNA) on routine paraffin embedded histologic sections of
30 oral biopsy specimens (six cases of leukoplakia with low-degree of dysplasia,
nine cases of leukoplakia with moderate-degree of dysplasia, nine cases of
leukoplakia with severe-degree of dysplasia, six cases of squamous microinvasive
carcinomas), tested for HPV-DNA by in situ hybridisation (ISH). The absolute
number of NORs per nucleus and the percentage of nuclear positivity for PCNA
were found to be different in each group of pathology, with further diversity due
to the presence or absence of HPV-DNA. In the major part of HPV-positive
lesions, the AgNOR number and percentage of cells positive for PCNA were
found to be generally lower than in corresponding negative forms. Conversely, a
few cases of HPV positive lesions showed significantly higher values both of
AgNOR and PCNA, on comparison to other cases of HPV-positive and HPV-
negative lesions. These data suggested that high values of AgNOR and PCNA, in
moderate and high-grade oral dysplasia, could represent an "alarm signal" of a
worse prognosis, and then a possible indication for a strict clinical management

59
and/or a stronger treatment of some HPV-associated preneoplastic lesions
71
.

AgNOR staining was performed on seventeen cases of pseudoepitheliomatous
hyperplasia of the oral cavity and genital tract, seventeen cases of squamous cell
carcinomas of the same regions, and nineteen cases of squamous cell carcinoma
of the cervix, to determine whether the stain could help to distinguish
pseudoepitheliomatous hyperplasia from squamous cell carcinoma. No constant
relationship of the AgNOR score to the grade of the lesion was determined
72
.

The proliferative activity of leukoplakia without dysplastic change (LP),
epithelial dysplasia (ED), and squamous cell carcinoma (SCC) in the oral mucosa
was examined by means of proliferating cell nuclear antigen (PCNA)
immunostaining, silver-binding argyrophilic nucleolar organizer region (AgNOR)
staining, and the frequency of mitotic figures. Significant differences in the
labeling index of PCNA immunostaining (PI) and mitotic index (MI) were noted
between LP and ED and between ED and SCC. The mean numbers of AgNORs
(AI) significantly differed between ED and SCC. There was a significant positive
correlation between PI and MI in samples of ED. However, there was no
significant correlation between AI and other indices. The number and the
distribution of PCNA-positive cells in ED varied among samples. This study
showed that increased number of mitotic figures used as the index of proliferating

60
activity were the main histological components related to severe ED of oral
mucosa. They would provide a useful means of deciding the histopathological
grade of oral ED
73
.
The relationship between argyophilic nucleolar organizer regions (AgNORs) and
the histologic effects of preoperative chemotherapy or external radiation on oral
squamous cell carcinoma was evaluated on thirty-three cases of oral squamous
cell carcinoma that were treated with chemotherapy (pepleomycin or 5-FU) or
60Co external radiation. The number of AgNORs per nucleus before therapy
ranged from 4.7 to 12.45. As the number of AgNORs per nucleus increased, the
histological effects of preoperative therapy were enhanced. These results
suggested that AgNORs could be used to predict the effects of preoperative
radiation and chemotherapy on oral squamous cell carcinoma
74
.
A study was conducted to determine whether AgNORs may be of value in
distinguishing various odontogenic cysts from unicystic amelobalstoma.15 cases
each of odontogenic keratocyst, residual cyst, Dentigerous cyst, unicystic
Ameloblastoma and conventional Ameloblastoma. Dentigerous cyst had
significantly higher counts than odontogenic keratocyst, residual cyst and
unicystic Ameloblastoma. They concluded that the AgNOR counts were of no
diagnostic significance and could not be used to distinguish the various
odontogenic cysts from unicystic Ameloblastoma
63
.


61
A study was conducted to assess the number of argyrophilic nucleolar organizer
regions (AgNORs) in oral carcinomas with or without human papillomavirus
(HPV) infection. The AgNOR counts of the HPV-positive samples were not
significantly higher than those of the HPV-negative ones. Furthermore, the lesions
infected with multiple HPV types had greater counts than those with HPV type
16/18 infection alone. Significant differences were observed between the mean
counts of the poorly, moderately and well-differentiated carcinomas. The mean
AgNOR numbers in the oral carcinomas at TNM stages III/IV were found to be
significantly higher than the numbers in corresponding stage II lesions.
Cytokinetics of the lesions assessed by the bromodeoxyuridine labelling index
showed a linear correlation with their respective mean AgNOR counts
24
.
The AgNOR content was studied at the invasive front of 80 squamous cell
carcinomas of the floor of the mouth/tongue with regard to prognosis and a
variety of clinico-pathological parameters. All standardized AgNOR parameters
[mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV)
of both AgNOR number and area] were statistically significantly associated with
the clinical course. It was concluded that standardized staining and computer-
aided analysis of AgNORs were the prerequisites for an objective and
reproducible AgNOR assessment, which had potential as a supplementary
diagnostic and prognostic tool in oral cancer
75


62
A study was conducted on sections from 51 (T1-2) Oral Squamous cell
carcinomas (SCC), 20 cases of dysplasia, and 8 specimens with normal
epithelium by 2 AgNOR counting methods: (1) the mean number of AgNORs per
nucleus (mAgNOR) and (2) the percentages of nuclei with >1, >2, >3, and >4
AgNORs (pAgNOR > 1, pAgNOR > 2, pAgNOR > 3, and pAgNOR > 4,
respectively). Both mAgNOR and pAgNOR counts enabled significant
discrimination between normal epithelium and dysplasia and between dysplasia
and SCC. For SCC, no correlation was found between AgNOR counts and clinical
classification. They concluded that AgNOR enumeration, in particular pAgNOR
>1, appears to be a useful tool in distinguishing between normal epithelium,
dysplasia, and SCC of the oral cavity. In this study, AgNOR counts were strong
prognostic markers for patients with SCC
76
.

A study was designed to assess the applicability and reproducibility of AgNOR
staining on archival oral squamous cell carcinoma specimens, with special
emphasis on the invasive tumor front. Standardized image cytometric AgNOR
analysis was performed at the invasive zone and central parts of the tumors as
well as in adjacent dysplastic and normal oral mucosa by applying modified silver
staining on routinely processed archival tissues after wet autoclave pretreatment
for protein retrieval. A statistically highly significant and reproducible difference
of all of the four standardized AgNOR parameters evaluated (mean AgNOR
number, mean AgNOR area, coefficient of variation (CV) of AgNOR number,

63
and CV of AgNOR area per nucleus) was observed between the different areas
assessed. There was increase of AgNORs at the invasive front of oral squamous
cell carcinomas which seemed to indicate a subgroup of tumor cells with
increased biosynthetic activity and malignant potential
77
.
A morphometric study of silver stained nucleolar organiser regions was conducted
on 21 cases of oral leukoplakia (13 low, 4 moderate, 4 severe degree of dysplasia)
and 5 cases of microinvasive carcinoma.insitu hybridization for HPV DNA was
performed on serial sections of the same samples. The following parameters were
studied: V NOR (single AgNOR volume per nucleus), TV NOR (total AgNOR
volume per nucleus), and R.I. (AgNOR's roundness index). TV NOR appeared
useful, while the other morphometric parameters appeared statistically
insignificant in differentiating between the different lesions. Their findings
suggested that high values neither of TV NOR in oral dysplasia could represent a
risk marker, identifying a subgroup of lesions with a worse prognosis, constituting
then a possible indication for rigorous clinical management and/or for complex
treatment of those HPV-associated preneoplastic lesions
78
.

A study was conducted to assess the expression of nucleolar protein p120 and
nucleolar organizer region counts (AgNOR) with that of nuclear proliferation
markers MIB-1 and PCNA on 10 cases of keratotic epithelial hyperplasia (KEH),
10 cases of epithelial dysplasia (ED), and 15 cases of squamous cell carcinoma

64
(SCC). Significant differences in p120 and AgNOR mean area values and PCNA
labeling index (LI) were recorded between KEH and ED, as well as ED and SCC.
All markers significantly differed between SCC grades I and III. Significant
differences were also noted in AgNOR mean area values between grade I and II
SCC and in p120 mean area values. MIB-1 and PCNA LI differed significantly
when grade II and III SCC were compared. There were significant correlations
between p120 and AgNOR, and between both of them and the proliferative
indexes. AgNOR correlated with tumor grade, stage, and lymph node status,
suggesting a prognostic role for that marker
79
.
A study was conducted to analyze comparatively by the AgNOR technique
twenty-two cases of ameloblastoma and ten cases of adenomatoid odontogenic
tumour (AOT). Ameloblastomas were distributed into three groups according to
their clinical behaviour: primary lesions without recurrences (PLWTR), 5 cases;
primary lesions with recurrences (PLWR), 4 cases; and recurrences, 13 cases. The
cases were also regrouped according to their histological pattern: follicular (9
cases), plexiform (7 cases), acanthomatous (4 cases) and unicystic (2 cases).
Considering histological patterns, there was a significant statistical difference
only between follicular and plexiform types. There were no significant differences
between the group of Ameloblastomas and the group of AOTs or between the
three groups of Ameloblastomas with different clinical behaviour. Their results
strongly suggest that the distinct clinical behaviour of Ameloblastomas and AOT
are not correlated with their cellular proliferation ratio. Thus, the infiltrative

65
ability of the Ameloblastomas is probably not related to the cellular proliferation
index of these tumours
80
.

A study was conducted on ten inflammatory fibrous hyperplasias, ten papillomas,
and nineteen oral squamous cell carcinomas by the AgNOR technique to
determine if different disturbances of oral epithelia presented different AgNOR
counts. The papilloma group showed higher mean AgNOR counts than the
hyperplasia group and smaller than the well-differentiated oral squamous cell
carcinoma group and poorly differentiated oral squamous cell carcinoma group.
Their findings suggested that the cellular proliferation ratio in papillomas is
greater than hyperplasias and smaller than carcinomas
81
.
A study was conducted to quantitatively analyze AgNORs in oral squamous cell
carcinomas as well as in dysplastic epithelial changes accompanied and not
accompanied by oral squamous cell carcinomas. Statistically significant
differences were found between groups with mild dysplasia and groups with
severe dysplasia as well as squamous cell carcinomas. Statistical analysis did not
show any differences in the number of AgNORs between squamous cell
carcinomas and epithelial lesions with severe dysplasia. Their results
demonstrated that the analysis of AgNOR expression could serve only as an
additional parameter to evaluate the potential of malignant transformation
82
.


66
A study to determine the prognostic importance of AgNORs and ki-67 in
premalignant and malignant lesions in the oral cavity using retroprospective
phases of 98 histologic sections of 26 mild epithelial dysplasia, 15 moderate
epithelial dysplasia, and 57 invasive oral squamous cell carcinoma was
conducted. They were stained with silver colloid stain to obtain the average
number and type of AgNORs, and immunologic marker Ki-67 to obtain the
percentage of cells Ki-67. In both phases the average number of AgNORs and
Ki-67 positive cells were significantly greater in the squamous cell carcinoma
cases, compared to the dysplasias. They concluded that the AgNOR tissue marker
could be used as a routine complementary histopathologic study, since the
variations in its number and distribution indicate existence of cell alterations in a
given lesion and the use of this technique was easy and inexpensive
83
.

The silver colloid technique was used on fifty cases, which comprised of 10 cases
of normal oral buccal mucosa epithelium and 40 cases of leukoplakia without
dysplasia and mild, moderate and severe leukoplakia. They were examined with
respect to their relationship between AgNOR counts and histologic grading. The
mean AgNOR count per nucleus increased from normal oral buccal mucosa
epithelium to leukoplakia without dysplasia to leukoplakia with dysplasia. Higher
counts, wider scatter and smaller size of AgNOR in the nuclei were seen as the
grading of dysplasia increased from mild to severe. It was suggested that this
method had potential in distinguishing between dysplastic and non-dysplastic

67
leukoplakias and for early diagnosis, prognosis and treatment planning of
dysplastic lesions
84
.

A study was conducted on 41 normal oral epithelia, 51 oral leukoplakia
(26 dysplastic, 25 non-dysplastic), and 51 cases of squamous cell carcinoma
specimens for their mean AgNOR counts using the silver staining technique.
Mean AgNOR counts increased gradually from normal epithelium to non-
dysplastic to dysplastic leukoplakia to squamous cell carcinoma. They concluded
that mean AgNOR count could be a valuable criterion for defining objective
parameters for diagnosis/determination of dysplasia distinguishing between
dysplastic and non-dysplastic leukoplakia
85
.

A retrospective study was conducted to assess the diagnostic accuracy of
AgNOR-analysis as an adjunctive diagnostic tool to conventional oral exfoliative
cytology taken from suspicious lesions. Cytological diagnoses obtained from
brush biopsies of macroscopically suspicious lesions of the oral mucosa from 75
patients (final diagnoses: 53 histologically proven squamous cell carcinomas, 11
leukoplakias and other inflammatory oral lesions) and from 11 patients with
normal mucosa as a negative control group were compared with histological
and/or clinical follow-ups. They concluded that AgNOR-analysis might be a
useful adjunct to other methods in routine cytological diagnosis of oral cancer that
can help to solve cytologically suspicious or doubtful cases
86
.

68
In a double blind clinical study, leukoplakias from 52 cases were diagnosed for
presence (DLK) or absence (NDLK) of epithelial dysplasia using hematoxylin
and eosin (H&E) stain as a gold standard criterion, and results were compared
against their mean silver stainable nucleolar organizer region (AgNOR) counts.
They used mean AgNOR count cut-point of 2.37 from their prior report as the
diagnostic threshold (mean 2.37 being DLK and mean <2.37 being NDLK). The
two methods (H&E and AgNOR) disagreed in 37% of the diagnoses. Both NDLK
and DLK had high AgNOR counts. They concluded that the mean AgNOR count
could be a useful tool in definitive diagnosis of epithelial dysplasia
87
.

A study was conducted to evaluate the diagnostic effectiveness of argyrophilic
staining of nucleolar organizer regions (AgNORs) in separating benign nevi from
Malignant Melanomas (MMs) by assessing 27 compound nevi (CN), 20
dysplastic nevi (DN), 10 Spitz nevi (SN), and 24 MMs. Both AgNOR count and
morphology variables were measured from the superficial, middle, and deep
zones of the lesions using video image analysis. Malignant melanomas had a
significantly greater AgNOR number per nucleus; mean AgNOR area per
nucleus, and variation in AgNOR area per nucleus compared with all types of
benign nevi. The results suggested that both AgNOR count and morphology
might help to separate benign and malignant melanocytic lesions and that the
combination of both sets of parameters might improve their discriminating
ability
88
.

69
A study was designed to evaluate oral exfoliative cytology of smokers without
any clinically evident lesion and smokers with leukoplakia or oral cancer using
AgNOR staining. Cytological smears of 30 smokers without lesion, 30 smokers
with leukoplakia, 30 smokers with oral cancer and 30 non-smokers (control
group) were studied using one step silver staining method. Analysis of AgNOR
suggested that smoking influenced proliferative activity in cells of smokers
without any clinical lesion and that oral cancer showed the highest proliferative
activity
89
.
A study was conducted to assess the usefulness of argyrophilic nucleolar
organizer regions (AgNORs) as quantitative criteria in the diagnosis of
odontogenic cysts and tumors using 37 archival paraffin blocks of 10 cases of
conventional ameloblastomas, 7 cases of unicystic ameloblastoma, 10
odontogenic keratocysts, and 10 dentigerous cysts. Their findings showed a
significant statistical difference among the 4 lesions. Conventional and unicystic
ameloblastomas had a significantly higher number of AgNORs than odontogenic
keratocysts and dentigerous cysts. AgNORs in conventional and unicystic
ameloblastomas were smaller but more broadly distributed, which might indicate
higher proliferative activity. They concluded that AgNORs could be useful in the
histopathologic differentiation of ameloblastomas from odontogenic cysts
20
.
A study was designed to evaluate the diagnostic value of silver staining NORs
(AgNORs) in preneoplastic epithelia and nasopharyngeal carcinoma (NPC) by the

70
quantitative assessment of AgNORs proteins by Silver-staining. The
morphometric features of AgNORs in preneoplastic epithelia and cancer cells
were analyzed by image cytometric analysis and then compared. They concluded
that the morphometric analysis of AgNORs reflected cell proliferative activity and
histologic differentiation of tumor rather than malignant transformation in
different nasopharyngeal epithelia and NPC. AgNOR area and AgNOR area-
count ratio were the most valuable features for differential diagnosis of normal,
preneoplastic, and cancer cells
90
.

A study was conducted to examine the AgNOR counts in normal, premalignant
and malignant oral mucosa and to evaluate their potential as a biological marker
for tumour progression and a prognostic predictor for treatment outcome in oral
carcinomas. The analysis between AgNOR counts and various stages of tumour
progression in oral mucosa exhibited a highly significant positive coefficient, thus
indicating the role of AgNORs in the early diagnosis of potentially malignant oral
leukoplakia. Along with AgNOR counts, the T-status of disease was also found to
be an independent predictor for treatment outcome. T3 and T4 tumours, with
mean AgNOR counts more than 2.8, were aggressive and might exhibit resistance
to current treatment protocols
91
.


71
Methodology
The present study was conducted in the Department of Oral Medicine and
Radiology, R.V. Dental College and Hospital, Bangalore and in Wellspring-
Deshpande Laboratory, Bangalore.
The Archival study specimens were taken from the Department outpatient
subjects of Oral Medicine & Radiology, and Oral and Maxillofacial Surgery, R.V.
Dental College and Hospital, Bangalore. The study specimens consisted of
Paraffin embedded tissue specimens, of which were 15 cases of Oral Leukoplakia
and 20 cases of Oral Squamous Cell Carcinoma.
Materials:
The paraffin embedded tissue specimens of Oral Leukoplakia (15 cases) and Oral
Squamous Cell Carcinoma (20 cases) were sectioned using a Soft tissue
microtome (LEICA RM 2025) (Fig 1) and subsequently deparafinised on a hot
plate (Fig 2) and through Xylene, and hydrated through alcohol and subsequently
stained with Hematoxylin and Eosin, and Silver stain and mounted on DPX.
Chemicals used for staining procedures:
Processing chemicals and materials:
Xylene
Alcohol (different grades)
DPX
Frosted microslide Blue star, 75mmlong, 25mm wide, 1.35 mm
thick
Microscopic cover glass, Blue star, 22mm x 22mm

72
Methodology
Chemicals for Hematoxylin and Eosin staining
Harris Hematoxylin stain
Eosin stain
Chemicals for nucleolar organiser region staining (Fig 3)
Silver Nitrate pure (Merk limited)
Gelatin
Formic Acid
Distilled water
Enumeration
The stained structures were seen under LYNX N-200M Trinocular
microscope(Fig 4).
Methodology:
The paraffin embedded Archival tissue specimens of 15 cases of Oral
Leukoplakia were grouped histologically as
29

No dysplasia
Mild dysplasia
Moderate dysplasia
Severe dysplasia
The paraffin embedded Archival tissue specimens of 22 cases of Oral Squamous
Cell carcinoma were grouped histologically as
30

Grade I Well differentiated
Grade II Moderately differentiated

73
Methodology
Grade III Poorly differentiated
Hematoxylin and Eosin Staining Procedure:
5 mm tissue section was cut from the paraffin block
Deparaffinised in two change of Xylene
Hydrated through descending grades of Alcohol
Stained with Harris Hematoxylin for 5 minutes
Rinsed in running distilled water for 5 minutes
Differentiation was done by one dip in acid alcohol
Rinsed in running distilled water
Stained with Eosin for 2 minutes
Dehydrated through ascending grades of Alcohol
Cleared in two changes of Xylene
Mounted in synthetic medium, DPX
Nucleolar organiser region staining procedure:
5 mm tissue section was cut from the paraffin block
Deparaffinised in two change of Xylene
Hydrated through descending grades of Alcohol
The slides were then incubated in fresh silver nitrate solution at room
temperature in a dark chamber for 40 minutes

74
Methodology
Dehydrated through ascending grades of Alcohol
Cleared in two changes of Xylene
Mounted in synthetic medium, DPX
The stained sections were seen under LYNX N-200M Trinocular microscope.
Preparation of AgNOR solution:
Prepration of the AgNOR solution was done according to the method proposed by
Ploton et al
20
.
The silver nitrate solution was prepared by mixing 1 part of Solution A with 2
parts of Solution B
Solution A Solution B
2g of Gelatin in 100ml of 1%
Formic acid

50% solution of Silver nitrate
in distilled water

The solution was poured immediately after mixing on the slides and placed in a
dark chamber for 40 minutes.
The silver nitrate solution deteriorates on standing, so the solution had to be
freshly prepared each time before use.
The stained sections were seen under LYNX N-200M Trinocular microscope.


75
Methodology
Counting procedure for AgNOR:
AgNOR counting was done as per the method proposed by Giri et al
21
,where in
counting of all the specimens was done independently by two observers.200 cells
were counted using a X100 objective, under oil immersion.
The number of individually discernable and separable block dots in each nucleus
was recorded and the average for each case computed; where two or more dots
were so closely aggregated within a nucleolus that the precise number within the
aggregate could not be counted, the aggregate was recorded as one.
Inter-observer differences were recorded and cases with a difference in excess of
1 was reassessed.
The average of that obtained by the two observers represented the final mean
AgNOR count for a given case.
The data obtained was entered in the proforma, which is enclosed (Master Charts
1 and 2)


76
Methodology
Statistical Analysis
The data collected was analyzed independently by two observers and was
tabulated in the master charts (1 and 2).
The average of both the values obtained by two observers was obtained for each
case, and this was tabulated as the average AgNOR count. The Mean AgNOR
count and the Standard deviation were calculated in cases of Oral Leukoplakia
and Oral Squamous cell carcinoma.
Standard deviation was calculated using the formula:
SD=d
2
/n-1
Sum of the square of the difference
SD= Square root of in mean and the individual sample
Number of the sample minus one

The interobserver variation between the two observers was calculated using the
Pearsons Co-relation coefficient (r) using the formula:
r =covariance between the two variables /product of the standard
deviation of the first and the second variable
The Mean AgNOR values of Oral Leukoplakia and Oral Squamous cell
carcinoma were compared using the t test, for comparing the means of
individual samples, using the formula:
t=ratio of difference between the two means to the standard error of
difference between the means.

77
Methodology
The mean AgNOR values of different grades of Oral Leukoplakia were calculated
and were compared using the Analysis of variance (ANOVA) test, using the
formula:
F=ratio of mean sum of squares between observations to mean sum of
squares in observations
The mean AgNOR values of different grades of Oral Squamous cell Carcinoma
were calculated and were compared using the t test, for comparing the means of
individual samples, using the formula:
t=ratio of difference between the two means to the standard error of
difference between the means.
Sensitivity = True positives (TP) X 100
True positives (TP) +False negatives (FN)

Specificity = True negatives (TN) X 100
True negative (TN) +False positives (FP)

Positive predictive value = True positives (TP) X 100
True positive (TP) +False positive (FP)

Negative predictive value = True negatives (TP) X 100
True negatives (TN) +False negative (FN)

78

Fig 1. LEICA RM 2025 Microtome

Fig 2. Thermostatic bath and Hot plate


79
Fig 3. Chemicals used in Silver staining

Fig 4. LYNX N-200M TrinocularMicroscope with Nikon CoolPix 4500 Digital
Camera


80
Fig 5.Photomicrographs of H & E stained sections of Oral epithelial hyperplasia
(40x)

Fig 6.Photomicrographs of Silver stained sections of Oral epithelial hyperplasia
(100x)


81

Fig 7.Photomicrographs of H & E stained sections of Oral Leukoplakia-
Mild Dysplasia (40x)

Fig 8.Photomicrographs of Silver stained sections of Oral Leukoplakia-
Mild Dysplasia (100x)


82

Moderate Dysplasia (40x)

Moderate Dysplasia (100x)


83

Severe Dysplasia (40x)

Severe Dysplasia (100x)


84

Fig 13.Photomicrographs of H & E stained sections of Oral Squamous Cell
Carcinoma- Well Differentiated (40x)

Fig 14.Photomicrographs of Silver stained sections of Oral Squamous Cell
Carcinoma- Well Differentiated (100x)


85

Fig 15.Photomicrographs of H & E stained sections of Oral Squamous Cell
Carcinoma-Moderately Differentiated (40x)

Fig 16.Photomicrographs of Silver stained sections of Oral Squamous Cell
Carcinoma-Moderately Differentiated (100x)


86

Results
The present study conducted in the Department Of Oral Medicine and Radiology,
R.V.Dental College and Hospital, Bangalore, comprised of 35 archival cases, of
which were 15 cases of Oral Leukoplakia and 20 cases of Oral Squamous Cell
Carcinoma. (Graph 1)
The main aim of the study was to analyze the AgNOR counts in Oral Leukoplakia
and Oral Squamous cell carcinoma and compare the counts in different grades of
Oral Leukoplakia and Oral Squamous cell carcinoma.
The archival study specimens were collected from the department outpatient cases
of Department of Oral Medicine and Radiology and Department of Oral and
Maxillofacial Surgery R.V.Dental College and Hospital, Bangalore during the
period of the year 2004-2005. The staining was done at Wellspring Deshpande
laboratory Bangalore, and the specimens were analyzed independently by the two
observers.

The data obtained from both the observers were tabulated in the master charts
1and 2 comprising of the various histological grades in Oral Leukoplakia and Oral
Squamous Cell Carcinoma and the AgNOR counts in each grade of the two
groups. The average counts obtained by both the observers was calculated for
each case and tabulated as the average AgNOR count for both cases of Oral
Leukoplakia and Oral Squamous cell Carcinoma.(master charts 1 and 2).This data
was further statistically analyzed.

87
Results
Interobserver variation:
The inter observer variation between the two observers was calculated for both
Oral Leukoplakia and Oral Squamous Cell Carcinoma after calculating the total
AgNOR count in each group for each observer in both Oral Leukoplakia and Oral
Squamous Cell Carcinoma.
Pearsons correlation coefficient was used to analyze the variation in counting
between the two observers, and the results were considered to be significant at 5%
level if the p value was 0.05, and highly significant at 1% and 0.1% if the p
value was 0.001 and 0.0001 respectively.
Interobserver variation in Oral Leukoplakia:
Observer 1: The total AgNOR count in Oral Leukoplakia was 42.69
Observer 2: The total AgNOR count in Oral Leukoplakia was 40.25
The difference in the total AgNOR count between the two observers in Oral
Leukoplakia was highly statistically significant. (p<0.0045)
Interobserver variation in Oral Squamous Cell Carcinoma:
Observer 1: The total AgNOR count in Oral Squamous Cell Carcinoma was
114.51
Observer 2: The total AgNOR count in Oral Squamous Cell Carcinoma was
109.75
Squamous Cell Carcinoma was highly statistically significant. (p<0.0001)


88
Results
The total AgNOR count obtained by the first observer was slightly higher in both
Oral Leukoplakia and Oral Squamous Cell Carcinoma but were statistically
significant in both the groups.
Irrespective of the diagnosis the interobserver variation in all the cases of both the
groups was highly statistically significant (p<0.0001). (Table 1)
Correlation of the mean AgNOR counts:
Oral Leukoplakia:
Of the 15 Archival cases of Oral Leukoplakia were 10 males and 5 females.
They were in the age range of 35 to 63yrs.
Mean AgNOR count:
The mean AgNOR count in 15 cases of Oral Leukoplakia was 2.80 with standard
deviation of 0.50. The range of the AgNOR count was 2.14-3.66. (Graph 2)
Degree of dysplasia and the Mean AgNOR count:
Of the 15 cases of Oral Leukoplakia, cases 5 cases were of mild dysplasia, and 9
cases were of moderate dysplasia and 1 case of severe dysplasia.

The AgNOR count in 5 cases of mild dysplasia was in the range of 2.14 3.66,
with a Mean of 2.59 and Standard deviation of 0.66.
The AgNOR count in 9 cases of moderate dysplasia was in the range of 2.45
3.63, with a Mean of 2.92 and Standard deviation of 0.43.
The AgNOR count in 1 case of severe dysplasia was 2.79 (Table 3)


89
Results
Oral Squamous Cell Carcinoma:
Of the 20 Archival cases of Oral Leukoplakia were 5 males and 15 females.
They were in the age range of 41 to 67yrs.
Mean AgNOR count:
The mean AgNOR count in 20 cases of Oral Squamous Cell Carcinoma was 5.71
with standard deviation of 1.08. The range of the mean AgNOR count was 3.48
8.55. (Graph 3)
Histological grade and the Mean AgNOR count:
Of the 20 cases of Oral Squamous Cell Carcinoma, 12 cases were well
differentiated and 8 cases were moderately differentiated.
The AgNOR count in 12 cases of well differentiated Oral Squamous Cell
Carcinoma was in the range of 3.48-8.55, with Mean of 5.73 and Standard
deviation of 1.62.
The AgNOR count in 8 cases of moderately differentiated Oral Squamous Cell
Carcinoma was in the range of 4.33-8.27, with a Mean of 5.67 and Standard
deviation of 1.19. (Table 4)
Comparison of AgNOR count in Oral Leukoplakia and Oral Squamous Cell
Carcinoma:
The AgNOR count in 15 cases of Oral Leukoplakia ranged from 2.14-3.66
with a mean of 2.80 and standard deviation of 0.50 and in 20 cases of Oral
Squamous Cell Carcinoma ranged from 3.48 8.55 with a mean of 5.71 with
standard deviation of 1.08. (Table 2) (Graph 4)

90
Results
The mean AgNOR counts between the two groups were compared using the t
test, for comparing the means of individual samples, the results were considered
to be significant at 5% level if the p value was 0.05, and highly significant at
1% and 0.1% if the p value was 0.001 and 0.0001 respectively.
The mean AgNOR counts between the two groups showed statistically highly
significant results (p<0.0001)

Comparison of AgNOR count in different grades of Oral Leukoplakia:
Of the 15 cases of Oral Leukoplakia, cases 5 cases were of mild dysplasia, and 9
cases were of moderate dysplasia and 1 case of severe dysplasia.
The AgNOR count in 5 cases of mild dysplasia was 2.59 0.66, in 9 cases of
moderate dysplasia was 2.92 0.43 and in 1 case of severe dysplasia was 2.79
(Table 3) (Graph 5)
The mean AgNOR counts between the three groups was compared using the
ANOVA test.
The mean AgNOR counts between the three groups showed statistically no
significant results (p>0.51)

Comparison of AgNOR count in different Histological grades of Oral
Squamous Cell Carcinoma:
Of the 20 cases of Oral Squamous Cell Carcinoma, 12 cases were well
differentiated and 8 cases were moderately differentiated.

91
Results
The AgNOR count in 12 cases of well differentiated Oral Squamous Cell
Carcinoma was 5.73 1.62, and in 8 cases of moderately differentiated Oral
Squamous Cell Carcinoma was 5.67 1.19.
(Table 4) (Graph 6)
The mean AgNOR counts between the two groups was compared using the t
test, for comparing the means of individual samples.
The mean AgNOR counts between the two groups showed statistically no
significant results (p>0.97)

Diagnostic validity of AgNOR counts in predicting cancerous lesions:
Cutoff value of AgNOR in Precancer and cancer
AgNOR value of more than 3.5 is suggestive of cancer in our study. Cutoff value
of >3.5 AgNOR count is suggestive to differentiate between precancer and
cancer. In our study the cutoff value of >3.5 AgNOR count showed a sensitivity
of 95%, specificity of 80%, positive predictive value of 86.36%, negative
predictive value of 92.31% and efficiency of 88.57%. (Table 5)


92
Results
Table 1 -Interobserver variation
Groups Total AgNOR
count
Observer 1
Total AgNOR
count
Observer 2
Statistical
Analysis
Oral
Leukoplakia
42.69 40.25

p<0.0045
Oral Squamous
Cell Carcinoma
114.51 109.75 p<0.0001
Irrespective of the diagnosis (p<0.0001)

Table 2 - Groups and comparison
Groups No. Of
cases
AgNOR
Range
Mean AgNOR and
Standard
Deviation
Oral Leukoplakia 15 2.14-3.66 2.800.50
Oral Squamous Cell
Carcinoma
20 3.48
8.55
5.711.08
t test (p<0.0001)

Table 3- Histological grades of Oral Leukoplakia
Histological Grading No. Of
Cases
AgNOR
Range
Mean AgNOR and
Standard
Deviation
Mild Dysplasia 05 2.14
3.66
2.59 0.66
Moderate Dysplasia 09 2.45
3.63
2.92 0.43
Severe Dyspalsia 01 2.79 2.79
ANOVA (p>0.51) (F= 0.72)


93
Results

Table 4- Histological grades of Oral Squamous Cell Carcinoma
Histological Grading No. Of
Cases
AgNOR
Range
Mean AgNOR and
Standard
Deviation
Well Differentiated 12 3.48-8.55 5.73 1.62
Moderately
Differentiated
08 4.33-8.27 5.67 1.19
t test (p>0.97)

Table 5 Cutoff value (95
th
percentile of Precancer)
AgNOR
count
Oral Squamous Cell
Carcinoma
Oral
Leukoplakia
Total
3.5 19 O3 22
<3.5 01 12 13
20 15 35
Chi-Square (p<0.0001)

Sensitivity =19/20=95%
Specificity =12/15=80%
Positive predictive value =19/22=86.36 %
Negative predictive value =12/13=92.31 %
Efficiency =31/35=88.57 %


94
Results

Graph 1
Number of cases i n the Gr oups included i n the st udy
1
5
2
0
0
5
10
15
20
25
1
Gr oups
C
a
s
e
s
Oral Leukoplakia Oral Squamous Cell Carcinoma

Graph 2
Range of AgNOR Counts in cases of Oral Leukoplakia
0
0.5
1
1.5
2
2.5
3
3.5
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cases
AgNOR Counts
Oral Leukoplakia


95
Results

Graph 3
Range of AgNOR Counts in cases of Oral Squamous Cell Carcinoma
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Cases
AgNOR Counts
Oral Squamous Cell Carcinoma

Graph 4

Mean AgNOR counts in Oral Leukoplakia and Oral Squamous Cell Carcinoma
2.8
5.71
0
1
2
3
4
5
6
1
Groups
M
e
a
n

a
g
N
O
R

c
o
u
n
t
s
Oral Leukoplakia
Oral Squamous Cell Carcinoma


96
Results

Graph 5

Mean AgNOR Counts in different grades of Dysplasia in Oral Leukoplakia
2.59
2.92
2.79
2.4
2.5
2.6
2.7
2.8
2.9
3
1
Groups
Mean AgNOR
counts
Mild Dysplasia Moderate Dysplasia Severe Dysplasia

Graph 6

Mean AgNOR Counts in Different Histological Grades Of Oral
5.73
5.67
5.64
5.65
5.66
5.67
5.68
5.69
5.7
5.71
5.72
5.73
5.74
1
Grades
Mean AgNOR
counts
Well Differentiated Moderately Differentiated


97
Discussion
Oral Cancer forms about 10% of the estimated 644,600 new cancers that occur in
all parts of the body each year in India. Amongst oral cancer, Squamous cell
carcinoma accounts for over 90% of the cases.
As most oral cancer arise from precancerous lesions or conditions, which
accounts to about 70%, the methods of primary prevention have to be stressed.
Despite improved surgical approaches, reconstruction techniques and advances in
radiation and medical oncology, the single most effective route of improving the
long term out come of oral cancer is early diagnosis, coupled with appropriate
treatment.
Therefore for early diagnosis, assessment of etiologic and risk factors like
tobacco, alcohol and precancerous lesions are important. A routine biopsy and
histopathological examination should be made for all precancerous lesions to
eliminate any dysplastic changes.
Several methods have been used in the past few years for the identification of
proliferating cells in tissue sections and these have been applied especially to pre
malignant lesions with the aim of using them as a marker for an impending
malignancy.
The silver binding Nucleolar Organiser Regions (AgNOR) is one such technique
and has been used in assessing the precancerous and cancerous lesions of breast
lumps parathyroid gland disorders and recently has been extended to oral lesions.


98
Discussion
The use of silver solutions for staining cells and chromosomes has been used in
histological studies of normal and degenerating nerve tissue as well as in
cancerous tissues
39
.
Howell and Black developed the one step procedure for AgNOR staining; thereby
reducing the careful controlled requirements of the earlier methods their method
gave good results for staining AgNORs on chromosome preparations
39
.

Nucleolar Organiser regions are chromosomal segments in which ribosomal RNA
(rRNA) is encoded, and they are thus responsible for the development of RNA
containing nucleolus or nucleoli into which the NORs project on large loops of
DNA
6
.
In the human karyotype the NORs are located on each of the short arms of the
acrocentric chromosomes 13,14,15,21 and 22.Although activated NOR are found
in the nucleolus, inactive NORs may also be detected in the extranucleolar
nuclear compartment
6

NORs have recently attracted much attention because of the claims that their
frequency within the nuclei is significantly higher in malignant cells than in
normal, reactive or benign neoplastic cells
6
.
It has been suggested that the number of AgNORs in a nucleus may reflect the
state of activation of activation and the proliferation activity of the cells
22
.
There is now some evidence that the morphology, intensity, and spatial
relationships of AgNOR on chromosomes vary during cell cycle phases. Since

99
Discussion
they are reported to be markers of rRNA transcriptional activities, an analysis of
their number and form is of great importance
6
.
The silver staining technique identifies neither rRNA nor rDNA but the acidic
designated as B23, C23,AgNOR protein and RNA polymerase I. In practice,
however, the AgNORs in a normal cell are usually tightly aggregated within the
one or two nucleoli evident in histological or cytological preparations. The
individual AgNORs are often not discernable
6
.
The nucleolus is not a constant structure, for it disperses before mitotic cell
division and reorganizes afterwards. The abundance and intensity of AgNORs are
an indication of not only their absolute number and dispersion but also their
transcriptional activity; indeed it is possible that the residual AgNOR proteins
persist even after transcriptional activity has ceased
6
.

The present study conducted in the Department Of Oral Medicine and Radiology,
R.V.Dental College and Hospital, Bangalore, comprised of 35 archival cases, of
which were 15 cases of Oral Leukoplakia and 20 cases of Oral Squamous Cell
Carcinoma. (Graph 1)


100
Discussion

The archival study specimens were collected from the department outpatient cases
of Department of Oral Medicine and Radiology and Department of Oral and
Maxillofacial Surgery R.V.Dental College and Hospital, Bangalore during the
period of the year 2004-2005.The staining was done at Wellspring Deshpande
laboratory Bangalore, and the specimens were analyzed independently by the two
observers.

Two sections were made of each of the archival tissue specimen and one was
stained with Hematoxylin and Eosin stain to confirm the diagnosis, followed by
Modified Silver staining method of Ploton et al for the Nucleolar Organiser
Regions
20
. The sections were incubated in freshly prepared Silver colloid solution
in a dark chamber for 40 minutes in accordance with other studies.
AgNOR counting was done as per the method proposed by Giri et al
21
where in
counting of all the specimens was done independently by two observers .200 cells
were counted using a X100 objective, under oil immersion, inter-observer
differences were recorded. The number of individually discernable and separable
block dots in each nucleus was recorded and the average of the counts of each
observer for each case computed in the master charts (1 and 2).


101
Discussion
The inter observer variation between the two observers was calculated for both
Oral Leukoplakia and Oral Squamous Cell Carcinoma after calculating the total
AgNOR count in each group for each observer in both Oral Leukoplakia and Oral
21

The total AgNOR count in Oral Leukoplakia in Observer 1 was 42.69, and in
Observer 2 was 40.25.
Leukoplakia was highly statistically significant. (p<0.0045)
The total AgNOR count in Oral Squamous Cell Carcinoma was in Observer 1
was 114.51, and in Observer 2 was 109.75.
Squamous Cell Carcinoma was highly statistically significant. (p<0.0001)
The total AgNOR count obtained by the first observer was slightly higher in both
Oral Leukoplakia and Oral Squamous Cell Carcinoma but were statistically
significant in both the groups.
Irrespective of the diagnosis the interobserver variation in all the cases of both the
groups was highly statistically significant (p<0.0001). (Table 1)
Based on these observations the counting protocol used in our study may be
termed reliable, as the interobserver variation in our study was highly statistically
significant.


102
Discussion
The Mean AgNOR count in 15 cases of Oral Leukoplakia in our study was 2.80
with standard deviation of 0.50. The range of the AgNOR count was 2.14-3.66.
(Graph 2)
The mean AgNOR count in Oral Leukoplakia in our study is similar to the study
conducted by other investigators which was 2.690.49
92
in one study and
2.650.38 in another study in dysplastic Leukoplakia
,85
.
The Mean AgNOR counts reported by others in Oral Leukoplakia is in contrast to
our study, of 3.890.433
89
in one study and 3.390.78
87
in another study.

In our study the Mean AgNOR count in cases of mild dysplasia was 2.59 0.66,
in moderate dysplasia was 2.92 0.43 and in severe dysplasia was 2.79.
The Mean AgNOR count was higher in cases of moderate dysplasia as compared
to mild dysplasia in cases of Oral Leukoplakia as evident in the (Graph 5)
(Table 3). There was only one case of severe dysplasia with AgNOR count of
2.79.There was no statistically significant difference between the three grades in
Oral Leukoplakia in our study (p>0.51).
The Mean AgNOR count of 2.80 0.50 in our study in dysplastic Leukoplakia
correlates with that of the study done by one investigator of 2.650.38
85
in
dysplastic Leukoplakia and 2.14 0.37
85
in nondysplastic Leukoplakia.


103
Discussion
The Mean AgNOR count in 20 cases of Oral Squamous Cell Carcinoma in our
study was 5.71 with standard deviation of 1.08. The range of the mean AgNOR
count was 3.48 8.55. (Graph 3)
The Mean AgNOR count in our study in cases of Oral Squamous Cell Carcinoma
correlates with the studies conducted by other investigators of 6.161.89
24
,
4.741.04
93
, 4.960.467
89
, 6.290.91
94
and 6.21.5
76
.
The Mean AgNOR counts reported by others in Oral Squamous Cell Carcinoma is
in contrast to our study of 8.712.26
74
, 8.376.11
65
and 3.860.75
85
.

The Mean AgNOR count in our study in cases of well differentiated Oral
Squamous Cell Carcinoma was 5.73 1.62 and in cases of moderately
differentiated Oral Squamous Cell Carcinoma was 5.671.19.There was no
statistically significant difference in the Mean AgNOR counts (p>0.97) in cases of
well differentiated and moderately differentiated Oral Squamous Cell Carcinoma
as shown in the (Graph 6) (Table 4).
The Mean AgNOR count of 5.73 1.62 in cases of well differentiated Oral
Squamous Cell Carcinoma correlates with the studies done by some investigators
of 4.731.04
93
in grade I Oral Squamous Cell Carcinoma (and 7.040.95 in grade
II); 6.561.25
81
in well differentiated (7.071.60 in poorly differentiated) Oral
Squamous Cell Carcinoma; 6.391.67
7
in grade I (9.741.72 in grade II and
6.192.37 in grade III) Oral Squamous Cell Carcinoma;5.120.85
24
in well

104
Discussion
differentiated (7.311.07 in moderately differentiated;10.500.54 in poorly
differentiated) Oral Squamous Cell Carcinoma.
The results obtained by other studies is in contrast to our study of 8.291.47
8
in
well differentiated (9.490.74 in poorly differentiated) Oral Squamous Cell
Carcinoma.
The Mean AgNOR count of 5.671.19 in cases of moderately differentiated Oral
Squamous Cell Carcinoma in our study is in contrast with the studies done by
others 9.741.72
7
in grade II Oral Squamous Cell Carcinoma; 7.040.95
93
in
grade II Oral Squamous Cell Carcinoma; 7.311.07
24
in moderately differentiated
Oral Squamous Cell Carcinoma.
The variation in the AgNOR counts between our study and other studies cited
above may be due to the differences in the staining and processing procedures
employed, and may also be due to the section thickness employed. As the section
thickness increases the number of AgNORs also increases.
In our study two cases of well differentiated (8.55, 8.55) and one case of
moderately differentiated (8.27) Oral Squamous Cell Carcinoma showed high
Mean AgNOR counts, which were well beyond the Mean AgNOR count of 5.73
1.62 for well differentiated and 5.671.19 for moderately differentiated Oral
Squamous Cell Carcinoma. The Hematoxylin and Eosin sections showed high
mitotic figures even though they were well and moderately differentiated Oral
Squamous Cell Carcinomas.


105
Discussion
Sano et al
7
proposed that the 5yr survival rate of cases with high AgNOR counts
(6.5) was significantly lower than that of counts (<6.5). Therefore they proposed
that AgNOR counts might be a useful marker in assessing the prognosis of the
malignant lesion.
Based on this proposition the AgNOR counts in our study showing very high
values may be marked as having poor prognosis
In our study the mean AgNOR count in cases of Oral Leukoplakia was 2.80 0.50
and in the range of 2.14-3.66.The mean AgNOR count in cases of Oral Squamous
Cell Carcinoma was 5.71 1.08 and in the range of 3.48 8.55. (Table 2)
(Graph 4)
The AgNOR count was high in cases of Oral Squamous Cell Carcinoma as
compared to cases of Oral Leukoplakia, and showed statistically significant
difference (p<0.0001).
The AgNOR counts were higher in cases of moderate dysplasia than in cases of
mild dysplasia in Oral Leukoplakia, but were not statistically significant (p>
0.51). There was only one case of severe dysplasia in our study, so the correlation
in the AgNOR count could not be established with mild and moderate dysplasia
cases.
The difference in AgNOR counts between well-differentiated and moderately
differentiated Oral Squamous Cell Carcinoma did not show statistically
significant results (p>0.97).


106
Discussion
The AgNOR count has been thought to be a proliferation marker; therefore one
would find a difference in normal mucosa, Oral Leukoplakia and Oral Squamous
Cell Carcinoma.
In one study an increase in the Mean AgNOR counts in Oral Squamous Cell
Carcinoma was found to be higher as compared to Papilloma and Normal Oral
mucosa
23
; and in another study there was an increase in the Mean AgNOR counts
in Oral Squamous Cell Carcinoma as compared to candidial dysplasia, epithelial
dysplasia to benign keratosis
65
.
In another study an increase in the Mean AgNOR count in Oral Squamous Cell
Carcinoma (3.480.48) was found as compared to Oral Leukoplakia (2.690.49)
and normal mucosa (2.260.32)
92

An increase in the Mean AgNOR count in Oral Squamous Cell Carcinoma
(6.21.5) as compared to Oral Dysplasia (3.80.8) and Normal oral mucosa
(2.30.4)
76
was found in another study.
An increase in the Mean AgNOR count in Oral Squamous Cell Carcinoma
(4.960.467) as compared to Oral Leukoplakia (3.890.433)
89
was found in
another study.
An increase in the Mean AgNOR count between Oral nondysplastic Leukoplakia
(2.140.37) and Oral dysplastic Leukoplakia (2.650.38), and Oral Squamous
Cell Carcinoma (3.860.75) was found in a study
85
.


107
Discussion
A similar increase in the Mean AgNOR counts were seen in our study between
Oral Leukoplakia and Oral Squamous Cell Carcinoma.

Diagnostic validity of AgNOR counts in predicting cancerous lesions was
estimated by determining the Cutoff value of AgNOR between Precancer and
cancer. In our study a cutoff value of 3.5 has been suggested, an AgNOR value
greater than 3.5 is suggestive of cancer. Therefore a cutoff value of >3.5 AgNOR
count is suggestive to differentiate between Oral Leukoplakia and Oral Squamous
Cell Carcinoma.
In one study the cutoff value of 4.8 was proposed to differentiate between Benign
and Malignant lesions in Brush biopsy cases of suspicious lesions of Oral
cavity
86
.
In another study the proposed a cutoff point was 2.37 to differentiate between
nondysplastic and dysplastic Oral Leukoplakia
85
.
In one study it was proposed that a mean AgNOR count >2.8 concurred with poor
prognosis in Oral Squamous Cell Carcinoma and that T3 and T4 tumors >2.8
Mean AgNOR counts were aggressive and may exhibit resistance to current
treatment protocols
91
.

The higher AgNOR counts found in many cancers may be due to dispersion of
AgNORs in the nucleoplasm, so the AgNOR type may be a helpful in making


108
Discussion
such a distinction, whether this indicates increased risk of malignant
transformation awaits longitudinal study
65
.

The AgNOR dots in benign lesions shows a large, central, regularly contoured
nucleolus containing single or multiple AgNOR dots, where as the malignant
neoplasms had multiple small nucleoli with prominent AgNORs distributed
throughout the nucleus
21
.
As with most histochemical markers of malignant transformation, AgNOR would
evidence the metabolic alterations associated with malignant transformation rather
than characterize malignancy as such
21
.
AgNORs detect cellular alterations before morphologic expression.

In one study the percentage of the number AgNORs present in the nucleus was
analyzed, and was proposed that the pAgNOR >1 or>2 reflects the size of the
growth fraction as well as its proliferative activity. This is consistent with the
observation that high proliferative activity coincides with poor prognosis. They
proposed that the pAgNOR>1 were strong indicators with regard to disease
recurrence and short survival in T1 and T2 Oral Squamous Cell Carcinoma
76
.

Image analysis methods have improved the efficiency of AgNOR analysis.
Morphometric studies done by several researchers have analyzed the single
AgNOR volume, total AgNOR volume per nucleus, contour index of nuclei,

109
Discussion
contour index of AgNOR, volume of tissue occupied by AgNOR
23
. These studies
have given a clear picture of AgNOR configuration and are rapid and less affected
by observer variations, but still could not eliminate the drawbacks of processing
time and staining procedure.

Therefore for interstudy comparisons a standardized staining technique and
staining procedure has to be established.

To draw substantial conclusions as to whether AgNOR counts indicate increased
risk of malignant transformation awaits longitudinal study with larger samples.

A follow up study is required to assess the prognosis of the three cases which
showed high AgNOR counts, as well as in other cases of Oral Squamous Cell
Carcinoma, to know if cases with high AgNOR counts indicate poor prognosis
and their 5yr survival rate.

A further follow up study is required to assess the malignant potentiality of cases
with high AgNOR counts in Oral Leukoplakia


110
Conclusion
The following conclusions were drawn
The Mean AgNOR count was higher in cases of Oral Squamous Cell
Carcinoma as compared to cases of Oral Leukoplakia, and showed
statistically significant difference
The Mean AgNOR count showed no statistically significant difference in
different grades of Oral Squamous Cell Carcinoma and Oral Leukoplakia,
but the AgNOR counts increased with the grade of dysplasia.
Therefore the AgNOR method can be used to provide information on the
malignant potentiality in premalignant lesions and aggressiveness of the
malignant lesions.
Three cases of Oral Squamous Cell Carcinoma which showed increased AgNOR
count could be marked as having poor prognosis though their grade of
differentiation was well and moderate.
A follow up study is required to assess the prognosis of these three cases, as well
as in other cases with high AgNOR counts to assess their prognosis.


111
Summary
Nucleolar Organiser regions are loops of DNA that encode ribosomal RNA and
are located on each of the short arms of the acrocentric chromosomes 13,14,15,21
and 22
6
.
The study specimens comprised of 35 archival cases, of which 15 cases were of
Oral Leukoplakia and 20 cases of Oral Squamous Cell Carcinoma. The specimens
were stained by Hematoxylin and Eosin and Modified Silver staining method of
Ploton et al for the Nucleolar Organiser Regions. The specimens were analyzed
independently by the two observers and was further statistically analyzed. The
results were analyzed as follows
In our study the mean AgNOR count in cases of Oral Leukoplakia was
2.80 0.50 and in cases of Oral Squamous Cell Carcinoma was 5.71 1.08.
The Mean AgNOR count was higher in cases of Oral Squamous Cell
Carcinoma as compared to cases of Oral Leukoplakia, and showed statistically
significant difference (p<0.0001).
The Mean AgNOR count was higher in cases of moderate dysplasia as
compared to mild to moderate dysplasia.


112
Summary
There was no significant difference in the Mean AgNOR counts in cases of
well-differentiated and moderately differentiated Oral Squamous Cell
Carcinoma.
Leukoplakia and Oral Squamous Cell Carcinoma was highly statistically
significant. (p<0.0001)
In our study two cases of well differentiated (8.55; 8.55) and one case of
moderately differentiated (8.27) Oral Squamous Cell Carcinoma showed high
Mean AgNOR counts, which were well beyond the Mean AgNOR count of
5.71 1.08 for Oral Squamous Cell Carcinoma. These AgNOR counts in our
study showing very high values may be marked as having poor prognosis
In our study a cutoff value of 3.5 has been suggested, an AgNOR value
greater than 3.5 is suggestive of cancer. Therefore a cutoff value of >3.5
AgNOR count is suggestive to differentiate between Oral Leukoplakia and
Oral Squamous Cell Carcinoma.


113
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125
Annexure
MASTER CHART 1

Leukoplakia

No Histological
diagnosis
Histologic
al
Grading
AgNOR
count
Observer
1
AgNOR
count
Observer 2
AgNO
R
Count
Average
AgNOR
Count
1 Leukoplakia Moderate 303 239 271 2.71
3 Leukoplakia Severe 290 268 279 2.79
6 Leukoplakia Mild 206 223 214 2.14


126

Annexure
MASTER CHART 2


N
o
Histological
diagnosis
Histological
Grading
AgNOR
count
Observer 1
AgNOR
count
Observer 2
AgNOR
Count
Average
AgNOR
Count
1 Squamous Cell
Carcinoma
Well
Differentiated
448 375 411 4.11
2 Squamous Cell
Carcinoma
Well
Differentiated
624 579 601 6.01
3 Squamous Cell
Carcinoma
Moderately
Differentiated
585 531 558 5.58
4 Squamous Cell
Carcinoma
well
Differentiated
602 527 564 5.64
5 Squamous Cell
Carcinoma
Moderately
Differentiated
628 553 590 5.90
6 Squamous Cell
Carcinoma
Well
Differentiated
818 892 855 8.55
7 Squamous Cell
Carcinoma
Well
Differentiated
673 640 656 6.56
8 Squamous Cell
Carcinoma
Well
Differentiated
863 847 855 8.55
9 Squamous Cell
Carcinoma
Well
Differentiated
396 301 348 3.48
10 Squamous Cell
Carcinoma
Well
Differentiated
518 507 512 5.12
11 Squamous Cell
Carcinoma
Moderately
Differentiated
843 812 827 8.27
12 Squamous Cell
Carcinoma
Well
Differentiated
411 467 644 6.44
13 Squamous Cell
Carcinoma
Moderately
Differentiated
601 567 584 5.84
14 Squamous Cell
Carcinoma
Moderately
Differentiated
513 511 512 5.12
15 Squamous Cell
Carcinoma
Well
Differentiated
430 485 457 4.57
16 Squamous Cell
Carcinoma
Well
Differentiated
413 457 435 4.35
17 Squamous Cell
Carcinoma
Moderately
Differentiated
582 549 565 5.65
18 Squamous Cell
Carcinoma
Moderately
Differentiated
472 394 433 4.33
19 Squamous Cell
Carcinoma
Moderately
Differentiated
453 483 468 4.68
20 Squamous Cell
Carcinoma
Well
Differentiated
578 498 538 5.38


127


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Nucleolar Organiser Regions (AgNORs), a quantative criteria in the

diagnosis of Human Oral Squamous Cell Carcinoma and Oral

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