Ministry of Higher Education and Scientific Research-University University of Sulaimani/School of Dentistry

Immunohistochemical Expression of PTCH1 and Laminin in Oral Mucosal hyperplastic, Potentially malignant lesions lesions, Oral Squamous Cell Carcinoma and Recurrence Samples. (A Clinicopathological Study)

A Thesis Submitted to the Council of the College of Dentistry/Faculty /Faculty of Medical Science at the University of Sulaimani in Partial Fulfillment of the Requirements Requirement for the Degree egree of Master in Oral Surgery

By Kanar Abdaladhim Hamaamin B.D.S Supervised by Dr. Falah A. Hussein

Dr. Dena na N. Mohamma Mohammad

Assistant professor B.D.S, H.D.D, F.I.C.M.S 2017 A.D

Lecturer B.D.S, M.Sc., Ph.D 2716 Kurdi

1438 A.H

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‫صدق ﷲ العظيم‬

Declaration This is to certify that the organization and preparation of this thesis have been made by the graduate student Kanar Abdaladhim Hamaamin under our supervision in the College of Dentistry, University of Sulaimani, in partial fulfillment of the requirements for the Degree of Master of Science in Oral Surgery.

Signature Dr. Falah A. Hussein Assistant professor B.D.S, H.D.D, F.I.C.M.S Supervisor

Signature Dr. Dena N. Mohammad Lecturer B.D.S, M.Sc., Ph.D Supervisor

Dedication This thesis is dedicated to: My dear mother, sisters and brothers My wonderful and beloved husband who has given me confidence to finish my work My lovely son Azo

Language evaluation certificate

This is to certify that I, Ary Mohammed Abdulrahman Abdulrahman, have proofread this thesis entitled “Immunohistochemical Immunohistochemical Expression of PTCH1 and Laminin in Oral Mucosal Hyperplastic, Potentially malignant lesions, Oral Squamous Cell Carcinoma and Recurrence Samples” by Kanar Abdaladhim Hamaamin. After marking and correcting the mistakes, the thesis was handed again to the researcher to make the corrections in the last copy.

Signature: Proofreader: Ary Mohammed Abdulrahman Date: 22/11/2016 Department of English, School of Languages, Faculty of Humanities, University of Sulaimani.

Acknowledgements First, I thank and praise Allah The Almighty who has illuminated the path for me to complete this work and I pray that His blessings upon me continue throughout my life. I would like to express my great respect and appreciation to Dr. Aras Maruf Rauf, the Dean of College of Dentistry and for his guidance, and encouragement throughout the study. I would like to express my great respect to my supervisor Dr. Falah A. Hawramy for his support in completing this work. I would like to express my great respect and gratitude to my co supervisor Dr. Dena Nadhim Mohammad who kindly and faithfully helped me to finish my work. My deep thanks go to Dr. Ibrahim S. Gataa for his help and support . Special thanks are due to the Oral Pathology Department, especially Dr. Balkees Taha Garib for her care and faithfulness. My special thanks are extended to all members in histopathology department in Shorsh Hospital for their help during my work. Special thanks and respect go to Mr. Ary M. Abdulrahman for his linguistic evaluation of this thesis. Deep thanks for Dr. Saeed A. Kareem for his help in translating the abstract to Kurdish language. Finally, my deepest thanks are due to my lovely mother, sisters, brothers and my dear husband for their continuous support and encouragement.

I

ABSTRACT Background: Oral premalignant lesions display a variety of dysplastic changes that precede the development of invasive oral squamous cell carcinoma; unfortunately there are no histopathological criteria for staging premalignancy. Oral squamous cell carcinoma (OSCC) is considered as one of ten commonest cancers of the world. Laminin is one of most important basal membrane glycoproteins with a specific adhesion function. The immunohistochemical expression of laminin showed progressive loss of continuity from dysplasia to invasive carcinoma. The transmembrane protein receptor Patched (PTCH1) inhibits the activity of Smoothened (SMO), a transmembrane protein. The transcription factor GLI1, a downstream component of Hh signaling, is prevented from entering the nucleus through interactions with cytoplasmic proteins, resulting in a repression of the transcriptional activation of Hh target genes. Hedgehog signaling pathway plays an important role in the proliferation and differentiation of certain stem cells during embryonic development. The dysregulation of hedgehog signaling has been implicated in tumorigenesis of various cancers, including OSCC. Aims: (1) Illustrating the relation between clinical parameters in both oral premalignant and OSCC. (2)Evaluating the immunohistochemical expression of PTCH1 and laminin in oral hyperplastic, premalignant, primary OSCC and recurrence cases. (3)Finding the correlation between the markers in oral premalignant and OSCC.

II

Materials and methods: This study consisted of 35 paraffin blocks of different groups of lesions (4 hyperplastic, 11 oral premalignant lesions, 15 OSCC and 5 recurrence cases), that were collected from Sulaimani Histopathological Centers. Slides were stained immunohistochemically by both of PTCH1 and laminin antibodies and they were evaluated microscopically .Chi square and Pearson’s correlations were used and the p<0.05 was considered to be statistically significant. Results: The oral premalignant lesions revealed male predominance (63.6%).A higher number of cases were found in >50 years age group .The lesions were mostly found in buccal mucosa and erythroleukoplakia was the most clinical feature. In OSCC, non significant relation was found among various clinical data, while most oral squamous cell carcinoma patients had a significant advanced stage (stage ІV, 60%). PTCH1 showed 100% expression in oral hyperplastic lesions, it was distributed equally (50% each) within the nucleous and/or mixed localization (cytoplasmic & nucleus). 81.8% of oral premalignant lesions demonstrated basal and parabasal distribution that pocess high mixed intracellular localization (8/11, 72.7%). On the other hand (93.3%) OSCC, showed positive expression with high score 2(7/15, 46.6%) and most positive cases had mixed cellular localization (10/14, 71.4%).Regarding recurrent oral cancer, 20% of cases were within the score 0.5

III

Laminin showed continuous linear expression in normal oral mucosa, while only half of cases (50%) revealed such expression in oral hyperplastic lesions. The oral premalignant lesions expressed 36.3% of absent expression with a 54.5% of discontinuous patterns. In OSCC, a major defect of laminin was seen (11/15, 73.3%).A relation was found in laminin expression between OSCC and recurrence samples. Finally, a relation was detected between PTCH1 localization and laminin scoring in oral premalignant lesions. Conclusion: The PTCH1 over expression in all of the studied groups of lesions might give impression of the active role of this biomarker in progression toward malignancy. Additionally, laminin defragmentation which started from dysplastic lesions extending to OSCC could emphasize the role of this marker from early precancerous stage. The expression of laminin in the basement membrane may be a useful parameter to evaluate tumor histological differentiation and aggressiveness. The hedgehog pathway plays an important role in OSCC progression and should be considered as a potential therapeutic target.

IV

List of contents Title NO.

Page No.

Subject Verse Language evaluation certification Declaration Examining committee certification Dedication Acknowledgements Abstract List of contents List of tables List of figures List of abbreviations Introduction Aims of the study

1 1.1 1.1.A 1.1.B 1.1.C. 1.1.D 1.1.E 1.2 1.3 1.4 1.4.1 1.5 1.6 1.6.1 1.7 2.1 2.2

Chapter One: Review of Literature The oral premalignant lesions Clinical background Leukoplakia Erosive lichen planus Oral submucous fibrosis Actinic cheilitis Oral erythroplakia Etiology of potentially malignant lesions. Diagnostic aids in detection of potentially malignant disorders Oral squamous cell carcinoma (OSCC). Clinical background of oral squamous cell carcinoma. The role of hedgehog pathway in premalignant lesions and cancers. Markers associated with basement membrane alteration in oral premalignant lesions and cancers. Analysis of laminin chain distribution. The role of laminin in premalignant lesions and cancers Chapter Two: Materials and Methods Study design Study sample

V

I II V VIII IX XI 1 3 4 4 4 6 7 8 9 10 10 11 12 16 19 23 25 27 27

2.3 2.4 2.5 2.6 2.6.1 2.6.2 2.7 2.7.1 2.7.2 2.8 2.9 2.9.1 2.9.1.1 2.9.1.2 2.9.2 2.9.2.1 2.9.2.2 2.10 2.11 3.1 A. B. 3.2. 3.2.1 3.2.1.1 3.2.1.2 3.2.2 3.2.2.1 3.2.2.2 3.3 3.3.1 3.3.2 4.1

Inclusion criteria Materials Instruments and equipment Staining methods Histological evaluation Immunohistochemical staining Controls Positive controls Negative controls Assessment of immunoreactivity Interpretation of staining. Staining of PTCH1 receptor Staining in oral hyperplastic and premalignant lesions Staining in OSCC and its recurrence Staining of laminin. Oral hyperplastic and premalignant lesions Staining in oral squamous cell carcinoma and OSCC recurrence Ethical issue Statistical analysis Chapter Three: Results Clinical data description Oral premalignant lesions Oral squamous cell carcinoma Immunohistochemical findings Immunohistochemical expression of PTCH1 Expression of PTCH1 in oral; normal mucosa, hyperplastic and premalignant lesions. Immunohistochemical expression of PTCH1 in oral squamous cell carcinoma and recurrence samples. Immunohistochemical expression of laminin Immunohistochemical expression of laminin in normal oral mucosa, hyperplastic and premalignant lesions. Immunohistochemical expression of laminin in primary and recurrence oral squamous cell carcinoma samples. The relation between laminin and PTCH1. In oral premalignant lesions In oral squamous cell carcinoma Chapter Four: Discussion Clinical findings

VI

27 27 28 29 29 30 34 34 34 34 34 34 34 35 35 35 35 36 36 37 37 42 48 48 48 54 58 58 61 64 64 65 67

4.1.1 4.1.2 4.2 4.2.1 4.2.2 4.3 4.3.1 4.3.2

Oral premalignant lesions Oral squamous cell carcinoma Immunohistochemical findings of PTCH1 Assessment of PTCH1 immunohistochemical expression in oral normal ,hyperplastic and premalignant lesions Assessment of PTCH1 immunohistochemical expression in oral squamous cell carcinoma and recurrence lesions Immunohistochemical finding of laminin Assessment of laminin immunohistochemical expression of oral normal, hyperplastic and premalignant lesions Assessment of laminin immunohistochemical expression of oral squamous cell carcinoma and recurrence Chapter Five: Conclusions and Suggestions Conclusions Suggestions References Appendix 1 @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ó–þ©a@ @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ón‚íq

VII

67 68 70 70 71 72 72 73

76 77 76

List of tables Table No. Title Page No. 1.1 Oral squamous cell carcinoma classification using the 15 standard Tumor, Nodes and Metastasis (TNM) classification system, given by AJCC. 1.2 An overview of the staging system of oral squamous cell 16 carcinoma 2.1 Bryne,s et al(1989,1992) invasive front grading system 30 2.2 Description of primary antibodies. 33 3.1 Clinical data description of oral premalignant lesions 39 3.2 Clinical data distribution of oral squamous cell carcinoma 44 3.3 PTCH1 expression pattern , localization and intensity in 53 oral hyper plastic and premalignant lesions 3.4 PTCH1 correlation according to expression pattern, 54 localization and intensity between oral hyperplastic and premalignant lesions. 3.5 PTCH1 expression and localization in oral squamous cell 57 carcinoma and recurrence 3.6 PTCH1 correlation in response to the expression pattern 58 and localization between oral squamous cell carcinoma and recurrence cases. 3.7 Laminin expression pattern and correlation in oral 61 hyperplastic and premalignant lesions 3.8 The expression and correlation of laminin in both primary 64 and recurrence oral squamous cell carcinoma samples. 64 3.9 The relation between PTCH1 expression and laminin scoring in oral premalignant lesions 3.10 The relation between intensity of PTCH1 with score of 65 laminin in oral premalignant lesions. 3.11 Relation between localization of PTCH1 and laminin score 65 in oral premalignant lesions. 3.12 The relation between PTCH1 expression and laminin 66 scoring in oral squamous cell carcinoma samples. 3.13 The relation of localization of PTCH1 and laminin scoring 66 in oral squmous cell carcinoma.

VIII

List of figures Figure No. Title 1.1 The role of the hedgehog signaling pathway in normal physiology and the pathogenesis of cancer 1.2 Basement membrane layer in normal oral mucosa 1.3 Electron micrograph of hemidesmosomes(HD) of a basal epithelial cell from a rat salivary gland excretory duct. 1.4 Basement membrane in normal, hyperplastic, premalignant and cancer 1.5 Laminin ultra structure 2.1 Scheme of two-step method in immunohistochemistry 2.2 Steps of immunohistochemistry work 3.1 Box and whisker plot showing age of oral premalignant lesions samples. 3.2 The distribution of different sites regarding the two age groups in oral premalignant lesions. 3.3 The distribution of different sites regarding both sexes in oral premalignant lesions. 3.4 The distribution of clinical presentations among two age groups in oral premalignant lesions. 3.5 The distribution of clinical presentations in different sites in oral premalignant lesions. 3.6 The distribution of different clinical presentations regarding both sexes in oral premalignant lesions. 3.7 Box and whisker plot showing age of oral squamous cell carcinoma samples. 3.8 The distribution of different sites among two age groups in oral squamous cell carcinoma sample. 3.9 The site distribution and sex in oral squamous cell carcinoma 3.10 68 years old male patient had ulcer at the ventral surface og the tongue 3.11 47 years old male presented with ulcerated mass at the right lateral side 3.12 The distribution of clinical presentations regarding two age groups in oral squamous cell carcinoma. 3.13 Distribution of clinical presentation and site of lesion in

IX

Page No. 17 20 21

22 24 32 33 37 40 40 41 41 42 43 45 45 46 46 46 47

3.14 3.15 3.16 3.17 3.18 3.19 3.20 3.21 3.22 3.23 3.24 3.25

oral squamous cell carcinoma Distribution of clinical presentation and sex in oral squamous cell carcinoma. PTCH1 expression (weak) in normal oral mucosa PTCH1 expression in oral hyperplastic lesions. Intensity of PTCH1 in oral hyperplastic samples. PTCH1 expression in oral premalignant lesions Cellular localization and intensity of PTCH1in oral premalignant lesions Cellular localization of PTCH1 in OSCC Cellular localization of PTCH1 in recurrence OSCC Continuous laminin expression Expression of laminin in oral premalignant lesions Laminin expression in OSCC Laminin expression in recurrence OSCC

X

47 49 50 50 51 52 55 56 59 60 62 63

List of abbreviations Abbreviation AC AJCC APC BM EBV EMT H&E Hh HPV IHC IQR LOH MDOSCC MMP-9 mRNA OE OLP OSCC OSMF PBS PTCH1 SCC SMO TNM WDOSCC

Meaning Actinic chelitis American joint committee on cancer Adenomatous polyposis coli Basement membrane Epstein Barr virus Epithelial mesenchymal transition Hematoxylin and eosin Hedgehog Human papilloma virus Immunohistochemistry Inter quartile rate Loss of heterozygosity Moderately differentiated oral squamous cell carcinoma Matrix metalloproteinase Messenger Ribonucleic acid Oral erythroplakia Oral lichen planus Oral squamous cell carcinoma Oral sub mucous fibrosis Phosphate buffered saline Patched receptor Squamous cell carcinoma Smoothened Tumor, Nodes and Metastasis Well differentiated oral squamous cell carcinoma

XI

Introduction Oral precancerous lesions, known as potentially malignant disorders, are group of diseases, which should be diagnosed in the early stage. Clinically, these diseases may sometimes simulate each other. Thus, final

diagnosis should be

confirmed by biopsy. Although there are various etiological factors, the etiology of almost all of these diseases is not fully understood. Early diagnosis is very important and can be lifesaving as neglected lesions may be progressed to severe dysplasia and even become carcinoma in situ and/or squamous cell carcinoma (Yardimci

et al., 2014).

Squamous cell carcinoma (SCC) is defined as “a malignant epithelial neoplasm exhibiting squamous differentiation as characterized by the formation of keratin and/or the presence of intercellular bridges”. (Rajendran, 2009) It can occur as a primary lesion in any of the oral sites. However, most oral cancers have been detected in the tongue and buccal mucosa (Zulfiqar et al., 2013). OSCC is considered among the 10th commonest cancers of the world (Sharma et al., 2010). Most of them are preceded by some premalignant lesions mostly leukoplakia and/or erythroleukoplakia(Speight et al., 1997). Carcinogenesis is the process by which normal cells undergo malignant transformation by accumulation of several genetic and epigenetic alterations (Inoue et al., 2005).So, the transition of normal epithelium to invasive cancer is a progressive process that is characterized by proliferation, angiogenesis, local invasion and, eventually, distant metastasis(Tsantoulis et al., 2007).

1

Laminin is a typical component of epithelial basement membrane and is considered as a biochemical equivalent of the anchoring filaments fixing basal keratinocytes to the basement membrane. (Kosmehl et al., 1999) The immunohistochemical expression of laminin in OSCC demonstrated a progressive loss of continuity as tissues progressed from dysplasia to OSCC (Garcia et al., 2006).

The hedgehog (Hh) pathway is essential for normal embryonic development. It plays a critical role in adult tissue maintenance, renewal and regeneration. PTCH1 receptor; on the cell membrane; activation may initiate a series of cellular responses that range from survival, proliferation, cell fate specification and differentiation (Wilson and Chuang, 2010). Alteration in hedgehog signaling pathway plays a role in malignant transformation among a subset of carcinomas, including pancreatic, lung, and esophageal cancers (Watkins et al., 2003, Thayer et al., 2003 and Sims et al., 2006). Wang et al. (2011) showed PTCH1 over expression in OSCC with a significant role of this marker in tumor growth, lymphatic metastasis, tumor recurrence, and patient’s prognosis.

2

Aims of the study

1. To identify the clinical feature of both oral premalignant lesions and OSCC. 2. To Evaluate the PTCH1 and laminin immunohistochemical expression regarding different groups of lesions (in oral mucosal hyperplastic, potentially malignant lesion, OSCC and recurrence) samples. 3. To find the correlation between the mentioned markers distribution regarding different lesions groups. 4. To find the relation of both markers in oral premalignant and OSCC.

3

Chapter one

Review of literature

1-The oral premalignant lesions: They are groups of diseases that act as precursors for the squamous cell carcinoma. Premalignant lesions are clinically and histologically distinct lesions preceding malignant changes. However, not all malignancies arise from premalignant lesions (Parkin et al., 2002, Shafer et al., 2006). Oral precancerous is an intermediate clinical state with increased cancer risk, which can be recognized and treated, obviously with a much better prognosis compared to the full blown malignancy (Neville et al., 2002, Amagasa, 2011).

1.1 Clinical background : 1.1. A. Leukoplakia: It refers to a clinical entity as a white patch or plaque that cannot be characterized clinically or histologically as any other disease (Martorell et al., 2009).It cannot be removed by simple scraping, thereby distinguishing it from pseudomembranous candidiasis(Sudbø and Reith, 2005).The prevalence of oral leukoplakia in Sulaimani was (0.3%) of the population (Gaphor and Sabri ,2014) and in Iraq was 0.074%.( Abid and Majeed 2009)

Petti (2003) found high prevalence of oral leukoplakia, which is worldwide approximately estimated to be 1%-2% for all age groups and in general, leukoplakias are usually diagnosed after the fourth decade of life and are six times more common among smokers than among non-smokers. The distribution by sex is variable and reported in high frequency in patients with smoking or drinking habits (Banoczy et al., 2001).

Syrjanen et al. (2011) found that alcohol may be an independent or synergistic risk factor. Both tobacco and alcohol are probably acting as predisposing factors (Reichart and Philipsen, 2004).

4

Chapter one

Review of literature

A study done by Paricio et al. (2002) showed that tobacco smoke is a powerful carcinogen and considered along with chronic alcoholism as the most important risk factor for developing head cancers.

There is much debate about whether candida species are implicated in the etiology or progression of leukoplakias. Different types of nitrosamine-producing candida species (other than the common albicans variant) have been isolated from clinically nonhomogeneous leukoplakias with dysplastic changes.Regarding the causal association between HPV and oral potentionally malignant disorder, it was found that the high-risk HPV types are more frequently associated with oral squamous cell carcinoma than low-risk types; it can therefore be concluded that HPV-16 could be a risk factor for malignant change in oral leukoplakia (Martorell et al., 2009).

Although some studies revealed Epstein Barr virus to have an etiological role in oral squamous cell carcinoma, no evidence was found of a causal link between proliferative verrucous leukoplakia and EBV (Bagan et al., 2008).

The homogenous leukoplakia refers to lesions with a regular, smooth and well-defined edge. This pattern shows a low risk of long-term malignant transformation (5%). The nonhomogenous patterns include leukoplakias with an erythematous component (erythroleukoplakia), nodular, erosive, ulcerated, and verrucous exophytic types. Malignant transformation occurs in 25% of them, and they are considered as high-risk lesions. Proliferative verrucous leukoplakia is currently considered as an independent entity with a high risk of malignant changes

5

Chapter one

Review of literature

and might transform into verrucous carcinomas or squamous cell carcinomas (Reibel, 2003).

Leukoplakia was distributed equally on buccal mucosa, tongue and alveolar ridge(Gaphor and Sabri ,2014), it may be affect any part of the oral cavity(van der Waal, 2009)

The

epithelial

dysplasia

is

often

correlating

with

a

clinically

nonhomogeneous, erythroleukoplakic subtype that is regarded as the most important indicator of malignant potential. Nevertheless, it should be recognized that some dysplastic lesions may remain unchanged or may even show complete regression (Holmstrup et al., 2006, Torres et al., 2009).

1.1. B. Erosive lichen planus: Lichen planus is a frequent mucocutaneous disease that affects mostly women between the fifth and sixth decades of life (Eisen, 2002).However, the disease may also affect individuals aged below 18 years with clinical features identical to those that present in older adults, but the prognosis is more benign (Laeijendecker et al 2005).Its prevalence is 1% to 2% in the general population with strong female preference (Gorsky et al., 2004). Lichen planus usually present in one of two main forms either the reticular and or erosive forms, although other forms are not rare (Edwards and Kelsch, 2002).

The erosive form is not as common as the reticular one, but it is more significant for patients, as the lesions are commonly symptomatic. Clinically, the erosive lichen planus manifests as atrophic and erythematous areas frequently surrounded by radiating thin striae. In certain severe cases, the epithelium may separate, resulting in a relatively rare form of the disease known as bullous lichen

6

Chapter one

planus(Neville

Review of literature

et al., 2004). The erosive lesions are mainly confined to the

gingival(Mignogna et al., 2005).

An Australian study stated that “women are nearly four times more affected by this condition than men, with white individuals are five and a half times more likely to develop this disease compared to other races” (Sugerman and Savage, 2002).

Malignant transformation of oral lichen planus remains a very controversial issue, approximately 0.2 % of those patients develop OSCC (Scully et al., 1998).

1.1.C. Oral Submucous Fibrosis(OSMF):It is a chronic, debilitating disease characterized by inflammation and progressive fibrosis of the sub mucosal tissues resulting in marked rigidity and an eventual inability to open the mouth. The buccal mucosa is the most commonly involved site and it may occur in other parts of oral cavity. The condition is well recognized for its malignant potential and it is particularly associated with areca nut chewing, the main component of betel quid (Ho et al., 2012).

Hsue et al. (2007) found the malignant transformation rates to be 1.9% for oral submucous fibrosis in Solomon Islands. Finally, Ho et al. (2012) in Southern Taiwan did not report any malignant transformation for this condition.

Worldwide, OSMF is confined to Indians and Southeast Asians, with overall prevalence rates about 0.2% to 0.5 %. The prevalence by gender is varying from 0.2-2.3% in males and 1.2-4.57% in females, with a wide age range of 20 and 40 years (Reddy et al., 2011).

7

Chapter one

Review of literature

The male: female ratio of patients with OSMF is 2.6:1 with most patients presenting in their 3rd decade of life. Most patients were using the areca nut for 5-10 years at presentation, the burning sensation in the mouth was the commonest complaint, and the inner cheek mucosa was the most commonly involved site (Chatterji and Mehrotra, 2014)

1.1.D. Actinic cheilitis(AC) :It is characterized by atrophy of the vermilion border of the lower lip, loss of elasticity, and the presence of keratotic plaques ranging from thin to thick, which may be rough and scaly and may overlap irregular erythematous areas, in addition to ulcers and fissures. A relevant aspect is the loss of a clear separation between the labial mucosa and the vermilion border (Pinera et al., 2010 and Souza et al., 2012).

AC of the lip is related to the exposure to solar radiation. This lesion can progress to (SCC) of the lip (Warnakulasuriya et al., 2007, Cavalcante et al., 2008). The prevalence of AC ranges from 0.45 to 2.4% of the population (Ntomouchtsis et al., 2010, Souza et al., 2012). In a study done in Brazil investigating population groups engaged in outdoor activities, the prevalence have varied between 4.2 and 43.2%.This discrepancy could be related to demographic, clinical, and histopathologic findings (Zanetti et al., 2007).

This lesion occurs predominantly in middle-aged and light-skinned men who have been excessively or chronically exposed to ultraviolet radiation. The most common anatomical site is the lower lip as it has higher exposure to solar radiation. In tropical countries such as Brazil, which experiences the highest levels of ultraviolet radiation in the world, exposure to sunlight can be extremely harmful,

8

Chapter one

Review of literature

especially during outdoor activities(Junqueira et al., 2011, Vieira et al., 2012 and Souza et al ., 2012). In the early stages of AC, the slow evolution of the condition usually is attributed to the process of aging and leads to neglect the lesion until it reaches a more advanced stage. This may lead to the development of SCC. So, special attention should be given in dealing with this disease because of its malignant potential (Markopoulos et al., 2004, and Pinera et al, 2010).

Relating the clinical features to the histopathological finding, the changes in AC range from atrophy to hyperplasia of the squamous cell epithelium of the vermilion border, with varying degrees of keratinization, disordered maturation, increased mitotic activity, cytological atypia and drop-shaped epithelial ridges. However, the basement membrane is intact. Accordingly, AC should be regarded as an intra-epithelial, or in-situ neoplasm (Nico et al., 2007, Rossi et al., 2007).

1.1. E. Oral erythroplakia (OE): OE is considered as a rare potentially malignant lesion of the oral mucosa. A case control study done in India reported a prevalence of 0.2%. The condition is mostly found in the middle aged and elderly. Erythroplakia is a flat macule or patch in which the mucosal surface has a red velvety or finely granular surface appearance. The borders of a true erythroplakia are usually well demarcated in contrast to those of an inflammatory process, in which the borders tend to merge with the surrounding normal tissue (Reichart and Philipsen, 2004). Lesions may be symptomatic when patients eat hot or spicy foods and most often occur at high-risk sites for OSCC. Other clinical entities must be ruled out by

9

Chapter one

Review of literature

clinical history combined with the results of histopathological findings that exclude infection, physical or chemical injury, dermatoses or immune reactions (Neville and Day, 2002). Reibel et al. (2003) and Neville and Day (2002) demonsrated that the average age of population affected with OE is 50-69years, occurring about five years earlier than oral cancer. However, other studies showed that 1- 5% of this lesion may affect a younger age group of 30 years This may be due to the fact that various extrinsic and intrinsic etiological factors are now more prelevant in today’s younger population(Holmstrup et al., 2006).

A specific type of OE occurs in chutta smokers in India. Lesions are typically less than 1.5 cm in diameter, with a strong association with tobacco consumption and the use of alcohol (Reichart and Philipsen, 2004). Histopathological examination of these clinically apparent OE revealed different distribution with 9% mild or moderate dysplasia, 40% carcinoma in situ and 51%invasive carcinoma (Villa et al., 2011). 1.2Etiology of potentially malignant lesions(Cabay et al., 2007) • • • • •

Tobacco (smoke and smokeless) Alcohol Candida albicans Viral antigens (HPV strains 16, 18) Levels of Vit A, B12, C, beta-carotene, folic acid and syphilis are associated.

1.3 Diagnostic aids in detection of potentially malignant disorders: Development and use of diagnostic aids that would help the oral health care professionals to readily identify persistent oral lesions of uncertain biologic

10

Chapter one

Review of literature

significance are essential to improve their ability to detect these diseases at early stage. These include the following: (Gillenwater et al., 2006, Lingen et al., 2008) 1. Clinical Methods: a. Conventional oral examination. b. Vital staining. 2. Optical Methods: fluorescence spectroscopy. 3. Imaging methods: a. Computed Tomography (CT). b. Magnetic Resonance Imaging (MRI). 4. Histopathological methods: a. Scalpel biopsy. b. Oral brush test. c. Cytology. 5. Molecular methods: Immunohistochemistry 1.4 Oral squamous cell carcinoma (OSCC): OSCC constitutes a major health problem in developing countries, representing a leading cause of death. The survival index continues to be small, as compared to the progress in diagnosis and treatment plan. According to World Health Organization, OSCC in males in developing countries is considered as the sixth commonest cancer after lung, prostrate, colorectal, stomach and bladder cancers, while in females, it is the tenth commonest type of cancer after breast, colorectal, lung, stomach, uterus, cervix, ovary, bladder and liver (Landis et al., 1999). In Iraq, although OSCC is a major problem, there is no complete registry of all cases. Also, it did not fall within or close to the common tenth cancers (AlReyahi, 2004). The frequency of occurrence recorded was 2% of all cancer of the body (Al–Niaimi, 2006).

11

Chapter one

Review of literature

In Sulaimani city, during the period of (1995-2004), OSCC showed rising trend in number and this increase virtually reached its peak in the year 2004(Ahmed, 2006)

The oral cavity is more accessible to complete examination, and could be used in early detection of precancerous and cancerous lesions, but the ignorance and or inaccessibility of medical care make the disease diagnosed in the later stages. Thus, there is a need for improvement in early detection of oral carcinomas in the initial stages, in order to ensure more effective treatment and make the morbidity minimal (Mehrotra et al., 2010).

Worldwide, approximately 275,000 oral cavity malignancies are diagnosed each year and the predominant one is SCC(Funk et al., 2002,Bray et al., 2013).In Sri Lanka, India, Pakistan and Bangladesh, nearly a quarter of total cancers diagnosed are OSCC. In western countries, OSCC represents approximately 3 %( Warnakulasuriya, 2010). 1.4.1 Clinical background of oral squamous cell carcinoma: OSCC presents with different clinical aspects which are related with the location of the tumor, evolution time, precancerous lesions and risk factors. The most frequent clinical aspects are: mass, ulcer, verrucous, leukoplakia, erythroplakia and erythroleukoplakia. The lesion can progress and develop an exophytic, irregular lobulated lesion or adopt an endophytic growth pattern characterized by a depressed ulcer with grayish-white edges with elevated and indurate borders. Tongue carcinoma is the most commonly observed intraoral carcinoma. It represents 30-40%, with the lateral border being the most frequent situation (80%), followed by ventral and dorsum regions (Brandizzi et al.,

12

Chapter one

Review of literature

2008).The lip is the most common location of OSCCs; despite that those patients are likely to consult a physician or a dermatologist (Neville et al., 2002).

Carcinomas of the dorsum of the tongue are generally linked to lichen planus or leukoplakia. They clinically appear as ulcerated forms that tend to expand on the surface rather than go deeper into it. The lateral border of the tongue and the floor of the mouth have high risk for cancer development associated with bad prognosis (Bolesina et al., 2012). Generally speaking the lateral border, the ventral surface of the tongue and the lips are the most commonly affected sites, followed by the floor of the mouth, the gingival, the alveolar mucosa and the palate (Neville and Day, 2002, Shafer et al., 2006).

Patients with OSCC of the gingivo-buccal region are mostly present with advanced stages (stages III and IV) and have a very high mortality despite best multimodal treatment (Walvekar, 2009).

OSCC primarily affects males (1.5:1).However, in regions where tobacco and betel nut consumption is common in females, the gender difference normalizes (Warnakulasuriya, 2010), and worldwide the mortality rate is estimated to be nearly 50% (Bray et al., 2013). OSCC more frequently affects men than women most probably because more men than women indulge in high-risk habits. The probability of developing OSCC increases with the period of exposure to risk factors, and increasing age adds the further dimension of age-related mutagenic and epigenetic changes (Feller and Lemmer, 2012).

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In the United States the median age of diagnosis of OSCC is 62 years. However, its incidence of oral SCC in persons under the age of 45 is increasing (Warnakulasuriya, 2009)

On the other hand, studies done in Iran showed that a low percentage of oral cancers has been presented in patients under the age of 40 years(Falaki et al., 2011).Although there has been a declining trend in the overall incidence of OSCC, the incidence in young adults seems to be increasing worldwide(Llewellyn et al., 2004,Siriwardena et al., 2006).A study in the United States reported an alarming increase in the incidence of oral cancer, particularly tongue cancer, in young white males under the age of 40 (Shiboski et al., 2000).Likewise, the biological behavior and etiogenesis of oral cancer in young patients are unlike older age groups with more aggressive behavior(Iamaroon et al., 2004).

In Iraq, OSCC occurs most commonly in the old age group 51–60 years as reported (Al-Kawaz, 2010 and Museedi and Younis, 2013).

OSCC is characterized by

epithelial-mesenchymal interaction

with

breakdown of basement membrane and further invade connective tissue with distant metastasis(Shruthy et al., 2013).The American Joint Committee on Cancer (AJCC) has detailed a comprehensive staging system for oral squamous cell carcinoma using the standard Tumor, Nodes and Metastasis (TNM) classification system. An overview of this system is presented in Table 1.2 and Table 1.3 (Edge, 2010).

In addition to TNM staging, histologic tumor depth has been shown to be an important prognostic variable. Tumors with less than 2 mm and with more than 2 mm of invasion have 13% and 46% chance of nodal metastasis at diagnosis

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respectively. Furthermore, their respective rates of disease-free survival are 95% and 85% respectively at 2 years (Layland et al., 2005).

Table 1.1: Oral squamous cell carcinoma classification using the standard Tumor, Nodes and Metastasis (TNM) classification system, given by AJCC. Primary Tumor

Regional Lymph Nodes

Distant Metastasis

TX

Cannot be assessed

NX

Cannot be assessed

M0

Absent

T0

No evidence of tumors

N0

None

M1

Present

Tis

Carcinoma in situ

N1

Single, ipsilateral, < 3 cm

T1

< 2cm

N2a Single, ipsilateral, 3-6 cm

T2

2-4 cm

N2b Multiple, ipsilateral, < 6 cm

T3

> 4 cm

N2c Bilateral or contralateral, < 6 cm

T4a

Invades adjacent structures

T4b

Invades distant structures

N3

> 6 cm

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Table 1.2: An overview of the staging system of oral squamous cell carcinoma Stage

T

N

M

0

Tis

N0

M0

I

T1

N0

M0

II

T2

NO

M0

III

T1-3

N1

M0

T3

NO

M0

T1-3

N2

M0

T4

N1-2

M0

T1-3

NO-3

M0

T1-3

N0-3

M1

IV

1.5 The role of hedgehog pathway in premalignant lesions and cancers: The hedgehog (Hh) pathway is a signaling pathway that transmits information to embryonic cells required for cell differentiation of embryo. It plays a critical role in adult tissue maintenance, renewal and regeneration. The secreted proteins of this pathway act to initiate a series of cellular responses that range from survival, proliferation, cell fate specification and differentiation (Wilson and Chuang, 2010).

Proper levels of Hh signaling require the regulated production, processing, secretion and trafficking of Hh ligands which include Sonic (Shh), Indian (Ihh) and Desert (Dhh).These ligands are synthesized as precursor proteins that undergo autocatalytic cleavage and concomitant cholesterol modification at the carboxy terminus and palmitoylation at the amino terminus, resulting in a secreted, dually-

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lipidated protein (Goetz and Anderson , 2010).Hh proteins initiate signaling through binding to the canonical receptor patched (PTCH1), which results in derepression of the protein smoothened (SMO) that mediates downstream signal transduction with dissociation of Gli proteins in response to activation of Hh signaling(Briscoe and Thérond, 2013),as shown in Fig1.1

Fig 1.1: The role of the hedgehog signaling pathway in normal physiology and the pathogenesis of cancer (Merchant and Matsui, 2010).

The secreted ligands bind to the membranous receptor patched (PTCH1) then promote the nuclear translocation and activation of the Gli family of proteins (Gli-1, Gli-2, and Gli-3), which then facilitate the transcription of several target genes, including those controlling the cell cycle, cell migration, angiogenesis, and apoptosis. The dysregulation of hedgehog signaling has been implicated in tumorigenesis(Watkins et al., 2003,Thayer et al., 2003 and Watkins and Peacock,

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2004). Aberrant Hh signaling is detected in various cancers (Falkenstein and Vokes, 2014). In the absence of a ligand, the Hh signaling pathway is inactive in those Hh responsive cells. In this case, the transmembrane protein receptor Patched (PTCH1) inhibits the activity of Smoothened (SMO), a transmembrane protein. The transcription factor GLI1, a downstream component of Hh signaling, is prevented from entering the nucleus through interactions with cytoplasmic proteins, including Fused (FU) and Suppressor of fused (SUFU), resulting in a repression of the transcriptional activation of Hh target genes(Pasca and Hebrok, 2003)

The expression of PTCH1 is increased significantly in the pancreatic premalignant lesions (Wang et al., 2014). A progressive activation in Hh pathway has been observed in lesions with greater cytological atypia toward pancreatic carcinoma (Onishi and Katano, 2014).

The PTCH1 expression was seen in dysplastic lesions with severe dysplasia or carcinoma in situ but not in those with mild/moderate dysplasia and an increasing trend of PTCH1 expression was associated with squamous dysplasia and esophagus carcinoma. Most staining was detected in epithelial cells and some staining was observed in stroma of esophagus carcinoma (Wang et al., 2012).

There is emerging evidence that aberrant hedgehog signaling may be related to breast carcinogenesis(Zardawi et al., 2009).A study done by Watkins et al.(2003) demonstrated that dysregulation of the Hh signaling plays a role in lung cancers.

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In prostate cancer, the level of hedgehog activity correlates with the severity of the tumor and is necessary for metastatic behavior. Blockade of the signaling leads to tumor shrinkage. Thus, hedgehog signaling might represent a novel pathway in prostate cancer that offers opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring (Datta, 2006).

In oral premalignant lesions, there is a significant increase in expression of all proteins of the hedgehog pathway, both in the epithelium and in the connective tissue. In actinic chelitis, it suggests that the hedgehog pathway in tumour transformation influences SCC (Gonzalez et al., 2016).

Wang et al. (2011) showed that PTCH1 was rarely and weakly expressed in the normal oral mucosa, whereas the OSCC counterparts demonstrated frequent and high expression of these proteins. Abnormal activation of Hh pathway has a putative role in MMP-9 and E-cadherin in order to induce EMT in OSCC. Furthermore Hh/GLI inhibitors which could effectively inhibit GLI expression, decrease cell growth, promote G1 arrest, increase apoptosis, and inhibit migration of OSCC. Thus demonstrating not only that activation of this pathway is important in OSCC progression, but also that a subset of OSCC patients may benefit from anti-Hh/GLI therapies (Yan et al., 2011).

1.6 Markers associated with basement membrane alteration in oral premalignant lesions and cancers: The demarcation between two different signal intensity of epithelium and lamina propria represents the basement membrane. This junction may appear as linear of undulated structure due to tissue formalin shrinkage effect. Small

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projections towards the lamina propria may be seen which represent ret pegs. Intact basement membrane is seen in benign and dysplastic cases, while complete or partial loss (breach) of the basement membraneis usually evident in cases of invasive carcinoma (Hamdoon et al., 2012)(Fig 1.2)

Fig 1.2: Basement membrane layer in normal oral mucosa (Squier et al., 2001)

The basement membrane offers mechanical support that influences cell proliferation, differentiation, adhesion, migration and gene expression. It acts as a factor in maintaining polarity and organization of epithelial cells and it is an active regulator in epithelial-mesenchymal interactions during epithelial cell development. Furthermore, it regulates the activity of growth factors which is, in turn, considered as important regulators in epithelial cell differentiation and development. Among BM components the laminins are especially functional molecules in regulation of epithelial cell development and differentiation (Lohi, 2001).

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The basal lamina is a physicochemical barrier to tumor invasion. It is a highly specialized structure, consisting of a set of molecules with different sensitivity to proteolytic degradation. Its components are synthesized and secreted by epithelial cells and connective tissue (Pereira et al., 2005).It consists of a dense network of collagen type IV and other macromolecular components such as proteoglycans, glycoproteins, glycosaminoglycans, laminin, fibronectin, and tenascins(Fenyvesi, 2003).The transmembrane adhesive molecules present in hemidesmosomes(Fig 1.3 )are the integrin α, β4, which binds specifically to the basal lamina glycoprotein,laminin, and collagen type XVII (also identified as BP180).

Fig 1.3: Electron micrograph of hemidesmosomes(HD) of a basal epithelial cell from a rat salivary gland excretory duct. BL, Basal lamina; COL, collagen fibrils; IF, intermediate filaments. B, Diagram of a hemidesmosome(Antonio, 2003). Basement membrane is seen in benign and dysplastic cases. However, a dysplastic lesion showed epithelial thickening without loss of the basement

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membrane while complete or partial loss with breakdown of the basement membrane is usually evident in invasive carcinoma (Hamdoon et al., 2012).as seen in (Fig 1.4)

Fig 1.4: Basement membrane in normal, hyperplastic, premaliganat and cancer (Squier et al., 2001). Interaction between tumor cells and extracellular matrix (ECM) components is essential for tumor growth and for the onset of cell spreading and subsequent metastatic activity (Fenyvesi, 2003).

Collagen type IV is a helical, trimeric protein.It is important as a structural backbone of the basement membrane. Oral premalignant lesions showed loss of continuity and alteration in the distribution (Tosios et al., 1998).

In OSCC, collagen type IV is one of the most affected molecules and its degradation is mediated by metalloproteinase that are secreted by neoplastic cells. In advanced stages of invasion, the degradation is further increased and it mediates the discontinuity of the basal lamina with an increased probability of metastasis and a poor prognosis (Pereira et al., 2005). Agarwal and Ballabh (2013) showed a

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definite alteration in distribution of collagen type IV with a significant loss in all 35 cases of OSCC.

In normal epithelium, perlecan is found in the basal layer of epithelia while in in oral dysplastic lesions, it shifts to suprabasal layers, with the increase in severity of dysplasia; its expression is more detected in suprabasal layers (Mishra et al., 2013). In OSCC, perlecan is present in stroma and tumor islands. It breaks down and releases growth factors, which help in tumor progression, angiogenesis, and metastasis of the carcinoma and are highly expressed in oral squamous cell carcinoma (Hasegawaa et al., 2016).

Wilson et al. (1999) showed that the proteoglycans decreased in OSCC compared to the normal counterpart. 1.6.1 Analysis of laminin chain distribution: Laminin are large, extra cellular, cross shaped adhesion proteins consisting of three polypeptide chains and are synthesized and secreted by keratinocytes. Laminin is a heterotrimeric molecule with an a, b and g chain (Fig 1.5). Laminin isoforms arise from an exchange of single chains and can be identified in situ by chain-specific antibodies. Laminin isoforms are deposited in a maturation and differentiation-dependent manner.The BM of normal oral squamous epithelium comprises the laminin chains: α 3, α5, β1, β3, γ1 and γ2.The α 1, α 2 and β 2 laminin chains were not present in the epithelial BM. A re-expression of the laminin a2 and b2 chains could be shown in hyperproliferative, dysplastic lesions.

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Fig 1.5: Laminin ultra structure Laminins (heterotrimers composed of α, β, and γ chains), are multifunctional glycoproteins present in basement membranes. Integrins, dystroglycan, syndecans, and several other cell surface molecules are cellular receptors for laminins. The globular domains located in the N- and Cterminus of the laminin α chains are critical for interactions with the cellular receptors. Integrin α6β1 binds to most of the laminin isoforms. Integrin α3β1 interacts with laminin-5 and -10/11 more specifically than the other isoforms. Integrins α1β1, α2β1, and α7β1 show binding activity to laminin-1 and -2. Interaction of integrin α6β4 with laminin-5 forms hemidesmosomes in the skin. αdystroglycan strongly binds to the laminin α1 and α2 chains and moderately interacts with the α5 chain(Martin and Timple,1987)

In oral squamous cell carcinoma (OSCC), multifocal breaks of the BM are present as indicated by laminin chain antibodies. These breaks correlate to malignancy grade in their extent. The profile of laminin chain expression was similar to that observed in hyperplastic and dysplastic oral squamous epithelium, namely the expression of the laminin a3, a3, a5, b1, b2), b3, g1 and g2 chains which could be demonstrated around the tumour cell nests. Whereas the a1 chain could not be demonstrated in the BM of the carcinomatous epithelium, An a2 laminin chain staining could be observed only focally. The correlation between the morphological pattern of invasive tumour clusters and a laminin-5 immunostaining in the adjacent stroma may suggest, first, that a laminin-5 deposition outside the BM is an immunohistochemical marker for invasion and second, that OSCC invasion is guided by the laminin-5 matrix(Kosmehl et al., 1999).

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1.7 The role of laminin in premalignant lesions and cancers: Laminin is a glycoprotein that is involved in cell adhesion, migration, proteolytic activity, cell proliferation, and metastatic growth (Engbring and Kleinman, 2003). It produces enzymes that degrade basal membrane components, consequently hindering new protein synthesis (Garcia et al., 2006).

Tumor cells bind to laminin receptors on the basal membrane and are subsequently stimulated to produce metalloproteinase, which begin fragmentation and degradation of the membrane (Souza et al., 2007). Laminin forms a rod-like structure with truncated short arms and its chains lack some domains present. The cell adhesion promoting activity of laminin is dependent on the coiled conformation of the molecule. It is a component of the epithelial cell adhesion complex containing hemidesmosomes, anchoring fibrils and anchoring fibers. Integrins connect the hemidesmosomes with the underlying connective tissue through type VII collagen (Lohi, 2001). Alteration in laminin expression and its distribution is related to the severity of cervical neoplasia (Wang et al., 2006).Furthermore, laminin is widely expressed in preinvasive area found to be increased progressively in colon adenoma ,where as no expression is detected in hyperplastic polyp or normal lining epithelial cells (Lenander et al., 2003).

Similarly, studies that were considering laminin expression in both laryngeal and renal premalignant lesions revealed marked over expression of this marker (Neves et al., 2004 and Savić et al., 2002).

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Alteration in laminin expression and its distribution is able to reflect changes in epithelial- mesenchymal adhesion and integrity. Such alterations have been reported in breast cancer (Henning et al., 1999), bladder carcinoma (Hindermann et al., 2003) and invasive cervical cancer(Skyldberg et al., 1999). In oral premalignant lesions, abnormal laminin expression was found and could be a prognostic marker for lesions at high risk for progression to SCC(Natarajan et al., 2003).There was a linear trend for discontinuous distribution of laminin from epithelial hyperplasia to epithelial dysplasia, with an increase in discontinuity accompanying the increase in dysplastic grades (Degen et al., 2012). Furthermore, OSCC revealed a progressive loss of continuity in the immunohistochemical expression of laminin, with a significant weak expression associated with the progression of the cancer. Aggressive carcinomas have an increased ability to produce enzymes that degrade basal membrane components, consequently hindering new protein synthesis (Garcia et al., 2006 and de Souza et al., 2007).

26

Chapter two

Materials and methods

Materials and Methods 2.1 Study design: This study was a cross sectional prospective and retrospective study conducted in Sulaimani governorate during the period from 1st December 2014 to the end of 2016. 2.2 Study sample: The sample included 4 gingival oral hyperplastic tissues, 11 oral premalignant lesions, 15 oral squamous cell carcinoma and 5 recurrence OSCC cases, that were collected from Sulaimani Teaching Hospital and Shorsh Hospital.The preparation and staining procedures were done in the College of Dentistry/University of Sulaimani. The prospective cases were processed into paraffin blocks in order to be ready for immunohistopathological procedure. 2.3 Inclusion criteria: For oral premalignant lesions, H and E stained slides should reveal dysplastic changes. For OSCCs, the TNM stages were recorded beside the available clinical information. 2.4 Materials: 1. Anti-Laminin antibody (abcam/UK). 2. Anti-Patched /PTCH1 antibody (abcam/UK). 3. Citrate buffer pH 6.0 (abcam/UK). 4. Expose Mouse and Rabbit Specific HRP/DAB Detection IHC kit (abcam/UK). 5. Harris, hematoxylin.

27

Chapter two

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6. Eosin, Syrbio,S.A.R. 7. Absolute ethanol,Scharlau,Spain 8. Xylene, Himedia, India. 9. Distilled water. 10. Absorbent wipes and sterile gauze. 11. DPX, Syrbio. 12. DAB chromogen. 13. PBS (Phosphate buffered saline) 2.5 Instruments and equipment: • Incubator, Binder, Germany. • Heater, Palson, Spain. • Microtome, (Leica, Germany) and disposable microtome blades. • Silanized slides, AFCO, China. • Tissue flotation bath, Thromolab industrie, Jordan. • Thermometer. • Pap pen, Dako, Denmark. • Coplin jars. • Staining racks. • Plastic containers. • Humidified chamber. • Micropipette, Xinking, China; 3sizes:0.5-10, 10-100,100-1000. • Micropipette (blue and yellow) tips, Citotest, China.

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Chapter two

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• Micropipette white tips 10µl, Gilson, China. • Eppendrof(500 and 100µl). • Eppendorf stand. • Dropper. • Graduated cylinders, Bomex. • Light microscope, Olympus CH30, Japan. •

Camera

• Personal computer /Sony VAIO. 2.6 Staining methods: Serial 4-5 µm tissue sections were cut from each block and mounted on 2 slides; normal and positively charged microscopic slides. After mounting the tissue section on the slide, it was air dried, deparaffinized, rehydrated, stained, dehydrated, cleared and finally covered with a cover slip sealed by mounting media. 2.6.1 Histological staining evaluation: The normal slide was stained with H&E for conforming histopathological features and grading. For oral premalignant lesions the dysplastic features were seen and graded into mild, moderate and severe (Barnes et al., 2005) For OSCC, the slides were graded according to deep invasive cell grading system (Bryne’s et al., 1989, 1992) with 4 morphologic parameters.(Tendency to keratinization, Nuclear pleomorphism, Pattern to invasion, Lymphoplasmacytic infilteration) there are points 1, 2, 3, 4 . The sum of scores (points) was grouped as

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follows: 4-8=grade I, 9-12=grade II, 13-16=grade III (Doshi et al., 2011 and Rastgoi et al., 2014). (Table 2.1) Table 2.1: Bryne’s et al (1989, 1992) invasive front grading system Morphologic

Points

parameter

1

2

3

4

Tendency to

>50% cells

20-50% cells

5-20% cells

0-5% cells

keratinization

keratinized

keratinized

keratinized

keratinized

Little nuclear

Moderately

Abundant

Extreme nuclear

pleomorphism

abundant

nuclear

pleomorphism

nuclear

pleomorphism

Nuclear pleomorphism

pleomorphism

Pattern to invasion

Pushing, well

Infiltrating,

Small groups or

Marked and wide

delineated

solid cords,

cords of

spread cellular

infiltrating

bands and/or

infiltrating cells

dissemination in

borders

strands

small groups and/or in single cells

Lymphoplamacytic

Marked

Moderate

Slight

None

infilteration

2.6.2 Immunohistochemical staining: The positively charged slide was used for immunohistochemical staining (one section for Laminin and the other for PTCH1). 1. The slides were put in oven (60oC) for 6 hrs.

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2. Tissue sections were deparaffinized by xylene twice (5 min for each), rehydrated by ethanol 100% twice (5 min for each), 90% and 70% ethanol 3 min for each respectively. 3. Antigen retrieval was done by boiling tissue sections in citrate buffer (1ml 100X citrate buffer: 100 ml distilled water) for 15 min at 95oC, then sections were cooled at room temperature for 15 min, and they were washed with PBS twice for 3 min each. The excess buffer was tapped off gently and the sections were wiped around by gauze pad and a circle around the section was made by pap pen. 4. Sections were incubated with hydrogen peroxidase at 37oC for 10 min in order to block endogenous peroxidase activity, and then they were washed twice with PBS and slides were tapped off carefully. 5. Protein block was applied and incubated for 10 min to block nonspecific background staining, and then it was washed once with PBS, and slides were tapped off and wiped as before. 6. Primary antibodies [Either laminin or PTCH1](Table 2.2) were applied and sections were incubated for 45min at 37C, then sections were washed 4 times with PBS. 7. Complement was added and sections were incubated for 10 min, and then sections were washed twice by PBS for 3min. 8. Sections were incubated with conjugate for 15 min and then they were washed with PBS and excess buffer was tapped off.

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9. Sections were stained with DAB chromogen and incubated for 5 min in a dark field. They were washed 4 times in buffer and the excess was tapped off and wiped from the slides. 10. Sections were counter stained with hematoxylin for 20 sec. Then the slides were washed with distilled water gently for 1 min. 11. The sections were then dehydrated in graded ethanol (70%, 90%) for 3 min each and twice in 100% ethanol for 5 min, and then they were transferred twice in xylene for 1 min. 12. Slides were mounted with DPX, dried and examined under a light microscope. (Fig 2.1 and 2.2)

Fig 2.1: Scheme of two-step method in immunohistochemistry. After antigen retrieval and proper blocking steps, the tissue section is first incubated with primary (1st) antibody. The polymeric conjugated secondary antibody (2nd) is applied afterward. In this complex, the molecules of the enzyme (horseradish peroxidase or alkaline phosphatase) and molecules of secondaryantibody. (Shi Z. and Stack MS., 2015)

32

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Slides were put in oven (60oC) for overnight. Primary antibodies [Laminin and PTCH1] incubated for 45min at 37C,

Complement was added and sections were incubated for 10 min

Slides were mounted with DPX, dried

deparaffinized by xylene twice (5 min for each), rehydration by ethanol 100%, 90% and 70%

Protein block was applied and incubated for 10 min

Antigen retrieval for 15 min at 95oC

hydrogen peroxidase at 37oC for 10

conjugate for 15 min

DAB chromogen and incubation for 5 min

dehydration in graded ethanol (70%, 90%) for 3 min each and

hematoxylin for 20 sec

Fig 2.2:Steps of immunohistochemistry work Table 2.2: Description of primary antibodies. Antiserum

Dilution

Purity

Clonality

Isotype

Anti-

(1/150)

Immunogen

Polyclonal

Rabbit IgG

Polyclonal

Rabbit IgG

Laminin

affinity

antibody

purified

Anti-Patched

(1/150)

Immunogen

/ PTCH1

affinity

antibody

purified

33

Chapter two

Materials and methods

2.7 Controls: 2.7.1 Positive controls: 1-Laminin: normal oral mucosa (Souza et al., 2007) 2-PTCH1: esophageal carcinoma (Yang et al., 2012) 2.7.2 Negative controls: 1-Laminin: The primary antibody is omitted, and diluent as a negative control. 2-PTCH1: normal oral mucosa (Wang et al., 2011) 2.8 Assessment of immunoreactivity: Using Image J software for windows, immunostained cells were counted from 5 high spot fields pictures taken from a light microscope (OLYMPUS, at 400X), then they were uploaded to the software and counted by grid. For each case, the positive brown staining within all sections was examined by two observers. 2.9 Interpretation of staining: 2.9.1 Staining of PTCH1 receptor: 2.9.1.1 Staining in oral hyperplastic and premalignant lesions: Expressions of PTCH1 were recorded as basal, basal and parabasal and full thickness in both oral hyperplastic and premalignant lesions. Regarding cellular localization, the stained slides were evaluated as nuclear, cytoplasmic and mixed. Finally, the intensity was recorded as faint, moderate and strong.

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2.9.1.2 Staining in OSCC and its recurrence: The overall IHC score (0, 0.5, 1, 1.5, 2, 3) was calculated by multiplying intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) by proportion positively stained cells (0=<10%; 0.5=10-30%; 1=30%). Furthermore, cellular localizations were recorded as nuclear, cytoplasmic and mixed (Yang et al., 2012). 2.9.2 Staining of laminin: The laminin staining was evaluated at 400X magnification along the basal membrane. 2.9.2.1Oral hyperplastic and premalignant lesions: Staining was classified as continuous when the brown line left along the epithelial and conjunctive border or discontinuous when there was fragmentary and finally absent (García et al., 2006). The areas of the epithelial disruption due to great inflammatory infiltrates of stroma were excluded from the study. Deposits of laminin around blood vessels were used as internal control (Tossios et al., 1998). 2.9.2.2 Staining in OSCC and recurrence: The basement membrane defects for the laminin chains were semi quantitatively evaluated at 400X and scored as follows: (Kobayashi et al (1995) •Score 0: continuous linear staining (no BM defects, score 0) •Score 1: loss of staining in less than 10% of the tumour–stromal interface per tumour cell nest (minor BM defects, score 1) • Score 2: loss of staining in less than 50% of the tumour–stromal interface per tumour cell nest (moderate BM defects, score 2)

35

Chapter two

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• Score 3: loss of staining in more than 50% of the tumour–stromal interface per tumour cell nest (BM defected to a large extent, score 3)

2.10 Ethical issue: This study was approved by the College of Dentistry Ethical Committee at University of Sulaimani.

2.11 Statistical analysis: The collected data were tabulated via using excel spread sheet then categorical data were statistically correlated and statistical analysis was performed by SPSS program, version 21 (IBM SPSS statistics package software program for statistical analysis).

The data were presented in tabular forms showing the frequency and relative frequency distribution of different variables among the different groups of patients. Chi-square tests were used to compare the categorical data between the four different groups of patients. P values of 0.05 were used as a cut off point for the significance of statistical tests.

36

Chapter three

Results

RESULTS 3.1 Clinical data description: The results presented in this study were based on the analysis consisted of 11 oral premalignant lesions, then 15 cases of OSCC then 4 hyperplastic cases and 5 recurrence cases.

A.Oral premalignant lesions: The oral premalignant lesions involved 7 males and 4 females (Table 3.1). The age ranged from 27 to 71 years with a median of 57 years old, IQR was 27 years. Box and whisker plot showed age distribution of oral premalignant lesions cases. The ends of whisker present the lowest and highest values, the box presents the line dividing the box into two parts presents the median (Fig 3.1).

Fig 3.1: Box and whisker plot showing age of oral premalignant lesions samples.

37

Chapter three

Results

Regarding the site involved by oral premalignant lesions, the buccal mucosa was the most affected site comprising 45.5%(5 cases,3 females and 2 males),followed by tongue comprising 27.3% (3 cases).The age and the sex did not have any effect on the site predominance as p values were 0.29 and 0.56 respectively (Fig 3.2 and 3.3). The most common clinical presentation of oral premalignant lesions in this study was erythroleukoplakia, which was 36.4 %( 4 males), two were found on the tongue and the patients were more than 50 years old. The second clinical feature that was detected in this study were the lichen planus and leukoplakia each were 27.3%(3cases).Nevertheless, the clinical presentation did not relate to age and site variations as the p values were 0.92 and 0.12 respectively (fig 3.4, 3.5), while there was a significant relation between clinical presentation and sex as the p value was 0.04(Fig 3.6).

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Chapter three

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Table 3.1: Clinical data relation of oral premalignant lesions.

Sex

Age

Site

Clinical presentation

Male Female Total ≤50 >50 Total Mean ± SD Range Median IQR Buccal mucosa Tongue Lower lip Upper alveolar ridge Upper lip Total Actinic chelitis Lichen planus Erythroleukoplakia Leukoplakia Total

*NS: no significant relation

39

N0. 7 4 11 3 8 11 55.45 ± 13.51 27 to 71 years 57 27 5 3 1

% 63.6 36.4 100 27.3 72.7 100

P value 0.37(NS)

0.13(NS)

45.5 27.3 9.1 0.21(NS)

1 1 11 1 3 4 3 11

9.1 9.1 100 9.1 27.3 36.4 27.3 100

0.63(NS)

Chapter three

Results

6 5 4 3 >50

2

<50 1 0 Tongue

Buccal mucosa

Upper alveolar ridge

Lower lip

Upper lip

Site

Fig 3.2: The distribution of different sites regarding the two age groups in oral premalignant lesions. 6 5 4 3 Female

2

Male 1 0 Tongue

Buccal mucosa

Upper alveolar ridge

Lower lip

Upper lip

Site

Fig 3.3: The distribution of different sites regarding both sexes in oral premalignant lesions.

40

Chapter three

Results

<50

>50

3 2

1

2

1

1

1

Lichen planus

Erythroleukoplakia

Leukoplakia

0 Actinic chelitis

Clinical presentation

Fig.3.4: The distribution of clinical presentations among two age groups in oral premalignant lesions. 6 5 4 3 2 1 0

Leukoplakia Erythroleukoplakia Lichen planus Tongue

Buccal mucosa

Upper Lower lip Upper lip alveolar ridge

Actinic chelitis

Site Fig 3.5: The distribution of clinical presentation presentations in different sites in oral premalignant lesions.

41

Chapter three

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Actinic chelitis

Lichen planus

Erythroleukoplakia

Leukoplakia

Clinical presentation Female

0

3

0

1

Male

1

0

4

2

Fig 3.6: The distribution of different clinical presentations regarding both sexes in oral premalignant lesions.

B.Oral ral squamous cell carcinoma: In this study, maless were predominantly affected (73.3%),however (73.3%) it did not reach the level of significance as the p value was 0.071.The The age ranged from 29 to 85 years with a median of 68 years old, IQR was 29 years old (Fig 3. 3.7).

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Fig 3.7: Box and whisker plot showing age of oral squamous cell carcinoma samples. The tongue was the most frequent site comprising 40%(6 cases;5 males and 1 female),followed by buccal mucosa comprising 20%(3 cases).The age and sex seemed to have no effect on the site predominance as the p values were 0.63,0.15 respectively(Fig 3.8,3.9,Table 3.2). The ulcer was most clinical presentation found in this study which was 46.7 %( 7 cases; 6 males and 1 female) (Fig 3.10). Two of these lesions were presented on the tongue in ≤50 years age group. The second clinical presentation was the mass (4 cases) and ulcerated mass (4 cases) (Fig 3.11). Two of mass lesions were detected in tongue in the old age group and two of ulcerated mass lesions were seen in tongue also in old age group. Nevertheless, the clinical presentation did not relate to the age, site and sex variations as the p values were 0.77, 0.51 and 0.43 respectively (Fig 3.12, 3.13 and 3.14). In this study, most OSCC patients were found in an advanced stage (stage ІV) which were 9 cases (60%) with significant difference as the p value was 0.02 (table 3.2).

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Table 3.2: Clinical data distribution of oral squamous cell carcinoma. NO. Sex

Age

Site

Clinical presentation

TNM stage

Male Female Total ≤50 >50 Total Mean ± SD Range Median IQR Tongue Buccal mucosa Upper alveolar ridge Lower alveolar ridge Retromolar region Vestibular region Palate Floor of the mouth Total Ulcer Mass Ulcerated mass Total Stage І Stage ІІ Stage ІІІ Stage ІV Total

11 4 15 5 10 15 60.07 ± 17.01 29-85 68 29 6 3 1 1 1 1 1 1 15 7 4 4 15 3 2 1 9 15

*NS: no significant relation. *S: significant relation

44

% P value 73.30% 0.071(NS) 26.70% 100 33.30% 0.20(NS) 66.70% 100

40 20 6.7 6.7 6.7 6.7 6.7 6.7 100 46.7 26.7 26.7 100 20 13.3 6.7 60 100

0.09(NS)

0.55(NS)

0.02(S)

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7 6 5 4 3 2

>50

1

<50 Floor of mouth

Palate

Vestibular region

Retromolar region

Lower alveolar ridge

Upper alveolar ridge

Buccal mucosa

Tongue

0

Fig 3.8: The distribution of different sites among two age groups in oral squamous cell carcinoma sample.

Floor of mouth

Palate

Vestibular region

Retromolar region

Lower alveolar ridge

Upper alveolar ridge

Buccal mucosa

Tongue

7 6 5 4 3 2 1 0

Site Fig 3.9:The :The site distribution and sex in oral squamous cell carcinoma

45

Female Male

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Fig 3.10: 68 years old male patient had ulcer at the ventral surface of tongue

Fig 3.11: 47 years old male presented with ulcerated mass at the right lateral side

8 7 6 5 4 >50

3

<50 2 1 0 Mass

Ulcer

Ulcerated mass

Clinical presentation

Fig 3.12 The distribution of clinical presentations regarding two age groups in oral squamous cell carcinoma.

46

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Ulcerated mass

Floor of mouth

Palate

Vestibular region

Retromolar region

Lower alveolar ridge

Upper alveolar ridge

Buccal mucosa

Tongue

6 5 4 3 2 1 0

Ulcer Mass

Site of the lesion

Fig 3.13: Distribution of cclinical presentation and site of lesion in oral squamous cell carcinoma 8 7 6 5 4 Female

3

Male

2 1 0 Mass

Ulcer

Ulcerated mass

clinical presentation

Fig 3.14: Distribution of clinical linical presentation and sex in oral squamous cell carcinoma.

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3.2.Immunohistochemical findings: 3.2.1Immunohistochemical expression of PTCH1: 3.2.1.1 Expression of PTCH1 in oral; normal mucosa, hyperplastic and premalignant lesions: PTCH1 receptor was rarely and weakly expressed in the basal layer of normal oral mucosa (Fig 3.15). Considering the oral hyperplastic samples, 3/4 of cases (75%) revealed basal and parabasal expression with only one case (25%) showed full thickness Regarding cellular localization, two cases had nuclear localization and the other two showed mixed expression (Fig 3.16A, B), with 50% for each of moderate and strong intensity (Fig 3.17A,B)(Table3.3). In oral premalignant lesions,PTCH1 positive expression was detected in the entire 11 cases (9 mild and 2 moderate).The expression was basal and parabasal in 9 cases(7 mild and 2 moderate)which composed 81.8% of all cases, while 2mild cases showed full thickness expression(Fig 3.18 A,B ). Three cases (2 mild and 1moderate)(18.18%) had nuclear localization(Fig3.19A,B),while mixed (nuclear and cytoplasmic) localization was seen with most frequent expressions (72.7%)(Fig 3.19 C,D )(Table 3.3) Faint intensity is shown in 18.2% of cases, while moderate intensity was seen in 7 cases (63.6%)(Fig 3.19B). Finally, strong intensity was detected in 2 mild cases (18.2%) (Table 3.3). (Fig3.19A, C and D)

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No significant relations were found between hyperplastic and oral premalignant lesions regarding expression, localization and intensity as p values were 0.77, 0.09 and 0.38 respectively (Table 3.4).

Fig 3.15: PTCH1 expression (weak) in normal oral mucosa (IHC,400X)

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(A)

(B)

Fig 3.16:PTCH1 expression in oral hyperplastic lesions. (A)Basal and parabasal and Nuclear localization (B) Full thickness expression with mixed localization (cytoplasmic and nuclear) (IHC, 400X)

(A)

(B)

Fig 3.17: Intensity of PTCH1 in oral hyperplastic samples. (A)Moderate intensity. (B)Strong intensity (IHC, 400X).

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(A)

(B)

Fig 3.18: PTCH1 expression in oral premalignant lesions (A) Basal and parabasal expression of PTCH1 in mild cases.(B) Full thickness expression in moderate degree. (IHC, 400X)

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(A)

(B)

(C)

(D)

Fig 3.19: Cellular localization and intensity of PTCH1in oral premalignant lesions. (A)Nuclear localization and strong intensity in mild oral premalignant lesion. (B)Nuclear localization in moderate case with faint intensity.(C) Mixed localization (cytoplasmic and nuclear) with strong intensity in mild case of oral premalignant lesion. (D)Mixed localization and strong intensity of PTCH1 in moderate case of oral premalignant lesion.(IHC, 400X).

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Table 3.3: PTCH1 expression pattern, localization and intensity in oral hyper plastic and premalignant lesions.

Hyperplastic(4 cases)

Expression

Localization

Intensity

Basal Basal and parabasal Full thickness Total Nuclear Cytoplasmic Mixed Total Faint Moderate Strong Total

No.

%

0 3 1 4 2 0 2 4 0 2 2 4

0 75 25 100 50 0 50 100 0 50 50 100

53

premalignant lesions(11 cases) (9)Mild (2)Moderate No. % No. % 0 0 0 0 7 77.7 2 100 2 22.2 0 0 9 100 2 100 2 22.2 1 50 0 0 0 0 7 77.7 1 50 9 100 2 100 1 11.1 1 50 6 66.6 1 50 2 22.2 0 0 9 100 2 100

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Table 3.4: PTCH1 correlation according to expression pattern, localization and intensity between oral hyperplastic and premalignant lesions.

NO.

%

Oral premalignant NO.

3

75

9

81.80

1 4 2 2 0 4 0 2 2 4

25 100 50 50 0 100 0 50 50 100

2 11 8 3 0 11 2 7 2 11

18.20 100 72.70 27.30 0 100 18.20 63.60 18.20 100

Hyperplastic

Expression of PTCH1

Localization of PTCH1

Intensity of PTCH1

Basal and parabasal Full length Total Mixed Nuclear Cytoplasmic Total Faint Moderate Strong Total

%

P value

3.2.1.2 Immunohistochemical expression of PTCH1 in oral squamous cell carcinoma and recurrence samples: PTCH1 showed positive expression in 14/15cases (93.3%) of oral squamous cell carcinoma. No case was with the score 0.5(10-30%) and a high percentage was detected in score 2 (46.7%) ,followed by score 3

composing 26.7% (Table

3.5).According to localization of PTCH1,a higher number of cases(10cases,71.4%) were within the mixed localization(Fig 3.20A) followed by cytoplasmic which composed 28.5% of all positive cases(Fig 3.20,B)(Table 3.5)

54

0.77

0.09

0.38

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(A)

(B)

Fig 3.20: Cellular localization of PTCH1 in OSCC .A. Mixed localization. B.Cytoplasmic localization (IHC, 400X).

In recurrence oral squamous cell carcinoma, score 0 and score 2 represented 40.0% of entire recurrence cases, followed by score 0.5(10-30%) which was seen in 20.0% (Table 3.5).The nuclear expression was seen in 60.0% followed by mixed expression composing 40.0% of all cases(Fig 3.21 A,B).There was no cytoplasmic expression (Table 3.5). The correlation between oral squamous cell carcinoma and recurrence cases in response to both the expression and localization of PTCH1 revealed nonsignificant relation as p values were 0.15 and 0.09 respectively. (Table 3.6)

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(A)

(B)

Fig 3.21: Cellular localization of PTCH1 in recurrence OSCC. A: Nuclear localization .B: Mixed localization (IHC, 400X)

56

e

n )

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Table 3.5:PTCH1 expression and localization in oral squamous cell carcinoma and recurrence

Score

Localization(14cases) Total

0

0.5

1

1.5

2

3

NO.&%

NO.&%

NO.&%

NO.&%

NO.&%

NO.&%

0(0.0%)

0(0.0%)

1(14.2%)

0(0.0%)

5(71.4%)

1(14.2)%

1(14.2%)

0(0.0%)

1(14.2%)

1(14.2%)

2(28.5%)

2(28.5)%

0(0.0%)

0(0.0%)

0(0.0%)

0(0.0%)

0(0.0%)

1(100%)

2(40.0%)

1(20.0%)

0(0.0%)

0(0.0%)

2(40.0%)

0(0.0%)

57

No.&% 7(100%) 7(100%) 1(100%) 5(100%)

nuclear

cytoplas mic

mixed

NO.&%

NO.%&

NO.

0(0.0%

2(28.5%)

5(71.4%)

0(0.0%

2(28.5%)

4(57.1)%

0(0.0%

0(0.0%)

1(100.0%)

3(60.0%)

0(0.0%)

2(40.0%)

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Table 3.6: PTCH1 correlation in response to the expression pattern and localization between oral squamous cell carcinoma and recurrence cases.

Expression of PTCH1

Localization of PTCH1

0 0.5 1 1.5 2 3 Total Mixed Nuclear Cytoplasmic Total

OSCC No. % 1 (6.7) 0 (0.0) 2 (13.3) 1 (6.7) 7 (46.7) 4 (26.7) 15 (100) 10 (66.7) 0 (0) 4 (26.7) 14 (100)

Recurrence NO. % 2 (40) 1 (20) 0 (0) 0 (0) 2 (40) 0 (0) 5 (100) 2 (40) 3 (60) 0 (0) 5 (100)

P value

0.15

0.09

3.2.2 Immunohistochemical expression of laminin: 3.2.2.1 Immunohistochemical expression of laminin in normal oral mucosa, hyperplastic and premalignant lesions: In normal oral mucosa, laminin showed a continuous linear pattern of expression (Fig 3.22 A). In oral hyperplastic lesions, laminin appeared continuous in 2 cases (50.0%) (Fig 3.22B), while one case showed absent (25.0%) and another one showed discontinuous expression (25.0%). In oral premalignant lesions, laminin was detected as a discontinuous line in 6 cases (5 mild and 1 moderate) (Fig 3.23 A), while 4 cases (3 mild, 1 moderate)

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showed absent expression (3.23B), and finally one mild case showed continuous expression (Fig 3.23C). Regarding the correlation of the pattern of expression of laminin in both oral hyperplastic and premalignant lesions, no significant relation was found as p value was 0.21(Table 3.7).

(A)

(B)

Fig 3.22: Continuous laminin expression A. In normal oral mucosa. (B). In oral hyperplastic samples. (IHC, 400X)

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Results

(B)

(C) Fig 3.23: Expression of laminin in oral premalignant lesions. A. Discontinuous expression. B. Continuous expression (C) Absent expression (IHC, (IHC 400X).

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Table 3.7: Laminin expression pattern and correlation in oral hyperplastic and premalignant lesions.

premalignant lesions(11 cases) Hyperplastic lesions(4cases)

Expression pattern

Mild(9cases) Moderate(2cases) No.

%

No.

%

No.

%

Absent

1

25

3

33.3

1

50

Continuous

2

50

1

11.1

0

0

Discontinuous

1

25

5

55.5

1

50

Total

4

100

9

100

2

100

P value 0.21

3.2.2.2Immunohistochemical expression of laminin in primary and recurrence oral squamous cell carcinoma samples: In oral squamous cell carcinoma, no case showed score 2(loss of staining in less than 50% of the tumour–stromal interface per tumour cell nest (moderate BM defects), while11/15(73.3%) (4 well, 6 moderate, 1poor) were within score 3 (loss of staining in more than 50% of the tumour–stromal interface per tumour cell nest (BM defects to a large extent) (Table 3.8)(Fig3.24A). Only 2 cases (1 well and 1 moderate) were found in score 0 (continuous linear staining, no BM defects) (Fig 3.24B) (Table 3.8).Finally, 2 well differentiated squamous cell carcinoma cases revealed score 1 (minor BM defects) (Fig 3.24C) (Table 3.8)

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(A)

Results

(B)

(C) Fig 3.24: Laminin expression in OSCC A. Discontinuous pattern (Score 3) B. Continuous pattern(no defect) (score 0) (IHC, 400X).(C). Minor defect (score 1) in OSCC

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In recurrence squamous cell carcinoma,40.0% of the cases were with the score 1 (minor BM defects))(Fig 3.25A) and score 2 (moderate BM defects)(Fig 3.25B)(Table3.8),while score 3 was detected in 20.0% of all cases(Fig 3.25C)(Table 3.8). In laminin expression, the relation between oral squamous cell carcinoma and recurrence reached the level of significance as the p value was 0.021(Table 3.8).

A

B

C

Fig 3.25: Laminin expression in recurrence OSCC. (A): Minor defect (score 1). (B): Moderate defect (score 2).(C) Major defect(score 3)(IHC,400X)

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Table 3.8:

Results

The

expression and correlation of laminin in both primary and

recurrence oral squamous cell carcinoma samples. Score 0 Grade No. Well(7cases) 1 Moderate(7cases) 1 Poor(1 case) 0 Recurrence(5 0 cases)

1

2

Total

3

% 14.2 14.2 0

No. 2 0 0

% 28.5 0 0

No. 0 0 0

% 0 0 0

No. 4 6 1

% 57.1 85.7 100

No. 7 7 1

% 100 100 100

0

2

40

2

40

1

20

5

100

P value 0.021

3.3 The relation between laminin and PTCH1: 3.3.1In oral premalignant lesions: the relation between PTCH1 expression and laminin score did not reach the level of significance as the p value was 0.36(Table 3.9) Table 3.9: The relation between PTCH1 expression and laminin scoring in oral premalignant lesions.

Laminin score Absent Continuous

PTCH1 expression Basal and parabasal Full thickness 4 0 1 0

Discontinuous

4

2

Total

9

2

P value 0.36

Similarly, the relation between intensity of PTCH1 with score of laminin did not reach the level of significance as the p value was 0.92(Table 3.10)

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Table 3.10: The relation between the intensity of PTCH1 with the score of laminin in oral premalignant lesions. Laminin score

Faint 1 0 1 2

Absent Continuous Discontinuous Total

Intensity of PTCH1 Moderate 2 1 4 7

Strong 1 0 1 2

P Value 0.92

On the other hand, the relation between localization of PTCH1 and laminin score in oral premalignant lesions reached the level of significance as the p value was 0.03(Table 3.11) Table 3.11: Relation between localization of PTCH1 and laminin score in oral premalignant lesions. Localization of PTCH1

Laminin score

Mixed 1 1 6

Absent Continuous Discontinuous Total

8

Nuclear 3 0 0

P Valu e 0.03

3

3.3.2 In oral squamous cell carcinoma: the relation between PTCH1 expression and laminin scoring was not at the level of significance as the p value was 0.72 (Table 3.12)

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Table 3.12: The relation between PTCH1 expression and laminin scoring in oral squamous cell carcinoma samples. PTCH1 expression

Laminin score

2

P Value

Zero

1

1.5

3

Continuous linear staining Loss of staining in less than 10% Loss of staining in More than 50%

0

1

0

0

1

0

0

0

1

1

1

1

1

6

2

Total

1

2

1

6

4

0.72

Similarly, the relation of localization of PTCH1 and laminin scoring did not reach the level of significance as the p value was 0.53(Table 3.13).

Table 3.13: The relation of localization of PTCH1 and laminin scoring in oral squamous cell carcinoma. Laminin score Continuous linear staining(score 0) Loss of staining in less than 10%(score 1) Loss of staining in more than 50%(score 3) Total

66

Localization of PTCH1 Mixed Cytoplasmic 1 1 2 0 7 3 10 4

P Value 0.53

Chapter four

Discussion

DISCUSSION 4.1 Clinical findings: 4.1.1 Oral premalignant lesions: Today, cancer is one of the leading threats to human life. Studies on precancerous lesions are very important since it is known that oral cancers still cannot be diagnosed adequately in early stages. The role of the dentist in detecting oral carcinomas and premalignant lesions is crucial. The main way to deal with this problem is to educate people about the importance of regular dental checkups and associated factors with the development of oral premalignancy and malignancy (Goyal et al., 2012). Correct diagnosis and timely treatment of potentially malignant lesions with high risk of malignant transformation may help to prevent this transformation. Premalignant lesions can be treated depending on the clinical and histological staging with various treatment modalities from cessation of the habit to surgical removal (Akman et al., 2003).

The male to female ratio reported in the premalignant condition in this study study was 1.75:1.This was in agreement with the finding of Gaphor and Abdullah (2011) and Najm (2013) in Iraq and a study in Turkey by Akman et al. (2003), while it disagreed with the finding of a study by Jabar and Majeed (2009) in south of Iraq and other studies by Almobeeriek and Aldosari (2009) in Saudi Arabia in which oral lesions were more prevalent in females than in males. The finding of our study could be due to that females were more aware about their dental care and oral mucosal health; however the difference in the ratio was not significant.

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These lesions were common in more than 50 years old people. This was in accordance with the study done by Takuji et al. (2011) in Japan, and Ghafour and Sabri (2014) in Sulaimani. This could be attributed to the time dependent factors that result in the initiation and promotion of cellular dysplstic changes (Carnelio et al., 2011).

Erythroleukoplakia is the most common oral premalignant lesion found in this study. This was in agreement with the study done by Carnelio et al. (2011) in Indian population, but disagreed with other studies done in Iraq and Turkey as they found that lichen planus to be the most common lesion(Ghafour and Sabri 2014, Akman et al. 2003). This difference could be due to difference in sample and/or geographic variations with environmental factors.

The most common site for this finding was buccal mucosa which was compatible with the study done by Mehrotra et al. (2010) in India.

4.1.2 Oral squamous cell carcinoma: The male to female ratio was 2:1 in this study, as men were more exposed to known oral carcinogenic factors such as tobacco and alcohol. This finding was in accordance with previous Iraqi studies (Al-Talabani et al., 2010; Al-Qazaz, 2012; Mohammad, 2013, Jabar, 2014 and Hamied, 2015)and agreed with the study of Ahmed, (2006) in Sulaimani city and other studies in different parts of world (Nemes et al., 2008 in Northeastern Hungary, Albuquerque et al., 2011in Brazil, and Kruse et al., 2011 in Switzerland).

Most of the patients detected in this study were in the old age group which was in line with other Iraqi studies (Abed, 2012; Jubair, 2013, Mohammad,

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2013),and with the studies in Sulaimani city (Ahmed, 2006 and Gaphor and Sabri, 2014)and a study in Iran done by Falaki et al. (2011). Because the accumulation of carcinogenic features might be related to chance, but most commonly the life time of environmental exposure that results in DNA damage would accumulate the genetic alteration that leads to head and neck squamous cell carcinoma (Patrick et al., 2009).

The most predominant feature in this study was an ulcer. This finding was in agreement with other Iraqi studies (Sarkis et al., 2010, Mohammad, 2013 and Hamid, 2015) and a study in Iran done by Falaki et al. (2011), but it was in disagreement with the finding of Abed (2012) as he found that the mass was the most clinical presentation our finding might be explained due to different sample size and geographic variation.

The most prevalent site reported in this study was tongue. This finding was in accordance with most previous Iraqi studies (Al-Qazaz, 2012: Jubair, 2013 and Mohammad, 2013), and it was in agreement with various studies in different countries done by Iype et al. (2001) and Jahanbani et al. (2009), as the ventral surface the tongue is lined by thin non-keratinised epithelium that allowed exogenous carcinogen to penetrate through and reach the progenitor cell compartment (Neville and Day, 2002). At the same time, the proliferation of basal keratinocytes and risk of cytogenetic mutation are high (Feller and Lemmer, 2012). But our finding was in disagreement with the finding of Iraqi studies (Al-Reyahi, 2004, Ahmed, 2006 and Al-Talabani et al. 2010) as they reported that the lip was the most predominant site. While other Asian countries revealed that buccal mucosa and gingiva were the most frequent sites (Iamaroon et al., 2004, Jainkittivong, et al. 2009, Krishna Rao et al., 2013, Varshitha, 2015).The reason for

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this variation might be due to the different sample size included in the studies and associated habits (life styles). Unfortunately, the precise location on the tongue could not be considered in this study because of limited data information in the available case sheets.

The clinical staging OSCC at the time of diagnosis is the most important prognostic factor (Scully and Bagan, 2009). Most cases in the findings of this study were presented in the advanced stage (stage ІV, 60%). This was less than the finding of Al-Rawi et al. (2014) which half of cases were at stage IV (14 case; 45.16%). This finding might be related to the fact that affected persons failed to seek professional advice timeously, either because they did not understand the significance of early signs and symptoms, or they are ignorant of the health implications and misdiagnosed by dental professional staff.

Most of the lesions in recurrence oral squamous cell carcinoma in our study were found in lower alveolar ridge and our finding disagreed with the result of Camisasca et al., (2011) they have concluded that tongue cancer recurrence. Or finding might be related with the infiltrative pattern of mandibular invasion which is significantly associated with a worse prognosis and recurrence (Wong et al., 2000) 4.2 Immunohistochemical findings of PTCH1: 4.2.1 Assessment of PTCH1 immunohistochemical expression in oral normal, hyperplastic and premalignant lesions:

This study reported low expression of PTCH1 in normal oral mucosa; this was in agreement with a study done by Wang et al. (2011).

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All of four oral hyperplastic cases showed positive expression (100%). This could be explained by the types of the samples as all of them were from gingiva that might be affected by a mastictory force and plaque accumulation.

In oral premalignant lesions, most cases showed basal and parabasal expression (81.8%). This finding was in disagreement with the study done by Yang et al. (2012) in esophageal dysplastic lesions as they found 21% of dysplastic lesions with basal and parabasal pattern. Our study revealed PTCH1 expression in both mild and moderate dysplastic lesions (9mild, 2moderate). Which was in contrast with a study done by Yang et al. (2012) as they found that PTCH1 expression was only seen in severe dysplastic lesions. This could be attributed to different sample size as their sample size was 131 cases. In our one case of actinic chelitis there was over expression of PTCH1 however, Gonzalez et al. (2016) found over expression of PTCH1 in actinic chelitis.

This study showed a great percentage of mixed expression of PTCH1 (8/11, 72.7%). This could be explained by the different stages of activation of this marker that could have a role in the progression from dysplastic lesions to cancers.

4.2.2 Assessment of PTCH1 immunohistochemical expression in oral squamous cell carcinoma and recurrence lesions:

PTCH1expression was detected in 14/15(93.3%) of OSCC. This finding was in disagreement with the study done by Wang et al. (2011) on OSCC as they found positive expression in 47.5%. This could be due to the difference in sample size. The most prominent score for positively stained cells in this study was score 2 which composed 46.6%.This was in agreement with the study done by Wang et al.

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(2011) as they reported score 2 to be 47.5%. Most cases of OSCC presented with mixed (cytoplasm and nuclear) localization (10/14, 71.4%) which was in disagreement with the finding of Leovic et al. (2012) as they reported high membranous localization (49.1%). The finding of this study could be explained by signal activation that facilitates the transcription of certain genes that contribute to cell migration, angiogenesis and then promote tumor progression.

In this study, score 2 represents 2/5(40%) of all recurrence cases. This result disagreed with the study done by Wang et al. (2011), which found 68.7% of all recurrence samples. The correlation between OSCC and recurrence in response to expression and localization did not reach the level of significance; this finding disagreed with the finding of Wang et al. (2011) as they demonstrated correlation of Gli biomarker between OSCC and recurrence samples.

4.3 Immunohistochemical finding of laminin: 4.3.1 Assessment of laminin immunohistochemical expression of oral normal, hyperplastic and premalignant lesions: In normal oral mucosa, laminin showed a continuous linear pattern of expression. This was in accordance to the study done by Garcia et al. (2006). In oral hyperplastic lesions, laminin pattern had continuous expression in 50% of cases. However, Firth and Reade (1996) showed that laminin distribution was continuous in all of oral hyperplastic samples, while Kannan et al. (1994) in India reported laminin discontinuity in all of their oral hyperplastic cases. This difference in results might be reasoned to differences in sample size and type of lesions used in this study.

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In oral premalignant lesions, laminin was seen more with discontinuous distribution in 6 cases (54.5% of all cases).This was in contrast with other studies in oral premalignant lesions as they found discontinuous distribution of laminin in all of their samples (Kannan et al. (1994), Tosios et al. (1998), Garcia et al., 2006). The high laminin expression with both discontinuous and/or absent pattern indicate the role of this marker in early diagnosis of potentially malignant lesions and the prediction of biological progression toward malignancy.

4.3.2 Assessment of laminin immunohistochemical expression in oral squamous cell carcinoma and recurrence: The basement membrane is considered as resistant, dynamic barrier that has an important role in controlling cell behavior. One of its components is laminin, which is involved in cell adhesion, migration, proteolytic activity, proliferation, and metastatic growth. During neoplastic invasion, glycoprotein synthesis in the basal membrane is decreased and disorganized, leading to permanent loss of continuity (Kannan et al., 1994).

The basal membrane has a role in the activation of signals that play a role in invasion of neoplastic cells, as various angiogenesis modulating molecules, growth factors and cytokines are stored in and appeared to be released and activated when the basal membrane is breached (Engbring and Kleinman, 2003 and Theocharis et al., 2012). Furthermore, tumor cells bind to laminin receptors on the basement membrane and are subsequently stimulated to produce metalloproteinase, which begin fragmentation and degradation of the membrane and help in tumor invasion (Souza et al., 2007 and Kinoshita et al., 2013).

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In this study, laminin showed high major basement membrane defect (score 3)(73.3%). This finding was in line with the results of Harada et al. (1994), Kobayashi et al. (1995) and Garcia et al. (2006) as they found prominent major basement membrane defect in their samples. This finding was in contrast with Tosios et al. (1998), Mostafa et al. (2007) and shruthy et al. (2013) studies in which high percentage of their cases had continuous staining of laminin around the basement membrane.

The major defect distribution of laminin was mainly seen in both moderately and poorly differentiated OSCC which were 85.7% and 100% respectively. This result was in contrast with the study done by Souza et al., 2007 in OSCC in which 50% of poorly differentiated SCC were with major basement defect expression. This finding supports the fact that laminin fragmentation gradually is increased with loss of differentiation of OSCC (Garcia et al., 2006). Continuous expression of laminin was only seen in well and moderately differentiated SCC (14% for each).The expression is less than that found by other researchers in well differentiated OSCC as they reported the continuous expression to be 52.6% and 90% (Souza et al., 2007, Shruthy et al., 2013).This difference could be related to different numbers of evaluated islands in selected samples of OSCC and the sample size.

Regarding the recurrence of OSCC, 40% of cases were recorded in both score 1 and score 2. This finding disagreed with the results of Stoltzfus et al. (2004) in which they detected score 2 to be the most frequent score (75%) in their recurrence cases and also disagreed with the study done by Yamamoto et al. (2001) who found that score 3 was 44% in their recurrence samples of esophageal squamous cell carcinoma. The high minor and moderate defect of laminin

74

Chapter four

Discussion

distribution in recurrence OSCC might be related to the ability of this marker to facilitate the development of the second primary tumor and could be used as a predictive biomarker for aggressiveness of oral cancer and distant metastasis, as a significant correlation between score of laminin in oral squamous cell carcinoma and recurrence samples was found in this study (the p value was 0.021).

Finally, the relation between laminin scoring and PTCH1 localization in oral premalignant lesion reached the level of significance because erythroleukoplakia was the most frequent clinical presentation and discontinuous pattern of laminin and mixed localization of PTCH1 were the most common and this might illustrate the combined role of these two markers in the initial invasion of basement membrane with neoangiogenesis that could pave the way for further invasions later on.

75

Chapter five

Conclusions and Suggestions

Conclusions 1. Oral premalignant lesions reported male predominance (63.6%), most of them with the old age group (72.7%), affecting buccal mucosa, with 1/3 erythroleukoplakia clinical presentation (36.4%). 2. In oral squamous cell carcinoma, most of patients had a significant advanced stage (stage IV) 60%. 3. The PTCH1 over expression in all of the studied groups of lesions might give impression of the active role of this biomarker in progression toward malignancy. The hedgehog pathway plays an important role in OSCC progression and should be considered as a potential therapeutic target. 4. Laminin defragmentation which started from dysplastic lesions extending to oral squamous cell carcinoma and recurrence could emphasize the role of this marker from early precancerous stage. .Furthermore, a significant relation was detected in its expression between oral squamous cell carcinoma and recurrence cases as p value was 0.021. 5. The expression of laminin in the basement membrane may be a useful parameter to evaluate tumor histological differentiation and aggressiveness. 6. Regarding the correlation of both laminin and PTCH1, a significant relation was found regarding laminin scoring and PTCH1 localization in oral premalignant lesions as p value was 0.03.

76

Chapter five

Conclusions and Suggestions

Suggestions

1. Further studies should be done concerning other hedgehog pathway ligands in order to identify the relation of these different biomarkers in progression from premalignant lesions toward malignancy. 2. Other mesenchymal repeated markers could be considered with laminin evaluation in order to verify the epithelial-mesenchymal transition role in carcinogenesis process. 3. Expanding the size of the sample and utilizing questionnaire sheets to identify possible associated precancerous causative factors that aid in preventing the subsequent oral cancer.

77

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Appendix1 Study form Case No……………. Patient information Name ……………………………. Age………………………….. Sex…………………………….Occupation………………………. Address……………… Telephone number…………………………………..Mobile…………..

Clinical presentation (Oral premalignant lesion) Actinic chelitis…………….. Leukoplakia ………………. Erythroleukoplakia……………… Lichen planus ………………

Degree of dysplasia Mild………… Moderate……….. Severe…………

Site of the lesion Buccal mucosa……………..

Tongue………………. Lower lip………….. Upper alveolar ridge……………. Upper lip………………..

Clinical presentation(Oral squamous cell carcinoma) Mass……….. Ulcer……….. Ulcerated mass………..

Grade Well………… Moderate……….. Poor……….

Site of the lesion Tongue…………. Buccal mucosa……………… Upper alveolar ridge…………… Lower alveolar ridge………….. Retromolar region……………. Vestibular region……………

Palate…………….

TNM stage Stage І……….. Stage ІІ………. Stage ІІІ…….. Stage ІV……….

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‫ ميالدي‬2017

‫ كردي‬2716

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‫ ھجري‬1438

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Immunohistochemical Expression of PTCH1 and Laminin in Oral ...

Immunohistochemical Expression of PTCH1 and Lamin ... quamous Cell Carcinoma and Recurrence Samples.pdf. Immunohistochemical Expression of PTCH1 ...

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