1. health and fitness
2. disease
3. cancer

“AN EVALUATION OF SERUM TOTAL SIALIC ACID IN ORAL
LEUKOPLAKIA AND ORAL SQUAMOUS CELL CARCINOMA”
By
DR. BHANUSHREE.R
Dissertation Submitted to the
Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka.
In partial fulfillment
Of the requirements for the degree of
MASTER OF DENTAL SURGERY
In
ORAL MEDICINE AND RADIOLOGY
Under the guidance of
Dr. M. MANJUNATH
PROFESSOR AND HEAD,
DEPARTMENT OF ORAL MEDICINE,
DIAGNOSIS AND RADIOLOGY,
V.S. DENTAL COLLEGE,
BANGALORE.
2006-2009
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA.
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation/thesis entitled “ AN
EVALUATION OF SERUM TOTAL SIALIC ACID IN ORAL
LEUKOPLAKIA AND ORAL SQUAMOUS CELL CARCINOMA”
is a bonafide and genuine work carried out by me under the
guidance of Dr. M. MANJUNATH PROFESSOR AND HEAD,
DEPARTMENT
OF
ORAL
MEDICINE,
DIAGNOSIS
RADIOLOGY, V.S. Dental College, Bangalore.
Date:
Dr. BHANUSHREE.R
Place: Bangalore
I
AND
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA.
CERTIFICATE BY THE GUIDE
This
is
to
certify
that
the
dissertation
entitled
“AN
EVALUATION OF SERUM TOTAL SIALIC ACID IN ORAL
LEUKOPLAKIA AND ORAL SQUAMOUS CELL CARCINOMA”
is a bonafide research work done by Dr. BHANUSHREE.R
in partial fulfillment of the requirement for the degree of
MASTER OF DENTAL SURGERY IN ORAL MEDICINE AND
RADIOLOGY.
Date:
Dr. M. MANJUNATH.
Guide and Head,
Department of Oral Medicine,
Diagnosis and Radiology,
V.S .Dental College &
Hospital, Bangalore.
Place: Bangalore
II
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA.
CERTIFICATE BY THE CO GUIDE
This
is
to
certify
that
the
dissertation
entitled
“AN
EVALUATION OF SERUM TOTAL SIALIC ACID IN ORAL
LEUKOPLAKIA AND ORAL SQUAMOUS CELL CARCINOMA”
is a bonafide research work done by Dr. BHANUSHREE.R
in partial fulfillment of the requirement for the degree of
MASTER OF DENTAL SURGERY IN ORAL MEDICINE AND
RADIOLOGY.
Dr. K.L.MAHADEVAPPA
Professor and Head,
Department of Biochemistry,
KIMS Hospital, Bangalore.
Date:
Place: Bangalore
III
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA.
ENDORSEMENT BY THE HEAD OF DEPARTMENT, PRINCIPAL/HEAD OF
THE INSTITUTION
This
is
to
certify
that
the
dissertation
entitled
“AN
EVALUATION OF SERUM TOTAL SIALIC ACID IN ORAL
LEUKOPLAKIA AND ORAL SQUAMOUS CELL CARCINOMA”
is
a bonafide research work done by Dr. BHANUSHREE.R
under the guidance of Dr.M.MANJUNATH. PROFESSOR AND
HEAD, DEPARTMENT OF ORAL MEDICINE, DIAGNOSIS AND
RADIOLOGY, V.S. DENTAL COLLEGE, BANGALORE.
Dr. M. MANJUNATH.
PROF. AND HEAD
DR. USHA MOHAN DAS
PRINCIPAL
Date:
Date:
Place: Bangalore
Place: Bangalore
IV
COPYRIGHT
Declaration by the Candidate
I hereby declare that the Rajiv Gandhi University of Health Science,
Karnataka, shall have the right to preserve, use and disseminate this dissertation /
Thesis in print or electronic format for academic/research purpose
Date:
Dr.Bhanushree.R
Post graduate student,
Department of Oral medicine
Diagnosis and Radiology
VS Dental college and Hospital
Bangalore-560004
Place:
©Rajiv Gandhi University Of Health Sciences, Karnataka
V
ACKNOWLEDGEMENT
Life can never be a success without the Grace of Almighty, support from family,
Guidance from teachers and well wishes from elders.
It is of great pleasure to express my gratitude at this juncture to THE ICON OF
PATIENCE and my guide to Dr.M.Manjunath, Professor and Head, Department of
Oral medicine and Radiology who has been unstintingly guiding me through the
itinerary study.
I would like to express my gratitude to Dr .Usha Mohan Das, Principal, V. S. Dental
College & Hospital for providing me an opportunity to pursue my post graduation in
Department of Oral Medicine & Radiology
I would like to extend my gratitude to my Co-Guide Dr. H. L. Mahadevappa,
Professor and Head, Department of Biochemistry, KIMS Hospital, Bangalore for his
constant support and encouragement and guidance during the entire course of this
study.
My sincere thanks to Dr. Deepak T.A. Associate professor, for his kind support,
constant backup and guidance in the upcoming of this work and to Dr.Sowmya
Krishna, Senior lecturer ,Dr.Annaji.A.G, Senior lecturer , Department of Oral
Medicine and Radiology without whose encouragement, it would have been difficult
to finish my work.
VI
I Acknowledge with sincer gratitude to Dr.Ramamurthy, former Associate professor,
Department of Oral Medicine and Radiology who had given guidance in my
academics even in short period of time.
I sincerely thank Mr.Tejeswi (Statistician) for his speedy work in analysing the result
and compilation of the Statistical data for this study.
The acknowledgements would not be complete without thanking the many people who
have stayed behind the scenes but have made significant contribution for this work.
Date:
DR. BHANUSHREE.R
Place: Bangalore
VII
LIST OF ABBRIVIATIONS
ƒ
AK-Actinic Keratosis
ƒ
ALV-Alveolus
ƒ
ANOVA-Analysis of Variants
ƒ
BCC-Basal cell carcinoma
ƒ
bFGF-basic fibroblast growth factor
ƒ
BM-Buccal Mucosa
ƒ
BMI-Body mass index
ƒ
CEA- carcinoembryogenic antigen
ƒ
COL-Commisure Of Lip
ƒ
CYFRA 21-1-Cytokeratin 19 fragment
ƒ
DPA- Diphenylamine
ƒ
FGFR-1-Fibroblast growth factor receptor 1.
ƒ
FOM-Floor Of Mouth
ƒ
GBS-Gingivo Buccal Sulcus
ƒ
HIF-1α –Hypoxia-inducible factor 1α
ƒ
HNSCC- Head and Neck Squamous Cell Carcinoma
ƒ
HPA-Helix pomatia
ƒ
HPV-Human Papilloma Virus
ƒ
IEC- intestinal epithelial cell line
ƒ
LASA-Lipid Associated Sialic Acid
ƒ
LP-Leukoplakia
ƒ
MC-Mast Cells
ƒ
MCM2 LI-Mini Microsome maintenance 2 Labeling Index
ƒ
MN-Micronuclei
VIII
ƒ
NANA- N-Acetyl Neuraminic Acid
ƒ
NF-kB-Nuclear Factor kB
ƒ
OC-Oral Cancer
ƒ
OC3-Oral Carcinoma 3
ƒ
OSCC-Oral Squamous Cell Carcinoma
ƒ
PA-Para cortical Area
ƒ
PBH- protein bound hexoses
ƒ
PC- precancerous
ƒ
PHA- phytohemagglutinin
ƒ
RIA-Radioimmunoassay
ƒ
RMA-Retro molar area
ƒ
SA-Sialic Acid
ƒ
SCC-Squamous Cell Carcinoma
ƒ
SP-Soft Palate
ƒ
ST-Smokeless Tobacco
ƒ
S-TSA- serum total Sialic acid
ƒ
TBA- Thiobarbituric acid
ƒ
TNF-α –Tumor Necrosis Factor- α
ƒ
TP-Total Protein
ƒ
TSA –Total Sialic Acid
ƒ
WGA-Wheat germ agglutinin
IX
ABSTRACT
BACKGROUND
AND
OBJECTIVES:
Altered
glycosylation
of
glycoconjugates is among the important molecular changes that accompany malignant
transformation. Various studies in the past have shown the direct relation of Total
Sialic acid in premalignant lesions and different malignancies. The purpose of this
study is to investigate clinical usefulness of circulatory levels of Total Sialic acid in
Leukoplakia, Squamous cell carcinoma and healthy controls of age and sex matched
subjects.
METHOD: Blood samples were collected from 25 untreated Leukoplakia, 25
untreated Squamous cell carcinoma and 25 healthy subjects. Total serum Sialic acid
were evaluated by the simplified quick method by G Sydow and measured
spectrophotometrically at 525nm.
RESULTS: Serum levels of Total Sialic acid were significantly elevated
(P<0.001) in untreated Oral cancer patients as compared to healthy controls and
Leukoplakia. Our data also inferred significant difference between 1)control group &
Leukoplakia group, 2)control group & squamous group and 3)Leukoplakia group &
squamous group with respect to the mean Sialic acid levels (P<0.001). The mean
Sialic acid is found to be more in squamous group compared to Leukoplakia and
control group and this difference is statistically significant. The mean Sialic acid in
Leukoplakia group is higher than control group and this difference is also statistically
significant.
X
CONCLUSION: The data revealed direct relation between significant
elevation of serum Total Sialic acid levels in Oral cancer patients and also an
ascending order of increase of serum Total Sialic acid levels from healthy controls to
Leukoplakia to Squamous cell carcinoma and suggested that potential utility of these
parameters in initial diagnosis of Leukoplakia and Squamous cell carcinoma.
Key words: Glycoprotein, Leukoplakia, Oral cancer, Sialic acid, Squamous
cell carcinoma.
XI
TABLE OF CONTENTS
SI.NO.
CONTENTS
1.
INTRODUCTION
1-3
2.
OBJECTIVES
4-4
3.
4.
REVIEW OF LITERATURE
METHODOLOGY
PAGE NO.
5-36
37-46
5.
RESULTS
47-62
6.
DISCUSSION
63-68
7.
CONCLUSION
8.
SUMMARY
71-71
9.
BIBILOGRAPHY
72-82
10.
ANNEXURES
83-88
XII
69-70
LIST OF TABLES
SI.NO.
1.
2.
TABLES
Table 1- Age distribution in 3 different
groups
Table 2- Gender distribution between 3
groups
PAGE NO.
48
49
3.
Table 3- Habit distribution between 2
Groups
52
4.
Table 4- Site distribution between 2
Groups
54
5.
Table 5- Staging in Leukoplakia, GroupI
55
6.
Table 6- Staging in Oral Cancer, GroupII
56
7.
Table 7- Tumor size in oral cancer
57
8.
Table 8- Nodal involvement in Oral
Cancer
58
9.
Table 9- Descriptive statistics
59
10.
Table 10- ANOVA-Sialic acid
61
11.
Table 11- Bonferroni Test
62
XIII
LIST OF GRAPHS
SI.NO.
GRAPHS
PAGE NO.
1.
Graph 1- Age distribution in Three Groups
48
2.
Graph 2- Gender distribution in Three Groups
50
3.
Graph 3- Habit distribution in Three Groups
52
4.
Graph 4- Site distribution in Three Groups
54
5.
Graph 5- Staging in Leukoplakia
55
6.
Graph 6- Staging in Oral Cancer
56
7.
Graph 7- Tumor size in Oral Cancer
57
8.
Graph 8- Nodal involvement in Oral Cancer
9.
Graph 9- Sialic acid in Three Groups
60
Graph 10- Mean Sialic acid in Three Groups
60
10.
XIV
58
LIST OF PHOTOGRAPHS
Fig.
No
Page
No.
PHOTOGRAPHS
1.
CLINICAL
PHOTOGRAPH
SHOWING
LEUKOPLAKIA ON LEFT BUCCAL MUCOSA
42
2.
PHOTOMICROGRAPH
LEUKOPLAKIA
42
3.
PHOTOMICROGRAPH SHOWING MILD
EPITHELIAL DYSPLASIA
43
4.
CLINICAL
PHOTOGRAPH
SHOWING
CARCINOMA ON THE RIGHT BUCCAL
MUCOSA
43
5.
PHOTOMICROGRAPH
SHOWING
WELL
DIFFERENTIATED
CARCINOMA
44
6.
CENTRIFUGE MACHINE
44
7.
INCUBATOR
45
8.
SPECTROPHOTOMETER
45
9.
REAGENTS
USED
IN
THE
ESTIMATION OF TOTAL SIALIC ACID
46
OF
ESTIMATED SERUM TOTAL SIALIC ACID IN
10. DIFFERENT SAMPLES
XV
46
INTRODUCTION
INTRODUCTION
Cancer meaning Crab in Greek as these adheres to any part, which it seizes upon
it as an obstacle manner. It was Hippocrates the father of medicine coined the term
“Karkinos” (for non-healing neoplastic Ulcers) and “Karkinoma” (for solid malignant
ulcers).1
Oral cancer is a serious health problem world wide accounting not only for
mortality but also responsible for extensive disfigurement, loss of function,
behavioural changes, financial and sociologic hardship. Frustration abounds because
the cure rate is dismally low for such an accessible tumor.2
Despite improvement in imaging and therapy, the survival rate for patients with
these has not changed substantially for many years.3
Oral cancer is the Sixth cause of Cancer related morbidity and mortality
globally4 with the incidence reaching high proportion in India, where there is 11.2%
Prevalent in males and 11% in females with the site predilection of oral cavity as the
second place in males and third in females (Hospital based cancer registry, Kidwai
Memorial Institute Of Oncology, Bangalore 2004-05).Clinical, epidemiology and
laboratory studies suggest direct etiological relationship with prolonged tobacco use
with Oral cancer.4
Despite the recent advances in tumour surgery and multimodal treatment
regimes, the prognosis of head and neck squamous cell carcinoma (HNSCC) is still
relatively poor. This may be because the symptoms that indicate the presence of the
carcinoma often appear when the tumour is in an advance stage.5
Oral cancer is usually preceded in many cases by precancerous lesions or
conditions like Leukoplakia or Sub mucous oral fibrosis, is attributed to different
types of tobacco chewing.6
1
INTRODUCTION
Therefore the early diagnosis of Squamous Cell Carcinomas (SCC) would
improve survival and quality of life, avoiding the mutilation that physician often have
to make to save patients lives. The search for biological marker that could predict the
changes in the pre-malignant Lesion would be useful in detecting patients with high
risk for malignancy.6
Studies of malignant cells have revealed alteration in cell surfaces and
membranes. Many properties of mammalian cells are expressed at, or mediated
through, cell surfaces. Glycoproteins and gylcolipids, which form the major
constituents of cells, have been implicated in cellular invasiveness, adhesiveness and
immunogenesity. They are released in to circulation through increased turnover,
secretion, and / or shedding from malignant cell. Changes in serum glycoprotein
levels are characteristic of many pathological conditions including malignancy.6
Over the past several decades, experienced gained from the studies of
tumours had lead to the recognition of the significant role of glycoconjugates in
malignant transformation. Terminal epitopes of carbohydrates have been proposed to
play a significant role in cell – cell interactions in the development of cell adhesion
and in malignant transformation. One of the most common changes in
glycoconjugates during malignant transformation is the increase in size of
oligosaccharides resulting in branching sites for incorporation of Sialic acid. The
occupancy of Sialic acid at the terminal or near to terminal position underlies its vital
role in determing surface characteristic of cells and secreted Glycoproteins. Being
non-reducing terminal Sialic acid gained outstanding importance in cancer research6.
Total Sialic acid (TSA) consists of Glycoproteins and Gylcolipids bound
Sialic acids. These glycoconjugates are released in to circulation through increase
turnover, secretions and / or shedding from malignant cells.7
2
INTRODUCTION
Sialic acids (N-acetyl neuraminic acid, NANA) frequently occupy the
terminal, non-reducing position on membrane glycoproteins. The negative charge due
to presence of a carboxyl group enables Sialic acid to mediate a wide array of cellular
functions such as transport of positively charged molecular, cell-cell and cell-matrix
interactions, influence the confirmation of membrane glycoproteins and masking
antigenic sites of receptors. The presence of Sialic acid at the terminal or near the
terminal position underlies its importance in determining chemical and biological
diversity, and characteristic of cell surface and secreted glycoproteins. Human and
experimental model based studies have documented clinical significance of this sugar
moiety. Numerous investigators have reported possible relation of increased Sialic
acid levels with various malignancies.8
The idea of screening and following patients with malignancy by blood – based
test is appealing from several point of view including its ease, economic advantage,
non-invasiveness and possibility of repeated sampling. Therefore, the present study is
an attempt to investigate the serum levels of Glycoconjugate-the Sialic acid in patients
with Leukoplakia and Squamous cell carcinoma for its early diagnosis.
3
AIMS AND OBJECTIVES
AIMS AND OBJECTIVES
¾ To Asses serum Total Sialic acid levels in Leukoplakia.
¾ To Asses serum Total Sialic acid levels in Squamous cell carcinoma.
¾ To Asses serum total Sialic acid levels in controls.
¾ To correlate the Total Sialic acid levels in Leukoplakia and Squamous cell
carcinoma with that of controls.
4
REVIEW OF LITERATURE
REVIEW OF LITERATURE
LEUKOPLAKIA:
The oral cavity is a unique environment. Oral mucosa is a critical
protective interface between external and internal environments and serves as a
physical, chemical and microbial barrier.9
Preleukoplakia was defined as a low grade or a very mild reaction of the
mucosa, appearing as a gray or grayish white, but never completely white, area with a
slightly lobular pattern and with indistinct borders blending in to the adjacent normal
mucosa.10
“Leukoplakia is the most common precancerous lesion of the oral mucosa.11
DEFINITION AND TERMINOLOGY:
Leukoplakia and definable lesions:
Leukoplakia (1969) is a white patch of the oral mucosa measuring 5mm or more
which can not be scraped off and which cannot be attributed to any other diagnostic
disease.10
The World Health Organization (1978) has defined oral Leukoplakia as a white
patch or plaque which cannot otherwise be characterized clinically or pathologically
as any other disease.12
For many decades, oral Leukoplakia has been defined as a clinical term for ‘‘a
predominantly white lesion of the oral mucosa that cannot be characterized as any
other definable lesion.’’
In addition, the recommendation has been made to make a distinction between a
provisional clinical diagnosis of oral Leukoplakia and a definitive one.
5
REVIEW OF LITERATURE
A provisional clinical diagnosis is made when a lesion at the initial clinical
examination cannot be clearly diagnosed as any other definable lesion of the oral
mucosa with a white appearance.
A definitive clinical diagnosis of oral Leukoplakia is made as a result of the
identification, and if possible elimination, of suspected etiological factors and, in the
case of persistent lesions (no signs of regression within 2–4 weeks), histopathological
examination. As a result one recognizes several levels of certainty with which a
diagnosis of oral Leukoplakia has been made.
The use of a certainty (C) factor is analogous to the use of the C in the TNM
Classification for Cancer.
C1 Evidence from a single visit, applying inspection and palpation as the only
diagnostic means (Provisional clinical diagnosis).
C2 Evidence obtained by a negative result of elimination of suspected etiologic
factors, e.g. mechanical irritation, during a follow-up period of 2–4 weeks (Definitive
clinical diagnosis).
C3 as C2, but complemented by pretreatment biopsy in which, histopathologically, no
definable lesion is observed (Histopathologically proven diagnosis).
C4 Evidence following surgery and pathologically examination of the resected
specimen.
A 2–4 week interval to observe the possible regression or disappearance of a
white lesion after elimination of possible causative factors seems an acceptable period
of time for the general practitioner before taking a biopsy or before referring the
patient to a specialist for further advice. It is well recognized that the time for a ‘‘reactive’’ white lesion to regress may in some cases be much longer than the 2–4 weeks
mentioned earlier. However, it may be hazardous to just observe a leukoplakic lesion
6
REVIEW OF LITERATURE
longer than such period without having taken a biopsy or having the patient referred
for further advice.11
The sites of oral cavity affected by Leukoplakia were buccal mucosa and the
commissure area (76%), buccal mucosa and alveolar sulcus (19%), and tongue (5%).
Most of the patients suffered from ulcerative (62%) followed by homogeneous (35%)
and nodular (3%) types of Leukoplakia. Fifty-two percent of the cancer sites were
from buccal mucosa and alveolar sulcus and the remaining sites were distributed
equally among lip, tongue, retromolar area, and buccal sulcus.17
CLINICAL CLASSIFICATION:
The clinical features have been classified into;
1. The homogenous type
2. The ulcerated type
3. The specked type
The homogenous type is characterized by raise plaque formation consisting of plaques
varying in size and with irregular edges. These lesions are predominantly white, but
may have areas of a grayish-yellow color.
The ulcerated type of Leukoplakia gives the impression that ulceration has been
caused by trauma either by trauma either of chewing or of burning in case of reverse
smoking. The affected area is usually uniformly red, but yellowish areas of fibrin may
be present.
The specked Leukoplakia has the characteristic of white patches on an erythematous
base.10
7
REVIEW OF LITERATURE
CLASSIFICATION AND STAGING SYSTEM FOR ORAL LEUKOPLAKIA
(OLEP) STAGING SYSTEM:
L (size of the Leukoplakia)
L1 Size of single or multiple leukoplakias together <2 c m
L2 Size of single or multiple leukoplakias together 2–4 cm
L3 Size of single or multiple leukoplakias together >4 c m Lx Size not specified
P (pathology)
P0 No epithelial dysplasia (includes ‘‘no or perhaps mild epithelial dysplasia’’;
equals OIN grade 0)
P1 Distinct epithelial dysplasia (includes ‘‘mild to moderate’’ and ‘‘moderate to
possibly severe’’ epithelial dysplasia; equals OIN grades 1 and 2)
P‘x Absence or presence of epithelial dysplasia not specified in the pathology report
OLEP staging system
Stage I L1P0
Stage II L2P0
Stage III L3P0 or L1L2P1
Stage IV L3P1
General rules of the OLEP staging system
1. If there is doubt concerning the correct L or P category to which a particular case
should be allotted, than the lower (i.e. less advanced) category should be chosen. This
will also be reflected in the stage grouping.
2. In case of multiple biopsies of single Leukoplakia or biopsies taken from multiple
leukoplakias the highest pathological score of the various biopsies should be used.
8
REVIEW OF LITERATURE
3. For reporting purposes the oral subsite according to the ICD-DA should be
mentioned (World Health Organization, International Classification of Diseases.
Tenth Revision. Application to Dentistry and Stomatology,
(ICD-DA, Geneva,
1992).11
Various etiological factors for Leukoplakia have been mentioned in the
literature. They are Tobacco, Candida infection, alcohol, dietary and deficiency states,
syphilis, viruses and galvanism. The overwhelming majority of leukoplakias,
especially in the tropics, are associated with tobacco usage- so much so that tobacco is
considered to be the single most important etiological factor.-textbook.12
Oral Leukoplakia frequently precedes oral cancer and has similar etiologic
factors. To investigate the relationship between tobacco use and oral Leukoplakia, as
well as the role of tobacco use in the malignant transformation of oral Leukoplakia in
the European and, specifically, the central European and Hungarian context, the
following points will be discussed:
• Epidemiologic patterns of tobacco habits,
• prevalence of oral Leukoplakia,
• prevalence of smoking and oral Leukoplakia,
• effect of smoking on clinically healthy oral mucosa, and
• Evaluation of the data and conclusions.13
9
REVIEW OF LITERATURE
Pindborg JJ et al and Schepman KP et al have put forth the classification and staging of
oral Leukoplakia as: Provisional (Clinical) diagnosis:20
L Æ Extent of
Leukoplakia.
S Æ Site of Leukoplakia C Æ Clinical aspect
LO
SI
LI
No evidence of
lesion
</=2cm
L2
2-4cm
L3
>/=4cm
Lx
Not specified
S2
Sx
All sites excluding CI
floor of mouth,
C2
tongue
Cx
Floor of mouth
and/or Tongue
Not specified
Homogenous
Non homogenous
Not specified
Definitive (Histopathologic) diagnosis:
P
Æ Histopathologic features
Staging
PI,
no dysplasia
1: any L, SI, CI, PI orP2
P2,
mild dysplasia
2: any L, SI or S2, C2, PI or P2
P3,
moderate dysplasia
3: anyL,S2,C2,PlorP2
P4,
severe dysplasia
4: any L, any S, any C, P3 or P4.
Px
not specified.
PREVALENCE OF LEUKOPLAKIA:
Early descriptive studies (performed mainly in India and Denmark) have shown that
the frequency of oral Leukoplakia among smokers is so high that, in the absence of
controls, the habit could be considered as causative.
In a survey of 50,915 Indian villagers, found a prevalence of oral Leukoplakia
between 0.2 and 4.9 percent and that intraoral locations varied depending upon the
chewing and smoking habits involved.
10
REVIEW OF LITERATURE
Studies of the role of smoking in the development of oral Leukoplakia is, strong
evidence that;
• both oral cancer and oral Leukoplakia can be induced and promoted by tobacco;
• cigarette smoking shows a steep increase in the central European countries, and in
these countries the incidence and mortality from oropharyngeal cancer ranks among
the highest in the world for both men and women;
• the proportion of tobacco users (both smoking and smokeless tobacco) among
individuals with leukoplakias is high, and a relationship is evident between the
tobacco habit and the anatomical location of the Leukoplakia;
• cross-sectional studies show a higher prevalence of Leukoplakia among smokers
than among non-smokers;
• a dose-response relationship exists between tobacco use and oral Leukoplakia; and
• intervention studies show a regression of oral Leukoplakia after tobacco cessation.13
RISK OF MALIGNANT TRANSFORMATION:
Epithelial dysplasia is usually divided into three categories: mild, moderate and
severe.
Commonly used histopathological features of epithelial dysplasia
1. Loss of polarity of the basal cells
2. Presence of more than one layer of cells having a basaloid appearance
3. Increased nuclear–cytoplasmic ratio
4. Drop-shaped rete processes
5. Irregular epithelial stratification
6. Increased number of mitotic figures (a few abnormal mitoses may be present)
7. Presence of mitotic figures in the superficial half of the epithelium
11
REVIEW OF LITERATURE
8. Cellular pleomorphism
9. Nuclear hyperchromatism
10. Enlarged nucleoli
11. Reduction of cellular cohesion
12. Keratinization of single cells or cell groups in the prickle layer11.
It was suggested that the metallothionein may be a marker to moderate dysplasia
and may play a role in oral carcinogenesis14.
It has been proved that GSTM1 null genotype may increase the risk for oral
Leukoplakia development.15
The development of oral Leukoplakia is strongly associated with exogenous
exposure to carcinogens, mainly smoking, chewing tobacco and betel nut. The use of
tobacco is the most important, and has been present in 80% of the cases. Oral
Leukoplakia shows predilection for man (70%), generally up to 40 years old and may
occur as a single or multiple lesion.15
The risk factors include tobacco and excess alcohol use and diets low in antioxidants.16
Tobacco chewing and smoking has been identified as the major risk factors for oral
cavity precancer and cancer in India. Polycyclic aromatic hydrocarbons, aldehydes,
aromatic amines, nitrosamines, etc., are thought to be carcinogenic components
present in tobacco. But chewing of tobacco with betel quid increases the
concentrations of carcinogenic tobacco-specific nitrosamines and reactive oxygen
species in the mouth.17
The fact that some oral leukoplakias (OLs) undergo malignant transformation during
their clinical evolution and that white lesions are found on the borders of some oral
squamous cell carcinomas lends support to the concept that OL is a potentially
12
REVIEW OF LITERATURE
malignant lesion. Those patients with OL associated with oral cancer presented with
tumours at a less advanced stage than those where no associated Leukoplakia
existed.18
13
REVIEW OF LITERATURE
ORAL CANCER:
Carcinoma of the oral cavity is one of the most frequent malignant tumors
worldwide, with major predominance in South-East Asia and India. In India, cancer
of the oral cavity is one of the five leading sites of cancer and accounts for 19% of
the total cancer cases in men and 7% of that in women.21, 34, 39
Among the oral tumors, 90% are squamous cell carcinoma (SCC), which
arises from the mucosal lining. This high incidence of oral cancer (OC) in India is
due to the widespread habits of tobacco chewing, betel nut chewing and smoking and
alcohol.27, 32,33,34,39
The clinicopathological profile of Indian oral cancers shows significant
differences from the oral cancers in several developed countries of the world,
including the USA, UK, France and Japan, where it is associated with tobacco
smoking with/without alcohol consumption. A variety of tobacco habits are prevalent
in India and they differ from region to region.39
Many people extensively use smokeless tobacco in the form of nass, naswar,
khaini, mishri, pan masala, gutkha, betel-quid (betel leaf coated with slaked lime
wrapped around areca nut and catechu) or smoke tobacco in the forms of bidi, chutta,
the reverse type of smoking and hooka.39
There are about 300 carcinogenic compounds present in tobacco out of which
tobacco-specific nitrosamines have been identified as the most important carcinogens
in tobacco. The other carcinogenic compounds in tobacco metabolites are polycyclic
aromatic hydrocarbons, a-particle-emit-ting
210
Po (polonium), trace metals, carbon
monoxide, hydrogen cyanide, and phenols.39
In India, the buccal mucosa (cheek) is the primary site for cancer
development as against the tongue and the floor of the mouth in Western countries,
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REVIEW OF LITERATURE
which may be due to the habit of keeping the betel-quid and tobacco in contact with
the cheek for a long time.39
CLINICAL CLASSIFICATION: 20
The staging of oral cancer has been given based on the size, lymphnode involvement
and the presence or absence of metastasis.
MÆ Distant
TÆ Size of the NÆCervical
Lymphnode metastasis metastasis
primary tumor
Tls: carcinoma in B NO: no node
situ
involvement
detected
Tl: tumor <2cm
Nl: single ipsilateral
T2: tumor >2cm
:
node<3cm
and<4cm
T3: tumor>4cm
T4: tumor >4cm
with invasion of
adjacent structures
(ie, through cortical
bone, deep into
extrinsic muscles of
tongue, maxillary
sinus, and skin)
MO: no known
metastases
Staging:
Stage
T1,N0,M0
Ml: metastases
Stage
present
T2,N0,M0
1:
2:
N2a: single ipsilateral
H:
node <6cm
Stage 3: T3,N0,M0
Tl,T2orT3,Nl, MO
N2b: multiple
ipsilateral nodes >3cm
and <6cm
Stage 4:
T4. any N.M0
N2c: bilateral or
contra lateral
AnyT5N2orN3, MO
Any TorN with Ml
lymphnode
<6cm
N3a: ipsilateral
node >6cm
N3b: bilateral nodes
Xicm
The TNM classification of tumor of oral cavity.21
Assessment of the size of the primary lesion, the number of regional lymph node
metastasis and the presence of distant metastasis (TNM) are widely used to define the
extent of tumour load and determine treatment options for patients with Oral
squamous cell carcinoma (OSCC). One of the criticisms of the TNM system is that it
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ignores individual histological characteristics of tumours, and it has been proposed
that a combined assessment of histological grading as well as clinical staging might
provide a more precise measure for predicting the outcome of the neoplasms and
deciding the best treatment for each patient.21
Brynes’s IFG system: 21
Morphologic
feature
score
1
Degree of
Highly
Keratinization keratinized
(> 50% of
cells)
Nuclear
Little nuclear
polymorphism polymorphism
(>75% mature
cells)
Number of
mitosis
(high power
field)
Pattern of
invasion
0-1
2
Moderately
keratinized
(20-50% of
cells)
Moderately
abundant
nuclear
polymorphism
50-755
mature cells)
2-3
3
Minimal
keratinization
(5-20% of
cells)
Abundant
nuclear
polymorphism
(25-50%
mature cells)
4
No
keratinization
(0-55 of cells)
4-5
>5
Pushing, well
delineated
infiltrating
borders
Infiltrating,
solid cords,
bands and/or
strands
Small groups
or cords of
infiltrating
cells
Marked and
wide –spread
cellular
dissociation in
small groups
and / or in
single
cells(n<15)
None
Host response
Marked
Moderate
Slight
Extreme
nuclear
polymorphism
(0-25%
mature cells)
In OSCC, the presence of tumour metastasis in regional lymph nodes at presentation
is the most significant adverse prognostic factor and a major determinant of poor
survival and precise indicators to predict local recurrence and/or metastasis might be
very valuable.21
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Significant association present between the pattern of invasion of the tumour
and metastasis of the tumour to regional lymph nodes. Tumours that invaded in
small groups or with widespread cellular dissociation showed a higher tendency to
metastasize to regional lymph nodes compared with tumours that invaded in pushing
fronts or in solid cords and/or strands. These ‘aggressive’ patterns of invasion might
imply that tumour cells have already reached a stage in their evolution where they
exhibit the molecular events that permit metastasis, such as the expression of cellular
receptors and the production of enzymes to facilitate their access to lymphatic
vessels.21
There was also a significant association between the degree of keratinization
and lymph node metastasis. Patients with tumours that showed minimal or no
keratinization had significantly higher lymph node metastasis compared with patients
with tumours that showed high or marked degree of keratinization. Evaluation of and
specific comment on the degree of keratinization and/or the pattern of invasion in
diagnostic biopsies could help in planning the extent of surgical treatment.21
Angiogenesis or neovascularization has long been known to aid progression and
metastasis of malignant tumors22. This biological process is complex and mediated by
several stimulators, for example fibroblast growth factor (FGF), transforming growth
factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, and vascular endothelial
growth factor (VEGF), and inhibitors, for example angiostatin, platelet factor IV
(PF4), and thrombos-pondin-1 (TSP-1; 3). Since angiogenesis increases with disease
progression in several malignant tumors including malignant melanoma, breast
cancer, and oral SCC.Potentially, angiogenesis can be used as an independent
prognostic marker in human cancers.22
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A significant correlation between the MC and micro vascular counts in oral SCC.
Angiogenesis as well as the number of MCs appeared to show a linear increase from
normal oral mucosa, hyperkeratosis, and pre-malignant dysplasia to invasive SCC.
These findings suggest that MCs may up regulate angiogenesis in oral squamous cell
carcinogenesis, perhaps via the release of MC tryptase. Further, the numbers of MCs
and micro vessels may be used as indicators for disease progression.22
HPV 16/18 infection or/and over expression of p53 protein play an important role in
developing some OSCCs. And the presence of HPV sequences and mutant p53 are not
necessarily mutually exclusive23. HPV is probably unimportant in the pathogenesis
of OSCC37.There are conflicting data on the potential role and incidence of HPV in
oral carcinogenesis.38
It has been demonstrated that the tumor size, histologic differentiation, and the
mode of carcinoma invasion correlate with patient prognosis and tumor metastasis.
Furthermore, MCM2 LI provides useful information in predicting patient prognosis
with oral SCCs.24
Considerable progress has been made in understanding the genetic basis of the
development of oral squamous cell carcinoma.25
Early genetic changes do not necessarily correlate with altered morphology.25
The presence of a field with genetically altered cells is a risk factor for oral cancer.
The presence of a large number of pre-neoplastic cells is likely to increase the risk for
another cancer. Detection and monitoring of a field at risk and the development of a
targeted molecular intervention may have profound implications for oral cancer
prevention.25
The initiation of oral SCC, increasing vascularity is observed at the periphery of
the tumor. As the tumor continues to grow, further increases of intratumoral
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vascularity and the presence of tumor-lined vessels are associated with cancer
progression.26
The co-expression of p53 and Ki67 may play roles in carcinogenesis of OSCC and
p53 over expression may promote cell proliferation in OSCC. Furthermore, EBV does
not appear to be a risk factor for OSCC particularly in the population of northern
Thailand.27
OC3 cell line could be valuable in understanding the genetic impairments and
phenotypic changes associated with areca in oral keratinocytes.28
The OC3 is the first documented OSCC cell line derived from a primary tumor in
an areca-chewing patient without exposure to tobacco. It will be an exploratory tool in
identification of the pathogenetic impacts of areca through, the NF-kB pathway.28
Formation of Micronuclei in the Eukaryotic cells is an end point of chromosomal
damage or segregation errors. The presence of micronuclei reflects a genotoxic or
carcinogenic exposure. The study concluded that a gradual increase in micronuclei
frequency from normal to precancerous to cancerous lesions. The MN frequency was
increased in preoperative cancer cases and decreased in postoperative cases.29
The pro-inflammatory cytokine tumor necrosis factor-a (TNF-α) is a central
mediator of the immune response involved in a wide range of immuno-inflammatory
and infectious diseases and is the first report that the TNF-α polymorphism is
associated with the risk for OSCC.30
The study concludes that vascular distribution and properties of endothelial cells
appear to be closely associated with metastasis.31
The study suggests that the process of repeat blood and lymph vessel destruction
and neogenesis with the invasion of cancer in the oral cavity helps to maintain the
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tumor. The inhibitors of angiogenesis and lymphogenesis may lead to novel
therapies.32
The clinicopathological features of OSCC <40 years of age did not show different
from older population. For all age groups, clinical stage and the pattern of invasion
were the most significant prognostic factors.33
The observation suggest the possibility of that cellular immune response in
particular T-cell function, plays a key role in developing lymph node metastasis and
the mean PA% is a useful prognostic factor in predicting survival in patients with
SCC of the oral cavity.35
HIF-1α has an important role to play in pathophysiology of oral cancer, both under
normal and hypoxic conditions, the pharmacological manipulation of HIF-1α has
marked effects on tumor growth, and it could prove to be an important target for drug
therapy, both in oral cancer and in other hypoxia- dependent disease states.36
The study showed that bFGF and FGFR-1 expressions in fibroblasts at the invasive
front are linked to the mode of invasion and the prognosis in OSCC.40
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SIALIC ACID:
The name Sialic acid comes from the Greek word Silon (Saliva), consistent
with the discovery of these carbohydrates in bovine sub maxillary mucin and brain
matter (neuroamine) by the German biochemists Blix in 1936 and Klenk in 1941.41
Although Blix in 1938 assumed the presence of ‘Sialic Acid’ in certain of
serum proteins, it was not until 1955 that Bohm and Baumeister, by preparing a
crystalline derivative from human serum, established the existence of Sialic acid as a
component of the prosthetic groups of the serum proteins.
The intensive study of the Sialic acids and closely allied substances isolated
from a variety of animal tissues indicated that these in fact formed a family of
compounds with a common structure which may be considered to be the product of N
acetylhexosamine and pyruvic acid. To avoid confusion Blix, Gottschalk, and Klenk
in 1957 have suggested that the primary non-acetylated compound should be used as a
collective term for acetyl and glycocyl derivatives of neuraminic acid.
It is generally considered by Gottschalk in 1960 that human serum contains
no free Sialic acid and that 90% of the serum Sialic acid is bound is bound to the α
and β globulins. Moreover, the only member of the ‘Sialic’ family so for identified in
human serum is the N acetyl derivative of neuraminic acid.42
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H
O
Ac-NH
COO¯
CHOH
|
CHOH
|
CH20H
H
H
OH
OH
H
STRUCTURE OF SIALIC ACID43
Molecular weight of Sialic acid (C11H9O9)-309.2744
The study aimed at evaluation of Serum Sialic acid in health and disease by
Svennerholm using standard containing 18% N acetyl neuraminic acid and observed
that increase in serum Sialic acid levels are usually associated with an increase in
circulating serum proteins normally rich in Sialic acid. Two classes may be
exceptions to this statement, namely, those nephrotics in whose sera high Sialic acid
levels are found and the β myelomata. The 19S component of the Sialic acid there is
no substantial difference between the Sialic acid content of these globulins in health
and disease.42
Several techniques have been developed for the determination of Sialic acids
like the tryptophen and perchloric acid (Siebert, 1948), diphenylamine (HCSS, 1956),
the Bial and direct Ehrlich Test (Warner & Odin, 1952), Sulphuric acid – acetic acid
mixtures (Hess, Coben, Bates 1957), Resorcinol (Svennerholm, 1958), Orcinol
22
REVIEW OF LITERATURE
(Reichl (1879) and Thiobarbituric Assay (warren, 1959), all requiring heating the
sample with strong acids to bound SA to their free forms.45
SA is acetylated derivatives of neuraminic acid. They are attached to non
reducing residues of the carbohydrate chains of glycoproteins and gylcolipids.The
suggested biological functions of SA include: (a)stabilizing the confirmation of
glycoproteins and cellular membranes; (b)assisting in cell to cell recognition and
interaction; (c)contributing to membrane transport; (d)affecting the function of
membrane receptors by providing binding sites for ligands; (e)influencing the
function, stability and survival of blood glycoproteins; and (f) regulating the
permeability of the basement membrane of glomeruli.72
A protein bound carbohydrate, the Sialic acid in serum values were estimated
in healthy Indian adults by both Diphenylamine method and Thiobarbituric acid
method. Only N-acetyl neuraminic acid has been isolated from the human serum. The
average values were 68±2.6 mg percent by DPA and 56±5mg percent by TBA
method. Age and sex showed no influence on Sialic acid levels in Serum.46
A Study was done to determine Sialic acid levels in malignancy by using two
known Biochemical methods (DPA and TBA).It was inferred that there was
significant rise in Sialic acid levels in malignancy. It was also concluded that the
Sialic acid levels was significantly more in metastasis case than without metastasis.47
The study was undertaken with the view to establish any significant
relationship between the level of Sialic acid present in the serum and the activity of
the disease process and also to find out if its estimation can be of the use in the
diagnosis and prognosis of different bone disorders. The values of serum Sialic acid in
different bone disorders were much more above the normal values in tuberculous and
pyogenic infection of the bones and joints, Rheumatoid arthritis and malignant bone
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REVIEW OF LITERATURE
tumors i,e Osteogenic sarcoma, Osteoclastoma, Chondrosarcoma, Adamantinoma,
Multiple myeloma and secondaries. Serum Sialic acid level in healthy Indian children
and adults are independent of sex but higher in adults.48
A periodiate resorcinol method for the Quantitative estimation of free Sialic
acids and their glycosides was undertaken which offers a number of advantages for
detecting and determining the Sialic acids. First under experimental conditions,
substances generally associated with the Sialic acids, such as sugars, lipids and
aminoacids do not react with nor interfere with the method. Second prior acid
hydrolysis, a prerequisite for analysis by the thiobarbituric acid procedures is not
required for the determination by the periodiate resorcinol method. Third, the method
can be used to determine free, glycosidically bound, or total Sialic acid. Fourth, the
present procedure is 3 to 6 times more sensitive than the standard or chromatographic
resorcinol procedures and thirteen times more sensitive than the specific enzymatic
and Thiobarbituric procedures. Micro quantities of the Sialic acids or their glycosides
can be detected on paper chromatograms.49
An automated method based on the Svennerholm resorcinol method was
successfully used to determine glycosidically bound Sialic acid and free Sialic acid.
This method is directly applicable to sera without any previous treatment which is a
considerable advantage.50
A comparative study of estimation of Serum protein bound hexose, methyl
pentose (fucose), Sialic acid, hexoseamine and seromucoids have been estimated in
patients of Ischemic heart disease between age group 40 and 50 years and adult age
and sex matched, male volunteers of the armed forces personnel with the normal
controls and proposed that raised Glycoproteins may form the earliest artherogenic
index.51
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The work was to investigate the potential utility of protein bound
carbohydrates Mannose, Galactose, Fucose as biochemical markers of breast cancer
and to guide chemotherapeutic treatment in maximizing their destruction. The result
did show a good relations of increasing protein bound fucose with progressive breast
cancer and decreasing fucose with responsive malignancy. Changes in protein bound
Sialic acid; mannose and galactose show only limited correlation with the clinical
status of the malignancy. Of necessity, because of a delay in developing methodology,
the samples which were analysed for Sialic acid had previously been thawed analyzed
for fucose, mannose and galactose, then refrozen. Although the effect of one freezethaw cycle on Sialic acid analysis seems relatively small, it would be desirable to
perform all analyses at the time the sample was first thawed.52
A study was conducted to determine the Level of glycoprotein- associated
carbohydrates(neutral hexoses, hexoseamine, Sialic acid and fucose) in the serum of
patients with either local, regional and metastatic cancer, patients clinically cured of
cancer, and controls(smokers and non-smokers) and compared with normal serum
glycoproteins, carcinoembryogenic antigen(CEA), and with lymphocytic activity to
phytohemagglutinin(PHA).Tumor burden was directly related to protein- bound
carbohydrate levels in patient groups. Levels of bound carbohydrates reflect the sum
of all the changes in serum glycoproteins, but primarily changes in the acute phase
proteins (α1-acid glycoprotein, α1-antitrypsin, haptoglobin, and ceruloplasmin) found
in the α-globulin fraction of serum. Increase in protein bound carbohydrates in tumor
bearers were not related to increase in CEA. Increased levels of the acute phase
protein occurred in individuals in depressed in vitro lymphocyte reactivity to PHA. A
significant positive correlation was found between lymphocyte reactivity and levels of
α2HS-glycoprotein.The results suggest that serum protein-bound carbohydrates or
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glycoproteins may be of adjunctive value in assessing tumor burden and immune
reactivity in cancer patients.53
A study was done to determine serum Sialic acid level in malignant
melanoma and association with tumor burden and concluded that increased serum
Sialic acid could prove to be a valuable clinical monitor of change in tumor burden as
a result of therapy or change due to recurrence of disease following treatment.54
The study was emphasised on the circulating levels of Glycoprotein in serum
of Human cancer. Patients with either localised or advanced metastatic malignancy
have significantly elevated mean serum values. The highest levels occur in patients
with lung, GI, Gynaecologic cancer, lymphoma and malignant melanoma. Patients
with leukaemia and multiple myeloma have slightly elevated values, but they were
not significantly different from. Patients following curative surgery have normal
values while patients in clinical remission following chemotherapy have elevated
mean serum protein and NANA levels. Elevated levels were also occurred in patients
with benign tumors and 12% of patients with non malignant disease. Tumor cells
appear to shed macromolecules which contribute to the observed elevation of serum
protein and Sialic acid levels.55
A study was done to correlate the Glycoprotein between circulating level and
disease status and to correlate NANA levels with response to therapy. In the
metastatic disease, after debulking surgery had a drop in their serum NANA value to
normal levels following a transient rise in the intermediate postoperative period.
Chemotherapeutically treated cases showed tumor regression or stable disease and
drop in their serum NANA level. In metastatic progression cases showed rise of
NANA in serum. But there was no rise of NANA with local relapse of the disease(two
chest wall, one lung mass and brain metastasis) and concluded that serial serum
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glycoprotein titres deserve further consideration as a monitor of response to
chemotherapy of metastatic disease.56
The study was taken up to examine the potential diagnostic value of serum
NANA in malignant melanoma and compare this with other suggested predictors of
tumor recurrence in patients with stage I disease and stage II lymphatic extension and
inferred that patients with lesions > 1.75mm and NANA <2µmol/ml have a more than
12-fold greater risk of recurrence by 2 years than those with lesions ≤ 1.75mm and
NANA ≤µmol/ml.57
The study was undertaken to determine the potential clinical application of
Sialic acid in the evaluation of the extent of neoplastic disease and its response or lack
of therapy in a group of patients with advanced malignancy of a variety of histology
types. It has been demonstrated that serum NANA correlates both with disease
progression and remission. They emphasise the importance of serial measurements in
evaluating a patient’s course. The changes in serum NANA appear to be related to the
overall changes in tumor mass i,e, minor responses were associated with very small
changes in NANA, while tumor progression and partial remission demonstrated larger
changes and inferred as a biologic marker for malignant disease.58
A simple procedure is described for the detection of Sialic acid in serum.
After a direct addition of Ehrlich reagent to serum and incubation at 560C for eight
hours, the resulting mixture is diluted with saline. After centrifugation, the colour in
the supernatant is determined at 525nm in a spectrophotometer. Serum Sialic acid was
significantly greater in cancer patients than in normals. Cancer patients with
metastases had significantly greater in cancer patients than in normals. Cancer
patients with metastases had significantly greater Sialic acid than cancer patients
without metastases. In two cancer patients, Sialic acid levels returned to normal after
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surgery. The diagnostic usefulness of 95.6% was similar to that report with a limited
spectrum of tumor. However, Patients with inflammatory diseases such as arthritis,
Crohn’s disease and psoriasis also showed elevated Sialic acid levels.59
A modification of the method reported by R.J.Shamberger for detection of
serum Sialic acid is described. The whole procedure takes less than one hour. It is
based on the release of bound Sialic acid by heating with 5% perchloric acid. After
cooling and brief centrifugation, the supernatant is heated for 15 min with Ehrlich’s
reagent at 1000C. Thereafter the absorbance of the developed in the sample is
measured at 525nm. The values obtained were within the estimated serum values of
different methodology.60
A study was conducted to evaluate use and limitation of serum Total and
lipid-bound Sialic acid concentrations as marker for Colorectal cancer by high
performance liquid chromatography method and compared to carcinoembryogenic
(CEA) as markers for colorectal cancer. Significant elevation of NANA and LSA was
found compared to CEA. NANA and LSA showed as promising supplemental
markers for staging and monitoring colorectal cancer and could be used as a valuable
marker in patients whose initial tumors donot produce CEA.61
A prospective study was done to evaluate the roles of serum NANA and
LSA concentration in the detection and staging of cancer and the follow-up of
treatment in patients with genitourinary malignancies including prostate cancer,
bladder cancer, renal cancer, urologic diseases and compared with normal volunteers.
The results showed a low incidence of elevated values in patient with early stages of
cancer and a high incidence of false positive values in serum NANA concentrations in
patients with benign urologic diseases, especially prostatitis. Serum NANA and
LASA concentrations were highly correlated with the stage and grade in patients with
28
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advanced urologic cancer, and may be used as markers of tumor activity during
follow-up under treatment.62
A study was conducted for Biochemical marker for Tumor diathesis and
early cancer and indicated that 1) serum α-1 acid glycoprotein and Sialic acid were
increased in lung cancer. But 20% pulmonary tuberculosis patients were also
positive.2) Serum polyamines determined by Radioimmunoassay (RIA) were
increased in cancer patients. The positive rates for cancer of lung and oesophagus
were 84% and 100% respectively. However, whether serum polyamine can be used
for early detection of oesophageal cancer awaits further studies. 3) An unknown
fluorescent compound in urine found in normal people but was very much decreased
in cancer patients. This compound showed cystostatic effect on tumor cells in vitro. 4)
Serum antibodies against EBV associated DNAse could be used as a marker for
Nasopharyngeal carcinoma (NPC).63
Study was conducted for management of patients with Gynaecologic cancer
by serum Sialic acid determination by an enzymatic technique and inferred that the
Sialic acid level became larger in ascending order of uterine myoma, benign ovarian
cancer, cervical cancer, corpus cancer and ovarian cancer. The SA level proved to be
significantly higher in cancer patients showing poor prognosis than those having good
prognosis irrespective of the category of therapy and it reflected well the clinical
course of cancer patients. In patients who receive a combination therapy and need to
be followed up for a long term, complete follow-up is sometimes impossible with
tumor-derived markers alone. Even in such cases, the Sialic acid level will work as a
useful follow-up marker because it is non-specific to histologic types of cancer.64
The study was carried out for usefulness of Serum Glycoconjugates in
Precancerous and cancerous diseases of the Oral cavity. These results suggest that
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combined evaluations of biomarkers like TSA, LSA, mucoid proteins and PBH may
provide useful biochemical indices for clinical assessment of the spread and
invasiveness of disease in oral cavity malignancy and is important in monitoring the
large population of tobacco and arecanut chewers who may develop OC, the
predominant types of cancer among men in India. In addition to their value in early
detection, they can also help in staging of the disease.8
The study was aimed at determining the sensitivity and specificity of serum TSA and
LSA as markers for carcinoma of the Uterine cervix. The result showed TSA
concentrations in patients with cervical cancer were found to be correlated with
advanced-stage disease. LSA concentrations were only in stage IV of the disease and
concluded that measurement of TSA or LSA appear to be of no value for the early
detection of cervical cancer or to complement the clinical staging of this tumor.65
The study was to asses individual and combined usefulness of serum levels of
TSA, LSA, and LDH in diagnosis and management of patients with cervical
carcinoma highly specific spectrophotometric methods. The levels of all markers were
significantly higher in untreated cervical cancer patients compared to the controls.
TSA was found to be the most sensitive (90.74%) marker for diagnosis of cervical
cancer. Combined use of the markers revealed maximum (100%) sensitivity. In
comparison between early (stage I + II) and advanced (stage III + IV) malignant
disease, the markers showed significant changes. TSA and LSA values in patients
who did not respond to radiotherapy were significantly higher than those of the
responders. The result suggests that combined evaluation of the markers is helpful for
diagnosis as well as treatment monitoring of cervical carcinoma patients.66
The study was under taken to evaluate the relative utility of TSA, LASA, TP,
TSA/TP factor for detecting and monitoring the progression of cancer stomach,
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breast, colorectal region and gall bladder and their reliability as cancer marker in these
diseases. A significant rise in TSA and TSA/TP factor and a decrease in serum TP
levels were found in all cases of cancer. This increase was directly proportional to the
degree of metastases. i.e., tumour burden. Serum TP levels were found decreased in
all patients. Rise in LASA levels was not significant. Raised TSA/TP levels became
lower to certain extent after effective therapy. Hence it can be inferred that all the
TSA and TSA/TP are not specific, they are however sensitive markers regarding
detection of malignancy and useful in monitoring cancer progression and/or recurrent
while evaluating the efficacy of various cancer therapies.67
The concentration of serum total Sialic acid (S-TSA) is risk marker for
cardiovascular mortality and atherosclerosis. Since the mortality from coronary heart
disease is higher in the united states than in Japan. One could expect the S-TSA to be
higher among Caucasian US citizens than among Japanese citizens, a hypothesis that
is tested in this study. And concluded that higher S-TSA level in Caucasians living in
Minneapolis compared to Japanese living in Akita, Japan is in concordance with the
higher cardiovascular mortality in the US. Differences in S-TSA levels may reflect
international differences in the prevalence of atherosclerosis.68
The study was conducted accepting serum Sialic acid as a reputed
cardiovascular risk factor, and its relationship to lipids, plasma fasting insulin, blood
pressure and body mass index in normal individuals. In females there was a strong
univariate correlation between serum total Sialic acid and plasma fasting insulin and
glucose concentrations, although in males there was a weaker univariate correlation
between serum total Sialic acid and fasting plasma glucose and the insulin resistance
index. In addition, serum total Sialic acid significantly correlated with systolic blood
pressure, fasting serum cholesterol and triacylglycerol concentrations and body mass
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index in the females. In males serum total Sialic acid significantly positively
correlated with fasting serum cholesterol and triacylglycerol concentrations and
correlated inversely with the hip/waist ratio. The study concludes by accepting further
research to explore serum TSA as a reputed cardiovascular risk factor due to lack of
relevant factors for varience.69
The role of Sialic acid in the neoplastic process prompted to investigate the
TSA, LSA, TP and TSA/TP values of plasma in BCC and AK. Data analysis
indicated a significant increase in TSA and TSA/TP values in the BCC and AK
(pathologic controls) group when compared with the healthy controls. When AK and
BCC groups were compared, no difference between TSA, LSA,TP and TSA/TP
values were observed. No significant difference was observed between the groups in
terms of LSA values. The results indicate that TSA and TSA/TP along with other
clinical and histopathological criteria may be valuable in establishing diagnosis of
BCC.70
The study was conducted to investigate the circulatory levels in serum TSA, LSA
and fucose in Precancerous lesion and cancer of the Oral cavity and noticed
significant elevation in the serum levels of TSA and LASA in patients with the
precancerous and cancer lesions when compared with the controls. Serum TSA levels
were elevated significantly in patients with cancer when compared with those with
precancerous lesions. Circulating TSA and LASA levels were found to reflect tumor
burden and correlated well with stage of the disease. However, serum fucose levels
did not show an increase corresponding to stage of the disease. The result suggests
that combined evaluation of these markers may be useful in predicting early
malignant change and also in assessing the spread and invasiveness of the disease in
cancer of the oral cavity.6
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The study was conducted to find out the relationship between Sialic acid and
nephropathy in type 2 diabetes mellitus (DM). The four groups according to the
presence or absence of type 2 DM and degree of albuminuria were; Group A- patients
with DM and no albuminuria; Group B- patients with DM and microalbuminuria;
Group C- patients with DM and albuminuria; and Group D – control group. The
following parameters were assessed: body mass index, lipidemic profile, creatinine
clearance, Sialic acid, presence of coronary artery disease and presence of diabetic
neuropathy. Type 2 DM patients had higher levels of Sialic acid compared to the
control group. Among diabetic patients, those with signs of nephropathy (groups B
and C) had higher levels of Sialic acid than DM patients without the signs of
nephropathy (group A). Sialic acid showed a strong negative correlation with
creatinine clearance in diabetic patients with albuminuria. It is concluded that Sialic
acid could be used as a marker of renal dysfunction in various stages of diabetic
nephropathy.71
The study was aimed to determine reference values for serum Sialic acid in
women and men among well characterised healthy population in a random sample and
showed that blood pressure and serum SA are associated in both in women and men
independently of age and BMI. This implicates one mechanism behind the earlier
observation about the association between SA and cardiovascular risk. A possible
explanation of these findings is that serum SA reflects acceleration of the
atherosclerotic process by increased blood pressure which is a known independent
risk factor for atherosclerosis. In women, age and the use of contraceptive pills are
factors increasing serum SA. In men smoking increases serum SA. The biological
mechanism(s) causing increased serum SA in various diseases including
cardiovascular disease are far from clear. Furthermore, because contraceptives,
33
REVIEW OF LITERATURE
smoking, BMI, and other factors, including menopause, increase SA, it is difficult to
give reference values of serum SA separately for different age groups. On the other
hand, the present study supported the conclusion that SA is relative stable among men
25-64 years of age and among women 25-54 years of age.72
The study was aimed to evaluate the total Sialic acid (TSA) content in
endometrial cancer tissue in relation to normal and Hyperplastic human
Endometrium. TSA content was significantly higher in endometrial cancer than in the
control tissue obtained from women in both the proliferative and the secretory phase
of menstrual cycle. Tissue TSA content was higher in the secretory phase than in the
proliferative phase though the differences were not significant. This supported the
view that endometrial cancer is associated with the increasing content of SA in the
tumor tissue.73
The study was conducted to analyse the diagnostic value of the novel tumor
markers α-L-fucosidase activity (AFU) and TSA in HNSCC. And compared the
results for these novel markers to those of SCC antigen, CYFRA 21-1 and CEA,
paying special attention to earlier TNM stages of the carcinoma. Finally the
combinations among these entire tumor markers have been analysed to asses the
possible improvement of the diagnostic sensitivity using a panel of tumor markers for
HNSCC. The results concluded that the combined use of AFU and TSA/TP with
CYFRA 21-1 may be useful in predicting early malignant transformation, thus
improving the dictation rates of premalignant lesions of head and neck and HNSCC at
early stages.5
The study was to investigated the levels of expression of Sialic acid and Nacetlygalactosamine reduces on the cell surface of a normal intestinal epithelial cell
line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic
34
REVIEW OF LITERATURE
potential Caco-2 and HCT-116. The study demonstrated that the level of expression
of WGA-and HPA-binding glycoproteins varied among the three cell lines used and
appeared to be associated with the origin of the cell types. On the basis of these
observations it is possible to suggest that WGA-binding but not HPA binding, appears
to be associated with metastatic potential, at least in these cell lines and the results
support the concept that cell membrane sialylation is important in determining the
metastatic potentials of cancer cells.74
The relationship between Sialic acid and metabolic variables in Indian type 2
diabetic patients was evaluated in this study and found that elevated serum and
urinary Sialic acid and micro albumin concentration were strongly related to the
presence of micro vascular complications like diabetic nephropathy and retinopathy
and cardiovascular risk factor in Indian type 2 diabetic subjects.75
The study was undertaken to findout the serum total Sialic acid levels in
smokers and users of smokeless tobacco in the form of oral powder (Maras powder)
and compared with the healthy controls. The TSA concentrations were significantly
higher in the sera of smokers and Maras powder users than those of control subjects.
The mean serum TSA level was found to be lowest in the control group and highest in
the mars powder users. But, there was no significant difference in serum TSA levels
between smokers and maras powder users. The results concluded that serum TSA was
affected by ST use as seen in smokers. This finding may be an indication of harmful
effects of ST use as maras powder as well as cigarette smoking.76
The study was carried to find out Total and Lipid bound serum Sialic acid
levels in cancer patients with different primary sites and differing degrees of
metastatic involvement and indicated significant increases in the mean TSA and
TSA/TP values in the cancer patients and in the pathologic controls (non malignant
35
REVIEW OF LITERATURE
diseses) when compared to the normal controls and a significant decrease in the mean
TP values in the cancer patients and pathologic controls when compared to normal
controls. No significant difference was observed between groups in LASA values.
And concluded that TSA, TSA/TP was the most useful of the markers tested for
detecting malignancies and monitoring therapeutic intervention and disease
progression.77
The investigation was aimed at determining the clinical significance of TSA
and LSA in Oral precancerous conditions and Oral cancer. The investigative data
revealed significant elevations of TSA and LSA in untreated oral cancer patients as
compared to healthy individuals as well as patients with Oral precancerous conditions
and suggested potential utility of these parameters in diagnosis as well as determining
clinical stage of the malignant disease.7
The study was intentionalised to investigate towards tissue and serum α2-3and α2-6-linkage specific sialylation changes in oral carcinogenesis. The findings
suggest that increased sialylation of tissue and serum glycoproteins in patients with
oral cancer and OPC are associated with oral cancer development and treatment
monitoring of the patients. The increased levels of α2-6- and α2-3-sialoproteins and
their enzyme activities are found to be associated with various stages of neoplasia.
The data also suggests that malignant transformation in oral epithelium is
accompanied by α2-3-Sialylation, whereas, α2-6-sialylation may be associated with
disease progression and metastatic potentials. These results also provide a foundation
for in depth studies in to the role of sialylation-associated early changes in OPC cells.4
36
MATERIALS AND METHODS
MATERIALS AND METHODOLOGY
SOURCE OF THE DATA:
This study was carried out in Department of Oral Medicine and Radiology,
Vokkaligara Sanga Dental College and Hospital, Bangalore and Kempegowda
Institute of Medical Sciences, Bangalore.
METHODS OF SELECTION OF DATA:
I. Sample Size:
1. Total number of subjects: 75
2. Patients with Leukoplakia: 25
3. Patients with Squamous cell carcinoma: 25
4. Age and sex matched controls for comparison of results: 25
II. Selection of Cases:
a. Inclusion Criteria:
1. 25 patients of Oral Leukoplakia (Histopathologically proven)
2. 25 patients of Squamous cell carcinoma (Histopathologically proven)
b. Exclusion Criteria:
Patients were referred to General physician for opinion to evaluate for any systemic
disease status like diabetes mellitus, Ischemic heart disease, different bone disorders
like pyogenic arthritis, Rheumatoid arthritis, malignant bone tumors.Subjects with any
of the above mentioned diseases were excluded from the study.
III. Selection of Controls:
Included age and sex matched 25 healthy individuals with the same exclusion
criteria as that of selection of cases.
37
MATERIALS AND METHODOLOGY
SAMPLE COLLECTION, STORAGE AND HANDLING:
1. The subject is seated comfortably with the arm supported. Aseptic measures
are used and tourniquet is applied 2 inches above the elbow of the upper arm.
The site of the puncture is cleaned using sterile gauze dipped in 100% alcohol.
Using a 5ml syringe with the needle size of 0.55 x 25mm. 5ml of blood is
drawn from the anticubital vein.
2. The blood is allowed to clot and the serum separated by centrifugation.
3. Serum Sialic acid is estimated through a simplied quick method from
G.Sydow.
MATERIALS REQUIRED:
1. Sialic acid Powder (Only for working Standard solution)
2. Ehrlich’s Reagent (p-dimethylamino benazaldehyde solution)
3. 5% perchloric acid (HClo4 solution)
4. Adjustable Micropipette of 1000µl.
5. Centrifuge
6. Incubator
7. Spectrophotometer
ESTIMATION OF SERUM SIALIC ACID:
PRINCIPLE:
Free Sialic acid in serum reacts with Paradimethyl aminobenzaldehyde
(Ehrlich’s Reagent) to form a pink colored solution. The absorbance of the color
38
MATERIALS AND METHODOLOGY
developed in the sample at 525nm is proportional to the total Sialic acid
concentration in the serum.
STOCK STANDARD SOLUTION:
25 mg of SA powder was measured by physical balance and was dissolved in
10 ml of deionized water. The dissolved solution was transferred to a 25 ml
volumetric flask. Water was added to make up the volume up to 25ml.
(conc:1mg/ml).
WORKING STANDARD SOLUTIONS:
1. 100mg% solution:
The concentrated solution of 100 mg % was prepared by pipetting out 5 ml
of stock solution via micropipette (1000µl x 5).
2. 80 mg% solution:
4ml of primary standard solution was pipetted out from remaining 20ml of
stock solution into a second conical test tube. 1 ml of deionized water was added
to it. Therefore 4mg of SA is present in 5ml solution concentration=80mg%
3. 60 mg% solution:
3ml of stock solution was pipetted out from remaining 16 ml of stock
solution in to third conical test tube. 2ml of deionized water was added to it.
Therefore 3mg of SA is present in 5 ml solution concentration = 60 mg%
4. 40mg%solution:
2ml of stock standard solution was pipetted out from remaining 13 ml of
stock solution in a fourth conical test tube. Therefore, 3ml of deionized water was
added
to
it.
Therefore
2mg
of
concentration=40mg%.
39
SA
is
present
in
5ml
solution
MATERIALS AND METHODOLOGY
5. 20 mg% solution:
1ml of stock standard solution was pipetted out from remaining 1ml of
stock solution in to fifth test tube. 4mg of deionized water was added to it.
Therefore 1mg of SA is present in 5ml solution concentration = 20mg%
PROCEDURE:
Simplified Quick method by G sydow.
Preparation of Standard curve:
In another set of 6 test tubes,
♦ 0.5 ml of standard with 2.0 ml of 5% Perchloric Acid and incubation
for 5 min at 100oC.
♦ Cool down; centrifugation at 2500 x g for 4 mins.
♦ 1.0 ml of clear supernatant added to 0.2 ml of Ehrlich's reagents;
Heated for 15 min at 100oC.
♦ Cooling the mixture and addition of 1.0 ml of water.
♦ Optical density measurement at 525 nm against reagent blanks in
spectrophotometer.
♦ The OD values were plotted in a graph paper against Sialic acid
concentrations of the standard solution to obtain a linear curve.
CALCULATION:
The serum (0.5 ml) collected from all 50 cases and 25 controls were
subjected to similar treatment. The OD values were plotted in the standard
40
MATERIALS AND METHODOLOGY
graph (curve) to obtain the serum levels of Sialic acid in controls and
cases60.
SIGNIFICANT FIGURES:
Suggested Significance: 0.05<P<0.10
Moderately significant: 0.01<P≤0.05
Strongly significant: P≤0.01
41
MATERIALS AND METHODOLOGY
PHOTOGRAPH 1: LEUKOPLAKIA OF LEFT BUCCAL MUCOSA
PHOTOGRAPH 2: PHOTOMICROGRAPH OF LEUKOPLAKIA
42
MATERIALS AND METHODOLOGY
PHOTOGRAPH 3: PHOTOMICROGRAPH OF MILD EPITHELIAL DYSPLASIA
PHOTOGRAPH 4:
CARCINOMA OF THE RIGHT BUCCAL MUCOSA
43
MATERIALS AND METHODOLOGY
PHOTOGRAPH 5: PHOTOMICROGRAPH OF WELL DIFFERENTIATED
CARCINOMA
PHOTOGRAPH 6: CENTRIFUGE MACHINE
44
MATERIALS AND METHODOLOGY
HOTOGRAPH 7: INCUBATOR
PHOTOGRAPH 8: SPECTROPHOTOMETER
45
MATERIALS AND METHODOLOGY
PHOTOGRAPH 9: REAGENTS AND STANDARD USED IN THE ESTIMATION
OF TOTAL SIALIC ACID
PHOTOGRAPH 9: ESTIMATED SERUM TOTAL SIALIC ACID IN DIFFERENT
SAMPLES
46
RESULTS
RESULTS
In our comparative study, Serum Sialic acid were estimated in three groups
consisting of 25 subjects in Leukoplakia designated as Group-I, 25 subjects in
Squamous cell carcinoma as Group-II, and age and sex matched 25 subjects as
controls in Group III.
I. Age distribution-Result:
The age distribution in three different groups are; In Group I with
Leukoplakia, the age distribution was between 20-70 years, 7 cases (28%) were in the
age group of 20-30 years, 6 cases (24%) were in the age group of 31-40 years, 3cases
(12%) in the age group of 41-50 years and also in 51-60 years, 6 cases (24%) were in
the age group of 61-70 years(Table 1, Graph 1).
It was inferred from our study that peak occurrence of Leukoplakia was
between the age group of 20-30 years.
In Group II with 25 cases of Oral Cancer (Squamous Cell Carcinoma), the
distribution of age was between 30-75 years. 4 cases(16%) were between the age of
31-40 years, 10 case(40%) were in the age group of 41-50 years, 7 cases(28%) were
in the age group of 51-60 years, 4 cases(16%) were in the age group of 61-70 years
(Table I, Graph I).
It was noticed from our study that peak occurrence of SCC was between the
age group of 41-50 years.
The age distribution among Group III was between 20-75 with 5 cases (20%)
between the age of 20-30 years, 6 cases (24%) were within the age of 31-40 years, 4150 years, 51-60 years respectively , 1 case(4%) was in the group of 61-70 years and
above 71 years respectively( Table 1, Graph 1).
47
RESULTS
Table 1: Age distribution in three different groups
Age
Lp-Group-I
No
SCC Group-II
%
No
Controls
Group-III
%
%
No
20-30
7
28.00
0
0.00
5
20.00
31-40
6
24.00
4
16.00
6
24.00
41-50
3
12.00
10
40.00
6
24.00
51-60
3
12.00
7
28.00
6
24.00
61-70
6
24.00
4
16.00
1
4.00
>71
0
0.00
0
0.00
1
4.00
Total
25
100
25
100
25
100
Graph 1:
LP Group-I
Age distribution in the three groups
SCC Group-II
45
Control Group-III
40.00
40
% o f p atien ts
35
30
28.00
28.00
24.00
25
24.00
24.00
24.00
24.00
20.00
20
16.00
15
16.00
12.00
12.00
10
4.00
5
0.00
4.00
0.00 0.00
0
20-30
31-40
41-50
51-60
Age group (yrs)
48
61-70
>71
RESULTS
II. Gender distribution-Result:
The Gender distributions between Three Groups are;
In Group I, Gender predilection was noticed. Of the 25 patients, 21 cases
(84%) were males and 4 cases (16%) were females (Table 2, Graph2).
It was inferred from our study that Leukoplakia occurrence was
predominant in males.
Of the 25 cases in Group II, 12 cases (48%) were males and 13 cases (52%)
were females (Table 2, Graph 2).
It was noticed from our study that almost equal predilection for men and
women are noted.
In Group III of 25 subjects, 15 cases (60%) were males and 10 cases (40%)
were females (Table 2, Graph 2).
Table 2: Gender distribution between Three Groups
Gender
LP-Group-I
No
%
SCC-Group-II
No
Controls
%
No
%
Male
21
84.00
12
48.00
15
60.00
4
16.00
13
52.00
10
40.00
25
100
25
100
25
100
Female
Total
49
RESULTS
Graph 2:
Gender distribution in the three groups
90
Male
Female
84.00
80
70
% o f p a tie n t s
60.00
60
52.00
48.00
50
40.00
40
30
20
16.00
10
0
LP Group-I
SCC Group-II
Group
50
Control Group-III
RESULTS
III. Habits distribution-Results:
Habits distributions between three Groups are;
The entire Group I subjects had deleterious oral habits. Of which 11
case(23.40%)
had
smoking
habits,
20
cases
(42.55%)
had
smokeless
tobacco(chewing) habit, 4cases (8.51) were alcoholic, 6 case (12.77%) had the habit
of smoking and smokeless tobacco habit, 4 cases(8.51%) had ST and alcohol habits, 2
cases(4.26%) had Smoking, ST and alcohol habits(Table 4, Graph 3).
In Group II , only 1case (2.86%) did not had any oral deleterious habits. 9
cases (25.71%) had smoking habit, 20 cases(57.14%) had smokeless tobacco habit. 5
cases (14.29%) had both smoking and smokeless tobacco habit.(Table 3,graph 3)
In both Group I and Group II subjects smokeless tobacco habit (chewing) was
the predominant deleterious habit.
The subjects in Group III did not have any deleterious oral habits.
51
RESULTS
Table 3: Habits distribution between Two Groups
LP Group - I
SCC Group II
Habit
No
%
No
%
None
0
0.00
1
2.86
Smoking-A
11
23.40
9
25.71
ST-B
20
42.55
20
57.14
Alcohol-C
4
8.51
0
0.00
A+B
6
12.77
5
14.29
B+C
4
8.51
0
0.00
C+A
0
0.00
0
0.00
A+B+C
2
4.26
0
0.00
Graph 3:
Series1
Habit distribution in two groups
60
Series2
57.14
50
% o f p atien ts
42.55
40
30
25.71
23.40
20
12.77
14.29
8.51
10
8.51
4.26
2.86
0.00
0.00
0.00
0.00 0.00
0.00
0
None
Smoking-A
ST-B
Alcohol-C
A+B
Habit
52
B+C
C+A
A+B+C
RESULTS
IV. Site distribution –Result:
The Site distribution between Group I and Group II are;
In Group I, the lesion showed different site of involvement in the oral cavity.
23 cases (52.27%) involved buccal mucosa, 10 (22.73%) cases involved commissure
of lip, 3(6.82%) cases in retromolar area, 2cases (4.55%) each in Gingivo buccal
sulcus, Buccal mucosa and gingivo buccal sulcus, Lip and Gingiva respectively
(Table4, Graph 4).
The lesions in Group II were distributed at different sites in the Oral cavity. 14
cases (31.82%) involved buccal mucosa. 2 cases (4.55%) in alveolus, 8 cases
(18.18%) in retromolar area, 2 cases (4.55%) in floor of the mouth and Tongue, 4
cases(9.09%) in Gingivobuccal sulcus, 5 cases(13.36%) in Gingivobuccal sulcus and
Buccal mucosa, 3 case(6.82%) in commissure of lip, 2 cases(4.55%) in gingiva,
1case(2.27%) in Tongue and floor of the mouth. 1case(2.27%) in palate (Table 4,
Graph 4).
It was noticed from our study that both in Group I and Group II, Buccal mucosa
is the site of predilection for LP and SCC respectively.
53
RESULTS
Table 4: Site distribution between Two Groups
LP Group - I
SCC Group II
No
%
No
%
BM
23
52.27
14
31.82
ALVEOLUS
0
0.00
2
4.55
RMA
3
6.82
8
18.18
RMA+SP
0
0.00
0
0.00
FOM
0
0.00
2
4.55
TONGUE
0
0.00
2
4.55
GBS
2
4.55
4
9.09
Sites
BM+GBS
2
4.55
5
11.36
ALV+FOM
0
0.00
0
0.00
COL
10
22.73
3
6.82
LIP
2
4.55
0
0.00
GINGIVA
2
4.55
2
4.55
TONGU+FOM
0
0.00
1
2.27
PALATE
0
0.00
1
2.27
Graph 4:
LP Group-I
Sites distribution in two groups
SCC Group-II
60
52.27
40
31.82
30
22.73
18.18
20
10
4.55
11.36
9.09
6.82
4.55
0.00
0.000.00
4.55
0.00
4.55
6.82
4.55
0.00
4.55
0.000.00
4.554.55
2.27
0.00
0.00
2.27
0.00
Sites
54
TE
LA
PA
U+
NG
TO
GI
NG
FO
IV
M
A
P
LI
L
CO
M
FO
V+
AL
+G
BS
S
BM
GB
UE
NG
M
TO
FO
SP
A+
RM
A
RM
VE
OL
US
BM
0
AL
% of patients
50
RESULTS
V. Staging in Leukoplakia -Result:
Of the 25 Subjects in Group-I, 24 cases (96%) were in stage I and 1case (4%)
was in Stage II of clinical staging (Table 5, Graph 5).
Our study showed the out numbering of LP in clinical Staging I.
Table 5: Staging in Leukoplakia – Group-I:
LP
No
%
Stage 1
24
96.00
Stage 2
1
4.00
Total
25
100
Graph 5:
Stages of Leukoplakia
4%
96%
Stage 1
Stage 2
55
RESULTS
VI. Staging in SCC-Result:
Clinical Staging of Oral cancer in 25 cases of Group II showed, Stage III of
17 cases(68%) and 8 cases(32%) of Stage IV(Table 6, Graph 6).
Our study showed the out numbering of SCC in Clinical Staging III.
Table 6: Staging in Oral Cancer- Group-II:
SCC
No
%
Stage I
0
0.00
Stage II
0
0.00
Stage III
17
68.00
Stage IV
8
32.00
Total
25
100
Graph 6:
Staging in Oral cancer - Group II
17
18
16
N o . o f s a m p le s
14
12
8
10
8
6
4
0
2
0
0
Stage I
Stage II
Stage III
Stage
56
Stage IV
RESULTS
VII. Tumor size in SCC-Result:
TNM staging in 25 cases of Group II revealed 5 cases (20%) reported with Tumor
size of T1, 13 cases(52.00%) with Tumor size of T2, 4 cases(16%) with Tumor size of
T3, 3 cases(12%) with Tumor size of T4 (Table 7,Graph 7).
The study showed the Outnumbering of SCC subjects in T3 Tumor size.
Table 7: Tumor size in Oral Cancer.
SCC-Group-II
Tumor size-T
No
T1
5
20.00
T2
13
52.00
T3
4
16.00
T4
3
12.00
%
Graph 7:
Tumor size in Oral cancer
13
14
N o . o f tu m o r s
12
10
8
5
6
4
3
4
2
0
T1
T2
T3
Tumor size
57
T4
RESULTS
VIII. Nodal status in SCC – Result:
The Nodal status of group II showed a significant number of 20 cases (80%) in N1
stage, 1 case(4%) in N2a stage, 2 cases (8%) in N2b stage , 2 cases(8%) in N2c stage
(Table 8 , Graph 8).
The study showed the Outnumbering of SCC subjects in N1 Nodal state.
Table 8: Nodal involvement in Oral cancer.
SCC-Group-II
Nodal
involvement-N
No
%
N1
20
80.00
N2a
1
4.00
N2b
2
8.00
N2c
2
8.00
Graph 8:
Nodal involvement in Oral cancer
20
20
18
N o. of nodes
16
14
12
10
8
6
4
2
1
2
2
0
N1
N2a
N2b
Nodal involvement
Metastasis was not noticed in any of the cases.
58
N2c
RESULTS
IX. Serum Total Sialic acid –Result:
The Serum Total Sialic acid levels were estimated in all the three Groups and
the arithmetic mean along with Standard deviation was calculated. (Table 9, Graph 9,
Graph 10).
Group I showed the mean of 76.18 mg/dl with the standard deviation of 17.08
mg/dl; the interval of mean between 69.13 to 83.23 mg/dl, with the minimum of 45.00
mg/dl to a maximum of 107.00 mg/dl.
Group II showed the mean of 95.68 mg/dl with the standard deviation of
18.45mg/dl; the interval of mean between 88.06 to 103.30 mg/dl, with the minimum
of 54.00 mg/dl to a maximum of 125.00 mg/dl.
Group III showed the mean of 60.22 mg/dl with the standard deviation of
4.92mg/dl; the interval of mean between 58.19 to 62.25 mg/dl, with the minimum of
51.50mg/dl to a maximum of 69 mg/dl.
Our study showed the ascending order of Serum Total Sialic acid levels in
Controls, LP and SCC.
Table 9: Descriptive Statistics:
Group
N
Mean
Std.
Deviation
95% Conf Interval for
Mean
Lower
Bound
Upper
Bound
Min
Max
Control
25
60.22
4.92
58.19
62.25
51.50
69.00
Leukoplakia
25
76.18
17.08
69.13
83.23
45.00
107.00
Squamous
25
95.68
18.45
88.06
103.30
54.00
125.00
59
RESULTS
Graph 9: Sialic acid in three groups:
140.00
Sialic Acid (mg/dl)
120.00
100.00
80.00
60.00
40.00
Control
Leukoplakia
Squamous
Group
Graph 10:
Mean Sialic acid (mg/dl) in the 3 groups
95.68
100
76.18
S ia lic a c id ( m g /d l)
90
80
60.22
70
60
50
40
30
20
10
0
Control
Leukoplakia
Group
60
Squamous
RESULTS
X. ANOVA – Result:
ANOVA (Analysis of variants) was carried out between three groups and the
results was conclude that there is a significant difference in the mean Sialic acid levels
in the 3 groups (P<0.001) (Table 10).
Table 10: ANOVA
ANOVA
Sialic Acid (mg/dl)
Between Groups
Within Groups
Total
Sum of
Squares
15769.860
15750.920
31520.780
df
2
72
74
61
Mean Square
7884.930
218.763
F
36.043
Sig.
.000
RESULTS
XI. Multiple group comparison - Result:
In order to find out among which pair of groups there exist a significant
difference, Bonferroni’s test was carry out between three Groups with multiple
comparison and inferred that; there is a significant difference between control group
& Leukoplakia group, control group & squamous group and Leukoplakia group &
squamous group with respect to the mean Sialic acid levels (P<0.001).
The mean Sialic acid is found to be more in squamous group compared to
Leukoplakia and control group and this difference is statistically significant. The
mean Sialic acid in Leukoplakia group is higher than control group and this difference
is also statistically significant (Table 11).
Table 11: Multiple Comparisons- Bonferroni Test
Multiple Comparisons
Dependent Variable: Sialic Acid (mg/dl)
Bonferroni
Mean
Difference
(I-J)
(I) Group
(J) Group
Std. Error
Control
Leukoplakia -15.96000* 4.18342
Squamous -35.46000* 4.18342
Leukoplakia Control
15.96000* 4.18342
Squamous -19.50000* 4.18342
Squamous Control
35.46000* 4.18342
Leukoplakia 19.50000* 4.18342
*. The mean difference is significant at the .05 level.
62
Sig.
.001
.000
.001
.000
.000
.000
95% Confidence Interval
Lower Bound Upper Bound
-26.2144
-5.7056
-45.7144
-25.2056
5.7056
26.2144
-29.7544
-9.2456
25.2056
45.7144
9.2456
29.7544
DISCUSSION
DISCUSSION
Carcinoma of the oral cavity is one of the most frequent malignant tumors
worldwide, with major predominance in South-East Asia and India22, 39.
Among the oral tumors, 90% are squamous cell carcinoma (SCC), which
arises from the mucosal lining. This high incidence of oral cancer in India is due to
the widespread habits of tobacco chewing and smoking and alcohol 28,33,34,39.
The transformation rate from premalignant to malignant varies from 0.6 to
36 % 1. Leukoplakia is the most common premalignant, potentially malignant or
precancerous lesion of the oral mucosa11.
In our present study conducted in VS Dental College and Hospital, Bangalore;
included 25 Leukoplakia cases, 25 Squamous cell carcinoma cases and 25 age and
sex matched controls.
Among 25 Leukoplakia cases the age distribution was between 20-70 years.
The existence of Leukoplakia even in earlier ages up to 15 years was noticed which
was related with the usage of tobacco10.
Our study showed gender predilection for men in Leukoplakia up to 84%
which was almost similar to other study16. This can also vary depending on the
geographical variation and usage of tobacco10, 16.
Every case of Leukoplakia had the habit of usage of tobacco either as smoking
or smokeless (chewing) form or in some with addition usage of alcohol. Smokeless
tobacco usage was most common.
Oral Leukoplakia frequently precedes oral cancer. They both has similar
etiologic factors8 and can be induced and promoted by tobacco; 14, 79
There are about 300 carcinogenic compounds present in tobacco out of which
tobacco-specific nitrosamines have been identified as the most important carcinogens
63
DISCUSSION
in tobacco. The other carcinogenic compounds in tobacco metabolites are polycyclic
aromatic hydrocarbons, a-particle-emit-ting
210
Po (polonium), trace metals, carbon
monoxide, hydrogen cyanide, and phenols18, 39.
The site of predilection for Leukoplakia being buccal mucosa and then
commissure of lip in different studies18 correlates with our study. Leukoplakia in stage
I clinical staging has outnumbered.
In Oral Cancer of Group II including 25 patients were between the age
distribution of 31-40 years with 40% of the patients were in the age group of 41-50
years and 28% were in the age group of 51-60 years. The similar study was showing
the wider range of age distribution between 14-80 years of age with the median age of
43 years7.
Almost equal predilection for males and females in our study which is
contradictory to the sex predilection in India which in turn depends on the usage of
tobacco4, 7, 10, 22, 39.
In India, the buccal mucosa (cheek) is the primary site for cancer development
as against the tongue and the floor of the mouth in Western countries, which may be
due to the habit of keeping the betel-quid and tobacco in contact with the cheek for a
long time4, 18, 39 which is similar to our study. Only one patient did not have any oral
deleterious habit.
In our study, majority of the patients were in Stage III clinical staging of SCC
with T2 tumor size, including Lymph node involvement in all the cases. This may be
due to lack of awareness as majority of the cases are poorly educated and are from
rural background6.
Tumor markers are substances specific for certain tumor or cancer cells and
thus could be of appreciable diagnostic and prognostic value in cancer patients67.
64
DISCUSSION
Many properties of the mammalian cells are expressed at or mediated through,
the cell surface. Among these properties are those which distinguish a malignant cell
from normal cells. As neoplastic changes are expressed at the cell surface, altered
surface characteristics are essential for the abnormal growth and behaviour of
malignant cells8.
Substances like Glycoproteins and Gylcolipids are major constituents of cell
membrane, and hence, cell-surface Glycoconjugates are important in malignancy8.
Glycoproteins are usually defined as protein- carbohydrate complexes in
which oligosaccharides and / or polysaccharides are joined by covalent linkage to
specific amino acids of proteins. The carbohydrate portion contains amino sugars
(glucosamine, galactosamine or Sialic acid) and hexoses (galactose, mannose) or
fucose8.
Sialic acid is thought to be important in determining the surface properties of
cells and has been implicated in cellular invasiveness, adhesiveness and
immunogenesity8.
Sialic acids (N-acetylneuraminic acid, NANA) frequently occupy the terminal,
non-reducing position on membrane glycoproteins. The negative charge due to
presence of a carboxyl group enables Sialic acid to mediate a wide array of cellular
functions such as transport of positively charged molecules, cell-cell and cell-matrix
interactions, influence the confirmation of membrane glycoproteins and masking
antigenic sites of receptors. The presence of Sialic acid at the terminal or near the
terminal position underlies its importance in determining chemical and biologic
diversity, and characteristics of cell-surface and secreted glycoproteins. Human and
experimental model based studies have documented clinical significance of this sugar
moiety4.
65
DISCUSSION
These Glycoconjugate are released into the circulation through increased
turnover, secretion. And / or shedding from malignant cells. Increased quantities of
Glycoconjugate like total Sialic acid (TSA) have been detected in serum of patients
with different types of malignancies, indicating their usefulness in diagnosis or
monitoring therapy8.
In our present study, Total serum Sialic acid was estimated using a simplified
quick method by G Sydow. The OD values were plotted in the standard graph (curve)
to obtain the serum levels of Sialic acid in controls and cases. The values obtained are
within the range which was noticed in different studies60.
The interval of mean was between 69.13 to 83.23 mg/dl in Group I, between
88.06 to 103.30 mg/dl in Group II and between 58.19 to 62.25 mg/dl in Group III.
Analysis of variants was carried out between three groups and the results was
conclude that there is a significant difference in the mean Sialic acid levels in the 3
groups (P<0.001) (Table 10).
In order to find out among which pair of groups there exist a significant
difference, Bonferroni’s test was carry out between three Groups with multiple
comparison and inferred that; there is a significant difference between control group
& Leukoplakia group, control group & squamous group and Leukoplakia group &
squamous group with respect to the mean Sialic acid levels (P<0.001).
The mean Sialic acid is found to be more in squamous cell carcinoma group
compared to Leukoplakia and control group and this difference is statistically
significant. The mean Sialic acid in Leukoplakia group is higher than control group
and this difference is also statistically significant (Table 11).
66
DISCUSSION
Our results are correlating with the different studies done by different people
corresponding to the level of Total serum Sialic acid in different untreated
malignancies and oral precancerous lesion of the oral cavity6, 7, 8.
SCC of different sites other than oral cavity also showed significant increase
in the level of Total Sialic acid due to its increased turn over, secretion and shedding
from malignant cells4,5,47,52,54,55,56,58,59,61,62,63,64,65,66,67,70,73,74,77.
In our study, Group III did not have any deleterious habits compared to Group
I and II except only one patient in Oral cancer, who had either smoking or smokeless
tobacco oral habit- which is one of the risk for developing Leukoplakia and Oral
cancer in India. The rise in the level of Sialic acid was also noticed in both smoking
and smokeless tobacco habits indicating its harmful effects76.
The changes in serum TSA was noticed in different systemic diseases like
diabetes mellitus, Ischemic heart disease, and different bone disorders like pyogenic
arthritis, Rheumatoid arthritis, malignant bone tumors43,48,69,71,75. The patients in our
study did not have any systemic disease. So, the increase in the TSA in both LP and
SCC is due to increased turnover, secretion, loss of adhesiveness and / or shedding
from Premalignant and malignant cells.
Serum TSA can be used for initial diagnosis, monitoring therapy of
premalignant and malignant lesions and even recurrence of malignancy4, 5, 6,7,57. The
idea of screening and following patients with malignancy by blood test using
simplified quick methodology by G Sydow is appealing from several point of
including its ease, economic advantage, non-invasiveness, and possibility of repeated
sampling.
But the disadvantage is that since TSA is elevated in different disease entities,
it can not be considered as specific Tumor marker. Further studies are required to
67
DISCUSSION
include Total Sialic acid would be helpful as a specific Tumor marker in the initial
diagnosis of Leukoplakia and Squamous cell carcinoma.
68
CONCLUSION
CONCLUSION
Our Present study has gathered following Conclusions from the Assessing of
Serum Total Sialic acid in Oral Leukoplakia, Squamous cell carcinoma and age and
sex matched controls:
¾ When the patients age and sex attributes were considered, the highest
incidence was observed in the 3rd decade in Leukoplakia and 5th decade in
OSCC. Males out numbered in Leukoplakia and almost equal sex
predilection in OSCC.
¾ Definitive association was found between the harmful habits of tobacco
chewing with smoking and alcohol consumption to the incidence of LP
and SCC.
¾ The Smokeless tobacco usage was found to be related to the occurrence of
LP and SCC with the site predilection of Buccal mucosa.
¾ Significant elevation of serum Total Sialic acid in Leukoplakia and
Squamous cell carcinoma was assessed. So TSA in serum can be used as
Tumor marker in LP and SCC.
¾ The level of Serum Sialic acid in LP with mild dysplasia (1 case) is not
significant compared to LP without dysplasia but the level of serum TSA
in poorly differentiated OSCC compared to well differentiated and
moderately differentiated OSCC is significant. So more sample
assessment of LP with dysplasia may be significant in initial diagnosis.
¾ The Simplified quick methodology by G.Sydow in the assessment of
serum TSA is less time consuming, less invasive, cost effective and values
were within the levels found by different methodology. So, this method
69
CONCLUSION
can be considered as a standardized method for the evaluation of TSA in
serum for initial diagnosis of LP and SCC.
Further studies are required to confirm the clinical usefulness of serum TSA
as an early diagnostic tumor marker.
70
SUMMARY
SUMMARY
Oral cancer is becoming the great threat to public health due to its potentiality
of morbidity and mortality globally which is having almost equal predilection for
males and females due to increased usage of tobacco.
This demoralizing disease entity is well documented to be preceded by Oral
precancer lesion like Leukoplakia which has the same etiologic factors for both.
During carcinogenesis, any intracellular micro-environmental change may
lead to alteration in the cell surface membranes constituents, releases certain
molecules in the blood which are called Tumor markers. The Glycoconjugate - serum
Total Sialic acid is one of the Tumor markers.
The study involving serum TSA in Oral Precancerous Lesion and Oral
Squamous cell carcinoma is limited. In our present study, we had considered 25
patients with Leukoplakia, 25 patients with Oral cancer and 25 age and sex matched
subjects as controls for assessing serum Total Sialic acid levels in these three groups
and comparison between the same.
Betel nut chewing and smoking had an influence on the occurrence of
Leukoplakia and oral carcinoma.
The assessment of Total serum Sialic acid assay was carried out using a
simplified quick method by G Sydow for LP and SCC and in controls. The OD values
were plotted in the standard graph (curve) to obtain the serum levels of Sialic acid in
all three groups.
The serum TSA in controls, LP and OSCC were found to be progressively
increased in an ascending order and had statistically significant values. This suggested
that TSA is of great value in early assessment of Leukoplakia and Squamous cell
carcinoma.
71
BIBILOGRAPHY
BIBILOGRAPHY
1. Diamondopoulos, Meissner. Anderson’s pathology, 8th edition, vol 1, chapter
15, Neoplasia, 1985.
2. Norman k.wood, Paul W.Goaz. “Differential Diagnosis of Oral and
maxillofacial lesions”. Fifth edition, Elsevier publication, Chapter 35.Page
587-594.
3. James d.cox, k.kian ang. “Radiation Oncology”. Eighth edition, Chapter 10.
Page 219-254.
4. Manisha H.shah, shaila D.telang, Pankaj M.Shah, Prabhudas S.Patel. “Tissue
and serum α 2-3 and α 2-6- linkage specific sialylation changes in oral
carcinogenesis. Glycoconj J (2008) 25:279-290.
5. Daniel ayude, Gloria Gacico, Maria Paez De La Cadena, Esterella Pallas,
Vicenta Soledad Martinez-Zorzano et al. “Combined use of established and
novel tumor markers in the diagnosis of head and neck squamous cell
carcinoma. Oncology Reports 10: 1345-1350, 2003.
6. Vasanti R.Rao, Ph.D, Lakshmi Krishnamoorthy, Ph.D., S.V.Kumaraswamy,
M.D.S and Girija Ramaswamy, Ph.D. “Cancer Detection and Prevention,
22(3):237-240(1998).
7. Kinnari B.Rajpura, Prabhudas S.Patel, Jyoti G.Chawla, Raksha M.Shah.
“Clinical significance of total and lipid bound Sialic acid levels in oral
precancerous conditions and oral cancer. J Oral Pathol Med (2005) 34:263-7.
8. Bina R Baxi, Msc, Prabhudas S.Patel, Msc, Siddarth G.Adhvaryu, PhD, and
Pramod K.Dayal, MDS. “Usefulness of Serum Glycoconjugates in
Precancerous and Cancerous Diseases of the Oral Cavity.
72
BIBILOGRAPHY
9. Beverly A. Dale and L. Page Fredericks. “Antimicrobial Peptides in the Oral
Environment: Expression and Function in Health and Disease”. Curr. Issues
Mol. Biol. 7: 119–134.
10. Fali S. Mehta, DDS, DMD, J.J.Pindborg, DDS, Dr.Odont, P.C.Gupta, Msc,
and D.K.Daftary, DDS. “Epidemiologic and histologic study of oral cancer
and Leukoplakia among 50915 villagers in India. Cancer 1969.24:832-849.
11. Van Der Waal, T. Axell
“
Review Oral Leukoplakia: a proposal for uniform
reporting”. Oral Oncology 38 (2002) 521–526.
12. Prabhu.S.R, Wilson.D.F, Daftary.D.K, Johnson.N.W, “Oral Disease in the
Tropics”. Oxford university press, 1993, Chapter 36.Page 402-428.
13. Sciubba.J.J. “Oral Leukoplakia”. Crit Rev Oral Biol Med6 (2):147-160 (1995).
14. Jolán Bánóczy, D.Sc; Zeno Gintner, Ph.D.; Csaba Dombi, Ph.D. “Tobacco
Use and Oral Leukoplakia. Journal of Dental education2001; 65(4):322-326.
15. Aline Cristina Batista Rodrigues Johann, João Batista Da Silveira-Júnior Et
Al. “Metallothionein immunoexpression in oral Leukoplakia”. Med Oral Patol
Oral Cir Bucal. 2008 Mar1; 13(3):E156-60.
16. Eliza Carla Barroso Duarte, Marina Sena Lopes Da Silva, Marcus Vinicius
Gomez, Ricardo Santiago Gomez. “GSTM1 polymorphism and oral
Leukoplakia”. J Oral Pathol Med (2006) 35: 202–5.
17. Twj Poate, Warnakulasuriya.S. “Effective management of smoking in an oral
dysplasia clinic in London”. Oral Diseases (2006) 12, 22-26.
18. Mousumi Majumder, Nilabja Sikdar, Ranjan Rashmi Paul, and Bidyut Roy.
“Increased Risk of Oral Leukoplakia and Cancer Among Mixed Tobacco
Users Carrying XRCC1 Variant Haplotypes and Cancer Among Smokers
Carrying Two Risk Genotypes: One on Each of Two Loci, GSTM3 and
73
BIBILOGRAPHY
XRCC1 (Codon 280). Cancer Epidemiol Biomarkers Prev 2005; 14(9).
September 2005.
19. Haya-Fernandez.M.C, Bagan.J.V, Murillo-Cortes.J, R Poveda-Roda, C
Calabuig. “The prevalence of oral Leukoplakia in 138 patients with oral
squamous cell carcinoma”. Oral Diseases (2004) 10, 346–348.
20. Martin S. Greenberg, Michael Glick. “Burket’s Oral medicine Diagnosis and
Treatment”, Tenth edition, Chapter 8. Page194-234.
21. Faleh A. Sawair Christopher R. Irwin Derek J. Gordon Alan G.
Leonard Mike Stephenson Se´Amus S. Napier. “Invasive front
grading: reliability and usefulness in the management of oral
squamous cell carcinoma”. J of oral Pathol med 2003:32:1-9.
22. Anak Lamaroon, Surawut Pongsiriwet, sumana Jittidecharaks,
Komkham
Pattanaporn,
Sangsom
Prapayasatok,
Sitthichai
Wanachantararak. “Increase of mast cells and tumor angiogenesis in
oral squamous cell carcinoma”. J Oral Pathol Med 2003: 32: 195-9.
23. Xiaofei Tang, Lin Jia, Jie Ouyang, Minoru Takagi. “Comparative study of
HPV prevalence in Japanese and North-east Chinese oral carcinoma”. J Oral
Pathol Med 2003: 32: 393-8.
24. Isamu Kodani Mitsuhiko Osaki Kohei Shomori Kunio Araki Eizo Goto Kazuo
Ryoke Hisao Ito. “Minichromosome maintenance 2 expression is correlated
with mode of invasion and prognosis in oral squamous cell carcinomas”. J
Oral Pathol Med 2003: 32: 468-74.
25. Boudewijn J. M. Braakhuis, C. Rene Leemans, Ruud H. Brakenhoff. “A
genetic progression model of oral cancer: current evidence and clinical
implications”. J Oral Pathol Med (2004) 33: 317–22.
74
BIBILOGRAPHY
26. Yi-Shing Shieh, Herng-Sheng Lee, Shine-Gwo Shiah, Yi-Wen Chu, ChengWen Wu, Long-Chang Chang. “Role of angiogenic and non-angiogenic
mechanisms in oral squamous cell carcinoma: correlation with histologic
differentiation and tumor progression”. J Oral Pathol Med (2004) 33: 601–6.
27. Anak Iamaroon, Udomrat Khemaleelakul, Surawut Pongsiriwet, Jarupan
Pintong. “Co-expression of p53 and Ki67 and lack of EBV expression in oral
squamous cell carcinoma”. J Oral Pathol Med (2004) 33: 30-6.
28. Shu-Chun Lin, Chung-Ji Liu, Chun-Po Chiu, Shun-Ming Chang, Suu-Yi Lu,
Yann-Jang Chen. “Establishment of OC3 oral carcinoma cell line and
identification of NF-kB activation responses to areca nut extract”. J Oral
Pathol Med (2004) 33: 79-86.
29. Halder.A, Chakraborty.T, Mandal.K, Gure, P.K, Das.S, Raychowdhury.R,
Ghosh.A.K, and M. De. “Comparative Study of Exfoliated Oral Mucosal Cell
Micronuclei Frequency in Normal, Precancerous and Malignant Epithelium”.
Int J Hum Genet, 4(4): 257-260 (2004).
30. Chung-Ji Liu, Yong-Kie Wong, Kuo-Wei Chang, Hsiao-Ching Chang Et Al.
“Tumor necrosis factor-a promoter polymorphism is associated with
susceptibility to oral squamous cell carcinoma”. J Oral Pathol Med (2005) 34:
608–12.
31. Hitoshi Nagatsuka, Kazuteru Hibi, Mehmet Gunduz, Hidetsugu Tsujigiwa.
“Various immunostaining patterns of CD31, CD34 and endoglin and their
relationship with lymph node metastasis in oral squamous cell carcinomas”. J
Oral Pathol Med (2005) 34: 70–6.
32. Hiromitsu Nakaya, Shuichi Kawashiri, Akira Tanaka, Natsuyo Noguchi Et Al.
“Influences of angiogenesis and lymph angiogenesis on cancerous invasion in
75
BIBILOGRAPHY
experimentally induced tongue carcinoma”. J Oral Pathol Med (2005) 34: 87–
92.
33. Sasaki.T, MolesD.R, Imai.Y, P. M. Speight.P.M. “Clinico-pathological
features of squamous cell carcinoma of the oral cavity in patients <40 years of
age”. J Oral Pathol Med (2005) 34: 129–33.
34. Tu Ngoc Tran, Yenchun Liu, Minoru Takagi, Akira Yamaguchi, Hiroaki Fujii.
“Frequent promoter hypermethylation of RASSF1A and p16INK4a and
infrequent allelic loss other than 9p21 in betel-associated oral carcinoma in a
Vietnamese non-smoking/non-drinking female population”. J Oral Pathol Med
(2005) 34: 150–6.
35. Masaya Okura, Hajime Kagamiuchi, Gyo Tominaga, Seiji Iida, Yasuo
Fukuda, Mikihiko Kogo. “Morphological changes of regional lymph node in
squamous cell carcinoma of the oral cavity”. J Oral Pathol Med (2005) 34:
214–9.
36. Brennan.P.M, Mackenzie.N, Quintero.M. “Hypoxia-inducible factor 1α in oral
cancer”. J Oral Pathol Med (2005) 34: 385–9.
37. Sonja Boy, Estrelita Janse Van Ransburg, Susan Engelbrecht, Leonora Dreyer,
Marlene Van Heerden, Willie Van Heerden. HPV detection in primary intraoral squamous cell carcinomas – commensal, aetiological agent or
contamination?” J Oral Pathol Med (2006) 35: 86–90.
38. Furrer.V.E, Benitez.M.B, Furnes.M, Lanfranchi.H.E, N. M. Modesti.N.M.
“Biopsy vs. superficial scraping: detection of human papillomavirus 6, 11, 16,
and 18 in potentially malignant and malignant oral lesions”. J Oral Pathol Med
(2006) 35: 338–44.
76
BIBILOGRAPHY
39. Takashi Hase, Shuichi Kawashiri, Akira Tanaka, Shinichi Nozaki.
“Correlation of basic fibroblast growth factor expression with the invasion and
the prognosis of oral squamous cell carcinoma”. J Oral Pathol Med (2006) 35:
136–9.
40. Jane.C,
Nerurkar.A.V,
Shirsat.N.V,
Deshpande.R.B,
Mrapurkar.A.D,
Karjodkar.F.R. ” Increased survivin expression in high-grade oral squamous
cell carcinoma: a study in Indian tobacco chewers”. J Oral Pathol Med (2006)
35: 595–601.
41. Eric R. Vimr, Kathryn A.Kalivoda, Eric L. Deszo, and Susan M.Steenbergen.
“Diversity of Microbial Sialic acid metabolism”. Microbiology and molecular
biology reviews. Mar 2004.p.132-153.
42. Ann Carter and H.Martin. “Serum Sialic acid levels in health and disease”.
J.clin.Path, (1962), 15, 69.
43. Harper. “Textbook of Biocemistry”-25th edition; Page no: 158.
44. Y.Uchida,
Y.Tsukada
and
T
Sugimori.
Biochim.
Biophys.Acta,
350,425(1974).
45. Aminoff.D. “Methods for quantitative estimation of Acetylneuraminic acid
and their application to Hydrolysates of Sialomucoids”. Biochem .J. (1961),
81,384.
46. Singh.R
and
Ramraju.B.
“Serum
Sialic
acid
in
healthy
Indian
adults.”Ind.Jour.Med.Res, 55, 3, March, 1967.
47. Singh.R, Sur.B.K, Agarwal.S.N and Ramraju.B. “Serum Sialic acid in
malignancy”.Ind.Jour.Med.Res, 55, 3, March, 1967.
48. Gupta.A.K, Sur.B.K, Taneja.D.K. “Serum Sialic acid in different bone
disorders”.J.Indian M.A., vol.60, no.3, February 1, 1973.
77
BIBILOGRAPHY
49. George W.Jourdian, Lawrence Dean, And Saul Roseman. “The Sialic acids”.
The Journal of Biological Chemistry vol.246, No.2, 430-435; 1971.
50. Elisabeth Rey, Lucile Gerbaul and Christian Lombart. “Automated method for
determination
of
bound
N-Acetyl
neuraminic
acid
in
serum”.
Clin.Chem.21/3,412-414 (1975).
51. Dutt.M, Chatterjee.A.K, Khanade.J. M and Rajan.S.R “Serum Glycoproteins
in Patients of Ischemic Heart Disease and Normal Controls. Indian J.Med Res
63, 2, February 1975.
52. Mrochek.J.E, Dinsmore.S.R, Tormey.D.C, and Waalkes.T.P. “Protein –bound
Carbohydrates in Breast Cancer. Liquid – Chromatographic analysis for
Mannose, Galactose, Fucose and Sialic Acid in Serum. Clinical Chemistry,
Vol.22, No.9, 1976.
53. William P.Bradley,MS, Andrew P.Blaso,MS, Joseph F.Weiss,Phd et al.
“Correlation among serum protein bound carbohydrates, serum glycoproteins,
Lymphocyte reactivity and tumor burden in cancer patients”. Cancer 40:22642272, 1977.
54. Hulbert K.B.Silver, MD, PhD, Decio M. Rangel, MD, and Donald L.Morton,
MD. “Serum Sialic acid elevations in malignant melanoma patients”. Cancer
41: 1497-1499, 1978.
55. Allan Lipton,MD, Harold A.Harvey,MD, Sally Delong, Bs, Joseph Allegra,
Md et al. “Glycoproteins and human cancer I. Circulating levels in cancer
serum”. Cancer 43:1766-1771, 1979.
56. Harold A.Harvey, MD. Allan Lipton, MD.Deborah Whiter, and Eugene
Davidson, PhD. “Glycoproteins and Human Cancer: II. Correlation between
circulating level and disease status”. Cancer 47:324-327, 1981.
78
BIBILOGRAPHY
57. Helbert K.B.Silver, R.Nelvin Murthy, Ann J.Worth, Fernando A et al.
“Prediction of malignant melanoma recurrence by serum N-Acetylneuraminic
acid”. Int.J.Cancer:31, 39-43; 1982.
58. Ujjaini Khanderia, Ms, James H.Keller, PhD, H.Barton Grossman, MD.
“serum Sialic acid is a Biologic marker for malignant Disease”. Journal of
Surgical Oncology 23:163-166(1983).
59. Shamberger.R.J. “Serum Sialic acid in normals and in cancer patients”.
J.Clin.Chem.Clin.Biochem.Vol 22, 1984, 647-651.
60. Sydow.G. “Biomed.Biochem.Acta 44(1985)11/12, s.1721-1723.
61. Kemal M.Erbil, PhD, James D. Jones, PhD and George G.Klee, MD, PhD.
“Use and limitations of Serum Total and Lipid –bound Sialic Acid
concentrations as markers for colorectal cancer. Cancer 55:404-409, 1985.
62. Kemal M.Erbil,Phd, Sabri E.Sen, Md, Horst Zincke, MD And James D. Jones,
PhD. “ Significance of serum Protein and lipid- bound Sialic Acid as a Marker
for genitourinary Malignancies”. Cancer 57:1389-1394, 1986.
63. Run-Sheng Tan, Yu-Qing Chen, Zhen-Fu Fan, Shu-Zhen-Fu Fan, Shu-Zhong
et al. “Biochemical markers of Tumor Diathesis and Early Cancer”. Cancer
Detection and Prevention 9: 145-149(1986).
64. Yoshio Shimizu, M.D., Hatsuhiko Hasumi, M.D., Kazumasa Masubuchi,
M.D., And Yoshi Okudaira, M.D. “Management of patients with Gynecologic
cancer by serum Sialic acid determination”. Gynecologic Oncology 33, 231236(1989).
65. Irwin Vivas, Luis Spagnuolo and Priscilla Palacios. “Total and Lipid- bound
serum Sialic acid as markers for carcinoma of the Uterine Cervix”.
Gynecologic Oncology 46, 157-162(1992).
79
BIBILOGRAPHY
66. Prabhudas
S.Patel, PhD., Gira N.Rawal, Msc., M.Phil., And Damodar
B.Balar,M.D.,D.C.P
“Importance
of
serum
Sialic
acid
and
lactate
dehydrogenase in diagnosis and treatment monitoring of cervical cancer
patients”. Gynecologic Oncology 50, 294-299(1993).
67. Tewarson.S.L, D.C.P., M.D, Mittal.V.P, D.C.P., M.D. Singh.M, D.C.P., M.D,
Gupta.G.P, M.S “Serum Sialic acid-An important cancer marker”. Indian
Journal of Cancer, vol 30 (Sept.1993)125-131.
68. Gunnar Lindberg, Hiroyasu Iso, Lennart Rastam, Arne Lundablad and Aaron
R Folsom. “Serum Sialic acid and its correlates in community samples from
Akita, Japan and Minneapolis, USA”. International journal of Epidemiology
vol 26, No1, 58-63; 1997.
69. Crook.M, Lumb.P, Andrews.V and Swaminathan.R. “Serum total Sialic acid,
a reputed cardiovascular risk factor, and its relationship to lipids, plasma
fasting insulin, blood pressure and body mass index in normal individuals”.
Clinical Science (1998)95, 53-57.
70. Pervin Vural, Mukaddes Canbaz, Dilek Selcuki. “Total and lipid bound Sialic
acid levels in Actinic Keratosis and Basal call Carcinoma”. Tr.J.of Medical
Sciences 29 (1999)419-423.
71. Melidonis.A, Tournis.S, Hraklioti.S, Harklianou.S. “Serum Sialic acid
concentration and Diabetic nephropathy in type II diabetic mellitus”. Diabetic
unit, Tzanio general hospital, Piraeus, Greece-Professional paper.1998.
72. Maritta Ponnio, Hannu Alho, Seppo T. Nikkari et al “Serum Sialic acid in a
random sample of the General population”. Clinical Chemistry 45:10, 18421849(1999).
80
BIBILOGRAPHY
73. Alicja Paszkowska,Phd, Marek Cybulski, M.D, Andrzej Semczuk,M D,Phd,
Krzysztof Postawski M D,Phd et al. “Total Sialic acid content in endometrial
cancer tissue in relation to normal and hyperplastic human endometrium”.
Cancer detection and prevention, 24(5):459-463(2000).
74. Patricia De Albuquerque Gracia Redondo, Celso Vataru Nakamura,
Wanderley De Souza, Jose Andres Morgado-Diaz. “Differential expression of
Sialic acid and N-Acetylgalactosamine residues on the cell surface of the
intestinal epithelial cell according to normal or metastatic potential”. Journal
of Histochemistry and Cytochemistry, volume 52 (5):629-640, 2004.
75. Shivanand Nayak B and Geeta Bakta. “Relation between Sialic acid and
metabolic variables in India Type II Diabetic Patients”. Lipids in health and
diseases 2005, 4:15.
76. Naciye Kurtul, M. Yasar Cil and Sefa Dogruluk Pacac. “Serum total Sialic
acid levels in smokers and users of smokeless tobacco in form of Oral powder
(maras powder).Journal of Biomedical science (2005)12:559-563.
77. Mark C Plucinsky, PhD, W.Michael Riley, M S, Joseph J. Prorok, Md and
Jack A. Alhadeff, PhD. “Total and lipid associated serum Sialic acid levels in
cancer patients with different primary sites and differing degrees of metastatic
involvement”. Cancer 58:2680, 1986.
78. Nilabja Sikdar, Ranjan Rashmi Paul, Chnmay K. Panda, Sanat K. Banerjee,
Bidyut Roy. “Loss of heterozygosity at APC and MCC genes of oral cancer
and Leukoplakia tissues from Indian tobacco chewers”. J Oral Pathol Med
2003:32:450-4.
81
BIBILOGRAPHY
79. Shiu.M.N, Chen.T.H.H, Chang.S.H and Hahn.L.J. “Risk factors for
Leukoplakia and malignant transformation to oral carcinoma: a Leukoplakia
cohort in Taiwan”. British Journal of Cancer (2000)82(11), 1871-1874.
82
ANNEXURES
ANNEXURES
CASE REPORT
I.
DEMOGRAPHIC DATA:
1. Name
2. Age
3. Sex
4. Adress
5. Occupation
II.
CHIEF COMPLAINT:
III.
HISTORY OF PRESENTING ILLNESS:
IV.
HISTORY OF PAST ILLNESS:
1. Medical
2. Dental
3. Family History
V.
PERSONAL HISTORY:
VI.
CLINICAL EXAMINATION:
VII.
GENERAL EXAMINATION:
VIII. VITAL SIGNS:
1. Pulse
2. Temperature
3. Blood Pressure
4. Respiratory Rate
IX.
LYMPH NODE EXAMINATION:
X.
LOCAL EXAMINATION:
1. EXTRA ORAL EXAMINATION:
A. Inspection
B.Palpation
83
ANNEXURES
XI.
INTRA ORAL EXAMINATION:
1. SOFT TISSUE EXAMINATION:
A. Inspection
B.Palpation
2. HARD TISSUE EXAMINATION:
A. Inspection
B.Palpation
XII.
PROVISIONAL DIAGNOSIS:
XIII. INVESTIGATIONS:
1. Haematological
2. Biopsy
3. Serum Sialic Acid Estimation
XIV. PHYSICIAN OPINION:
84
ANNEXURES
CONSENT FORM
I
…………………………
son
/
daughter
of
…………………
aged…………………., resident of ………………………. Being under the treatment
of ……………………….. Do hereby give consent to the performance of diagnostic
procedure of…………………………… upon myself. The procedure has been
explained to me in my language and I will be solely responsible for any complication
arising out of such procedure. I agree that no responsibility will be attached to the
surgeon or the hospital staff.
Place:
Date:
Signature of the patient/ parent
Signature of witnesses
1.
2.
85
ANNEXURES
1. TOTAL SIALIC ACID IN LEUKOPLAKIA
SL
NO
NAME
AGE /
SEX
HISTOLOGICAL
DIAGNOSIS
OPTICAL
DENSITY
LEUKOPLAKIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITH MILD
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
LEUKOPLAKIA WITHOUT
DISPLASIA
0.332
0.25
SIALIC
ACID IN
mg/dl
78
60
0.19
45
0.41
97
0.383
90
0.21
50
0.27
64
0.35
83
0.245
57.5
0.45
107
0.21
50
0.25
60
0.395
93
0.412
97
0.36
85
0.26
62
0.31
74
0.38
90
0.35
83
0.32
76
0.29
69
0.38
0.4
90
90
0.27
64
0.38
90
1
2
BIMARAYA
SATTAR
58/M
68/M
3
YOGESH
28/M
4
GANGADHAR
37/M
5
RAJESH
30/M
6
SHARIFF
30/M
7
SAMPATH KUMAR
54/M
8
RAMACHANDRA
43/M
9
KUMAR
32/M
10
RAMACHANRACHARI
61/M
11
CHANDU
35/M
12
AMEER
25/M
13
ISMAIL
39/M
14
RAGHU
62/M
15
CHANDRAIAH
64/M
16
GOUTHAM
25/M
17
LAKSHMINARAYAN
63/M
18
SUKANYA
46/F
19
SHIVAKUMAR
36/M
20
SRINIVAS
30/M
21
AJAYKUMAR
30/M
22
23
LEELAVATHI
PARVATAMMA
49/F
65/F
24
SHAMANNA
38/M
25
SHARADAMMA
55/F
86
ANNEXURES
2. TOTAL SIALIC ACID IN SQUAMOUS CELL CARCINOMA (SCC)
SL
NO
NAME
AGE/
SEX
1
2
3
MAHADEV GOWDA
PUTTAMMA
BHASKAR
51/M
60/F
32/M
4
RANGASHREE
45/F
5
6
HANUMANTH RAJ
BABU
50/M
45/M
7
KVR SHETTY
69/M
8
RAMANJAPPA
55/M
9
AROGYAMMA
70/F
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
MODERATELY
DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
MODERATELY
DIFFERENTIATED SCC
POORLY DIFFERENTIATED SCC
MODERATELY
DIFFERENTIATED SCC
0.23
0.23
0.33
SIALIC
ACID IN
mg/dl
54
54
78
0.425
100
0.32
0.323
76
76
0.39
94
0.5
120
0.3
71
HISTOLOGIC DIAGNOSIS
OPTICAL
DENSITY
36/M
WELL DIFFERENTIATED SCC
0.402
95
11
12
13
14
SHANKAR
HUNDEKAR
MUNIYAMMA
MUNIYAMMA
KEMPAMMA
SHANTAMMA
45/F
55/F
55/F
50/F
0.41
0.42
0.42
0.45
98
100
100
107
15
SRINIVAS
40/M
0.52
125
16
17
18
19
ROJA
SHARADAMMA
BASAPPA
NAGARATNA
38/F
48/F
50/M
45/F
0.43
0.41
0.42
0.43
103
98
100
103
20
HONNEGOWDA
48/M
0.45
105
21
22
NARASAIAH
VICHITHI
68/M
65/M
0.49
0.5
117
120
23
PRABHAVATI
60/F
0.4
95
24
LAKSHMAMMA
48/F
0.41
98
25
VENKATAMMA
60/F
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
MODERATELY
DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
MODERATELY
DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
POORLY DIFFERENTIATED SCC
EARLY INVASIVE
DIFFERENTIATED SCC
WELL DIFFERENTIATED SCC
MODERATELY
DIFFERENTIATED SCC
0.44
105
10
87
ANNEXURES
SL
NO
NAME
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
KAMALA
LAKSHMIDEVI
USHA
CHANDRAKALA
SUSHEELA
SHASHIRABBAS
MAHALAKSHMI
VASANTH KUMAR
INDIRA
KRISHNA URTHY
SATISH
REVANNA
MADHUSHARMA
MANJUNATH
PADMA
SHAKUNTALA
RAJU
LATHA
MATHUR GOWDA
RAMARAJU
RAMAIAH
DORAISWAMY
CHANNAMMA
NARAYAN RAO
VEERANNA
3. TOTAL SIALIC ACID IN CONTROLS
AGE/
OPINION FROM
OPTICAL
SEX
PHYSICIAN
DENSITY
70/F
60/F
28/F
21/F
30/F
60/M
45/F
55/M
55/F
41/M
26/M
35/M
20/M
31/M
50/F
41/F
45/M
35/F
48/M
37/M
40/M
60/M
40/F
74/M
65/M
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
TAKEN
88
0.27
0.26
0.29
0.27
0.23
0.25
0.24
0.23
0.23
0.23
0.25
0.25
0.26
0.22
0.23
0.23
0.25
0.23
0.25
0.27
0.26
0.28
0.24
0.28
0.27
SIALIC
ACID IN
mg/dl
65
63
69
64
55
60
58
55
55
55
60
60
63
51.5
55
55
60
55
60
65
63
68
58
68
65