STUDY OF EXPRESSION OF HYPOXIA INDUCIBLE FACTOR - 1 …

STUDY OF EXPRESSION OF HYPOXIA INDUCIBLE

FACTOR - 1 ALPHA IN THE RISK PREDICTION OF ORAL

EPITHELIAL DYSPLASIA AND IN ORAL SQUAMOUS CELL

CARCINOMA - AN IMMUNOHISTOCHEMISTRY STUDY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfilment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH VI

ORAL PATHOLOGY AND MICROBIOLOGY

APRIL 2016

Acknowledgement

ACKNOWLEDGEMENT

I bow in gratitude to Almighty and my mother for all her showers of

blessings.

My heartfelt gratitude to my teacher Dr. K. Ranganathan, MDS., MS

(Ohio), Ph.D. , Professor and Head of Department of Oral and Maxillofacial

Pathology, Ragas Dental College and Hospital for his constant support and

encouragement throughout my course. I consider myself extremely lucky to

have had the opportunity to study under him. He has always been a source of

inspiration to perform better not only in academics but also in life. Thank you

so much sir.

I extend my sincere gratitude to Dr. UmaDevi. K. Rao, Professor,

Department of Oral and Maxillofacial Pathology, Ragas Dental College and

Hospital for her constant guidance, support and her advice in completion of

my work. I want to take this opportunity to acknowledge and thank her for the

help and support .Thank you mam.

My sincere thanks to Dr. Elizabeth Joshua, Professor, Department of

Oral and Maxillofacial Pathology, Ragas Dental College and Hospital for her

constant support and encouragement throughout my study. She has always

been like a friend in guiding me in academics. Thank you mam.

I earnestly thank Professor, Dr.T. Rooban, Department of Oral and

Maxillofacial Pathology, Ragas Dental College and Hospital for his

encouragement and concern in helping me to complete this study.

My sincere thanks to the principal Dr. S. Ramachandran and

Chairman Mr. Kanakaraj, Ragas Dental College and Hospital for their

permission to use the facilities of the institution.

I extend my sincere thanks to Readers Dr. N. Lavanya and Dr. C.

Lavanya, Department of Oral and Maxillofacial Pathology, Ragas Dental

College and Hospital for their encouragement and support .

I extend my sincere thanks to Senior lecturer Dr. Sudharsan,

Dr. Kavitha, Department of Oral and Maxillofacial Pathology, Ragas Dental

College and Hospital for his help throughout my project.

I am very grateful to our Geneticist and Lab Manager, Mrs. Kavitha,

who helped a lot throughout my dissertation procedure.

I extend my sincere thanks to our Biostatistician, Ms. Aarthi, Lab

Technician, Mr. Rajan, Department of Oral and Maxillofacial Pathology and

Attender Mrs .Vasanthi, Ragas Dental College and Hospital for their constant

help in completion of my study.

I acknowledge gratefully the help of my batchmates Dr.Deepasri,

Dr. Saranya, Dr. Divya, Dr. Angaiyarkkanni and Dr. Joseph. I also thank

Ambika mam, Jayasri, Saranya, Revathi, Roxana and Vasanthi for their

support and encouragement.

I am dedicating this work to my father Mr. Kamarajan and my

husband Mr. C.V. Lenin and to my kids Keona and Ronald. I thank them for

all their sacrifices, understanding, support and constant encouragement.

ABSTRACT

Background:

Oral squamous cell carcinoma (OSCC) is a multistep process which

includes combined effects of individual genetic predisposition and exposure to

environmental carcinogens. The rapidly proliferating tumor has insufficient

vascularization which limits oxygen diffusion resulting in the development of

hypoxic region. Hypoxic micro-environment of tumor cell is an important step

in the progression of OSCC with involvement of hypoxia inducible factor

1 alpha (HIF-1 alpha).

Aim and Objectives:

To study the expression of HIF-1 alpha in mild, moderate and severe

epithelial dysplasia, OSCC and normal mucosa by immunohistochemistry

(IHC).

Material and Methods:

Immunohistochemical detection of HIF-1 alpha was done using

polyclonal antibody and Poly Excel HRP/DAB chromogen detection system

on 60 samples, which included mild epithelial dysplasia (15 cases), moderate

and severe dysplasia (15 cases), oral squamous cell carcinoma

(OSCC-15 cases) and the expression was compared with that of normal

mucosa (15 cases). The positive control used for HIF-1 alpha was human

normal kidney.

Results:

The pattern of HIF-1 alpha staining in all the cases (N=60) was

cytoplasmic. All the cases of OSCC showed 100% positivity for HIF-1 alpha

expression. 93.3% of mild epithelial dysplasia and moderate to severe

dysplasia showed positive expression when compared to normal which was

80% positive. Tissue localization of HIF-1 alpha stain in the suprabasal,

keratin and connective tissue increased from normal mucosa to increasing

grades of epithelial dysplasia. All the cases of well differentiated and poorly

differentiated OSCC expressed HIF-1 alpha. 66.6% and 33.3% of moderately

differentiated carcinoma expressed mild and moderate intensity respectively.

Conclusion:

Compared to that of normal mucosa, HIF-1 alpha expression was

increased with increasing grades of epithelial dysplasia and increasing grades

of OSCC. This indicates that HIF-1 alpha expression was found to increase

with the severity of the lesion. Hence HIF-1 alpha could be considered as one

of the marker comparing the stages of dysplasia.

Key words: HIF-1 alpha, hypoxic micro-environment, oral epithelial

dysplasia

CONTENTS

1. INTRODUCTION 1

2. AIM AND OBJECTIVES 3

3. MATERIALS AND METHODS 5

4. REVIEW OF LITERATURE 15

5. RESULTS 45

6. DISCUSSION 55

7. SUMMARY AND CONCLUSION 64

8. BIBLIOGRAPHY 66

9. ANNEXURES

I - Institutional Review Board approval form

II - Dissertation protocol

III - Primary antibody

IV - Secondary antibody

V - Department declaration form

VI - Plagiarism check form

VII - Abbreviations

Introduction

Introduction

1

Head and neck cancer is a major problem globally and a significant

problem in the Indian subcontinent. More than two lakh new cases of head and

neck cancers are diagnosed each year in India. India contributes up to 7.8% of

the global cancer burden and 8.33% of global cancer deaths. In developed

countries around 40% of patients present with advanced disease whereas in

developing countries like India around 60% of patients have advanced disease

at the time of presentation1.

Epithelial dysplasia is a step that often precedes squamous cell

carcinoma2. The term “dysplasia” is generally employed in the sense of a

disordered development. In a stratified squamous epithelium, architectural

disturbances affecting normal maturation and stratification may occur. When

such alterations are accompanied by cytological atypia, the term “dysplasia” is

applied2.

Although it is established that oral potentially malignant lesions with

epithelial dysplasia are statistically more likely to progress to cancer, the

actual mechanisms are poorly understood3.

The degree of dysplasia is a best guide to the potential progression of

oral lesions. Severe epithelial dysplasia has an overall malignant

transformation rate around 7–50%4. Moderate dysplasias have a malignant

transformation potential of 3–15%, whereas mild epithelial dysplasia shows a

relatively low risk (5%). Potential markers of progression to malignancy

include analysis of p53 mutations and loss of heterozygosity. Gross genomic

Introduction

2

aberrations that can be assessed by DNA ploidy status also have promise as a

predictor of malignant progression4.

WHO (2005) defined leukoplakia as “a white plaque of questionable

risk having excluded (other) known diseases or disorders that carry no

increased risk for cancer.”

Hypoxia

Hypoxia is a condition in which the body or a region of the body is

deprived of adequate oxygen supply. Generalized hypoxia occurs in healthy

people at high altitudes, where it causes altitude sickness. The oxygen binding

capacity of hemoglobin is influenced by the partial pressure of oxygen in the

environment. A small amount of oxygen is transported in solution in the

blood.

HIF-1 alpha is upregulated in oral epithelial dysplasia and oral

submucous fibrosis at both protein and mRNA levels in hypoxic conditions.

A statistically significant correlation with the degree of epithelial dysplasia

and expression level of the gene HIF-1 alpha has been reported. HIF-1alpha is

a known transcription factor induced by hypoxia and has been proposed that

hypoxia together with HIF-1 alpha play a significant role in malignant

transformation5.

This study was done to ascertain the expression of HIF 1 alpha in

leukoplakia, OSCC and normal mucosa by immunohistochemistry (IHC).

Aim and Objectives

Aim and Objective

3

AIM

To evaluate the expression of hypoxia inducible factor (HIF) -1 alpha

in oral epithelial dysplasia and oral squamous cell carcinoma.

OBJECTIVES

1. To study the expression of HIF-1 alpha in formalin fixed paraffin

embedded tissue specimen of mild epithelial dysplasia by

Immunohistochemistry.


embedded tissue specimen of moderate and severe epithelial dysplasia

by Immunohistochemistry.


embedded tissue specimen of OSCC by Immunohistochemistry.


embedded tissue specimen of normal mucosa by


5. To compare the expression of HIF-1 alpha in mild, moderate, severe

epithelial dysplasia, OSCC and normal oral mucosa by


Aim and Objective

4

HYPOTHESIS (NULL)

There is no difference in expression of HIF-1 alpha in mild, moderate,

severe epithelial dysplasia and OSCC when compared to normal mucosa.

STUDY SETTING

The study was conducted in the Department of Oral and Maxillofacial

Pathology, Ragas Dental College and Hospital, Chennai, using archival

formalin-fixed-paraffin embedded blocks of mild, moderate, severe epithelial

dysplasia, OSCC and normal mucosa using IHC. This study was approved by

Institutional Review Board (IRB) of Ragas Dental College and Hospital,

Chennai (Annexure I).

STUDY SUBJECTS:

The study material comprised of 60 formalin-fixed, paraffin embedded tissue

specimens. The samples were divided into 4 groups namely: Group I, Group

II, Group III and Group IV.

Group I: 15 normal oral mucosal tissue specimens.

Group II: 15 histopathologically confirmed mild epithelial dysplasia

tissue specimens

Group III: 15 histopathologically confirmed moderate and severe

epithelial dysplasia tissue specimens

Group IV: 15 histopathologically confirmed OSCC tissue specimens

Materials and Methods


5

1. Tissue samples of normal oral mucosa (n=15), mild epithelial

dysplasia (n=15), moderate and severe dysplasia (n=15) and OSCC

(n=15) were taken from the archival blocks.

2. A detailed case history including patient’s age, gender, occupation,

past medical and dental history, history of drugs and trauma were

recorded.

3. Extra oral and intra oral examination was done.

4. Biopsy was performed from the lesional site. Normal oral mucosa was

obtained when the patients were undergoing minor surgery for

impacted teeth cases.

5. The tissue biopsied was immediately transferred to 10 % buffered

formalin.

6. After adequate fixation, tissues were embedded in paraffin.

7. From the paraffin embedded blocks 4 microns thick, sections were cut

and used for routine hematoxylin and eosin (H & E) staining and

immunohistochemical staining.

8. Tissue sections of normal kidney were used as positive control for

HIF-1 alpha positivity.


6

HEMATOXYLIN AND EOSIN STAINING:

REAGENTS

Harris hematoxylin

1% acid alcohol

Eosin

Xylene

Alcohol

PROCEDURE

The slides were dewaxed in xylene and hydrated through three grades

of alcohol and then to water. The sections on the slides were flooded with

Harris hematoxylin for 5 minutes. The slides were washed in running tap

water for 5 minutes. Then the slides were differentiated in 1% acid alcohol for

5 minutes followed by running tap water wash for 5 minutes. The tissue

sections on the slides were then stained in eosin for 30 seconds and washed

again with running tap water for 1minute. The slides were then dehydrated

through alcohol, cleared, mounted and viewed under light microscope.


7

IHC STAINING:

Armamentarium for HIF-1 alpha

Microtome

Autoclave

Hot air oven

Slide warmer

Couplin jars

Measuring jar

Weighing machine

APES coated slides (Amino propyl triethoxysilane)

Slide carrier

Aluminium foil

Micro-pipettes

Toothed forceps

Electronic timer

Beakers

Rectangular steel tray with glass rods

Sterile gauze

Cover-slips

Light microscope


8

REAGENTS USED

1. Concentrated HCl

2. APES (3 amino propyl tri ethoxysilane)

3. 1N sodium hydroxide

4. 1N HCl

5. Citrate buffer

6. Phosphate buffer solution

7. 3% H2O2

8. Deionized water

9. Distilled water

10. Hematoxylin

11. Absolute alcohol (Isopropyl alcohol)

12. Xylene

ANTIBODIES USED

1. Primary antibody – Anti HIF-1 alpha rabbit polyclonal antibody-

Abcam

2. Secondary antibody – Poly Excel-HRP Micro polymer IHC

detection system-Vkan Life Care

3. Chromogen – DAB-3, 3’- di amino-benzidine tetrahydrochloride

Vkan Life Care


9

PREPARATION OF PARAFFIN SECTIONS

After the slides were dried, tissue sections of 4 micron thickness were

made in a rotary manual microtome. The ribbons of tissue section were

transferred onto the APES coated slides from the tissue float bath such that

two tissue bits come onto each slide with a gap in between. One of the tissue

sections towards the frosted end of the slide was labeled positive to which the

primary antibody, secondary antibody and chromogen were added and the

tissue section away from frosted side is the negative to which primary

antibody is not added. Instead buffer is added to prevent the drying of tissue

which is followed by the addition of the secondary antibody and then the

chromogen was added.

IHC PROCEDURE

The slides with tissue sections were treated with three changes of

xylene to remove paraffin wax which is called as deparaffinisation. They were

put in descending grades of alcohol (100% absolute alcohol, 90% and 70%

alcohol) and then to phosphate buffer solution. The slides were transferred to

citrate buffer of pH 6 and steamed in a pressure cooker for antigen retrieval at

15 lbs pressure for 15 minutes. Slides were washed two times with phosphate

buffer solution and then treated with 3% hydrogen peroxide for 5 minutes to

quench endogenous peroxidase activity of cells that would otherwise result in

non-specific staining. Again it was washed 2 times with phosphate buffer


10

solution. Circles were drawn using a diamond marker around the tissues, so

that the antibodies added later on do not spread and are restricted to the circle.

The slides were wiped carefully without touching the tissue section. The

primary antibody, anti HIF 1 alpha antibody was diluted in 1: 100 dilutions

and was added to positive tissue on the slide and to the normal tissue citrate

buffer was added. The slides were incubated for overnight at 40C in the

refrigerator. Next day the slides were washed in phosphate buffer solution for

two times and then wiped carefully without touching the tissue section to

remove excess of antibody. Then target binder reagent is added and incubated

for 12 minutes. The slides washed with phosphate buffer solution for two

times in 2 minutes each and were wiped carefully without touching the tissue

section to remove excess buffer solution. Then a drop of poly horseradish

peroxidase was added on both the sections and the slides were incubated for

30 minutes. Later the slides washed with phosphate buffer solution for two

times in 2 minutes each and were wiped carefully without touching the tissue

section to remove excess buffer solution. A drop of DAB was added to the

sections and incubated for 10 minutes. Slides were then washed in phosphate

buffer to remove excess chromogen and counter stained with hematoxylin.

Then the slides were shown under tap water wash followed by drying and one

change of xylene. The tissue sections were mounted with DPX. The slides

were then observed under the microscope. Throughout the procedure care was

taken not to dry the tissues.


11

POSITIVE CONTROL

A case of normal kidney specimen known to express HIF-1 alpha

positive cells were fixed, processed, embedded, sectioned, stained in same

manner and used as positive control. One positive control tissue slide was

included for each batch of staining. The tissue section away from the frosted

end of the slide was taken as the negative control.

IHC PROCEDURE:

1. APES coated slides with 2 paraffin embedded tissue

2. Placed in xylene twice (5 minutes each)

3. Placed in 100% isopropanol twice (5 minutes)

4. Placed in 90% isopropanol (5 minutes)

5. Placed in 70% isopropanol (5 minutes)

6. Washed in phosphate buffer twice (2 minutes each)

7. Kept in citrate buffer at pH 6 for antigen retrieval

8. Placed in 3% hydrogen peroxide (5 minutes)

9. Washed with phosphate buffer twice (2-3 minutes)

10. Primary antibody added and incubated at 40C (overnight)


12

11. Washed in phosphate buffer (2-3 minutes)

12. Poly excel target binder reagent added and incubated (12 minutes)

13. Washed in phosphate buffer twice (2-3 minutes)

14. Poly excel HRP added and incubated (30 minutes)

15. Washed slides in phosphate buffer twice (2-3 minutes)

16. DAB added and incubated in an enclosed in hydrated container

(10 minutes)

17. Washed in phosphate twice (2-3 minutes)

18. Stained with Harris Hematoxylin (30 seconds)

19. Washed in tap water

20. Placed in xylene (1 dip)

21. Slides to be mounted using DPX

22. Slides to be observed under the LM and graded


13

CRITERIA FOR EVALUATION OF HIF-1 ALPHA STAINING:

TISSUE LOCALISATION:

HIF-1 alpha staining was present in basal, suprabasal, keratin and

connective tissue and hence its intensity was analyzed in the above mentioned

layers in the study groups.

CELLULAR LOCALIZATION OF STAIN

The normal localization of the staining includes both nucleus and

cytoplasm. In our study the stain is localized to the cytoplasmic region. The

stained slides were screened, examined systematically for HIF-1 alpha

expression in the cytoplasmic portion.

STAINING PATTERN OF HIF-1 ALPHA

The evaluation of expression included the staining intensity. The intensity was

evaluated based on the intensity in the control section of normal kidney using

IHC. Staining intensity evaluation was given using a 0 to 3 scale.

0 - negative

1- mild

2-moderate

3- intense


14

STATISTICAL ANALYSIS

Data were entered and analyzed using SPSSTM

software

(version 20.0). Pearson’s Chi-square test was done to compare intensity of

staining between the groups. p value ≤ 0.05 was considered statistically

significant. Kappa analysis was done to compare the intensity of HIF-1 alpha

staining as observed between three observers.

Review of Literature


15

ORAL EPITHELIAL DYSPLASIA

Dysplasia refers to a series of subtle changes in cell signifying that

anaplasia will soon develop. Dysplasia is a premalignant change. It is a change

in the tissue level whereas atypia is a change at cellular level. The alteration in

dysplasia includes genetic changes, epigenetic changes and surface alterations.

The genetic change involves complex process due to interaction of host with

the carcinogen in the environment and includes activation of proto-oncogenes

and inactivation of tumour suppressor genes and genomic instability genes.

Epigenetic changes include heritable changes in the gene expression that occur

without alteration in the DNA sequence.

The surface alteration includes cellular adaptations, reversible and

irreversible changes. The reversible changes are reversible if causative factors

are removed. If they persist, dysplastic cells escapes normal homeostatic

control and assume the autonomy of tumor cells. The irreversible changes is

characterised by accelerated cell division, which facilitates accumulation of

genetic damage and further drives toward path of transformation and lead to

cell death or neoplastic transformation6.

The diagnosis and grading of oral epithelial dysplasia is based on a

combination of architectural and cytological changes, but evaluation of these

is subjective and has been subject to considerable inter- and intra-observer

variations in the grading of lesions.


16

The architectural features that should be addressed are7

Loss of polarity

Disordered maturation from basal to squamous cells

Includes top-to-bottom change of carcinoma in situ

Increased cellular density

Basal cell hyperplasia

Dyskeratosis

Bulbous drop shaped rete pegs

Increased number of mitotic figures / abnormally superficial mitoses

The cytological alterations to be observed are as follows

Nuclear pleomorphism

Cellular pleomorphism

Anisonucleosis

Anisocytosis

Increased nuclear size

Increased nuclear-cytoplasm ratio


17

Atypical mitotic figures

Increased number and size of nucleoli

The observation of these alterations should be done considering the

epithelium divided into “thirds”. Accordingly, lesions should be classified into

five categories, as described below7, 8

.

1. Hyperplasia: describes a lesion showing an increase in cell number in

the spinous layer and/or in the basal/parabasal cell layers. There is

regular stratification and no cellular atypia.

2. Mild dysplasia: (grade I) demonstrates proliferation or hyperplasia of

cells of the basal and parabasal layers which does not extend beyond

the lower third of the epithelium.

3. Moderate dysplasia: (grade II) demonstrates a proliferation of

atypical cells extending into the middle one-third of the epithelium.

The cytological changes are more severe than in mild dysplasia and

changes such as hyperchromatism and prominent cell and nuclear

pleomorphism may be seen. Increased and abnormal mitoses may be

present, but these are usually located in the basal layers.

4. Severe dysplasia: (grade III) there is abnormal proliferation from the

basal layer into the upper third of the epithelium. Cytological and

architectural changes can be very prominent. Pleomorphism often with

abnormally large nuclei with prominent or even multiple nucleoli.


18

Prominent and suprabasal mitoses are usually evident and abnormal

tripolar or star-shaped forms may be seen. Apoptotic bodies may also

be prominent. Architectural changes are severe, often with complete

loss of stratification and with deep abnormal keratinisation and even

formation of keratin pearls.

5. Carcinoma in situ: Theoretically, indicates that malignant

transformation has occurred but invasion has not. Full or almost full

thickness architectural disturbance in viable cellular layers with

pronounced cellular atypia. Atypical mitotic figures and abnormal

superficial mitoses are common7 , 8

.

Risks associated with smoking and drinking are not necessarily

constant over the multistage pathway to oral cancer. They investigated

whether smoking and drinking patterns differ for persons with oral cancer

(OC) relative to those with oral epithelial dysplasia (OED), a precancerous

condition by conducting a questionnaire containing questions on smoking and

drinking. Odds ratios (ORs) compared the odds of smoking and drinking

among persons with OC relative to OED and this was found to be statistically

significant( Morse DE et al, 2007)9.

The effects of betel nut chewing, smoking and alcohol on the

occurrence of leukoplakia and its malignant transformation to oral carcinoma

were quantified in a leukoplakia cohort (n = 435) from one medical centre

between 1988 and 1998 in Taiwan. Sixty oral carcinomas were ascertained in


19

this cohort. A case–control study within the leukoplakia cohort was used to

study, risk factors. Using the Weibull survival model, the incidence of

malignant transformation of leukoplakia was shown to increase with follow-up

years. After adjustment for other relevant risk factors, betel nut chewing

(adjusted odds ratio (OR) = 4.59; 95% confidence interval (CI) 1.25–16.86)

remained a significant risk factor for malignant transformation. Results from

the case–control study showed that the adjusted odds ratios for betel nut

chewing and smoking on the occurrence of leukoplakia were 17.43

(95% Cl 1.94–156.27) and 3.22 (95% Cl 1.06–9.78), respectively. Similar

findings were observed when daily frequency and duration were taken into

account. This implies that cessation of smoking may reduce by

36% leukoplakia cases, while elimination of betel nuts may prevent 62% of

leukoplakia and 26% of malignant transformation to oral carcinoma in the

underlying population10

.

LEUKOPLAKIA

In 2012 Van Der Waal proposed as “A predominantly white lesion or

plaque of questionable behavior having excluded, clinically and

histopathologically, any other definable white disease or disorder”.

Only about 50% of biopsied leukoplakias show dysplasia and overall

the malignant transformation rate for leukoplakia is only about 0.1–2% per

year. Ironically rates are lower in the developing world where tobacco


20

chewing habits are most prevalent. In the west malignant transformation is

estimated at about 5% of leukoplakias. Higher rates of about 20% have been

reported in non-homogeneous lesions, which are also more likely to show

dysplasia on biopsy.

The cohort studies about oral leukoplakia are very rare, so it is difficult

to appreciate its real malignant transformation rate due to various regional

habits. Average of about 1% have been reported annually in different

populations11,12

.

The risk factors for malignancy are vicious habits (smoking, alcohol

intake), clinical form, location of lesions were studied. Among them, tobacco

cigarette smoking was reported to be the most important etiological factor for

the development of oral premalignant lesions and to their progression into oral

carcinoma13

.

According to Nasser W et al., 36% of dysplastic lesions and 16% of

non-dysplastic lesions progress to carcinoma. However it is known that

epithelial dysplasia is correlated with clinically heterogeneous lesions that are

considered to have the greatest risk. Expression of p53 and loss of expression

of p16 are shown to be the earliest events in the process of malignant

conversion. In non-dysplastic leukoplakia a combined alteration of

p53/Ki67/p16INK4a was proven to be a risk of progression14

.


21

Genetic Susceptibility to malignant transformation

Genomic instability denotes early genetic events during malignant

transformation causing Loss of Heterozygosity (LOH) and chromosomal copy

number abnormality. Considering the well known fact that chromosome 13q is

highly susceptible to genomic instability in HNSCC, they hypothesized that

genes within the 13q14–q33 LOH region may play essential roles in the

initiation of oral carcinogenesis. The other susceptibility regions in HNSCC

also include 3p24-p22, 6q26-q27, 9q22.3, 12p11.2, and 20p12-11. These LOH

loci were found to harbor numerous oncogenes and tumour suppressor genes

involved in the regulation of hypoxia, cellular adhesion/migration, matrix

remodeling, cell cycle/apoptosis, DNA repair and inflammation. This

acquisition of LOH may subsequently alter gene function and expression15,16

.

ORAL SQUAMOUS CELL CARCINOMA

Carcinogenesis can be divided conceptually into four steps: tumor

initiation, tumor promotion, malignant conversion, and tumor progression.

Carcinogenesis requires the malignant conversion of benign hyperplastic cells

to a malignant state, and invasion and metastasis are manifestations of further

genetic and epigenetic changes17

.

Molecular Changes in Oral Cancer

Cancer occurs through multiple steps, each characterized by the

sequential stimulation of additional genetic defects, followed by clonal


22

expansion. The genetic alterations observed in head and neck cancer are

mainly due to oncogene activation and tumor suppressor gene inactivation,

leading to deregulation of cell proliferation and death. These genetic

alterations include gene amplification and overexpression of oncogenes such

as myc, erbB-2, Epidermal Growth Factor Receptor (EGFR), cyclin D1 and

mutations, deletions and hypermethylation leading to p16 and p53 tumor

suppressor gene inactivation18

.

Regulators

Growth regulators and TSGs act as transducers of negative growth

signals. The TSG p53, is called as „Guardian of the Genome‟, having a role in

maintaining genomic stability, cell cycle progression, cellular differentiation,

DNA repair and apoptosis. The gene can be inactivated by several

mechanisms, including point mutations, deletions and binding with cellular

and viral proteins18

.

Genomic instability

The risk of progression from premalignancy to cancer is low, when no

genetic changes were seen, intermediate, if there is genetic loss on the short

arms of chromosomes 3 and 9 (3p and9p) and high, if there is 3p and 9p loss

accompanied by genetic loss on additional chromosome arms, including

4q, 8p, 11q, 13q and 17p18

.


23

Role of deregulated apoptosis in the pathogenesis of oral cancer

Evasion of apoptosis via abnormal expression of bcl-2, mcl-1 and p53,

may contribute to oral cancer pathogenesis18

.

Enhanced telomerase activity

Telomerase activity is not seen in any of the normal tissues, but it was

found in 100% of the cell lines, 90% of the invasive neoplasia and 100% of

the dysplastic lesions18

.

Neovascularization

The steps critical to successful angiogenesis include the degradation of

the extracellular matrix, endothelial cell proliferation, migration and assembly

of endothelial cells into higher order structures. In the majority of cancers,

highly vascularized tumors showed a poor prognosis18

.

HYPOXIA INDUCIBLE FACTOR – 1 ALPHA

The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key

regulator responsible for the induction of genes that facilitate adaptation and

survival of cells and the whole organism from normoxia (21% O2) to hypoxia

(1% O2)19

.


24

The Discovery of HIF-1

HIF-1 was discovered by the identification of a hypoxia response

element (HRE; 5-RCGTG-3) in the gene for erythropoietin (EPO), a hormone

that stimulates erythrocyte proliferation and undergoes hypoxia induced

transcription. HIF-1 alpha is also known as the aryl hydrocarbon nuclear

translocator (ARNT), which was originally identified as a binding partner of

the aryl hydrocarbon receptor. These proteins belong to the basic helix-loop-

helix– Per-ARNT-Sim (bHLH–PAS) protein family. The bHLH and PAS

motifs are required for heterodimer formation between the HIF-1 and HIF-

1subunits, and the downstream basic region affords specific binding to the

HRE DNA sequence19

.

Two transactivation (stimulation of transcription) domains, N terminal

(N-TAD) and C-terminal (C-TAD) was identified. HIF-1 alpha also contains

an oxygen-dependent degradation domain (ODDD) that mediates oxygen-

regulated stability. Later work revealed that HIF-1 is ubiquitously expressed in

human and mouse tissues and has a general role in multiple physiological

responses to hypoxia, such as erythropoiesis and glycolysis, which quickly

counteract oxygen deficiency and angiogenesis, which provides a long-term

solution19

.


25

Domain structure of human HIF

The Regulation of HIF-1

Although HIF-1 is constitutively expressed and its mRNA and protein

are maintained at constant levels regardless of oxygen availability, HIF-1

protein has a short half-life (t1/2-5 min) and is highly regulated by oxygen.

The transcription and synthesis of HIF-1 are constitutive and seem not to be

affected by oxygen. However, in normoxia, the HIF-1 proteins are rapidly

degraded, resulting in essentially no detectable HIF-1 protein. During hypoxia,

HIF-1 becomes stabilized and translocates from the cytoplasm to the nucleus,

where it dimerizes with HIF-1 and the HIF complex formed becomes

transcriptionally active. The activated HIF complex then associates with HREs

in the regulatory regions of target genes and binds the transcriptional

co-activators to induce gene expression. In addition, hydroxylation of an


26

asparagine residue in the C-TAD inhibits the association of HIF-1 with

CBP/p300 and thus inhibits its transcriptional activity19.

The Target Gene of HIF-1

Given that cells and organs need to adapt to changes in oxygen supply,

it would not be surprising to find that a significant variety of the HIF-1 target

genes are regulated in a tissue-specific manner. To date, there are more than

100 HIF-1 downstream genes identified with varying functions. HIF-1

activates the expression of these genes by binding to a 50-base pair cis-acting

HRE located in their enhancer and promoter regions19

.

Angiogenesis

Angiogenesis is a complex process that involves multiple gene

products expressed by different cell types. A large number of genes involved

in different steps of angiogenesis have been shown to increase by hypoxia

challenge. Among them, the vascular endothelial cell growth factor (VEGF) is

the most potent endothelial-specific mitogen and it directly participates in

angiogenesis by recruiting endothelial cells into hypoxic and avascular area

and stimulates their proliferation. Therefore, the induction of VEGF and

various other pro-angiogenic factors leads to an increase in the vascular

density and hence a decrease in the oxygen diffusion distance19

.


27

Glucose Metabolism

Under low oxygen supply, cells switch their glucose metabolism

pathway away from the oxygen-dependent tricarboxylic acid (TCA) cycle to

the oxygen-independent glycolysis. With only 2 ATP molecules from each

glucose molecule produced by glycolysis, instead of 38 ATP provided by TCA

cycle, hypoxic cells elevate their ability to generate ATP by increasing the

glucose uptake. This is achieved by up-regulating the expression of glycolytic

enzymes and glucose transporters. Hypoxia and HIF-1 increase virtually all

the enzymes in the glycolytic pathway, as well as the glucose transporters

1 and 3 (GLU1, GLU3). Furthermore, the glycolysis metabolic products such

as lactate and pyruvate have been reported to cause HIF-1 accumulation under

normoxia and regulate hypoxia-inducible gene expression hence establishing a

potential positive feedback loop19

.

Cell Proliferation/Survival

Hypoxia and HIF-1 induce growth factors such as insulin-like growth

factor-2 (IGF2) and transforming growth factor (TGF). Binding of such

growth factors to their cognate receptors activates signal transduction

pathways like MAPK and PI3K that lead to cell proliferation/survival and

stimulates the expression of HIF-1 itself. This leads to increased HIF-1

transcriptional activity of target genes including those encoding IGF2 and


28

TGF, thereby contributing to autocrine-signaling pathways that are crucial for

cancer progression19

.

Apoptosis

Hypoxia has been shown to induce apoptosis where HIF-1 plays a

complex role. Activation of caspase-3 and Apaf-1-mediated caspase-9, and the

release of cytochrome C, have been reported in several cell types under

hypoxic conditions. It has also been demonstrated that the expression of

HIF-1 alpha and HIF-1 beta significantly correlated with apoptosis and the

pro-apoptotic factors such as caspase-3, Fas, and Fas ligand. p53 has been

implicated in regulating hypoxia-induced apoptosis through induction of

apoptosis-related genes such as Bax, NOXA, PUMA and PERP. In addition to

the above classes of genes, HIF-1 also regulated many other target genes

implicated in diverse processes such as adipogenesis, carotid body formation,

B lymphocyte development and immune reactions19

.

The Role of HIF-1 in Development and Diseases

Development

Components of the HIF-1 system play essential roles in embryonic

development. Knockout of either HIF-1 or HIF-2 resulted in abnormal

vascular development and lethality in mice19

.


29

Cancer

Overexpression of HIF-1 alpha and HIF-2 alpha was found in various

human cancers, probably as a consequence of intratumoral hypoxia or genetic

alteration. Immunohistochemical analyses demonstrated that there are

detectable levels of HIF-1 protein in benign tumors, elevated levels in primary

malignant tumors, and a marked amount in tumor metastases, in contrast to its

absence in normal tissues. There is a remarkable frequency of common genetic

alterations in cancer cells associated with increased HIF-1 alpha expression19

.

Ischemic Disease

The levels of HIF-1 alpha and VEGF were increased in the

myocardium when patients developed acute coronary artery occlusion.

Effective vascular remodeling after ischemic injury depends on an integrated

program of HIF dependent gene expression. In addition, induction of HIF-1

alpha or HIF-2 alpha and their target genes has been shown in the

pre-eclamptic placenta, in the ischemic retina and as well as from wound

healing19

.


30

HIF-1 alpha and Metabolic Adaptation

HIF-1 alpha controls metabolic and pH-regulating pathways. Cells

respond to hypoxia by HIF- 1 alpha mediated upregulation of glucose

transporters (Glut-1 and Glut-3) and enzymes of glycolysis. Conversion of

pyruvate to lactic acid is facilitated by the induction of lactate dehydrogenase

(LDH). HIF-1 alpha also induces pyruvate dehydrogenase kinase-1 (PDK-1),

which inhibits the conversion of pyruvate into acetyl-CoA by pyruvate

dehydrogenase (PDH), thus preventing entry of pyruvate into the TCA cycle.

Subunit composition of cytochrome c oxidase (COX4) is influenced by HIF-1

alpha in hypoxia. COX4-2 is induced and COX4-1 is reciprocally reduced by

induction of the protease LON that degrades COX4-1. Switching the COX

subunits ensures optimal efficiency of mitochondrial respiration in hypoxia20

.


31

HIF-1 alpha and Angiogenesis

The three major processes involved in the formation of new blood

vessels are referred to as vasculogenesis, angiogenesis and arteriogenesis. If

the cell mass expands, angiogenic factors would be released, but the triggers

for the so-called angiogenic switch, a phenomenon in which a tumor

progresses from a non-angiogenic to an angiogenic phenotype, remained

obscure. The hypoxic microenvironment caused by the increased oxygen

consumption of hyperplasia and/or hypertrophy and the decreased oxygen

delivery due to the increase in diffusion distance was assumed to contribute to

the angiogenic switch. An important link between hypoxia and angiogenesis

was the discovery that the expression of the potent vascular endothelial growth

factor was induced by hypoxia. Angiogenesis is essential for development,

wound healing and tissue or organ regeneration. It is an intricate multistep and

temporally ordered process that involves a great number of genes, modifiers

and pathways. Many of these genes are directly induced by HIF-1 alpha, such

as nitric oxide synthases, angiogenic and vascular growth factors (VEGF) and

genes regulating matrix metabolism (urokinase type plasminogen activator

receptor; uPAR). Others are independently regulated by hypoxia and might be

influenced by secondary mechanisms, but a central role of HIF-1 alpha is well

established20

.

The individual steps of angiogenesis require distinct changes to a

variety of cells (e.g. endothelial cells or pericytes). Endothelial cells have to be


32

transformed from a stable growth-arrested state to a plastic proliferative

phenotype. The basement membrane has to be digested and the extracellular

matrix remodeled so that the endothelial cells are able to migrate. HIF-1 alpha

signaling pathways have been demonstrated to influence factors such as

uPAR, collagen prolyl-4-hydroxylases, matrix metalloproteinases (e.g. MMP-

2) and tissue inhibitors of matrix metalloproteinases (TIMP-1)20

.

HIF-1 alpha and Cancer

Many genes that are induced by HIF-1 alpha are expressed at higher

levels in cancer than in normal tissues particularly angiogenic growth factors

(such as VEGF) and enzymes of the glucose metabolism. The hallmark of

cancer metabolism is significantly influenced by HIF-1 alpha: increased

glucose uptake, lactate production and decreased respiration20

.

In malignant tissue, different stimuli activate HIF-1 alpha : Local

hypoxia due to increased proliferation or insufficient oxygen supply,

inactivation of tumor suppressors such as VHL, oncogenes, growth factors,


33

accumulation of TCA intermediates such as fumarate or succinate and

therapeutic irradiation20

.

Together with other cell types such as macrophages these factors

contribute to a tumor microenvironment that is capable of modulating the HIF

response itself. These complex interactions together influence the phenotype

and the behavior of the tumor in terms of progression, invasiveness or

metastatic potential20

.

HIF in Renal Development

Hypoxia occurs physiologically during embryogenesis, and

stabilization of HIF alpha subunits has been demonstrated during

nephrogenesis. Although the role of HIF signaling in renal development is

unclear, HIF alpha subunits exhibit a cell type and stage-specific expression

pattern during nephrogenesis. This correlated with the expression of important


34

angiogenic factors, such as VEGF and endoglin, supporting the notion that

HIF signaling has a regulatory role in the developing kidney. HIF-1 alpha

expression was predominantly found in the cortical and medullary collecting

ducts, S-shaped bodies, and glomerular cells. The expression of HIF-2 alpha

was detectable in podocytes, as well as in cortical and medullary endothelial

and interstitial cells, but was absent in the fully developed kidney.

Furthermore, a distinct role for HIF-1 alpha and HIF-2 alpha in glomerular

development has been proposed based on the finding that S- or comma-shaped

bodies expressed only HIF-1 alpha, whereas more mature glomeruli expressed

HIF-2 alpha21,22

.

HIF 1 alpha and breast cancer

Dales JP et al., 2010- Using real-time quantitative reverse

transcription PCR assays, mRNA concentrations of total HIF-1 alpha and 4

variants in breast tissue specimens in a series of 29 normal tissues or benign

lesions (normal/benign) and 53 primary carcinomas were measured. In breast

cancers HIF-1 alpha splice variant levels were compared to

clinicopathological parameters including tumour microvessel density and

metastasis-free survival. HIF-1 alpha isoforms containing a three base pairs

TAG insertion between exon 1 and exon 2 and HIF-1 alpha 736 mRNAs were

found expressed at higher levels in oestrogen receptor (OR)-negative

carcinomas compared to normal/benign tissues (p = 0.009 and p = 0.004

respectively). In breast carcinoma specimens, lymph node status was


35

significantly associated with HIF-1 alpha TAG mRNA levels (p = 0.037).

Significant statistical association was found between tumour grade and HIF-1

alpha TAG (p = 0.048) and total HIF-1 alpha (p = 0.048) mRNA levels. It also

showed that high HIF-1 alpha TAG mRNA levels correlated with shortened

metastasis free survival (p = 0.01)23

.

Kallergi G et al., 2009- The expression of pFAK, HIF-1 alpha, VEGF

and VEGF2 in CTCs of patients with metastatic breast cancer could explain

the metastatic potential. This was studied using peripheral blood mononuclear

cells (PBMCs) staining with a monoclonal A45-B/B3 pancytokeratin antibody

in combination with either VEGF or VEGFR2 or HIF-1 alpha or pFAK

antibodies24

.

Saponaro C et al., 2013- The expression of VEGF and HIF-1 alpha

and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX

compared to 77 sporadic breast cancers was analysed by

immunohistochemistry. The percentage of nuclear HIF-1 alpha expression and

MVD was higher in the BRCA1 carriers than in BRCAX cancers (p=0.05) and

in all familial than in sporadic tumor tissues (p=0.0045) suggesting that

angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers25

.

HIF 1 alpha and colorectal cancer

Simiantonaki N et al., 2008 – HIF-1 expression in normal colonic

mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA),


36

low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well

as in non-metastatic and metastatic colorectal adenocarcinomas were done

using immunohistochemistry and Western blot. Eight colorectal carcinoma

cell lines are tested for their HIF-1 alpha inducibility after lipopolysaccharide

(LPS) stimulation using western blot and immunocytochemistry. In normal

mucosa, HPP and TA-LGD HIF-1 alpha was not expressed. In contrast,

perinuclear protein accumulation and nuclear expression of HIF-1 alpha were

shown in half of the examined SSA and TA-HGD. In all investigated

colorectal carcinomas there was a significant nuclear HIF-1 alpha

overexpression compared to the premalignant lesions but a significant

correlation with the metastatic status was not found. Nuclear HIF-1 alpha

expression was strongly accumulated in perinecrotic and peri-inflammatory

regions26

.

HIF-1 alpha in lung cancer

In non-small cell lung carcinoma (NSCLC), high levels of expression

of HIF-1 alpha are associated with a poor prognosis and also Epidermal

growth factor receptor expression was closely associated with HIF-1 alpha.

Expression of HIF-1 alpha and CA IX was significantly associated with

shorter disease free survival in NSCLC. They also have longer median

survival times than patients with HIF-negative. In squamous cell carcinoma of

lung, HIF-1 alpha/ VEGF double positive tumors were associated with poor

survival27

.


37

HIF 1 alpha in Clear Cell Renal Cell Carcinoma

Klatte T et al., 2007- Immunohistochemical analysis was done on a

tissue microarray constructed from paraffin-embedded primary tumor

specimens from 357 patients treated by nephrectomy for renal cell carcinoma

(RCC). HIF-1 alpha expression was greater in RCC than in benign tissue.

Clear cell RCC showed the highest expression levels. In clear cell RCC, HIF-1

alpha was significantly correlated with markers of apoptosis (p21, p53), the

mammalian target of rapamycin pathway (pAkt, p27), CXCR3, and proteins of

the vascular endothelial growth factor family. HIF-1 alpha was correlated with

CAIX and CAXII in localized, but not in metastatic RCC. HIF-1 alpha

expression predicted outcome in metastatic patients. Patients with high HIF-1

alpha expression (>35%) had significantly worse survival than patients with

low expression. HIF-1 alpha and CAIX expression are the strongest

independent prognostic factors for patients with metastatic clear cell RCC28

.

HIF 1 alpha and tongue cancer

Kang FW et al., 2013 – In this study, immunohistochemistry and

reverse transcription-polymerase chain reaction (RT-PCR) were done in 49

tumor samples and 15 adjacent non-tumor samples. The expression of HIF-1

alpha was detected in 87.76% (43/49) of the TSCC samples and in 33.33%

(5/15) of the adjacent non-tumor tissues. The expression of vascular

endothelial growth factor (VEGF) was also observed in 83.67% (41/49) of the


38

TSCC samples and in only 20% (3/15) of the adjacent non-tumor samples at a

low level. RT-PCR revealed that the mRNA expression of HIF-1 alpha and

VEGF was present in the tumor tissues but not in adjacent normal tissues. The

overexpression of HIF-1 alpha was significantly associated with T

classification (p=0.01), lymphatic metastasis (p=0.05) and histological

differentiation (p<0.001). Furthermore, HIF-1 alpha overexpression was

significantly associated with poor overall (p=0.001) and disease-free survival

rates (p=0.01) independent of T stage and lymphatic metastasis. The Cox

proportional hazards regression model demonstrated that the level of HIF-1

alpha expression may be an independent prognostic factor for TSCC. HIF-1

alpha overexpression was observed in TSCC and its overexpression suggests a

poor prognosis. HIF-1 alpha may be a molecular marker for predicting the

prognosis of TSCC29

.

HIF 1 alpha and gastric cancer

Griffiths EA et al., 2007 – It was found that it is a weak prognostic

factor for survival when it is studied between gastric and gastro-oesophageal

adenocarcinoma in 177 specimens. HIF-1 alpha expression was not observed

in normal gastric mucosa but increased in density and intensity with

progression from H. pylori-associated gastritis, intestinal metaplasia, dysplasia

to adenocarcinoma when it was demonstrated using immunohistochemistry30

.


39

Liang X et al., 2008- HIF-1 alpha protein expression, VEGF-C protein

expression, lymphatic vessel density (LVD) and blood vessel density (BVD)

in OSCC were investigated by means of immunohistochemistry in samples

from 65 cases of OSCC. The density of the lymphatic microvessels and blood

microvessels immunohistochemically stained by LYVE-1 and CD34 antibody

respectively was calculated. The association between the HIF-1 alpha

expression and the clinicopathological parameters was evaluated. HIF-1 alpha

overexpression occurred in 43 out of the 65 tumor samples (66.2%), while

VEGF-C overexpression was observed in 34 out of the 65 tumor samples

(52.3%). Higher LVD was found in both high HIF-1 alpha and high VEGF-C

expression cases. HIF-1 alpha overexpression was significantly correlated

with VEGF-C overexpression (p=0.018, Chi-square test), higher LVD

(p<0.001, Mann–Whitney U-test), and regional lymph nodal involvement

(p=0.004, Chi-square test) as well as UICCTMN classification (p=0.043,

Chi-square test), respectively31

.

Wang Net al.,2014 – In this study they examined HIF-1 alpha,

metallothionein and SLUG expression in papillary thyroid carcinoma and

assessed association of their expression with clinicopathological indicators in

129 patients using immunohistochemistry of which 61 nodular hyperplasia

and 118 normal thyroid tissue specimens. Concomitant high expression of all

these three molecules is significantly associated with high TNM stage and

LNM32

.


40

LR Alves et al., 2012- Restriction fragment length polymorphism

analysis was used to investigate HIF-1 alpha C1779T and G1790A

polymorphisms in 48 patients with epithelial dysplasia (ED) and 40 patients

with OSCC. Additionally, 88 elderly individuals without head and neck

squamous cell carcinoma were enrolled as a control group. The frequency of

the TT, GA and AA genotypes was higher in patients with ED and OSCC

when compared with controls. However CT genotype was associated with

moderate epithelial dysplasia in ED patients, while TT genotype was more

frequent in OSCC patients33

.

Rodolico V et al., 2011- They studied tumor specimens from

62 OSCC; a higher prevalence of tumors in TNM stage II and also in T2 class

between OSCC infected positive HPV16 DNA than non-infected ones was

observed. HIF-1 alpha positivity was detected throughout the analysed fields,

not associated with areas of necrosis and also observed in cells immediately

adjacent to blood vessels. A significant increase in mean values of the HIF-1

alpha labeling indexes was observed for T1-T2, as well for stage I-II in the

infected positive HPV16 DNA tumors than non-infected ones. HIF-1 alpha

and HPV16 E7 labeling indexes showed a significantly positive correlation

which suggested a positive association between HPV16 E7 and HIF-1 alpha

expression34

.

Peyssonnaux C et al., 2005- HIF-1 alpha was induced by bacterial

infection even under normoxia and regulated the production of key immune


41

effector molecules, including granule proteases, antimicrobial peptides, nitric

oxide, and TNF-α. Mice lacking HIF-1 alpha in their myeloid cell lineage

showed decreased bactericidal activity and failed to restrict systemic spread of

infection from an initial tissue focus. Conversely, activation of the HIF-1

alpha pathway through deletion of von Hippel–Lindau tumor-suppressor

protein or pharmacologic inducers supported myeloid cell production of

defense factors and improved bactericidal capacity. HIF-1 alpha control of

myeloid cell activity in infected tissues could represent a novel therapeutic

target for enhancing host defense35

.

Shin MR et al., 2014- Isocudraxanthone K (IK) is a novel, natural

compound from a methanol extract of the root bark of Cudraniatricuspidata.

IK caused time dependent phosphorylation of Akt, p38, and ERK

(extracellular signal-regulated kinase). In addition, IK increased the cytosolic

to nuclear translocation of nuclear factor-𝜅B (NF-𝜅B) p65 and the degradation

and phosphorylation of I𝜅B-𝛼 in HN4 and HN12 cells. Furthermore, IK

treatment downregulated HIF-1 alpha and its target gene, vascular endothelial

growth factor (VEGF). Cobalt chloride (CoCl2), a HIF-1 alpha activator,

attenuated the IK-induced growth-inhibiting and apoptosis-inducing effects,

and blocked IK-induced expression of apoptosis regulatory proteins, such as

Bax, Bcl-2, caspase-3, caspase-8, and caspase-9 and cytochrome c.

Collectively, these data provide the first evidence of anti-proliferative and


42

apoptosis-inducing effects of IK as a HIF-1 alpha inhibitor and suggest it may

be a drug candidate for chemotherapy against oral cancer36

.

Lin PY et al., 2008- The expression of HIF-1 alpha in 57 specimens of

oral squamous cell carcinoma (OSCC), 41 specimens of oral epithelial

dysplasia (OED, 12 mild, 17 moderate, and 12 severe OED cases), and

14 specimens of normal oral mucosa (NOM) by immunohistochemistry was

analyzed. The mean nuclear HIF-1 alpha labeling indices (LIs) increased

significantly from NOM (9 +/- 6%) through mild OED (25 +/- 18%), moderate

OED (41 +/- 27%), and severe OED (42 +/- 22%) to OSCC samples

(55 +/- 23%, p < 0.001). A significant correlation was found between the

higher mean nuclear HIF-1 alpha LI and OSCCs with larger tumor size

(p< 0.001), regional lymph node metastasis (p< 0.001), or more advanced

clinical stages (p< 0.001). Only larger tumor size (p= 0.002) and nuclear

HIF-1 alpha LI >or= 60% (p= 0.048) were identified as independent

unfavorable prognosis factor by multivariate analyses with Cox regression

model. Kaplan-Meier curve showed that OSCC patients with a nuclear

HIF-1 alpha LI ≥ 60% had a significantly poorer cumulative survival than

those with a nuclear HIF-1 alpha LI < 60% (log-rank test, p= 0.022)37

.

Zhang X et al., 2013 - Expression of HIF-1 alpha, Glut-1 and CA9

were detected in clinical samples of eight normal oral mucosa, 85 transitional

areas of oral squamous cell carcinoma (OSCC) and 28 OED with or without

malignant conversion using immunohistochemistry and were also


43

comparatively detected in immortalised human oral keratinocyte (IHOK) and

OSCC cell lines under hypoxia using immunoblotting. Sequential expression

of HIF-1 alpha, Glut-1 and CA9 was found both in transitional areas of OSCC

and cell lines of IHOK and OSCC under hypoxia, supporting hypoxia-aerobic

and glycolysis-acidosis axis. Expression of all proteins showed significant

association with malignant conversion of OED and CA9 was an independent

risk factor of malignant transformation of OED. But the predictability of

malignant transformation was improved when all three proteins were applied

together38

.

HIF 1 alpha and prognosis

Filies T et al., 2005– In this study, immunohistochemistry and tissue

microarray were done to analyze the expression of HIF-1 alpha and its

prognosis in floor of the mouth in 85 patients. Using kaplan-Meier curves, log

rank tests and multivariate cox regression analysis it was found that HIF-1

alpha is overexpressed in disease free period, improved 5-year survival rate

independent from nodal status and tumour size. The absence of HIF-1 alpha

expression specified a subgroup of high-risk patients (p < 0.05)39

.

Dos Santos M et al., 2012 - In this study, HIF-1 alpha expression by

immunohistochemistry in tissue microarrays and its relationship with clinical

findings, histopathological results and survival of 66 patients with squamous

cell carcinoma of the lower mouth was done. They demonstrated that high


44

HIF-1 alpha expression is associated with local disease-free survival,

independently from the choice of treatment. Furthermore, high expression of

HIF-1 alpha in patients treated with postoperative radiotherapy was associated

with survival, therefore being a novel prognostic marker in squamous cell

carcinoma of the mouth. Additionally, it also showed that MVD was

associated with HIF1alpha expression and local disease relapse40

.

Results

Results

45

SAMPLE CHARACTERISTICS:

The study population includes 60 cases taken from the archival blocks.

They were categorized into four groups. Group I (n=15) comprised of normal

mucosa samples. Group II (n= 15) comprised of mild epithelial dysplasia

samples. Group III (n=15) comprised of both moderate and severe epithelial

dysplasia samples. Group IV (n=15) comprised of oral squamous cell

carcinoma samples. All the samples were analyzed for the immunoreactivity

of HIF-1 alpha stain.

DISTRIBUTION OF AGE IN THE STUDY GROUPS: (TABLE 1 &

GRAPH 1):

The distribution of age of the patients was divided into 3 groups:

20-40 years, 41-60 years and those above 61 years of age. Group I consisted of

14 (93.3%) cases in the age group 20-40 years and 1 (6.6%) case in the age

group of 41-60. Group II consisted of 5 (33.3%) cases in 20-40 years, 9 (60%)

cases in 41-60 years and 1 (6.6%) cases above 61 years. Group III consisted of

6 (40%) cases in 20-40 years, 7 (46.6%) cases in 41-60 year and 2 (13.3%)

case of above 61 years. Group IV consisted of 2 (13.3%) cases in 20-40 years,

8 (53.3%) cases in the 41-60 years and 5 (33.3%) cases above 61 years of age.

Results

46

DISTRIBUTION OF GENDER IN THE STUDY GROUPS: (TABLE 2 &

GRAPH 2):

In group I, 8 (53.3%) were males and there was 7 (46.6%) female. In

group II, all 15 (100%) were males. In group III, 11 (73.3%) were males and

4 (26.6%) were females. In group 1V, 9 (60%) were males and 6 (40%) were

females.

DISTRIBUTION OF HABITS IN THE STUDY GROUPS (TABLE 3 &

GRAPH 3):

Based on the prevalence of habits in the study groups, they were

categorized in to six groups. They were those without any habits, those with

habit of chewing tobacco and consuming alcoholic beverages, chewing

tobacco alone, smoking alone, consuming alcoholic beverages alone, chewing

tobacco, smoking & consuming alcoholic beverages. In group I (control

group) none of them had any habits. In group II, there were 7 (46.6%) with

habit of chewing tobacco and consuming alcoholic beverages, 4 (26.6%) had

chewing tobacco alone, 1 (6.6%) had smoking alone, 2 (13.3%) had

consuming alcoholic beverages alone and 1 (6.6%) had chewing tobacco,

smoking & consuming alcoholic beverages. In Group III, 8 (53.3%) of the

cases had no habit, 1 (6.6%) had the habit of chewing tobacco and smoking,

5 (33.3%) had the habit of chewing tobacco alone and 1 (6.6%) had the habit

of chewing tobacco, smoking and consuming alcoholic beverages.

Results

47

In group IV, 9 (60%) had no habits, 5 (33.3%) had the habit of chewing alone

and 1 (%) had the habit of smoking alone (p=0.000).

DISTRIBUTION OF SITE IN THE STUDY GROUPS (TABLE 4 &

GRAPH 4):

In group I, 15(100%) incisional biopsies were from retro-molar region.

In group II, 14 (93.3%) cases were from buccal mucosa and 1 (6.6%) were

from alveolar mucosa. In group III, 10 (66.6%) were from buccal mucosa and

5 (33.3%) were from lateral border of tongue. In group IV, 4 (26.6%) was

from buccal mucosa, 8 (53.3%) were from the lateral border of tongue,

2 (13.3%) were from palatal region and 1 (6.6%) cases were from commissure

of the mouth (p=0.007).

DISTRIBUTION OF STAINING OF HIF-1 ALPHA IN THE STUDY

GROUPS (TABLE 5 & GRAPH 5):

Of the total number of cases subjected to HIF-1 alpha staining, in

group I, 12 (80%) showed positivity while 3 (20%) were negative. In group II,

14 (93.3%) showed positivity and 1 (6.6%) were negative. In group III,

14 (93.3%) showed positivity and 1 (6.6%) were negative. Group IV showed

100% positivity (p=0.246).

Results

48

The following parameters were used to evaluate HIF-1 alpha staining in all the

4 groups:

- Staining intensity

- Staining pattern

- Tissue localisation of the stain

TISSUE LOCALIZATION OF THE STAIN (TABLE 6 AND GRAPH 6):

The HIF-1 alpha staining was demonstrated in basal, supra basal

layers, keratin and connective tissue. 3 (20%) of group I, 5 (33.3%) of group

II, 4 (26.6%) of group III and 1 (6.6%) of group IV showed positive staining

in all the four above mentioned layers. 4 (26.6%) of group I, 5 (33.3%) of

group II, 5 (33.3%) of group III and 9 (60%) of group IV showed positive

staining in three of the above mentioned layers except for basal layer. 1 (6.6%)

of group I, 2 (13.3%) of group II, 4 (26.6%) of group III and 4 (26.6%) of

group IV showed positive staining in keratin and connective tissue only.

1 (6.6%) of group II and IV showed positivity for suprabasal and connective

tissue staining and 1 (6.6%) of group III showed positivity for basal,

suprabasal and connective tissue staining. Only connective staining was

present in group I and group II of about 3 (20%) and 1 (6.6%) respectively

without staining in the epithelium. 3 (20%) of group I, 1 (%) of group II, 1 (%)

of group III showed no staining at all (p=0.027).

Results

49

STAINING INTENSITY

COMPARISON OF OVERALL HIF-1 ALPHA INTENSITY BETWEEN

THE STUDY GROUPS (TABLE 7 & GRAPH 7):

On comparing the HIF-1 alpha intensity between the study groups,

Group I showed 3 (20%), 10 (66.6%) and 2 (13.3%) of negative, mild and

moderate staining respectively. Group II showed 1 (6.6%), 11 (23.3%) and

3 (20%) of negative, mild and moderate staining respectively. In Group III,

1 (6.6%), 7 (46.6%) and 7 (46.6%) cases showed negative, mild and moderate

staining respectively. In group IV, 14 (93.3%) and 1 (6.6%) cases showed

mild and moderate staining respectively. There was no intense staining in any

of the groups (p=0.050).

COMPARISON OF BASAL LAYER INTENSITY BETWEEN THE

STUDY GROUPS (TABLE 8 & GRAPH: 8)

On analyzing the basal layer intensity of HIF-1 alpha in all the four

groups, in group I, 12 (80%) and 3 (20%) cases showed negative and mild

staining respectively. In group II, 10 (66.6%) and 5 (33.3%) cases showed

negative and mild staining respectively. In group III, 10 (66.6%), 3 (33.3%)

and 2 (13.3%) cases showed negative and mild and moderate staining

respectively. In group IV, 14 (93.3%) and 1 (6.6%) cases showed negative and

mild staining respectively. There was no intense staining in any of the groups

(p=0.141).

Results

50

COMPARISON OF SUPRABASAL LAYER INTENSITY BETWEEN

THE STUDY GROUPS (TABLE 9 & GRAPH 9):

On analyzing the supra basal layer intensity of HIF 1 alpha in all the

four groups, in group I, 8 (53.3%), 6 (40%) and 1 (6.6%) case showed

negative, mild and moderate staining respectively. In Group II, 4 (26.6%),

9 (60%) and 2 (13.3%) cases showed negative, mild and moderate staining

respectively. In group III, 5 (33.3%), 4(26.6%) and 6 (40%) cases showed

negative, mild and moderate staining respectively. In group IV, 4 (26.6%),

9 (60%) and 2 (13.3%) cases showed negative, mild and moderate staining

respectively. There was no intense staining in any of the groups (p=0.125).

COMPARISON OF KERATIN INTENSITY BETWEEN THE STUDY

GROUPS (TABLE 10 & GRAPH 10):

On analyzing the keratin layer and keratin pearl intensity of HIF 1

alpha in all the four groups, in group I, 6(40%), 7(46.6%) and 2(13,3%)cases

showed negative, mild and moderate staining respectively. In Group II,

3 (20%) and 12(80%) cases showed negative and mild staining respectively. In

group III, 2(13.3%), 8(53.3%) and 5(33.3%) cases showed negative, mild and

moderate staining respectively. In group IV, 1(6.6%), 11(73.3%) and 3(20%)

cases showed negative, mild and moderate staining respectively. There was no

intense staining in any of the groups (p=0.034).

Results

51

COMPARISON OF CONNECTIVE TISSUE INTENSITY BETWEEN

THE STUDY GROUPS (TABLE 11& GRAPH 11):

On analyzing the connective tissue intensity of HIF 1 alpha in all the

four groups, in group I, 4 (26.6%), 9 (60%) and 2 (13.3%) cases showed

negative, mild and moderate staining respectively. In Group II, 3 (20%) and

12 (80%) cases showed negative and mild staining respectively. In group III,

1 (6.6%), 9 (60%) and 5 (33.3%) cases showed negative, mild and moderate

staining respectively. In group IV, 14 (93.3%) and 1 (6.6%) cases showed

mild and moderate staining respectively. There was no intense staining in any

of the groups (p=0.034).

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN THE

GRADES OF DYSPLASIA (TABLE 12& GRAPH 12):

The grades of epithelial dysplasia were divided into three groups

namely mild (n=15), moderate (n=9) and severe dysplasia (n=6). In mild

epithelial dysplasia 1 (6.6%), 11 (73.3%) and 3 (20%) cases showed negative,

mild and moderate staining respectively. In moderate epithelial dysplasia 1

(11.1%), 4 (44.4%) and 4 (44.4%) cases showed negative, mild and moderate

staining respectively. In group III, 1 (6.6%), 9 (60%) and 5 (33.3%) cases

showed negative, mild and moderate staining respectively. In group IV,

3 (50%) and 3 (50%) cases showed mild and moderate staining respectively.

There was no intense staining in any of the groups (p=0.000).

Results

52

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN THE

GRADES OF OSCC (TABLE 13& GRAPH 13):

The grades of OSCC were divided into well differentiated (n=10),

moderately differentiated (n=3) and severely differentiated OSCC (n=2). In

well differentiated OSCC, 10 (100%) cases showed mild intensity. In

moderately differentiated OSCC, 2 (66.6%) and 1 (33.3 %) case showed mild

and moderate intensity respectively. In poorly differentiated OSCC, 2 (100%)

cases showed mild intensity (p=0.001).

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN GROUPS I

AND GROUP II (TABLE 14& GRAPH 14):

Group I showed 3 (20%) cases negative, 10 (66.6%) cases mild and 2

(13.3%) cases moderate intensity of staining. Group II showed 1 (6.6%) case

negative, 11 (73.3%) cases mild and 3 (20%) cases moderate intensity of

staining (p=0.536).

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN GROUPSI

AND GROUP III (TABLE 15 & GRAPH 15):

Group I showed 3 (20%) cases negative, 10 (66.6%) cases mild and 2

(13.3%) cases moderate intensity of staining. Group III showed 1 (6.6%) case

negative, 7 (46.6%) cases mild and 7(46.6%) cases moderate of intensity

(p=0.116).

Results

53

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN GROUP I

AND GROUP IV (TABLE 16 & GRAPH 16):

Group I showed 3 (20%) cases negative, 10 (66.6%) cases mild and

2 (13.3%) cases moderate intensity of staining. Group IV showed 14 (93.3%)

cases mild and 1(6.6%) case moderate intensity of staining (p=0.135).

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN GROUP II


Group II showed 1 (6.6%) case negative, 11 (73.3%) cases mild and 3

(20%) cases moderate intensity of staining. Group IV showed 14 (93.3%)

cases mild and 1(6.6%) cases moderate intensity of staining (p=0.300).

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN GROUP II

AND GROUP III (TABLE 18 & GRAPH 18):

Group II showed 1 (6.6%) cases negative, 11 (73.3%) cases mild and

3 (20%) cases moderate intensity of staining. Group III showed 1 (6.6%) case

negative, 7(46.6%) cases mild and 7(46.6%) cases moderate of intensity

(p=0.288).

Results

54

COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN GROUP III


Group III showed 1 (6.6%) case negative, 7(46.6%) cases mild and

7(46.6%) cases moderate intensity of staining. Group IV showed 14 (93.3%)

cases mild and 1(6.6%) case moderate intensity of staining (p=0.020).

KAPPA VALUE:

Three observers analyzed the slides for scoring the intensity of

staining. The overall Kappa value for the inter observer variation is 0.7.

TABLE 1: DISTRIBUTION OF AGE IN STUDY GROUPS (N=60)

GRAPH 1: DISTRIBUTION OF AGE IN STUDY GROUPS (N=60)

GROUP I: NORMAL MUCOSA

GROUP II: MILD DYSPLASIA

GROUP III: MODERATE & SEVERE DYSPLASIA

GROUP IV: OSCC

93.3%

33.3%

40%

13.3%

6.6%

60 %

46.6%

53.3%

6.6%

13.3%

33.3%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

GROUP I GROUP II GROUP III GROUP IV

DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%)

AGE GROUPS IN YEARS

20 - 40

41 - 60

ABOVE 61

AGE

GROUPS

IN YEARS

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15

20- 40 14 (93.3%) 5 (33.3%) 6 (40%) 2 (13.3%)

41 – 60 1 (6.6%) 9 (60%) 7 (46.6%) 8 (53.3%)

ABOVE 61 0 1 (6.6%) 2 (13.3%) 5 (33.3%)

TABLE 2: DISTRIBUTION OF GENDER IN STUDY GROUPS (N=60)

GENDER GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15

MALE 8 (53.3%) 15 (100%) 11 (73.3%) 9 (60%)

FEMALE 7 (46.6%) 0 4 (26.6%) 6 (40%)

GRAPH 2: DISTRIBUTION OF GENDER IN STUDY GROUPS (N=60)




GROUP IV: OSCC

53.3%

100%

73.3%

60%

46.6%

26.6%

40%

0%

20%

40%

60%

80%

100%

120%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%)

GENDER

MALE

FEMALE

TABLE 3: DISTRIBUTION OF HABITS IN THE STUDY GROUPS (N=60)

HABITS GROUP I

n=15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

NO HABITS 15 (100%) 0 8 (53.3%) 9 (60%)

0.000*

CHEWING+

ALCOHOL(C+A) 0 7 (46.6%) 1 (6.6%) 0

CHEWING ALONE (C) 0 4(26.6%) 5 (33.3%) 5 (33.3%)

SMOKING ALONE (S) 0 1 (6.6%) 0 1 (%)

ALCOHOL ALONE (A) 0 2 (13.3%) 0 0

CHEWING+SMOKING

+ ALCOHOL(C+S+A) 0 1 (6.6%) 1 (6.6%) 0

GRAPH 3: DISTRIBUTION OF HABITS IN THE STUDY GROUPS (N=60)

10

0%

46

.6%

26

.6%

6.6

%

33

.3%

6.6

%

53

.3%

6.6

%

33

.3%

6.6

%

60

.0%

33

.3%

6.6

%

0%

20%

40%

60%

80%

100%

120%

NO

HABITS

C+A C S A C+S+A

DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

HABITS

GROUP I

GROUP II

GROUP III

GROUP IV




GROUP IV: OSCC

* p value < 0.05 is significant

TABLE 4: DISTRIBUTION OF SITE OF LESION/BIOPSY IN THE STUDY

GROUPS (N= 60)

SITE OF

LESION

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

RETROMOLAR

AREA 15 (100%) 0 0 0

0.007*

BUCCAL

MUCOSA 0 14 (93.3%) 10 (66.6%) 4 (26.6%)

LATERAL

BORDER OF

TONGUE

0 0 5 (33.3%) 8 (53.3%)

ALVEOLAR

MUCOSA 0 1 (6.6%) 0 0

PALATAL

REGION 0 0 0 2 (13.3%)

COMMISSURE

OF MOUTH 0 0 0 1 (6.6%)

GRAPH 4: DISTRIBUTION OF SITE OF LESION/BIOPSY IN THE STUDY

GROUPS (N= 60)

10

0%

93

.30

%

6.6

0%

66

.60

%

33

.30

%

26

.60

% 5

3.3

0%

13

.30

%

6.6

0%

0%

20%

40%

60%

80%

100%

120%

RETROMOLAR

AREA

BUCCAL MUCOSA LATERAL

BORDER OF

TONGUE

ALVEOLAR

MUCOSA

PALATAL REGION COMMISSURE OF

MOUTH

DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

SITE OF LESION/BIOPSY

GROUP I

GROUP II

GROUP III

GROUP IV

GROUP I : NORMAL MUCOSA

GROUP II : MILD DYSPLASIA

GROUP III : MODERATE & SEVERE DYSPLASIA

GROUP IV : OSCC


TABLE 5: DISTRIBUTION OF STAINING OF HIF–1 ALPHA AMONG THE STUDY

GROUPS (N=60)

HIF-1ALPHA

STAINING

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15

p Value

PRESENT 12 (80%) 14 (93.3%) 14 (93.3%) 15 (100%) 0.246

ABSENT 3 (20%) 1 (6.6%) 1 (6.6%) 0 (%)

GRAPH 5: DISTRIBUTION OF STAINING OF HIF – 1 ALPHA AMONG THE STUDY

GROUPS (N=60)




GROUP IV: OSCC

20.0%

6.6% 6.6%

0%

20%

40%

60%

80%

100%

120%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

HIF 1 STAINING

PRESENT

ABSENT

TABLE 6: TISSUE LOCALISATION IN HIF-1 ALPHA POSITIVE

STAINED CELLS IN THE STUDY GROUPS

TISSUE

LOCALISATION

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

BASAL+ SUPRA

BASAL+KERATIN+

CONNECTIVE

TISSUE (B+S+K+C)

3 (20%) 5 (33.3%) 4 (26.6%) 1 (6.6%)

0.027*

SUPRA

BASAL+KERATIN+

CONNECTIVE

TISSUE (S+K+C)

4 (26.6%) 5 (33.3%) 5 (33.3%) 9 (60%)

BASAL +

SUPRABASAL+

CONNECTIVE

TISSUE (B+S+C)

0 (%) 0 (%) 1 (6.6%) 0 (%)

SUPRABASAL+

CONNECTIVE

TISSUE (S+C)

0 (%) 1 (6.6%) 0 (%) 1 (6.6%)

KERATIN+

CONNECTIVE

TISSUE (K+C)

1 (6.6%) 2 (13.3%) 4 (26.6%) 4 (26.6%)

CONNECTIVE

TISSUE (C) 3 (20%) 1 (6.6%) 0 (%) 0 (%)

NEGATIVE 3 (%) 1 (%) 1 (%) 0 (%)

GRAPH 6: TISSUE LOCALISATION IN HIF-1ALPHA POSITIVE STAINED

CELLS IN THE STUDY GROUPS (N=60)

20

.0%

26

.6%

6.6

%

20

.0%

20

.0%

33

.3%

33

.3%

6.6

% 13

.3%

6.6

%

6.6

%

26

.6%

33

.3%

6.6

%

26

.6%

6.6

%

6.6

%

60

.0%

6.6

%

26

.6%

0%

10%

20%

30%

40%

50%

60%

70%

B+S+K+C S+K+C B+S+C S+C K+C C NEGATIVE

DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%)

TISSUE LOCALIZATION OF HIF 1 ALPHA

GROUP I

GROUP II

GROUP III

GROUP IV




GROUP IV: OSCC


TABLE 7: COMPARISON OF HIF-1 ALPHA INTENSITY BETWEEN THE

STUDY GROUPS (N=60)

HIF-1

APLHA

INTENSITY

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

NEGATIVE 3 (20%) 1 (6.6%) 1 (6.6%) 0 (%)

0.050*

MILD 10 (66.6%) 11 (23.3%) 7 (46.6%) 14 (93.3%)

MODERATE 2 (13.3%) 3 (20%) 7 (46.6%) 1 (6.6%)

INTENSE 0 0 0 0

GRAPH 7: COMPARISON OF HIF-1ALPHA INTENSITY BETWEEN THE

STUDY GROUPS (N=60)




GROUP IV: OSCC

20

.0%

6.6

%

6.6

%

66

.6%

23

.3%

46

.6%

93

.3%

13

.3%

20

.0%

46

.0%

6.6

%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

HIF 1 ALPHA STAINING INTENSITY

NEGATIVE

MILD

MODERATE

INTENSE


TABLE 8: COMPARISON OF BASAL LAYER INTENSITY BETWEEN THE

STUDY GROUPS (N=60)

BASAL

LAYER

INTENSITY

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

NEGATIVE 12 (80%) 10 (66.6%) 10 (66.6%) 14 (93.3%)

0.141

MILD 3 (20%) 5 (33.3%) 3 (33.3%) 1 (6.6%)

MODERATE 0 0 2 (13.3%) 0

INTENSE 0 0 0 0

GRAPH 8: COMPARISON OF BASAL LAYER INTENSITY BETWEEN THE

STUDY GROUPS (N=60)




GROUP IV: OSCC

80

.0%

66

.6%

66

.6%

93

.3%

20

.0%

33

.3%

20

.0%

6.6

% 13

.3%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY IN BASAL LAYER

NEGATIVE

MILD

MODERATE

INTENSE

TABLE 9: COMPARISON OF SUPRABASAL LAYER INTENSITY BETWEEN THE

STUDY GROUPS (N=60)

SUPRABASAL

LAYER

INTENSITY

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

NEGATIVE 8 (53.3%) 4 (26.6%) 5 (33.3%) 4 (26.6%)

0.125 MILD 6 (40%) 9 (60%) 4 (26.6%) 9 (60%)

MODERATE 1 (6.6%) 2 (13.3%) 6 (40%) 2 (13.3%)

INTENSE 0 0 0 0

GRAPH 9: COMPARISON OF SUPRABASAL LAYER INTENSITY BETWEEN THE

STUDY GROUPS (N=60)




GROUP IV: OSCC

53

.3%

26

.6%

33

.3%

26

.6%

40

.0%

60

.0%

26

.6%

60

.0%

6.6

%

13

.3%

40

.0%

13

.3%

0%

10%

20%

30%

40%

50%

60%

70%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY IN SUPRABASAL LAYER

NEGATIVE

MILD

MODERATE

INTENSE

TABLE 10: COMPARISON OF KERATIN STAINING INTENSITY BETWEEN THE

STUDY GROUPS (N=60)

KERATIN

INTENSITY

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

NEGATIVE 6(40%) 3 (20%) 2(13.3%) 1(6.6%)

0.034*

MILD 7(46.6%) 12 (80%) 8(53.3%) 11(73.3%)

MODERATE 2 (13.3%) 0 5 (33.3%) 3 (20%)

INTENSE 0 0 0 0

GRAPH 10: COMPARISON OF KERATIN STAINING INTENSITY BETWEEN THE

STUDY GROUPS (N=60)




GROUP IV: OSCC

40

.0%

20

.0%

13

.3%

6.6

%

46

.6%

80

.0%

53

.3%

73

.3%

13

.3%

0.0

%

33

.3%

20

.0%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY IN KERATIN

NEGATIVE

MILD

MODERATE

INTENSE


TABLE 11: COMPARISON OF CONNECTIVE TISSUE INTENSITY BETWEEN THE

STUDY GROUPS (N=60)

CONNECTIVE

TISSUE

INTENSITY

GROUP I

n =15

GROUP II

n =15

GROUP III

n =15

GROUP IV

n =15 p Value

NEGATIVE 4 (26.6%) 3 (20%) 1 (6.6%) 0

0.034*

MILD 9 (60%) 12 (80%) 9 (60%) 14 (93.3%)

MODERATE 2 (13.3%) 0 5 (33.3%) 1 (6.6%)

INTENSE 0 0 0 0

GRAPH 11: COMPARISON OF CONNECTIVE TISSUE INTENSITY BETWEEN THE

STUDY GROUPS (N=60)




GROUP IV: OSCC

26

.6%

20

.0%

6.6

%

60

%

80

%

60

%

93

.30

%

13

.3%

33

.3%

6.6

%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY IN CONNECTIVE TISSUE

NEGATIVE

MILD

MODERATE

INTENSE


TABLE 12: COMPARISON OF INTENSITY OF HIF-1 ALPHA BETWEEN GRADES

OF EPITHELIAL DYSPLASIA (GROUP II & III) N=30

GRADES OF

EPITHELIAL

DYSPLASIA

NEGATIVE MILD MODERATE INTENSE p Value

MILD

EPITHELIAL

DYSPLASIA

1 (6.6%) 11 (73.3%) 3 (20%) 0

0.000*

MODERATE

EPITHELIAL

DYSPLASIA

1 (11.1 %) 4 (44.4 %) 4 (44.4%) 0

SEVERE

EPITHELIAL

DYSPLASIA

0 3 (50%) 3 (50%) 0

GRAPH 12: COMPARISON OF INTENSITY OF HIF-1 ALPHA BETWEEN GRADES

OF EPITHELIAL DYSPLASIA (GROUP II & III) N=30




GROUP IV: OSCC

6.6

%

73

.3%

20

.0%

11

.1%

44

.4%

44

.4%

50

.0%

50

.0%

0%

10%

20%

30%

40%

50%

60%

70%

80%

NOT EXPRESSED MILD MODERATE INTENSE

DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY IN GRADES OF DYSPLASIA

MILD DYSPLASIA

MODERATE DYSPLASIA

SEVERE DYSPLASIA


TABLE 13: COMPARISON OF INTENSITY OF HIF-1 ALPHA BETWEEN GRADES

OF OSCC (GROUP IV) N=15

GRADES OF OSCC NEGATIVE MILD MODERATE INTENSE p Value

WELL

DIFFERENTIATED 0 10 (100%) 0 0

0.001*

MODERATELY

DIFFERENTIATED 0 2 (66.6%) 1 (33.3 %) 0

POORLY

DIFFERENTIATED 0 2 (100%) 0 0

GRAPH 13: COMPARISON OF INTENSITY OF HIF-1 ALPHA BETWEEN GRADES

OF OSCC (GROUP IV) N=15




GROUP IV: OSCC

10

0%

66

.60

%

33

.30

%

10

0%

0%

20%

40%

60%

80%

100%

120%

NEGATIVE MILD MODERATE INTENSE

DIS

TR

IBU

TIO

N O

F S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY IN GRADES OF OSCC

WELL DIFFERENTIATED

MODERATELY

DIFFERENTIATED

POORLY DIFFERENTIATED


TABLE 14: COMPARISON OF INTENSITY BETWEEN GROUP I AND GROUP II

GROUPS NEGATIVE MILD MODERATE INTENSE p Value

GROUP I

(n=15) 3 (20%) 10 (66.6%) 2 (13.3%) 0

0.536 GROUP

II (n=15) 1 (6.6%) 11 (73.3%) 3 (20%) 0

GRAPH 14: COMPARISON OF INTENSITY BETWEEN GROUP I AND GROUP II



20

.0%

66

.6%

13

.3%

6.6

%

73

.3%

20

.0%

0%

10%

20%

30%

40%

50%

60%

70%

80%


DIS

TR

IBU

TIO

N

BE

TW

EE

N S

TU

DY

GR

OU

PS

(%

)

STAINING INTENSITY OF HIF 1 ALPHA

GROUP I

GROUP II

TABLE 15: COMPARISON OF INTENSITY BETWEEN GROUP I AND GROUP III


GROUP I

(n=15)

3 (20%) 10 (66.6%) 2 (13.3%) 0

0.116

GROUP III

(n=15)

1 (6.6%) 7 (46.6%) 7 (46.6%) 0

GRAPH 15: COMPARISON OF INTENSITY BETWEEN GROUP I AND GROUP III



20

.0%

66

.6%

13

.3%

0.0

%

6.6

%

46

.6%

46

.6%

0.0

%

0%

10%

20%

30%

40%

50%

60%

70%


DIS

TR

IBU

TIO

N B

ET

WE

EN

ST

UD

Y G

RO

UP

S (

%)


GROUP I

GROUP III

TABLE 16: COMPARISON OF INTENSITY BETWEEN GROUP I AND GROUP IV


GROUP I

(n=15) 3 (20%) 10 (66.6%) 2 (13.3%) 0

0.135 GROUP IV

(n=15) 0 14 (93.3%) 1 (6.6%) 0

GRAPH 16: COMPARISON OF INTENSITY BETWEEN GROUP I AND GROUP IV


GROUP IV: OSCC

20

.0%

66

.6%

13

.3%

93

.3%

6.6

%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


DIS

TR

IBU

TIO

N B

ET

WE

EN

ST

UD

Y G

RO

UP

S (

%)


GROUP I

GROUP IV

TABLE 17: COMPARISON OF INTENSITY BETWEEN GROUP II AND GROUP IV


GROUP II

(n=15) 1 (6.6%) 11 (73.3%) 3 (20%) 0

0.30 GROUP

IV (n=15) 0 14 (93.3%) 1 (6.6%) 0

GRAPH 17: COMPARISON OF INTENSITY BETWEEN GROUP II AND GROUP IV


GROUP IV: OSCC

6.6

%

73

.3%

20

.0%

93

.3%

6.6

%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


DIS

TR

IBU

TIO

N B

ET

WE

EN

ST

UD

Y G

RO

UP

S


GROUP II

GROUP IV

TABLE 18: COMPARISON OF INTENSITY BETWEEN GROUP II AND GROUP III


GROUP II

(n=15) 1 (6.6%) 11 (73.3%) 3 (20%) 0

0.288

GROUP III

(n=15) 1 (6.6%) 7 (46.6%) 7 (46.6%) 0

GRAPH 18: COMPARISON OF INTENSITY BETWEEN GROUP II AND GROUP III



6.6

%

73

.3%

20

.0%

6.6

%

46

.6%

46

.6%

0%

10%

20%

30%

40%

50%

60%

70%

80%


DIS

TR

IBU

TIO

N B

ET

WE

EN

ST

UD

Y G

RO

UP

S (

%)


GROUP II

GROUP III

TABLE 19: COMPARISON OF INTENSITY BETWEEN GROUP III AND GROUP IV


GROUP III

(n=15) 1 (6.6%) 7 (46.6%) 7 (46.6%) 0

0.020*

GROUP

IV(n=15) 0 14 (93.3%) 1 (6.6%) 0

GRAPH 19: COMPARISON OF INTENSITY BETWEEN GROUP III AND GROUP IV


GROUP IV: OSCC

6.6

%

46

.6%

46

.6%

93

.3%

6.6

%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


DIS

TR

IBU

TIO

N B

ET

WE

EN

ST

UD

Y G

RO

UP

S (

%)


GROUP III

GROUP IV


ANTIBODY KIT

PRIMARY ANTIBODY SECONDARY ANTIBODY

(HIF-1 ALPHA)

ARMAMENTARIUM

POSITIVE CONTROL – NORMAL KIDNEY (HUMAN)

10 X

40 X

CYTOPLASMIC EXPRESSION OF HIF-1 ALPHA

ORAL SQUAMOUS CELL CARCINOMA

10 X 40 X

10 X 40 X


SEVERE EPITHELIAL DYSPLASIA

10 X 40 X

10 X 40 X


NORMAL MUCOSA

10 X

40 X


MILD EPITHELIAL DYSPLASIA

4 X

10 X

40 X


MODERATE EPITHELIAL DYSPLASIA

10 X

40 X


Discussion

Discussion

55

Oral squamous cell carcinoma (OSCC) is the sixth most common

cancer worldwide with 300,000 new cases being diagnosed every year41

.

OSCC occurs as a result of multiple molecular events which develop from

combined effects of individual genetic predisposition and exposure to

environmental carcinogens42

.

The presence of hypoxic microenvironment which is caused by the

imbalance between oxygen demand and oxygen delivery in solid tumors is a

negative prognostic factor in head and neck squamous cell carcinoma

(HNSCC).The rapidly proliferating HNSCC has insufficient vascularization

which limits the oxygen diffusion, resulting in the development of localized

hypoxia. This leads to unregulated expression of hypoxia-inducible factor and

vascular endothelial growth factor, which promotes irregular vascular

endothelial cell proliferation and differentiation and growth of new blood

capillaries, leading to the development of neovascularization43

.

The threshold oxygen tension in solid tumor is (expressed as partial

pressure of oxygen) 10mmHg. HNSCC is characterized by low oxygen

tension44,45

. Acute hypoxic stress leads to the development of aggressive

cancer phenotype with high rate of metastasis, resistance to therapeutic agents

and high tumor recurrence rates. Prolonged deprivation of oxygen leads to

chronic hypoxic stress which results in tumor necrosis. These features

observed in HNSCC are regarded as a major contributing factor leading to

poor treatment outcome46, 47

.

Discussion

56

HIF-1 alpha targets the transcription of over 60 genes involved in

many aspects of cancer biology including cell survival, glucose metabolism,

cell invasion and angiogenesis. Among these genes, vascular endothelial

growth factor (VEGF) is essential as a regulatory gene of angiogenesis in the

adaptation to a hypoxic microenvironment which in turn has a critical role in

tumour development and progression in different types of malignances48

.

Given the fact that HIF-1 alpha has a vital role in the progression of

carcinogenesis, this study was done on oral epithelial dysplasia and OSCC

using immunohistochemistry to study the expression of HIF-1 alpha and was

compared with that of the normal mucosa.

Archival blocks were taken from the department for the study purpose.

The study population includes 60 cases. They were categorized into four

groups. Group I (n=15) comprised of clinically normal mucosa samples which

was obtained from retromolar region from the patients who came for the

removal of impacted teeth. Group II (n= 15) comprised of mild epithelial

dysplasia samples. Moderate and severe epithelial dysplasias have a high risk

for malignant transformation and so we have considered them as a single

group. Hence, Group III (n=15) comprised of both moderate and severe

epithelial dysplasia samples. Group IV (n=15) comprised of oral squamous

cell carcinoma samples.

In our study 60% of the cases in group II (mild epithelial dysplasia),

46% of cases in group III (moderate and severe dysplasia) and 53% of cases in

Discussion

57

group IV (oral squamous cell carcinoma) were in the age group between 20-40

years. The distribution of age of patients with oral epithelial dysplasia and oral

squamous cell carcinoma was statistically significant. The pattern of age

distribution for OSCC was also consistent with those of Massarelli. E et al in

whose study 89% of OSCC cases were seen above 40 years49

. Our observation

from the above data suggests that the occurrence of OSCC was predominantly

seen in the age group of above 40 years i.e.,53% in the group of 40 – 60 years

and 33% cases was in the age group of above 60 years. This was also

consistent with the study of Pires FR et al who stated that 46% of the patients

in their study were between 41 – 60 years and 41% of affected age group was

between 61 – 80 years50

. Youssef HE et al also found that mean age of 54

years old is common for the occurrence of OSCC51

. This could be probably

due to the fact that mutations are more common to occur in older people, who

could inturn activate oncogenes that encourage the expression of many

transcriptional factors such as HIF-1alpha and their activation52

.

In all the three groups (mild, moderate and severe epithelial dysplasia

and OSCC), males were more predominantly affected than females. The

overall male and female ratio was 7:2. This pattern of gender distribution was

statistically significant (p=0.024) and was consistent with the study of Lee CH

et al in whose study 94% of subjects with epithelial dysplasia were males53

.

ChenYJ et al in their study of 703 OSCC cases, reported that 93% of their

study population were males. The authors thus suggested that the prevalence

Discussion

58

of OSCC was common in males when compared to females41

. Waris S and

Nagi AH stated that male preponderance could be due to the fact that the habit

of pan chewing is more common in males. The habit is less prevalent in

females and this finding could be attributed to prevalent religious and

socio-cultural reasons where females avoid coming out and buying pan at pan

shops54

.

The oral epithelial dysplasia and OSCC onset is epidemiologically

related to the presence of habits. The etiology of leukoplakia includes various

smokeless tobacco habits such as snuffing, chewing and smoking55

.In our

study nearly 50% of epithelial dysplasia (group II) patients had the habit of

chewing tobacco and consuming alcohol when compared to the control groups

and this difference was statistically significant (p=0.000). This finding was

consistent with that of Jaber MA et al in whose case-control study sample

included 630 patients with oral epithelial dysplasia and 643 control subjects

selected in United Kingdom. They concluded that, while tobacco and alcohol

synergistically influenced the development of oral epithelial dysplasia,

exclusive tobacco consumption was more likely to give rise to oral epithelial

dysplasia than exclusive alcohol consumption56

.

About 26% of group II patients and 33% of group III and group IV

patients had the habit of chewing alone presented with moderate to severe

dysplasia and oral squamous cell carcinoma. This finding was consistent with

the study by Paymaster JC et al who proposed that an increased frequency of

Discussion

59

cancer in the oral cavity was often associated with the habit of the chewing of

tobacco with betel leaf, betel nut, and slaked lime than with other habits57

.

Squier CA et al suggested that the relative risk of developing oral cancer is

2.82 for smokers and 5.98 for chewers58

. Thus chewers are at a higher risk of

developing cancer which is proved in our study also.

In our study 53.3% of moderate to severe dysplasia and 60% of OSCC

cases did not have any habits and this finding was concurrent with the report

of Shan Ho Pet al, who in their prospective study of 148 cases with OSCC,

found that 40% did not have any habits59

.There was another study by Schmidt

BL et al, who in their study of 67 cases of OSCC reported that 33% of the

cases did not have the habit of smoking60

.

In our study, buccal mucosa was the common site of biopsy in

93% and 67% cases of mild and moderate to severe epithelial dysplasia and in

27% cases of OSCC. This finding was similar to that of Shan Ho Pet al who

stated that in their cohort of 148 cases, buccal mucosa was the common site of

occurrence for epithelial dysplasia and OSCC59

. Premalignant disorders are

usually found on the buccal mucosa, followed by gingiva, tongue and floor of

the mouth61,62

.

In OSCC 53% cases were from the lateral border of tongue followed

by 27% cases in the buccal mucosa. This was consistent with the study of

Pires FR et al who reported that the most common site of OSCC was the

Discussion

60

lateral border of the tongue (37%), followed by the alveolar mucosa and

gingiva (20%) and floor of the mouth and ventral tongue (19%)50

. In an

another study by Youssef HE et al, the tongue was the most commonly

affected site followed by buccal mucosa51

. This finding was also consistent

with Chen YJ et al in whose study nearly 35% of OSCC occurred on the

lateral border of tongue41

.

TISSUE LOCALIZATION OF HIF-1 ALPHASTAINING

In this study 80% of normal mucosa, 93% of mild, moderate and

severe epithelial dysplasia and all cases of OSCC expressed HIF-1 alpha

staining.

HIF-1 alpha expression in suprabasal layer, keratin and connective

tissue was seen to increase from normal mucosa to mild to moderate to severe

dysplasia to carcinoma which was statistically significant (p=0.027). This

pattern of distribution of HIF-1 alpha is due to the microenvironment that

affects the malignant progression by contributing to altered gene expression61

.

In our study, the expression of HIF-1 alpha is seen in about 80% of cases of

normal mucosa. This suggests that the HIF-1 alpha plays a role in maintaining

homeostasis at a cellular, local, and systemic level. It has been suggested that

HIF-1 alpha may be present in normal tissue for a basal induction of genes that

are necessary to provide the cellular energy requirements. In our study the

proportion of cases which expressed HIF-1 alpha increased in case of oral

epithelial dysplasias (93%) when compared to the normal mucosa and was

Discussion

61

found to be the highest in oral squamous cell carcinoma (100%) and this

finding could probably be due to the increase in hypoxic stress with

progression to malignancy63

.

STAINING PATTERN OF HIF 1 ALPHA

We observed the expression of HIF-1 alpha only in the cytoplasm in all

the cases. Nuclear expression was not seen. This observation was consistent

with that of Stroka DM who stated that, there is an increase in both nuclear

and cytoplasmic HIF-1alpha levels in hypoxic tissues. The strong cytoplasmic

presence of HIF-1alpha could be the result of enhanced protein stabilization

and accumulation before to nuclear translocation64

. Likewise, cytoplasmic

staining of HIF-1alpha was detected in various adenocarcinomas65

, hypoxic

differentiated U937 cells66

, and normoxic rat hepatocytes67

. This was also

consistent with the study by Youssef HE et al who demonstrated that

cytoplasmic HIF-1alpha was predominant than nuclear staining or the only

pattern of expression in 63% cases of well-differentiated OSCC in their

study51

.

In our study, there was increasing expression of HIF-1 alpha with the

increasing grades of epithelial dysplasia and in increasing grades of OSCC.

When those cases which showed moderate staining intensity were compared,

we observed that in mild epithelial dysplasia there were 20% of cases which

increased to 44.4% in moderate dysplasia and further increased to 50% of

cases in severe dysplasia. Similarly when comparing moderately and poorly

Discussion

62

differentiated carcinoma, HIF-1 alpha expression increased from 66.6% to

100% of the cases where the staining intensity was mild. This was similar to

the study by Kopfstein L et al 68

and Roh JL et al 69

who studied that

HIF-1alpha immunostaining and stated that it significantly increased with

advanced clinical stages of OSCC(p=0.002). In their study, 11 out of 20 cases

with stage III and IV of OSCC showed strong HIF-1alpha positivity, while

only one case out of 25 with stage I and II showed marked upregulation of

HIF-1 alpha expression. Costa AD et al in their retrospective study with 38

cases of OSCC found out that there is a significant correlation between TNM

clinical staging and histopathologic parameters70

. Thus the pattern of

expression between grades of dysplasia and OSCC in our study was

concurrent with that of Kopfstein L et al, Roh JL et al and Costa AD et al.

The presence of staining in the keratin and connective tissue in our

study is supported by the study of Kopfstein L et al and Roh JL et al who

proposed that HIF-1 alpha immunoreactivity can be presented in the peripheral

malignant cell layers, in the keratin and epithelial pearls. They also suggested

that HIF-1alpha expression was more pronounced around areas of necrosis and

keratinization and appeared more intense in areas away from the closest blood

vessels68, 69

. This was consistent with our study where the expression of HIF-1

alpha in keratin of OSCC cases was seen in 93% of the cases, whereas in

normal mucosa cases it was observed only in 60% of the cases.

Discussion

63

In our study population, 20% of normal mucosa cases and 7% of both

mild and moderate to severe cases had complete absence of staining for HIF-1

alpha. This could be due to the prolonged fixation of tissue specimens which

would compromise the antigen detection so that failure to stain these cases

might be artificial. In addition, this negativity could be also explained that

HIF-1alpha protein expression in these cases was at levels below the limits of

detection by IHC methodology71

.

When the staining intensity for the expression of HIF-1 alpha was

compared in the keratin between normal mucosa, mild, moderate and severe

dysplasia and OSCC, we observed that 47% cases of normal mucosa,

80% cases of mild dysplasia, 53% of moderate to severe dysplasia cases and

73% of OSCC cases showed mild staining intensity. When the staining

intensity for the expression of HIF-1 alpha was compared in the connective

tissue between normal mucosa, mild, moderate and severe dysplasia and

OSCC, we found that there was a statistical significance between the study

groups (p=0.034). This may due to the role of HIF-1 alpha activating the

VEGF and other growth factors resulting in connective tissue staining.

Furthermore, the diffuse HIF-1alpha expression observed in some

cases of OSCC irrespective with the distance from the closest blood vessel,

may be explained by the hypothesis that HIF-1alpha production is not only

stimulated by hypoxia, but also with other signals as growth factors as well as

the activation of other oncogenic signal transduction pathways that may play a

role in upregulation of HIF-1alpha expression in addition to hypoxia66

.

Summary and Conclusion


64

A total of 60 biopsies were included in this study, comprising of

15 cases of normal oral mucosa (group I), 15 cases of mild epithelial

dysplasia (group II), 15 cases of moderate and severe epithelial

dysplasia (group III) and 15 cases of oral squamous cell carcinoma

(group IV).

In group II, group III and in group IV, 60%, 46.6% and 53.3% of were

in the age group of 41- 60 years respectively. This age distribution was

statistically significant (p=0.000)

In group I, all the cases were taken from from retromolar region from

the patient who has come for impaction.

In group II, 93.3% of biopsies were taken from the buccal mucosa and

6.6% from alveolar mucosa.

In group III, 66.6% of biopsies were taken from the buccal mucosa and

33.3% from lateral border of the tongue.

In group IV 26.6% biopsies were taken from buccal mucosa, 53.3%

from lateral border of the tongue, 13.3% from palatal region and 6.6%

from commissure of mouth.

Out of 15 cases in group IV, 66.6% were well differentiated OSCC,

20% were moderately differentiated OSCC and 13.3% were poorly

differentiated.

The pattern of HIF-1 alpha staining in all the groups was cytoplasmic.


65

In mild, moderate and severe epithelial dysplasia 93.3% showed HIF 1

alpha positivity and 80% positivity was seen in normal mucosa.

All the cases in OSCC showed positivity. In OSCC, 100% of well-

differentiated and poorly differentiated cases expressed mild intensity

of staining. About 66.6% and 33.3% of moderately differentiated cases

expressed mild and moderate staining intensity respectively. There was

statistically significant difference of HIF-1 alpha expression seen in the

increasing grades of OSCC (p=0.001).

There was increase in the intensity of HIF-1 alpha with the increasing

grades of epithelial dysplasia.

Our study shows increased expression of HIF-1 alpha in supra basal,

keratin and connective tissue layer of epithelial dysplasias and an increase in

HIF-1 alpha staining intensity from mild to moderate towards increasing

grades of epithelial dysplasia and OSCC indicating that HIF-1 alpha

expression has potential to be a surrogate marker of malignant transformation.

In this study we have addressed the role of HIF-1 alpha in a study

group of oral epithelial dysplasia and oral squamous cell carcinoma. The result

of this study has highlighted that, there is increased expression of HIF-1 alpha

in increasing grades of dysplasia and OSCC when compared to that of normal

mucosa. Thus addressing the hypoxic condition could be one of the important

adjunctive methods in the treatment of OSCC.

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Annexures

Annexures

ANNEXURE I

Annexures

ANNEXURE II

DISSERTATION PROTOCOL

1. Title of the dissertation

To study of expression of HIF 1 alpha in the risk prediction of oral epithelial dysplasia and in

oral squamous cell carcinoma.

2. Name and designation of the principal investigator

Dr. K. Malarvizhi

II year postgraduate student

Department of Oral and Maxillofacial Pathology

3. Name of HOD and staff in charge

Dr. K.Ranganathan, MDS,MS (OHIO),Ph.D

Dr. Elizabeth Joshua, MDS

4. Department where the project is to be carried out

Study will be conducted in Department of Oral & Maxillofacial Pathology,

Ragas Dental College & Hospital

5. Duration of the project

1 year

6. Rationale

Carcinogenesis is a multistage process that occurs as a result of number of molecular events

which develop from combined effects of an individual, genetic predisposition and exposure to

environmental carcinogens. The presence of hypoxic microenvironment in solid tumors is caused

by the imbalance between oxygen demand and oxygen delivery. The rapidly proliferating tumour

Annexures

has insufficient vascularization which limits the oxygen diffusion, resulting in the development

of hypoxic region. This causes unregulated expression of a variety of oncogenes such as

hypoxia-inducible factor and vascular endothelial growth factor which enhance irregular vascular

endothelial cell proliferation and differentiation which enhance the growth of new blood

capillaries, leading to the development of neovascularized tumors in head and neck.

7. Hypothesis (null)

There is no difference in expression of HIF 1 alpha in oral epithelial dysplasia, oral squamous

cell carcinoma when compared to normal mucosa

AIM

To evaluate the expression HIF 1 alpha in the risk prediction of oral epithelial dysplasia and in

oral squamous cell carcinoma.

OBJECTIVES

1. To study the expression of HIF–1 alpha in paraffin embedded tissue specimen of mild

dysplasia by Immunohistochemistry.

2. To study the expression of HIF–1 alpha in paraffin embedded tissue specimen of

moderate and severe epithelial dysplasia by Immunohistochemistry.

3. To study the expression of HIF–1 alpha in paraffin embedded tissue specimen of Oral

Squamous Cell Carcinoma by Immunohistochemistry.

4. To study the expression of HIF–1 alpha in paraffin embedded tissue specimen of

normal mucosa by Immunohistochemistry

5. To compare the expression of HIF–1 alpha in mild, moderate, severe epithelial

dysplasia, OSCC and normal oral mucosa by Immunohistochemistry

Annexures

8. MATERIALS AND METHODS

Sample:

Group I: 15 normal oral mucosal tissue specimens.

Group II: 15 histopathologically confirmed mild epithelial dysplasia tissue specimens

Group III: 15 histopathologically confirmed moderate and severe epithelial dysplasia

tissue specimens

Group IV: 15 histopathologically confirmed OSCC tissue specimens

Procedure:

Immunohistochemistry (IHC)

Statistics to be used:

Chi-square test

Data analysis to be done using SPSS (Statistical Package of Social Science)

Version 20

Equipments and chemical reagents needed:

1. Microtome

2. Autoclave

3. Hot air oven

4. Slide warmer

5. Coupling jars

6. Measuring jar

Annexures

7. Weighing machine

8. APES coated slides

9. Slide carrier

10. Aluminium foil

11. Micro-pipettes

12. Toothed forceps

13. Electronic timer

14. Beakers

15. Rectangular steel tray with glass rods

16. Sterile gauze

17. Cover-slips

18. Light microscope

Reagents used:

1. Conc. HCl

2. APES (Amino propyl triethoxysilane)

3. Acetone

4. Citrate buffer

5. Phospho Buffer Saline (PBS)

6. 3% H2O2

7. Deionized distilled water

8. Hematoxylin

9. Absolute alcohol

10. Xylene

Annexures

Signature of principal investigator

Signature of Head of Department

Remarks of committee:

Permission granted YES/NO

Modifications / condition

ANNEXURE-III

Annexures

ANNEXURE IV

Annexures

Annexures

ANNEXURE VII

ABBREVIATIONS

1. HIF - Hypoxia Inducible Factor

2. OSCC - Oral squamous cell carcinoma

3. HNSCC - Head and neck squamous cell carcinoma

4. WHO - World Health Organisation

5. IHC - Immunohistochemistry

6. APES - Amino propyl triethoxysilane

7. H&E - Hematoxylin and Eosin

8. LM - Light Microscope

9. HCl - Hydrochloric acid

10. H2O2 - Hydrogen peroxide

11. DAB - Di amino benzidine

12. HRP - Horseradish Peroxidase

13. DPX - Dibutyl Phthalate in xylene

14. OED - Oral epithelial dysplasia

15. Ki67 - Kiel 67 protein

16. INK - Inhibitor Kinases

17. LOH - Loss of heterozygosity

18. DNA - Deoxy ribo nucleic acid

19. EGFR - Epidermal Growth Factor Receptor

20. erbB - 2 - Human epidermal growth factor

Annexures

21. TSG - Tumour suppressor gene

22. bcl2 - B cell lymphoma 2 gene

23. p53(or)TP53 - Protein 53 (or ) Tumour Protein 53

24. EPO - Erythropoietin

25. ARNT - Aryl hydrocarbon nuclear translocator

26. bHLH - Basic helix loop helix

27. ODDD - Oxygen dependent degradation domain

28. VEGF - Vascular endothelial growth factor

29. TCA - Tricarboxylic acid

30. ATP - Adenosine tri phosphate

31. GLUT 1 - Glucose transporter 1

32. IGF2 - Insulin like growth factor 2

33. MAPK - Mitogen activated protein kinases

34. TGF - Transforming growth factor

35. Bax - B cell lymphoma associated X protein

36. NOXA - BH3 only family

37. PUMA - p53 upregulated modulator of apoptosis

38. PERP - p53 apoptosis effector regulated PMP

39. LDH - Lactate dehydrogenase

40. PDH - Pyruvate dehydrogenase

41. COX - Cyclooxygenase

42. PCR - Polymerase chain reaction

Annexures

43. pFAK - Phosphorylated Focal adhesion Kinase

44. PBMCs - Peripheral blood mononuclear cells

45. MVD - Microvessel density

46. HPP - Hyperplastic polyps

47. LPS - Lipopolysaccharide

48. RCC - Renal cell carcinoma

49. p27 - Kinase Inhibitory Protein p27

50. CXCR3 - Chemokine ( C-X-C Motif) Receptor

51. CAIX - Carbonic anhydrases IX

52. SLUG - Zinc finger protein

53. HPV - Human papilloma virus

54. TNF - Tumour necrosis factor

55. VHL - Von Hippel–Lindau

56. ERK - Extracellular signal-regulated kinase

57. NF-𝜅B - Nuclear factor-𝜅B

STUDY OF EXPRESSION OF HYPOXIA INDUCIBLE FACTOR - 1 …

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