Expression of Cancer Stem Cell Marker CD44 in Oral Squamous Cell Carcinoma of Tongue

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EXPRESSION OF CANCER STEM CELL MARKER

CD44 IN ORAL SQUAMOUS CELL CARCINOMA OF

TONGUE

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

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ACKNOWLEDGEMENT

I bow in gratitude to the Almighty for all His shower of blessings on me.

Words are just not expressive enough to extend my sincere gratitude to my parents who helped me throughout all my endeavours.

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 active guidance, help, encouragement and ample time spent throughout my course. Without his conscientious support, I would not have made headway in this project. Thank you so much sir.

I am extremely thankful and pay my sinceregratitude to Professor, Dr. Uma Devi K. Rao, Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital whose expertise, consistent guidance and moral support helped me to succeed in my study. I take this opportunity to acknowledge her for her care which helped me to become responsible doctor.

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I am thoroughly grateful to Professor, Dr. T. Rooban, Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital for his immense interest in my topic of research, for providing me with material and links that I could not have possibly discovered on my own to complete this study. Thank you so much Sir.

I extend my personal gratitude to Readers Dr. N. Lavanya and Dr. C. Lavanya, and Senior lecturers Dr. Sudharsan, Dr. Kavitha and Dr. Joseph, Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital for giving the right advice at the right time and for being a source of motivation in completing the study.

I am grateful to our Geneticist and Lab Manager Mrs. Kavitha Wilson, for her advices to carry out my study successfully. Much of my experimental work would have not been completed without the assistance of our Lab Technician, Mr. Rajan, Department of Oral and Maxillofacial Pathology. Thank you so much sir.

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At this juncture, I would like to thank Dr. Vidhyarani Shyamsundar, Preventive Oncology Section, Cancer Institute, Adayar for lending her hands in the technical aspects of the study.

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Oral cancer is the eleventh most common cancer in the world1. Globally, the Age-Standardised Incidence Rate per 100,000 population for oral cancer in men and women is 5.5 and 2.5, respectively. India accounts for one-fifth of all oral cancer cases and one-fourth of all oral cancer deaths. Oral cancers include cancers of the mucosal lip, tongue, gum, floor of the mouth, palate and mouth1. In spite of significant advances in treatment, 5-year survival rate after diagnosis of OSCC remains low due to advanced stage at diagnosis and tumor recurrence2. The predominant cause of death in patients with cancer is loco-regional invasion, distant metastasis, and recurrence3. Even after therapies that aim to completely regress tumours, it is possible that Cancer Stem Cells (CSCs) are left behind allowing tumour recurrence. Therapies that are specifically directed against CSCs result in much more durable responses and eliminate metastatic tumours4.

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Introduction

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The most common CSC surface marker and a potential therapeutic target for many cancers is CD448,9. It is a transmembrane glycoprotein involved in cell-cell and cell-cell-matrix adhesion and in cell-cell signaling10. It is broadly distributed on hematopoietic cells, fibroblasts, and numerous tumor cells of melanoma, leukemias and cancers of breast, ovary, liver, pancreas, lung, colon, brain, vulva and prostate8,11.

CD44 interacts with Hyaluronic Acid (HA) to influence cytoskeleton function, cell survival, growth and migration through various processes such as RhoA signalling, Ankyrin co-localization and Ezrin recruitment12,13. Proﬁling of CD44 with respect to tumour type and subtype may help in determining tumor growth, survival and metastasis14.

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Aim and Objectives

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AIM:

To evaluate the expression of CD44 in Oral Squamous Cell Carcinoma.

OBJECTIVES:

1. To investigate CD44 expression in Formalin Fixed Paraffin Embedded tissues of Squamous Cell Carcinoma of Tongue using mouse monoclonal CD44 primary antibody and SS Polymer HRP/DAB detection kit by Immunohistochemistry.

2. To investigate CD44 expression in Formalin Fixed Paraffin Embedded tissues of Squamous Cell Carcinoma of Buccal mucosa using mouse monoclonal CD44 primary antibody and SS Polymer HRP/DAB detection kit by Immunohistochemistry.

3. To investigate CD44 expression in Formalin Fixed Paraffin Embedded tissues of normal mucosa using mouse monoclonal CD44 primary antibody and SS Polymer HRP/DAB detection kit by Immunohistochemistry.

4. To compare the CD44 expression in normal mucosa, Squamous Cell Carcinoma of Tongue and Squamous Cell Carcinoma of Buccal mucosa.

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Materials & Methods

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STUDY DESIGN:

This comparative study was done to evaluate the CD44 expression in normal mucosa, Oral Squamous Cell Carcinoma of Tongue and Buccal mucosa.

STUDY GROUPS:

Group 1 - Tissues from normal buccal mucosa (n=6)

Group 2 - Tissues from Oral Squamous Cell Carcinoma of Tongue (n=16)

Group 3 - Tissues from Oral Squamous Cell Carcinoma of Buccal mucosa (n=16) Immunohistochemistry Control : Human tonsil tissue 15

(previously known positive case for CD44)

STUDY SETTING:

This study was done at Ragas Dental College and Hospital and Cancer

Institute, Adayar and approved by the Institutional Ethics Committee of Ragas

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ARMAMENTARIUM USED:

• Microtome • Autoclave • Hot air oven • Slide warmer • Coplin jars • Measuring jar • Weighing machine

• APES (3 amino propyl triethoxysilane) coated slides • Slide box

• Micro-pipettes • Toothed forceps • Electronic timer • Beakers

• Rectangular steel tray with glass rods • Sterile gauze

• Cover slips • Light microscope

REAGENTS USED:

1) Xylene

2) Absolute alcohol (Isopropyl alcohol) 3) Harris Hematoxylin

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6 5) Eosin

6) APES

7) 1 N sodium hydroxide 8) 1 N Hydrochloric acid

9) Tris EDTA (Ethylene Diamine Tetra Acetate) buffer 10) 3% Hydrogen peroxide

11) Tris buffered Saline

12) Distilled water

ANTIBODIES USED:

• Primary antibody –Anti-CD44 mouse monoclonal antibody,

Clone: DF1485, Concentrated (1ml) CAT NO- MU310-UC, BioGenex

(ANNEXURE IV).

• Secondary antibody - SS Polymer-HRP/DAB IHC Detection system, BioGenex (ANNEXURE IV).

PROCEDURE:

1) A detailed case history including patient’s age, gender, past medical and dental

history, history of drug intake, deleterious habits and trauma was taken from records for control and study group.

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3) From the Formalin Fixed Paraffin Embedded tissues, 5 micron thick sections were cut and used for routine Hematoxylin and Eosin (H&E) staining and Immunohistochemical (IHC) staining.

4) Positive control for CD44 was a section of human tonsil tissue15 (previously known positive case for CD44)

H & E STAINING:

The slides were deparaffinized in xylene and hydrated through grades of alcohol

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. The slides were differentiated in 1% acid alcohol for 5 minutes. The slides were washed well in running tap water for 5 minutes. The tissue sections on the slides were then stained in eosin for 30 seconds. The slides were washed in running tap water for 1 minute. The slides were then dehydrated through alcohol, cleared, mounted and viewed under light microscope and tumors were graded into varying histologic gradings of malignancy as well, moderately and poorly differentiated.

IMMUNOHISTOCHEMICAL STAINING OF CD44:

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xylene to remove paraffin wax. They were put in descending grades of alcohol and then rehydrated with water. Circles were drawn using a diamond marker around the tissues, so that the antibodies added later are restricted to the circle. The slides were transferred to TRIS EDTA buffer of pH 9 and steamed in pressure cooker for antigen retrieval at 15 lbs pressure for 15 minutes. Slides were then treated with 3 % hydrogen peroxide for 7 minutes to quench endogenous peroxidase activity of cells that would result in non-specific staining. Then, the slides were dipped in Tris buffered saline with Tween 20 for 6 minutes. The slides were wiped carefully without touching the tissue section. The sections were incubated at room temperature with mouse monoclonal primary CD44 antibody (BioGenex). Primary antibody was detected using SS Polymer-HRP/DAB IHC Detection system (BioGenex). After thorough washing with Tris buffered saline at pH 7.4, sections were treated with super enhancer for 20 min at room temperature followed by incubation with SS Polymer-HRP reagent for 30 min at room temperature. After three washes with TBS, substrate DAB was applied to the sections for 2 min in the dark. Slides were then washed in distilled water to remove excess chromogen and counterstained with hematoxylin, dehydrated with ethanol and xylene and mounted permanently with DPX. The slides were then observed under the Light Microscope (LM).

POSITIVE AND NEGATIVE CONTROL:

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STEPS INVOLVED:

1. APES coated slides with 2 paraffin embedded tissue placed in warming table. 2. Placed in xylene twice (5 minutes each)

3. Placed in 100% isopropanol (5 minutes) 4. Placed in 90% isopropanol (5 minutes) 5. Placed in 70% isopropanol (5 minutes) 6. Washed in distilled water (2 minutes each)

7. Keep in Tris EDTA buffer at pH 9 under steam pressure for 15 min for antigen retrieval

8. Cooling of solution done for 10 minutes 9. Slides were transferred to distilled water. 10. Placed in 3% hydrogen peroxide (7 minutes) 11. Washed in Tris buffer saline (2-3 minutes)

12. Primary antibody added and incubated (30 minutes) 13. Washed in Tris buffer saline (2-3 minutes)

14. Poly excel target binder reagent added and incubated (12 minutes) 15. Washed in Tris buffer (2-3 minutes)

16. SS Polymer-HRP added and incubated (12 minutes) 17. Washed slides in Tris buffer (2-3 minutes)

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20. Stained with Harris Hematoxylin (20 seconds) 21. Washed in tap water

22. Placed in 70% alcohol (1 minute) 23. Placed in 100% alcohol (1 minute) 24. Placed in xylene (1 dip)

25. Slides to be mounted using DPX

26. Slides to be observed under the LM and graded

CRITERIA FOR EVALUATION OF STAINING:

Evaluation of H & E sections:

Tumors were graded as well, moderate and poorly differentiated tumors.

Evaluation for IHC16:

CD44 expression was evaluated as brown membranous staining in tumor nests & stromal cells (fibroblasts). The staining intensity was graded semi-quantitatively as follows:

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To calculate the percentage of CD44 positive cells, atleast four high-power fields were chosen randomly, and 1000 cells were counted for each slide. The percentage positivity was graded semi-quantitatively as follows:

o Grade I – 0-10 % of positive cells o Grade II – 11-24 % of positive cells o Grade III – 25-49 % of positive cells o Grade IV – 50-74 % of positive cells o Grade V – 75-100 % of positive cells16

Each case was evaluated by two blinded observers independently with respect to positive control.

Statistical analysis:

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Review of Literature

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ORAL CANCER:

Oral cancer is the eleventh most common cancer in the world. Globally, the Age-Standardised Incidence Rate per 100,000 population for oral cancer in men and women is 5.5 and 2.5, respectively. India accounts for one-fifth of all oral cancer cases and one-fourth of all oral cancer deaths. The observed trends in incidence and mortality among men and women are closely correlated with the patterns in tobacco and alcohol use.

Tobacco use, in any form is the major risk factor for oral cancer. With dietary deficiencies, these factors cause more than ninety percent of oral cancers which include cancers of the mucosal lip, tongue, gum, floor of the mouth, palate and mouth, corresponding to the ICD, 10th revision. The buccal mucosa is the most common site for oral cancer in South and Southeast Asia; in all other regions, tongue is the most common site.

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Personalised diagnostics using biomarker can guide treatment and consequently improve the chance of curing the disease19.

CAUSES FOR POOR PROGNOSIS IN OSCC20:

RECURRENCE, SECOND PRIMARY TUMOR (SPT) & SECOND FIELD

TUMOR:

Long-term exposure to tobacco and/ or alcohol causes “field cancerisation” of

the upper digestive tract, thereby predisposing patients towards tumor recurrence, second field tumor formation and development of SPT at a separate anatomic site from the index tumor. It is said to be synchronous if it is diagnosed within 6 months after the index tumor or metachronous if it is diagnosed more than 6 months.

LYMPH NODE METASTASIS:

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EXTRACAPSULAR SPREAD:

Metastases may penetrate the lymph node capsule and infiltrate the perinodal tissue which has been referred to as Extracapsular Spread. It is a significant predictor of regional recurrence and the development of distant metastases.

DISTANT METASTASIS:

In patients with head and neck cancer, distant metastasis is defined as metastases below the clavicle. It may be the result of lymphogenic or haematogenous spread. High incidence of distant metastases has been reported in hypopharyngeal SCC, followed by SCC of the tongue. The average survival once distant metastases have been diagnosed ranges between 4 and 7 months. Tumor differentiation, immune status and genetic susceptibility of the host also plays a role in determining prognosis20.

MARKER OVERVIEW:

A cancer biomarker is a biological molecule found in blood, other body fluids,

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MARKER BIOLOGICAL

PROCESSES

CANCER

CONTEXT

I.SUSTAINED PROLIFERATIVE SIGNALING19

Ki-67 Cell cycle, cell proliferation

Best prognostic factor of the survival rate and recurrence

CDKN2A Cell cycle, cell cycle arrest Tumor suppressor gene

HPV16 High-risk HPV type HPV(+) tumors demonstrated favorable outcomes compared to TP53 mutants

DLC1 Negative regulation of cell proliferation

Tumor suppressor

CYR61 Regulation of cell growth and adhesion

Function as an oncogene or a tumor suppressor, depending on the cancer origin

TP53 Cell cycle, cell cycle arrest Tumor-suppressor

CCND1 Cell cycle, cell division Biomarker of cancer phenotype and disease

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PROCESSES

CANCER CONTEXT

RB1 Cell cycle, cell cycle arrest Tumor-suppressor protein. Defects in this gene are a cause of retinoblastoma and osteogenic sarcoma

CA9 Response to hypoxia Intracellular pH maintenance by which cancer cells adapt to the toxic conditions of the extracellular environtment EGFR Positive regulation of cell

proliferation

Overexpression is associated with poor prognosis

II. EVADING GROWTH SUPPRESSORS

MYC Positive regulation of cell proliferation

Deregulation of cell growth, proliferation, metabolism and genome stability

ALDH1A1 Ethanol oxidation Regulates growth and differentiation of CSCs PROM1 Retina layer formation Maintains stem cell

properties by suppressing differentiation

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PROCESSES

CANCER

CONTEXT

MAP1LC3A Autophagy Positive expression had a shorter survival

FAS Apoptosis Promotes tumor growth

III. ANGIOGENESIS

HMOX1 Angiogenesis Highly expressed in cancer than in healthy tissues

PDPN Lymphangiogenesis Identifies lymphatic endothelial

differentiation in vascular endothelial neoplasms

CTTN Cell motility and focal adhesion assembly

Overexpressed in breast cancer and OSCC

PTK2 Angiogenesis Tumor progression and metastasis

CTNND1 Cell adhesion Downregulation

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PROCESSES

CANCER CONTEXT

MMP1 Proteolysis Angiogenesis, response to hypoxia and proteolysis progression of cancer

VIM Movement of cell EMT

CDH1 Cell adhesion Loss of function cause cancer proliferation and metastasis

VCAN Cell adhesion Associated with poor outcome

AMFR Movement of cell Tumor motility-stimulating protein

IV. REPROGRAMMING OF ENERGY METABOLISM

HIF-1α Angiogenesis and response to hypoxia

Anaerobic metabolism

V. TUMOR-PROMOTING INFLAMMATION

IL4R Regulation of cell proliferation and Immune system

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PROCESSES

CANCER

CONTEXT

CXCL8 Movement of cell, chemotaxis

Neovascularisation

CD163 Inflammatory response Anti-inflammatory myeloid marker SERPINB3 Regulates cell

proliferation

EMT

CRP Inflammatory response High baseline CRP patients had a greater risk of early death

STEM CELL MARKER IDENTIFIED IN OSCC:

STEM CELL

MARKER

FUNCTIONAL RELEVANCE IN OSCC

CD44 Cell surface glycoprotein which causes proliferation, differentiation, migration, invasion, tumour sphere formation and resistance to chemotherapy.

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STEM CELL

MARKER

CD133 Transmembrane glycoprotein displaying increased clonogenicity, EMT phenotype, tumour sphere formation, self-renewal and proliferation.

c-Met Tyrosine kinase receptor for HGF is associated with metastasis, tumour invasion and decreased survival. Side

populations

Subpopulations of Hoechst 33342 dye-resistant cells demonstrate tumourigenic, chemoresistance and self-renewal properties in vivo22.

Musashi-1(Msi-1)

RNA binding post transcriptional gene regulator whose over expression correlates with advanced stage and poor differentiation.

CD97 EGF - 7 transmembrane surface protein which is highly expressed in OSCC.

Cripto-1 Key regulator of embryonic development which plays a vital role in malignant transformation.

Bone

Morphogenetic Proteins (BMPs)

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STEM CELL

MARKER

CXCR4 Chemokine receptor promote migration and invasion by regulating MMP-9 &13 via ERK signaling pathway in OSCC.

CD166 Over expression demonstrate tumour formation ability in vivo and are positively correlated with poor patient outcome

SLC2A13 A solute carrier protein family member which facilitates glucose transport whose overexpression is observed in sphere forming cells of primary cultures of OSCC samples. Nestin Up-regulated in metastasizing samples of OSCC CD29/β1

integrin

An integrin unit associated with very late antigen receptors. When combined with CD44high cells, tumors exhibit molecular characteristics of EMT. p63 A member of the p53 family of transcription

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STEM CELL

MARKER

GRP78 Key role in both stem cell and cancer by mediating tumour proliferation, metastasis and conferring resistance to treatment. ALDH1+ cells in OSCC showed increased GRP78 anchored at the plasma membrane, exerting stemness properties.

HMGA2 A transcriptional factor initiating mesenchymal tumour formation in OSCC which initiates sphere and colony formation.

p75 A low affinity neurotrophic receptor that binds to NGF and protects stem cells from apoptosis.

Podoplanin Podoplanin positive tumours demonstrated better patient survival.

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STEM CELLS – REVIEW:

Stem cells are undifferentiated, self-renewing cells with the potential to

produce specialized differentiated cells24. Stem cells are divided into three categories:

 Embryonic Stem Cells (ESCs)  Germinal Stem Cells

 Somatic Stem Cells (SSCs)

ESCs: These cells originate from the inner cell mass of the blastocyst. They are

totipotent and have indefinite replicative potential.

Germinal stem cells: These are derived from primary germinal layers of embryo.

They differentiate into progenitor cells of specific organs.

SSCs: These cells are less totipotent than ESCs and exists in haematopoietic,

neural, gastrointestinal and mesenchymal tissues25.

Stem cell potency is defined as the potential to give rise to a range of new cell phenotypes.

1. Totipotent: Cells which can differentiate into all cells of the three embryonic germ layers and extraembryonic tissue. E.g., A fertilized egg.

2. Pluripotent: Cells which can differentiate into all cell types of the three germ layers, but they lose the ability to differentiate into cells of the extraembryonic tissue. E.g., ESCs

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4. Unipotent: It is capable of differentiation into one cell type only. E.g., Muscle stem cells24

EMBRYONIC STEM CELL MARKERS26:

STEM CELL

MARKERS

CHARACTERISTICS CLASSIFICATION

SSEA-1 Murine embryos, mouse ESCs, mouse and human germ cells, EC cells

Carbohydrate associated molecules SSEA-3, SSEA-4

Primate ESCs, human embryonic germ cells, human ESCs, EC cells

CD90 (Thy-1)

Human ES cells, mouse ES cells, hematopoietic stem

cells, EC cells Surface marker CD31, CD59, CD9,

CD 29

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STEM CELL

MARKERS

CHARACTERISTICS CLASSIFICATION

CD326 Human ES cells, mouse ES cells, EC cells

Surface marker CD133 Human ES cells, mouse ES

cells, embryonal carcinoma (EC) cells, Hematopoietic stem cells

TRA-1-81 Human ESCs, teratocarcinoma, embryonic germ cells, EC cells

Surface antigen TRA-1-60 Human ESCs, teratocarcinoma,

embryonic germ cells, EC cells

Cripto (TDGF-1) Mouse ES cells, human ESCs, cardiomyocyte, EC cells

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BEST STEM CELL MARKER IN VARIOUS TUMORS22,27:

STEM CELL MARKERS COMMON

CANCERS

CD44+, ALDH, CD133, c-Met, Side populations OSCC

CD44+, ESA+, CD24–/low lineage, ALDH-1high Breast cancer

CD44+, CD133+, EpCAM+, CD24+ Pancreatic cancer

CD44+, α2β1high, CD133+ Prostate cancer

CD44+, CD133+, ALDH-1, CD166+, CD29, CD24+, CD26, Msi-1, Lgr-5, Wnt activity/β-catenin

Colon cancer CD133+, BCRP1+, A2B5+, SSEA-1+ Brain

cancer CD34+, CD38–, HLA–DR–, CD71–, CD90–, CD117–,

CD123+

Leukemia

CD133+, CD49f+, CD90+ Liver

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CD44:

Synonyms: Pgp-1, Ly-24, Hermes, lymphocyte homing receptor, H-CAM, and HUTCH-110.

One of the most common CSC surface marker and a promising therapeutic target for many cancers8. It is a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesion and in cell signaling10. It was first described by Dalchau et al9. It is broadly distributed on hematopoietic cells, fibroblasts, and numerous tumor cells. It was first identified as gp85 which was then shown to be a HA receptor in placenta cells11. CD44 is encoded by the highly conserved CD44 gene on chromosome 11 in humans. The full-length CD44 gene consists of 20 exons and 19 introns. Ten of these exons are expressed in all isoforms (constant exons). The ten central exons (variable exons) undergo extensive alternative splicing via excision or inclusion in various combinations in the membrane proximal stem region to generate splicing variants (CD44v isoforms), which accounts for CD44 heterogeneity.

VARIANTS OF CD44:

1. CD44s - Smallest or standard isoform lacks all variant exons in the membrane proximal domain and is expressed on most vertebrate cells.

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CD44 molecular weight ranges from 85-230 kD for three reasons:

1. Variety of mRNAs are generated by alternative splicing of ten variant exons in its pre-mRNA.

2. Undergo various post-translational modifications

3. They get shortened in vivo, owing to partial cleavage by matrix metalloproteases, and this effect is most pronounced in human cancer of all types10.

CD44 also contributes to T cell activated extravasation of lymphocytes into sites of inflammation and plays substantial role in angiogenesis28.

MOLECULAR STRUCTURE OF CD4429:

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Hyaluronan binding domain:

This domain forms the primary ligand binding receptors for HA. Other CD44

ligand includes collagen, osteopontin, integrin, fibronectin, laminin, and MMPs.

Membrane proximal domain:

This is the most diverse region of the CD44 molecule in which introduction of new exons modulates HA-binding affinity by inducing conformational changes or allowing CD44 isoforms to function as a co-receptor by generating new binding sites for many growth factors, receptors, and non receptor protein-tyrosine kinases. For example, the sequence encoded by exon v6 contains a binding site for HGF and VEGF. The binding of HGF with CD44v6 induces CD44v6/HGF/cMet complex formation, leading to c-Met or HGF-induced Ras signaling activation. The inclusion of variant exons has been found to be dependent on mitogenic or oncogenic signals that regulate alternative splicing. Therefore, cancer cells express a variety of CD44 variants, particularly in an advanced stage.

Transmembrane (TM) domain:

It provides a platform for CD44v oligomer formation or coupling to cofactors, adaptor proteins, and receptor or non receptor protein-tyrosine kinases.

Cytoplasmic Domain:

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CD44 INTERACTIONS IN CANCER:

CD44 AND HA:

CD44-HA binding is tightly coupled with Leukemia-Associated Rho Guanine nucleotide exchange factor (LARG) which induces LARG-mediated RhoA activation. When activated by RhoA, Phospholipase C (PLC) hydrolyze inositol diphosphate into inositol trisphosphate (IP3), resulting in Ca2+ release from intracellular stores. This in turn activates Ca2+/calmodulin-dependent kinase-II. It then phosphorylates the cytoskeletal protein, filamin, leading to cytoskeleton reorganization and tumor cell migration.

CD44-HA binding activates one of the RhoGTPase i.e., ROK. It phosphorylates myosin phosphatase and myosin light chain, thereby activating myosin adenosine triphosphatase (ATPase) and generating actomyosin-mediated membrane motility and cell migration.

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CD44 AND ANKYRIN:

CD44-HA binding also promotes recruitment of the cytoskeletal protein, ankyrin, and inositol-1,4,5-triphosphate (IP3) receptor into cholesterol-containing lipid rafts. The Ankyrin Repeat Domain of ankyrin is responsible for binding IP3 receptor to CD44 at lipid rafts which subsequently triggers intracellular Ca2+ mobilization leading to HA-induced membrane–cytoskeleton interactions and tumor cell-specific behaviors (e.g., cell survival, growth and migration)12,30.

CD44 AND THE ERM (EZRIN, RADIXIN, AND MOESIN):

ERM proteins regulate the structural assembly and stabilisation of plasma membrane domain. They are concentrated at the surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. They also serve as linkers between molecules such as CD44 and ICAM-2 and the cytoskeleton. ERM proteins are recruited to the cytoplasmic tail of CD44 and so linking to the cytoskeleton, under the influence of growth factors, results in proliferation and invasion of tumors13.

CD44 REGULATION OF DRUG TRANSPORTER EXPRESSION:

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hyaluronan interactions, viz., hyaluronan oligomers, rapidly induces internalization of the transporters and CD44 into the cell29.

CD44 IN INFLAMMATION:

Binding between CD44 and E-selectin ligand support neutrophil slow rolling.

Interaction of CD44 with P-selectin causes initial tethering and fast rolling. These interactions play a key role in the pathogenesis of Leucocyte Adhesion Deficiency syndrome in which binding of CD44 with E-selectin is inhibited. Bi-molecular complex formation between CD44 and VLA-4 integrin is required for firm adhesion of neutrophils. Neutrophil transit across epithelial monolayers in vitro was found to be attenuated when neutrophil CD44 was activated. In contrast, visualization of neutrophil recruitment in vivo within the inﬂamed cremaster

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Activated cells present in tonsillar lymphocyte preparations possess CD44 which is capable of binding hyaluronan. It is this that accounts for lymphocytes to adhere under flow with tonsillar stromal cells, stromal cell extracellular matrix, purified hyaluronan, and reticular fibers in the T cell areas of secondary lymphoid

organs33. In the context of autoimmune disease, CD44 also enables rolling

subsequent to T cell stimulation, in the peripheral circulation during chronic inflammatory diseases which supports their role in the enhanced homing of activated lymphocytes to target sites of autoimmunity33.

CO-FACTORS INTERACTIONS WITH CD44:

HYALURONAN:

Hyaluronan is a major ligand for CD44 and can bind CD44v isoforms. Through binding of CD44, HA can activate cytoskeleton and MMPs signaling involved in tumor progression. It exists in high molecular weight or low molecular weight due to cleavage into varying sizes. High molecular weight HA is involved in tumorigenesis, anti-angiogenic and anti-inflammatory responses. However, low molecular weight HA promote cell motility, CD44 cleavage and angiogenesis. Therefore, the size of HA ligand determines biological function.

OSTEOPONTIN (OPN):

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N-terminal domain of CD44. OPN binding to CD44 variants/beta1-containing integrin promotes cell spreading, motility and chemotaxis. OPN increases expression of both CD44s and variant isoforms in human melanoma.

MATRIX METALLOPROTEASES (MMPS):

Extracellular matrix proteins that are involved in extracellular matrix degradation which are important during development, wound healing, bone resorption, and angiogenesis. MMP-9 and CD44 binding results in MMP9 activity localization on the cell surface. Ability of CD44 to localize proteolytically active MMP-9 to the tumor cell surface is important for tumor invasion34.

CD44 - THERAPEUTIC TARGET IN CANCER:

CD44 ANTIBODIES AND VACCINES:

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HYALURONAN OLIGOMERS:

Hyaluronan oligomers act in vivo by disrupting endogenous hyaluronan-CD44 interactions that are necessary for tumor progression. Hence, inhibits local tumor growth, primary tumor spreading and residual tumor growth following surgical removal of the tumor mass38. It also suppresses resistance to anticancer drugs, including, doxorubicin, taxol, vincristine and methotrexate29.

HYALURONAN -COATED NANOPARTICLE SYSTEMS:

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vessels to tumor tissue rather than their normal counterpart. Also, the lack of lymphatic vessels causes lymph circumfluence to suffocate. Under these circumstances, nanoparticles, accumulate in tumor tissues. Magnetic nanomaterials are good candidates for disease treatment because tissues rarely absorb magnetic waves, which penetrate deeper than visible light and infrared rays without side effects. It contains iron oxide, which can be locally heated by a magnetic field. The walls of microcapsules are integrated with 18 nm-diameter iron oxide nanotubes. Under an alternating magnetic field, magnetic nanotubes can heat their surroundings and destroy the walls of the microcapsules, thereby releasing the encapsulated material into the surrounding solution38.

E.g., HA– Iron Oxide Magnetite nanoparticles are efficient in labelling CD44 containing tumor cells in targeted drug delivery systems39.

HYALURONIDASES:

Hyaluronidases improve access of drugs to cancer cells through effects on cell adhesion and matrix barriers. Its oligosaccharide products inhibit hyaluronan-CD44 signaling, resulting in decreased cell survival and chemoresistance35.

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CD44 IN OSCCs AND OPMD:

An Immunohistochemical study by Flavia Paiva Prudente de Moraes et al

concluded that CD44 might be associated with patient outcome in OSCC in which evaluation of the expression of CD44, CD24, CD133, ALDH1, CD29 and Ki-67 in 52 OSCC specimens [Oropharynx not otherwise specified (n=4), Tonsils (n=10), Soft palate (n=2), Mobile tongue (n=20), Gingiva (n=5), Floor of the mouth (n=4), Hard palate (n=4), Retromolar trigone (n=2), Cheek mucosa (n=1)] and 21 metastatic lymph nodes and correlation with clinicopathologic features was done. The study shown CD44 positivity in 30 OSCC specimens (57.7 %), 7 metastatic lymph nodes (33.3%) and in the basal and parabasal layers of normal mucosa. In the study, a statistically significant association between CD44 expression and well-differentiated tumors (P=.045) was seen. Also, the median 5-year survival rate of patients who shown negative and positive expression for CD44 was 74% and 38%, respectively40.

Among the CD44 variants, CD44v6 proved to be a poor predictor of tumor

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subsites (n = 32) presented a high expression of this marker only in 22 cases (53.7%). However, CD44v6, p63 and podoplanin expression did not show signiﬁcant relationship with clinico–pathological variables. But, a signiﬁcant

relation was found between MMP-9 and tumour stage (p=0.002) and also with nodal metastasis (p=0.018)16.

CD44 was found to be useful in predicting the disease course in which

immunohistochemical expression of HIF-1α, CD44, p16, Ki67, and podoplanin in 35 OSCC specimens was investigated and correlation with clinical findings was done by

Johannes Dunkel et al. It was found that high CD44 expression and low HIF-1α

expression had a strong positive correlation with the 5-year disease-free survival (P < .001). Whereas, p16, Ki67 and podoplanin did not correlate with the course of the disease41.

CD44 was found to be useful in assessing the prognosis of OSCC in relation to different sub-sites of the oral cavity in an immunohistochemical study by

Michal Krump and Jiri Ehrmann which analyzed 29 OSCC samples {floor of

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39

statistically significant CD44 negativity in tongue tumour group (0%) compared with that of the floor of the mouth (85%; p=0.003) and other sites (100%; p=0.003). But, no statistically significant difference was found in histologic grades of differentiation42.

With regard to premalignant conditions, CD44v6 serves as a useful marker for detecting high-risk leukoplakias. This finding was demonstrated in a study by

Pournima Y Godge and Leena S Poonja which analyzed the

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40

CD44 appears to be the most useful among other CSCs markers in OSCC in a

study by Cl. Margaritescu et al which evaluated immunohistochemical expression of three CSC markers, namely, CD44, CD133 and CD117. Out of 30 specimens, CD44 positivity was seen in 80–100% of well-differentiated, 60–85% of moderate differentiated and 40–62% of poor differentiated cases. In normal mucosa, the strongest reaction was seen in basal and parabasal layers and became progressively weaker in the upper granular layers. Both in normal and tumor tissues, there was no expression for CD133 and CD11744.

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41

Taking into account the staining patterns, irregular staining of CD44 was found to predict more advanced disease and shortened survival of the patients in an immunohistochemical study by Kosunen A et al which evaluated CD44 and MMP-9 expression in OSCC. The study included 138 and 124 specimens for CD44 and MMP-9 analyses, respectively and correlated with clinicopathological factors and survival in OSCC. The results revealed association between irregular staining pattern of CD44 and poor tumour differentiation (p = 0.003), clinical stage III–IV (p = 0.049) and the presence of T3-4 tumour stage (p = 0.03). It was also found that MMP-9 intensity was strong in 21 (17%), moderate in 78 (63%) and weak in 25 (20%) cases and MMP-9 stromal staining intensity was strong in 31 (25%), moderate in 61 (49%) and weak in 32 (26%) cases. A significant association was found between irregular staining of CD44 in tumour cells and strong staining intensity of MMP-9 in stromal tissue (p < 0.001) whereas CD44 expression and MMP-9 intensity were not related to each other in tumour cells46.

CD44 IN OSCC OF TONGUE:

CD44 targeting was found to increase therapeutic success, thereby decreasing

morbidity and mortality of OSCC in an immunohistochemical study by Nasrollah

Saghravanian et al which investigated CD44 and p63 expression in two groups

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42

in high grade tumors (p < 0.001), advanced stage tumors (p = 0.009) and overall survival (p < 0.05) which was statistically significant. And, the death risk for cases with CD44 expression more than 30% was 2.08 fold higher than those with immunoreactivity less than 30%. Whereas, increased expression of p63 correlated with higher grades of disease and a significant difference was found between p63 expression and different grades of OSCC (p= 0.004)47.

CD44 was not found to be useful in predicting tumor prognosis in an

immunohistochemical study by Reza Kaboodkhani et al which evaluated the expression of CD44 in 51 OSCC specimens of tongue. The study did not show significant correlation with survival (p=0.77), age (p=0.4), clinical lymphadenopathy (p = 0.155), lymph node metastasis (p=0.87), sex (p=0.947), smoking (p= 0.287), and tumor size (p=0.813)48.

Irrespective of HPV status, CD44 was found to be a prognostic marker in

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43

(p=0.174 and p=0.345), E-cadherin (p=0.355 and p=0.395), COX-2 (p=0.944 and p=0.599), Ki-67 (p=0.628 and p=0.145) and p27 (p=0.690 and p=0.471). And, also expression and intensity of CD44, EGFR, COX-2, Ki-67, VEGF, E-cadherin and p27 was not correlated to HPV status. In the multivariate analyses, CD44, p16 and EGFR were included together with age, TNM stage, grade, tumor location and sex. CD44 was found to be statistically significant prognostic marker (p=0.026), while expression of p16 and EGFR failed to show a statistically significant correlation49.

CD44 was found to be a useful in predicting the invasive behaviour of OSCC

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44

CD44 expression was found to associate only with histological grading of

malignancy in OSCC of tongue in an immunohistochemical study by Maria

Carmen Fontoura Nogueira da Cruz et al which associated the expression of

CD44v6 and E-cadherin in 30 OSCC specimens [Tongue (n=15) and lower lip (n=15)] with the anatomic location, nodal metastasis and histological grading of malignancy. The results revealed OSCCs with low score of malignancy had a higher immunoexpression of E-cadherin (p = 0.005) and CD44v6 (p = 0.003). However, no significant difference in the expression pattern and number of positive cells for CD44v6 or E-cadherin in OSCCs of tongue and lower lip (p>0.05) and also with and without nodal metastasis (p>0.05) was found15.

Loss of CD44 expression was found to be an early event in lingual carcinogenesis and a marker of major alterations in non-tumour adjacent epithelium in an immunohistochemical study by Miguel Angel Gonzalez-Molesa

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45

Among the isoforms, CD44v9 proved to be a useful indicator in assessing

metastatic potential of OSCCs of Tongue in which the immunoexpression of CD44 and their correlation to lymph node metastasis and survival was done by S.

Sato et al. In 120 specimens, CD44v9 expression did not correlate with the

histological grading of the tumors (p=0.109), whereas positive correlation was seen between reduced expression of CD44v9 and without metastatic lesions. Also, shorter survival time (p=0.0125) showed CD44v9 reduction. Hence, reduced expression of CD44v9 serves in assessing tumor prognosis52.

OTHER METHODS OF STUDYING CD4453,54:

Other methods in demonstrating CD44 expression were as follows:

Fluorescence Activated Cell Sorting (FACS), Immunofluorescence, Polymerase Chain Reaction (PCR), Western Blot, Immunocytochemistry.

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46

expressed in higher levels in the cell lines of stage IV SCC which was resistant to the conventional treatment regimens (11A). Real time quantitative RT-PCR shown high levels of CD44v 4 and CD44v 6 in primary tumor cell lines rather than their corresponding recurrences. The levels of expression increased with the tumor stage. And, also CD44 v4 and CD44 v6 was high in CD44 enriched cells sorted by FACS by 4 and 2 fold correspondingly. CD44v 1 and CD44v 2 was expressed equally in CD44high and CD44low cells. Immunofluorescence staining with CD44s were shown to delineate the plasma membrane and cell-cell boundaries. CD44v 1, CD44v 2, CD44v 4, and CD44v 6 were seen in cells with mesenchymal morphology. Specifically, CD44v1 and CD44v 2 in higher magnification participated in philopodia. CD44 staining in dividing cells labeled the centers of organization of microtubules. Immunocytochemistry did not pick up any variant forms for this cell line, indicating that both FACS and real time qRT-PCR have higher sensitivity53.

CD44 does not confer a survival advantage in head and neck cancers, however its variants offers a survival advantage or stem cell properties. In a study

by M. Athanassiou-Papaefthymiou et al, In Silico Transcriptomics database

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47

six probes, respectively, whereas decreased CD44 was found for five out of six probes in disease and only one cell line i.e., SCC of tongue. In Immunocytochemistry, all cell lines stained 50% for CD44s, except for HN12 where only 10% cells were stained. All cell lines also stained 50% for CD44 variants namely, v3, v5, v6, and v9. CD44v10 had occasional staining in all cell lines except cell line 20. HN4 cell line did not stain for CD44v 7, while its metastasis-derived HN12 stained sparsely.

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48

expressed another band at 180–190 kDa.. Variants i.e., v5, v7/8, and v10 were expressed by all cell lines but there were no differences in their expression between them. Flow Cytometry analysis shown that all cell lines expressed total CD44 greater than 50% increase in mean channel fluorescence (MCF). The most frequently undetected variant exon was v7, followed by v10 and v4. OSC-20 cell line expressed the fewest variant exons. And, also HN4 lacked v7 while its metastasis-derived partner, HN12, lacked v10. Expression of v4 was infrequent in HN30 (50% increased MCF) but in HN31 it was highly expressed (50% increased MCF). However, HN22 could not be analysed54.

CD44 IN VARIOUS CANCERS:

CD44 also plays a key role in the prognosis of tumors such as Prostate cancer,

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Results

49

SAMPLE CHARACTERISTICS:

The study population comprised of 38 cases taken from the archival blocks.

They were categorized into three groups. Group 1 (n=6) comprising of normal mucosa samples, Group 2 (n=16) comprising of Oral Squamous Cell Carcinoma of Tongue and Group 3 (n=16) comprising of Oral Squamous Cell Carcinoma of buccal mucosa. All the samples were analyzed for immunohistochemical expression of CD44.

Distribution of age in the study groups (Table 1 & Graph 1):

The age of patients were divided into 3 groups: 20 – 40 years, 41 – 60 years and those above 61 years. Group 1 consisted of 6 (100%) cases. All were in the age group of 20 – 40 years. Group 2 consisted of 2(12.5%) cases in 20 – 40 years, 11(68.8%) cases in 41 – 60 years and 3(18.8%) cases above 61 years. Group 3 consisted of 3(18.8%) cases in 20 – 40 years, 7(43.8%) cases in 41 – 60 years and 6(37.5%) cases above 61 years. A significant difference was found with respect to age in the study groups (p=0.001*)

Distribution of gender in the study groups (Table 2 & Graph 2):

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50

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 with

1. No habits

2. Habit of tobacco (chewing only)

3. Habit of tobacco usage (smoking and chewing)

4. Habit of chewing tobacco in combination with alcohol usage 5. Habit of smoking in combination with alcohol consumption

6. Habit of tobacco (smoking and chewing) and alcohol consumption

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Results

51

All the cases enrolled in the study showed CD44 staining. In group 1, group 2 and group 3, CD44 staining was present in all of the 6(100%) cases, 16(100%) cases and 16(100%) cases, respectively.

The following parameters were used to evaluate CD44 in the study groups:  Staining intensity and

 Percentage of positive cells stained

Comparison of staining intensity of CD44 between the study groups (Table 4 &

Graph 4):

On comparing the staining intensity between the study groups, out of 6(100%) cases in group 1, 5 (83.3%) cases exhibited strong staining, whereas only 1(16.7%) case demonstrated weak staining. In group 2, 4(25%), 6(37.5%) and 6(37.5%) cases showed weak, moderate and strong staining, respectively. In group 3, 4(25%), 5(31.3%) and 7(43.8%) cases showed weak, moderate and strong staining. However, no significant difference was found with respect to CD44 staining intensity between the study groups (p=0.363*).

Comparison of percentage of CD44 positive cells between the study groups

(Table 5 & Graph 5):

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52

% and 0 to 10 % of positive cells, respectively. However, the results were statistically insignificant (p=0.177*).

Comparison of staining intensity of CD44 between histologic grades of

differentiation in OSCC (Table 6 & Graph 6):

Out of 32 cases of OSCC, 13, 10 and 9 were well, moderate and poorly differentiated tumors, respectively. Among the well differentiated tumors, strong and moderate intensity was present in 8(61.5%) and 5(38.5%) cases, respectively. Among the moderately differentiated tumors, strong, moderate and weak intensity was present in 4(40%), 4(40%) and 2(20%) cases. Within the poorly differentiated tumors, strong, moderate and weak intensity was found in 1(11.1%), 2(22.2%) and 6(66.7%) cases. When the sub-site of tumors were not taken into account, staining intensity of CD44 correlated with histologic grades of differentiation (p=0.009*)

Comparison of percentage of CD44 positive cells between histologic grades of

differentiation in OSCC (Table 7 & Graph 7):

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53

Comparison of CD44 staining intensity between histologic grades of group II

In group 2, i.e., Oral S quamous Cell Carcinoma of Tongue, out of 16(100%) cases, 7, 5, 4 cases were well, moderate and poorly differentiated tumors, respectively. Among well differentiated, 4(57.1%) and 3(42.9%) cases exhibited strong and moderate intensity. In moderately differentiated tumors, 2(40%), 2(40%) and 1(20%) cases exhibited strong, moderate and weak intensity. In poorly differentiated tumors, 1(25%) and 3(75%) cases exhibited moderate and weak intensity. But, no significant difference was found with respect to CD44 staining intensity in the group 2 (p=0.083*).

Comparison of CD44 staining intensity between histologic grades of group III

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54

group II (Table 10 & Graph 10):

In group 2, i.e., out of 16(100%) cases of Oral Squamous Cell Carcinoma of Tongue, 7, 5, and 4 were well, moderate and poorly differentiated tumors, respectively. In well differentiated type, 1(14.3%), 2(28.6%), 3(42.9%) and 1(14.3%) cases demonstrated 75 to 100 %, 50 to 74 %, 25 to 49 % and 11 to 24 % of positive cells, respectively. In moderately differentiated tumors, 1(20%), 1(20%), 2(40%) and 1(20%) cases exhibited 75 to 100 %, 50 to 74 %, 25 to 49 % and 0 to 10 % of positive cells, respectively. In poorly differentiated tumors, 1(25%) and 3(75%) cases exhibited 50 to 74 % and 11 to 24 % of positive cells, respectively. However, no significant difference was found (p=0.246*).

group III (Table 11 & Graph 11):

Out of 16(100%) cases of Oral Squamous Cell Carcinoma of buccal mucosa, 6, 5, and 5 were well, moderate and poorly differentiated tumors, respectively. Among the well differentiated type, 3(50%), 2(33.3%), and 1(16.7%) cases demonstrated 75 to 100 %, 50 to 74 % and 25 to 49 % of positive cells, respectively. In moderately differentiated tumors, 1(20%), 2(40%) and 2(40%) cases exhibited 75 to 100 %, 50 to 74 % and 25 to 49 % of positive cells, respectively. In poorly differentiated tumors, 1(20%), 2(40%) and 2(40%) cases exhibited 75 to 100 %, 25 to 49 % and 0 to 10% of positive cells, respectively. However, the results were insignificant (p=0.238*).

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[image:73.595.101.513.149.319.2]

Tables and Graphs

TABLE 1: DISTRIBUTION OF AGE AMONG THE STUDY GROUPS (N=38)

AGE GROUPS

IN YEARS

GROUP 1

(n=6)

GROUP 2

(n=16)

GROUP 3

(n=16)

p VALUE

20-40 _6(100%) _2(12.5%) _3(18.8%)

0.001*

41-60 _0(0%) _11(68.8%) _7(43.8%)

61 AND

ABOVE

0(0%) _3(18.8%) _6(37.5%)

*p Value < 0.05 is significant

GRAPH 1:DISTRIBUTION OF AGE AMONG THE STUDY GROUPS

GROUP I – Normal mucosa

GROUP II – Oral Squamous Cell Carcinoma of Tongue

[image:73.595.119.507.411.625.2]

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[image:74.595.100.516.168.285.2]

Tables and Graphs

TABLE 2: DISTRIBUTION OF GENDER AMONG THE STUDY GROUPS (N=38)

GENDER GROUP 1

(n=6)

GROUP 2

(n=16)

GROUP 3

(n=16)

p VALUE

MALE _4(66.7%) _13(81.3%) _12(75%)

0.763

FEMALE _2(33.3%) _3(18.8%) _4(25%)

GRAPH 2:DISTRIBUTION OF GENDER AMONG THE STUDY GROUPS

[image:74.595.110.517.391.615.2]

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[image:75.595.101.512.131.423.2]

Tables and Graphs

TABLE 3: DISTRIBUTION OF HABITS AMONG THE STUDY GROUPS (N=38)

HABITS GROUP 1

(n=6) GROUP 2 (n=16) GROUP 3 (n=16) p VALUE

NO HABITS _6(100%) _1(6.3%) _0(0%)

0.000*

TOBACCO CHEWING 0(0%) _4(25%) _5(31.3%)

TOBACCO

(SMOKING+CHEWING)

0(0%) _3(18.8%) _2(12.5%)

TOBACCO

CHEWING+ALCOHOL

0(0%) _0(0%) _4(25%)

SMOKING+ALCOHOL 0(0%) _1(6.3%) _2(12.5%)

SMOKING+TOBACCO

CHEWING +ALCOHOL

0(0%) _7(43.8%) _3(18.8%)

GRAPH 3:DISTRIBUTION OF HABITS AMONG THE STUDY GROUPS

[image:75.595.109.514.492.718.2]

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[image:76.595.100.508.159.302.2]

Tables and Graphs

TABLE 4: COMPARISON OF STAINING INTENSITY OF CD44 BETWEEN THE STUDY GROUPS (N=38)

STAINING

INTENSITY

GROUP 1

(n=6)

GROUP 2

(n=16)

GROUP 3

(n=16)

p VALUE

WEAK 1(16.7%) _4(25%) _4(25%)

0.363

MODERATE 0(0%) _6(37.5%) _5(31.3%)

STRONG 5(83.3%) _6(37.5%) _7(43.8%)

GRAPH 4:DISTRIBUTION OF STAINING INTENSITY OF CD44

BETWEEN THE STUDY GROUPS

[image:76.595.118.504.396.652.2]

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[image:77.595.102.512.135.369.2]

Tables and Graphs

TABLE 5: COMPARISON OF PERCENTAGE OF CD44 POSITIVE CELLS BETWEEN THE STUDY GROUPS (N=38)

PERCENTAGE OF

CD44 POSITIVE

CELLS

GROUP 1

(n=6)

GROUP 2

(n=16)

GROUP 3

(n=16)

p VALUE

0-10 _0(0%) _1(6.3%) _2(12.5%)

0.177

11-24 0(0%) _4(25%) _0(0%)

25-49 1(16.7%) _5(31.3%) _5(31.3%)

50-74 1(16.7%) _4(25%) _4(25%)

75-100 4(66.7%) _2(12.5%) _5(31.3%)

GRAPH 5:COMPARISON OF PERCENTAGE OF CD44 POSITIVE

CELLS BETWEEN THE STUDY GROUPS

[image:77.595.120.512.424.670.2]

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[image:78.595.107.505.134.334.2]

Tables and Graphs

TABLE 6: COMPARISON OF CD44 STAINING INTENSITY BETWEEN HISTOLOGIC GRADES OF DIFFERENTIATION (N=32)

STAINING

INTENSITY

OF CD44

HISTOLOGIC GRADES OF

DIFFERENTIATION

p VALUE

WELL

(n=13)

MODERATE

(n=10)

POOR

(n=9)

WEAK _0(0%) _2(20%) _6(66.7%)

0.009*

MODERATE 5(38.5%) _4(40%) _2(22.2%)

STRONG 8(61.5%) _4(40%) _1(11.1%)

GRAPH 6:COMPARISON OF CD44 STAINING INTENSITY BETWEEN

HISTOLOGIC GRADES OF DIFFERENTIATION (N=32)

[image:78.595.124.499.442.696.2]

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[image:79.595.110.518.135.395.2]

Tables and Graphs

TABLE 7: COMPARISON OF PERCENTAGE OF CD44 POSITIVE CELLS BETWEEN HISTOLOGIC GRADES OF DIFFERENTIATION (N=32)

PERCENTAGE

OF CD44

POSITIVE

CELLS

DIFFERENTIATION p VALUE WELL (n=13) MODERATE (n=10) POOR (n=9)

0-10 _0(0%) _1(10%) _2(22.2%)

0.277

11-24 1(7.7%) _0(0%) _3(33.3%)

25-49 4(30.8%) _4(40%) _2(22.2%)

50-74 4(30.8%) _3(30%) _1(11.1%)

75-100 4(30.8%) _2(20%) _1(11.1%)

GRAPH 7:COMPARISON OF PERCENTAGE OF CD44 POSITIVE CELLS

BETWEEN HISTOLOGIC GRADES OF DIFFERENTIATION

[image:79.595.119.512.453.693.2]

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[image:80.595.102.507.145.341.2]

Tables and Graphs

TABLE 8: COMPARISON OF CD44 STAINING INTENSITY BETWEEN HISTOLOGIC GRADES OF GROUP II (N=16)

STAINING

INTENSITY

OF CD44

DIFFERENTIATION

p VALUE

WELL

(n=7)

MODERATE

(n=5)

POOR

(n=4)

WEAK _0(0%) _1(20%) _3(75%)

0.083

MODERATE 3(42.9%) _2(40%) _1(25%)

STRONG 4(57.1%) _2(40%) _0(0%)

HISTOLOGIC GRADES OF GROUP II

[image:80.595.107.511.435.705.2]

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[image:81.595.102.513.145.341.2]

Tables and Graphs

TABLE 9: COMPARISON OF CD44 STAINING INTENSITY BETWEEN HISTOLOGIC GRADES OF GROUP III (N=16)

STAINING

INTENSITY

OF CD44

DIFFERENTIATION

p VALUE

WELL

(n=6)

MODERATE

(n=5)

POOR

(n=5)

WEAK _0(0%) _1(20%) _3(60%)

0.222

MODERATE 2(33.3%) _2(40%) _1(20%)

STRONG 4(66.7%) _2(40%) _1(20%)

HISTOLOGIC GRADES OF GROUP III

[image:81.595.110.516.431.706.2]

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[image:82.595.103.506.121.385.2]

Tables and Graphs

TABLE 10: COMPARISON OF PERCENTAGE OF CD44 POSITIVE CELLS BETWEEN HISTOLOGIC GRADES OF GROUP II (N=16)

PERCENTAGE

OF CD44

POSITIVE

CELLS

DIFFERENTIATION p VALUE WELL (n=7) MODERATE (n=5) POOR (n=4)

0-10 _0(0%) _1(20%) _0(0%)

0.246

11-24 1(14.3%) _0(0%) _3(75%)

25-49 3(42.9%) _2(40%) _0(0%)

50-74 2(28.6%) _1(20%) _1(25%)

75-100 1(14.3%) _1(20%) _0(0%)

GRAPH 10:COMPARISON OF PERCENTAGE OF CD44 POSITIVE CELLS

BETWEEN HISTOLOGIC GRADES OF GROUP II

[image:82.595.108.512.475.707.2]

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[image:83.595.103.505.131.386.2]

Tables and Graphs

TABLE 11: COMPARISON OF PERCENTAGE OF CD44 POSITIVE CELLS BETWEEN HISTOLOGIC GRADES OF GROUP III (N=16)

PERCENTAGE

OF CD44

POSITIVE

CELLS

DIFFERENTIATION

p VALUE

WELL

(n=6)

MODERATE

(n=5)

POOR

(n=5)

0-10 _0(0%) _0(0%) _2(40%)

0.238

11-24 0(0%) _0(0%) _0(0%)

25-49 1(16.7%) _2(40%) _2(40%)

50-74 2(33.3%) _2(40%) _0(0%)

75-100 3(50%) _1(20%) _1(20%)

GRAPH 11:COMPARISON OF PERCENTAGE OF CD44 POSITIVE

CELLS BETWEEN HISTOLOGIC GRADES OF GROUP III

[image:83.595.112.513.486.705.2]

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Photographs

ARMAMENTARIUM

ANTIBODY KIT

(86)

Photographs

CD44 EXPRESSION IN POSITIVE CONTROL

10 X 40 X

CD44 EXPRESSION IN NORMAL MUCOSA

10 X 40 X

(87)

Photographs

CD44 EXPRESSION IN OSCC – WELL-DIFFERENTIATED

10 X 40 X

CD44 EXPRESSION IN OSCC – MODERATELY-DIFFERENTIATED

10 X 40 X

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Photographs

CD44 EXPRESSION IN OSCC – POORLY-DIFFERENTIATED

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Expression of Cancer Stem Cell Marker CD44 in Oral Squamous Cell Carcinoma of Tongue