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Genetic Polymorphism of Cypiai, Gstm1 and Gstt1 Among Various Tobacco Habit Groups with Susceptibility to Oral Pre-Cancerous Lesions and Squamous Cell Carcinoma

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Genetic Polymorphism of Cypiai, Gstm1 and Gstt1 Among Various Tobacco Habit Groups with Susceptibility to Oral Pre-Cancerous Lesions and Squamous Cell Carcinoma GENETIC POLYMORPHISM OF CYPIAI, GSTM1 AND GSTT1 AMONG VARIOUS TOBACCO HABIT GROUPS WITH SUSCEPTIBILITY TO ORAL PRE-CANCEROUS LESIONS AND SQUAMOUS CELL CARCINOMA by MUHAMMAD MOHIUDDIN ALAMGIR MBBS, M.Phil (Path) Department of Pathology, Faculty of Medicine/ Ziauddin University A thesis submitted in partial fulfillment of the requirements for the degree of PhD in Pathology Karachi/ Pakistan December, 2016 i Dedicated to Late Prof. Dr. Naeem Aon Jafarey in the memory of his enthusiastic guidance and kind support vii Acknowledgement First I offer my thanks and gratitude to Allah Almighty, for providing the opportunity and means to do this work. All respect for his Last Prophet (PBUH). I am grateful to my supervisor Prof. Dr. Qamar Jamal for her able guidance and continued encouragement throughout my research. She always remained positive regarding accomplishment of this endeavor. Her efforts in critical reading of the thesis are commendable. My special thanks are to my co-supervisor Late Prof. Naeem Aon Jafarey. He was my main inspiration for initiation of this research project. His guidance and interest in this work remained throughout right from the beginning of my synopsis writing until his demise. He helped me a lot in retrieval of literature and critical evaluation of my results. I am grateful to my co-supervisor Prof. Dr. Talat Mirza, for the valuable feedback and limitless encouragement. She helped me not only in sample collection, but guided me in data analysis, paper writing and finally thesis writing. Without her support and interest it may not be possible to complete this work. My special thanks to Prof. Dr. Kamran Hameed, Prof. Dr. Masood Hameed Khan and Prof. Dr. Khalid Mahmood for their support in ethical approvals from respective Institutes. My sincere thanks are for Dr. Israr Nasir for his guidance and technical support. I carried out the tedious task of molecular analysis of my samples under his guidance. Without his technical expertise and trouble-shooting my work could not have been completed. I am also thankful to his supporting lab staff specially Miss Nazneen, Miss Maheen, Mr.Haris Lucky, Mr.Tariq, Mr.Nazuk and Mr.Waqar. I express my sincere thanks to Mr. Manzoor Asi for his help in retrieval of histopathology slides of cancer cases as well as their photomicrography. At this point, I would like to offer my gratitude to Dr. Mushtaq at DUHS and Prof. Dr. Saeeda Baig at ZU, who introduced me to the fascinating world of molecular biology and genetics. viii I am thankful to Professor Dr. Iqbal Khayani at CHK, Mr. Illtifat at Radiotherapy department of ZUH, North Nazimabad, and my students Salman and Shahjee who helped and supported me in collecting patient samples and data. In fact, there is along list of persons who supported me in collection of studied cases and I am thankful to all of them. I am also thankful to Mr. Ejaz (ZU) and Mr. Faisal Raza (Panjwani Centre, KU) for helping me in statistical analysis. I am thankful to Prof. Dr. Zahida Memon and Prof. Dr. Nikhat Siddiqui for their guidance and help. I am grateful to my colleagues, subordinates at BUM&DC, and friends for their invaluable cooperation, support and sacrifices. My special thanks go to my beloved family, my wife Dr. Hina Zaman and my daughter Maryam, who have been pillars of support throughout this project. I am indebted to them for being there when I felt depressed and tired. Finally, I am grateful to Higher Education Commission of Pakistan and Ziauddin University, Karachi, for providing me financial support to complete this project. ix List of Abbreviations AARs Age-adjusted rates AHUH Aga Khan University Hospital APCR Aga Khan University Pathology-based Cancer Registry ASRs Age-standardized incidence rates CDK Cyclin dependent kinase CDKI Cyclin dependent kinase inhibitor CHK Civil Hospital Karachi CISH Chromogenic In situ Hybridization CYP1A1 Cytochrome P4501A1 CYPs Cytochrome P450s DNA Deoxy-ribonucleic acid DPX Distrenedibutylphthalatexylene EBV Epstein Bar Virus EDTA Ethylenediaminotetra acetic acid EGFR Epidermal growth factor receptor FISH FluorecentIn situ Hybridization GAP GTPase-activating proteins GDP Guanine Diphosphate GEFs Guanine-nucleotide exchange factors GSTM1 Glutathione S-tranferase M1 GSTs Glutathione S-transferases GSTT1 Glutathione S-tranferase T1 GTP Guanine Triphosphate x HNSCC Head and neck squamous cell carcinoma HPV Human Papilloma Virus IARC International Agency on Cancer ICD International Classification of Disease JPMC Jinnah Post-Graduate Medical Centre KCR Karachi Cancer Registry KIRAN Karachi Institute of Radiotherapy and Nuclear Medicine KPK Khyber Pakhtunkhwa LOH Loss of heterozygosity NAACCR North American Association of Central Cancer Registries NATs N-acetyltransferases NCCP National Cancer Control Programme NCRP National Cancer Registry Programme NNK Nicotine-derived nitrosamine ketone NNN Nitrosonornicotine OPLs Oral precancerous lesions OR Odds ratio OSCC Oral squamous cell carcinoma OSF Oral submucous fibrosis PAHs Polycyclic aromatic hydrocarbons PBCRs Population-based Cancer Registries PCLs Pre-Cancerous Lesions PCR Polymerase Chain Reaction PCR-RFLP PCR- Restriction Fragment Length Polymorphism MDSCC Moderately Differentiated Squamous Cell Carcinoma xi PMRC Pakistan Medical Research Council PSCC Papillary squamous cell carcinoma qRT – PCR Quantitative Real Time PCR ROS Reactive oxygen species SAARC South Asian Association for Regional Cooperation SCE Sister chromatid exchange SKMCH & RC Shaukat Khanum Memorial Cancer Hospital and Research Centre SLT Smokeless tobacco SSCP Single Stranded Conformational Polymorphism ST Sulfotransferases TNM Tumor Node Metastases UGTs UDP-glucuronosyltransferases UP Uttar Pradesh VC Verrucous carcinoma XMEs Xenobiotic metabolizing enzymes ZU Ziauddin University xii List of Tables TABLE NO. DESCRIPTION PAGE 1.1 Top 3 malignancies by gender and age in an 18-year time period (1994-2012) at SKMCH & RC ………………………........................ 02 Epidemiological facts about cancer of oral cavity according to 1.2 World regions ...……………………………………………………... 04 1.3 ASRs per 100,000 in women, Karachi South ……………………….. 09 1.4 ASRs per 100,000 in men, Karachi South …………………………... 10 1.5 Basic constituents of a gutka ………………………………………... 29 1.6 Overview of CYP1A1 Polymorphism Nomenclature ……………….. 34 3.1 Characteristics of the study subjects ………………………………… 60 3.2 Ethnic distribution of PCLs, OSCC & Control cases ……………….. 61 3.3 OSCC Cases According to Age, Sex & Intra-oral Sub-site …... 62 3.4 Tobacco indices in OSCC cases and controls ………………………. 69 3.5 CYP1A1MspI, GSTM1 and GSTT1 genotype variants in OSCC cases and controls ……………………………………………..................... 70 3.6 Genotype distribution among different tobacco exposure groups for oral cancers and controls ……………………………………………. 72 3.7 Effect of genotype combinations on OSCC cases and control …….... 75 3.8 Risk analysis according to intra-oral sub-site (Cheek)………………. 80 3.9 Risk analysis according to intra-oral sub-site (Tongue)……………... 83 3.10 Mean and median tobacco indices in Controls and PCL cases ……... 86 3.11 CYP1A1, GSTM1 and GSTT1 genotype variants in PCLs and controls ………………………………………………………............ 88 3.12 Genotype distribution among different tobacco exposure groups in PCL cases and controls ……………………………………………… 90 3.13 Effect of genotype combinations on PCL cases and controls ………. 92 3.14 Distribution of genotypes according to ethnicity….......…………….. 97 xiii List of Figures FIG.NO. DESCRIPTION PAGE 1.1 Structures located in the Head and Neck region……………………… 3 1.2 Comparison of AARs for mouth cancer in males across all PBCRs – India …………………………………………………………………... 6 1.3 Comparison of AARs for mouth cancer in females across all PBCRs – India ………………………………………………………………… 7 1.4 Comparison of AARs for Tongue cancer in males across all PBCRs – India …………………………………………………………………... 7 1.5 Natural history of oral carcinogenesis ………………………………... 22 1.6 Tobacco culture of Karachi …………………………………………... 30 1.7 Polymorphism of human CYP1A1 gene ...…......................................... 34 2.1 Steps in Polymerase Chain Reaction ………………………................. 53 2.2 Exponential amplification in PCR cycling ………………………........ 53 3.1 Carcinoma of buccal mucosa, Stage III, Case ID-105………….. 63 3.2 Carcinoma of left cheek, Stage II, Case ID-122………………... 64 3.3 Carcinoma of the lip, Stage IV, Case ID-48……………………. 64 3.4 Metastatic carcinoma, Stage IV, Case ID-03…………………… 65 3.5 Moderately-differentiated oral squamous cell carcinoma (H&E;× 200 magnification), Case ID-142……………………... 65 3.6 Moderately-differentiated SCC of tongue showing muscle invasion (H&E; × 200 magnification), Case ID-132…………… 66 3.7 Intact stratified squamous epithelium, tumor pushing from below (H&E;× 200 magnification), Case ID-110………………. 66 Cont…... xiv 1. INTRODUCTION 1.1 Epidemiology In the past, a number of cancer registries have been established in Pakistan but there has been a problem as regards to their sustenance. In the first five decades after its creation the country lacked any substantive cancer data other than a few sporadic institution-based frequencies (Pakistan Medical Research Council, 1982). This data lacks complete demographic details and there was the problem of continuit. In subsequent decades, a number of proper cancer registries were established namely; The Karachi Cancer Registry (KCR) in 1995, The Aga Khan University Cancer Surveillance for Pakistan (ACSP) in 2000 at the Aga Khan University Pathology-based Cancer Registry (APCR), the hospital-based cancer registry
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