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Molecular epidemiology of gastric and esophageal cancer survival 2011

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MOLECULAR EPIDEMIOLOGY OF GASTRIC AND ESOPHAGEAL CANCER SURVIVAL  by   MORTEZA BASHASH        A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES   (Interdisciplinary Oncology)       THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   January 2011      © Morteza Bashash, 2011   ii ABSTRACT Introduction Gastric and esophageal cancers are among the deadliest forms of cancer. Studies of human cancer susceptibility examine factors associated with the incidence of disease. Studies of human cancer prognosis and prediction examine factors associated with disease outcomes. This dissertation is about molecular and other factors that affect survival of gastric and esophageal cancer patients. Methods Population-based registry data linked with patient outcome data was used to describe the epidemiology of gastric and esophageal cancers in BC; to compare survival of cancer patients in BC, and Ardabil, Iran and to describe differences in survival of BC patients of different ethnicity. The ethnicity of patients was determined based on lists of names corresponding to each ethnic group. A prospective cohort study was conducted to examine the effect of genetic polymorphisms in TIMP (1-4) and MMP (2, 7 and 9) genes. Results Analysis of cancer registry data points to several factors associated with gastric and esophageal cancer survival. Patients with gastric cardia experience worse survival compared to other gastric cancers. Ethnicity of gastric and esophageal cancer patients is associated with their survival. Gastric and esophageal cancer patients diagnosed in British Columbia have better survival compared to those daignosed in Adabil, Iran. Genetic polymorphisms are also associated with survival. My prospective study identified 4 genetic polymorphisms at TIMP-   iii 3 associated with survival of esophageal adenocarcinoma and gastroesophageal junction (GEJ). Conclusion Besides established prognostic indicators, other factors affect survival of gastric and esophageal cancers. Differences in survival by ethnicity support the importance of ethnicity as a prognostic factor. Survival differences between BC and Ardabil are likely due to disease characteristics and patient factors, in addition to differences in healthcare systems. TIMP3 genetic polymorphisms are promising prognostic factors for adenocarcinoma of esophagus and GEJ. Modeling prognosis based on host factors, including ethnicity and genetic polymorphisms, is an emerging field of translational cancer research. More research is needed to fully explore the functional effects of TIMP3 polymorphisms, and to identify both genetic and lifestyle factors underlying the effect ethnicity on survival.    iv PREFACE CHAPTER 2 A version of this chapter has been submitted for publication and is under review. Bashash M, Shah A, Hislop G, Le N, Bajdik C, Brooks-Wilson A.  Review:  Genetic variants associated with esophageal and gastric cancer survival (Submitted June 19, 2010) Author contribution: MB designed and conducted the study, and wrote 100% of this manuscript. AS assisted in the intepretation and reviewed the manuscript. GH assisted in the intepretation and reviewed the manuscript. NL assisted in the intepretation and reviewed the manuscript. CB and ABW supervised all aspects of the study, contributed to interpretation of the findings, and reviewed and edited the manuscript. Ethics approval: Not applicable CHAPTER 3 A version of this chapter has been published: Bashash M, Shah A, Hislop G, Brooks-Wilson A, Le N, Bajdik C. Incidence and survival for gastric and esophageal cancer diagnosed in British Columbia, 1990 to 1999. Can J Gastroenterol. 2008 Feb; 22(2):143-8. Author contribution: MB designed the study, extracted data, performed the analysis and wrote 100% of the manuscript. AS supervised clinical aspects of the study and reviewed the manuscript. GH supervised epidemiological aspects of the study and reviewed the manuscript. NL supervised statistical aspects of the study and reviewed the manuscript. CB and ABW supervised all aspects of the study, contributed to interpretation of the findings, and reviewed and edited the manuscript.   v Ethics approval: This study was approved by the UBC/BCCA Ethics Board (certificate number H07-2807) CHAPTER 4 A version of this chapter has been published: Bashash M, Yavari P, Hislop TG, Shah A, Sadjadi A, Babaei M, Le N, Brooks-Wilson A, Malekzadeh R, Bajdik C. Comparison of Two Diverse Populations, British Columbia, Canada, and Ardabil, Iran, Indicates Several Variables Associated with Gastric and Esophageal Cancer Survival. J Gastrointest Cancer. 2010 Nov 20. Author contribution: MB designed the study, collected the BC data, performed the analysis and wrote 100% of the manuscript. AS supervised clinical aspects of the study and reviewed the manuscript. GH supervised epidemiological aspects of the study and reviewed the manuscript. NL supervised statistical aspects of study and reviewed the manuscript. CB and ABW supervised all aspects of study, contributed to interpretation of the findings, and reviewed and edited the manuscript. PY and RM supervised the Iranian investigators and corresponding portions of the study. Ethics approval: This study was approved by the UBC/BCCA Ethics Board (certificate number H06-00025) CHAPTER 5 A version of this chapter has been submitted for publication and is under review. Bashash M, Shah A, Hislop G, Le N, Brooks-Wilson A, Bajdik C. The prognostic effect of ethnicity for gastric and esophageal cancer: the population-based experience in British Columbia, Canada (Submitted August 6, 2010)   vi Author contribution: MB designed the study, performed the analysis and wrote 100% of the manuscript. AS supervised clinical aspects of the study and reviewed the manuscript. GH supervised epidemiological aspects of the study and reviewed the manuscript. NL supervised statistical aspects of study and reviewed the manuscript. CB and ABW supervised all aspects of study, contributed to the interpretation of findings, and reviewed the manuscript. Ethics approval: This study was approved by the UBC/BCCA Ethics Board (certificate number H08-02486) CHAPTER 6 A version of this chapter will be submitted for publication. Bashash M, Shah A, Hislop G, Le N, Brooks-Wilson A, Bajdik C. Genetic polymorphism at TIMP-3 predicts survival for patients with adenocarcinoma of the esophagus and gastroesophageal junction. Author contribution: MB designed the study, performed patient recruitment, performed data collection, designed the genetic analysis, performed the statistical analysis and wrote 100% of the manuscript. AS supervised clinical aspects of the study and reviewed the manuscript. GH supervised epidemiological aspects of the study and reviewed the manuscript. NL supervised statistical aspects of study and reviewed the manuscript. CB and ABW supervised all aspects of the study, contributed to interpretation of the findings, and reviewed and edited the manuscript. All sample preparation, DNA extraction and genotyping preparation was performed in A Brooks-Wilson’s lab. Genotyping was sponsored by ABW. Ethics approval: This study was approved by the UBC/BCCA Ethics Board (certificate number H07-2807)   vii TABLE OF CONTENTS ABSTRACT ............................................................................................................................. ii  PREFACE ............................................................................................................................... iv  TABLE OF CONTENTS ....................................................................................................... vii  LIST OF TABLES .................................................................................................................. ix  LIST OF FIGURES ................................................................................................................ xi  LIST OF ABBREVIATIONS ................................................................................................ xii  ACKNOWLEDGEMENTS .................................................................................................... xv  DEDICATION ..................................................................................................................... xvii  CHAPTER 1: Introduction ....................................................................................................... 1  1.1 Background ......................................................................................................................... 1  1.2 Prognostic factors ................................................................................................................ 5  1.3 Research objectives ............................................................................................................. 8  1.4 The thesis sections .............................................................................................................. 8  CHAPTER 2: Review:  Genetic variants associated with esophageal and gastric cancer survival .................................................................................................................................... 14  2.1 Introduction ....................................................................................................................... 14  2.2 Material and methods ........................................................................................................ 15  2.3 Results ............................................................................................................................... 16  2.4 Discussion ......................................................................................................................... 23  2.5 Conclusion ........................................................................................................................ 24  CHAPTER 3: Incidence and survival of gastric and esophageal cancer diagnosed in British Columbia, 1990 to 1999  ......................................................................................................... 35  3.1 Introduction ....................................................................................................................... 35  3.2 Methods............................................................................................................................. 36  3.3 Results ............................................................................................................................... 37  3.4 Discussion ......................................................................................................................... 39  CHAPTER 4: Comparison of two diverse populations, British Columbia, Canada and Ardabil, Iran, indicates several variables associated with gastric and esophageal cancer survival  ................................................................................................................................... 49  4.1 Introduction ....................................................................................................................... 49  4.2 Methods............................................................................................................................. 50  4.3 Results ............................................................................................................................... 51  4.4 Discussion ......................................................................................................................... 52  4.5 Conclusion ........................................................................................................................ 55  CHAPTER 5: The prognostic effect of ethnicity for gastric and esophageal cancer: the population-based experience in British Columbia, Canada  ................................................... 62  5.1 Introduction ....................................................................................................................... 62  5.2 Methods............................................................................................................................. 63  5.3 Results ............................................................................................................................... 65  5.4 Discussion ......................................................................................................................... 67  CHAPTER 6: Genetic polymorphism at TIMP-3 predicts survival for patients with adenocarcinoma of the esophagus and gastroesophageal junction  ........................................ 79  6.1 Introduction ....................................................................................................................... 79  6.2 Methods............................................................................................................................. 80    viii 6.3 Results ............................................................................................................................... 83  6.4 Conclusion ........................................................................................................................ 85  CHAPTER 7: Conclusions ................................................................................................... 101  7.1 Opportunities and challenges .......................................................................................... 103  7.2 Future directions ............................................................................................................. 103  REFERENCES ..................................................................................................................... 105     ix LIST OF TABLES  Table 1.1 Stage category definitions for gastric cancer………………………………………9 Table 1.2 Staging group of gastric cancer…………………………………………………...10 Table 1.3 Stage category definitions for esophageal cancer………………………………...11 Table 1.4 TNM staging of esophageal squamous cell carcinoma…………………………...12 Table 1.5 TNM staging of esophageal and GEJ adenocarcinoma. …………………………13 Table 2.1. Studies of prognostic significance for genetic polymorphisms in esophageal cancer patients ……………………………………………...………………………………. 27  Table 2.2. Studies of prognostic significance of genetic polymorphisms gastric cancer patients ……………………………....................................................................................... 30  Table 3.1. Age standardized incidence rate per 100,000 (ASR) and estimated annual percentage change (EAPC), with 95% confidence interval (95% CI), for esophageal and gastric cancer by topology and histology ……….………………………………………….. 45  Table 4.1. Gastric cancer patients in BC (Canada) and Ardabil (Iran) ……………………. 58  Table 4.2. Esophageal cancer patients in BC (Canada) and Ardabil (Iran) …….................. 59  Table 4.3. One-year relative survival for gastric cancer patients in BC (Canada) and Ardabil (Iran) ……………………………………………………………………………………….. 60  Table 4.4. One-year relative survival for esophageal cancer patients in BC (Canada) and Ardabil (Iran) ……………………………………………………......................................... 61  Table 5.1. Descriptive characteristics for gastric cancer by ethnicity …………................... 71  Table 5.2. Hazard ratio (HR) and 95% confidence interval (CI) from Cox proportional hazards regression analysis for overall survival of gastric cancer patients ………………... 72  Table 5.3. Descriptive characteristics for esophageal cancer by ethnicity ………………… 73  Table 5.4. Hazard ratio (HR) and 95% confidence interval (CI) from Cox proportional hazards regression analysis for overall survival of esophageal cancer patients …………… 74  Table 6.1. SNPs in TIMP and MMP genes used for survival analyses ...………………….. 88   x  Table 6.2 Hazard ratios (HR) and 95% confidence intervals (CI) estimates for the association between TIMP and MMP gene variations and survival of study cohort................................ 90  Table 6.3 Hazard ratio (HR) and 95% confidence interval (CI) estimated for the association between TIMP-3 gene variations and survival of study cohort adjusted for patient age, tumour location, disease stage and treatments …………………........................................... 96   xi LIST OF FIGURES Figure 1.1. Anatomy of the esophagus with landmarks and recorded distance from the incisors used to divide the esophagus into topographic compartments ………....................... 4  Figure 2.1. Genetic polymorphism associated with esophageal and gastric cancer Survival……………………………………………………………………………………... 26  Figure 3.1. New diagnoses of esophageal (A) and gastric (B) cancer in BC during 1990-1999 by age and gender …………………………………………………………………...……... 44  Figure 3.2. Five-year survival of esophageal and gastric cancer ………………………….. 46  Figure 3.3. Five-year survival by gender for (A) esophageal and (B) gastric cancer……… 47 Figure 3.4 Five-year survival by tumour location for esophageal and gastric cancer …….. 48  Figure 4.1. Overall one-year age-standardized survival rates for gastric cancer cases in Ardabil (Iran) and British Columbia (Canada) …………………………………………….. 56  Figure 4.2. Overall one-year age-standardized survival rates for esophageal cancer cases in Ardabil (Iran) and British Columbia (Canada) …………………………………………….. 57  Figure 5.1. Survival of gastric cancer patients by ethnic group …………………………… 75  Figure 5.2. Survival of esophageal cancer patients by ethnic group ……………………….76  Figure 5.3. Survival of gastric cancer patients by ethnic group for non-metastatic disease………….…………………………………………………………... 77  Figure 5.4. Survival of esophageal cancer patients by ethnic group for non-metastatic disease ………………………………........................................................... 78  Figure 6.1. Detectable Hazard Ratio by power and MAF………………………………….98  Figure 6.2. Linkage disequilibrium (LD) plot for TIMP-3 gene…………………………... 99  Figure 6.3. Survival of study cohort by TIMP-3 variations, rs130274, rs1962223, rs5754312, rs715572 and rs5754312/rs715572 haplotype ……………………………………………..100     xii LIST OF ABBREVIATIONS 3' UTR Three prime untranslated region 5FU Fluorouracil ADAM Adamalysins ADAMTS Adamalysin-thrombospondin AJCC American Joint Committee on Cancer Arg Arginine ASR  Age-standardized incidence rate BC British Columbia BCCA British Columbia Cancer Agency BCCR British Columbia Cancer Registry BRCA Breast cancer (gene) CEU Utah residents with Northern and Western European ancestry cGy centi-Gray CI Confidence interval del Deletion polymorphism DNA  Deoxyribonucleic acid EAPC Estimated annual percent change ECM Extracellular matrix ECRG Esophageal cancer-related gene EGFR Epidermal growth factor receptor ERCC1 Excision repair cross-complementing FA Folinic acid   xiii GEJ Gastroesophageal junction GERD Gastro-esophageal reflux disease GI Gastrointestinal GSH  Glutathione GST Glutathione-S-transferases GWAS Genome-wide association studies HapMap Human haplotype map HR Hazard ratio HWE Hardy-Weinberg equilibrium ICDO International Classification of Diseases for Oncology IL Interleukin Ile Isoleucine ins Insertion polymorphism MDR  Multidrug resistance MMP Matrix Metalloproteinases MSP British Columbia Medical Services Plan MTHFR 5, 10-methylenetetrahydrofolate reductase mTOR Mammalian target of rapamycin NS Non-significant OGG1  8-oxoguanine glycosylase Pgp P-glycoprotein PI3K Phosphoinositide 3-kinases Pro Proline   xiv PTEN  Phosphatase and tensin homolog SCC Squamous cell carcinoma SD Standard deviation SMPBC Screening Mammography Program of British Columbia SNP Single nucleotide polymorphisms STR Short tandem repeat (microsatellite) polymorphism TIMP Tissue inhibitors of metalloproteinases TNF Tumour necrosis factor TYMS Thymidylate synthase Val Valine VEGFR Vascular endothelial growth factor receptor WHO World Health Organization WHOSIS WHO Statistical Information System XP Xeroderma pigmentosum XRCC X-ray repair cross complementing group           xv ACKNOWLEDGEMENTS  I would like to thank my supervisors Drs. Chris Bajdik and Angie Brooks-Wilson for their guidance, support and nurturing scientific environment during my PhD study. I am highly benefited from the expertise from both sides. Many thanks to my supervisory committee Drs. Amil Shah, Greg Hislop, Nhu Le and Victor Ling for their support, helpful comments, advice and encouragements. I am also grateful to Ms. Rozmin Janoo-Gilani who provided expertise and contributed lab work. This dissertation could not be completed without the support of Drs. Sharlene Gill, John Hay, Christian Kollmannsberger, Kong Khoo, Sanjay Rao, Pamela Leco, Marianne Taylor, Alex Agranovich, Barb Melosky, Caroline Lohrisch, Lyly Le, Ursula Lee, Roy Ma, Delia Sauciuc, Ed Hardy, Paris Ingledew, Howard Joe, Howard Lim, Milton Po and other members of the BC Cancer Agency’s Gastrointestinal Tumour Group. Thank you to BC Cancer Registry particularly Sharon Tamaro and Sherry Reid for providing BC registry data and Dr. Parvin Yavari for providing Ardabil data. Thank you to Drs. John Spinelli, David Huntsman, Isabella Tai and Rick Gallagher for constructive scientific comments and advice. Many thanks to Ms. Amy English and Agnes Lai for providing help in patient recruitment. Thank you to all freinds at the Cancer Control Research Program BCCA and the Cancer Genetics group at the Canada's Michael Smith Genome Sciences Centre for their support. Finally, I would like to thank all patients participated in the “Molecular epidemiology of gastric and esophageal cancer survival” study. Financial support for my graduate work was provided by the Canadian Cancer Society (STU- 08-019764) and the Michael Smith Foundation for Health Research (MSFHR) with the BC   xvi Cancer Foundation (ST-SGS-00843(06-1)POP). I am also recipient of PhD Tuition Fee Award, AACR-Aflac Scholar-in-Training Award and Scholar-in-Training, AACR Scholar- in-Training Grant Supported by Susan G. Komen for the Cure®.                       xvii DEDICATION  To Maryam, Deniz and Doreen!   In the loving memory of my mom Khadijeh, my dad Samad Bashash, My grandmother Robabeh and my sister in law Adeleh!                      1 CHAPTER 1: Introduction 1.1 Background It is estimated that in the year 2010 more than 4,210 cases of gastric and esophageal cancers will be diagnosed in Canada and more than 3,220 will die of this disease 1. In spite of being infrequent in Canada, these cancers have significant contribution to cancer mortality. After lung, prostate and colorectal, this combination of cancer is the 4th cause of cancer death among men in Canada. The morbidity of these cancers makes them extremely important to general health and of interest to researchers. Increased knowledge about early detection, better treatments and influences on survival are essential. This dissertation concentrates on factors that might influence survival of these cancers. Gastric cancer Overall, gastric cancer incidence and mortality have fallen dramatically over the past 70 years2. Despite its recent decline, gastric cancer is the fourth most commonly-diagnosed cancer and the second leading cause of cancer-related death worldwide 2. In 2000, about 880,000 people globally were diagnosed with gastric cancer and approximately 650,000 died of the disease 3. Several different types of cancer can occur in the stomach. Adenocarcinoma, which starts in the glandular cells, is the most common histological type, accounting for 90-95% of all gastric malignancies. It can spread to nearby lymph nodes and other areas of the body, such as the liver, pancreas, colon, lung, and ovaries 4. The two main sites of gastric adenocarcinoma are cardia (proximal) and non-cardia (lower). Despite a decline in lower gastric cancers, proximal tumours have been increasing in incidence since the 1970s, especially among males in the Western countries 5. Gastric cardia tumours now account for nearly half of all stomach cancers among men in the US and UK 6. Risk factors for gastric cancers include: Helicobacter pylori infection7;   2 heredity, genetic and individual immunological factors; diet and lifestyle; tobacco smoking; obesity; ionizing radiation, pernicious anemia, blood type A, prior gastric surgery for benign conditions, and Epstein-Barr virus 3. Gastric cancer is also classified based on the Lauren classification8 into two major types: (1) intestinal and (2) diffuse gastric cancer (DGC). It has been shown that germline  mutations in the E-cadherin (CDH1) gene are the cause of hereditary diffuse gastric cancer (HDGC). It is estimated that about 40% of HDGC families may harbour the CDH1 mutation 9. The mutation’s penetrance is reported to be about  70% 10 11. Esophageal cancer 80% of the worldwide esophageal cancers occur in developing countries with high incidence areas including Asia, south-eastern Africa, eastern South America and some areas of Western Europe. It includes, most commonly, squamous cell carcinoma (SCC) in the upper and mid esophagus and adenocarcinoma in the lower esophagus. The incidence of SCC of the esophagus is decreasing in western countries, while that of gastroesophageal adenocarcinoma is increasing 2,12. What is disturbing (but also fascinating) about esophageal cancer, is the remarkable variation in its geographic incidence in different parts of the world. While the incidence of this disease in North America is about 5 to 10/100,000, the corresponding figure in some areas of Iran is more than 100/100,000. It is  often lethal, most commonly presenting in an advanced stage as a swallowing disorder (dysphagia) in elderly patients 13. Risk factors for SCC include: a chronic interaction between tobacco smoking or chewing and alcohol intake together with low intake of fresh fruit and vegetables, resulting in vitamin and mineral deficiencies; repeated intake of high- temperature drinks such as tea in China, Calvados in France, or maté in Brazil; chronic exposure to tobacco, opium pipe residue, betel quid; contaminants such as mycotoxins, nitrosamines in foods and drinks; rare conditions, such as tylosis, a genetic change leading to thickening of the skin (hyperkeratosis) of the palms and soles; chronic esophageal stasis, e.g. achalasia, peptic   3 strictures; Plummer–Vinson syndrome; and previous or concomitant head or neck squamous cell cancer 2,12. Until recently, most epidemiological and biological studies of esophageal cancer have focused on SCC. Prior to 1980, approximately 90% of cases of esophageal cancers were of squamous cell histology. During the past two decades, however, there has been a dramatic increase in the incidence of adenocarcinoma of both the esophagus and gastroesophageal junction (GEJ) in North America and Western Europe 14. In many western countries, adenocarcinoma is now more common than SCC 15. The first report of an esophageal adenocarcinoma is credited to White in 1898 16. A review of the literature in 1900 revealed only six cases, and at the time most physicians believed that these cancers represented extension of gastric tumours into the esophagus 16. Once a rare tumour, adenocarcinoma of the esophagus currently has higher incidence than SCC in America. The rate of increase of adenocarcinoma of the esophagus is outpacing the next closest cancer, melanoma, by nearly three times 17. The current average yearly rise in incidence in the United States exceeds 20% and, among white men, the incidence has increased >800% since the mid-1970s18. Risk factors for adenocarcinoma include: gastroesophageal reflux, Barrett’s esophagus, asthma medications, LES (lower esophageal sphincter)-relaxing medications, removal of the gallbladder (cholecystectomy), obesity and cigarette smoking 16. The anatomy of the esophagus is shown in Figure 1.1 19. The organ bridges three anatomic regions: the neck, thorax, and abdomen. The esophagus extends from the cricopharyngeus muscle to the gastroesophageal junction (GEJ). The cervical esophagus is defined as the ~3 cm portion between the cricopharyngeus and the thoracic inlet. The remainder of the esophagus is commonly divided into thirds. This includes the upper third (upper thoracic ~ 6 cm) extending from the thoracic inlet to the carina, the middle third (mid thoracic ~ 8 cm) extending from the   4 carina to the inferior pulmonary veins, and the lower esophagus (lower thoracic/abdominal ~ 8 cm) traversing the remaining distance into the abdomen to the gastroesophageal junction. Adenocarcinoma predominates in the lower esophagus19,20. The cardia is the portion of the stomach surrounding the cardioesophageal junction, or cardiac orifice (the opening of the esophagus into the stomach).  Figure 1.1 Anatomy of the esophagus with landmarks and recorded distance from the incisors used to divide the esophagus into topographic compartments.   5 1.2 Prognostic factors A prognostic factor is used to help define patients with high and low risks of death. Knowledge of prognostic factors helps us understand the natural history of cancer 21. Prognostic factors (predictors) could be classified into three broad groups: i) tumour-related prognostic factors, ii) host-related prognostic factors, and iii) environment-related prognostic factors 21-23. Tumour-related prognostic factors Tumour biology includes a large and ever growing field of research that includes molecular and biological prognostic factors. These factors predict the natural history of the disease as well as the likelihood of response to treatment 22. Molecular cancer research has generated information concerning the progression of adenocarcinoma of the lower esophagus and GEJ 24. Molecular prognostic factors are still not included in current clinical prognostic models, such as the TNM classification. The main reason for this is that molecular biological research is rapidly evolving and an astonishing number of biomarkers have been described, but sufficiently large studies regarding the prognostic value of each specific gene or protein are still lacking 24. The histological grade of a tumour refers to an evaluation of whether a tumour is malignant and how aggressive it is likely to be. There can be variation in grade within the same tumour, and the highest (ie, worst) grade is usually recorded for prognostic purposes20. According to grade classification: grade 1 tumours are well differentiated with similar characteristics as the original tissue, grade 2 are moderately well differentiated, grade 3 are poorly differentiated, and grade 4 are undifferentiated tumours, which cannot be recognized as having any characteristics of their tissue of origin.   6 Tumour pathology is crucial to the determination of prognosis in cancer. The Lauren classification is an independent prognostic factor in adenocarcinoma of the esophagus and GEJ; some studies show worse survival of diffuse-type 24. Stage is a fundamental prognostic factor related to the anatomical extent of disease 21. Current staging systems (American Joint Committee on Cancer [AJCC] and Union Internationale Contre le Cancer [UICC]), for gastric esophagus and adenocarcinoma of the esophagus and GEJ are TNM based 25,26. (T), or increasing depth of tumour invasion is associated with lymphatic involvement and is a known important independent prognostic parameter 20. Pathologic lymph node (pN) staging was evaluated according to the number and site of nodal metastasis. The number-based classification is based on criteria provided by the UICC and AJCC rules 25,26. Stage groupings are strong independent prognostic parameters, with a higher stage implying more advanced disease 25,26. Recently, adenocarcinoma of the lower esophagus and GEJ are staged following the same rules as esophageal cancer 25,26. Before that, the UICC suggests classifying adenocarcinoma of the GEJ as esophageal carcinoma if more than 50% of the tumour mass involves the esophagus and as gastric carcinoma if more than 50% involves the stomach 27,28.This obviously caused  problem for classification of GEJ tumours because of the borderline location. GEJ tumours were staged as esophageal cancer by some authors and as gastric cancer by others 20. For gastric cancer AJCC definedprimary tumour (T), regional lymph node (N) and distant metastasis (M) status is shown in    table 1.1 25,26. TNM staging group of gastric cancer based on AJCC guidelines is shown in table 1.2 25,26. For Esophageal cancer AJCC categorizes primary tumour (T), regional lymph node (N)  and distant metastasis(M) status as described in table 1.3 25,26. AJCC recommends separate staging systems for squamous cell carcinoma and   7 adenocarcinoma. In addition to the TNM classifications, for squamous cell carcinoma, the classification is subdivided based on the location of the original tumour as well as the grading of tumour. TNM staging of esophageal squamous cell carcinoma is  shown in table 1.425,26. For adenocarcinoma, AJCC uses the T, N, and M classifications, as well as the tumour grade. Adenocarcinomas of the GEJ are staged following the same rules as esophageal adenocarcinoma. TNM staging of esophageal and GEJ adenocarcinoma is shown in table 1.5:  In BC, the general treatment for esophageal cancer is surgery in stage I. 50% of esophageal cancer patient with stage II/III disease receive radiation (4500 cGy in 25 fractions) and chemotherapy (5-fluorouracil [5FU] + cisplatin) followed by surgery, followed by chemotherapy (5FU + cisplatin). The other 50% of patients first undergo surgery followed by radiation (4500 cGy in 25 fractions) and chemotherapy (5FU + cisplatin). 75% of Stage IV esophageal cancer patients recieve chemotherapy (5FU + cisplatin), the other 25% receives only symptomatic care. Generally, treatment for gastric cancer is surgery in stage I, II, III; chemotherapy (5FU or 5FU+cisplatin) in 65% of stage IV cases; and symptomatic care only in the other 35% of stage IV. Host-related prognostic factors Patient (or host-related) prognostic factors include inherent and demographic characteristics such as age, gender, and ethnicity. Other factors, such as performance status, comorbidity, and immune status, are also important. All of the host-related factors are also significantly impacted by constitutional genetic polymorphisms. Even the complex process of metastasis might be significantly impacted by host genetics 29. Ethnicity is a possible prognosis factor for cancer in upper GI other than GEJ 30.     8 Environment-related prognostic factors Environment-related prognostic factors are those that are external to the patient, such as choice of treatment, quality of treatment, access to care, health-care policy, and access to drugs or technology that may impact outcome 22. Treatment is likely the greatest determinant of cancer patients’ survival. These factors lend themselves to immediate modification in the interest of improved outcome. 1.3 Research objectives Our primary objective was to assess genetic polymorphism of TIMPs (1,2,3,4) and their prognostic effects in esophagus and GEJ adenocarcinomas in newly-diagnosed patients with esophageal and GEJ adenocarcinomas in BC. Our secondary objectives were to assess the prognostic effects of i) genetic polymorphisms in MMPs 2, 7, and 9; and ii) the environmental and healthcare variables, and iii) ethnicity. 1.4 The thesis sections Chapter 2 reviews genetic studies of gastric and esophageal cancer prognosis and prediction. Chapter 3 describes the incidence and survival for gastric and esophageal cancer in the population of BC, Canada between 1990 and 1999. Chapter 4 compares one-year survival of gastric and esophageal cancers between the populations of British Columbia (BC), Canada and Ardabil, Iran. Chapter 5 compares survival of gastric and esophageal cancer patients among Chinese, South Asian and Iranian and other ethnic groups in BC. Chapter 6 assesses genetic polymorphism of TIMPs and MMPs and their prognostic effects for esophagus and GEJ adenocarcinomas.   9 Table 1.1 Stage category definitions for gastric cancer 25,26. PRIMARY TUMOR (T) TX Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ: intraepithelial tumor without invasion of the lamina propria T1 Tumor invades lamina propria, muscularis mucosae, or submucosa T1a Tumor invades lamina propria or muscularis mucosae T1b Tumor invades submucosa T2 Tumor invades muscularis propria T3 Tumor penetrates subserosal connective tissue without invasion of visceral peritoneum or adjacent structures*,**,*** T4 Tumor invades serosa (visceral peritoneum) or adjacent structures**,*** T4a Tumor invades serosa (visceral peritoneum) T4b Tumor invades adjacent structures  *A tumor may penetrate the muscularis propria with extension into the gastrocolic or gastrohepatic ligaments, or into the greater or lesser omentum, without perforation of the visceral peritoneum covering these structures. In this case, the tumor is classified T3. If there is perforation of the visceral peritoneum covering the gastric ligaments or the omentum, the tumor should be classified T4. **The adjacent structures of the stomach include the spleen, transverse colon, liver, diaphragm, pancreas, abdominal wall, adrenal gland, kidney, small intestine, and retroperitoneum. ***Intramural extension to the duodenum or esophagus is classified by the depth of the greatest invasion in any of these sites, including the stomach. REGIONAL LYMPH NODES (N) NX Regional lymph node(s) cannot be assessed N0 No regional lymph node metastasis * N1 Metastasis in 1 to 2 regional lymph nodes N2 Metastasis in 3 to 6 regional lymph nodes N3 Metastasis in 7 or more regional lymph nodes  * A designation of pN0 should be used if all examined lymph nodes are negative, regardless of the total number removed and examined. DISTANT METASTASIS (M) M0 No distant metastasis M1 Distant metastasis    10     Table 1.2 Staging group of gastric cancer 25,26. Group T N M 0   Tis N0 M0  IA   T1 N0 M0  IB T2 N0 M0 T1 N1 M0  IIA T3 N0 M0 T2 N1 M0 T1 N2 M0  IIB T4a N0 M0 T3 N1 M0 T2 N2 M0 T1 N3 M0  IIIA T4a N1 M0 T3 N2 M0 T2 N3 M0  IIIB T4b N0 M0 T4b N1 M0 T4a N2 M0 T3 N3 M0  IIIC T4b N2 M0 T4b N3 M0 T4a N3 M0  IV   Any T Any N M1     11 Table 1.3 Stage category definitions for esophageal cancer25,26. PRIMARY TUMOR (T) TX Primary tumour cannot be assessed T0 No evidence of primary tumour Tis High-grade dysplasia * T1 Tumour invades lamina propria, muscularis mucosae, or submucosa T1a Tumour invades lamina propria or muscularis mucosae T1b Tumour invades submucosa T2 Tumour invades muscularis propria T3 Tumour invades adventitia T4 Tumour invades adjacent structures T4a Resectable tumour invading pleura, pericardium, or diaphragm T4b Unresectable tumour invading  *High-grade dysplasia includes all non-invasive neoplastic epithelium that was formerly called carcinoma in situ, a diagnosis that is no longer used for columnar mucosae anywhere in the gastrointestinal tract. REGIONAL LYMPH NODES (N) NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Regional lymph node metastases involving 1 to 2 nodes N2 Regional lymph node metastases involving 3 to 6 nodes N3 Regional lymph node metastases involving 7 or more nodes DISTANT METASTASIS (M) M0 No distant metastasis M1 Distant metastasis     12 Table 1.4 TNM staging of esophageal squamous cell carcinoma25,26. GROUP T N M Grade Tumour Location 0 Tis N0 M0 1 Any IA T1 N0 M0 1, X Any IB  T1 N0 M0 2-3 Any T2-3 N0 M0 1, X Lower, X IIA  T2-3 N0 M0 1, X Upper, middle T2-3 N0 M0 2-3 Lower, X IIB  T2-3 N0 M0 2-3 Upper, middle T1-2 N1 M0 Any Any IIIA   T1-2 N2 M0 Any Any T3 N1 M0 Any Any T4a N0 M0 Any Any IIIB T3 N2 M0 Any Any IIIC   T4a N1-2 M0 Any Any T4b Any M0 Any Any Any N3 M0 Any Any IV Any Any M1 Any Any        13 Table 1.5 TNM staging of esophageal and GEJ adenocarcinoma25,26.   GROUP  T    N    M    Grade  0    Tis   N0    M0    1, X  IA    T1    N0    M0    1-2, X  IB   T1    N0    M0    3  T2    N0    M0    1-2, X  IIA    T2    N0    M0    3  IIB   T3    N0    M0    Any  T1-2    N1    M0    Any  IIIA    T1-2    N2    M0    Any  T3    N1    M0    Any  T4a    N0    M0    Any  IIIB    T3    N2    M0    Any  IIIC    T4a    N1-2    M0    Any  T4b    Any    M0    Any  Any    N3    M0    Any  IV    Any    Any    M1    Any      14  CHAPTER 2: Review:  Genetic variants associated with esophageal and gastric cancer survival1 2.1 Introduction Esophageal and gastric cancers are among the most aggressive and deadliest malignancies in the world. Esophageal cancer is the eighth most common cancer worldwide, and the sixth most common cause of death from cancer 2. Gastric cancer is the fourth most common cancer worldwide, and the second most common cause of death from cancer 2. During 2009, it is estimated that 16,470 cases of esophageal cancer and 21,130 cases of gastric cancer were diagnosed in the US, and more than 25,000 people died because of these cancers 31. Prognostic factors estimate the risk of various outcomes based on  clinical and non-clinical characteristics 32, but do not consider  treatment. In contrast, predictive markers consider which patients may benefit from a specific treatment 33 and classify populations into groups for which different treatment options can be recommended. The majority of prognostic factors in cancer are directly related to the tumour 22,33, including tumour stage, tumour size and, in some cases, other markers related to the presence of a tumour. Molecular research has generated an immense amount of information about the progression of cancers, including gastric and esophageal cancers 24, but molecular markers are not yet routinely included in prognostic models. Other variables affecting the  outcome include host-related and environment-related factors 34. Environment-related factors are external to the patient, such as choice of treatment, quality of treatment, access to care, health-care policy and access to drugs or technology 22. Host-related  1 A version of this chapter has been submitted for publication and is under review. Bashash M, Shah A, Hislop G, Le N, Bajdik C, Brooks-Willson A. (Submitted  June 19, 2010 )   15 prognostic factors include inherent and demographic characteristics such as age, gender and ethnicity. Other factors such as performance status, co-morbidity and immune status are also important 22. Many host factors are inherited and likely to be, at least in part, due to genetic variants 35. Some of these factors directly affect tumour cell growth and may modulate the host immune response to allow tumour growth 36. For example, genes influence the risk of progression and/or metastasis from cancer (permissive metastasis genotypes), and similar cancers in different patients may exhibit different tendencies to metastasize 29. Genetic studies of human cancer susceptibility determine alleles associated with the incidence of disease. Genetic studies of human cancer prognosis try to determine alleles associated with the outcomes of disease. This article reviews genetic studies of esophageal and gastric cancer prognosis. 2.2 Material and methods Using PubMed, MEDLINE has been searched for the keywords “gastric cancer”, “esophageal cancer”, “survival”, “outcome”,  “polymorphism”, “SNP” and “genetic”. Searches were limited to English language articles on human studies published between 1957 and 2009. Abstracts of articles were reviewed and those that were only related to disease incidence excluded. Studies of mutations in tumour DNA were also excluded, as were studies that did not consider genetic variants in any way. Genetic polymorphisms were divided into two classes: single nucleotide polymorphisms (SNPs) and structural variants 35. The dbSNP database was used to standardize variation in genetic names 37.   16 2.3 Results The literature search identified 36 articles that examined 129 polymorphisms in 63 genes for their effect on esophageal and gastric cancer survival. These included 20 articles on gastric cancer, 14 on esophageal cancer and two regarding the gastroesophageal junction. Of 14 articles about esophageal cancer, 5 specifically studied adenocarcinoma 38,39 and 2 studied squamous cell carcinoma 40 41. A summary of the esophageal cancer studies is given in Table 2.1; a summary of the gastric cancer studies is given in Table 2.2. Note that one study considered esophageal and gastric cancer patients combined 42and is included in both Tables 2.1 and 2. The gastric cancer articles included studies of between 44 43 and 503 44 cases. For esophageal cancer, the corresponding numbers of cases were 39 38 and 371 45. Blood was the source of DNA in 10 esophageal cancer studies 40,42,45-52 and 14 gastric cancer studies 43,44,48,53-63; the other studies used normal tissue from tumour samples (blocks, slides or fresh tissue). Most of the articles reported on North American studies 38-40,42,45,46,49,64-68 although 6 articles were about German populations 41,47,51,63,69,70 and 5 were about Chinese populations 43,55-57,61. All of the samples were collected as part of routine clinical procedures and some patients already had received adjuvant and neoadjuvant treatment 43,48,51,55-57,63,70. Results from these studies indicate that 6 polymorphisms in 5 genes were consistently associated with esophageal cancer prognosis, 7 polymorphisms in 7 genes showed an association in some but not all studies, and 55 polymorphisms in 41 genes did not show any effects. For gastric cancer, 9 polymorphisms in 8 genes showed a prognostic effect, 10 polymorphisms in 7 genes showed discordant results and 39 polymorphisms in 21 genes did not influence gastric cancer patients’ survival. Finally, 8 polymorphisms in 8 genes had prognostic effects on both gastric and   17 esophageal tumours (Figure 2.1). This includes 3 genetic polymorphisms in 3 genes for esophageal and stomach cancer combined. These polymorphisms can be categorised according to the genetic pathways in which they are involved. Cytokines Cytokines are small intercellular signalling peptides that act within the autocrine, paracrine or endocrine system to promote growth, differentiation and activation of cells. The anti- inflammatory activity and immunosuppressive activity of cytokines depends on the microenvironment 71. Cytokines that are predominantly produced by monocytes include tumour necrosis factor (TNF) and interleukin (IL) molecules IL1, IL6, IL8, IL12, IL15, IL18 and IL23 71. Genetic polymorphisms of cytokines have been shown to be important in cancer. In a review of 161 meta-analyses and pooled analyses of candidate SNPs in 99 genes, nearly one-third (98/344) of variants were significantly associated with various cancers, including 6 cytokine gene variants 72. Among patients with advanced cancer, increased expression of pro- inflammatory cytokines also are associated with anorexia and cachexia, pain, toxicity and resistance to treatment 72. Tumour necrosis factor-α (TNFα) is a highly multifunctional cytokine involved in immune and inflammatory responses and affecting angiogenesis and tumour growth 73. Deans et. al. conducted a study of 203 gastroesophageal (i.e., gastric, esophageal or gastroesophageal junction) cancer patients showing that interaction of three genotypes (IL6 −174 G/C, IL10 -1082A/G and TNFα -308G/A) resulted in a cumulative reduction in survival 42. In a small study of 44 gastric and gastroesophageal junction cancer patients, Jatoi et al. examined IL1β and found a survival benefit for patients with +3954C/T or +3954T/T genotypes (hazard ratio (HR) 0.3; p=0.04) compared to patients with the C/C genotype 66. In a study of 123 patients   18 with relapsed or metastatic gastric cancer, Graziano et al. reported that short tandem repeat (microsatellite) polymorphism (STR) of IL1RN, IL1β -511C/T and IL1β -31T/C polymorphisms influence prognosis 74. In an earlier study, Shimura et al. examined an NcoI restriction fragment length polymorphism of the tumour necrosis factor-β (TNFβ) gene among gastric cancer patients and reported better 3-year survival among patients homozygous for the 10.5 kb fragment (87.1% 3-year survival) compared to other alleles (5.5 kb homozygote, 52.5% 3-year survival; heterozygote, 79.1% 3-year survival) 75. There was no prognostic significance of IL1β -31T/C, IL1β -511C/T or IL1RN STR 66, IL1β -511C/T, LTα +252 42, IL8 -251, IL1β -511, IL1RN or TNFα -857 62 on gastric and esophageal cancer patients. Matrix Metalloproteinases (MMPs) and inhibitors MMPs contribute to multiple steps of tumour progression including invasion, promotion, angiogenesis, and the establishment and growth of metastatic lesions in distant organ sites 76. It is recognized that MMPs can be synthesized by tumour cells, but are frequently produced by surrounding stromal cells including fibroblasts and infiltrating inflammatory cells 76. Finally, MMPs solubilize cell surface and matrix-bound factors that can influence cellular properties such as growth, death, migration and metastasis 76. An important mechanism for the regulation of MMP activity is binding to a family of homologous proteins referred to as the tissue inhibitors of metalloproteinases (TIMPs). These are natural inhibitors of MMP proteolytic activity in vitro as well as in vivo 77. Tang et al. conducted a study of 74 patients in China showing an association between the MMP9 Gln279Arg-Pro574Arg haplotype and 1 year postoperative mortality of gastric cancer patients 52. Kubben et al. showed association of MMP7 -181A/G and TIMP2 303C/T with poor outcome in patients from the Netherlands 78. In contrast, a recent study on 313 esophageal cancer patients in the U.S did not find any association between polymorphisms of MMP1, MMP3 and MMP12 and outcome 45. There were no significant associations between   19 MMP2 -1306C/T, MMP7 -153C/T, MMP8 -799C/T, MMP8 +17C/G, MMP9 -1562C/T, TIMP1 372C/T and TIMP2 -418G/C  78, MMP9 R279Q 52 polymorphisms and survival of gastric cancer patients. Xenobiotic metabolism Glutathione-S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to a wide variety of xenobiotics 79. Glutathiones play a role in detoxifying, and consequently protecting cells from alkylating agents and products of reactive oxidation. GSTP1 is known to detoxify platinum compounds including oxaliplatin 80 and cisplatin 79. Polymorphisms of these genes are well studied in the literature, however, only a few studies have found significant results for GST gene variants and survival 57 69 51 59. Huang  et al. conducted a study of 102 gastric cancer patients who were treated with oxaliplatin-based adjuvant chemotherapy, and showed an association between GSTP1 Ile105 Ile  genoytpe and survival 57. Goekkurt et al., in a study of 52 gastric cancer patients who received at least one complete cycle of 5FU/cisplatin/FA as first-line chemotherapy, showed that patients possessing the GSTP1 Val105 Val genotype demonstrated a significantly superior median survival time of 15.0 months (95% CI 7.8-22.0) compared to 6.0 months (95% CI 5.1-7.0) in patients with at least one GSTP1 105 Ile allele 69. In a study of 110 patients with locally advanced gastric carcinomas who received preoperative chemotherapy, Ott et al. examined the effect of GST polymorphism on outcome and found improved survival for patients with the GSTM1- (non- conjugators) genotype compared to patients with the GSTM1 null 51. Finally, Lee et al. conducted a study of 233 patients with esophageal cancer and showed that patients carrying GSTP1 105 Val variants had poorer survival 59. In these and other studies there were no statistically significant associations between survival and GSTP1/rs1138272, GSTP1/rs1695, GSTP1 Ile105Val, GSTP1 Ala114Val, GSTT1 +/- or GSTM1 +/-  polymorphisms 51 57 68 61 69 70 59 57.   20 A polymorphism of the thymidylate synthase (TYMS) gene was one of the most studied variants for prognostic and predictive effect on gastric and esophageal cancer. TYMS is the main target of 5FU 80, a major chemotherapeutic agent for gastric and esophageal cancers. One of the most highly studied polymorphisms is a tandem repeat, of which TSER*2 and TSER*3 are the most common alleles 80. Huang et al. conducted a study of 116 patients with gastric cancer treated with 5FU-based adjuvant chemotherapy and reported significantly better survival among patients with TYMS 3′UTR (1494del6)  ins6/ins6 compared with patients with the del6/del6 or heterozygous genotypes 57. Keam et al. conducted a study of 73 patients with metastatic or relapsed gastric adenocarcinoma who were enrolled in a prospective phase II clinical trial. The study reported that the del6/del6 variant in TYMS 3'UTR was significantly associated with prolonged overall survival 48. In the same and other studies, the TYMS variants rs2790, rs699517 70 68 57 39, TYMS  tandem repeat polymorphisms, rs45445694 41 81 57 39 and TYMS 3’UTR del6/del6 67 69 57 39 did not show significant prognostic effects. Multidrug resistance (MDR) describes the phenomenon of simultaneous resistance to unrelated drugs, possibly because of reduced drug accumulation involving the P-glycoprotein (Pgp; mdr1 gene) 82. Wu et al. studied four MDR variants (3435C/T, Ala892Ser, Ala892Ser, 3425C/T) of the MDR1 gene and reported a significant association of the MDR1 3435C/T variant allele with reduced recurrence risk among esophageal cancer patients receiving platinum-based drugs 68. 5, 10-methylenetetrahydrofolate reductase (MTHFR) is a key enzyme for intracellular folate homeostasis and metabolism. Because activity of 5FU is dependent on a competitive interaction with folate metabolism, the MTHFR polymorphism has been suggested to have an effect on 5FU-based chemotherapies 57. Wu et al. examined the effect of polymorphism of this gene among esophageal cancer patients and concluded that MTHFR 677C/T (Ala222Val, rs1801131)   21 and 1298A/C (rs1801131) SNPs influence disease recurrence and survival; and patients with variant alleles at both loci had a significantly reduced recurrence risk and better survival 68. Huang et al. also reported that MTHFR 677CC genotype was associated with shorter overall survival among gastric cancer patients 57. Cell cycle Alteration of the p53 gene is found in about half of human cancers, and most other cancers deactivate the p53 pathway by increasing its inhibitors, reducing its activators or inactivating its downstream targets 83. p53 is best characterized as a transcription factor that binds to specific DNA sequences and transactivates a number of genes with a variety of functions including cell cycle arrest, apoptosis and metabolism 83. As a result, this gene might be prognostic for gastric and esophageal cancer patients. Huang et al. conducted studies on 110 gastric cancer patients treated with 5FU-based adjuvant chemotherapy 43 and 102 with oxaliplatin-based adjuvant chemotherapy 57, showing a roughly two-fold prognostic decrease for patients carrying p53 codon Pro72Pro. The same result has been shown by Cescon et al. in a recent study of 371 esophageal carcinoma patients 45. Other studies failed to show significant prognostic effects of p53 +62 A/G(rs1625895),  p53 Pro72Arg 68 or other p53 variants (rs1788332, rs1042522, rs1625895 or rs1801173/rs2273953) 53 among gastric and esophageal cancer patients. The p53 binding protein homolog 2 (MDM2) is a key element in the activation of p53. In two relatively large studies, Ohmiya et al. and Cescon et al. studied the prognostic effect of MDM2. In one, analysis of 410 gastric cancer patients showed that MDM2 309G/G is an independent marker of poor overall survival in advanced carcinomas 62. In the other study, of 371 patients with esophageal carcinoma, MDM2 309T/G was associated with markedly reduced survival in squamous cell carcinoma 45.   22 The cyclin D1 proto-oncogene is a powerful control element that regulates the mitotic cell cycle, and excessive cyclin D1 expression and/or activity is common in human cancers. Polymorphism of cyclin D1 is also suggested to influence cancer risk and outcome in general 84. In a study of 124 esophageal cancer patients, Izzo et al. observed polymorphism of cyclin D1 870G/A was statistically associated with overall survival 65. However, in a smaller study of 69 esophageal squamous carcinomas patients, no significant association of this polymorphism was observed 40. The esophageal cancer-related gene 2 (ECRG2) has been shown to be related to cell proliferation and induction of apoptosis in esophageal cancer cells 85. Short tandem repeat polymorphism of ECRG2 have been studied in 86 patients with primary esophageal cancer, showing that the ECRG2 TCA3/TCA3 genotype was the factor most strongly associated with negative outcome 85. DNA repair genes Cancer cells are often defective in a DNA repair pathways such as  mismatch repair, base excision repair, nucleotide excision repair, homologous recombination, nonhomologous end- joining and trans-lesion synthesis 86. Polymorphisms in OGG1, XRCC1, ERCC1, XPC, XPD, XPF, BRCA2 and XRCC3 have been shown to affect other cancer risks outcomes 87. Wu et al. conducted a study of genetic polymorphisms on clinical outcomes in 210 esophageal cancer patients and reported that variant alleles of XRCC1 Gln399Arg were significantly associated with poor survival 68. In another study of 62 gastric cancer patients, Liu et al. determined that patients with the XRCC1 Gln399Arg genotype demonstrated a significant worse survival 61. Other studies of ERCC1/rs3212986, ERCC1/rs11615, MGMT/rs12917, XRCC1/rs25487, XRCC1/rs1799782, XRCC3/rs861539 70, ERCC1 3'UTR, ERCC6 Met1097Val, ERCC6 Arg1230Pro, APEX1 Asp148Glu, XRCC1 Gln399Arg 68, ERCC1 Asn118Asn, ERCC1 8092C/A 48, ERCC1 118C/T,   23 55,57, ERCC1 C118C/T ERCC2 Gln751Lys 69 polymorphisms did not find significant associations with survival. Signalling pathways and growth factor genes Signalling through the PI3K/PTEN/AKT/mTOR pathway is responsible for balancing cell survival and apoptosis 64. The signal is initiated by growth factors and hormones that bind receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) 64. Hildebrandt et al. studied polymorphisms of the PI3K/PTEN/AKT/mTOR pathway in 210 esophageal cancer cases who received chemotherapy. They described an association of mTOR pathway polymorphisms FRAP1/rs11121704 and FRAP1/rs2295080 on the overall study group and AKT1/rs1130214 and AKT2/rs892119 on patients who received taxane 64. Jain et al. conducted a study of 69 patients and reported that EGF 61A/G genotype independently influenced survival in squamous cell esophageal cancer 40. In a recent study Bradbury et al. reported the variant allele of VEGF 936C/T was associated with improved overall survival compared with the wild type allele among esophageal cancer patients 88. Kim et al. examined 503 gastric cancer patients for polymorphism of VEGF and reported that the +936C/T variant TT allele was associated with worse overall survival compared to the C/C allele 89. Other studies did not show any association between  AKT1/rs4375597 70, AKT1/rs3803304, AKT1/rs2498804, AKT1/rs2494738, AKT2/rs8100018, PIK3CA/rs7651265, PIK3CA/rs7640662, PIK3CA/rs7621329, PIK3CA/rs6443624 64, VEGF -460T/C, VEGF 405G/C 88 polymorphisms and survival. 2.4 Discussion Gastric and esophageal cancer are among the most deadly of all gastrointestinal malignancies worldwide, with 5-year mortality rates exceeding 80% 90. This review examined 36 articles in   24 which prognostic effects of 62 genes were studied and among them, 31 polymorphisms affected gastric and esophageal cancer survival. In general, the literature review identified some recurring problems with published studies. First, most studies concentrated on a group of gastric and esophageal cancer patients that was not population-based. Second, most studies described patients who received a specific treatment and, as a result, reported prognostic effects might  be predictive. Third, the small number of cases in most studies limits statistical power. Fourth, most studies did not discriminate between cardia and lower gastric cancers, and some studies did not discriminate between squamous cell carcinoma and adenocarcinoma of the esophagus. Two large studies considered all gastric, esophageal and gastroesophageal junction cancer cases as a single group. Finally, several different kinds of biological samples were used in these studies and therefore comparisons between the studies might not be optimal. Generally, most of the papers summarized here examined only one or a very small number of polymorphisms in each gene. This candidate polymorphism strategy fails to examine the bulk of the genetic variation. Newer studies examine genes more comprehensively using haplotype tags based on HapMap data and provide more meaningful results. For single SNP studies, a negative result can mean that the SNP examined is not involved in disease or outcome, but the gene could still have an effect. No genome-wide association studies (GWAS) of survival in gastric and esophageal cancer have been reported. 2.5 Conclusion This review shows that genetic polymorphisms in cell cycle (Cyclin D1, ECRG2, MDM2 and p53), DNA repair (XRCC1), signalling and growth factors (AKT1, AKT2, EGF, FRAP1, and VEGF) and xenobiotic metabolism (MDR1, GSTP1, and MTHFR) are associated with esophageal cancer survival. Similarly, genetic polymorphisms in cell cycle (p53, MDM2),   25 cytokines (IL1β, IL1RN, and TNFβ), DNA repair (XRCC1), matrix metalloproteinases and their inhibitors (MMP7, MMP9 and TIMP2), signalling and growth factor genes (VEGF) and xenobiotic metabolism (TYMS, GSTM1, GSTP1 and MTHFR) had prognostic value for gastric cancer. Genetic polymorphisms in the cytokines IL10, IL1β, IL6 and TNFα were significant in gastric and esophageal cancer patients combined (Figure 2.1). Our results indicate that polymorphisms in genes associated with cell cycle, xenobiotic metabolism, DNA repair and signalling and growth factors have prognostic significance for both gastric and esophageal cancer. Understanding the mechanism of each polymorphism, and pathway-based analyses, might help identify markers for gastric and esophageal cancer survival. There is an increasing interest in the effect of host genetic polymorphisms on the survival of cancer patients. Conventional techniques generally do not adequately predict the heterogeneity of patient outcomes. In many cancers, tumour markers have been used as a factor for survival models and guiding treatment decisions. However in gastric and esophageal cancer, adequate tumour samples for these assays may not be easily available. Modeling prognosis based on host factors including genetic polymorphisms is an emerging field of translational research. Compared to tumour, constitutional genetic material is relatively easy to obtain, and can be assessed before treatment is started. To definitively evaluate prognostic biomarkers, however, a large sample is required. A consortium of research groups with a large numbers of samples would allow the optimal detection of predictive and prognostic effects. 26   1conjugators vs non- conjugators,*gastric and esophageal cancer combined, ** adenocarcinoma only, *** squamous cell carcinoma only  Figure 2.1: Genetic polymorphism associated with esophageal and gastric cancer survival  27   Table 2.1: Studies of prognostic significance for genetic polymorphisms in esophageal cancer patients.   Gene Polymorphism1 SNP ID Risk allele Effect2 Study population3 Reference  VEGF -460T/C rs833061 361 esophageal carcinoma patients (293 adenocarcinoma, 56 SCC, 12 poorly differentiated/other) enrolled during 1999-2004, Boston, USA Bradbury et al., 200988 VEGF 405G/C rs2010963 VEGF 936C/T rs3025039  OS HR=0.70 (95% CI: 0.49-0.99) p53 Pro72Arg rs1042522 Pro/Pro OS HR=2.05 (95% Cl: 1.30-3.24) 371 esophageal carcinoma patients (300 adenocarcinoma, 63 SCC, 8 poorly differentiated/other) enrolled during 1999-2004, Boston, USA Cescon et al., 200945 MDM2 309T/G rs2279744 GG OS HR=7.9 (95% Cl: 2.4-26.0) in SCC AKT1 SNP rs4375597 52 patients (21 adenocarcinoma, 31 SCC) with locally advanced resectable esophageal cancer (cT2–4, Nx, M0) from a prospective neoadjuvant trial, Cologne, Germany  Warnecke- Eberz et al., 200970 C-ERBB-2 Ile655Val rs1801200 ERCC1 8092C/A rs3212986 ERCC1 Asn118Asn rs11615 CC response to neoadjuvant therapy FGFR4 Gly388Arg rs351855 GSTP1 Ala114Val rs1138272 GSTP1 Ile105Val rs1695 MDR1 3435C/T rs1045642 MGMT 16286C/T rs12917 MTHFR Glu429Ala rs1801131 TERT SNP rs6882077 TYMS 227A/G rs2790 TYMS 157C/T rs699517 XRCC1 Gln399Arg rs25487 XRCC1 Arg194Trp rs1799782 AA response to neoadjuvant therapy XRCC3 18067C/T rs861539 MMP1 1G/2G rs1799750 313 esophageal adenocarcinoma cases, Boston, MA Bradbury et al., 200946 MMP3 6A/5A rs3025058 MMP12 –82A/G rs2276109 MMP12 1082A/G rs652438 AKT1 SNP rs3803304 174 patients with resectable adenocarcinoma and 36 squamous cell carcinoma patients, Houston, TX Hildebrandt et al., 200964 AKT1 SNP rs2498804 AKT1 SNP rs2494738 AKT1 SNP rs1130214 TT OS HR=8.92 (95% CI: 1.56-51.17) in taxane-treated patients AKT2 SNP rs892119 AG+GG OS HR=3.5 (95% 28  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population3 Reference  CI: 1.43-8.78) in taxane-treated patients AKT2 SNP rs8100018 FRAP1 SNP rs11121704 TT OS HR=3.53 (95% CI: 1.48-8.39) FRAP1 SNP rs2295080 TT OS HR=4.19 (95% CI: 1.83-9.61) PIK3CA SNP rs7651265 PIK3CA SNP rs7640662 PIK3CA SNP rs7621329 PIK3CA SNP rs6443624 Cyclin D1 870G/A rs17852153   39 esophageal adenocarcinoma cases, Pittsburgh, PA Gupta et al., 200838 EGF 61A/G rs4444903   312 esophageal adenocarcinoma cases, Boston, MA Lanuti et al., 200849 TYMS STR rs34743033   82 esophageal adenocarcinoma patients who underwent esophagectomy, Los Angles, CA Kuramochi et al., 200839 BCL2 Ala43Thr rs1800477 69 esophageal squamous carcinoma patients, Lucknow, India Jain et al., 200740 FAS -670A/G rs1800682 Cyclin D1 870G/A rs17852153 EGF 61A/G rs4444903 61GG OS HR=31.1 (95% CI: 4.1-224.3) EGFR Arg497Lys rs11543848 ECRG2 STR  TCA3/TCA3 OS RR=2.56 (95% CI: 1.53–4.29) 86 patients (48 SCC, 38 adenocarcinoma), Hamburg-Germany Kaifi et al., 200785 Cyclin D1 870G/A rs17852153 870 AA OS HR=3.48 (95% CI: 1.94-6.23) 124 esophageal adenocarcinoma patients, Houston, TX Izzo et al., 200765 MTHFR 677C/T rs1801133 68 patients with locally advanced esophageal (SCC) cancer, Essen, Germany Sarbia et al., 200641 MTR Asp919Gly rs1805087   TYMS STR rs34743033 GSTP1 Ile105Val rs1695 Val/Val, lle/Val OS HR=1.36 (95% CI: 1.01-1.84) 233 patients with esophageal cancer (200 SCC, 20 adenocarcinoma, 10 other), Taipei, Taiwan Lee et al., 200559 GSTT1 GSTT1+/- GSTM1 GSTM1+/- MTHFR Glu429Ala rs1801131 Glu429Ala OS HR=0.56 (95% CI: 0.35-0.89) 174 adenocarcinoma and 39 SCC esophageal cancer patients, Houston, TX Wu et al., 200668 MTHFR 1298A/C rs1801133 MTR Asp919Gly rs1805087 TYMS 157C/T rs699517 TYMS 227A/G rs2790 MDR1 3435C/T rs1045642 3435CT OS HR=1.92 (95% CI: 0.23-0.85) MDR1 Ala892Ser rs2032582 GSTP1 Ile105Val rs1695 29  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population3 Reference  GSTP1 Ala114Val rs1138272 MPO -764T/C rs2243828 P53 62A/G rs1625895 P53 Pro72Arg rs1042522 FAS -670A/G rs7089946 FasL -844C/T rs763110 NQO1 Pro187Ser rs1800566 XPA 23A/G rs1800975 XPC Lys939Gln rs2228001 XPD (ERCC2) Lys751Gln rs13181 XPG (ERCC5) Asp1104His rs17655 ERCC1 8092C/A rs3212986 ERCC6 Met1097Val rs2228526 ERCC6 Arg1230Pro rs4253211 CCNH Val270Ala rs2230641 RAD23B Ala249Val rs1805329 hOGG1 Ser326Cys rs1052133 APEX1 Asp148Glu rs1130409 ADPRT Val762Ala rs1136410 XRCC1 Gln399Arg rs25487 Gln399Arg OS HR=1.92 (95% CI: 1.00-3.72) IL1β −511T/C rs16944 91 esophageal, 37 gastroesophageal junction and 75 gastric cancer patients, UK Deans et al., 200742 IL6 −174 G/C rs1800795 CC compared with GG or GC median survival 256 vs. 431 days IL10 -1082A/G rs1800896 GG compared with AA/AG median survival 310 vs. 389 days TNFα -308G/A rs1800629 AA OS HR=2.5 (95% CI: 1.3–4.9) LTa 252A/G rs909253  1 SNP is an unspecified Single Nucleotide Polymorphism; STR is short tandem repeat (microsatellite) polymorphism; +/- is conjugators vs. non-conjugators 2 HR is hazard ratio; CI is confidence interval; RR is relative risk; OR is odds ratio; OS is overall survival; PFS is progression free survival 3 SCC is squamous cell carcinoma      30  Table 2.2:  Studies of prognostic significance of genetic polymorphisms gastric cancer patients.  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population2 Reference MMP9 Gln279Arg rs17576 Haplotype double homozygotes OR=6.5 (95% CI: 1.18-35.7) for 1 year survival 74 patients with gastric carcinoma, Fuzhou, China Tang et al., 200852 MMP9 Pro574Arg rs2250889 GSTT1 GSTT1+/- 110 patients with locally advanced gastric carcinomas who received preoperative chemotherapy, Heidelberg, Germany Ott et al., 200851 GSTM1 GSTM1+/-  GSTM1+ RR=0.32 (95% CI: 0.12–0.86) for completely resected patients GSTP1 Ile105Val rs1695 IL6 -634C/G rs1800796 194 stages II and III gastric adenocarcinoma patients, Taichung, Taiwan Liao et al., 200850 IL6 −174G/C rs1800795 TYMS STR rs34743033 73 advanced gastric cancer patients administered a modified FOLFOX-6 regimen Seoul, South Korea Keam et al., 200848 TYMS 1494del6 rs34489327 6-bp deletion in 3'UTR OS HR=0.55 (95% CI: 0.29–1.07)  GSTP1 Ala114Val rs1138272 ERCC1 Asn118Asn rs11615 ERCC1 8092C/A rs3212986 XPD (ERCC2) Arg156Arg rs238406 XPD (ERCC2) Asp312Asn rs1799793 XPD (ERCC2) Lys751Gln rs13181 XRCC Gln399Arg rs25487 Pro/Pro OS HR=2.58 (95% CI: 1.05–6.33) p53 Arg72Pro rs1042522   110 gastric cancer who were treated with 5FU-based adjuvant chemotherapy, Jiangsu, China Huang et al., 200843 TYMS STR rs34743033 116 patients with gastric cancer who were treated with 5FU-based adjuvant chemotherapy, Jiangsu, China Huang et al., 200956 TYMS 1494del6 ins6/ins6 vs. del6/del6 and ins6/del6 OS HR=2.44 (95% CI: 1.04–5.72) 31  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population2 Reference MTHFR C677C/T rs1801133 TT and CT vs. CC OS HR=1.68 (95% CI: 0.99–2.86) p53 Pro72Arg rs1042522 Pro/Pro RFS HR=2.48 (95% CI: 1.44–4.27) 102 patients with gastric cancer treated with oxaliplatin-based adjuvant chemotherapy, Jiangsu, China Huang et al., 200957 OS HR=2.0 (95% CI: 1.11–3.64) ERCC1 Asn118Asn rs11615 GSTP1 Ile105Val rs1695 Ile/Ile RFS HR=2.00 (95% CI: 1.15–3.48) OS HR=2.13 (95% CI: 1.14–4.00) GSTM1 GSTM1+/- XRCC1 Gln399Arg rs25487 Gln/Gln and Arg/Gln OS HR=2.13 (95% CI: 1.14–4.00) p53 SNP rs1788332 115 gastric cancer patients who underwent curative gastrectomy, Rome, Italy De Feo et al., 200953 p53 Pro72Arg rs1042522 p53 62A/G rs1625895 p73 14C/T rs1801173 p73 -81C/T rs2273953 RANTES -403G/A rs 2107538 177 gastric cancer patients, Taiwan Liou et al., 200860 RANTES -28G/C rs2280788 CCR2 Val64Ile rs1799864 ERCC1 Asn118Asn rs11615   82 patients with gastric cancer treated with oxaliplatin-based adjuvant chemotherapy, Jiangsu, China Huang et al., 200855 IL1β −31C/T rs1143627 44 gastric and gastroesophageal junction adenocarcinoma patients collected by North Central Cancer Treatment Group (NCCTG), Mayo clinic, Rochester, MN Jatoi et al., 200766 IL1β −511T/C rs16944 IL1β +3954C/T rs1143634 CT and TT compared with CC OS HR=0.3; P=0.04 IL1RN STR rs2234663   32  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population2 Reference XPD (ERCC2) Lys751Gln rs13181 62 gastric adenocarcinoma patients, Nanjing, China Liu et al., 200761 GSTP1 Ile105Val rs1695 XRCC1 Gln399Arg rs25487 G allele median survival 337 days vs. 370 days (p=0.03) VEGF VEGF -460T/C rs833061   503 gastric cancer patients who underwent surgical gastrectomy, Kim et al., 200789 VEGF –116G/A    Daegu, Korea VEGF VEGF 405G/C rs2010963 VEGF 936C/T rs3025039 TT OS HR=3.23 (95% CI: 1.13–9.25) NAT2 Arg64Gln rs1801279 100 gastric cancer patients, Oman Al- Moundhri et al., 200744 NAT2 282C/T rs1041983 NAT2 Ile114Thr rs1801280 NAT2 481C/T rs1799929 NAT2 Arg197Gln rs1799930 NAT2 Arg268Lys rs1208 NAT2 Gly286Glu rs1799931 MTHFR SNP rs1801133 135 gastric cancer patients that received preoperative chemotherapy, Munich, Germany Ott et al., 200663 TYMS STR rs34743033 3rpt/3rpt OS HR=4.57 (95% CI: 1.88-11.14) MDM2 MDM2 T309G rs2279744 GG OS HR=3.16 (95% CI: 1.22-8.20) in stage IB-IV disease 410 gastric cancer patients, Nagoya, Japan Ohmiya et al., 200662 IL8 IL8 −251A/T rs4073 IL1β −511T/C rs16944 IL1RN STR rs2234663 TNFα SNP rs1799724 TYMS 1494del6 rs34489327   146 Caucasian patients with adenocarcinoma of the esophagus, Houston, TX Liao et al., 200667   33  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population2 Reference MMP2 -1306C/T rs243865 79 patients underwent resection for primary gastric adenocarcinoma Leiden, The Netherlands Kubben et al., 200658 MMP7 -181A/G rs11568818 AA vs. AG/GG OS HR=1.72 (95% CI: 0.97–3.06) MMP7 -153C/T rs12184413 MMP8 -799C/T rs11225395 MMP8 +17C/G MMP9 -1562C/T rs3918242 TIMP1 372C/T rs4898 TIMP2 303C/T rs2277698 CC vs. CT/TT OS HR=3.22 (95% CI: 1.57–6.62) TIMP2 -418G/C rs2277698 GSTP1 Ile105Val rs1695 Val/Val median survival 15.0 months (95% CI: 7.8- 22.0) vs.  6.0 months (95% CI: 5.1-7.0) 52 Caucasian patients with advanced gastric cancer, Hamburg Eppendorf, Germany Goekkurt et al., 200669 GSTT1 GSTT1+/- TYMS STR rs34743033  TYMS 1494del6 rs34489327 MTHFR 677C/T rs1801133 ERCC1 Asn118Asn rs11615 XPD (ERCC2) Lys751Gln rs13181 IL1β −511T/C rs16944 wild-type genotypes and IL1RN 2R allele vs. IL1β - 511T/IL1β - 31C carriers with wild- type IL1RN OS HR=3.01 (95% CI: 2.15-4.23) 123 patients with relapsed or metastatic gastric cancer, Italy Graziano et al., 200554 IL1β −31C/T rs1143627 PFS HR=3.33 (95% CI: 2.35-4.46) IL1RN STR rs2234663 34  Gene Polymorphism1 SNP ID Risk allele Effect2 Study population2 Reference TNFβ NcoI 10.5-kb homozygote vs. 5.5 kb homozygote and heterozygote OS 143 patients with gastric cancer, Japan Shimura et al., 199575 IL1β −511T/C rs16944 91esophageal, 37 gastroesophageal junction and 75 gastric cancer patients, UK Deans et al., 200742 IL6 −174 G/C rs1800795 CC compared with GG or GC median survival 256 vs. 431 days IL10 -1082A/G rs1800896 GG compared with AA/AG median survival 310 vs. 389 days TNFα -308G/A rs1800629 AA OS HR=2.5 (95% CI: 1.3–4.9) LTa 252A/G rs909253   1 SNP is an unspecified Single Nucleotide Polymorphism; STR is short tandem repeat (microsatellite) polymorphism; +/- is conjugators vs. non-conjugators 2 HR is hazard ratio; CI is confidence interval; RR is relative risk; OR is odds ratio; OS is overall survival; PFS is progression free survival; SCC is squamous cell carcinoma    35  CHAPTER 3: Incidence and survival of gastric and esophageal cancer diagnosed in British Columbia, 1990 to 1999 2 3.1 Introduction Changing patterns of esophageal and gastric cancer incidence over recent decades have made this subject of increasing interest in cancer epidemiology. Geographic and temporal trends in incidence have been reported to vary according to both tumour morphology and organ subsite 91-93. Esophageal cancer incidence shows striking variation in different parts of the world .  About 80% of esophageal cancers occur in developing countries. High incidence areas include parts of Asia, south-eastern Africa, eastern South America and Western Europe 91,92,94. Esophageal cancer incidence in North America is about 5 to 10 per 100,000, whereas it is more than 100 per 100,000 in areas of the Iranian east Caspian littoral 13. Prior to 1980, approximately 90% of all cases of esophageal cancers were squamous cell carcinoma (SCC). Over the past two decades, however, the incidence of esophageal SCC has decreased and that of adenocarcinoma increased 5,95. In many western countries, adenocarcinoma is now more common than SCC 15. Esophageal cancer is one of the deadliest types of cancers and the sixth leading cause of death from cancer worldwide 96. Gastric cancer incidence and mortality have fallen dramatically over the past 70 years 2,93. Nonetheless, gastric cancer remains the fourth most commonly diagnosed cancer, and the second most common cause of cancer-related death worldwide 2,93. Adenocarcinoma is the most common histological type of gastric cancer, accounting for 90-95% of all gastric  2 A version of this chapter has been published: Bashash M, Shah A, Hislop G, Brooks-Wilson A, Le N, Bajdik C. Can J Gastroenterol. 2008 Feb;22(2):143-8.   36  malignancies 4,93,94. Despite a decline in lower gastric cancers, proximal tumour incidence has been increasing since the 1970s, especially among men in Western countries 5. Tumours of the gastric cardia now account for nearly half of all gastric cancers among men in the US and UK 6.Gastric cancer is a disease of poor prognosis and high mortality. In general, countries with higher incidence rates of gastric cancer have better survival rates than countries with lower incidence 3. In this paper, incidence and survival of gastric and esophageal cancer in the population of BC, Canada between 1990 and 1999 have been described. 3.2 Methods Data Cancer incidence data for invasive primary esophageal and gastric cancers were obtained from the BC Cancer Registry (BCCR) for the period 1990-1999. The topography and histology of cases were coded according to the International Classification of Diseases for Oncology, Second Edition 97. The topography of esophageal cancers was grouped into four anatomic subsites: esophagus upper third (C15.0-C15.3), esophagus middle third (C15.4), esophagus lower third (C15.5), overlapping lesion and esophagus unknown (C15.8 and C15.9). The topography of gastric cancer was grouped into three subsites: proximal (cardia) in the gastroesophageal junction or upper third of the stomach (C16.0-C16.1), lower stomach or lower two-thirds of the stomach (C16.2-C16.7), and unknown or unspecified/overlapping regions (C16.8-C16.9). Histological categories for esophageal and gastric cancers were squamous cell carcinoma (8050-8082), adenocarcinoma (8140-8573) and others (mainly 8000- 8020) (16). Diffuse gastric tumours were defined by histology codes 8142, 8145 and 8490 98. Five years of follow-up information was available for each patient. The stage of   37  diagnosis was defined according to American Joint Committee on Cancer (AJCC) TNM classification 99. Statistical analysis Annual age-adjusted incidence rates were computed by anatomic subsite, histological type and gender. All rates were standardized to the 1996 Canadian population. The Estimated Annual Percent Change (EAPC) was used to measure trends or the change in rates over time by fitting a regression line to the natural logarithm of the rates using calendar year as an independent variable 100. Overall survival was calculated as the time between cancer diagnosis and death. Cases diagnosed at the time of a patient’s death were excluded because they were probably they registered through autopsy or death certificate only. Survival curves were calculated using the Kaplan-Meier method and log-rank statistics were used to compare survival differences between groups. p-values less than 0.05 were considered statistically significant. 3.3 Results Incidence Between 1990 and 1999, 1741 cases of esophageal cancer and 3431 cases of gastric cancer were diagnosed in BC. Figure 3.1 shows the frequency of esophageal and gastric cancers according to the age of diagnosis and gender. The average age of diagnosis was 69.0 years (standard deviation (SD) 11.5) for esophageal cancer and 69.5 years (SD 13.2) for gastric cancer. In both esophageal and gastric cancers, men were more commonly affected with 71.0% and 64.5% of diagnoses, respectively. Staging information for 60% (1132) of esophageal and 20% (696) of gastric cancer patients was available. For esophageal cancer, 10% of patients had stage I, 56% had stage II, 11% had stage III and 23% had stage IV   38  disease. For gastric cancer, 14% of patients had stage I, 16% had stage II, 23% had Stage III (18% III a, 5% IIIb) and 47% had stage IV disease. Table 3.1 shows the incidence rates for gastric and esophageal cancers by topography, histology and gender. For esophageal cancer, 55% were diagnosed in the lower third, 22% in the middle third, and 10.5% in upper third. Incidence of men’s cancers in the lower esophagus increased during the study period (EAPC=4.6 with average incidence 3.7/100,000). 57% of esophageal cancers were SCC, 43% were adenocarcinoma, and 12% were other histological types. There was a substantial increase in incidence of esophageal adenocarcinoma over the study period among men (EAPC=9.0 with average incidence 3.3/100,000). There was almost no increase in esophageal adenocarcinoma incidence among women (EAPC=0.2 with average incidence 0.2/100,000). For gastric cancer, 29% of cases were diagnosed in the proximal third, 33% in the lower two-thirds, and 38% had uspecified topology. There was an increase in incidence of proximal gastric cancers over time for both men (EAPC=3.8 with average incidence 4.4/100,000) and women (EAPC=9.2 with average incidence 1.0/100,000). 84% of gastric cancer patients were diagnosed with adenocarcinoma and 16% had other histological types. Among gastric adenocarcinoma, 20% were the diffuse type (including signet ring cell carcinoma). The rates of diffuse gastric cancer in both men (EAPC=7.5 with average incidence 1.6/100,000) and women (EAPC=8.1 with average incidence 1.3/100,000) increased substantially from 1990 to 1999. Survival Figure 3.2 shows overall 5-year survival curves for esophageal and gastric cancer. The patients had overall 5-year survival rates of 8.8% for esophageal cancer and 16.2% for gastric cancer, and gastric cancer survival was significantly better than that for esophageal cancer. Gender did not have any significant effect on gastric cancer survival, but women had   39  significantly better survival than men for esophageal cancer (Figure 3.3). There was no significant difference in survival for cancers affecting the lower, middle and upper third of the esophagus, however, patients with cancer of the upper third had a slightly better survival when compare with patients having cancer in the lower third (Figure 3.4A). For gastric cancer, lower tumours had a significantly better survival than proximal tumours (p<0.001) (Figure 3.4B) however proximal gastric (cardia) cancers had significantly better survival compared to adenocarcinoma tumours in the lower third of the esophagus (p<0.001). There were no significant differences by tumour histology for esophageal cancer survival. 3.4 Discussion Over the last decades the epidemiology of upper GI cancers has changed. There are some similarities and interesting differences between changing pattern of cancers in the gastroesophageal junction area. The most notable similarity was the incidence of proximal gastric cancer and esophageal adenocarcinoma. Major differences in survival and incidence of these cancers were seen between men and women. Increased incidence of esophageal adenocarcinoma is only substantial in men, but women had a greater incidence than males for proximal gastric cancer. This trend is different than in other parts of Canada 101. The incidence of esophageal cancer in men showed an increase over the study period. The pattern of histological changes for esophageal cancer in BC is compatible with patterns in other western countries. The incidence of esophageal adenocarcinoma in men is rising in most countries, although this trend is highly varied among ethnicities 102,103. Our study did not analyze ethnicity because the BC registry does not collect this information. The highest incidence rates for white men in the year 2000 were found in Great Britain (5.0- 8.7 cases per 100,000 population) and Australia (4.8 cases per 100,000 population) followed by The   40  Netherlands (4.4 cases per 100,000 population), the US (3.7 cases per 100,000 population), and Denmark (2.8 cases per 100,000 population) (21). Regional differences between esophageal adenocarcinoma incidence rates have been reported in the US (22). BC has shown a decrease in incidence of esophageal SCC, however this decrease was not significant and different than the trend in Ontario, Canada 101. Gastric cancer incidence has decreased over the past several decades worldwide 94. In 1900, gastric cancer was the leading cause of death in United States 104. The incidence of lower gastric in BC have been almost steady over time, however the incidence of proximal (cardia) cancer has increased. The increasing incidence rate for proximal gastric in men follows an increasing trend elsewhere in Ontario 101. Like adenocarcinoma of the esophagus, the incidence of gastric cardia cancer has increased significantly since the 1970s 16. In the US, this increasing trend has stabilized since the late 1980s 105. Our study indicates the incidence and increase in incidence of proximal gastric is higher in BC than in the US and Ontario. Unlike other major tumours of the upper GI tract, incidence of diffuse gastric cancer was not influenced by gender in BC. The increase of diffuse gastric cancer in the US increased from 0.3 cases per 100,000 persons in 1973 to 1.8 cases per 100,000 persons in 2000 98. The 5-year overall survival for esophageal cancer in BC (9%) is very poor. In our study, women had better esophageal cancer survival compared with men. This result agrees with a report from Europe 106. It should be reminded that rates in this paper should be compared with caution to those in other reports because of the possible effects of standardizing with different populations. Topology in esophageal cancer patients did not significantly influence survival. However, cancers in the upper esophagus have a small survival benefit compared with cancer in the lower esophagus, possibly because they are detected or present earlier.   41  According to our data, there was no significant difference in survival between SCC and adenocarcinoma of esophagus. These results are in contrast with studies in UK (1987-2000) and Germany (1982-2000) that have shown adenocarcinoma has a more favorable prognosis than SCC 107,108 in esophageal cancer patients. In general, gastric cancer 5-year survival (16%) is poor but it was significantly better than that for esophageal cancer. This result shows a slightly better survival comparing with a previous report of gastric cancer patients treated in BC between 1978 to 1997 30. In our study, a significant difference in 5-year survival was observed between lower and proximal cancers of the stomach with a worse outcome for patients with cancer of cardia. This agrees with other studies, which have shown the same prognosis pattern for cardia cancers compared with other gastric cancers 109,110. The strength of this study was the availability of population-based data with details of histology and pathology. Furthermore, the Gastrointestinal Tumour Group at the BC Cancer Agency (BCCA) provides province-wide treatment guidelines in BC so that most patients receive similar treatment. This is important because treatment is one of the greatest determinants of cancer survival. Over the study period, the general treatment for esophageal cancer was surgery in stage I cancer; preoperative radiation (4500 cGy in 25 fractions  and chemotherapy (5-fluorouracil [5FU] and cisplatin) followed by surgery and further chemotherapy (5FU+cisplatin) in 50% of stage II and III cancers, or surgery followed by radiation (4500 cGy in 25 fractions) and chemotherapy (5FU+cisplatin) for the other 50%; and chemotherapy (5FU+cisplatin) in 75% of stage IV with the remainder receiving symptomatic care only. General treatment for resectable gastric cancer (Stages I, II or III) was surgery; chemotherapy chemotherapy with 5FU or 5FU plus cisplatin was prescribed for about 65% Stage IV pateints, while the rest had symptomatic care only. A weakness of this   42  study is the number of cases with unspecified histology and pathology, and the lack of ethnicity information. Although gastric and esophageal cancers are relatively infrequent in Canada, their epidemiology is changing. The trends are most evident when tumours are classified by histology and anatomic location. While incidence of squamous cell carcinoma of the esophagus and adenocarcinoma of the lower stomach appears to be stable or decreasing, the incidence of esophageal adenocarcinoma and gastric cardia cancer are increasing.There are three possible explanations for this trend: (i) increased exposure to one or more risk factors, (ii) misclassification and over-diagnoses, and (iii) immigration and the changing population of BC. Risk factors for gastric cancer include Helicobacter pylori infection 7, heredity, genetic and immunological variables 4, diet and lifestyle 111, tobacco smoking 112, obesity 113, ionizing radiation 114 and exposure to the Epstein-Barr virus 115. Risk factors for esophageal cancer include gastroesophageal reflux 116, Barrett’s esophagus 117, asthma medication use, LES (lower esophageal sphincter-relaxing) medication use 118, cholecystectomy 119, obesity 113, and cigarette smoking 16. It is unlikely that misclassification or over-diagnosis is responsible for the temporal changes because all of the cancers were invasive, and different survival patterns between cardia and esophageal adenocarcinoma indicate that these are separate diseases. Finally, the number of immigrants living in BC in the 1996 Census was 903,190 120. This is a 25% increase since 1991 120. Among these, immigrant Chinese was the largest ethnic group, representing 14% of the BC population. Esophageal SCC and lower gastric cancers are more common in China, but there is no indication of an increase in the incidence rate of cardia or lower esophagus adenocarcinoma in China.   43  Tumours in the upper gastro-intestinal (GI) tract are heterogeneous but share some epidemiological features. Gastric and esophageal cancers arise in advanced age, both predominantly occur in men, they are silent until advanced stages and therefore they have very poor survival. Gastric cardia cancers share epidemiologic features with adenocarcinoma of the lower esophagus and gastroesophageal junction. Both gastric and esophageal cancer patients have poor survival. Factors associated with the poor survival rates are the absence of symptoms in early cancer, lack of effective screening tools, and lack of effective treatment options. Examining these cancers together might elucidate new etiologic and prognostic factors.   44      3.1 A   3.1 B  Figure 3.1: New diagnoses of esophageal (A) and gastric (B) cancer in BC during 1990-1999 by age and gender 0-64 65-74 75+ Men 700 725 789 Women 322 317 578 0 100 200 300 400 500 600 700 800 N um be r o f N ew  C as es Age Group Esophageal Cancer by Age and Gender Men Women 0-64 65-74 75+ Men 700 725 789 Women 322 317 578 0 100 200 300 400 500 600 700 800 N um be r o f N ew  C as es Age Group Gastric Cancer by Age and Gender Men Women   45    Table 3.1: Age standardized incidence rate per 100,000 (ASR) and estimated annual percentage change (EAPC), with 95% confidence interval (95% CI), for esophageal and gastric cancer by topology and histology, BC 1990-1999 .  Gender  Cancer Men Women ASR (95% CI)  EAPC (95% CI)   ASR (95% CI)  EAPC (95% CI) ESOPHAGEAL TOPOLOGY Upper Third 0.6 (0.5,0.7) -1.2 (-8.8,7.1) 0.4 (0.3,0.4) 6 (-1.5,14.1) Middle Third 1.3 (1.1,1.5) 0.4 (-5,6.1) 0.5 (0.5,0.6) -1.9 (-6.1,2.5) Lower Third 3.7 (3.3,4.1) 4.6  (1.1,8.3) 0.7 (0.7,0.8) 0.1 (-4.6,5.1) GASTRIC TOPOLOGY Cardia 4.4 (4,4.8) 3.8 (0.3,7.4) 1 (0.8,1.2) 9.2 (2.6,16.2) Lower 3.8 (3.6,4) 0.4 (-1.9,2.7) 2.2 (2,2.4) -1.8 (-6,2) ESOPHAGEAL HISTOLOGY SCC 2.9 (2.6,3.2) -2.9 (-6.6,1) 1.7 (1.6,1.8) 0.4 (-2.8,3.7) AC 3.3 (2.7,3.9) 9.5 (5.1,14.2) 0.2 (0.2,0.3) 6.7 (-4.9,19.7) GASTRIC HISTOLOGY AC Diffuse 1.6 (1.3,1.9) 7.5 (1,14.4) 1.3 (1.1,1.5) 8.1 (2.5,14) AC (other)  9.1 (8.6,9.5) -0.3 (-2.3,1.8) 2.9 (2.6,3.2) -2.7 (-6,1) SCC (Squamous Cell Carcinoma), AC (Adenocarcinoma)   46     Figure 3.2: Five-year survival of esophageal and gastric cancer in BC.                47      Figure 3.3: Five-year survival by gender for (A) esophageal and (B) gastric cancer in BC.   48       Figure 3.4 Five-year survival by tumour location for (A) esophageal and (B) gastric cancer in BC.   49    CHAPTER 4: Comparison of two diverse populations, British Columbia, Canada and Ardabil, Iran, indicates several variables associated with gastric and esophageal cancer survival 3  4.1 Introduction Geographic variation and temporal trends in the epidemiology of esophageal and gastric cancers vary according to tumour morphology and organ subsite 121. Both diseases are among the deadliest forms of cancer. Gastric cancer incidence and mortality have fallen dramatically over the past 70 years in western countries, but it is the fourth most commonly diagnosed cancer and the second most common cause of cancer related death worldwide 2. The majority of esophageal carcinoma patients in the world die within a year of diagnosis and only 8-20% are alive after 5 years 122. Gastric and esophageal cancers are relatively infrequent in Canada, but common in Iran 123. This study compares one-year survival of gastric and esophageal cancers between the populations of British Columbia (BC), Canada and Ardabil, Iran. BC and Ardabil have been choosen because both areas have high-quality population-based cancer registries. The BC Cancer Registry has been in existence since 1969 (www.bccancer.bc.ca/HPI/CancerStatistics accessed November 4, 2009) and the Ardabil Cancer Registry is the first such registry in the Islamic Republic of Iran 124. This study does not compare survival rates for different types of esophageal or gastric cancer within Ardabil or within BC.  3 A version of this chapter is accepted for publication: Bashash M, Yavari P, Hislop G, Shah A, Sadjadi A, Babaei M, Le N, Brooks-Wilson A, Malekzadeh R, Bajdik C. Journal of Gastrointestinal Cancer (In press)   50    4.2 Methods Data for invasive primary esophageal and gastric cancer patients diagnosed in 2004 were obtained from the cancer registries for BC and Ardabil. For the BC registry, completeness of case ascertainment was 86.8% and completeness of other information was 99.8%125. For the Ardabil registry, overall completeness was 89% based on reports from pathology centers, identity information, demographic information and percentage of coded cancer cases 126. Dates in the Ardabil registry were converted to equivalent values in the western calendar. For both registries, the topography and histology of cases were coded according to the International Classification of Diseases for Oncology, Third Edition (ICD-O) 127. Similar methods were used for the collection and classification of breast cancer data from BC and Ardabil data in an earlier report 128. Esophageal cancers were grouped into four anatomic subsites: upper third (ICD-O codes C15.0-C15.3), middle third (C15.4), lower third and overlapping lesions (C15.5), and unknown (C15.8 and C15.9). Gastric cancers were grouped into three anatomic subsites: proximal third (i.e., cardia) in the gastroesophageal junction or upper third of the stomach (C16.0 and C16.1), lower stomach or lower two thirds of the stomach (C16.2–C16.7), and unknown or unspecified/overlapping lesion (C16.8 and C16.9). Histological categories for esophageal cancers were squamous cell carcinoma (ICD-O codes 8050-8082), adenocarcinoma (8140-8573) and others (mainly 8000-8020). Gastric cancers were categorized as diffuse or intestinal according to the Lauren classification system 128. Diffuse gastric tumours are defined by ICD-O histology codes 8142, 8145 and 8490 8; other gastric tumours are defined as intestinal. In BC, the vital status and date of death for cancer patients is routinely collected from government statistics. At least one year of follow-up information was available for each patient in BC. In Ardabil, information on a patient’s survival and date of death was obtained by   51    interviewing cases or their families. Interviews were conducted by members of the Ardabil Cancer Registry whenever possible. The death registry in Ardabil was used to confirm this information and obtain data for cases that could not be interviewed. Based on this approach, 83.3% of Ardabil patients had complete one-year follow-up information. Survival time was defined as the time between cancer diagnosis and death. The relative survival rate 129 was calculated for various subgroups of each population using WHO Statistical Information System (WHOSIS) life-tables for each country 130. Chi-square and Fisher's exact tests were used to compare differences in one-year survival proportions between BC and Ardabil. T-tests, chi-square tests and Fisher’s exact test were used to compare patient characteristics and tumour factors between the populations. p-values less than 0.05 were considered statistically significant. Other p-values were denoted non-significant (NS). 4.3 Results In 2004, 357 and 261 cases of gastric cancer were diagnosed in BC and Ardabil, respectively. Characteristics of the cases are summarized in Table 4.1. The mean age of patients was 69.1 years in BC and 66.1 years in Ardabil (p<0.01). Women comprised about one third of gastric cancer patients in both BC and Ardabil (NS). Approximately 34.5% of gastric cancer cases (49% of cases with known topography) in BC and 41.8% (60% of cases with known topography) in Ardabil were diagnosed with proximal disease (p<0.05). Adenocarcinoma was the predominant histological type of gastric tumour, accounting for 87.4% and 79.7% of cases in BC and Ardabil, respectively (p<0.01). About 16.0% of gastric tumours in BC and 30.3% in Ardabil were the diffuse type (p<0.05). In 2004, 232 and 124 cases of esophageal cancer were diagnosed in BC and Ardabil, respectively. Characteristics of cases are summarized in Table 4.2. The mean age of cases was   52    69.7 years in BC and 63.3 years in Ardabil (p<0.01). Women accounted for about one third of cases in BC and half of cases in Ardabil (p<0.01). Most tumours in BC cases were located in the lower third of the esophagus, while the lower and middle thirds of the esophagus had nearly equal incidence in Ardabil (p<0.01). Adenocarcinoma was the leading type of tumour in BC cases (50% of all cases) while only 10% of cases in Ardabil had this histology type (p<0.01). Figures 4.1 and 4.2 show the overall one-year age-standardized relative survival of gastric and esophageal cancers in BC and Ardabil. Details of the survival rates for gastric and esophageal cancer in BC and Ardabil are shown in Tables 4.3 and 4.4. Overall and separately for each gender, age group, tumour location and histology, there was greater one-year survival of gastric cancer patients in BC compared to Ardabil. Patients under age 65, patients with tumours in the middle or upper third of the esophagus, and patients with squamous cell carcinoma had significantly better esophageal cancer survival in BC than in Ardabil. In BC, there were significant 1-year survival differences among age and tumour location groups. In Ardabil there were no significant differences in one-year survival within groups. However, in esophageal cancer patients one-year survival difference have seen in age and tumour location groups among Ardabil patients , but not in BC patients. 4.4 Discussion Based on available registry and follow-up information, this study compares one-year survival of gastric and esophageal cancer in two populations. Results indicate major differences and some interesting similarities between the populations. In general, overall one-year relative survival was better in BC than Ardabil. There were significant differences between the populations in gastric cancer survival according to patient gender, age, tumour location and tumour histology. For esophageal cancer; patients under age 65, patients with tumours in the middle or upper third of   53    esophagus, and patients with squamous cell carcinoma had significantly better survival in BC than in Ardabil. Survival differences between the populations might be based on other tumour- related factors, other patient characteristics, cancer control measures and treatment factors. Stage at diagnosis is likely the main tumour-related factor affecting a patient’s prognosis, and stage at diagnosis determines the course of a patient’s treatment22. Unfortunately, stage could not be included in our analysis because both cancer registries provided only limited information about it. Cell histology is another tumour-related factor that might affect patient survival 108. In this study, the Lauren classification (based on tumour histology) did not have prognostic significance for one-year survival of gastric cancer patients in either population. Several clinical studies report better survival for adenocarcinoma of esophagus 107,108,131 but it has not been observed at this study. In both BC and Ardabil, tumour histology did not have a substantial influence on the survival of esophageal cancer patients. In Ardabil, tumours in middle third of the esophagus were associated with worse survival than tumours located in lower third. Low number of cases with tumour at the upper third of esophagus in Ardabil makes it impossible to make any statistical conclusion. This result is consistent with a recent report from Turkey132. However, results from BC and other North American studies 121,122 indicate that survival of patients with cancers in the upper, middle and lower third of the esophagus are similar. Ethnicity has been suggested to be a possible prognosis factor for cancer in upper GI tract 22,30. BC has an ethnically diverse population. The 2006 census reported that only 52% of people living in BC at that time had a single ethnic origin 133. In contrast, the population of Ardabil is homogeneous, with 95% being of Azeri ethnic background, which is of Aryan Caucasoid ancestry 124. Family history also has been shown to be a prognostic factor in gastric and esophageal cancer. Gastric cancer patients with a family history of this tumour have unique   54    clinicopathologic characteristics 134. Poor survival among young patients from Ardabil could be explained by the presence of higher proportion of familial cases of the disease. This is consistent with reports from other high incidence areas 135. Also, the high incidence of diffuse gastric cancer in the ethnically homogeneous Ardabil population is consistent with some cases having inherited mutations in the E-cadherin gene that underlie hereditary diffuse gastric cancer 136. Treatment is likely to be the greatest external determinant of cancer patients’ survival, and province-wide treatment guidelines in BC result in nearly uniform treatment (http://www.bccancer.bc.ca/HPI/CancerManagementGuidelines/Gastrointestinal/default.htm). Patient characteristics include inherent and demographic characteristics such as age, sex, ethnicity, physical performance status, comorbidity and immune status. These variables are usually unrelated to the presence of tumour but may have a profound impact on treatment choices as well as direct influence on survival. The general treatment for esophageal and respectable gastric cancer was surgery. Some patients also received radiation and chemotherapy depending on the stage of disease 121. In Ardabil, guidelines do not exist and treatment is not uniform. Based on previous reports, only 28% of patients with gastric and esophageal cancer in Ardabil received curative resectional surgery and about 25% of patients did not receive any treatment 137. tumourOne of the interesting differences between BC and Ardabil is the survival pattern for proximal and lower gastric cancers. In BC, patients with proximal gastric tumours had poorer survival than patients with lower ones. In Ardabil, there was very little difference between survival for proximal and lower gastric cancers. In BC, only about one-third of tumours occurred in the middle and upper third of the esophagus. In Ardabil, more than half of tumours with known topology were located in this anatomic region. More men than women had gastric and   55    esophageal cancers in BC; however, the incidence of esophageal cancer in Ardabil seemed independent of gender (i.e., the same in both sexes). The strength of this study was the availability of population-based data with details of tumour histology and pathology. This study’s limitations include the lack of complete staging information, incomplete follow-up data, and the relatively large proportion of esophageal tumours with unspecified histology. Differences in the quality of registry data between two populations could also have influenced survival comparisons. As noted in the Report of National Cancer Registration in Iran 126, there are challenges in interpreting registry information regarding the health care system in Iran. There is vast, uncontrolled population movement in and out of Ardabil, an uncoordinated medical services system, and inconsistent referrals to different centers for diagnosis and treatment 126. It is also possible that patients with better socioeconomic status are referred to better medical facilities in central cities. Population-based survival studies cannot assess specific treatments but can quantify the effect of cancer control measures at population level 138. Neither BC nor Ardabil has a screening program for gastric and esophageal cancers. In BC, these cancers are infrequent and feasibility of screening is questionable. However, Ardabil has the highest rates of gastric and esophageal cancers in the world, and a screening program should be considered. 4.5 Conclusion Gastric and esophageal cancers are heterogeneous diseases, but they share important features. They remain clinically asymptomatic until late in the disease process with consequent poor prognoses and high mortality rates. This study points to differences in disease characteristics and patient factors, not solely differences in healthcare systems, as being responsible for the survival   56    difference in these populations. Even so, the outcomes of these cancers are poor for both populations and improvements in diagnosis and management are urgently needed.          Figure 4.1. Overall one-year age-standardized survival rates for gastric cancer cases in Ardabil (Iran) and British Columbia (Canada). Bars are ± standard error.    57     Figure 4.2. Overall one-year age-standardized survival rates for esophageal cancer cases in Ardabil (Iran) and British Columbia (Canada). Bars are ± standard error.    58    Table 4.1. Gastric cancer patients in BC (Canada) and Ardabil (Iran).    British Columbia Ardabil Gender Women 119 (33.3%) 75 (28.7%) Men 238 (66.7%) 185 (70.9%) Unknown   1 (0.4%)  Age Group Less than 65 118 (33.1%) 108 (41.4%) 65 or more 239 (66.9%) 153 (58.6%)  Location Lower 127 (35.6%) 72 (27.6%) Proximal 123 (34.5%) 109 (41.8%) NOS / Overlapping lesion 107 (30.0%) 80 (30.7%)  Lauren Classification* Intestinal 262 (84.0%) 145 (69.7%) Diffuse 50 (16.0%) 63 (30.3%)  NOS = not otherwise specified.  *Adenocarcinomas only    59    Table 4.2. Esophageal cancer patients in BC (Canada) and Ardabil (Iran).    British Columbia Ardabil Gender Women 64 (27.6%) 61 (49.2%) Men 168 (72.4%) 62 (50.0%) Unknown   1 (0.8%)  Age Group Less than 65 76 (32.8%) 67 (54.0%) 65 or more 156 (67.2%) 57 (46.0%)  Tumour Location Upper third 23 (9.9%) 6 (4.8%) Middle third 40 (17.2%) 35 (28.2%) Lower third 112 (48.3%) 38 (30.6%) NOS / Overlapping lesion 57 (24.6%) 45 (36.3%)  Tumour Histology Squamous Cell Carcinoma 88 (37.9%) 89 (71.8%) Adenocarcinoma 116 (50.0%) 13 (10.5%) Other 28 (12.1%) 22 (17.7%)  NOS = not otherwise specified.  *Adenocarcinomas only    60    Table 4.3. One-year relative survival for gastric cancer patients in BC (Canada) and Ardabil (Iran).     BC Ardabil p Gender Male 0.48 ± 0.074 0.18 ± 0.09 <0.01  Female 0.46 ± 0.09 0.26 ± 0.08 <0.01 p  NS NS Age Less than 65 0.62 ± 0.068 0.22 ± 0.09 <0.01  65 or more 0.41 ± 0.04 0.20 ± 0.05 <0.01 p  <0.01 NS Tumour Location Lower 0.61 ± 0.07 0.23 ± 0.06 <0.01  Proximal 0.45 ± 0.08 0.19 ± 0.06 <0.01 p  <0.05 NS Lauren Classification* Intestinal 0.48 ± 0.07 0.18 ± 0.04 <0.01  Diffuse 0.50 ± 0.10 0.24 ± 0.04 <0.05 p  NS NS  *Adenocarcinomas only Values are age-standardized rates ± standard error NS=Non-significant.     61    Table 4.4. One-year relative survival for esophageal cancer patients in BC (Canada) and Ardabil (Iran).      BC Ardabil p Gender Male 0.32 ± 0.07 0.25 ± 0.1 NS  Female 0.34 ± 0.07 0.18 ± 0.06 NS p  NS NS Age Less than 65 0.44 ± 0.11 0.19 ± 0.06 <0.01  65 or more 0.27 ± 0.03 0.26 ± 0.05 NS p  NS <0.05 Tumour Location Upper third 0.50 ± 0.14 0 <0.05  Middle third Lower third 0.42 ± 0.12 0.33 ± 0.10 0.1 ± 0.05 0.33 ± 0.09 <0.05 NS p  NS <0.01 Histology SCC 0.40 ± 0.10 0.20 ± 0.06 <0.01  Adenocarcinoma 0.34 ± 0.08 0.38 ± 0.16 NS p  NS NS  Values are age-standardized rates ± standard error NS=Non-significant.     62    CHAPTER 5: The prognostic effect of ethnicity for gastric and esophageal cancer: the population-based experience in British Columbia, Canada 4  5.1 Introduction Gastric and esophageal cancers are among the most lethal human malignancies. Worldwide, gastric cancer is the fourth most common cancer, but the second most common cause of death from cancer 2. Esophageal cancer is the eighth most common cancer, but the sixth most common cause of cancer death 2. The epidemiology of these cancers is geographically diverse. Incidence rates for gastric cancer vary from 3.4 per 100,000 among women in North America to 26.9 per 100,000 among men in Asia. The 5-year survival is usually about 20% 139; however, countries with higher incidence rates of gastric cancer generally have better survival rates than countries with lower incidence 3. Incidence rates for esophageal cancer range from 5-10 per 100,000 in North America to more than 100 per 100,000 in Eastern Iran near the Caspian Sea 13. These differences between populations reflect different environmental and lifestyle (including healthcare) factors, as well as different genetic profiles of the tumours and the patients. In order to investigate population characteristics such as ethnicity and to reduce or eliminate environmental confounding, it is preferable to conduct a study in a single geographic area with a heterogenous population rather than to conduct international comparisons 140. British Columbia (BC), Canada, has a multi-ethnic population. Based on 2006 census data, about one in every four 4,428,400 British Columbians (24.8%) belongs to a visible minority, representing about one million people in the province. Of these, approximately 75% were born outside Canada, and about 60% immigrated to BC from 1991 to 2006 133. That indicates about 676,000 immigrants  4 A version of this chapter has been submitted for publication and is under review. Bashash M, Shah A, Hislop G, Le N, Brooks-Wilson A, Bajdik C.   63    and 297,000 non-immigrants in BC belonged to a visible minority group in 2006 133. Chinese was the largest group, accounting for 40% of all visible minorities in the province, followed by South Asians (26%) 133. Iranians represent a relatively small but growing percentage of the BC population (0.5%, or 19,000 people) in 2001 141, although they originate from a geographic region with the world’s highest incidence of gastric and esophageal cancers 124,142.  This study compares survival of gastric and esophageal cancer patients among Chinese, South Asian and Iranian and other ethnic groups in BC. 5.2 Methods Cancer incidence and survival data for invasive primary esophageal and gastric cancers were obtained from the population-based BC Cancer Registry for all BC patients diagnosed between 1984 and 2006. The topology and histology of cases were coded according to the International Classification of Diseases for Oncology, Third Edition (ICD-O) 127. The topography for esophageal cancers was then grouped into four categories: esophagus upper third (ICD-O codes C15.0-C15.3), esophagus middle third (ICD-O codes C15.4), esophagus lower third and overlapping lesions (ICD-O codes C15.5), and esophagus unknown (ICD-O codes C15.8 and C15.9). The topography for gastric cancer was grouped into three categories: proximal third (cardia) in the gastroesophageal junction or upper third of the stomach (ICD-O codes C16.0 and C16.1), lower stomach or lower two thirds of the stomach (ICD-O codes C16.2–C16.7), and unknown or unspecified/overlapping lesion (ICD-O codes C16.8 and C16.9). Histological categories for esophageal cancers were squamous cell carcinoma (ICD-O codes 8050-8082), adenocarcinoma (ICD-O codes 8140-8573) and others (mainly ICD-O codes 8000-8020). Histology for gastric cancer was also categorized based on the Lauren classification system as diffuse or intestinal type 8 (diffuse gastric tumours defined by histology codes 8142, 8145 and   64    8490) 98. For both esophageal and gastric cancers, nonepithelial tumours (ICD-O codes 8800- 9759) were excluded. Primary treatment was categorized as surgery, chemotherapy and radiotherapy, with only therapeutic (i.e., not diagnostic) surgeries being considered as treatment. Overall survival was the primary study outcome, and was calculated as the time between diagnosis and death. Complete follow-up information was available for all patients to 31 August 2007. The ethnicity of patients was determined according to their names and categorized as Chinese, South Asian or Iranian. This method for identification of ethnicity was necessary because the BC Cancer Registry does not record ethnicity; the method has been described elsewhere 30,98,141,143- 145. Patients not classified as belonging to any of these three ethnic groups were categorized as “Other.” Based on the ethnic distribution of the BC population, more than 80% of “Other” are British and Western Europeans 120. British and Western Europeans could not be separated as a group because corresponding name lists do not exist. Univariate comparisons of demographic, tumour and treatment variables between ethnic groups were performed using Chi-square tests. Survival was calculated using the Kaplan-Meier method and log-rank tests were used to compare survival differences among groups. All analyses were performed separately for nonmetastatic (Stage I-III) and metastatic (Stage IV) disease. Cox proportional hazards regression was used to estimate the effect of ethnicity adjusted for patient sex, age (less than 55 years, 55-64 years, 65-74 years and 75+ years), date of diagnosis (1984- 1990, 1991-1995, 1996-2000, 2001-2006), tumour histology (intestinal and diffuse for gastric cancer; adenocarcinoma and squamous cell carcinoma for esophageal cancer), tumour location, disease stage and treatment received. The hazard ratio (HR) was calculated for each ethnic group   65    and is the ratio of the hazard rate in each ethnic group compared to the “Other” group. For each HR, a 95% confidence interval (95%CI) was calculated. p-values less than 0.05 were considered statistically significant. 5.3 Results Gastric cancer 3136 cases of invasive gastric cancer were diagnosed during the study period. Descriptive information for the cases is shown by ethnicity in Table 5.1. The age and sex distributions were significantly different among the ethnic groups (p<0.01). A higher proportion of Chinese and South Asian gastric cancer patients were female as compared to the other ethnic groups. The average age at diagnosis was 61.0 years for Iranians, 62.6 years for Chinese, 61.7 years for South Asians, and 65.4 years for Other ethnicities. There were significant differences among the year of diagnosis by ethnicity (p<0.01). Tumour location was significantly different among the ethnic groups (p<0.01). Tumours in the proximal 1/3 of the stomach were more common in South Asians and Other ethnicities as compared to Chinese and Iranians. Histology based on the Lauren classification was also significantly different among ethnic groups (p=0.03). The diffuse type of gastric cancer was most common among the Chinese compared to the other ethnic groups. The distribution of stage and proportion with metastatic disease was not significantly different among the ethnic groups; however, treatment by surgery and chemotherapy were significantly different among the ethnic groups. The Chinese and Iranian groups received surgery more often than the South Asian or Other ethnicities group (p<0.01), and the South Asian and Iranian groups received chemotherapy more often than Chinese or Others (p<0.01). Figure 5.1 shows survival curves for gastric cancer patients according to ethnic group. Survival was significantly different between ethnic groups (p<0.01). When considered separately by presence   66    or absence of metastatic disease, significant differences were only found for non-metastatic disease (p<0.01), as shown in Figure 5.3. Furthermore, the association between survival and ethnicity was only significant for patients with non-metastatic disease who received therapeutic surgery (p<0.01). In multivariate analyses adjusting for patient factors, disease factors and treatment, there was a significant difference among ethnic groups. Only Chinese had significantly longer survival as compared to the Other ethnicities, as shown in Table 5.2. This survival advantage in Chinese was only seen for non-metastatic disease (HR=0.78, 95% CI=0.64-0.95). Esophageal cancer 2873 cases of esophageal cancer were diagnosed during the study period. Descriptive characteristics of these patients are presented by ethnicity in Table 5.3. The majority of South Asians were women whereas the majority in the other ethnic groups was men (p<0.01). There was no significant difference in age at diagnosis among the ethnic groups, the average age being 73.0 years, 68.0 years, 65.5 years and 68.4 years for Iranians, Chinese, South Asians and Other ethnicities, respectively. There was no significant difference among ethnic groups based on date of diagnosis. Tumour location was significantly different among ethnic groups (p<0.01). More than half of tumours in Iranians and Other ethnicities were located in the  lower third of esophagus whereas this location was less common in Chinese and South Asians. Histology was significantly different among the ethnic groups (p<0.01), with Chinese and South Asians having higher proportions of squamous cell carcinoma compared to Iranians and Other ethnicities. There were no significant differences in stage or the proportion with metastatic disease among ethnic groups. Treatment received was not different, except for chemotherapy which had   67    significant differences among the ethnic groups (p<0.01), with the Chinese, Iranian and South Asian patients accessing chemotherapy more often than Other ethnicities. Figure 5.2 shows the survival curves for esophageal cancer patients by ethnic group (p=0.029). In multivariate analyses, there was no overall significant difference among ethnic groups, however South Asians showed better survival compared to the Other ethnicity group, as shown in Table 5.4. A significant survival difference only was observed among ethnic groups for patients with non- metastatic disease (p=0.0498), as shown in Figure 5.4. Again, South Asians showed better survival compared to the Other ethnicity group (HR=0.74, 95%CI=0.56-0.97) in the multivariate analysis. 5.4 Discussion An earlier population-based study in BC reported overall five-year survival rates of 8.8% for esophageal cancer and 16.2% for gastric cancer 121. The current study was conducted to examine the effect of ethnicity on survival. Our results indicate that patient ethnicity is a prognostic factor for both gastric and esophageal cancer; however ethnicity is only an independent prognostic factor for gastric cancer patients. Prognostic factors can be classified into three broad groups: i) tumour-related, ii) host-related, and iii) environment-related (health care, treatment, lifestyle) factors 22,34. Among tumour-related prognostic factors, disease stage is the most important 34 and often strongly influences the treatment plan. There were no significant differences in the stage distributions among ethnic groups; however, survival differences among ethnic groups were only significant for non- metastatic (i.e., stage I-III) disease. After adjustment for other factors (such as stage), the prognostic effect of ethnicity was significant only for gastric cancer patients.   68    Tumour topography is another prognostic factor, and there were significant differences in tumour location among different ethnic groups. It has been shown previously in Western countries that gastric cardia tumours are associated with worse survival as compared to lower gastric tumours 146-148. In addition, for studies of esophageal cancer, the location of tumours also showed differences in survival. Tumours in the middle 1/3 of esophagus 132,149 showed worse survival for Turkey and Ardabil (Iran), but for BC and the United States where tumours in the lower 1/3 of esophagus are reported to have worse survival 121,122. Among host-related prognostic factors, ethnic differences were found for sex and age in both gastric and esophageal cancer. Of environment-related factors, treatment is likely the most powerful determinant of survival. There were significant ethnic differences in the proportions of gastric cancer patients who received surgery and chemotherapy. The reason for treatment differences among ethnic groups is not clear in a system where all patients have equal access to cancer care, but the differences might be explained by disease factors, other patient characteristics or patient preferences. It has been suggested that lower quality care and disparities in treatment are major contributors to differences in survival between minority and non-minority populations 150. BC residents have access to publicly-funded healthcare, and the BC Cancer Agency (BCCA) has developed province-wide treatment guidelines and protocols 151. The main strength of this study is the availability of reliable population-based data with details on tumour histology and pathology, treatment, disease stage and survival outcomes. The main weakness is the lack of self-reported ethnicity information, requiring the use of a proxy method (i.e., name lists) to assign ethnicity. The weakness of using name lists as proxy for ethnicity is   69    greater for women, who may change their surnames after marriage. This method has been developed, however, based on the names of women participating in the Screening Mammography Program of British Columbia (SMPBC) 143. Further, women account for only 30% of gastric and esophageal cancer cases. The difference between proportion of ethnicities in this study and general population might be due to age distribution differences. Our study investigated ethnicity as a prognostic factor for gastric and esophageal cancer patients. It has been shown that for gastric cancer, patient ethnicity is significant and Chinese patients experience better survival than other people in BC. It has also been shown that, for esophageal cancer, South Asian patients experience better survival than others in BC. The result for gastric cancer is consistent with several US studies in which all other ethnic groups had better survival compared to the non-Hispanic white population 152, and a Los Angeles study that showed that Asians with gastric adenocarcinoma had superior outcomes compared to other ethnic groups 153. Our study also confirms the findings of an earlier study in BC that reported better survival outcomes for gastric cancer patients with Asian ethnicity compared to the general population 30. Our findings are consistent with international population-based cancer survival data that indicate that the 5-year survival for gastric cancer in China is higher than in India 154. A comparison between registries from Shanghai (China) and Madras (India) shows that the 5-year relative survival for gastric (20% versus 7.5%) and esophageal cancer (9.0% versus 6.9%) is better in Shanghai 155. These survival rates for both cancers are also higher than those reported in Iran 137. Ethnicity may represent biological characteristics of patients. Genetic variation may be responsible for differences in tumour-host interactions, such as the micro-architecture of tumours 156 and the complex process of metastasis, both of which are influenced by host genetic polymorphisms 29. Ethnicity may also determine lifestyle and environmental characteristics   70    including cultural, socioeconomic, and religious practices. Such differences are expected to be less apparent with increasing generations after immigration. The difference observed in patient survival is not likely to be due to healthcare disparities among minority groups, as all BC residents are covered for healthcare through the BC Medical Services Plan (MSP).  Interestingly, survival was found to be better in minority groups compared to the BC general population. Gastric and esophageal cancers are deadly diseases that are often diagnosed at a stage when the treatment options are limited and less effective. It have been shown that for gastric and esophageal cancers, there are significant differences in survival among ethnic groups. Ethnicity may represent underlying genetic factors. Such factors could influence host-tumour interactions by altering tumour etiology and therefore its chance of spreading. Alternatively, genetic factors may determine response to treatments. Finally, ethnicity may represent non-genetic factors that affect survival. Differences in survival by ethnicity support the importance of ethnicity as a prognostic factor, and may provide clues for the future identification of genetic or lifestyle factors that underlie these observations.    71    Table 5.1.  Descriptive characteristics for gastric cancer by ethnicity  * NES not elsewhere specified; NOS not otherwise specified.       Iranian Chinese South Asian Other p Sex (N=3136) Male 15 (78.9%) 168 (62.2%) 57 (58.8%) 1974 (71.8%) 4.4x10-4 Female 4 (21.1%) 102 (37.8%) 40 (41.2%) 776 (28.2%) Age in years (N=3136) Less than 55 7 (36.8%) 86 (31.9%) 26 (26.8%) 515 (18.7%) 8.0x10-6 55-64 3 (15.8%) 49 (18.1%) 20 (20.6%) 652 (23.7%) 65-74 7 (36.8%) 65 (24.1%) 35 (36.1%) 884 (32.1%) 75 and More 2 (10.5%) 70 (25.9%) 16 (16.5%) 699 (25.4%) Years of Diagnosis (N=3136) 1984-1990 0 (0.0%) 32 (11.9%) 11 (11.3%) 643 (23.4%) 3.3x10-5 1991-1995 7 (36.8%) 54 (20.0%) 16 (16.5%) 481 (17.5%) 1996-2000 4 (21.1%) 63 (23.3%) 27 (27.8%) 626 (22.8%) 2001-2006 8 (42.1%) 121 (44.8%) 43 (44.3%) 1000 (36.4%) Tumour Histology - Lauren classification (N=3136) Intestinal 14 (73.7%) 205 (75.9%) 74 (76.3%) 2188 (79.6%) 0.032 Diffuse 3 (15.8%) 55 (20.4%) 13 (13.4%) 382 (13.9%) Other 2 (10.5%) 10 (3.7%) 10 (10.3%) 180 (6.5%) Tumour Location (N=3136) Proximal 1/3 6 (31.6%) 52 (19.3%) 47 (48.5%) 1302 (47.3%) 1.93 x 10-22 Lower 2/3 10 (52.6%) 171 (63.3%) 28 (28.9%) 894 (32.5%) NES/NOS* 3 (15.8%) 47 (17.4%) 22 (22.7%) 554 (20.1%) Tumour Stage (N=2567) I 1 (5.6%) 14 (6.1%) 3 (3.7%) 108 (4.8%) 0.85 II 6 (33.3%) 65 (28.5%) 29 (35.8%) 702 (31.3%) III 6 (33.3%) 96 (42.1%) 29 (35.8%) 829 (37.0%) IV 5 (27.8%) 53 (23.2%) 20 (24.7%) 601 (26.8%) Surgery (N=3080) Yes 14 (73.7%) 178 (66.7%) 56 (57.7%) 1502 (55.7%) 0.0027 No 5 (26.3%) 89 (33.3%) 41 (42.3%) 1195 (44.3%) Chemotherapy (N=3065) Yes 10 (52.6%) 116 (43.6%) 44 (45.4%) 906 (33.8%) 5.3x 10-4 No 9 (47.4%) 150 (56.4%) 53 (54.6%) 1777 (66.2%) Radiotherapy (N=3058) Yes 6 (31.6%) 99 (37.1%) 43 (44.3%) 1203 (45.0%) 0.061 No 13 (68.4%) 168 (62.9%) 54 (55.7%) 1472 (55.0%)   72    Table 5.2. Hazard ratio (HR) and 95% confidence interval (CI) from Cox proportional hazards regression analysis for overall survival of gastric cancer patients adjusted for patient sex, patient age, year of diagnosis, tumour histology (Lauren), tumour location, tumour stage and treatment.   Ethnicity N HR 95% CI p Iranian 16 0.64 0.34 1.18  P=0.006 Chinese 214 0.76 0.65 0.90 South Asian 72 0.88 0.68 1.14 Other 2038 Reference     73    Table 5.3.  Descriptive characteristics for esophageal cancer by ethnicity   *SCC  squamous cell carcinoma **AC adenocarcinoma *** NES not elsewhere specified; NOS not otherwise specified    Iranian Chinese South Asian Other P Sex (N=2873) Male 10 (71.4%) 94 (74.6%) 57 (47.9%) 1821 (69.7%) 5.0 x 10-6 Female 4 (28.6%) 32 (25.4%) 62 (52.1%) 793 (30.3%) Age in years (N=2873) Less than 55 0 (0.0%) 14 (11.1%) 21 (17.6%) 314 (12.0%) 0.12 55-64 1 (7.1%) 35 (27.8%) 32 (26.9%) 610 (23.3%) 65-74 9 (64.3%) 41 (32.5%) 35 (29.4%) 858 (32.8%) 75 and More 4 (28.6%) 36 (28.6%) 31 (26.1%) 832 (31.8%) Years of Diagnosis (N=2873) 1984-1990 3 (21.4%) 16 (12.7%) 22 (18.5%) 486 (18.6%) 0.164 1991-1995 1 (7.1%) 26 (20.6%) 15 (12.6%) 580 (22.2%) 1996-2000 3 (21.4%) 38 (30.2%) 33 (27.7%) 637 (24.4%) 2001-2006 7 (50%) 46 (36.5%) 49 (41.2%) 911 (34.9%) Tumour Histology (N=2874) SCC * 5 (35.7%) 103 (81.7%) 81 (68.1%) 1389 (53.1%) 1.53 x 10-11 AC ** 7 (50.0%) 19 (15.1%) 27 (22.7%) 1101 (42.1%) Other 2 (14.3%) 4 (3.2%) 11 (9.2%) 124 (4.8%) Tumour Location (N=2874) Upper 1/3 2 (14.3%) 23 (18.3%) 17 (14.3%) 314 (12.0%) 7.35 x 10-4 Middle 1/3 1 (7.1%) 45 (35.7%) 34 (28.6%) 605 (23.1%) Lower 1/3 9 (64.3%) 40 (31.7%) 51 (42.9%) 1383 (52.9%) NES/NOS*** 2 (14.3%) 18 (14.3%) 17 (14.3%) 312 (12.0%) Tumour Staqe (N=2594) I 1 (8.3%) 12 (10.3%) 8 (7.6%) 212 (9.0%) 0.84 II 6 (50.0%) 66 (56.9%) 56 (53.3%) 1363 (57.8%) III 3 (25.0%) 27 (23.3%) 26 (24.8%) 459 (19.4%) IV 2 (16.7%) 11 (9.5%) 15 (14.3%) 326 (13.8%) Surgery (N=2830) Yes 2 (15.4%) 24 (19.2%) 35 (29.9%) 630 (24.5%) 0.23 No 11 (84.6%) 101 (80.8%) 82 (70.1%) 1944 (75.5%) Chemotherapy (N=2820) Yes 0 (0.0%) 39 (31.2%) 25 (21.6%) 526 (20.5%) 0.0084 No 13 (100.0%) 86 (68.8%) 91 (78.4%) 2039 (79.5%) Radiotherapy (N=2853) Yes 13 (100.0%) 112 (89.6%) 111 (93.3%) 2240 (86.3%) 0.052 No 0 (0.0%) 13 (10.4%) 8 (6.7%) 355 (13.7%)   74    Table 5.4. Hazard ratio (HR) and 95% confidence interval (CI) from Cox proportional hazards regression analysis for overall survival of esophageal cancer patients adjusted for patient sex, patient age, year of diagnosis, tumour histology, tumour location, tumour stage and treatment.       Ethnicity N HR 95 % CI P Iranian 10 1.13 0.61 2.12 0.14  Chinese 95 0.90 0.72 1.13 South Asian 81 0.80 0.59 0.98 Other 1947 Reference   75      Figure 5.1. Survival of gastric cancer patients by ethnic group    76        Figure 5.2. Survival of esophageal cancer patients by ethnic group.    77       Figure 5.3 . Survival of gastric cancer patients by ethnic group for non-metastatic disease.       78          Figure 5.4. Survival of esophageal cancer patients by ethnic group for non-metastatic disease.     79    CHAPTER 6: Genetic polymorphism at TIMP-3 predicts survival for patients with adenocarcinoma of the esophagus and gastroesophageal junction 5  6.1 Introduction During the past two decades, there has been a dramatic increase in the incidence of adenocarcinoma of both the esophagus and gastroesophageal junction (GEJ) in North America and Western Europe 5,18,96,121. Because of this increase and also generally poor survival, lower esophageal and GEJ adenocarcinomas are clinically important cancers 18,121. Similarities and shared prognostic factors suggest these cancers can be considered a single neoplastic entity in many contexts 5. Adenocarcinomas of the lower esophagus and GEJ originate from the approximately 10 cm segment around the GEJ, including the lower 5 cm of the esophagus and proximal 5 cm of the stomach 25. Lesions with their centre between 1 and 5 cm above the GEJ are classified as type I or esophagus tumours; lesions between 1 cm above or 2 cm below GEJ are type II or cardia tumours; and lesions between 2 and 5 cm below GEJ are type III or subcardia tumours 157,158 . In spite of this subclassification, the American Joint Committee on Cancer (AJCC) Cancer Staging Manual uses “single-stage grouping” across the entire lower esophagus and GEJ area 25. Cancer occurs more frequently at the junction of two different tissue microarchitectures, and anatomic sites that have this characteristic are highly influenced by microarchitecture disruption156. Loss of structure is a prerequisite and defining characteristic of most cancers and precancerous lesions 159. The extracellular matrix (ECM) influences tissue and organ architecture, as well as the growth of neoplastic cells 160. Malignant cells acquire the ability to remodel the ECM and modulate the expression of ECM receptors 161. The balance between  5 A version of this chapter will be submitted for publication. Bashash M, Shah A, Hislop G, Le N, Brooks-Wilson A, Bajdik C.   80    activated matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) controls  ECM remodelling  162. MMPs also contribute to the invasion, promotion, angiogenesis, and the establishment and growth of metastatic lesions in distant organ sites 76. MMPs can be synthesized by tumour cells, but are frequently produced by surrounding stroma, including fibroblasts and infiltrating inflammatory cells 76. Finally, MMPs solubilize cell surface and matrix-bound factors that can influence cellular properties, such as growth, death, and migration and metastasis 76. It is possible that a tissue microenvironment giving rise to malignancy also could allow a malignant cell to metastasize. Many phenotypes, including cancer, are significantly affected by constitutional genetic polymorphisms, and the complex process of metastatic dissemination might be significantly affected by host genotypes 29. As a result, assessment of genetic polymorphisms in the TIMP gene family has been chosen to evaluate prognosis in esophagus and GEJ adenocarcinoma patients. This study used a prospective cohort of patients with adenocarcinoma of the esophagus and GEJ in British Columbia (BC) who were admitted to the BC Cancer Agency. Our objective was to assess genetic polymorphisms of TIMP genes for association with prognosis for adenocarcinomas of the esophagus and GEJ. This study was reviewed and approved by the joint University of British Columbia / British Columbia Cancer Agency Research Ethics Board. All subjects provided written informed consent. 6.2 Methods Eligible patients were patients between ages 20 and 90 years who were diagnosed with primary invasive adenocarcinoma of esophagus and GEJ between January 1, 2008 and April 30 2009, admitted to the BCCA, and able to provide written informed consent. Patient recruitment started January 1, 2008 and finished July 31 2009, this includes a two-month recruitment follow-up   81    period. Eligible patients were identified using the electronic appointment list of new gastrointestinal (GI) tumour group patients. Pathology reports of all gastric and esophageal cancer patients were reviewed to identify eligible patients for the study. The anatomic sites esophagus and cardia were defined as International Classification of Diseases for Oncology (ICDO-3) site codes C150-C160, and adenocarcinoma was defined as ICDO-3 histology codes 8140/3-8573/3. Eligibility of identified patients and capability to participate was assessed by a BCCA GI Tumour Group oncologist.  Eligible patients were invited to participate in the study at their next BCCA visit. The BCCA operates clinics in five major centres across the province of British Columbia. A patient’s participation required their written consent to provide blood or saliva for genetic analysis, and access to their medical chart for demographic and clinical information. Germline DNA was extracted from blood or saliva (saliva was collected using Oragene® DNA sample collection kit).  To minimize the potential for population stratification bias, only patients who identified themselves as white Canadian, British, and Western European were included for genotyping. TagSNPs 163 representing genetic variation in each gene were chosen using Haploview Ver. 4.1164 on HapMap 165 (phase 3 release 2) western European ancestry (CEU) data. TagSNPs with minimum minor allele frequency of 0.1 were chosen within ≤500  kb of each gene using an r2 threshhold of 0.9. We also included SNPs reported to be associated with cancer in the literature, and non-synonymous coding SNPs in these genes. The minor allele frequencies of these SNPs were obtained using HapMart (BioMart ver 7. using HapMap release 27) on CEU population data. The list of SNPs genotyped is shown in Table 6.1. In total, 63 SNPs were genotyped using two Sequenom multiplex iPLEX Gold assays 166 on a single 96-well plate of 90 subject DNA samples. 88 out of 90 samples (98%) were genotyped   82    successfully, with an average call rate of 96%. For quality control of genotyping, clustering of observed genotypes was reviewed by transferring intensity data to MassArray Typer software (version 7.0.2.5).  15 SNPs with less than 95% call rate were excluded from analysis . Concordance between duplicate samples was 100%. All of the X chromosome markers (rs6609533, rs4898) were consistent with the sex of patients. After all quality control restrictions, 48 SNPs were used in the final analysis. Patient characteristics and clinical information were obtained from BCCA medical charts and preadmission questionnaires. Patient age was categorized into two groups (<70 and 70+) and BMI was categorized into three groups (normal: 18.5 to <25, overweight: 25 to <30, and obese: 30+). Tumour location was categorized as esophagus (i.e., Siewert I) or gastric cardia (i.e., Siewert II/III). Disease stage was defined according to recent AJCC Guidelines 25 for lower esophagus and GEJ cancers and categorized as metastatic or non-metastatic cancer. Primary treatment was categorized as surgery, chemotherapy and radiotherapy, with only therapeutic (i.e., not diagnostic) surgeries considered as treatment. Overall survival was the primary study outcome, and calculated as the time between diagnosis and death. April 30, 2010 was the end of follow-up and all patients had at least 1 year follow-up information on this date (median follow- up = 16.7 months). Univariate comparisons of demographic, tumour and treatment variables between living and deceased patient groups were performed using Chi-square tests. Survival estimates were calculated using the Kaplan-Meier method; log-rank tests were used to compare survival differences.  Haplotype analysis was performed using HAPSTAT software167. SNPs were each fit using additive, dominant, recessive and codominant models. Cox proportional hazards regression was used to estimate the effect of SNPs adjusted for patient age, tumour location,   83    disease stage and treatment received. For each hazard ratio (HR), a 95% confidence interval (95%CI) was calculated. P-values less than 0.05 were considered statistically significant. The false discovery rate (FDR) method 168 was applied to address multiple comparisons, and FDR values of <0.2 were considered as the least likely to be due to a false positive finding. Given 85 cases and assuming 80% power, 0.05 type I error probability, a range of MAF values was used to calculate the minimum detectable hazard ratios (Figure 6.2). 6.3 Results During the study period 202 esophageal and gastroesophageal junction (GEJ) adenocarcinomas patients diagnosed from Jan 1 2008 to 30 April 2009 were assessed for eligibility. Fifty-five of these 202 patients were excluded: 15 (24%) patients who were deceased at the time of assessment, 18 (33%) patients who were deceased at time of contact and 22 (40%) patients who were unable to consent because of poor health. The total number of eligible cases for the study was 147 patients.  Of these, 4 (2.7%) could not be contacted, 31 (21%) did not reply to after repeated study invitations, and 8 (5.4%) refused to participate. Biological samples appropriate for DNA extraction were received for 93 cases. Genotyping results were available for 88 (98%) of the sample; 85 cases satisfied quality control restrictions and thus were available for analysis. Men accounted for 91% of the sample, and the median age of diagnosis was 63 years. With regard to BMI, 27% of cases were normal, 43% were overweight and 30% were obese. About half of the patients (48%) had a tumour in the esophagus (Siewert I) and the others had a tumour in the GEJ or cardia. The majority of patients received chemotherapy (65%) and radiation (65%) as their primary treatment, and 45% underwent surgery before the time of recruitment. Stage could be assessed for 94% of the patients: 59% had local/regional disease and 41% had   84    metastatic disease. The stages of non-metastatic cases were: 6.4% IA, 4.3% IIA, 34.0% IIB, 42.6% IIIA, 6.4% IIIB and 6.4% IIIC. Table 6.1 lists the SNPs genotyped. Estimates from a Cox model of survival associated with TIMP genetic variations are shown in table 6.2. Variation in MMP2 markers (rs243842 and rs243847) showed significant association with patient survival in the codominant model. Other models and other MMP gene SNPs were not significant. Among TIMP genes, significant association of variations in TIMP3 with the survival of adenocarcinoma of the esophagus and GEJ cancer patients was observed. The SNPs and the linkage disequilibrium (LD) structure plot for TIMP3 is shown in Figure 6.2. Of 14 SNPs tested in TIMP3, there were significant associations for rs130274, rs1962223, rs5754312, rs715572 in all (i.e., codominant, additive, recessive, or dominant) adjusted and unadjusted models. These four SNPs also passed the multiple testing comparison criterion (i.e., FDR < 0.2).  Table 6.3 shows the survival model for TIMP3 variations after adjusting for age, tumour location, disease stage and treatment. A genetic polymorphism in the promoter region of TIMP3 (rs1962223) was associated with about a 3-fold increase in the HR for patients who carried the CG genotype. This association was observed after adjustment for patient age, tumour location, disease stage and treatment received. rs1962223 and rs130274 were in moderate LD (r2=0.56). rs130274 showed a more than 3-fold increased HR for the dominant and codominant models and 2-fold increased HR for the additive model. rs715572 was associated with about a 3-fold increased HR for the dominant and codominant models and a 2-fold reduction in the HR for the additive model; rs5754312 was associated with a 4-fold reduction in the HR in the recessive and codominant model, and a 2-fold reduction in the HR for the additive model. In part because of the low number or lack of homozygotes for the minor allele at rs130274, rs715572 and rs1962223, a   85    dominant model best describes the association of these SNPs  with the survival of cancer patients. For rs5754312, a recessive model best describes the association. In haplotype analysis, a block including rs5754312, rs715572 and showed significant association with the patients’ survival (p=0.002). Figure 6.3 shows the survival of patients by SNPs and haplotype. There were also associations for rs137485 and rs9619311 with the HR in the dominant and additive models respectively. 6.4 Conclusion Adenocarcinoma of the esophagus and GEJ is one of the most aggressive forms of human malignancies. In spite of improvements in the management of this disease, the 5-year overall survival after treatment hardly exceeds 25% 20. Patients with adenocarcinoma of the GEJ and gastric cardia (Siewert II and III) have distinctively worse survival than patients with lower gastric and lower esophageal adenocarcinoma  169. TNM cancer staging systems predict survival on the basis of the anatomic extent of the tumour. Other factors, however, including host factors, should be considered for determining and predicting the outcome of these patients. We demonstrated that polymorphisms at TIMP3 are prognostic factors for this cancer. The TIMP gene products are important in human cancers.  The family of homologous proteins corresponding to the tissue inhibitors of metalloproteinases are expressed by a variety of cell types and present in most tissues and body fluids. TIMP proteins are natural inhibitors of proteolysis activity MMP, and adamalysins (ADAMs and ADAMTS) proteins170. Numerous reports suggest that TIMP proteins act at various steps of tumour progression including invasion, metastasis, growth and angiogenesis 77. The protein product of TIMP3 is a 24-kDa protein that, unlike other TIMP protein family members, binds to the ECM. The protein corresponding to TIMP3 acts as a suppressor in some cancers by affecting tumour growth, angiogenesis, invasion   86    and the development of metastases 76,171,172. TIMP3 also been reported to induce apoptosis of cancer cells 173. The study has several strengths. A prospective design allows study of current treatments for esophagus and GEJ adenocarcinomas. It also permits the collection of biological samples, allows the verification of clinical and patient information, and greatly simplifies ethical issues regarding patient participation and use of personal information. This study includes patients from the entire province of British Columbia, and is not restricted to a single hospital or clinic. Additionally, our provence-wide approach is not biased by treatment disparity because all BC residents are covered for healthcare through the BC Medical Services Plan (MSP). Moreover, the GI Tumour Group at the BC Cancer Agency (BCCA) provides care for all patients in the province, and devises province-wide treatment guidelines and protocols. Our use of a candidate gene design addresses genetic pathways of known biological relevance, and is based on a prior hypothesis for each gene. This approach simplifies interpretation of findings based on the biological plausibility of each gene and minimizes loss of study power due to correction for multiple tests. A limitation of the study is that patients with esophagus and GEJ adenocarcinomas have poor survival and are often diagnosed with significant comorbidities or poor physical performance. Because of this limitation, our results do not apply to patients with very short survival (i.e. less than 2 months) or additional substantial health problems. Compared to other cancers, adenocarcinoma of the esophagus and GEJ is a rare disease and a consortium of research groups would be required to obtain enough samples to detect lower predictive and prognostic effect size (i.e., smaller HRs). This study shows that one polymorphism in the promoter region and three intronic tagSNPs of TIMP3 predict survival of patients with adenocarcinoma of the esophagus and GEJ.. This   87    conclusion is consistent with other studies that showed  associations of the methylation status of TIMP3 with cancer outcomes 174-178. Some reports suggest that reduced expression of the TIMP3 protein has a dual role in esophageal adenocarcinoma. There seems to be both an early role in the development of tumours via Barrett’s esophagus, and a later role in the invasive and metastatic phases, the latter leading to worse patient survival 178,179. It has been also shown that aberrant hypermethylation of the human TIMP3 is directly responsible for the transcriptional inactivation of its expression in gastric cancer cell lines. This supports the recent extensive accumulation of evidence about TIMP3 and tumour progression/invasion 180. SNPs in TIMP3 have been associated with breast cancer prognosis 181,182. To our knowledge, this study is the first report of the association of genetic polymorphisms of TIMP3 with survival of gastric or esophageal cancers. Adenocarcinoma of the esophagus and GEJ are deadly diseases that are often diagnosed at a stage when the treatment options are limited and have limited effectiveness. Our results do not establish an exact mechanism by which TIMP3 affects survival. Other reports suggest that TIMP3 is involved in variety of steps affecting cancer progression, including the induction of apoptosis183 and anti-angiogenesis184 The latter might involve directly binding to VEGF receptor 2 or  inhibiting ADAM-17 activity 185. Regardless of the mechanisms, factors that affect regulation of TIMP3 expression, including promoter methylation or genetic variation, could be a promising prognostic factor or therapeutic target for this cancer. Modeling prognosis based on host factors including genetic polymorphisms is an emerging field of translational research. Compared to tumour, constitutional genetic material is relatively easy to obtain, and can be assessed before treatment is started.  88   Table 6.1. SNPs in TIMP and MMP genes used for survival analyses.  Gene Name Reference ID Chromosome Genomic position Alleles Location in Gene HapMap MAF HWE p* GENOTYPE %* MAF* Alleles* MMP2 rs11541998 16 54094264 C/G SYNONYMOUS CODING 0.102 1 100 0.1 C/G MMP2 rs11639960 16 54090771 A/G INTRONIC 0.35 0.905 98.8 0.286 T/C MMP2 rs17301608 16 54076111 C/T INTRONIC 0.385 0.563 98.8 0.304 C/T MMP2 rs1992116 16 54085392 G/A INTRONIC 0.469 0.437 100 0.347 C/T MMP2 rs243842 16 54084923 T/C INTRONIC 0.36 0.324 100 0.418 T/C MMP2 rs243847 16 54081499 T/C INTRONIC 0.398 0.397 100 0.441 A/G MMP2 rs243865 16 54069307 C/T UPSTREAM 0.243 1 100 0.2 C/T MMP2 rs7201 16 54097115 A/C 3PRIME UTR 0.451 0.945 97.6 0.355 T/G MMP7 rs10502001 11 101903803 C/T NON SYNONYMOUS CODING 0.205 0.074 100 0.253 G/A MMP7 rs11225308 11 101904688 T/G INTRONIC 0.21 0.056 98.8 0.25 A/C MMP7 rs12184413 11 101894798 C/T DOWNSTREAM 0.102 1 100 0.118 G/A MMP7 rs12285347 11 101901817 T/C INTRONIC 0.467 0.162 100 0.453 A/G MMP7 rs1996352 11 101901457 C/T INTRONIC 0.23 1 100 0.206 T/C MMP7 rs495041 11 101895398 C/T DOWNSTREAM 0.128 1 100 0.165 G/A MMP7 rs880197 11 101910881 A/T UPSTREAM 0.235 1 100 0.206 A/T MMP9 rs17576 20 44073632 A/G NON SYNONYMOUS CODING 0.363 0.153 100 0.353 A/G MMP9 rs3918261 20 44076999 A/G INTRONIC 0.164 1 100 0.165 T/C TIMP1 rs4898 X 47329929 T/C SYNONYMOUS CODING 0.485 0 100 0.494 C/T TIMP1 rs6609533 X 47330230 A/G INTRONIC 0.485 0 100 0.494 C/T TIMP2 rs12452379 17 74427053 C/A INTRONIC 0.496 1 100 0.429 G/T TIMP2 rs12600817 17 74413060 G/A INTRONIC 0.487 1 100 0.476 A/G TIMP2 rs2277700 17 74378306 C/T INTRONIC 0.254 1 100 0.188 T/C TIMP2 rs2377004 17 74382054 C/T INTRONIC 0.382 0.65 100 0.335 T/C TIMP2 rs2889529 17 74409070 A/G INTRONIC 0.429 0.749 100 0.459 A/G TIMP2 rs4789932 17 74435870 G/A UPSTREAM 0.442 0.447 98.8 0.393 C/T 89  Gene Name Reference ID Chromosome Genomic position Alleles Location in Gene HapMap MAF HWE p* GENOTYPE %* MAF* Alleles* TIMP2 rs4789936 17 74409569 C/T INTRONIC 0.496 1 100 0.471 A/G TIMP2 rs6416835 17 74420830 A/G INTRONIC 0.433 1 100 0.394 C/T TIMP2 rs7211674 17 74410660 C/A INTRONIC 0.438 0.779 100 0.447 A/C TIMP2 rs7212662 17 74429726 T/G INTRONIC 0.456 0.704 100 0.494 A/C TIMP2 rs8064344 17 74388569 C/T INTRONIC 0.25 1 100 0.188 T/C TIMP2 rs8068674 17 74419040 C/T INTRONIC 0.425 0.779 100 0.447 C/T TIMP3 rs130274 22 31534334 C/T INTRONIC 0.23 0.974 100 0.259 C/T TIMP3 rs135029 22 31570290 A/G INTRONIC 0.314 1 98.8 0.31 C/T TIMP3 rs137485 22 31584283 T/A INTRONIC 0.279 0.905 98.8 0.286 A/T TIMP3 rs137487 22 31589104 A/G DOWNSTREAM 0.473 0.907 100 0.459 G/A TIMP3 rs137489 22 31592935 T/C DOWNSTREAM 0.235 0.855 100 0.241 A/G TIMP3 rs1427378 22 31582041 A/G INTRONIC 0.254 1 100 0.276 A/G TIMP3 rs1962223 22 31523905 G/C UPSTREAM 0.183 0.139 100 0.165 C/G TIMP3 rs2040435 22 31593431 C/T DOWNSTREAM 0.288 1 100 0.165 C/A TIMP3 rs242072 22 31565517 C/T INTRONIC 0.478 0.712 100 0.482 T/C TIMP3 rs242077 22 31559685 T/C INTRONIC 0.392 0.984 98.8 0.417 C/T TIMP3 rs5754312 22 31574421 A/T INTRONIC 0.473 0.948 100 0.482 A/T TIMP3 rs715572 22 31564931 G/A INTRONIC 0.221 0.657 100 0.188 C/T TIMP3 rs738992 22 31540005 C/T INTRONIC 0.5 0.162 100 0.471 C/T TIMP3 rs9606994 22 31523050 G/A UPSTREAM 0.483 0.642 100 0.429 G/A TIMP3 rs9619311 22 31526693 T/C UPSTREAM 0.292 0.235 100 0.312 A/G TIMP4 rs308952 3 12129428 A/G INTRONIC 0.107 1 97.6 0.133 G/A TIMP4 rs3755724 3 12175906 C/T REGULATORY REGION 0.35 0.898 100 0.341 G/A  *Observed at study cohort     90   Table 6.2. Hazard ratios (HR) and 95% confidence intervals (CI) estimates for the association between TIMP and MMP gene variations and survival. (Unadjusted)   Gene SNP ID Alleles NO (Freq) Codominant model HR(95%CI) P Additive model HR(95%CI) Dominant Model HR(95%CI) Recessive model HR(95%CI) MMP2 rs11541998 CC 69(81.2%) REF 0.709 0.726(0.316,1.667) 0.747(0.315,1.773) 0.049(0,78936.855)   CG 15(17.6%) 3183.68(0.00, E)   GG 1(1.2%) 2502.46(0.00, E)  rs11639960 TT 42(50.0%) REF 0.412 0.709(0.426,1.179) 0.69(0.374,1.275) 0.518(0.125,2.147)   TC 36(42.9%) 0.74(0.39, 1.39)   CC 6(7.1%) 0.45(0.11, 1.92)  rs17301608 CC 39(46.4%) REF 0.823 0.867(0.525,1.43) 0.884(0.485,1.609) 0.667(0.161,2.768)   TC 39(46.4%) 0.92(0.50, 1.70)   TT 6(7.1%) 0.64(0.15, 2.74)  rs1992116 CC 34(40.0%) REF 0.825 0.862(0.537,1.384) 0.835(0.455,1.533) 0.827(0.295,2.318)   TC 43(50.6%) 0.85(0.45, 1.60)   TT 8(9.4%) 0.76(0.26, 2.24)  rs243842 CC 26(30.6%) REF 0.032 0.995(0.608,1.628) 0.649(0.349,1.208) 1.992(0.953,4.162)   TC 47(55.3%) 0.52(0.26, 1.02)   TT 12(14.1%) 1.33(0.59, 3.02)  rs243847 AA 24(28.2%) REF 0.043 0.988(0.61,1.599) 0.638(0.34,1.197) 1.811(0.891,3.681)   AG 47(55.3%) 0.51(0.26, 1.01)   GG 14(16.5%) 1.18(0.53, 2.64)  rs243865 CC 54(63.5%) REF 0.814 0.841(0.463,1.526) 0.811(0.421,1.56) 0.992(0.135,7.301)   TC 28(32.9%) 0.80(0.41, 1.57)   TT 3(3.5%) 0.92(0.12, 6.86)  rs7201 AA 35(42.2%) REF 0.793 0.857(0.546,1.345) 0.819(0.445,1.509) 0.815(0.32,2.077)   AC 37(44.6%) 0.84(0.44, 1.61)   CC 11(13.3%) 0.75(0.28, 2.01) 91  Gene SNP ID Alleles NO (Freq) Codominant model HR(95%CI) P Additive model HR(95%CI) Dominant Model HR(95%CI) Recessive model HR(95%CI)  MMP7 rs10502001 GG 51(60.0%) REF 0.988 0.971(0.618,1.526) 0.975(0.524,1.814) 0.921(0.329,2.583)   AG 25(29.4%) 0.99(0.50, 1.97)   AA 9(10.6%) 0.92(0.32, 2.64) rs11225308 CC 51(60.7%) REF 0.834 1.041(0.672,1.613) 1.133(0.613,2.093) 0.888(0.317,2.488)   AC 24(28.6%) 1.21(0.62, 2.36)   AA 9(10.7%) 0.94(0.33, 2.71)  rs12184413 GG 66(77.6%) REF 0.021 0.779(0.365,1.666) 0.669(0.298,1.505) 8.823(1.118,69.648)   AG 18(21.2%) 0.58(0.25, 1.38)   AA 1(1.2%) 8.01(1.01, 63.45)  rs12285347 AA 29(34.1%) REF 0.218 0.868(0.586,1.285) 1.083(0.56,2.093) 0.567(0.262,1.227)   AG 35(41.2%) 1.37(0.69, 2.74)   GG 21(24.7%) 0.68(0.28, 1.66)  rs1996352 TT 53(62.4%) REF 0.841 0.893(0.519,1.537) 0.926(0.497,1.724) 0.559(0.076,4.095)   TC 29(34.1%) 0.97(0.51, 1.83)   CC 3(3.5%) 0.55(0.07, 4.10) rs495041 GG 59(69.4%) REF 0.756 0.932(0.502,1.731) 0.879(0.451,1.714) 1.774(0.24,13.099)   AG 24(28.2%) 0.84(0.42, 1.68)   AA 2(2.4%) 1.69(0.23, 12.58)  rs880197 AA 53(62.4%) REF 0.841 0.893(0.519,1.537) 0.926(0.497,1.724) 0.559(0.076,4.095)   TA 29(34.1%) 0.97(0.51, 1.83)   TT 3(3.5%) 0.55(0.07, 4.10) MMP9 rs17576 AA 32(37.6%) REF 0.84 1.145(0.705,1.859) 1.211(0.639,2.294) 1.098(0.391,3.081)   AG 46(54.1%) 1.21(0.63, 2.33)   GG 7(8.2%) 1.23(0.41, 3.76)  rs2274755 CC 59(69.4%) REF 0.183 0.543(0.279,1.058) 0.556(0.273,1.13) 0.047(0,82.314)   AC 24(28.2%) 0.61(0.30, 1.25)   AA 2(2.4%) 0.00(0.00, E) 92  Gene SNP ID Alleles NO (Freq) Codominant model HR(95%CI) P Additive model HR(95%CI) Dominant Model HR(95%CI) Recessive model HR(95%CI) MMP9 rs3918261 TT 59(69.4%) REF 0.183 0.543(0.279,1.058) 0.556(0.273,1.13) 0.047(0,82.314)   TC 24(28.2%) 0.61(0.30, 1.25)   CC 2(2.4%) 0.00(0.00, E ) TIMP1 rs4898 CC 40(47.1%) REF 0.983 1.025(0.752,1.397) 1.055(0.579,1.922) 1.04(0.571,1.894)   TC 6(7.1%) 1.09(0.32, 3.66)   TT 39(45.9%) 1.05(0.57, 1.95) rs6609533 CC 40(47.1%) REF 0.983 1.025(0.752,1.397) 1.055(0.579,1.922) 1.04(0.571,1.894)   TC 6(7.1%) 1.09(0.32, 3.66)   TT 39(45.9%) 1.05(0.57, 1.95) TIMP2 rs12452379 GG 28(32.9%) REF 0.603 0.927(0.606,1.416) 1.143(0.508,2.57) 0.765(0.392,1.492)   TG 41(48.2%) 1.39(0.70, 2.79)   TT 16(18.8%) 1.08(0.42, 2.74)  rs12600817 AA 23(27.1%) REF 0.962 0.955(0.623,1.464) 0.912(0.476,1.75) 0.978(0.468,2.043)   AG 43(50.6%) 0.91(0.45, 1.82)   GG 19(22.4%) 0.92(0.39, 2.16)  rs2277700 TT 56(65.9%) REF 0.243 1.231(0.7,2.167) 1.115(0.595,2.091) 3.224(0.761,13.657)   TC 26(30.6%) 1.02(0.53, 1.96)   CC 3(3.5%) 3.24(0.75, 13.94)  rs2377004 TT 36(42.4%) REF 0.663 1.249(0.771,2.025) 1.285(0.692,2.388) 1.38(0.489,3.891)   TC 41(48.2%) 1.25(0.66, 2.36)   CC 8(9.4%) 1.56(0.52, 4.72)  rs2889529 AA 26(30.6%) REF 0.733 1.154(0.762,1.748) 1.316(0.663,2.612) 1.114(0.548,2.266)   AG 40(47.1%) 1.31(0.64, 2.71)   GG 19(22.4%) 1.32(0.56, 3.12)  rs4789932 CC 33(39.3%) REF 0.614 0.806(0.52,1.25) 0.746(0.404,1.377) 0.764(0.322,1.815)   TC 36(42.9%) 0.78(0.40, 1.50)   TT 15(17.9%) 0.67(0.27, 1.69)   93  Gene SNP ID Alleles NO (Freq) Codominant model HR(95%CI) P Additive model HR(95%CI) Dominant Model HR(95%CI) Recessive model HR(95%CI) TIMP2 rs4789936 AA 24(28.2%) REF 0.872 0.924(0.604,1.415) 0.847(0.447,1.604) 0.978(0.468,2.043)   AG 42(49.4%) 0.83(0.42, 1.65)   GG 19(22.4%) 0.88(0.38, 2.03) rs6416835 CC 31(36.5%) REF 0.542 0.993(0.648,1.521) 1.178(0.628,2.206) 0.702(0.276,1.785)   TC 41(48.2%) 1.30(0.68, 2.50)   TT 13(15.3%) 0.82(0.30, 2.25)  rs7211674 AA 27(31.8%) REF 0.344 1.345(0.883,2.047) 1.664(0.82,3.379) 1.329(0.652,2.706)   AC 40(47.1%) 1.61(0.77, 3.38)   CC 18(21.2%) 1.81(0.75, 4.35)   rs7212662  AA 23(27.1%) REF 0.349 1.055(0.704,1.579) 1.455(0.698,3.035) 0.81(0.399,1.647)     AC 40(47.1%) 1.65(0.77, 3.55) CC 22(25.9%) 1.13(0.46, 2.79)  rs8064344 TT 56(65.9%) REF 0.243 1.231(0.7,2.167) 1.115(0.595,2.091) 3.224(0.761,13.657)   TC 26(30.6%) 1.02(0.53, 1.96)   CC 3(3.5%) 3.24(0.75, 13.94)  rs8068674 CC 27(31.8%) REF 0.882 1.092(0.711,1.678) 1.062(0.544,2.072) 1.199(0.589,2.44)   TC 40(47.1%) 1.01(0.49, 2.05)   TT 18(21.2%) 1.20(0.52, 2.79) TIMP3 rs130274 CC 46(54.1%) REF 0.025 1.961(1.191,3.228) 2.113(1.149,3.884) 2.632(0.785,8.82)   TC 34(40.0%) 2.00(1.07, 3.75)   TT 5(5.9%) 3.68(1.04, 12.97)  rs135029 CC 40(47.6%) REF 0.335 1.34(0.846,2.12) 1.291(0.694,2.402) 1.856(0.779,4.424)   TC 36(42.9%) 1.16(0.60, 2.24)   TT 8(9.5%) 2.00(0.79, 5.08)  rs137485 AA 42(50.0%) REF 0.423 1.329(0.817,2.161) 1.297(0.705,2.384) 1.851(0.657,5.218)   AT 36(42.9%) 1.206(0.638, 2.281)   TT  6(7.1%)  2.026(0.685, 5.996)  94  Gene SNP ID Alleles NO (Freq) Codominant model HR(95%CI) P Additive model HR(95%CI) Dominant Model HR(95%CI) Recessive model HR(95%CI) TIMP3 rs137487 GG 24(28.2%) REF 0.127 1.406(0.912,2.167) 2.247(0.999,5.053) 1.151(0.55,2.412)   AG 44(51.8%) 2.088(0.775, 5.623)   AA 17(20.0%) 2.306(1.003, 5.300)  rs137489 AA 48(56.5%) REF 0.152 0.839(0.503,1.398) 1.022(0.553,1.887) 0.044(0,8.164)   AG 33(38.8%) 1.25(0.676, 2.32)   GG 4(4.7%) 0(0.00, )  rs1427378 AA 44(51.8%)  0.258 0.735(0.45,1.2) 0.818(0.449,1.492) 0.22(0.03,1.603)   AG 35(41.2%) 0.22(0.03, 1.60)   GG 6(7.1%) 0.96(0.52, 1.76)  rs1962223 CC 57(67.1%) REF 0.012 2.155(1.17,3.967) 2.155(1.17,3.967) NA   CG 28(32.9%) 2.16(1.17, 3.97)   GG 0(0%)  rs242072 TT 24(28.2%) REF 0.869 0.898(0.598,1.35) 0.886(0.461,1.7) 0.837(0.412,1.7)   TC 40(47.1%) 0.93(0.46, 1.88)   CC 21(24.7%) 0.80(0.35, 1.83)  rs242077 CC 29(34.5%) REF 0.833 1.127(0.749,1.696) 1.212(0.64,2.296) 1.135(0.543,2.37)   TC 40(47.6%) 1.19(0.60, 2.37)   TT 15(17.9%) 1.25(0.54, 2.90) rs5754312 AA 22(25.9%) REF 0.036 0.633(0.41,0.979) 0.773(0.395,1.514) 0.281(0.1,0.787)   TA 44(51.8%) 1.03(0.52, 2.05)   TT 19(22.4%) 0.29(0.09, 0.89)  rs715572 CC 57(67.1%) REF 0.01 1.709(1.092,2.674) 2.367(1.296,4.322) 1.095(0.264,4.537)   TC 24(28.2%) 2.53(1.36, 4.71)   TT 4(4.7%) 1.49(0.35, 6.35)  rs738992 CC 20(23.5%) REF 0.913 1.106(0.696,1.757) 1.134(0.543,2.369) 1.136(0.545,2.372)   TC 50(58.8%) 1.11(0.52, 2.37)   TT 15(17.6%) 1.22(0.48, 3.09)  95  Gene SNP ID Alleles NO (Freq) Codominant model HR(95%CI) P Additive model HR(95%CI) Dominant Model HR(95%CI) Recessive model HR(95%CI) TIMP3 rs9606994 GG 26(30.6%) REF 0.305 1.037(0.672,1.6) 1.414(0.696,2.873) 0.692(0.291,1.644)   AG 45(52.9%) 1.59(0.77, 3.28)   AA 14(16.5%) 0.95(0.34, 2.62)  rs9619311 AA 43(50.6%) REF 0.082 0.586(0.362,0.949) 0.578(0.313,1.066) 0.272(0.066,1.125)   AG 31(36.5%) 0.71(0.38, 1.34)   GG 11(12.9%) 0.23(0.06, 0.99) TIMP4 rs308952 GG 62(74.7%) REF 0.65 1.149(0.614,2.15) 1.219(0.631,2.356) 0.049(0,89202.191)   AG 20(24.1%) 1.26(0.65, 2.44)   AA 1(1.2%) 0.00(0.00,E.)  rs3755724 GG 36(42.4%) REF 0.383 1.077(0.672,1.727) 0.911(0.498,1.666) 1.705(0.717,4.054)   AG 40(47.1%) 0.80(0.42, 1.53)   AA 9(10.6%) 1.52(0.61, 3.83)                    96  Table 6.3.. Hazard ratio (HR) and 95% confidence interval (CI) estimated for the association between TIMP3 gene variations and survival of the study cohort adjusted for patient age, tumour location, disease  stage and treatments.  SNP ID Alleles No. (Freq) Codominant model HR(95%CI)p Additive *model HR(95%CI) p Dominant model HR(95%CI) p Recessive model HR(95%CI) P rs130274 CC 42 REF 1.918(1.157, 3.179) 0.012 3.039(1.455, 6.348) 0.003 1.363(0.39, 4.766) 0.628   TC 33 3.273(1.503, 7.126)   TT 5 3.68(1.04, 12.97) rs135029 CC 39 REF 1.464(0.883, 2.427) 0.14 1.359(0.706, 2.615) 0.359 2.555(0.932, 7.001) 0.068   TC 33 1.163(0.578, 2.34)   TT 7 2.724(0.949, 7.82) rs137485 AA 41 REF 1.381(0.826, 2.309) 0.218 1.305(0.68, 2.505) 0.423 2.367(0.779, 7.187) 0.129   AT 32 1.142(0.57, 2.29)   TT 6 2.478 (0.794, 7.738) rs137487 GG 22 REF 1.633(0.956, 2.79) 0.072 2.689(0.99, 7.303) 0.052 1.368(0.613, 3.055) 0.444   AG 41 3.09(0.941, 10.145)   AA 17 2.608(0.949, 7.167) rs137489 AA 46 REF 0.875(0.485, 1.58) 0.658 1.022(0.517, 2.019) 0.951 0(0, .) 0.976   AG 30 1.195(0.60, 2.38)   GG 4 0(0.00, ) rs1427378 AA 42 REF 0.612(0.332, 1.131) 0.117 0.673(0.333, 1.364) 0.272 0.229(0.028, 1.867) 0.169   AG 33 0.724(0.361, 1.45)   GG 5 0.179(0.0, 1.565) rs1962223 CC 53 REF 2.722(1.373, 5.398) 0.004 2.722(1.373, 5.398) 0.004 NA NA   CG 27 2.72(1.373, 5.4)   GG 0 - rs242072 TT 23 REF 0.977(0.615, 1.552) 0.922 1.032(0.48, 2.216) 0.936 0.903(0.404, 2.018) 0.804   TC 38 0.948(0.37, 2.435)   CC 19 1.087(0.473, 2.5) 97  SNP ID Alleles No. (Freq) Codominant model HR(95%CI)p Additive *model HR(95%CI) p Dominant model HR(95%CI) p Recessive model HR(95%CI) P rs242077 CC 28 REF 1.408(0.889, 2.228) 0.144 1.737(0.851, 3.546) 0.13 1.417(0.597, 3.366) 0.43   TC 38 1.687(0.789, 3.608)   TT 13 1.874(0.708,4.963) rs5754312 AA 22 REF 0.577(0.349, 0.954) 0.032 0.677(0.332, 1.378) 0.282 0.251(0.078, 0.812) 0.021   TA 40 0838(0.412, 1.706)   TT 18 0.222(0.062, 0.790) rs715572 CC 53 REF 2.238(1.328, 3.77) 0.002 2.79(1.455, 5.349) 0.002 2.086(0.477, 9.118) 0.329   TC 23 2.752(1.415, 5.351)   TT 4 3.25(0.714, 14.785) rs738992 CC 18 REF 1.013(0.593, 1.728) 0.963 1.316(0.552, 3.136) 0.535 0.777(0.326, 1.853) 0.569   TC 48 1.387(0.579, 3.322)   TT 14 1.016(0.326, 3.172) rs9606994 GG 24 REF 0.969(0.607, 1.547) 0.896 1.395(0.64, 3.042) 0.403 0.591(0.237, 1.471) 0.258   AG 44 0.832(0.283, 2.452)   AA 12 1.666(0.744, 3.731) rs9619311 AA 41 REF 0.561(0.323, 0.977) 0.041 0.51(0.255, 1.019) 0.057 0.342(0.076, 1.536) 0.161   AG 29 0.586(0.280, 1.223)   GG 10 0.290(0.064, 1.312)  * The effect of the minor allele on the risk of disease is changed by a factor equal to the number of copies         98    Figure 6.1 Minimum detectable Hazard Ratio by different MAF. Calculation is based on 85 patients, 80% power, Type I error probability =0.05. 0.8 0.85 0.9 0.95 1 1.5 2.0 2.5 3.0 3.5 4.0 Detectable HR 0.1 0.2 0.3 0.4 0.5 MAF 99     Figure 6.2. Linkage disequilibrium (LD) plot for TIMP3. Numbers in the plot are r2 values; boxes with dark color illustrate high LD; lighter shading represent weak LD. Circled SNPs show significant association with survival in the study cohort.   100   Figure 6.3. Survival of study cohort by TIMP3 variations, rs130274, rs1962223, rs5754312, rs715572 and rs5754312/rs715572 haplotype rs130274 rs196223 rs5754312 rs715572 rs5754312/rs715572 101  CHAPTER 7: Conclusions  Studies of human cancer susceptibility examine factors associated with the incidence of disease. Studies of human cancer prognosis and prediction examine factors associated with the outcomes of disease. This dissertation is about molecular and related factors that affect gastric and esophageal cancer patients’ prognosis (i.e., survival). Chapter 2 indicated that polymorphisms in genes associated with cell cycle, xenobiotic metabolism, DNA repair and signalling and growth factors have prognostic significance for both gastric and esophageal cancer. Understanding mechanisms of each polymorphism and pathway- based analyses might help identify markers for gastric and esophageal cancer patient survival. There is an increasing interest in the effect of host genetic polymorphisms on the survival of cancer patients. Generally, conventional techniques do not adequately predict the heterogeneity of patient outcomes. In many cancers, tumour markers have been used as a factor for survival models and guiding treatment decisions. However in gastric and esophageal cancer, adequate tumour samples for these assays may not be easily available. Modeling prognosis based on host factors, including genetic polymorphisms, is an emerging field of translational research. Based on population-based registry data (chapter 3), it is concluded that gastric and esophageal cancers are relatively infrequent in Canada, although their epidemiology is changing. The trends are most evident when tumours are classified by histology and anatomic location. While incidence of squamous cell carcinoma of the esophagus and adenocarcinoma of the lower stomach appears to be stable or decreasing, the incidence of esophageal adenocarcinoma and gastric cardia cancer are increasing. Both gastric and esophageal cancer patients have poor survival. 102  In an international comparison of gastric and esophageal cancer (chapter 4), differences in survival between patients in BC, Canada and Ardabil, Iran have been found. Based on this study, it is concluded that disease characteristics and patient factors, not solely differences in healthcare systems, are likely responsible for the survival difference in these populations. Even so, the outcomes of these cancers are poor for both populations and improvements in diagnosis and management are urgently needed. It has shown that for gastric and esophageal cancers, there are significant differences in survival among ethnic groups in BC (chapter 5). Ethnicity may represent underlying genetic factors. Such factors could influence host-tumour interactions by altering the tumour’s etiology and therefore its chance of spreading. Alternatively, genetic factors may determine response to treatments. Finally, ethnicity may represent non-genetic factors that affect survival. Differences in survival by ethnicity support the importance of ethnicity as a prognostic factor, and may provide clues for the future identification of genetic or lifestyle factors that underlie these observations. Based on a prospective cohort study, it demonstrated that genetic polymorphism in the promoter region and three intronic TagSNPs of TIMP3 predict survival of patients with adenocarcinoma of esophagus and GEJ. Regardless of the mechanisms, factors that affect regulation of TIMP3 expression, including promoter methylation or genetic variation, could be a promising prognostic factor or therapeutic target for this cancer. Modeling prognosis based on host factors including genetic polymorphisms is an emerging field of translational research. Compared to tumour, the constitutional genetic material is easy to obtain, and can be assessed before treatment is started. 103  7.1 Opportunities and challenges In BC, population-based registry data with outcome information are available and make possible studies about prognostic factors at the population level. Additionally, all BC residents are covered for healthcare through the BC Medical Services Plan (MSP) and the BC Cancer Agency (BCCA) provides care for all cancer patients in the province (using province-wide treatment guidelines and protocols). The biggest challenge with BC registry data was the lack of ethnicity information. This required us to use a proxy method (i.e., name lists) to assign ethnicity. With the collaboration of Iranian scientists, access to population-based registry data for Ardabil, Iran was made posible. Ardabil has the highest incidence of gastric and esophageal cancer in the world. There are some meaningful challenges in registry information and health care in Ardabil; including vast immigration, an uncoordinated system of medical services and patients’ repeated referral to different centers for diagnosis and treatment. Additionally, lack of complete information did not allow us to adjust our results for staging or follow-up, giving only a very limited view of prognosis. The biggest challenge in our prospective study was the lack of operational funding thereby causing difficulties in recruitment and genotyping. Despite this, the support of GI tumour group physician, made it possible to recruit patients and collect biological samples with minimal cost. On the other hand, improvements in technology made it possible to genotype our samples at a substantially lower cost. 7.2 Future directions The dissertation leaves some unanswered questions that would benefit from subsequent research. First, there is a need for prospective studies with longer recruitment period. The prospective GE study successfully set up a patient recruitment system. Continuation of patient ascertainment 104  would produce a valuable cohort of patient to allow detection of more prognostic markers. In addition, genetic results from this dissertation should be validated in other, national and international, gastric and esophageal patient cohorts. It would be useful to study of TIMP3 expression in tumour to understand the correlation between TIMP3 polymorphism and gene expression (using a tissue microarray (TMA)) on survival of GEJ adenocarcinoma. One potential study would be the comparison of TIMP3 polymorphism and expression between BC and Iran. Currently, Iranian collaborators are collecting DNA and tumour specimens from cardia and esophageal cancers. Comparing specimens from the BC and Iranian populations could produce valuable information about these cancers. Finally, there is a need to study factors underlying the effect of ethnicity on survival of gastric and esophageal cancer. This requires studies of both genetic and environment, including quantitative studies on lifestyle and behavior to address the complex concept of ethnicity.  105  REFERENCES  1. 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