The Role of DNA Methylation in the Development of Colorectal Neoplasia

The role of DNA methylation in the development of colorectal neoplasia

Justin Jong Leong Wong

BSc (Hons)

July 2008

A thesis submitted for the degree of Doctor of Philosophy in the School of Medical Sciences, University of New South Wales, Australia

Acknowledgements

The completion of this PhD thesis would not have been possible without the support and assistance of the following individuals.

First of all, I would like to thank my principal supervisor Professor Nicholas John Hawkins for his consistent support and constructive criticisms throughout my candidature. I truly appreciate his efforts in organising regular meetings with me to ensure that I am always on the right track. I am also grateful to my co-supervisor Dr Megan Hitchins for her continuous encouragement and technical advice. A special thank you also goes out to Professor Robyn Ward for providing me the facilities to undergo my research and her intellectual input.

I am also grateful to the past and present members of the Molecular and Cellular Oncology group for their constant support, assistance, company and friendship. I would like to express my special thanks to the past and present students in the laboratory; Andrew, Nerida, Chau-To, Joice and James for being such a good company especially during the odd hours.

Finally, I am indebted to my family, especially my mom and dad for their continuous encouragement. A big thank you also goes out to my special friend, Katherine for her cherished love, patience and friendship. Contents Contents………………………………………………………………………….………...i

List of Tables……………………………………………………………………….…….ix

List of Figures………………………………………………………………….……...... xiii

Abbreviations…………………………………………………………………………..xviii

Abstract………………………………………………………………………………....1

CHAPTER 1: Literature review…………………………………………...... 3

1.1 Colorectal neoplasia...... 4 1.1.1 Epidemiology of the disease...... 4 1.1.2 Aetiology of the disease...... 5 1.1.3 Pathology of the disease ...... 5 1.1.3.1 Non-invasive colorectal neoplasms...... 6 1.1.3.1.1 Aberrant crypt foci ...... 6 1.1.3.1.2 Hyperplastic polyps...... 8 1.1.3.1.3 Serrated adenomas ...... 8 1.1.3.1.4 Colorectal adenomas ...... 9 1.1.3.2 Colorectal carcinomas...... 10 1.1.3.3 The adenoma-carcinoma sequence ...... 11 1.1.3.4 The serrated neoplasia pathway ...... 12 1.1.3.5 Pathological staging of colorectal cancer...... 13 1.2 Genetic mechanisms of colorectal neoplasia ...... 15 1.2.1 Chromosomal instability...... 16 1.2.2 Microsatellite instability...... 16 1.2.2.1 Diagnosis and features of microsatellite instability ...... 17 1.3 Genetic basis of colorectal neoplasia ...... 18 1.3.1 Familial cancer syndromes ...... 18 1.3.1.1 Familial adenomatous polyposis (FAP) syndrome ...... 19 1.3.1.2 Other FAP variants...... 20 1.3.1.3 MYH-associated Polyposis (MAP)...... 21 1.3.1.4 HNPCC syndrome...... 22 1.3.2 Sporadic colorectal cancer...... 23

i 1.4 Genetic alterations in cancer development ...... 24 1.4.1 Adenomatous polyposis coli (APC) ...... 24 1.4.2 KRAS...... 26 1.4.3 Loss of 18q ...... 26 1.4.4 p53 ...... 27 1.4.5 BRAF ...... 28 1.5 The “Epigenetic Code” and its changes in colorectal neoplasia...... 29 1.5.1 Epigenetic events in normal human cells ...... 30 1.5.1.1 Chromatin modifications...... 30 1.5.1.2 DNA methylation...... 32 1.5.1.3 RNA modification...... 34 1.5.2 Epigenetic basis of colorectal neoplasia...... 34 1.5.2.1 Global DNA hypomethylation in colorectal cancer...... 35 1.5.2.2 Hypermethylation of CpG islands in colorectal cancer ...... 36 1.5.2.3 MLH1 methylation in MSI colorectal cancer as a classic example of promoter hypermethylation in colorectal cancer ...... 39 1.5.2.4 Dysregulation of chromatin modifications ...... 40 1.6 Patterns of gene methylation in colorectal cancer...... 41 1.6.1 CpG island methylator phenotype (CIMP) and the serrated neoplasia pathway...... 41 1.6.2 Long-range epigenetic silencing...... 44 1.7 CpG island methylation as a marker for the predisposition of colorectal neoplasia...... 46 1.8 Germline epimutation and colorectal cancer...... 47 1.9 Methods for detection of DNA methylation at candidate gene promoters ...... 49 1.9.1 Traditional methods...... 49 1.9.2 Quantitative methods...... 55 1.10 Research aims and outline...... 56

CHAPTER 2 : Materials and methods………….....………………………………….59

2.1 Materials...... 60 2.1.1 Suppliers ...... 60 2.1.2 Oligonucleotide primers ...... 60

ii 2.1.3 Reagents...... 62 2.1.4 Buffers and solutions ...... 63 2.1.5 Kits...... 64 2.1.6 Restriction enzyme ...... 64 2.1.7 DNA modification enzyme and substract...... 64 2.1.8 Growth media for cell line...... 65 2.2 Methods...... 65 2.2.1 Sample collection...... 65 2.2.1.1 Handling of tissue samples ...... 65 2.2.1.2 Handling of blood samples ...... 66 2.2.2 Patient clinical details...... 66 2.2.3 Histopathological data of patients ...... 66 2.2.4 Cell lines...... 67 2.2.5 Cell culture...... 67 2.2.6 Extraction and quantification of DNA...... 68 2.2.6.1 DNA extraction from blood samples ...... 68 2.2.6.2 DNA extraction from tissue and cell lines...... 69 2.2.6.3 DNA quantification...... 69 2.2.7 Microsatellite instability testing ...... 70 2.2.8 Immunohistochemistry ...... 70 2.2.9 CpG Methylase (M.SssI) treatment of DNA ...... 70 2.2.10 Bisulfite treatment of genomic DNA...... 71 2.2.10.1 Denaturation...... 71 2.2.10.2 Bisulfite conversion ...... 71 2.2.10.3 Desalting ...... 71 2.2.10.4 Desulfonation ...... 72 2.2.10.5 Neutralisation and precipitation ...... 72 2.2.11 Construction of the standard curve for quantitative PCR assay ...... 72 2.2.11.1 Plasmid concentration...... 72 2.2.11.2 Serial dilution of plasmid DNA ...... 73 2.2.12 Quantitative PCR assay for methylation detection...... 74 2.2.13 Assessment of assay sensitivity and linearity using artificially mixed DNA with varying percentages of methylation...... 75

iii 2.2.14 Agarose gel electrophoresis...... 75 2.2.15 Purification of PCR product ...... 76 2.2.16 Clonal sequencing...... 76 2.2.16.1 Ligation of PCR product ...... 76 2.2.16.2 Transformation...... 76 2.2.16.3 Selection of colonies with desired insert...... 77 2.2.16.4 Sequencing of plasmid DNA ...... 77 2.2.16.5 Analysis of DNA sequence ...... 77 2.2.17 Combined bisulfite restriction analysis (COBRA)...... 78 2.2.17.1 PCR amplification of fragments with CpG islands within promoter regions of genes ...... 78 2.2.17.2 Restriction enzyme digestion ...... 78 2.2.18 BRAF mutation analysis ...... 78 2.2.19 KRAS mutation analysis...... 79 2.2.20 Statistical analyses...... 79

CHAPTER 3: MLH1 methylation in individuals with colorectal cancers...... 80

3.1 Introduction...... 81 3.2 Methods...... 85 3.3 Results...... 85 3.3.1 Development and comparison of assays for the detection of methylation at the MLH1-C promoter region ...... 85 3.3.1.1 Specificity of quantitative assays for methylation detection at the MLH1-C region...... 85 3.3.1.2 Sensitivity of quantitative assays for the detection of methylation at MLH1-C...... 88 3.3.1.2.1 MethyLight assay...... 88 3.3.1.2.2 Quantitative Methylation-specific PCR (qMSP) ...... 89 3.3.1.3 Intra- and inter-assay variations of the MethyLight and qMSP assays...... 91 3.3.1.4 Linearity of the MethyLight and qMSP assay for MLH1-C methylation detection ...... 93

iv 3.3.2 qMSP is superior to MethyLight in the detection of mosaic allelic methylation ...... 95 3.3.3 MLH1 methylation analyses in tissue samples by qMSP...... 97 3.3.3.1 Study population - Individuals with sporadic colorectal cancer and controls ...... 97 3.3.3.2 MLH1 methylation in sporadic colorectal cancers...... 98 3.3.3.3 MLH1 methylation in normal colonic mucosa...... 100 3.3.3.3.1 Clinicopathological characteristics of individuals showing MLH1 methylation in normal colonic mucosa ...... 104 3.3.3.4 MLH1 methylation in peripheral blood mononuclear cells and lymph nodes...... 106 3.3.3.5 MLH1 methylation in cases of germline epimutation...... 108 3.3.3.5.1 Study group...... 108 3.3.3.5.2 Assessment of MLH1 methylation...... 108 3.4 Discussion ...... 113 3.4.1 Real-time PCR assay for the detection of MLH1 methylation ...... 113 3.4.2 MLH1 methylation in biological samples...... 114 3.4.2.1 MLH1 methylation in sporadic MSI colorectal cancers...... 114 3.4.2.2 MLH1 methylation in normal colonic mucosa as a field defect in colorectal cancer development ...... 115 3.4.2.3 MLH1 methylation in lymphocytes from peripheral blood or lymph nodes...... 118 3.4.2.4 MLH1 methylation and germline epimutation...... 119 3.4.3 Summary...... 121

CHAPTER 4: Clinicopathological and molecular significance of CpG island methylation of gene promoters within the 3p22 chromosomal region ……………………………………………………………………….…..122

4.1 Introduction...... 123 4.2 Methods...... 126 4.2.1 Sporadic colorectal cancer cohort...... 126 4.2.2 Methylation analyses...... 126 4.2.3 Hierarchical clustering analysis...... 128

v 4.2.4 Statistical analyses...... 128 4.3 Results...... 129 4.3.1 Clinicopathological and molecular characteristics of the study group...... 129 4.3.2 Promoter methylation of 3p22 genes in the study group...... 132 4.3.3 Association between promoter methylation of individual 3p22 genes, and BRAF V600E mutation and MSI status...... 136 4.3.4 Association between simultaneous promoter methylation of multiple 3p22 genes, and BRAF V600E mutation and MSI status ...... 139 4.3.5 Association between promoter methylation of individual 3p22 genes and other clinicopathological and molecular features of colorectal cancer...... 141 4.3.6 Regional 3p22 methylation and the clinicopathological and molecular features of sporadic colorectal cancers...... 144 4.3.7 Distribution of regional 3p22 methylation by BRAF mutation, CIMP and MSI status ...... 146 4.3.8 Correlation between promoter methylation of 3p22 genes and survival of individuals in the entire study group ...... 147 4.3.9 Regional 3p22 methylation, BRAF V600E mutation, CIMP and the survival of individuals with sporadic colorectal cancer ...... 149 4.4 Discussion ...... 152 4.4.1 Methylation of 3p22 genes in sporadic colorectal cancers...... 152 4.4.2 Methylation of 3p22 genes, BRAF mutation and MSI status...... 153 4.4.3 Methylation of 3p22 genes and common clinicopathological and molecular features of colorectal cancers...... 155 4.4.4 Methylation of 3p22 genes and patient survival...... 157 4.4.5 Summary...... 158

CHAPTER 5: Methylation of 3p22 genes and BRAF V600E mutation in apparently normal colonic mucosa adjacent to colorectal cancers……………... 160

5.1 Introduction...... 161 5.2 Methods...... 163 5.2.1 Normal colonic mucosa samples ...... 163 5.2.2 Methylation profiling of 3p22 genes and BRAF mutation analysis...... 165

vi 5.2.3 Statistical analyses...... 165 5.3 Results...... 166 5.3.1 Methylation of 3p22 genes in normal colonic mucosa...... 166 5.3.1.1 Confirmation of presence of methylation in normal colonic mucosa...... 169 5.3.2 BRAF mutation in the normal colonic mucosa ...... 173 5.3.3 BRAF mutation is associated with a higher frequency and intensity of methylation at 3p22 genes in normal colonic mucosa samples...... 176 5.3.4 Clinicopathological characteristics of individuals with BRAF V600E mutant and regional 3p22 methylation-positive normal colonic mucosa ...... 179 5.3.5 Concomitant BRAF V600E mutation and regional 3p22 methylation in normal colonic mucosa is associated with a higher frequency of neoplastic polyps in the colorectum ...... 181 5.4 Discussion ...... 183 5.4.1 Summary...... 187

CHAPTER 6: The role of APC methylation in the development of familial and sporadic polyposis...... 188

6.1 Introduction...... 189 6.2 Methods...... 192 6.2.1 Collection of samples ...... 192 6.2.2 Analysis of APC-1A promoter methylation ...... 194 6.2.3 Statistical analyses...... 195 6.3 Results...... 196 6.3.1 Screening for germline epimutations of APC in polyposis cases using combined bisulfite restriction analysis (COBRA)...... 196 6.3.2 Assessment of low level methylation of the APC-1A promoter using quantitative methylation-specific PCR (qMSP) assay...... 197 6.3.2.1 APC-1A methylation in PBMC samples ...... 197 6.3.2.2 APC-1A methylation in colorectal tumours and normal mucosa samples ...... 200

vii 6.3.3 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours in individuals with polyposis...... 203 6.3.4 The extent of APC-1A methylation in the normal colonic mucosa and clinicopathological features of individuals in the study group...... 207 6.3.5 APC-1A methylation in the normal colonic mucosa and the molecular features of matched tumours...... 209 6.4 Discussion ...... 211 6.4.1 Germline epimutation of APC is not associated with familial and sporadic colorectal polyposis...... 211 6.4.2 Somatic APC methylation in the tissues of individuals with a polyposis condition...... 212 6.4.3 APC promoter methylation is inversely correlated with BRAF V600E mutation and CIMP+ve phenotype in colorectal tumours...... 214 6.4.4 Age-related methylation of APC promoter in the normal colonic mucosa of individuals with polyposis...... 214 6.4.5 Summary...... 216

Conclusions from this thesis...... 217

Future research directions ...... 220 Appendix 1...... 221 Appendix 2...... 222 Appendix 3...... 223 References...... 224

viii List of Tables

Table 1.1 TNM staging system ...... 15

Table 1.2 Genes commonly silenced by promoter methylation in colorectal neoplasia...... 38

Table 2.1 Oligonucleotide primers used for methylation assays in this thesis ...... 60

Table 2.2 Oligonucleotide primers for microsatellite instability testing...... 61

Table 2.3 Oligonucleotide primers for BRAF and KRAS mutation analyses ...... 61

Table 2.4 Oligonucleotide primers for sequencing of PCR products cloned into vectors ...... 61

Table 2.5 Fluorescence probes...... 61

Table 2.6 Common reagents and chemicals...... 62

Table 2.7 Buffers and solutions ...... 63

Table 2.8 Kits...... 64

Table 2.9 Restriction enzymes ...... 64

Table 2.10 DNA modification enzyme and substrate ...... 64

Table 2.11 Cell lines used in this study...... 67

Table 2.12 Dilution of plasmid standards ...... 74

Table 2.13 DNA mixture of varying percentages of methylation...... 75

Table 3.1 Intra-assay variation of threshold cycle (Ct) measured by either MethyLight or qMSP ...... 92

Table 3.2 Inter-assay variation of threshold cycle (Ct) measured by either MethyLight or qMSP ...... 93

Table 3.3 Intensity of MLH1-C methylation (PMR) detected by each quantitative PCR assay...... 95

Table 3.4 Comparison between the quantitative methylation specific PCR (qMSP) and MLH1 immunohistochemistry in the detection of the loss of MLH1 protein in colorectal cancer...... 100

ix Table 3.5 Clinicopathological characteristics of individuals harbouring low levels of MLH1-C methylation in their normal colonic mucosa...... 104

Table 3.6 Clinicopathological characteristics of individuals with colorectal cancer stratified by the presence of MLH1-C methylation in their normal colonic mucosa ...... 105

Table 3.7 The frequencies of different types of neoplastic polyps in individuals with sporadic colorectal cancer by the presence of MLH1-C methylation in their normal colonic mucosa...... 106

Table 3.8 Histopathological assessment of available lymph node samples in individuals in whom MLH1-C methylation was found in one of their lymph nodes ...... 108

Table 3.9 Percentages of MLH1-C methylation in tissues of individuals with a germ-line epimutation and their family members...... 111

Table 4.1 Summary of pathological characteristics of all colorectal tumours in the study group ...... 129

Table 4.2 Summary of molecular features of all colorectal tumours in the study cohort ...... 130

Table 4.3 Multivariate Cox proportional hazards regression model showing significant prognostic factor in sporadic colorectal cancers ...... 132

Table 4.4 Frequency of colorectal cancers showing promoter methylation at each gene in the 3p22 chromosamal region ...... 132

Table 4.5 The frequency of association between promoter methylation of each 3p22 gene and that of other 3p22 genes in colorectal cancers ...... 134

Table 4.6 The associations between promoter methylation of individual 3p22 genes and BRAF V600E mutation, as well as MSI status in colorectal cancers...... 137

Table 4.7 The frequency of association between regional 3p22 methylation and BRAF V600E mutation, as well as MSI status in colorectal cancers ...... 140

Table 4.8 Clicopathological associations of the methylation of individual 3p22 genes in colorectal cancers...... 142

x Table 4.9 Clinicopathological associations of regional 3p22 methylation in colorectal cancers...... 144

Table 4.10 Multivariate logistic regression analysis showing significance predictors of regional 3p22 methylation ...... 146

Table 4.11 Multivariate Cox proportional hazards regression model comparing the disease-specific risk of mortality in individuals with microsatellite stable colorectal cancers...... 151

Table 4.12 Kaplan-Meier estimates of mean survival in relation to various stratifications of MSS tumours by combinations of BRAF mutation, CIMP and regional 3p22 methylation status...... 151

Table 5.1 Normal colonic mucosa samples included in the present study...... 164

Table 5.3 Clinicopathological details of the four individuals harbouring concomitant BRAF mutation and methylation of 3 of 5 3p22 genes in their normal colonic mucosa samples...... 179

Table 5.4 Clinical characteristics of individuals with BRAF V600E mutant/regional methylation-positive tumours as well as the pathological features of their tumours stratified by the presence of concomitant BRAF mutation and methylation of 3 of 5 3p22 genes in their normal mucosa...... 180

Table 6.1 Individuals with a polyposis condition recruited from St. Vincent’s Hospital, Sydney and their clinical diagnosis ...... 193

Table 6.2 Density of allelic APC-1A methylation in PBMC samples...... 200

xi Table 6.3 Comparison of the levels of APC-1A methylation in the normal mucosa samples of individuals with polyposis or those without neoplasia...... 202

Table 6.4 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours arising in individuals with polyposis ...... 204

Table 6.5 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours arising in individuals with hyperplastic polyposis...... 205

Table 6.6 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours arising in individuals with sporadic polyposis...... 206

Table 6.7 Absence of correlation between the levels of APC-1A methylation in the normal colonic mucosa samples of individuals in the study group and clinicopathological features...... 209

xii List of Figures

Figure 1.1 The adenoma-carcinoma sequence...... 19

Figure 1.2 The epigenetic code ...... 30

Figure 1.3 A model of epigenetic modifications and their effect on transcription...... 32

Figure 1.4 Methylation of cytosine residues...... 33

Figure 1.5 Organisation and consequences of CpG methylation in normal and cancer cells ...... 36

Figure 1.6 Map of the MLH1 promoter showing the 3’-most (proximal) and 5’- most (distal) regions CpG island methylation at the proximal region but not the distal region correlates invariably with the loss of MLH1 expression...... 39

Figure 1.7 Pathways for colorectal tumourigenesis ...... 42

Figure 1.8 DNA methylation profile of the 4-Mb region on chromosome 2q14.2 in a colorectal cancer and paired normal colonic mucosa...... 44

Figure 1.9 Methylation profile of gene promoters within the chromosome 3p22 region in colorectal cancer and adenoma ...... 45

Figure 1.10 Schematic diagram showing chemical reactions involved in the conversion of unmethylated cytosine to uracil by sodium bisulfite ...... 50

Figure 1.11 Sodium bisulfite conversion followed by PCR amplification...... 51

Figure 1.12 Methylation Specific PCR (MSP) and Combined Bisulfite Restriction Analysis (COBRA) ...... 53

Figure 1.13 Bisulfite sequencing analysis...... 54

Figure 1.14 MethyLight assay ...... 55

Figure 1.15 Quantitative methylation specific PCR ...... 56

Figure 3.1 Optimisation of the annealing temperatures for MLH1-C and MyoD amplifications ...... 86

xiii Figure 3.2 Real time PCR amplification plot of MLH1-C and MyoD by qMSP or MethyLight assays...... 87

Figure 3.3 Limits of detection of MLH1-C methylation using the MethyLight assay...... 88

Figure 3.4 Real-time PCR amplification plot for MLH1-C methylation by MethyLight ...... 89

Figure 3.5 Limits of detection of MLH1-C methylation using the quantitative methylation-specific PCR (qMSP) assay ...... 90

Figure 3.6 Real-time PCR amplification plot for MLH1-C methylation detection using the qMSP assay ...... 91

Figure 3.7 Linearity of the MethyLight and the quantitative methylation- specific PCR (qMSP) assays for the detection of MLH1-C promoter methylation ...... 94

Figure 3.8 Clonal bisulfite sequencing of MLH1-C fragments generated by either MethyLight or qMSP assays for one methylated sample...... 96

Figure 3.9 MLH1 methylation levels in sporadic colorectal cancers...... 98

Figure 3.10 Receiver Operating Characteristic (ROC) curve of MLH1-C methylation levels versus MLH1 expression by immunohistochemistry ...... 99

Figure 3.11 Intensity of MLH1 methylation in the normal colonic mucosa and matched cancers...... 101

Figure 3.12 Methylation at individual CpG dinucleotides of the MLH1-C promoter region studied...... 102

Figure 3.13 MLH1 expression in the normal colonic mucosa of a representative case with MLH1-C methylation...... 103

Figure 3.14 Intensity of MLH1 methylation in the lymph nodes and matched colorectal cancers ...... 107

Figure 3.15 Pedigrees of patients A and B showing intergenerational transmission of an MLH1 epimutation and haplotypes ...... 110

xiv Figure 4.1 Map of genes and CpG islands flanking MLH1 within the 3p22 chromosomal domain ...... 123

Figure 4.3 Survival analysis in SVH cohort...... 131

Figure 4.4 COBRA results for the detection of promoter methylation of 3p22 genes ...... 133

Figure 4.5 Dendrogram and heat maps showing the relationship between concomitant methylation of genes the 3p22 cluster ...... 135

Figure 4.6 Frequency of colorectal cancers with promoter methylation at 0-5 genes within the 3p22 chromosomal domain ...... 136

Figure 4.7 Distribution of colorectal cancers that were methylated at each 3p22 gene, stratified by BRAF mutation and MSI status...... 138

Figure 4.8 Histogram showing the frequency of BRAF mutant and wild-type colorectal cancers with promoter methylation of 0, 1, 2, 3, 4 and 5 genes within 3p22 chromosomal domain ...... 139

Figure 4.9 Histogram showing the frequency of MSI and MSS colorectal cancers with promoter methylation at 0, 1, 2, 3, 4 and 5 genes within 3p22 chromosomal domain ...... 140

Figure 4.10 Distribution of regional 3p22 methylation in colorectal cancers stratified by BRAF V600E mutation and MSI status into four distinct groups...... 141

Figure 4.11 Distribution of regional 3p22 methylation in colorectal cancers stratified by BRAF V600E mutation, CIMP and MSI status ...... 147

Figure 4.12 Kaplan-Meier plots showing colorectal cancer survival by methylation status of 3p22 genes individually and as a group...... 148

Figure 4.13 Kaplan-Meier plots showing the association between various molecular features and prognosis of individuals with MSS cancers...... 150

xv Figure 4.14 Conventional and alternate conceptions of CpG island methylator phenotype (CIMP) that incorporate the phenomenon of regional methylation ...... 154

Figure 5.1 The presence of regional 3p22 methylation in normal colonic mucosa samples ...... 166

Figure 5.2 The methylation profiles of 3p22 genes in normal colonic mucosa samples of individuals with colorectal cancer...... 168

Figure 5.3 The methylation profiles of 3p22 genes of normal colonic mucosa samples from control subjects without colorectal neoplasia ...... 169

Figure 5.4 Methylation at individual CpG dinucleotides of the AB002340 promoter region in normal colonic mucosa samples...... 170

Figure 5.5 Methylation at individual CpG dinucleotides of the ITGA9 promoter region in normal colonic mucosa samples...... 171

Figure 5.6 Methylation at individual CpG dinucleotides of the PLCD1 promoter region in normal colonic mucosa samples...... 172

Figure 5.7 Methylation at individual CpG dinucleotides of the DLEC1 promoter region in normal colonic mucosa samples...... 173

Figure 5.8 Real-time PCR amplification plots for the detection of the BRAF V600E mutation in cancer and normal mucosa samples...... 175

Figure 5.9 Comparison of the median and range of methylation level (PMR value) at 3p22 loci in normal colonic mucosa with or without BRAF V600E mutation...... 178

Figure 6.1 Regions of APC-1A promoter analysed by the COBRA or qMSP assay...... 194

Figure 6.2 APC-1A qMSP shows linearity in the detection of methylated DNA...... 195

Figure 6.3 Combined bisulfite restriction analysis of APC-1A promoter ...... 196

Figure 6.4 Distribution of APC-1A methylation levels in the peripheral blood mononuclear cell samples measured using the qMSP assay ...... 198

Figure 6.5 Confirmation of qMSP result using clonal bisulfite sequencing...... 199

xvi Figure 6.6 Frequency of APC-1A hypermethylation in tumours of individuals with a polyposis condition...... 201

Figure 6.7 Distribution of APC-1A methylation in apparently normal colonic mucosa stratified by methylation status of paired tumour...... 203

Figure 6.8 Scatter plot of APC-1A methylation levels in the normal colonic mucosa samples against the age of individuals with a polyposis condition ...... 207

Figure 6.9 Scatter plot of APC-1A methylation levels in the normal colonic mucosa samples against the age of individuals without a polyposis condition in this study cohort ...... 208

xvii Abbreviations

ACF aberrant crypt focus AFAP attenuated familial adenomatous polyposis AJCC American Joint Committee on Cancer APC adenomatous polyposis coli bp base pair CIMP CpG island methylator phenotype CIN chromosomal instability COBRA combined bisulfite restriction analysis CRC colorectal cancer Ct threshold cycle Ct difference in threshold cycle DNA deoxyribonucleic acid DNMT DNA methyltransferase EDTA ethylenediamine tetra-acetic acid F female FAP familial adenomatous polyposis HAT histone acetyl transferase HDAC histone deacetylase H3 histone 3 H3K9 lysine 9 residue of histone 3 H4 histone 4 HNPCC hereditary non-polyposis colorectal cancer HP hyperplastic polyp IEL intraepithelial lymphocyte LCL lymphoblastoid cell line LOH loss of heterozygosity M male MAP MYH-associated polyposis MBD methyl-CpG binding protein MCO Molecular and Cellular Oncology MMR mismatch repair

xviii MSI microsatellite instability/unstable MSI-L microsatellite instability-low MSP methylation-specific polymerase chain reaction MSS microsatellite stable MsSNUPE methylation-specific single nucleotide primer extension Mut mutant NCM normal colonic mucosa n number na not available or assessible nd not done NSW New South Wales OR odds ratio PBMC peripheral blood mononuclear cell PCR polymerase chain reaction PMR percentage of methylated reference qMSP quantitative methylation-specific PCR ROC receiver operating characteristic SA serrated adenoma SD standard deviation SNP single nucleotide polymorphism SSA sessile serrated adenoma TNM Tumour Node Metastasis (system) TSA traditional serrated adenoma UICC International Union Against Cancer WT wild-type +ve positive -ve negative

xix Abstract

DNA methylation is increasingly recognised as a significant epigenetic event that may initiate and drive the process of neoplasia in humans. In the colon, DNA methylation of key genes is common in a subset of colorectal cancers. The extent to which DNA methylation at various genes contributes to initiation of colorectal neoplasms is less clear. This study sought to clarify the biological and clinicopathological significance of methylation of various genes in the development of sporadic and familial colorectal neoplasia.

Quantitative methylation-specific PCR (qMSP) assays (capable of detecting down to a measureable proportion of 0.1% of the total input DNA) were developed to determine the presence of CpG methylation at a given gene.

Methylation of MLH1-C was found in the apparently normal mucosa samples from seven of 104 (7%) of individuals with sporadic colorectal cancer (CRC) showing microsatellite instability (MSI). No methylation of MLH1-C was found in the biological samples of individuals with microsatellite stable (MSS) counterparts (n=131). MLH1-C methylation may be a field defect that predisposes to the development of sporadic colorectal neoplasia, particularly those demonstrating MSI.

Methylation of three of five genes within the 3p22 region including AB002340, MLH1, ITGA9, PLCD1 and DLEC1 (regional 3p22 methylation) was found in 83% of sporadic MSI (n=86) and 12% of MSS cancers demonstrating BRAF V600E mutation (n=42). Regional 3p22 correlated strongly with CpG island methylator phenotype (CIMP), and other clinicopathological characteristics typical of CIMP. Thus, regional 3p22 methylation and CIMP may be overlapping phenomena.

Regional 3p22 methylation and the BRAF V600E mutation were found in normal colonic mucosa of four individuals with sporadic MSI CRC, and these cases also had multiple synchronous serrated polyps. These molecular aberrancies may predispose some individuals to the development of metachronous serrated neoplasia.

Germline epimutations of APC do not contribute towards the development of FAP, AFAP, or hyperplastic polyposis syndromes. However, APC methylation in normal colonic mucosa of these individuals may represent a field defect in the development of futher neoplasms.

1 In conclusion, different patterns of DNA methylation in normal colonic mucosa may represent a field defect important in the development of different subtypes of colorectal neoplasia.

CHAPTER 1

Literature review

1.1 Colorectal neoplasia

Colorectal neoplasia is the clonal proliferation of cellular components of the large bowel to form an abnormal mass of tissue. In practice, neoplastic changes within the large bowel are almost invariably epithelial in origin, and hence the term colorectal neoplasia as used in this thesis will be synonymous with colorectal epithelial neoplasia. This phenomenon of colorectal neoplasia is characterised by rapid cell growth, failure of apoptosis and loss of cellular differentiation. The term encompasses a range of non- invasive entities, including aberrant crypt foci, serrated polyps and adenoma. Less commonly, colorectal neoplasms can take the form of invasive lesions, known commonly as bowel cancer, but more correctly as colorectal carcinoma. The morphology of these lesions is discussed in detail in Section 1.1.3.

1.1.1 Epidemiology of the disease

Over the past few decades, colorectal carcinoma has remained the third most common cancer globally (Greenlee et al., 2001; Hawk et al., 2002; Rupnarain et al., 2004), and the fourth largest cause of cancer mortality (Weitz et al., 2005). Approximately nine percent of the 6.35 million invasive cancers that occur worldwide each year are attributed to colorectal cancer (CRC), with the highest incidence documented in North America, Australia and New Zealand (Rupnarain et al., 2004).

The most up to date information on the incidence of CRC in New South Wales, Australia was published by the New South Wales Cancer Council and is discussed here (Tracey et al., 2006). Of all cancer cases reported in New South Wales in 2004, CRC was ranked second highest in incidence, for both males and females, as well as overall. The crude incidence rate of this disease per 100,000 individuals was 74.6 overall, whereas for males and females, it was 74.6 and 60.6, respectively. The median age of diagnosis is 69 for males and 72 for females, and thus CRC can be regarded as a disease of the elderly. By the age of 75, 1 in 17 males and 1 in 26 females will have developed CRC, increasing to 1 in 10 males and 1 in 14 females by the age of 85 years.

Between 1995 and 2004, the mortality rates of CRC in New South Wales fell by 21% in males and 19% in females. However, this disease was still ranked third among all cancer deaths reported in 2004. Deaths from CRC in that year accounted for 12.2% and

4 13.4% of all cancer deaths in males and females respectively. Males in New South Wales are reported to be 1.5 times more likely to suffer and die from CRC than females.

1.1.2 Aetiology of the disease

The variability in the incidence of CRC between regions and ethnic groups indicates that the disease is highly influenced by both genetics and environmental factors. Of the two, twin studies support the contention that environmental factors are more influential (Lichtenstein et al., 2000). Lifestyle and dietary factors including red meat intake, alcohol consumption, and smoking have all been associated with the development of CRC and these have been reviewed extensively (Ahmed, 2004; Hashibe et al., 2005; Gonzalez and Riboli, 2006; van den Brandt and Goldbohm, 2006). In addition, inflammatory bowel diseases such as Crohn’s and ulcerative colitis also increase the risk of CRC (Eaden et al., 2001; von Roon et al., 2007).

Hereditary syndromes such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) account for approximately 5% of CRCs (Bodmer, 2006). Importantly, they are responsible for a significant percentage of the CRCs that arise in younger individuals. Studies of these hereditary syndromes have revealed several highly penetrant genetic mutations that have also been implicated in sporadic CRC when somatically acquired. These syndromes will be discussed more broadly under a later section that described the genetic basis of CRC.

1.1.3 Pathology of the disease

Colorectal neoplasia is a process that shows variable progression through successive lesions, each with distinctive pathological features. Most colorectal carcinomas develop via the “adenoma-carcinoma sequence” that centres upon the clonal evolution of adenomatous polyps into carcinoma. Another pathway termed the “serrated neoplasia pathway” underpins the development of cancer from serrated polyps, a group of lesions that includes hyperplastic polyps, sessile serrated adenomas and serrated adenomas. In order to understand these pathways, it is important to recognise various lesions involved in the transition from normal epithelium to carcinoma. In this section, characteristics of various neoplastic lesions and their association with cancer progression are first

5 discussed, followed by a grouping of these lesions and their underlying genetic defects into several recognised pathways of colorectal carcinogenesis.

1.1.3.1 Non-invasive colorectal neoplasms The vast majority of colorectal neoplasms are non-invasive. Nevertheless, they are critical to our understanding of the processes of colorectal carcinogenesis, and they will be described here in some detail.

Aberrant crypt foci are not evident macroscopically. However, most other non-invasive neoplasms of the colon and rectum form macroscopically identifiable lesions often referred to as colorectal polyps, and the taxonomy of these lesions is complex and still evolving. Before the 1980s, the majority of colorectal polyps were considered to fall into two major groups; hyperplastic polyps and adenomas. The former lacked dysplasia, while the latter showed clear evidence of cytological dysplasia and were considered to be the precursor lesion of colorectal carcinoma. This concept was challenged by the discovery of lesions with the architectural features of hyperplastic polyps (including serrated luminal profiles) but the cytological dysplasia typical of adenomas (Urbanski et al., 1984; Longacre and Fenoglio-Preiser, 1990). These lesions became known as serrated adenomas (SAs). In recent years, a further subgroup of hyperplastic polyps was recognised that showed some of the atypical features of serrated adenomas but which lacked cytological dysplasia. The term sessile serrated adenoma (SSA) has been proposed for these lesions, to distinguish them from serrated adenomas which are now termed traditional serrated adenomas (TSAs) (Torlakovic et al., 2003). Thus a current classification of neoplastic colorectal polyps includes both adenomas and a group of “serrated polyps” that includes hyperplastic polyps, sessile serrated adenomas and traditional serrated adenomas.

1.1.3.1.1 Aberrant crypt foci Aberrant crypt foci (ACF) are now well recognised as the earliest identified precursor lesion of colorectal neoplasia though only a very small number of these will eventually develop into carcinoma. First described in rodents (Bird, 1987) and later in humans (Pretlow et al., 1991; Roncucci et al., 1991a), ACF are clusters of enlarged colonic crypts that exhibit increased proliferative capacity. They are macroscopically flat and

6 therefore cannot be identified without magnification. Under the microscope, they appear to be slightly elevated, larger, and have a thicker epithelium than adjacent normal crypts (Pretlow et al., 1991; Roncucci et al., 1991a; Otori et al., 1995). The size of human ACF may vary from just one to hundreds of crypts (Roncucci et al., 1991a; Otori et al., 1995)

ACF have been reported at a higher frequency in the distal or left colon (Pretlow et al., 1991; Roncucci et al., 1991a; Takayama et al., 1998), and are particularly common in individuals with CRC or polyps (Pretlow et al., 1991; Yokota et al., 1997; Takayama et al., 1998; Nascimbeni et al., 1999; Adler et al., 2002; Rudolph et al., 2005). In addition, the number and size of ACF increase with age (Takayama et al., 1998; Rudolph et al., 2005).

From a histopathological perspective, ACF are associated with both hyperplastic and dysplastic changes within the epithelial cells of the focus. In general terms, hyperplasia refers to an increase in the number of cells within a tissue, without evidence of loss of differentiation of individual cells. On the other hand, dysplasia is a term used to describe changes in nuclear morphology and patterns of differentiation that are associated with neoplasia. Based on these histological features, ACF are generally classified into non-dysplastic and dysplastic types even though mixed type ACF have been observed (Pretlow et al., 1994; Otori et al., 1995; Nascimbeni et al., 1999). In relation to their role as the initial lesion of colorectal neoplasia, different types of ACF have been linked to the subsequent development of phenotypically distinct lesions (reviewed in Cheng and Lai, 2003) (Cheng and Lai, 2003).

Importantly, increasing evidence has demonstrated a link between ACF and the development of colorectal carcinoma. ACF were initially hypothesized as precursor lesions of colorectal carcinoma since they were first observed in the carcinogen-treated colons of rodents (Bird, 1987), and later at a higher frequency in the colons of individuals with carcinoma (Pretlow et al., 1991; Takayama et al., 1998). Molecular evidence including both genetic and epigenetic changes has since emerged to support this hypothesis, as discussed later in this chapter.

7 1.1.3.1.2 Hyperplastic polyps Hyperplastic polyps are small foci of epithelial proliferation (usually <5mm in diameter) that are thought to represent neoplasms with limited or no capacity for tissue invasion and thus no significant role in cancer development (Jass, 2001; Jass et al., 2002). While their name suggests a hyperplastic process, there is compelling molecular evidence that hyperplastic polyps are derived from the clonal expansion of colonic epithelial cells (Iino et al., 1999). In addition, it has been speculated that hyperplastic polyps act as “sanctuary neoplasms” that exhibit failure of apoptosis and will accumulate genetic changes over time (Jass et al., 2002). Biologically, they have been seen as lesions with epithelial hypermaturation as a consequence of delayed migration of mature cells up the colonic crypt (Kaye et al., 1973; Hayashi et al., 1974). Histologically, they are characterised by elongated crypts that have a serrated or “saw- tooth-like” profile, and cytologically they exhibit maturation towards the upper crypt and surface epithelium (Huang et al., 2004).

Based on worldwide autopsy data, the prevalence of hyperplastic polyps appears to range from <5% to 50% in various populations (reviewed in Huang et al., 2004) (Huang et al., 2004). Hyperplastic polyps occur at an earlier age than adenomas, and do not increase in frequency with age (Clark et al., 1985; Morimoto et al., 2002). Like adenomas, they are more frequently observed in the distal colon and rectum (DiSario et al., 1991; Weston and Campbell, 1995; Farraye and Wallace, 2002). More recent studies have now provided evidence with regards to variants of hyperplastic polyps that have a more clearly defined malignant potential. These lesions are termed serrated adenomas (either sessile or traditional).

1.1.3.1.3 Serrated adenomas First observed by Urbanski and co-workers (1984) and later termed serrated adenomas by Longacre and Fenoglio-Preiser (1990), these lesions bear an architectural resemblance to hyperplastic polyps but simultaneously exhibit the dysplastic epithelial characteristics of adenomas (Urbanski et al., 1984; Longacre and Fenoglio-Preiser, 1990). In contrast to hyperplastic polyps, the dysplastic or adenomatous features of serrated adenomas suggest their potential for malignant transformation. They are thought to arise either from hyperplastic polyps or directly from aberrant crypt foci

8 (Huang et al., 2004), and there is clear evidence of their ability to progress to adenocarcinoma (Hawkins and Ward, 2001).

In terms of prevalence, serrated adenomas are rather uncommon. Based on a review by Huang et al., they occur in approximately 1-7% of individuals with CRC and are twice as common in males than females, with a mean age at diagnosis of 60 to 65 years (Huang et al., 2004). They are reported to be distributed throughout the colon, albeit more frequent in the rectosigmoid (Matsumoto et al., 1999; Yao et al., 1999; Fogt et al., 2002). Nevertheless, these reports may be subject to selection bias in view of the changes in endoscopic practices (Hawkins et al., 2002a). In contrast to other studies, Makinen et al reported a high preponderance of serrated adenoma in the caecum, particularly in women (Makinen et al., 2001).

As mentioned earlier, the term sessile serrated adenoma (SSA) has been proposed by Torlakovic et al (2003) to distinguish a subgroup of hyperplastic polyps which showed atypical features, but which lacked the cytologic dysplasia seen in serrated adenomas (Torlakovic et al., 2003). To avoid confusion, the term traditional serrated adenoma (TSA) has been proposed to identify conventional serrated adenomas. It can be difficult to distinguish histologically between SSA and hyperplastic polyps, as well as between SSA and TSA. Nevertheless, these distinctions can be of clinical importance (reviewed in Snover et al., 2005 (Snover et al., 2005).

1.1.3.1.4 Colorectal adenomas The term colorectal adenoma or adenomatous polyp refers to a benign neoplasm of colorectal epithelium characterised cytologically by epithelial dysplasia. These lesions have long been recognised as the precursor lesions of CRC, and this morphological progression has been referred to as the adenoma-carcinoma sequence. In the general population, the frequency of colonic adenomas is associated with increased age (Morimoto et al., 2002; Okamoto et al., 2002). This contrasts with hyperplastic polyps, which develop earlier and do not increase in frequency with age (reviewed in Huang et al, 2004). Adenomas are distributed throughout the colon and rectum, although they are more frequent distal to the splenic flexure (Gillespie et al., 1979; Shinya and Wolff, 1979; Clark et al., 1985).

9 The degree of loss of differentiation in adenomas is typically expressed as low or high grade, or more conventionally as mild, moderate or severe dysplasia (Morson et al., 1990). This progressive loss of differentiation reflects neoplastic progression.

On the basis of epithelial architecture, adenomas can be subdivided into three types, namely tubular, villous and tubullovillous adenomas (Kumar et al., 2005). As their names suggest, tubular adenomas show obvious tubular architecture (>80%), villous adenomas exhibit more than 80% villous architecture, whereas tubulovillous adenomas show a mixture of tubular and villous architecture (>20% of each villous and tubular architecture) (Jass and Sobin, 1989). Most adenomas are tubular, followed by tubulovillous (5-10%), with villous adenomas least common (1%). Nevertheless, it is the villous adenoma that has the greatest potential to undergo malignant transformation. As documented by Burgart (2002), the risk of malignancy is strongly correlated with both the size of polyp and the degree of dysplasia (Burgart, 2002). Notably, villous adenomas tend to be larger, and to exhibit more severe dysplasia than tubular adenomas.

It is also important to mention that not all adenomas are morphologically protuberant or polypoid. Non-polypoid adenomas are morphologically flat, slightly depressed or slightly elevated in comparison to surrounding normal colonic mucosa. These adenomas also exhibit a predilection to the right colon (Hurlstone et al., 2003) in association with a greater rate of severe dysplasia (Tada et al., 1995; Teixeira et al., 1996; Rembacken et al., 2000). Biologically, non-polypoid adenomas differ from their polypoid counterparts in terms of a lower rate of KRAS mutations and a higher rate of LOH at chromosome 3 (where mismatch repair gene MLH1 resides) (Yashiro et al., 2001). Flat and polypoid adenomas have distinct chromosomal imbalances, and it has been suggested that this may reflect involvement of different pathways for tumour development (Richter et al., 2003). Convincing evidence to support this hypothesis is not yet available.

1.1.3.2 Colorectal carcinomas Colorectal neoplasms that show evidence of invasion through the muscularis mucosae are referred to as colorectal carcinomas. Carcinomas of the colon and rectum almost always show evidence of secretory differentiation, and are therefore termed adenocarcinomas. Approximately 35% of all colorectal carcinomas arise in the rectosigmoid, with the remainder occurring in the caecum and ascending colon (34%),

10 transverse colon (10%), descending colon (7%) and other sites (14%) (Rupnarain et al., 2004). Their macroscopic appearance varies with location. In the proximal colon, carcinomas are typically polypoid, while distal tumours are often annular, encircling lesions that constrict the lumen.

Histologically, the majority of CRCs are adenocarcinomas of no specific type. Mucinous tumours, account for 10-20% of cases, and are defined as having more than 50% extracellular mucinous component. Occasionally, intracellular accumulation of mucin can occur and give rise to a signet-ring cell appearance. Other recognised histological subtypes include medullary and undifferentiated carcinomas (Ponz de Leon and Di Gregorio, 2001). While histological type has relatively little impact on prognosis, mucinous carcinomas are associated with poorer outcome (Consorti et al., 2000), and the medullary type are said to have a better outcome (Lanza et al., 1999). This observation needs to be taken with caution as mucinous carcinomas with microsatellite instability tend to show better outcome than microsatellite stable counterparts (Kakar et al., 2004).

The grade, or degree of histological differentiation of CRCs, is typically reported as well, moderate or poor (Bosman, 1995), based on the extent of gland formation. While invariably reported, tumour grade is generally of little prognostic significance (Ponz de Leon and Di Gregorio, 2001).

Other histological characteristics of prognostic significance in CRC include the nature and extent of lymphocytic infiltration, and the presence of vascular space invasion (reviewed in Ponz de Leon and di Gregario, 2001) (Ponz de Leon and Di Gregorio, 2001). Tumours with infiltrating lymphocytes have been found to be associated with microsatellite instability and show improved survival (Guidoboni et al., 2001; Quinn et al., 2003).

1.1.3.3 The adenoma-carcinoma sequence The adenoma-carcinoma sequence describes the transition of precursor adenomatous lesions into carcinoma. This process is known to underlie the development of most CRCs, and this section will discuss briefly the epidemiological and clinicopathological evidence that support it. Further evidence based on molecular genetics of CRC is discussed in sections 1.3 and 1.4.

11 The concept of an adenoma-carcinoma sequence was first introduced by Schmeiden (Schmieden, 1926). However, it was several more decades before this concept became acknowledged as a valid model for the development of CRC. The research led by Morson at St Mark’s Hospital found epidemiological evidence that supported the existence of the adenoma-carcinoma sequence (Morson, 1966; Muto et al., 1975). This encouraged further studies by other researchers and led eventually to a better understanding of the molecular basis underlying this process (Vogelstein et al., 1988).

From epidemiological and clinicopathological perspectives, there are several lines of evidence that support the existence of the adenoma-carcinoma sequence. Firstly, the prevalence of adenomas, although varied from one geographical region to another, correlated well with the incidence of CRCs in respective regions (Clark et al., 1985). Furthermore, both adenomas and carcinomas shared a similar predilection to the distal colon (Gillespie et al., 1979; Shinya and Wolff, 1979; Clark et al., 1985; O'Brien et al., 1990). Based on age distribution, the peak incidence of adenomas was shown to precede that of carcinomas by at least 5 years (Muto et al., 1975). These observations suggested that adenomas may evolve into carcinomas. Furthermore, polyps that were left in situ were shown to possess the capacity to grow, and for carcinoma to develop at the site of the index polyp (Stryker et al., 1987).

Histologically, foci of malignancy have been found within colorectal adenomas in 0.2- 8.3% of cases (Gillespie et al., 1979; Shinya and Wolff, 1979; Colacchio et al., 1981; Cranley et al., 1986). Adenomas were also shown to co-exist with carcinomas in approximately 30% of cases (Chu et al., 1986; Arenas et al., 1997). More importantly, a higher risk of synchronous and metachronous cancer was reported in individuals who had CRC and simultaneous adenomas when compared to those without (Chu et al., 1986). Likewise, endoscopic removal of adenomatous polyps was shown to significantly reduce the risk of developing CRC (Winawer et al., 1993).

1.1.3.4 The serrated neoplasia pathway The serrated neoplasia pathway is an alternative morphological pathway for the development of CRC in which colorectal polyps demonstrating serrated architecture rather than conventional adenomas serve as the precursor lesion. While the distinction between the serrated neoplasia pathway and the conventional adenoma-carcinoma

12 sequence has subsequently been clarified through molecular studies, this section will focus on the clinicopathological aspect of this pathway.

Serrated polyps consist of hyperplastic polyps, traditional serrated adenoma and sessile serrated adenoma. Although the malignant potential of hyperplastic polyps remains contentious, an early suggestion that serrated polyps may progress to carcinoma stemmed from the observation that hyperplastic polyps were present in conjunction with significant percentages of adenomas (Goldman et al., 1970). They are also more commonly observed in the population at risk of CRC (Eide, 1986), and occasionally they can become larger and coincide with adenocarcinoma (Warner et al., 1994). Subsequently, it was shown that lesions of the serrated neoplasia pathway were commonly associated with the development of microsatellite unstable CRC, and that more hyperplastic polyps and serrated adenomas were observed in individuals with this type of cancer (Makinen et al., 2001; Hawkins et al., 2002a). On this basis, it was postulated that hyperplastic polyps may progress into serrated adenomas, which eventually become adenocarcinomas (Hawkins and Ward, 2001).

Further evidence supporting the concept of the serrated neoplasia pathway comes from the study of hyperplastic polyposis syndrome, a condition in which individuals develop numerous large serrated polyps. Initially thought to lack malignant potential, this syndrome is now known to increase the risk for malignancy (Leggett et al., 2001; Hyman et al., 2004). Individuals with this syndrome have a mean age at diagnosis of 52 years (Ferrandez et al., 2004), and present with 40-100 serrated polyps within the colorectum including hyperplastic polyps and conventional, as well as serrated adenomas (Lage et al., 2004). Hyperplastic polyposis particularly in cases with large or dysplastic polyps, has been associated with an increased risk of synchronus adenocarcinoma of the colorectum (Warner et al., 1994; Abeyasundara and Hampshire, 2001). However, it remains uncertain as to whether this syndrome is familial in nature (reviewed in Young and Jass, 2006) (Young and Jass, 2006).

1.1.3.5 Pathological staging of colorectal cancer Pathological staging of CRC is important to clinicians as it allows an assessment of the prognosis of disease and guides treatment. As reviewed by Greene (2006), the staging of CRC has evolved in conjunction with the development of better techniques for

13 tumour evaluation (Greene, 2006). The first staging system for CRC was the Dukes system, introduced in 1926 by Lockhart-Mummery (Lockhart-Mummery, 1926) and revised by Dukes in 1929 (Dukes, 1929). It was initially applied to the staging of rectal cancer, but was also found to be useful in the context of colon cancer. This system was based on the extent of invasion of the bowel wall, together with local lymph node spread. Several modifications were later made to describe more specifically the extent of tumour invasion. The most useful modification was the introduction by Turnbull (1967) of a stage to describe cases with known distant metastases (Turnbull et al., 1967).

For decades, the Dukes system was instrumental for CRC staging as it correlated well with patient survival and was simple in its application. However, it has become apparent now that the more comprehensive tumour, node, metastasis (TNM) system offers greater advantages over the Dukes system. The TNM system, initially proposed by the American Joint Committee on cancer (AJCC) and the International Union Against Cancer (UICC) is now the staging system recommended by the College of American Pathologists, the Royal College of Pathologists, the Commission on Cancer of the American College of Surgeons, and the National Cancer Institute (reviewed in Compton and Green, 2004). In this system, the severity of CRC is determined based on the extent of tumour at the primary site (T), the presence/absence and extent of lymph node metastases (N), and the presence/absence of distant metastases (M). The TNM staging system based on the 6th edition of the AJCC staging manual (2002) (Greene et al., 2002) is outlined in Table 1.1.

TNM staging nomenclature

Primary Tumour (T) Tx Primary tumour cannot be assessed T0 No evidence of primary tumour Tis Carcinoma in situ, intraepithelial or invasion of lamina propria T1 Tumour invades submucosa T2 Tumour invades muscularis propria T3 Tumour invades through the muscularis propria into the subserosa or the nonperitonealised pericolic or perirectal soft tissues T4 Tumour directly invades other organs or structures and/or penetrates the visceral peritoneum

14 Regional Lymph Node (N) Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in 1 to 3 regional lymph nodes N2 Metastasis in 4 or more regional lymph nodes Distant metastasis (M) Mx Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis

Stage T N M Dukes 0 Tis N0 M0 N/A IT1N0M0A T2 N0 M0 A II A T3 N0 M0 B II B T4 N0 M0 B III A T1-T2 N1 M0 C T3-T4 N1 M0 C III C Any T N2 M0 C IV Any T Any N M1 N/A

Table 1.1 TNM staging system Stages and definitions of the TNM staging system. Corresponding Dukes stages are shown in the right hand-most column.

To date, CRC staging is based entirely on clinical and pathological features of the disease. As our understanding of the molecular basis of CRC improves, it is possible that biological markers may be incorporated within the existing staging taxonomy, in order to better predict responses to treatment and disease outcomes.

1.2 Genetic mechanisms of colorectal neoplasia

In terms of their underlying biology, colorectal neoplasia can be considered in terms of the mechanisms that drive mutations within the clone and thus give rise to genomic instability. Studies on hereditary and sporadic CRCs suggest at least two distinct patterns of genomic instability, one that is characterised by chromosomal instability and the other by microsatellite instability. The former is associated with the development of cancers via the adenoma-carcinoma sequence. The latter, while mostly underpinning the development of cancers arising via the serrated neoplasia pathway, also involves a small proportion of cancers that develop via the adenoma-carcinoma sequence. These patterns of genomic instability are summarised in brief below.

15 1.2.1 Chromosomal instability

Chromosomal instability (CIN) describes CRCs that show an accumulation of gross chromosomal rearrangements, including a high frequency of allelic loss, and thus exhibit abnormal chromosome number (aneuploidy). Phenotypically, CIN cancers are more prone to be distally-located and behave more aggressively. Up to 85% of all CRCs, especially those that progress via the adenoma-carcinoma sequence, exhibit CIN. These cancers include the majority (70-80%) of sporadic CRCs, as well as those associated with familial cancer syndromes including FAP and MYH associated polyposis (MAP). Much of our knowledge of this common pathway comes from lessons learned in the context of FAP. Mechanistically, CIN cancers progress from adenoma to carcinoma via the accumulation of mutations and loss of tumour suppressor genes including APC (chromosome 5q), p53 (chromosome 17p), DCC or SMAD4 (chromosome18q), as well as mutations of the oncogene, KRAS (chromosome 12p).

1.2.2 Microsatellite instability

Microsatellite sequences are highly abundant tandem repeats found throughout the genome. These sequences are commonly mono- or di-nucleotide repeats interspersed between coding and non-coding regions. Due to the repetitive nature of these sequences, they are prone to strand slippage and DNA replication error. Normally, these “replication errors” are repaired by enzymes of the mismatch repair (MMR) system. Failure of these enzymes results in a state of mismatch repair deficiency, and leads to the condition called microsatellite instability (MSI), typically characterised by an alteration in the length of microsatellite sequences. In CRC, mismatch repair deficiency is the result of defects in the genes in that comprise the mismatch repair machinery; MSH2, MLH1, PMS1, PMS2 or PMS6 (Fishel et al., 1993; Bronner et al., 1994; Nicolaides et al., 1994; Liu et al., 1996; Akiyama et al., 1997).

Many of the lessons about this cancer phenotype were learned from the familial cancer predisposition syndrome, HNPCC (Aaltonen et al., 1993; Konishi et al., 1996; Gryfe et al., 2000). MSI has also long been recognised as a feature of sporadic CRCs (Ionov et al., 1993; Thibodeau et al., 1993), and indeed it is known to occur in up to 15% of sporadic CRCs (Kim et al., 1994; Feeley et al., 1999; Gryfe et al., 2000), particularly those developing via the serrated neoplasia pathway.

16 1.2.2.1 Diagnosis and features of microsatellite instability Under current practice, microsatellite instability is assessed using a set of microsatellite markers, which can inform an altered pattern of microsatellite repeat sequences in the tumour when compared to a normal tissue in a particular individual. Although the set of markers used may vary from one study to another, the recommended panel by Boland et al. (1998) comprises five markers including two mononucleotide repeats, BAT25 and BAT26 and three dinucleotide markers, D5S346, D2S123 and D17S250 (Boland et al., 1998). Typically, cancers are classified as microsatellite unstable (MSI) when two or more ( 40%) markers exhibit instability. When there is none or only one marker that shows instability, the cancer is classified as microsatellite stable (MSS). There is however contention as to whether some cancers should be labeled microsatellite instability-low (MSI-L) if one marker demonstrates instability. The National Cancer Institute Workshop has previously recommended the use of more markers to distinguish this phenotype (20-40% of unstable markers) from MSI and MSS (Boland et al., 1998).

While MSI-L cancers do not exhibit clinicopathological features that differ from MSS cancers, there are apparent genetic differences such as a higher incidence of KRAS mutation (Whitehall et al., 2001) and downregulation of Bcl-2 (Biden et al., 1999) in these cancers. Silencing of the DNA-repair gene, MGMT has also been strongly associated with MSI-L cancers (Whitehall et al., 2001).

As opposed to CIN tumours that are typically aneuploid, MSI tumours are usually diploid. In terms of clinicopathological features, MSI tumours are strongly associated with right-sidedness, high grade, mucinous type and infiltration with intraephitelial lymphocytes (Jass et al., 1998). In cancers that occur sporadically, MSI is also predominant in elderly females (Jass et al., 1998). Perhaps the most important feature of MSI cancers is its strong association with a better prognosis. This has been demonstrated by independent groups of researchers (Elsaleh et al., 2000; Gafa et al., 2000; Gryfe et al., 2000; Wright et al., 2000) and more recently a systematic review has confirmed the improved outcome of MSI cancers in comparison to MSS cancers of similar stage and grade (Popat et al., 2005). However, a recent study found that MSI was not a significant prognostic marker when included in a multivariate analysis with cancer stage (Malesci et al., 2007).

17 1.3 Genetic basis of colorectal neoplasia

Aberrancies in numerous genes, classified as tumour suppressors or oncogenes, have been shown to play important roles in neoplastic development or progression. Loss of both copies of a gene with tumour suppressor activity is necessary for tumourigenesis, whereas only a single copy of an oncogenic mutation is required to promote tumour development. The presence of these genetic aberrancies across a wide range of successive lesions of colorectal neoplasia, from aberrant crypt foci to carcinoma, provided the opportunity to compare the frequencies and chronology of genetic changes within these lesions. In combination, these data have facilitated the understanding of the multi-step progression of normal colonic epithelium to carcinoma. This section will consider the various genes whose dysregulation leads to the main genetic pathways in CRC, firstly in the context of familial cancer syndromes with high penetrance mutations, followed by sporadic cancers, in which alterations of the same genes occur, although the mechanisms may differ.

1.3.1 Familial cancer syndromes

There are several familial cancer syndromes which predispose to CRC due to highly penetrant heritable mutations of tumour suppressor genes. The autosomal dominant cancer syndromes are caused by heterozygous loss-of-function mutations in the germline. Loss of function of the remaining wild-type allele in somatic cells gives rise to tumour development. This process is known as “Knudson’s two-hit hypothesis”. While cancers with a strong familial predisposition comprise <5% of all CRCs, study of these syndromes has provided a better understanding of the genetic pathways leading to the majority of CRCs, including those that occur sporadically.

A classic genetic model for CRC development was proposed by Fearon and Vogelstein (1990), and later modified by Fearon (1996) (Figure 1.1) based on the understanding of the two most common familial cancer predisposition syndromes; FAP and HNPCC. While this model also underlies the development of CRCs predisposed by other hereditary syndromes, the initial genetic alterations that underlie these syndromes are distinct.

18 Normal epithelium 5q loss / APC mutation / ß-catenin mutation Hyperproliferating epithelium

DNA hypomethylation

Early adenoma KRAS mutation

Intermediate adenoma 18q loss / DCC mutation / Mismatch repair SMAD2 and 4 mutation gene inactivation

Late adenoma

17p loss / p53 mutation

Carcinoma

Other alterations

Metastasis

Figure 1.1 The adenoma-carcinoma sequence A schematic diagram of the molecular changes involved in the adenoma-carcinoma sequence. Modifications (in red) were made by Fearon (1996) to the original model by Fearon and Vogelstein (1990) to explain the role of mismatch repair gene inactivation that accelerates somatic mutations of oncogenes and tumour suppressor genes.

1.3.1.1 Familial adenomatous polyposis (FAP) syndrome Familial adenomatous polyposis (FAP) syndrome is an autosomal dominant disorder characterised by the formation at a relatively young age of hundreds to thousands of adenomatous polyps throughout the colon and rectum (Kinzler and Vogelstein, 1996). Polyps typically develop in the teens and twenties, and some of these will lead to carcinoma within 10 to 15 years unless they are resected surgically. FAP accounts for less than 1% of all cases of CRC (Burt et al., 1990).

19 Following a linkage study in 1987 (Bodmer et al., 1987), the gene responsible for this disease was mapped to the long arm of chromosome 5. This subsequently led to the recognition of an inherited mutation in one allele of the adenomatous polyposis coli (APC) gene, localized at 5q21 (Groden et al., 1991; Kinzler et al., 1991). According to Knudson’s two hit hypothesis, both alleles of a tumour suppressor gene need to be silenced in order to lose its function. Notably, truncating germline mutations causing monoallelic silencing of the APC tumour suppressor gene were found in 70-80% of FAP cases (Powell et al., 1992; Nagase and Nakamura, 1993; Laurent-Puig et al., 1998). Subsequent somatic mutation or deletion of the other allele is the typical second hit that inactivates APC expression.

While APC loss results in the development of adenomatous polyps in FAP, these polyps do not necessarily progress into carcinoma. Other genetic aberrancies are required for the transition from a polyp to carcinoma. Vogelstein and co-workers were the first to report four molecular alterations that were strongly implicated in both adenomas and carcinomas in individuals with FAP (Vogelstein et al., 1988). These molecular alterations, including allelic deletions on chromosomes 5, 17, and 18, as well as mutation of the KRAS oncogene are now accepted as the basis of the adenoma- carcinoma sequence (Vogelstein et al., 1988). In subsequent studies, important tumour suppressor genes APC (chromosome 5), p53 (chromosome 17) and DCC or SMAD4 (chromosome 18) were found to reside in these frequently deleted alleles. Fearon and Vogelstein went on to propose that sequential inactivation of these genes paralleled the progression of colorectal neoplasia, from early adenoma to carcinoma (Figure 1.1) (Fearon and Vogelstein, 1990a). While this seems to correlate with the multistep progression of CRC, accumulation of all these changes was suggested to be more important than the order in which they occurred (Leslie et al., 2002).

1.3.1.2 Other FAP variants Attenuated familial adenomatous polyposis syndrome (AFAP), Gardner syndrome and Turcot syndrome are variants of FAP syndrome, and like FAP, they are underpinned by germline APC mutation. Curiously, Turcot syndrome is HNPCC-like as well, as evident by the presence of germline MLH1 and MSH2 mutations in some cases. Gardner and Turcot syndromes are more often described within the context of extracolonic cancers,

20 and will not be discussed here. Synonymous with its given name, attenuated adenomatous polyposis syndrome is a milder phenotype of FAP characterised by less than a hundred adenomatous polyps in the colon of affected individuals. On average, the age of disease onset is 15 years later than FAP and often, it is not characterised by extracolonic features (Sieber et al., 2000). Germline mutation of APC associated with AFAP has been detected in the 5’ end (codons 78-167), in exon 9 and in the 3’end (codons 1581-2843) of the gene, yet these mutations were only accountable for 5-15% of AFAP cases (reviewed in Lipton and Tomlinson, 2006) (Lipton and Tomlinson, 2006). More importantly, most of these mutations result in in-frame deletions that still encode almost the entire full-length protein, thus providing a potential explanation for a milder phenotype in comparison to FAP, in which the latter tends to involve frameshift mutations that result in protein truncation (reviewed in de la Chapelle, 2004) (de la Chapelle, 2004).

1.3.1.3 MYH-associated Polyposis (MAP) Recently, a recessive form of FAP called MYH-associated polyposis (MAP) was described and synonymous with its name, this disease is associated with germline mutations of the base-excision-repair gene MYH (Al-Tassan et al., 2002; Sieber et al., 2003). MAP is characterised by the presence of a few to several hundred colorectal polyps and therefore, the distinction between MAP and FAP or AFAP is clinically difficult. Individuals with MAP have a mean age at diagnosis of 44-53 years (Enholm et al., 2003; Sampson et al., 2003; Sieber et al., 2003; Gismondi et al., 2004; Aretz et al., 2006; Bouguen et al., 2007), which is similar to that of AFAP (Soravia et al., 1998) but later than that of classical FAP. This syndrome, in contrast to APC mutation-related polyposis syndrome is autosomal recessive and caused by bi-allelic mutations of MYH. The risk of inheritance is 25% but this syndrome can appear as sporadic if both parents are recessive carriers (reviewed in Lipton and Tomlinson, 2006) (Lipton and Tomlinson, 2006).

Bi-allelic MYH mutation was first described as the factor contributing to the loss of oxidative repair mechanisms, resulting in downstream somatic GCTA substitutions in APC (Al-Tassan et al., 2002) and subsequently KRAS (Lipton et al., 2003; Jones et al., 2004). The first two MYH mutations discovered were Y165C and G382D (Al-Tassan et

21 al., 2002). Ever since, more than 20 variants of MYH mutation have been identified, of which just a few are definitively pathogenic (Alhopuro et al., 2005; Kim et al., 2007a). Mutations of MYH demonstrate ethnic bias, as evident by the high incidence (80%) of Y165C and G382D in the caucasian population with MAP, while in Pakistani, Indian, Italian and Portuguese populations with this syndrome, the common mutations are Y90X, E466X, 1395delGGA and 1187insGG respectively (Jones et al., 2002; Gismondi et al., 2004; Isidro et al., 2004; Venesio et al., 2004). Overall, pathogenic MYH mutations have been found in approximately 25% of individuals diagnosed with FAP or AFAP and who do not have a germline APC mutation (Varesco, 2004).

Consistent with the fact that some MAP cases occur in the absence of a family history, bi-allelic MYH mutations have been reported in 0.2-1% of sporadic CRC cases (Enholm et al., 2003; Croitoru et al., 2004; Fleischmann et al., 2004; Wang et al., 2004; Farrington et al., 2005; Peterlongo et al., 2005; Colebatch et al., 2006). Conversely, mono-allelic MYH mutations have been reported both in cancer (0.5-2.4%) and control populations (0-1.9%) but the consequence of this remains unclear (Enholm et al., 2003; Croitoru et al., 2004; Fleischmann et al., 2004; Wang et al., 2004; Kambara et al., 2004a; Farrington et al., 2005; Peterlongo et al., 2005; Colebatch et al., 2006). While individual studies do not suggest a significantly increased risk of cancer for monoallelic MYH mutation carriers (Enholm et al., 2003; Fleischmann et al., 2004; Wang et al., 2004; Kambara et al., 2004a; Farrington et al., 2005; Peterlongo et al., 2005; Webb et al., 2006), a meta-analysis based on these studies shows a marginal increase in risk (pooled odds ratio =1.4, CI, 1.0-2.0) (Jenkins et al., 2006). Therefore, monoallelic MYH mutation is unlikely to confer a strong predisposition to CRC (Lipton and Tomlinson, 2006).

1.3.1.4 HNPCC syndrome Hereditary non-polyposis colorectal cancer (HNPCC) is also known as the Lynch syndrome, named after its founder, Henry Lynch. Between 1 and 3% of colorectal carcinoma cases arise in individuals with HNPCC (Mecklin, 1987; Aaltonen et al., 1998; Salovaara et al., 2000; Cunningham et al., 2001). From a clinical perspective, this syndrome is characterised by an increased risk of multiple colorectal and gastric carcinomas as well as extra-colonic cancer, including endometrial carcinoma.

22 Individuals with this syndrome often develop cancer at an early age, with a median onset of 44 years, and display a relatively good prognosis (Craanen et al., 1996). Unlike FAP, adenomas are relatively uncommon in HNPCC, and are usually solitary (Jass, 1995). In terms of pathological features, CRCs arising in the setting of HNPCC are typically right-sided, and show a mucinous phenotype, poor differentiation and prominent lymphocytic infiltration. However, the hallmark of HNPCC cancers, and indeed the feature that allowed recognition of the biological basis of this syndrome, is the phenomenon of microsatellite instability (MSI).

Individuals with HNPCC are predisposed to the development of MSI cancers because they harbour heterozygous loss-of-function germline mutations of one of the DNA mismatch repair genes, most commonly MLH1 and MSH2 (Kinzler and Vogelstein, 1996). Germline mutations of other MMR genes such as MSH6 and PMS2 have also been reported in individuals with HNPCC, albeit to a lesser extent (Cunningham et al., 2001; Peltomaki and Vasen, 2004; Hampel et al., 2005). Colorectal cancers that arise in individuals with HNPCC often harbour somatic mutations that inactivate the remaining wild-type allele of the respective mismatch repair gene bearing a germline mutation. Biallelic inactivation of a mismatch repair gene leads to the increase of replication error which may accelerate the accumulation of mutations in tumour suppressor genes and oncogenes, and which may eventually lead to malignancy (Kinzler and Vogelstein, 1996). Based on this finding, Fearon and Vogelstein’s original model of genetic changes in the adenoma-carcinoma progression was modified by Fearon (1996) to incorporate the “mutator” role of aberrant mismatch repair machinery in colorectal carcinogenesis (Figure 1.1) (Fearon, 1996).

1.3.2 Sporadic colorectal cancer

In the sporadic setting, colorectal neoplasia can be initiated by the somatic loss of both copies of a tumour suppressor or DNA repair gene. This is followed by additional genetic changes as the lesion progresses from adenoma to carcinoma. However, the mechanisms of inactivation of tumour suppressor and DNA repair genes in sporadic colorectal carcinomas differ from their familial counterparts, in that large deletions or somatic methylation of tumours suppressor or DNA repair genes occur more frequently. Oncogenic point mutations also frequently occur in hotspots as exemplified by those of

23 BRAF and KRAS. In addition, the frequencies and characteristics of genetic aberrancies are distinct between cancers demonstrating chromosomal and microsatellite instability.

In sporadic MSI cancers, mutations of tumour suppressor genes, APC, p53, and SMAD4 were found at a lower frequency than in CIN cancers (Ionov et al., 1993; Kim et al., 1994; Konishi et al., 1996; Olschwang et al., 1997; Salahshor et al., 1999; Woodford- Richens et al., 2001). Similarly, loss of heterozygosity at these gene loci was uncommon (Thibodeau et al., 1993; Konishi et al., 1996; Olschwang et al., 1997). While there is a low frequency of APC mutations in MSI cancers, APC mutations observed in these tumours often result in a frameshift (Huang et al., 1996). This is in contrast to the high frequency of point mutations in CIN tumours that are microsatellite stable (MSS). Since MSI is caused by a defect in the mismatch repair machinery, it is not surprising that coding regions with simple repeats are also susceptible to deletion that results in frameshift mutation. High rates of mutations have also been detected within the repetitive region of genes including TGFBRII, BAX and IGFIIR, MSH3 and MSH6 in MSI cancers (Calin et al., 2000).

1.4 Genetic alterations in cancer development

In light of the genetic basis underlying the development of familial and sporadic CRCs discussed in the previous section, this section will outline the functional consequences of genetic alterations and the stages at which they occur in cancer development.

1.4.1 Adenomatous polyposis coli (APC)

The adenomatous polyposis coli (APC) tumour suppressor gene maps to the chromosomal location of 5q21-22 (Groden et al., 1991; Kinzler et al., 1991). It encodes a large 312 kDa protein comprising 2843 amino acids that interacts with a number of other proteins including ß-catenin (Rubinfeld et al., 1993; Su et al., 1993), glycogen synthase kinase (GSK) 3ß (Rubinfeld et al., 1996), end binding protein (EB) 1 (Su et al., 1995), and Bub kinases (Kaplan et al., 2001). APC is well known as a multi- functional protein involved in many cellular processes including chromosome segregation, cell adhesion, cell migration, signal transduction and apoptosis (Sieber et al., 2000; Fearnhead et al., 2001).

24 Of all these functions, the tumour prevention property of APC is established through its interaction with ß-catenin (Korinek et al., 1997; Morin et al., 1997). As a major component of the Wnt signalling pathway, ß-catenin acts as a growth regulator required for activation of oncogenes such as c-myc, matrylycin and c-jun. Under normal circumstances, APC serves as a regulator of ß-catenin levels in the cell by mediating the degradation of ß-catenin through the formation of a complex that includes ß-catenin, axin and GSK-3ß. Loss of APC results in the overexpression of ß-catenin that in turn, constitutively upregulates the transcriptional activities of downstream oncogenes. This eventually triggers the hyperproliferation of cells in colonic mucosa.

In a less well-recognised manner, APC may also be important in preventing tumour development through interaction with microtubules. It has been shown that wild-type but not mutant APC protein potentiates microtubule assembly (Munemitsu et al., 1994; Smith et al., 1994b). Wild-type APC is localized at kinetochore microtubules and may have a role in promoting their stability and attachment to chromosomes. Supporting this is the observation that cells with mutant APC display defective chromosomal segregation in conjunction with chromosomal instability (Fodde et al., 2001; Kaplan et al., 2001). Of particular note, APC interacts with the mitotic checkpoint proteins Bub-1 and Bub-3 when localized to kinetochore microtubules (Kaplan et al., 2001). Since inactivation of Bub-1 has been associated with chromosomal instability, the loss of APC may also render the same consequences. More importantly, APC also interacts with the microtubule plus end–binding protein, EB1, and recently it was shown that APC mutations inhibit the interaction of APC with EB1, thus leading to improper alignment of chromosomes, and therefore chromosomal aneuploidy (Green et al., 2005; Caldwell et al., 2007).

APC has been regarded as the “gatekeeper” of the multistep adenoma-carcinoma sequence as the entry of a cell into this pathway is largely determined by the integrity of this gene. As well as being a feature of cancers associated with FAP, APC mutations or allelic losses of 5q have been observed in up to 80% of CRCs (Bodmer et al., 1987; Solomon et al., 1987; Vogelstein et al., 1988; Cottrell et al., 1992; Powell et al., 1992). More importantly, this same frequency of APC mutations has been observed in adenomas, including those as small as 0.5 cm, (Vogelstein et al., 1988; Powell et al., 1992; Jen et al., 1994; Miyaki et al., 1994), and ACF (Jen et al., 1994; Smith et al.,

25 1994a; Otori et al., 1998) highlighting the involvement of this mutation at the earliest stages of the adenoma-carcinoma sequence.

1.4.2 KRAS

The family of RAS genes encodes three functional proteins, H-RAS, K-RAS and N- RAS. These proteins are important signal transducers in multiple signaling pathways critical for normal cell proliferation, differentiation and apoptosis (Bos, 1989; Bourne et al., 1990). The cancer-causing role of mutated ras genes was first identified through transformation of the normal fibroblast NIH/3T3 cell line following transfection with mutant RAS DNA (Krontiris and Cooper, 1981; Perucho et al., 1981; Shih and Weinberg, 1982). Ever since, mutations of ras genes have been found to be strongly implicated in various human cancers (Bos, 1989). These mutations are typically oncogenic and therefore result in autonomous stimulation of growth or differentiation.

In CRC, mutation of HRAS and NRAS is rare. However, KRAS mutations, predominantly in codon 12 and 13 have been reported in approximately 31-40% of CRCs (McLellan et al., 1993; Andreyev et al., 1998; Nagasaka et al., 2004). KRAS mutations have also been found in adenomas (Scott et al., 1993; Eppert et al., 1996; Maltzman et al., 2001; Takayama et al., 2001; Adler et al., 2002; O'Brien et al., 2006) and aberrant crypt foci (Pretlow et al., 1993; Jen et al., 1994; Smith et al., 1994a; Yamashita et al., 1995; Losi et al., 1996; Shivapurkar et al., 1997; Takayama et al., 2001; Rosenberg et al., 2007), thus indicating this gene defect occurs as an early event in colorectal carcinogenesis.

However, higher incidences of KRAS mutation have been detected in larger compared to smaller adenomas (Vogelstein et al., 1988; Maltzman et al., 2001; O'Brien et al., 2006), suggesting that KRAS mutation is not the initiating event in the adenoma-carcinoma sequence.

1.4.3 Loss of 18q

As documented in the initial report by Vogelstein and co-workers, 18q is the second most common region at which allelic loss is observed in CRC (Vogelstein et al., 1988). While the loss of 18q has been observed in approximately 70% of CRCs, it has also

26 been reported in 10-30 % of early adenomas; and up to 60% of late adenomas (Vogelstein et al., 1988; Boland et al., 1995).

While defective tumour suppressors or oncogenes are clearly recognised in other regions with frequent LOH, there is considerable contention pertaining to the altered tumour suppressor gene in 18q. Initially, the deleted in colorectal cancer (DCC) gene was thought to be the likely candidate (Fearon et al., 1990b). While DCC is likely to be a tumour suppressor gene as it encodes a protein that acts as a receptor for netrin 1, which is involved in axonal guidance(Keino-Masu et al., 1996), point mutations of DCC have only been identified in approximately 6% of sporadic colorectal carcinomas (Cho et al., 1994). Moreover, mutant mice with biallelic loss of DCC do not show alteration in the colonic epithelium, casting doubt on the role of this gene as a tumour suppressor (Cho et al., 1994).

Other candidate tumour suppressors SMAD2 and SMAD4 have also been mapped to 18q (Eppert et al., 1996; Hahn et al., 1996). These genes are members of a gene family that encodes proteins which interact with the transforming growth factor (TGF)-ß receptors, and exert an essential role in the regulation of cell growth, differentiation, matrix production and apoptosis (Heldin et al., 1997; Duff and Clarke, 1998). Mutation of SMAD4 has been reported in 16-25% of CRC cases and is closely linked to cancer progression (Takagi et al., 1996; Thiagalingam et al., 1996; Ando et al., 2005). However, alteration of SMAD2 has only been found in 6% of cases (Eppert et al., 1996).

In a recent study, SMAD4 was suggested to have a tumour-suppressive function through regulation of the potential metastatic modulator, Claudin-1 (Shiou et al., 2007). Others have also found a significant correlation between SMAD4 loss with metastasis (Miyaki et al., 1999; Tanaka et al., 2006, Losi , 2007), advanced stage of colorectal carcinogenesis (Miyaki et al., 1999; Kouvidou et al., 2006) and poorer prognosis (Xie et al., 2003).

1.4.4 p53

The tumour suppressor p53 is a key protein in cell cycle regulation. It has been labeled the ‘guardian of the genome’ based on its crucial role in maintaining the integrity of the cell during proliferation (Lane, 1992). In the presence of DNA damage, p53 will stimulate DNA repair or, when necessary, apoptosis. Loss of this protein will allow

27 propagation of damaged cells and inevitably give rise to human cancer. Therefore it is not at all surprising that p53 is the most commonly mutated gene in human cancers (Caron de Fromentel and Soussi, 1992). p53 mutation was initially linked to CRC due to the frequent allelic loss of chromosome 17p on which this gene resides (Vogelstein et al., 1988). Existing studies based on immunohistochemistry, DNA sequencing and loss of heterozygosity (LOH) at 17p reported p53 mutation or allelic loss of 17p in 4-26% of early adenomas, in approximately 50% of late adenomas and in 50-75% of carcinomas (reviewed in Leslie et al., 2002) (Leslie et al., 2002). Therefore, p53 loss is postulated to occur during the transition from late adenoma to carcinoma, prior to metastasis (Ilyas et al., 1999).

1.4.5 BRAF

The RAF family of protein kinases is a component of the MAPK signalling pathway, which is crucial in connecting extracellular signals to transcriptional regulation. The three members of the RAF family: ARAF, BRAF and CRAF are downstream effectors of RAS and upon activation by RAS, they phosphorylate MEK1 and 2, which in turn activate ERK 1 and 2 (Wellbrock et al., 2004). This chain reaction involving RAS/RAF/MEK/ERK activates several transcription factors such as c-jun and c-myc, which then signals cell division.

Under normal circumstances, this multi-step pathway is modulated by different factors to ensure integrity of cell proliferation, differentiation and survival. In human cancers, regulation of this pathway is often disrupted, mainly due to activating mutations affecting two components of this pathway, the RAS and RAF oncogenes. In CRC, mutations of KRAS and BRAF are common, but almost always mutually exclusive (Rajagopalan et al., 2002; Chan et al., 2003). The former is often found in cancers demonstrating CIN, while the latter is more frequent in MSI cancers. Curiously, the frequencies of KRAS mutations were also found to be higher in HNPCC than sporadic MSI cancers (Fujiwara et al., 1998; Oliveira et al., 2004). In contrast, the mutation of BRAF is common in sporadic MSI cancers but rare in HNPCC cases (Deng et al., 2004; Kambara et al., 2004b; Domingo et al., 2005). Consistent with this, KRAS mutations are more appropriately associated with cancers that develop via the adenoma-carcinoma

28 sequence (both familial and sporadic), whereas BRAF mutations are integral to sporadic MSI cancers associated with the serrated neoplasia pathway (Chan et al., 2003).

The activating V600E change caused by a T to A transversion within exon 15 accounts for over 90% of BRAF mutations in CRC (Deng et al., 2004). This mutation has also been observed in aberrant crypt foci (with serrated morphology), hyperplastic polyps, as well as serrated adenomas (mostly SSA and some TSA) (Kambara et al., 2004b; Beach et al., 2005; Rosenberg et al., 2007), but is rare in conventional adenomas (Koinuma et al., 2004; Kambara et al., 2004b). These findings are consistent with those found in CRCs in that BRAF mutation is useful as a marker for the serrated neoplasia pathway. This suggestion however needs to be taken with caution as recent observations have suggested a distinct subtype of hyperplastic polyp, termed the goblet cell type, that shows low frequencies of BRAF mutation, but high frequencies of KRAS mutation (Yang et al., 2004). Furthermore, it has become clear that traditional serrated adenomas lack BRAF mutation when compared to sessile serrated adenomas (Jass et al., 2006). Again, KRAS mutations are more common in TSAs (Yang et al., 2004; Kambara et al., 2004b; Jass et al., 2006).

1.5 The “Epigenetic Code” and its changes in colorectal neoplasia

Epigenetics refers to modifications of DNA that can be maintained through cell division but do not entail the alteration of the DNA sequence itself. As a whole, they are referred to as the “epigenetic code” (Figure 1.2) which includes histone variants, modifications to histone tails, methylation of DNA, as well as the more recently described phenomenon of RNA modifications. These epigenetic factors complement nucleotide sequences in the regulation of protein synthesis to maintain the integrity of cellular function and homeostasis.

Figure 1.2 The epigenetic code Schematic of the inter-related cellular processes that constitute the epigenetic code. (Wong et al., 2007).

1.5.1 Epigenetic events in normal human cells

In every normal mammalian cell, the DNA sequence remains essentially the same. However, epigenetic modifications can vary from one cell type to another and play a fundamental role in maintaining the integrity of biological processes such as embryonic development and cell differentiation. Epigenetic changes are dynamic, and involve several key modifications, as summarised below.

1.5.1.1 Chromatin modifications The basic unit of human chromatin, the nucleosome, is made up of 146 bp of DNA looped twice around an octamer of core histones (two H2A/H2B dimers and a H3/H4 tetramer). Covalent modifications of histone proteins can change chromatin from a densely compacted inactive structure to an open, active configuration, and vice versa (Figure 1.3). These modifications, which include acetylation, methylation, phosphorylation, and ubiquitinylation, are reversible epigenetic events that occur at the N-terminal domains of all core histones. Histone modification is made possible by the activities of a number of enzymes including histone acetyl transferases (HATs), histone deacetylases (HDACs), histone methyltransferases, DNA methyltransferases (DNMTs) and methyl-CpG binding proteins (MBDs). The balanced activity of this series of

30 enzymes is pivotal to normal cellular function, and alteration in their function is known to cause diverse and often profound disorders (Egger et al., 2004).

Generally, the active chromatin structure corresponding to increased transcriptional activity is associated with increased histone acetylation. HATs such as P300 and cellobiose phosphorylase (CBP) are known to catalyze acetylation of lysine residues on H3 and H4 (Ogryzko et al., 1996). Acting antagonistically to HATs, histone deacetylases (HDACs) as a general rule produce transcriptional repression by a complicated mechanism that involves formation of a complex that comprises DNA methyltransferases (DNMTs) (Fuks et al., 2000; Rountree et al., 2000) and methyl- CpG-binding proteins (MBDs) (Ng et al., 2000; Saito and Ishikawa, 2002). Likewise, methylation and phosphorylation of histones are also involved in regulation of the chromatin state (Egger et al., 2004). A classic example of histone tail modifications that determine transcriptional state is the acetylation and di or trimethylation of lysine 9 on H3 (Figure 1.3), which has been associated with gene activation and silencing respectively (Kondo and Issa, 2004). Taken together, the pattern of histone modifications constitutes the ‘histone code’ which complements the primary DNA sequence in defining transcription states (Jenuwein and Allis, 2001).

Following some recent studies (Santos-Rosa et al., 2002; Santos-Rosa et al., 2003; Schneider et al., 2004), it has become clear that active genes are highlighted with a trimethylation mark on lysine 4 of histone 3 (designated H3K4me3). More importantly, gene activation is found to be mediated through binding of nucleosome remodelling factors to nucleosomes carrying H3K4me3 marks (Pray-Grant et al., 2005; Li et al., 2006; Wysocka et al., 2006). Nucleosome remodelling factors are enzymes that can cause disruption to DNA-histone interactions, facilitate the focal opening of chromatin and hence, the potential for gene activation (Becker, 2006). This reveals the involvement of another level of chromatin modification termed nucleosome remodelling in the regulation of gene function.

Figure 1.3 A model of epigenetic modifications and their effect on transcription The nucleosome is assembled from DNA and histones, and the chemical modification of histone tails induces conformational changes that can cause either activation or repression of transcription. Repressive modifications include methylation of the lysine 9 residue of histone 3 (H3K9), lysine 27 residue of histone 3 (H3K27) and lysine 20 residue of histone 4 (H4K20) in association with DNA methylation. Changes such as acetylation at H3K9 (shown) are associated with open chromatin (euchromatin) formation. (Wong et al., 2007).

1.5.1.2 DNA methylation Among the various epigenetic modifications, DNA methylation is perhaps the most widely studied and the best understood. DNA methylation is the covalent modification of cytosine residues in DNA to form 5-methylcytosine (Figure 1.4). In the mammalian genome, this occurs most commonly at 5’-CG-3’ dinucleotides (also termed CpG dinucleotides) and occasionally at 5’-CA-3’ or 5’-CT-3’ dinucleotides (Ramsahoye et al., 2000). Approximately 70-80% of all CpG dinucleotides in the human genome are methylated (Ehrlich et al., 1982) with the exception of CG rich regions that usually span the promoters and sometimes the first exons of approximately 60% of all human genes (Robertson, 2005). These regions, termed CpG islands are defined as sequences greater than 500 bp in length, with a GC content greater than 55% and an observed GC ratio greater than 0.65 (Takai and Jones, 2002).

32 O O

2 3 2 3 SAM 1 4 NH2 1 4 NH2 DNMT 6 5 6 5

H CH3 Unmethylated Methylated Cytosine Cytosine

Figure 1.4 Methylation of cytosine residues DNA methyltransferases (DNMTs) catalyse this reaction in the presence of S-adenosylmethionine (SAM) as the donor of a methyl group.

The genome-wide pattern of DNA methylation is reprogrammed in the early embryo. However, it requires constant maintenance to ensure the faithful execution of its functional role in somatic cells. Thus far, DNA methylation is known to regulate the differential expression of genes, including the silencing of genes on the inactive X chromosome, as well as the regulation of age related and tissue specific gene expression (Plass and Soloway, 2002). In addition, DNA methylation plays a significant role in the maintenance of genomic imprinting. As reviewed extensively by others (Plass and Soloway, 2002; Wilkins, 2005), genomic imprinting is solely an epigenetic phenomenon by which DNA methylation permits the monoallelic expression of genes in a parent-of-origin specific manner. To date, more than 80 imprinted loci have been described, and these are typically charaterised by tissue-specific and stage-specific patterns of expression (Robertson, 2005).

The regulation of DNA methylation is brought about by the activity of enzymes termed DNA methyltransferases. To date, several enzymes have been classified into this group including DNMT1, DNMT2, DNMT3A and DNMT3B (Goll and Bestor, 2005). DNMT1 is well recognised as the maintenance methyltransferase that executes faithful propagation of DNA methylation patterns in adult mammalian cells during mitosis (Bestor et al., 1988; Yen et al., 1992). This enzyme exhibits strong activity towards hemimethylated CpG dinucleotides generated from semiconservative DNA replication and catalyses the methylation of unmethylated CpG sites on the daughter strand following DNA replication (Yoder et al., 1997). In some studies, the role of DNMT1 in de novo methylation has also been shown (Okano et al., 1998; Jair et al., 2006). Meanwhile, DNMT3A and 3B are de novo methyltransferases (Okano et al., 1998) that

33 together play an important role in the setting of imprints in gametes and restoring genome-wide methylation patterns following a wave of demethylation in early embryogenesis (Okano et al., 1999). The role of DNMT2 is poorly understood. DNMT2 exhibits weak methyltransferase activity and is not essential for maintenance or de novo methylation of DNA in embryonic stem cells (Okano et al., 1999).

1.5.1.3 RNA modification In recent years, it has become apparent that some untranslated RNAs (non-coding RNAs) which do not encode any functional protein play essential roles in the regulation of epigenetic phenomena (Bernstein and Allis, 2005). For instance, the Xist RNA was found to have a hand in regulating the dosage compensation of the X chromosome (Heard, 2004). This is clearly an epigenetic event as it involves long-term X- inactivation that can be transmitted through cell division (Bernstein and Allis, 2005). In addition, mitotically heritable RNA modification can occur in the form of antisense RNA transcription that causes DNA methylation-induced silencing of genes. An example of this phenomenon is described in a case of -thalassemia, in which antisense transcription results in DNA methylation and silencing of the globin gene, HBA2 (Tufarelli et al., 2003).

1.5.2 Epigenetic basis of colorectal neoplasia

A wealth of existing literature has now pinpointed particular epigenetic events as the hallmark of human cancers. Changes in DNA methylation patterns have long been recognised as a sign of cancer development. More recent studies have now revealed the role of chromatin remodelling, which colludes with changes in DNA methylation patterns to promote carcinogenesis. In colorectal neoplasia, there is considerable interplay between epigenetics and genetic events, which can be detected across a wide range of lesions from aberrant crypt foci to carcinomas. Furthermore, CRCs generally demonstrate two common patterns of genomic instability, namely microsatellite and chromosomal instability, in which both genetic and epigenetic components are involved.

While the role of epigenetic changes may be examined in isolation, it is perhaps more informative when discussed in the context of established pathways of colorectal

34 neoplasia. These aberrancies further explain how inappropriate gene reactivation or silencing can occur, depending on the nature of the epigenetic change. These changes and their effects on gene expression are discussed below.

1.5.2.1 Global DNA hypomethylation in colorectal cancer Global hypomethylation of DNA is a typical feature of many human cancers, by which a global reduction of 5-methylcytosine level is observed in conjunction with the loss of CpG methylation within sequences that are heavily-methylated in normal cells. The cancer specific progressive and global decrease in 5-methylcytosine levels was first demonstrated within tumour cells in rats (Lapeyre and Becker, 1979). In the experiment by Lapeyre, a significant loss of 5-methylcytosine was found in the acetylaminofluorene-induced rat hepatocellular carcinoma when compared to the normal liver tissue of rat (Lapeyre and Becker, 1979). This phenomenon was subsequently observed in human tumours, both benign and malignant (Feinberg and Vogelstein, 1983; Flatau et al., 1983; Riggs and Jones, 1983). Particularly in CRC, global hypomethylation of DNA has been implicated in lesions across the neoplastic spectrum, from polyps to carcinomas (Goelz et al., 1985; Bariol et al., 2003).

Hypomethylation has been linked to several mechanisms that could result in neoplastic progression. While absent in normal cells, hypomethylation occurs typically at repetitive sequences residing in satellite or pericentromeric regions within the genome of tumour cells and was associated with the occurrence of chromosomal instability (Esteller, 2006). Such chromosomal instability includes breakage and recombination of pericentromeric DNA as reported previously in chromosome 1 and 16 of tumour cells (Ji et al., 1997; Qu et al., 1999) Hypomethylation can also result in the reactivation of previously silenced retrotransposons and leads to the disruption of normal gene structure and function (Esteller and Herman, 2002 ; Suter et al., 2004a). In addition, hypomethylation can lead to the activation of oncogenes, as exemplified by the activation of S100A4 metastasis-associated gene in colorectal carcinoma (Nakamura and Takenaga, 1998) and that of the cyclin D2 (Oshimo et al., 2003) and maspin (Akiyama et al., 2003) genes in gastric carcinoma.

Last but not least, the loss of DNA methylation has been associated with the loss of imprinting (LOI), an event that leads to cellular proliferation in cancer. The best

35 example of this phenomenon is the LOI of the IGF2 gene due to hypomethylation at the differentially methylated region of IGF2 (Cui et al., 2002). This event, reported in about 40% of CRCs, has been associated with a positive family history (Cui et al., 1998). Therefore, it is likely to be due to a gene-specific DNA hypomethylation rather than a consequence of global hypomethylation.

1.5.2.2 Hypermethylation of CpG islands in colorectal cancer In concert with global hypomethylation, hypermethylation at CpG islands is also recognised as a crucial event in cancer development (Figure 1.5). This phenomenon is commonly associated with the silencing of tumour suppressor genes.

Figure 1.5 Organisation and consequences of CpG methylation in normal and cancer cells The upper panel shows a normal cell, in which a cluster of CG dinucleotides (CpG island) remains unmethylated, (pale pins) where as scaterred cytosines elsewhere are methylated (red pins). In the absence of methylation of this CpG island, DNA in the promoter region remains accessible to transcription factors, and the gene is expressed. In the lower panel, a cancer cell shows characteristic CpG island methylation, with concomitant compact chromatin structure in the promoter region, causing silencing of gene expression. (Wong et al., 2007).

The retinoblastoma (RB) gene was probably the first tumour suppressor gene found to be silenced by promoter hypermethylation in cancer (reviewed by Feinberg and Tycko, 2004). Ever since, a long list of tumour suppressor and DNA repair genes have been identified as targets of this epigenetic alteration, including p16ink4A, VHL (Von-Hippel- lindau), APC, CDH1 (E-cadherin), and MLH1 (reviewed by Feinberg and Tycko, 2004). More recently, DNA hypermethylation has also been recognised as a mechanism that induces silencing of microRNAs with tumour-suppressor functions (Saito et al., 2006;

36 Lujambio et al., 2007), thus indicating an additional contribution of this phenomenon to the development of cancer.

Furthermore, DNA hypermethylation has been recognised as another cause underlying dysregulation of imprinting in cancer. Particularly in Wilm’s tumour, IGF2/H19 loss of imprinting is caused by the hypermethylation of a second differentially methylated region of IGF2 (Ogawa et al., 1993; Rainier et al., 1993), distinct from the region frequently hypomethylated in CRC. Similarly, hypermethylation leads to the loss of imprinting at other imprinted loci including p73 in haematological malignancies (Corn et al., 1999) as well as ARH1 in follicular thyroid carcinoma (Weber et al., 2005).

In CRC, a rapidly growing list of hypermethylated genes has been described including those involved in apoptosis, angiogenesis, cell-cycle regulation, differentiation, DNA repair, metastasis, signal transduction and transcription (Table 1.2). This list is likely to grow as large scale methods for the detection of methylation are improved. Of important note, DNA hypermethylation was thought to have a seminal role in the earliest step of colorectal carcinogenesis (Baylin and Ohm, 2006; Feinberg et al., 2006). As postulated in recent reviews (Baylin and Ohm, 2006; Feinberg et al., 2006; Jones and Baylin, 2007), a series of genes termed “epigenetic gatekeepers” may prevent stem/precursor cells from becoming cancerous. Among the speculated “epigenetic gatekeepers” are p16ink4A, SFRPs, GATA-4 and -5, as well as APC, all of which are pivotal in maintaining the fidelity of cell renewal. Hypermethylation induced silencing of these genes has been demonstrated in preinvasive lesions of colon and other cancers (Jones and Baylin, 2007). The inappropriate silencing of these genes may potentiate abnormal growth and clonal expansion of stem/precursor cells to form cancerous lesions.

37 Gene Function Frequency (%) Reference

APC Signal transduction, beta- 10-50 (Esteller et al., 2000a; catenin regulation Kim et al., 2003; Bai et al., 2004; Lee et al., 2004; Kim et al., 2005) CDH13 Cell signalling (cell 30-40 (Hibi et al., 2005) recognition and adhesion) CDKN2A Cell cycle regulation 15-30 (Merlo et al., 1995; Hawkins and Ward, 2001; Lee et al., 2004) CHFR Mitotic stress checkpoint 30-40 (Corn et al., 2003; Toyota et al., 2003) HIC1 Regulation of DNA damage ~80 (Maekawa et al., 2001; responses Chen et al., 2005) HPP1 Transmembrane TGF-beta ~80 (Young et al., 2001a) antagonist

LKB1 Cell signalling, cell polarity 5-10 (Esteller et al., 2000b) MGMT Repair of DNA guanosine 30-40 (Lee et al., 2004; Kim methyl adduct et al., 2005; Shen et al., 2005; Fox et al., 2006; Wynter et al., 2006) MLH1 Mismatch repair 10-20 (Herman et al., 1998; Miyakura et al., 2001; Nakagawa et al., 2001; Kim et al., 2005) P14ARF Cell cycle regulation 20-30 (Esteller et al., 2001; Shen et al., 2003; Lee et al., 2004) RASSF1A DNA repair, cell cycle >50 (Lee et al., 2004; regulation Sakamoto et al., 2004; Oliveira et al., 2005) SOCS-1 Cell signaling 5-10 (Hibi et al., 2005) THBS1 Angiogenesis 10-20 (Lee et al., 2004; Kim et al., 2005) TIMP3 Matrix remodeling, tissue 10-30 (Lee et al., 2004; invasion Brueckl et al., 2005)

Table 1.2 Genes commonly silenced by promoter methylation in colorectal neoplasia

38 1.5.2.3 MLH1 methylation in MSI colorectal cancer as a classic example of promoter hypermethylation in colorectal cancer MLH1 represents a classic example of gene promoter hypermethylation resulting in transcriptional silencing in CRC. Of all the epigenetically silenced genes in cancer, MLH1 has probably been studied in the most detail and is a major focus of this thesis.

MLH1 is a mismatch repair gene that maintains the integrity of DNA sequences following DNA replication. The loss of MLH1 protein leads to replication errors which are marked by microsatellite instability. Silencing of MLH1 due to bi-allelic hypermethylation of the MLH1 promoter is the hallmark of sporadic MSI CRCs (Kane et al., 1997; Herman et al., 1998; Veigl et al., 1998; Wheeler et al., 2000). This event, rarely if ever, occurs in HNPCC tumours that harbour germline mutations of mismatch repair genes, including MLH1 (Wheeler et al., 2000; Young et al., 2001b; Deng et al., 2004; Domingo et al., 2005; Lubomierski et al., 2005). However, monoallelic hypermethylation of the MLH1 promoter has been suggested as the second hit in some HNPCC cases (Young et al., 2001b; Deng et al., 2004).

Of important note, a specific pattern of MLH1 methylation correlates with the expression of this gene. As reported by Deng et al., methylation at the MLH1 promoter particularly at the 3’-most (proximal or C) region but not that of the 5’-most (distal or A) region correlates invariably with the loss of MLH1 expression (Figure 1.6) (Deng et al., 1999; Deng et al., 2002). The same group also identified the methylation of a specific CpG site in the proximal region of the MLH1 promoter that resulted in inhibition of the binding of the transcription factor CBF, to cause MLH1 silencing (Deng et al., 2001).

Distal or A Proximal or C -796 region -547 -322 region +56 MLH1 Exon 1

-800 -700 -600 -500 -400 -300 -200 -100 +1 +100 +200

CpG methylation has no CpG methylation correlates with the effect on MLH1 silencing loss of MLH1 expression

Figure 1.6 Map of the MLH1 promoter showing the 3’-most (proximal) and 5’-most (distal) regions CpG island methylation at the proximal region but not the distal region correlates invariably with the loss of MLH1 expression.

39 In recent years, much interest has been placed on elucidating the association between MLH1 methylation and known genetic mutations. Notably, MLH1 methylation is strongly associated with the V600E activating mutation of BRAF in sporadic MSI cancers (Deng et al., 2004). While MLH1 silencing is unlikely to induce BRAF mutation (Wang et al., 2003), a recent study has suggested the role of BRAF in potentiating the methylation of MLH1 (Minoo et al., 2007). The precise mechanism for this remains unclear.

1.5.2.4 Dysregulation of chromatin modifications While much is now known about the involvement of CpG island methylation in human cancer, the role of chromatin modifications, either independently or in collaboration with DNA methylation is increasingly being recognised. First and foremost, cancer cells are marked with histone modifications that are unusual in normal cells. These include changes in the chemical modifications of histone tails. These modifications have all been linked to the alteration of gene expression, as exemplified by the silencing of tumour suppressor and DNA repair genes (Fahrner et al., 2002; Kondo et al., 2003; McGarvey et al., 2006).

Key modifications to histone tails, namely deacetylation and bi- or trimethylation of the lysine 9 residue of histone H3 (H3K9), loss of histone H3 lysine 4 (H3K4) trimethylation and gain of histone H3 lysine 27 (H3K27) trimethylation have all been found to occur concomitantly with the aberrant methylation of genes (Fahrner et al., 2002; Bachman et al., 2003; Ballestar et al., 2003; Kondo et al., 2003; McGarvey et al., 2006; Schlesinger et al., 2007). Importantly, these changes are common in CRC (Fahrner et al., 2002; Bachman et al., 2003; Kondo et al., 2003; McGarvey et al., 2006). In addition, a more recent study has proposed changes in the core histones H4, characterised by the loss of both monoacetylation of lysine 16 and trimethylation of lysine 20, as a universal marker for malignant transformation (Fraga et al., 2005).

In recent years, the role of chromatin remodelling proteins in cancer development has been rigorously explored. For instance, the EZH2-containing polycomb complex usually expressed in the embryo, but absent in adult somatic cells, was found to be expressed in cancer cells (Schlesinger et al., 2007). More significantly, the presence of this complex promoted the recruitment of DNA methyltransferases and de novo

40 methylation of CpG islands of genes packaged within nucleosomes containing H3K27 trimethylation (Schlesinger et al., 2007). This evidence reflects the important collaboration between chromatin remodelling, histone modification and DNA methylation in the silencing of genes in cancer.

1.6 Patterns of gene methylation in colorectal cancer

Promoter hypermethylation induced-silencing of individual genes is a well established phenomenon in cancer development. However, this phenomenon is not necessarily a focal event affecting single genes. Hypermethylation of CpG islands can occur concomitantly at the promoter region of multiple genes, as characterised by the CpG Island Methylator phenotype (CIMP), and the newly-introduced concept of long range epigenetic silencing.

1.6.1 CpG island methylator phenotype (CIMP) and the serrated neoplasia pathway

The CpG island methylator phenotype or CIMP was a concept first proposed by Toyota and co-workers in 1999 to describe the concomitant and extensive methylation of multiple distinct CpG islands at different loci within the genome in CRCs (Toyota et al., 1999). They identified two distinct entities of CRCs, those with and those without multiple methylation events, termed CIMP+ and CIMP- respectively. In subsequent studies of CRC cohorts, CIMP+ cancers are suggested to be clinically, pathologically and genetically distinct from CIMP- cancers. CIMP+ cancers are characterised by many features of MSI cancers including right sidedness, high grade, mucinous type and high prevalence in elderly people particularly in females (van Rijnsoever et al., 2002; Hawkins et al., 2002b; Samowitz et al., 2005a). Therefore, there has been contention as to whether CIMP+ cancers exist as a biologically distinct subgroup of CRC or just an artificially selected group of cancers with microsatellite instability showing methylation at multiple loci. However, a small proportion of microsatellite stable cancers has also been associated with the CIMP+ phenotype and recently, it has become clear that CIMP+ve phenotype underlies cancers that develop via the serrated neoplasia pathway. The overlaps between CIMP and other known features of CRC are shown in a working model that underlies pathways by which CRC can develop (Figure 1.7).

Figure 1.7 Pathways for colorectal tumourigenesis A working model depicting the dichotomy between chromosomal and microsatellite instability pathways in colorectal carcinogenesis, and the common morphological and genetic changes that accompany each subtype. A subgroup of tumours is shown in the centre of the figure that are characterised by CpG island methylation (CIMP +) and microsatellite stability (MSS). It is not clear whether these tumours arise from either or both of the main pathways, or whether they develop separately. MSI - microsatellite instability; Serrated polyp - hyperplastic polyp or serrated adenoma. (Wong et al., 2007).

On a separate issue, there has been considerable argument regarding the heritability of CIMP+ cancers. While CIMP+ cancers were initially shown to be associated with a positive family history (Frazier et al., 2003), subsequent studies on larger cohorts have suggested otherwise (Ward et al., 2004; Samowitz et al., 2005a). Also of importance is

42 the clear distinction in clinical outcomes between CIMP+ and CIMP- cancers when co- stratified by microsatellite status. Particularly in microsatellite stable tumours, the CIMP+ phenotype is associated with a poorer prognosis (Ward et al., 2003; Samowitz et al., 2005b; Ogino et al., 2007b).

In relation to known genetic changes, CIMP+ cancers are strongly associated with the V600E activating mutation of BRAF (Kambara et al., 2004b; Samowitz et al., 2005a; Weisenberger et al., 2006), and inversely correlated with p53 mutations (Toyota et al., 2000; van Rijnsoever et al., 2002; Hawkins et al., 2002b). Contradictory findings have however been reported in the association between KRAS mutations and CIMP status. Some studies have demonstrated a high frequency of KRAS mutations in CIMP+ tumours (Toyota et al., 2000; Samowitz et al., 2005a) while others have only found a weak correlation between the two (van Rijnsoever et al., 2002; Nagasaka et al., 2004; Kambara et al., 2004b). Importantly, overexpression of c-fos has been associated with the upregulation of DNMT1 that can increase global DNA methylation (Bakin and Curran, 1999). While this event can typically be induced by both KRAS and BRAF mutations, it is fascinating that the latter is predominantly observed in cancers with the CIMP+ phenotype. The significance of the link between BRAF mutation and CIMP+ phenotype remains to be elucidated.

Although the concept of CIMP has been around for more than a decade, there is still controversy regarding a consensus panel of markers to define CIMP status. In part, this is due to the lack of knowledge as to which markers best characterise CIMP. While the classic panel of markers (Issa, 2004) comprising MINT1, MINT2, MINT31, MLH1 and p16 are most widely used, this panel may not necessarily be the one that most accurately defines CIMP status. In a recent study, Weisenberger et al have proposed a new panel of CIMP markers which consists of CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 (Weisenberger et al., 2006). Despite being claimed as superior to the classic panel and displaying excellent correlation with V600E mutation of BRAF, several genes in this panel including CACNA1G, NEUROG1 and SOCS1 have little known relationship to colorectal carcinogenesis. Recently, the performance efficiency of this new panel of CIMP markers was evaluated in a large population-based study (Ogino et al., 2007a). Although good performance was achieved using these markers, an alternative set of

43 markers that comprise at least RUNX3, CACNA1G, IGF2 and MLH1 has also been suggested as good surrogate markers for CIMP status (Ogino et al., 2007a).

At present, the lack of an operational definition of CIMP is clearly a limiting factor in elucidating the biological importance of this concept. For instance, the lack of a consensus definition for CIMP results in the usage of markers which do not correlate with CIMP and can ultimately lead to the conclusion that CIMP does not exist (Anacleto et al., 2005).

1.6.2 Long-range epigenetic silencing

Long-range epigenetic silencing was first reported by Frigola et al who demonstrated hypermethylation as a regional event that affects contiguous CpG islands on chromosome 2q14 domain in CRC (Frigola et al., 2006). This phenomenon has also been reported in the 3p22 chromosomal region in a small group of MSI CRC (Hitchins et al., 2007b). These findings have challenged the dogma that hypermethylation is confined to discrete CpG islands. Rather, epigenetic silencing can involve hypermethylation and ultimately the silencing of large stretches of chromosomal DNA affecting multiple contiguous genes, as shown in Figures 1.8 and 1.9.

Normal colonic Colorectal mucosa Carcinoma

Figure 1.8 DNA methylation profile of the 4-Mb region on chromosome 2q14.2 in a colorectal cancer and paired normal colonic mucosa Coordinate methylation of contiguous CpG islands represented by grey-coloured squares was found in a colorectal carcinoma, while absent in the paired normal colonic mucosa. (Modified from Frigola et al., 2006).

A Locus map of the 3p22 chromosomal region

)

) )

1 1

P P

(a)(a)(a) P

1 1

C C

4 4 4

2 2

I I I

0 0 0

/ / P P

2 2 2

9 9

I I

- - - P P P2

6 6

A A A 4 4 4

C C

A A

I I

3 3 3

A A A

P P

(

( (

A A

D D D

G G G

A A A

F F FI

2 2 2

1 1

2 2

P P PP

G G G

C C C

L L L

T 0 0 0

R R R

H H H

R R R

T T

0 0 0

ST ST

S M

M M

D D

O O O

I I I

R R R PLCD1

DLEC1

L L L ACAA1

OXSR1

A A A

MYD88

B B

B P

P P

CTDSPL

P P PD

G G G

L L L

E E

A A A

M M M

(b)B MSI CRC

(c)C MSS CRC

(d)D Adenoma

Figure 1.9 Methylation profile of gene promoters within the 3p22 chromosomal region in colorectal cancer and adenoma A, Locus map of the 3p22 chromosomal region. B, C and D show typical patterns of CpG island methylation within the 3p22 chromosomal region in MSI and MSS CRC as well as adenoma. Black and white squares indicate methylated and unmethylated loci respectively. MSI, microsatellite stable; MSS, microsatellite unstable; CRC, colorectal cancer.

In addition, unmethylated genes residing between methylated ones were also down- regulated due to H3K9 di-methylation. Overall, long-range epigenetic silencing involving DNA and H3K9 methylation results in the inactivation or suppression of genes residing within large regions of chromosomes. This phenomenon may be important as an epigenetic equivalent to the genetic deletion of large chromosomal regions, termed loss of heterozygosity (LOH).

Inevitably, long-range epigenetic silencing of the 3p22 chromosomal domain has a functional significance as this region encompasses several cancer-causing genes including the mismatch repair gene MLH1 and tumour suppressor genes such as CTDSPL and DLEC1. On the contrary, genes residing in the 2q14 region are not well- associated with cancer and therefore, long-range epigenetic silencing of this region could be a “by-stander” effect of cancer.

45 1.7 CpG island methylation as a marker for the predisposition of colorectal neoplasia

As mentioned in previous sections, methylation of CpG islands within the promoter region of genes has been reported in precursor lesions of CRC such as aberrant crypt foci and colorectal polyps. CpG island methylation has also been observed in the apparently normal colonic mucosa of individuals with CRC. This may occur as a result of germline epimutation (discussed in the next section) or potentially, a field defect in the development of synchronous or metachronous CRC.

The concept of field defect or field cancerisation was first introduced by Slaughter et al (1953) to describe the presence of apparently normal cells with abnormal histological characteristic in close proximity to malignant cells (Slaughter et al., 1953). In a well- designed study, elevated levels of methylation at the promoter of the DNA repair gene MGMT has been observed in the normal colonic mucosa of individuals with sporadic colorectal carcinoma demonstrating MGMT methylation in their tumours (Shen et al., 2005). This finding suggested the role of MGMT methylation as a field defect in the development of CRC. On a broader perspective, a field defect in the development of CRC may also be marked by the methylation of other genes in the normal colonic mucosa adjacent to cancer cells.

The mismatch repair gene MLH1 is perhaps the gene most intensively studied for methylation in normal colonic mucosa. In part, methylation of this gene has been found in the majority of sporadic CRCs with microsatellite instability. Several studies have now documented the presence of MLH1 methylation in histologically normal colonic mucosa, although the pattern of methylation is generally different from that observed in tumours (Miyakura et al., 2001; Nakagawa et al., 2001; Furukawa et al., 2002).

Particularly in sporadic MSI cancers with MLH1 loss, methylation at the MLH1 promoter is often dense and encompasses the entire promoter region of this gene (Miyakura et al., 2001; Nakagawa et al., 2001; Deng et al., 2002; Furukawa et al., 2002). MLH1 methylation when present in the normal colonic mucosa is more mosaic and often affects the 5’-most region, with no significant correlation to gene silencing (Miyakura et al., 2001; Nakagawa et al., 2001; Deng et al., 2002; Furukawa et al., 2002). The dense methylation of the entire MLH1 promoter has however been reported

46 in a significant number of normal colonic mucosae (Nakagawa et al., 2001) though this finding was not confirmed by others (Miyakura et al., 2001; Deng et al., 2002; Furukawa et al., 2002).

Interestingly, MLH1 methylation detected in normal colorectal tissue has been thought to be non-specific for susceptibility to MSI cancers (Minoo et al., 2006). The dense methylation of this gene has been observed in approximately 20% of normal colonic mucosa from individuals with microsatellite stable cancers (Nakagawa et al., 2001). Inevitably, methodologies applied in each investigation may vary in terms of sensitivity and specificity and thus contribute to this discrepancy in data.

There has been speculation that MLH1 methylation may spread from the 5’-most to the 3’-most region of the promoter (Miyakura et al., 2003), yet there is insufficient evidence to support this. The presence in the normal colonic mucosa of methylation at the 5’- most region of the MLH1 promoter does not necessarily indicate an increased chance of spreading to the 3’-most promoter region. Furthermore, many individuals with MSS colorectal carcinoma harbour 5’-most methylation of MLH1 in both tumour and matched normal colorectal tissue (Miyakura et al., 2001; Deng et al., 2002).

Aberrant methylation in the normal colonic mucosa of individuals with CRC is not restricted to the MLH1 promoter alone. In a more recent study, low levels of CpG methylation have been found at the promoter regions of tumour suppressor genes including P16, DAPK, APC and TIMP3 in normal colonic mucosa adjacent to colorectal carcinoma (Kawakami et al., 2006). Such low level methylation in the normal colonic mucosa may serve as a risk marker for the development of colorectal neoplasia, yet this hypothesis would need to be tested through a longitutional study on individuals at risk of developing CRC (Kawakami et al., 2006).

1.8 Germline epimutation and colorectal cancer

The term epimutation generally refers to the abnormal activation or silencing of genes as a result of aberrant epigenetic modification. Epimutation can occur as a germ-line event as documented across different species from plants (Das and Messing, 1994; Jacobsen and Meyerowitz, 1997; Chandler et al., 2000) to mammals (Morgan et al., 1999 ; Rassoulzadegan et al., 2006). Germ-line epimutation has now been recognised as

47 a predisposing factor to CRC and clear evidence has been provided by studies on germline epimutation that affects the mismatch repair gene MLH1.

Germline epimutation of MLH1 is a rare event but has since been reported in 23 individuals with colorectal and other cancers (Gazzoli et al., 2002; Miyakura et al., 2004; Suter et al., 2004b; Hitchins et al., 2005; Valle et al., 2007; Hitchins et al., 2007a; Morak et al., 2008). These individuals manifest a somawide monoallelic hypermethylation of MLH1, in conjunction with the early onset of cancers typical of hereditary nonpolyposis colorectal cancer (HNPCC). Like HNPCC tumours, cancers from these individuals exhibit microsatellite instability as a result of biallelic MLH1 loss, following the somatic loss of the wild type MLH1 allele (Gazzoli et al., 2002; Miyakura et al., 2004; Suter et al., 2004b; Hitchins et al., 2005).

More importantly, germline epimutation of MLH1 has recently been shown to be transmissible across generations, consistent with the concept of transgenerational epigenetic inheritance (Hitchins et al., 2007a). As reported by our group, the germline epimutation of MLH1 was transmitted from a mother with CRC to a son who had inherited the same allele. Interestingly though, the epimutation was not present in the spermatozoa of the affected son. In addition, the epimutation was present in neither of two other siblings who also inherited the same maternal allele associated with the epimutation in their mother. Therefore, this event has been suggested to have occurred due to a failure in epigenetic reprogramming in their mother by which the epimutated allele failed to undergo complete demethylation and transcriptional reactivation during oogenesis.

In a recent study by Chan et al, germline epimutation of the mismatch repair gene MSH2 has also been reported in a family affected by HNPCC (Chan et al., 2006). Surprisingly, the epimutation was shown to be faithfully transmitted with the same genetic allele in an autosomal dominant fashion across three generations affecting multiple members. Intriguingly, only a low level of methylation was found in leucocytes, while a higher level was detected in buccal and colonic mucosae. This mosaic methylation pattern is distinct from the more consistent somawide monoallelic methylation seen in individuals with an MLH1 epimutation. The mosaicism and tissue specificity of MSH2 epimutation as well as its strong heritability has invoked controversy (Chong et al., 2007; Horsthemke, 2007; Suter and Martin, 2007). This

48 mosaic methylation pattern was thought to mimic the segregation of an allele that is susceptible to epigenetic silencing in the soma as a consequence of an in cis alteration of the DNA sequence. As shown previously in the fragile X mental retardation syndrome, the unstable CGG repeat in the FMR1 gene can predispose to somatic hypermethylation and silencing of the gene (Malter et al., 1997). While Chan et al did not find any sequence mutation within the exons or promoter of MSH2, changes of DNA sequence further upstream or downstream (Horsthemke, 2007) or the presence of an unlinked modifier (Chong et al., 2007) may yet be identified. Furthermore, the strong heritability of the MSH2 epimutation is indicative of a linked genetic alteration rather than a purely epigenetic one, which would more likely demonstrate stochastic inheritance patterns. Meanwhile, germline epimutation of MLH1 is more likely to represent a true epigenetic event that contributes to a small number of cases with the HNPCC phenotype, suggesting distinct molecular mechanisms that underlie various types of epimutation.

1.9 Methods for detection of DNA methylation at candidate gene promoters

1.9.1 Traditional methods

Prior to the development of the protocol for sodium bisulfite modification, methylation assays relied solely on the use of restriction enzymes that are methylation sensitive or insensitive isochizomers. When directed against genomic DNA, these enzymes recognise the same nucleotide sequences but induce cleavage that distinguishes between methylated and unmethylated recognition sites. For instance, both MspI and HpaII recognise the sequence CCGG but only MspI can cleave the sequence when the internal CpG is methylated. This approach is affected by many drawbacks, including the incompleteness of enzyme cutting and the limitation of available enzymes to study certain regions. Moreover, this approach often involves Southern Blotting, an assay that requires large quantities of high molecular weight DNA, and thus a difficult task to perform when samples are limited.

49 The introduction of sodium bisulfite modification (Figure 1.10), which changes unmethylated cytosines to uracils and leaves methylated cytosines unchanged (Figure 1.11), revolutionised the study of DNA methylation.

NH2

CH3 N

O NH Methylated Cytosine

NH2 NH2 O O

+ - H2ONH4 - N HSO3 N HN HSO3 HN

OH- OH- O O NH O NH SO3- O NH SO3- NH Unmethylated Uracil Cytosine

Step 1: Step 2: Step 3: Sulfonation Hydrolytic Alkali deamination Desulfonation

Figure 1.10 Schematic diagram showing chemical reactions involved in the conversion of unmethylated cytosine to uracil by sodium bisulfite The first step, sulfonation, involves the addition of sodium bisulfite, which leads to the formation of cytosine sulfonate under acidic pH and optimal temperature (50-60 °C). Under inappropriate pH and temperature, this reaction is reversible and leads to non-conversion which in turn can give rise to a false positive result. In the second step, cytosine sulfonate is converted to uracil sulfonate via hydrolytic deamination. The final step of the reaction involves treatment with an alkali such as sodium hydroxide to remove the bisulfite adducts and stabilise the reaction, with uracil as the end product. Methylated cytosine is unreactive with sodium bisulfite and so remains unconverted.

50 Methylated CpG Unmethylated CpG

CCTACGCGTAGCCCG CCTACGCGTAGCCCG

Bisulfite

UUTACGCGTAGUUCG UUTAUGUGTAGUUUG

PCR

TTTACGCGTAGTTCG TTTATGTGTAGTTTG

Figure 1.11 Sodium bisulfite conversion followed by PCR amplification All unmethylated cytosines within any given sequence, whether or not occurring within CG dinucleotides (in black) are converted by sodium bisulfite to uracils, which are then incorporated as thymines (in blue) during PCR. Methylated cytosines within CpG dinucleotides (in red) remain unmodified.

51 Combining bisulfite conversion of DNA with methylation specific PCR (MSP), restriction digestion (combined restriction bisulfite analysis, COBRA) or DNA sequencing (Figure 1.12 and 1.13) allows studies of DNA methylation using not only minute amounts of DNA, but also archival material. However, any bisulfite-based method has its vagaries, principally due to non-conversion of cytosine during the bisulfite conversion step (see step 1 of Figure 1.10). This can lead to a failure in PCR amplification since PCR primers are complementary to bisulfite-converted sequences, in which all unmethylated cytosines have been converted to thymines. However, false positive results can also be generated in MSP due to non-specificity of primer binding, and in COBRA, due to the digestion of non-converted CpG sites. Therefore, it is always necessary to include a methylated and an unmethylated sample for the DNA sequence of interest as the positive and negative controls respectively. MSP and COBRA determine the methylation status at a limited number of CpG sites that are recognised by complementary PCR primers or within restriction enzyme recognition sites, and therefore these assays do not convey the precise pattern of DNA methyation in the test sample. DNA sequencing allows interrogation of the specific methylation pattern at a number of CpG dinucleotides within the amplified fragment. Clonal bisulfite sequencing enables heterogenous patterns of methylation present in biological samples to be revealed. However, this method is more time consuming and therefore unsuitable for high-throughput analysis.

52 A B GCGC GCGC Unbiased primers Unbiased primers 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ CGCG TGTG PCR TCGA TTGA GCGC ACAC PCR 3’ 5’ 3’ 5’ 3’ AGCT 5’ 3’ AACT 5’ CGCG CGCG Unbiased primers Unbiased primers TaqI 5’ 3’ 5’ 3’ TCGATCGA TTGA Digest 3’ AGCT 5’ 3’ AACT 5’

TaqI M U

M U

MSP Unmethylated DNA fragments

Methylated DNA fragments

USP

Figure 1.12 Methylation Specific PCR (MSP) and Combined Bisulfite Restriction Analysis (COBRA)

A, Top: Schematic diagram demonstrating the principal of Methylation specific PCR (MSP). Primers are specific to methylated alleles following bisulfite conversion as they are complementary to CG dinucleotides within bisulfite converted DNA, which have been converted to TG in unmethylated sequences. Bottom: Gel picture showing an example of MSP result. Methylation specific primers detected methylated fragments in a partially methylated sample (M) but not in the completely unmethylated sample (U). The USP primers amplified unmethylated fragments in both samples M and U, thus confirming the integrity of DNA templates used for PCR, and allow semi-quantitative assessment of methylated alleles in these samples. B, Top: Schematic diagram showing the principal of combined bisulfite restriction analysis (COBRA). A restriction enzyme that recognises particular CpG sites following bisulfite conversion cleaves the amplified product from methylated templates. For example, the restriction enzyme TaqI recognises and cleaves the recognition site TCGA preserved in methylated DNA fragments. This recognition site is converted to TTGA in unmethylated DNA fragments and would not be cleaved. Bottom: In the gel picture, the partially methylated DNA sample (M) was partially excised into two smaller fragments whereas the completely unmethylated sample (U) remained as a larger fragment. 53

A M U

B Methylated allele Unmethylated allele

Figure 1.13 Bisulfite sequencing analysis DNA sequencing provides direct analysis of the methylation status at individual CpG sites. Following bisulfite conversion and PCR, sequencing can be performed (A) directly on purified PCR products (direct sequencing) or (B) on individual amplicons representing individual alleles (clonal bisulfite sequencing). Using direct sequencing, methylated CG dinucleotides were detected in the partially methylated sample (M) while in the completely unmethylated sample (U), all cytosines were converted to uracil and hence detected as thymines. Direct sequencing detected heterogeneity of methylation at individual CpG sites (C/T peaks) in sample M. However, it does not inform heterogenous patterns of methylation of individual alleles in that sample, which can be examined using clonal bisulfite sequencing. An example of clonal bisulfite sequencing is shown in (B) by which, CGs were detected in a cloned methylated allele while absent in an unmethylated one. Sequencing 10 to 20 alleles from a given tissue sample can determine heterogeneous patterns of methylation in different alleles of cells that constitute the tissue.

54 1.9.2 Quantitative methods More recently, quantitative PCR-based methods have been developed to quantify the amount of DNA methylation in biological specimens. These methods are sensitive and can quantify minimal amounts of methylated alleles. These include the widely used MethyLight (Eads et al., 2000) as well as the quantitative methylation specific PCR (Chan et al., 2004) assays. These methods also rely on the principal of bisulfite conversion and subsequent PCR. Both methods utilise methylation specific oligonucleotides that detect methylated sequences containing CG but not TG dinucleotides. The MethyLight assay (Figure 1.14) uses primers and a fluorescent- labelled probe that specifically detect and measure the levels of methylated sequences. The high-specificity of primers and probe based on differential CGs allows accurate detection of DNA sequences with dense methylation. Quantitative methylation specific PCR (Figure 1.15) utilises the intercalating dye SybrGreen to detect and measure methylated amplicons generated by primers specific for methylated DNA sequences. This assay does not rely on a probe that recognises fully methylated CpG sites within its binding region, and thus allows detection of DNA sequences with more heterogeneous patterns of methylation. However, it requires absolute specificity in primer binding as SybrGreen can intercalate into any double stranded DNA, including primer dimers and non-specific products, which have the potential to generate false positive results.

Figure 1.14 MethyLight assay Methylation-specific primers are used in combination with a methylation-specific probe to detect fully methylated alleles. A, Annealing of primers and probe to methylated sequences containing CGs but not TG dinucleotides. B, Exonuclease activity of Taq polymerase cleaves the probe, separating the fluorescein from its quencher, generating a fluorescent signal proportional to the amount of fully methylated alleles in the starting template.

55 Sybergreen Methylation- 5’ 3’ specific CGCG CGCG annealing 3’ 5’ Sybergreen

5’ 3’

Taqpolymerase Extension Taqpolymerase

3’ 5’ Figure 1.15 Quantitative methylation specific PCR Primers are methylation specific and therefore anneal to methylated DNA only. At defined annealing temperatures, SybrGreen dye intercalates with newly synthesised double-stranded DNA fragments generating a fluorescence signal proportional to the amount of starting template.

1.10 Research aims and outline

The broad aim of this thesis is to further examine DNA methylation and associated genetic events such as BRAF mutation in the development of colorectal neoplasia, including those that occur in sporadic or familial settings. This aim has been addressed through the analysis of specimens from individuals with sporadic CRCs enrolled in the St. Vincent’s Hospital cohort. This cohort consists of a consecutive series of 1040 individuals who had undergone curative resection of CRCs at the St. Vincent’s Hospital from January 1993 to June 2006. Analysis was also performed on specimens from individuals with familial CRCs referred to St. Vincent’s Hospital’s Familial Cancer Clinic and or supplied by collaborators. This broad aim has been subdivided into four specific aims.

Aim 1. Determine the frequency of MLH1 methylation in various tissue samples from individuals with sporadic colorectal cancer, including more accessible samples such as blood.

Hypermethylation of the mismatch repair gene MLH1 underlies the development of sporadic microsatellite unstable CRC. However, it is unclear whether MLH1 methylation is present in other tissue samples from individuals with this type of cancer, and if present, its significance. A sensitive real-time PCR assay was developed and used

56 to screen for MLH1 methylation in over 300 biological specimens from 235 individuals enrolled in the St. Vincent’s Hospital cohort including colorectal carcinomas, normal colonic mucosae, leukocytes and lymph nodes. The findings will provide a better understanding of the biology of MLH1 methylation and its potential utility as a biomarker for the development of sporadic CRCs, particular those demonstrating microsatellite instability. In addition, the role of MLH1 methylation in the predisposition to germline epimutations of MLH1 was also investigated.

Aim 2. Determine the association between hypermethylation of CpG islands spanning the promoter of genes across the 3p22 chromosomal region encompassing MLH1, and the presence of the V600E mutation of BRAF and other clinicopathological and molecular features in sporadic colorectal cancer.

Recently, our laboratory demonstrated the hypermethylation of a cluster of 3p22 genes encompassing MLH1 in a small group of sporadic MSI CRCs occuring in conjunction with the V600E mutation of BRAF. The association between this phenomenon and BRAF mutation needs to be confirmed in a larger cohort. No study to date has examined the association between the hypermethylation of multiple 3p22 genes and other clinicopathological and molecular characteristics of CRC, as well as patients’ survival. Using a combination of COBRA and qMSP assays, a study group consisting of 874 consecutive sporadic CRCs from the St Vincents Hospital cohort was examined for the presence of methylation among key loci across the 3p22 chromosomal region. The pattern and density of methylation at these loci were also evaluated. Subsequently, the clinicopathological and molecular characteristics of tumours showing concomitant methylation across the 3p22 region as well as the association between hypermethylation of 3p22 genes and patients’ survival were determined. This work may provide insight into the the mechanism that leads to concomitant methylation across the 3p22 region and the relationship of this phenomenon with other cancer-related genetic or epigenetic phenomena. In turn, this may facilitate better characterization of the pathway of colorectal tumourigenesis in tumours showing concomitant methylation across the 3p22 region, and suggest better management strategies for individuals with tumours demonstrating this phenomenon.

Aim 3. Determine the presence of CpG island methylation within the 3p22 chromosomal region or V600E mutation of BRAF in the normal colonic mucosa of individuals with sporadic colorectal cancer.

Following directly from Aim 2, the presence and level of methylation at key 3p22 loci and BRAF mutation was assessed in approximately 60 normal colonic mucosa samples from individuals with or without CRC. Sensitive real-time PCR assays were developed to detect low levels of methylation in these samples. The findings of this work will better define the potential utility of both concomitant methylation of 3p22 loci and mutation of BRAF as biomarkers for the identification of individuals at risk of sporadic CRC. They may also better define the relationship between BRAF mutation and regional methylation, including their chronology in cancer development.

Aim 4. Investigate the role of APC methylation in the development of hereditary and sporadic polyposis syndromes.

Germline epimutations were recognised in the mismatch repair gene MLH1 in 2004 and two years later, evidence was presented suggesting involvement of MSH2. It is not known if germline epimutation occurs in the tumour suppressor gene APC, in individuals with hereditary polyposis syndromes. It is also unclear whether low levels of somatic APC methylation may be present in normal colonic mucosa to predispose to the development of hereditary or sporadic polyposis conditions. The potential for germline and somatic APC methylation in the development of hereditary and sporadic polyposis warrants further investigation. This investigation was approached in two ways. Firstly, constitutional DNA from a cohort of over 100 individuals with either FAP, attenuated FAP or other polyposis syndromes and in whom no germline sequence mutations of APC had been identified by standard genetic screening was examined for the presence of germline epimutation of the APC gene. Secondly, the presence of somatic APC methylation was determined within colonic mucosal samples in these individuals as well as additional individuals with sporadic polyposis. Work in this section may provide insights into whether an epigenetic error may serve as an alternative mechanism in the aetiology of these polyposis conditions.

CHAPTER 2

General Methods

2.1 Materials

2.1.1 Suppliers

The addresses of the suppliers for reagents, hardware and software are listed in Appendix 1.

2.1.2 Oligonucleotide primers

Marker Assay Sequence Product size Tm (bp) (°C) Forward 5’-TATTTTAGTAGAGGTATATAAGTTYGG-3’ COBRA 1 321 52 MLH1-C Reverse 5’-CCTTCAACCAATCACCTCAATACC-3’ region methylation Forward 5’-CGTTAAGTATTTTTTTCGTTTTGCG-3’ qMSP 2 214 61 Reverse 5’-TAAATCTCTTCGTCCCTCCCTAAAACG-3’

Forward 5’-GGAAGGTTAGAGATGTTTTAGTGTTTTT-3’ COBRA 1 387 54 Reverse 5’-CAAACACACAATAAATACTTAATATTC-3’ AB002340 methylation Forward 5’- GCGGTTCGGTTATTTAGTCG-3’ qMSP 146 61 Reverse 5’- CCACTCAACAACGACGTACAC- 3’

Forward 5’-GTAGGGTTTACGGTTCGTAGGGGG-3’ COBRA 1 338 59 Reverse 5’-CTTCCCCTCTACCCGACGAACAAAAC-3’ ITGA9 methylation Forward 5’-GTCGTTTTTGTGTTCGTTTTTAGC -3’ qMSP 109 64 Reverse 5’-CCGAAACGAAAACTCTACGCCTAAAC-3’

Forward 5’-TTTTTAGYGTAGTAGTTYGGTTGGG-3’ COBRA 1 198 57 Reverse 5’-AATAACCAAATTCCTCCAACTTCCT-3’ PLCD1 methylation Forward 5’-GGGCGTCGGATTTTATACG-3’ qMSP 133 61 Reverse 5’-GAACCGCGAACCCTATCATTAC-3’

Forward 5’-TAGTAGTTTTAGTTAGTTTATTTGGA-3’ COBRA 1 228 49 Reverse 5’-ATAACCTCRACTAAATACAAATTAC-3’ DLEC1 methylation Forward 5’-GTAGTTTGCGTTGGCGTAGC -3’ qMSP 103 63 Reverse 5’- ACGAAAAACGCCGATAAACA-3’

Forward 5’-GGGTTAGGGTTAGGTAGGTTG-3’ COBRA 3 229 55 Reverse 5’-ACACCTCCATTCTATCTCCAATAA-3’ APC methylation Forward 5’-TGTTTTATTGCGGAGTGCGGGTCG-3’ qMSP 143 67 Reverse 5’-TACAACCACATATCGATCACGTACG-3’

MyoD Forward 5’-CCAACTCCAAATCCCCTCTCTAT-3’ qMSP 2 163 61 control Reverse 5’-TGATTAATTTAGATTGGGTTTAGAGAAGGA-3’

Table 2.1 Oligonucleotide primers used for methylation assays in this thesis Primers used in combined restriction bisulfite analysis (COBRA) and quantitative methylation specific PCR (qMSP). As denoted in the table, some primers have previously been published: 1 Hitchins et al., 2007b, 2 Hitchins et al, 2007a and 3 Hitchins et al 2006.

60 Marker Assay Characteristic Sequence/ GenBank accession no. Product size (bp) Mononucleotide BAT 25 MSI testing 9834508 110-130 repeat marker

Mononucleotide BAT 26 MSI testing 9834505 100-120 repeat marker

Dinucleotide D5S346 MSI testing 181171 100-130 repeat marker

Dinucleotide D2S123 MSI testing 187953 200-230 repeat marker

Dinucleotide D17S250 MSI testing 177030 140-170 repeat marker

Table 2.2 Oligonucleotide primers for microsatellite instability testing MSI: Microsatellite instability.

Marker Assay Characteristic Sequence/ GenBank accession no. Product Tm size (bp) (°C) KRAS codons Pyro- Forward 5’-GGCCTGCTGAAAATGACTGA-3’ 82 58 12 and 13 sequencing mutation Reverse 5’-TTAGCTGTATCGTCAAGGCACTCT-3’

Forward 5’-AGGTGATTTTGGTCTAGCTACAGT-3’ wild-type BRAF Allelic V600E Forward 149 66 discrimination mutation mutant 5’-AGGTGATTTTGGTCTAGCTACAGA-3’ Reverse 5’-TAGTAACTCAGCAGCATCTCAGGGC-3’

Table 2.3 Oligonucleotide primers for BRAF and KRAS mutation analyses Primers used in the pyrosequencing assay to detect KRAS codons 12 and 13 mutation and in the quantitative PCR assay to detect the V600E mutation of BRAF.

Marker Assay Characteristic Sequence/ GenBank accession no. Tm (°C) SP 6 Sequencing Universal primer 5’-GATTTAGGTGACACTATAG-3’ 55

Table 2.4 Oligonucleotide primers for sequencing of PCR products cloned into vectors

Probe Sequence qMLH1C-probe 5’-(FAM)CGGATAGCGATTTTTAACGCGTAAGCG-(BHQ1)-3’ qMyoD-probe 5’-(FAM)TCCCTTCCTATTCCTAAATCCAACCTAAATACCTCC-(BHQ1)-3’

Table 2.5 Fluorescence probes Fluorescence probes (Trilink biotechnologies) used in the quantification of MLH1 promoter methylation by MethyLight assay, in combination with MLH1-C qMSP Forward and Reverse, as well as MyoD qMSP Forward and Reverse respectively.

61 2.1.3 Reagents

Reagent Supplier Absolute ethanol (molecular biology grade) Sigma Acetic acid Sigma Agar Bacto Agarose powder (molecular grade) Probiogen Agarose powder (high resolution) Probiogen Ammmonium acetate Sigma Ammonium chloride Sigma Bovine serum albumin (BSA) New England Biolabs Chloroform BDH DNA molecular marker pUC19/MspI Fermentas dNTP mix New England Biolabs EDTA (ethylenediamine tetraacetic acid) Sigma EDTA Disodium salt Ajax Ethidium Bromide Sigma Fetal calf serum (FCS) Gibco D-glucose BDH Glycogen Applichem GmbH HEPES buffer solution (1 M) Gibco Hydroquinone (1,4-Benzenediol) Sigma Isopentane BDH IPTG Probiogen L-glutamine Gibco Magnesium Chloride Roche Mineral oil Sigma N,N’-dimethyl-formamide Sigma Orange G Sigma Phosphate buffer saline (PBS) Invitrogen, USA Potassium hydrogen carbonate BDH Phenol/chloroform/isoamylalcohol (25:24:1) Amresco Proteinase K Fermentas Ribonuclease A Fermentas RPMI medium 1640 Gibco Sodium acetate BDH Sodium bisulfite Sigma Sodium chloride BDH Sodium dodecyl sulphate (SDS) Sigma Sodium hydroxide Sigma Sucrose Sigma Taq DNA polymerase (FastStart Taq) Roche Trypsin Gibco TRISMA base Sigma Water Baxter X-gal Probiogen

Table 2.6 Common reagents and chemicals

62 2.1.4 Buffers and solutions

Buffer and solution Constituents Genomic DNA extraction : Ortholysing agent 0.15 M Ammonium Chloride, 10 mM KHCO3, 1 mM EDTA disodium salt

TESS lysing agent 10 mM Tris pH 7.8, 1 mM EDTA, 100 mM Nacl, 1% SDS

TE solution 10X 10 mM Tris-HCl, 1 mM EDTA disodium salt pH 7.5

DNA amplification : IQ supermix (Bio-Rad) 100 mM KCl, 40 mM Tris-HCL pH 8.4, 0.4 mM of each dNTP (dATP, dCTP, dGTP and dTTP), iTaq DNA polymerase, 50 units/ml 6 mM Mg Cl2, and stabilizers

IQ sybergreen supermix (Bio- 100 mM KCl, 40 mM Tris-HCL pH 8.4, 0.4 mM of each dNTP Rad) (dATP, dCTP, dGTP and dTTP), iTaq DNA polymerase, 50 units/ml 6 mM Mg Cl2, SYBR Green 1 20 nM fluoresein and stabilizers

FastStart buffer, 10X without 500 mM Tris/HCl, 100 mM KCl, 50 mM [NH4]SO4, pH 8.3/25 MgCl2 (Roche) °C

Gel electrophoresis: TBE buffer 89 mM Tris, 89 M boric acid, 2 mM EDTA in H20

6X Orange G Gel loading dye 0.25% Orange G, 50% sucrose in H20

Bacteria transformation growth and selection: LB (Luria-Bertani) Broth 1% bacto tryptone, 0.5% yeast extract, 0.5% sodium chloride, with carbenicillin 0.08% sodium hydroxide 5 M, 100 g/ml carbenicillin

SOC media 2% Bacto tryptone, 0.5% Yeast extract, 0.05% sodium chloride, 0.02% potassium chloride, 2 M glucose, 2 M Mg2+ stock

LB (Luria-Bertani) Agar with 1% bacto tryptone, 0.5% yeast extract, 0.5% sodium chloride, carbenicillin, x-gal and IPTG 0.08% sodium hydroxide 5 M, 100 g/ml carbenicillin, 0.08 mg/ml X-gal, 5 M IPTG, 1.5% Agar

Buffer for enzyme: Buffer EcoRI 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100 and 0.1 mg/ml BSA

NEBuffer 1 10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1 nM dithiothreitol (pH 7.0 at 25°C)

NEBuffer 2 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9 at 25°C)

NEBuffer 3 100 mM NaCl, 50 mM Tris-HCl, 10mM MgCl2, 1 mM dithiothreitol (pH 7.9 at 25°C)

NEBuffer 4 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9 at 25°C)

Promega Buffer B 50 mM NaCl, 6 mM Tris-HCl, 6 mM MgCl2, 1 mM dithiothreitol (pH 7.5 at 37°C)

Table 2.7 Buffers and solutions

63 2.1.5 Kits

Kit Manufacturer Quant-iT ™ dsDNA Assay Kit, High Sensitivity Invitrogen, USA pGEM-T Easy Vector System Promega QIAquick DNA Purification Kit Qiagen

Plasmid Miniprep96 Kit Milipore BigDye Terminator v 3.1 Cycle Sequencing Kit Applied Biosystem

Table 2.8 Kits Molecular biology kits used in the analysis of gene promoter methylation in biological samples.

2.1.6 Restriction enzyme

Enzyme Manufacturer Recommended Buffer AatII New England Bioloabs NEBuffer 4 Csp45I Promega Promega Buffer B EcoR1 Fermentas Buffer EcoRI MluI New England Bioloabs NEBuffer 3 RsaI New England Bioloabs NEBuffer 1 SnaBI New England Bioloabs NEBuffer 4 TaqI New England Bioloabs NEBuffer 3

Table 2.9 Restriction enzyme Restriction enzyme used in the miniprep screening for correct insertion in PCR product cloning and the combined bisulfite restriction analysis (COBRA).

2.1.7 DNA modification enzyme and substract

Enzyme Manufacturer CpG Methylase (M.SssI) New England Biolabs S-adenosylmethionine (SAM) New England Biolabs

Table 2.10 DNA modification enzyme and substrate DNA methylase and substrate used for methylating cytosines within CpG dinucleotides in a sequence independent manner. The resulting methylated DNA is used as the positive control in COBRA analysis.

64 2.1.8 Growth media for cell line All cell lines used in this study were grown in complete RPMI media. This media was prepared by adding 10% heat-inactivated fetal calf-serum and two mM L-glutamine into RPMI medium 1640 with 10mM HEPES buffer solution.

2.2 Methods

2.2.1 Sample collection

Tumour and blood samples were procured from a consecutive series of individuals enrolled in the Molecular and Cellular Oncology (MCO) group colorectal cancer study cohort, unless stated otherwise. All individuals had undergone curative surgical resection of colorectal adenocarcinoma at St. Vincent’s Hospital, Sydney between January 1993 and November 2006. Individuals known to have FAP, HNPCC and inflammatory bowel disease at the time of operation were not enrolled. Corresponding normal colonic mucosa was also procured from the surgical resection specimen, usually as far as possible from the tumour site. Blood and tissue samples were also collected from control populations without cancer. For blood samples, the control group consisted of 161 males and 139 females with ages ranging from 17 to 71 years (mean age, 46.5±13.5 years) recruited from consenting donors at the NSW Red Cross Blood Bank, Sydney. For tissue samples, the control group consisted of 11 males and 11 females with ages ranging from 33 to 82 years (mean age, 58.4±17.5 years). Biopsy samples of normal mucosa were procured from each of these individuals during a colonoscopic or a surgical procedure for a non-cancer-related disorder. This study was approved by the Human Research and Ethics Committee of St. Vincent’s Hospital, Sydney. Informed consent was obtained from all participating individuals.

2.2.1.1 Handling of tissue samples Tissue samples from freshly resected colorectal adenocarcinoma and adjacent normal colonic mucosa were procured from the Department of Anatomical Pathology at St. Vincent’s Hospital, Sydney. Samples of normal colonic mucosa from the control group without neoplasia were obtained during colonoscopic procedure by Dr. Alan Meagher of St. Vincent’s Hospital. In both cases, tissues were frozen immediately in liquid

65 nitrogen or snap frozen in isopentane immersed in liquid nitrogen before storage at - 80°C (prior to DNA or protein extraction) or in liquid nitrogen (prior to RNA extraction).

2.2.1.2 Handling of blood samples Samples of peripheral blood were obtained from consented individuals and stored at 4°C prior to DNA extraction.

2.2.2 Patient clinical details

The clinical details of patients including their age, sex, tumour site and date of cancer resection were obtained from their medical records by members of the Molecular and Cellular Oncology Group, St. Vincent’s Hospital. Vital status and date and cause of death were procured from patient medical records, the NSW Registry of Births, Deaths and Marriages, and through active communications with patients themselves or their attending practitioners. All patients in this study were followed-up for a maximum of five years or until death.

2.2.3 Histopathological data of patients

Histopathological assessment of resected specimens, including the type, grade, stage, and site of each carcinoma were procured from the Department of Anatomical Pathology, St. Vincent’s Hospital. Right-sided tumours were defined as those located within the caecum through to the transverse colon, while those occurring within the splenic flexure, descending colon, sigmoid colon and rectum were defined as left-sided. Based on the information of the histopathological report, carcinomas were staged by the AJCC/UICC TNM system, as described in section 1.1.3.5.

Further histopathological review of haematoxylin and eosin stained carcinoma sections was performed in association with Professor Nicholas Hawkins, School of Medical Sciences, University of New South Wales. This review confirmed the grade of carcinoma, as well as the presence of mucinous differentiation. In addition, other histopathological features were determined including the extent of lymphocytic infiltration.

66 A tumour was described as poorly differentiated when less than 10% of cells formed glandular structures with identifiable lumens. Carcinomas were classified as mucinous when extracellular mucin occupied more than 50% of the carcinoma area on representative sections. Intraepithelial lymphocytes were considered present when more than 30 lymphocytes were seen within tumour epithelium per ten high power fields (x40 objective).

The details of other colorectal polyps in individuals with colorectal cancer were based on the histopathological report obtained at the time of resection. For the purposes of analysis in this thesis, tubular, villous and tubullovillous adenomas were grouped together as conventional adenomas. Whenever the term multiple polyps appeared in a pathology report, then in the abence of further specific information, the number of polyps was recorded as four for statistical purposes.

2.2.4 Cell lines

Colorectal cancer and lymphoblastoid cell lines used in this study are listed in Table 2.11. Lymphoblastoid cell lines were generated via immortalization of B-lymphocytes by Epstein-Barr virus. This procedure was performed by staff of the Molecular and Cellular Oncology Group, St. Vincent’s Hospital.

Cell line Type Source Growth Characteristic RKO colorectal ATCC Monolayer MSI, Biallelic MLH1 methylation ST LCL LCL MCO Suspension (Proband C)

Germ-line epimutation, monoallelic TT LCL LCL MCO Suspension (Proband D) MLH1 methylation

VT LCL LCL MCO Suspension (Proband E)

Table 2.11 Cell lines used in this study LCL: lymphoblastoid cell line, ATCC: American Type Culture collection.

2.2.5 Cell culture

All cell lines were grown in continuous culture as described below in a Forma Scientific

Incubator at 37°C, 5% CO2 and 100% humidity.

67 Cell suspension from liquid nitrogen stocks were thawed by shaking in a 37°C water bath before transferred to a 15 ml centrifuge tube. Five ml of culture media was slowly added drop wise, with continuous swirling to mix the cell suspension. Cells were centrifuged at 900 g for five minutes with no brake. The supernatant was removed and the cell pellet was resuspended in ten ml of fresh culture media. The cell suspension was transferred to a T25 tissue culture flask (Corning) and incubated in the environment described above.

Cells were passaged when 90% confluency was achieved. Used culture media was removed and cells were washed with PBS. For monolayer culture, attached cells were further removed by incubation in Trypsin for five minutes. Cells were transferred to a 50 ml centrifuge tube and centrifuged at 900 g for five minutes. The supernatant was removed and cells were resuspended in 1-5 ml of complete culture media. Where necessary, cells were counted using a haemocytometer, and an approximate number reseeded to reach confluency in 3-5 days. Remaining cells were centrifuged and the DNA pellet was stored at -80°C until DNA extraction was performed.

For long term storage of cell lines, cells were resuspended in culture media to approximately 106 cells/ml. Equal volume of freezing solution containing 70-80% RPMI, 10% DMSO and 10-20% FCS was added to the resuspended cells drop wise, with swirling to mix. One ml of this cell suspension was added to pre-cooled cryotubes (Nunc). The cryotubes were placed in insulated freezing racks and allowed to cool down at a rate of 1°C per minute in a -80°C freezer. Once equilibrated at -80°C, they were transferred to a liquid nitrogen tank for long term storage.

2.2.6 Extraction and quantification of DNA

2.2.6.1 DNA extraction from blood samples Ten ml of whole blood was mixed with 40-45 ml of the ortho-lysing (OTL) agent and mixed well by flicking from time to time for ten minutes at room temperature. Peripheral blood mononuclear cells (PBMCs) were spun down at 900 g for ten minutes and the supernatant was discarded. Another 30 ml of OTL was added and the above steps were repeated. PBMCs were washed in PBS and centrifuged at 900 g for ten minutes. Four ml of TESS lysing agent and seven l of 0.5 mg/ml proteinase K (20

68 mg/ml) were added to the resuspended pellets. Protein was degraded by incubation at 50°C overnight. Following overnight incubation, six l of RNase (10 mg/ml) were added to each tube and incubation was carried out at 37°C for 45 minutes. The RNase treated DNA was transferred into 15 ml phase lock tubes (Eppendorf GmbH, Germany). Four ml of 25:24:1 phenol/chloroform/ isoamylalcohol were added into each tube, mixed by gentle invertion several times in a fume exhaust hood and centrifuged at 4000 g for five minutes. This was repeated with a further four ml of chloroform after which the aqueous phase containing only DNA but no protein was transferred to a ten ml tube. DNA was precipitated by adding 1/10 volume of 3 M Na-Acetate and two volume of cold absolute ethanol. Each reaction was mixed gently until a fluffy precipitate of genomic DNA formed. The fluffy precipitate was transferred to a clean, sterile 1.5 ml microcentrifuge tube. The DNA was washed with 70 % ethanol and centrifuged at 15000 g for five minutes to remove the supernatant. The DNA was air-dried before being resuspended in water or 1X TE buffer. The DNA was stored at -80°C until use.

2.2.6.2 DNA extraction from tissue and cell lines Approximately 100 mg of tissues or cells pellet were added to 500 l of TESS lysing agent in 1.5 ml Eppendorf tubes and cut into small pieces using sharp scissors. For protein degradation, five microlitres of proteinase K (20 mg/ml) was added into each tube, after which overnight incubation was carried out at 55°C. Following overnight incubation, phenol/chloroform extraction and ethanol precipitation was performed as described in section 2.2.6.1. DNA was extracted from buccal and hair follicles using the BuccalAmp DNA Extraction Kit (Epicentre Biotechnologies) according to manufacturer’s instructions.

2.2.6.3 DNA quantification Concentration and quality (260/280 ratio) of the DNA were checked using NanoDrop® ND-1000 Spectophotometer (NanoDrop Technologies Inc., USA) according to the manufacturer’s instructions. A given DNA sample was considered good in quality when the 260/280 ratio ranged between 1.7-1.8.

69 2.2.7 Microsatellite instability testing

Microsatellite instability testing of each adenocarcinoma specimen was carried out by the staff of the Molecular and Cellular Oncology group, St. Vincent’s Hospital. Briefly, a panel of 5 microsatellite markers including two mononucleotide markers BAT25 and BAT26 as well as three dinucleotide markers D5S346, D2S123 and D17S250 were screened for instability by PCR. A carcinoma sample was classified as having microsatellite instability when two or more markers demonstrated instability in the tumour DNA compared to the corresponding constitutional DNA.

2.2.8 Immunohistochemistry

Immunohistochemical analysis for MLH1 and P53 was carried out on paraffin- embedded tissue sections by the staff at the Molecular and Cellular Oncology group, St. Vincent’s hospital.

Expression of MLH1 proteins was determined using the avidin-biotin peroxidase immunohistochemical technique as previously described (Ward et al., 2005). MLH1 expression was considered lost when lack of nuclear staining was observed in the epithelial cells within the tumour, in the presence of staining in the adjacent normal mucosal epithelium and lamina propria lymphocytes.

P53 immunohistochemical staining was performed using the mouse monoclonal antibody DO7 (DAKO) as previously described (Ward et al., 1997). Nuclear accumulation of p53 protein (p53 mutation) was considered present when more than 20% of carcinoma cells showed nuclear staining of moderate to high intensity, in the absence of staining in stromal cells and normal epithelium.

2.2.9 CpG Methylase (M.SssI) treatment of DNA

CpG methylase (M.SssI) treatment was performed on leukocyte DNA extracted from healthy donors. Briefly, ten g of genomic DNA was incubated overnight at 37°C in a 50 l reaction containing 1-2 units of CpG methylase (M.SssI), 160 M S- adenosylmethionine (SAM), 1X NEBuffer 2, and H20. S-adenosylmethionine was replenished every four hours. Methylated DNA was stored at -20°C prior to use.

70 2.2.10 Bisulfite treatment of genomic DNA

Bisulfite treatment was performed on genomic DNA using a standard protocol developed in the MCO laboratory. For each batch subjected to bisulfite treatment, a negative control (water that contained no DNA template) was included to exclude contamination.

2.2.10.1 Denaturation Genomic DNA (1-2 g) was diluted into a final volume of 18 l 1X TE buffer. Two l of 3 M NaOH was added to the DNA to a final concentration of 0.3 M. The DNA was overlayed with 200 l mineral oil and incubated at 50°C for 20 minutes after which the sample was placed on ice so that the DNA remained single stranded.

2.2.10.2 Bisulfite conversion Twelve l of hydroquinone (75 mM) and 210 l sodium bisulfite (3.5 M, pH 5.0) were added to the denatured DNA beneath the mineral oil and mixed well with pipetting. The sample was heated to 95°C for five minutes, and then transferred immediately to 52°C for 16-18 hours.

2.2.10.3 Desalting This process was carried out using the Qiaquick PCR purification kit (Qiagen) following the manufacturer’s instructions. Briefly, one ml of buffer PB was added to the sample and mixed by vortexing. 700 l of sample mix was loaded into a prelabelled spin column assembly and spun for 0.5-1 minutes at 15000 g. Flow-through was discarded. Remaining sample mix was added into the same column and spun again as above. Sample was subjected to washing by adding 750 l PE wash buffer and spun at 15000 g for one minute. Flow-through was discarded after which the empty column was spun for an additional one minute to dry the column. The column was transferred to a clean 1.5 ml Eppendorf tube. Elution was carried out by adding 50 l elution buffer (EB) into the column, allowed to stand for two minutes and spun for two minutes at 15000 g.

71 2.2.10.4 Desulfonation 1.6 l 10 M NaOH was added to the eluted DNA to a final concentration of 0.3 M and incubated at 37°C for 15 minutes.

2.2.10.5 Neutralisation and precipitation One l of glycogen (10 g/ml), 35 l ammonium acetate (7.5 M, pH 7.0) and 200 l absolute ethanol was added to each DNA sample. The reaction was mixed by vortexing. DNA was precipitated at -20°C for more than one hour. Following incubation at room temperature, the DNA was spun down for 30 minutes at 15000 g. The resulting supernatant was gently decanted off the DNA pellet, which was washed with 70% ethanol and then precipitated again by spinning for ten minutes at 15000 g. The 70% ethanol was removed and the DNA was dried at ambient temperature before being resuspended in 25 l 1X TE buffer. The bisulfite-treated DNA was stored at 4°C for immediate use or -20°C for long term storage.

2.2.11 Construction of the standard curve for quantitative PCR assay

2.2.11.1 Plasmid concentration Respective pGEM®-T Easy vectors (Promega) harbouring the completely methylated MLH1 fragment and the internal control DNA fragment Myod were kindly provided by Dr. Megan Hitchins, Molecular and Cellular Oncology group, St. Vincent’s Hospital, Sydney. pGEM®-T Easy vectors habouring the completely methylated fragments corresponding to the promoter region of AB002340, ITGA9, PLCD1, DLEC1 and APC were kindly provided by Dr. Vita Lin, Molecular and Cellular Oncology group, St. Vincent’s Hospital, Sydney. Concentration of plasmid stocks was measured using the Quant-iT TM dsDNA Assay Kit (Invitrogen, USA) according to the manufacturer’s instructions. In brief, 200 l of HS buffer was added to one microlitre of DNA HS reagent in each micoplate well. Ten ul of Quant-iT DNA HS standards as well as plasmid DNA were then added into respective wells. The relative fluorescence unit (RFU) generated by each well was measured using endpoint analysis on the MyIQTM Single-Color Real-Time PCR machine (Bio-Rad). Based on the RFU readings, the concentration of plasmid stocks was determined from a standard curve.

2.2.11.2 Serial dilution of plasmid DNA Working stocks containing 5x106 copy number of respective plasmid per microlitre were prepared according to Applied Biosystems technical notes available on-line at www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf . Firstly, the size and concentration of the plasmid DNA of interest was determined, after which the mass of DNA per plasmid was calculated using the following formula: M = [n] [1 mole/6.023x1023 molecules (bp)] [660 g/mole] Where: n = DNA size (bp) M = mass Avogadro’s number = 6.023x1023 molecules/mole Average molecular weight of a double-stranded DNA = 660 g/mole

Mass of plasmid DNA containing the copy number of interest in the working stock was determined by multiplying the mass of DNA per plasmid with the copy number of interest. Concentration of the working stock that contained the copy number of interest was determined as the mass needed per microlitre. Working stock containing 5x106 copy number of plasmid per microlitre was prepared based on following formula:

C1V1 = C2V2 Where:

C1 = Concentration of plasmid stock C2 = Concentration of working stock

V1 = Volume of diluent (H20) V2 = Final volume desired

Dilutions were then performed to obtain a series of plasmid dilutions shown in Table 2.12. Two microlitres of each dilution 1-8 was used in subsequent quantitative PCR assays to construct a standard curve with copy number ranging from 10 to 106.

73 Dilution Source of Initial Volume of Volume of Final Resulting copy plasmid DNA copy plasmid diluent (ul) volume number of for dilution number DNA (ul) (ul) plasmid/ul 1 Working stock 5x106 10 90 100 5x105 2 Dilution 1 5x105 10 90 100 5x104 3 Dilution 2 5x104 10 90 100 5x103 4 Dilution 3 5x103 10 90 100 500 5 Dilution 4 500 10 90 100 50 6 Dilution 5 50 50 50 100 25 7 Dilution 6 25 50 50 100 12.5 8 Dilution 4 50 10 90 100 5

Table 2.12 Dilution of plasmid standards Dilutions of plasmid DNA into a series of standards with decreasing copy numbers.

2.2.12 Quantitative PCR assay for methylation detection

Quantitative PCR reactions were performed using either the MethyLight or the quantitative methylation specific PCR (qMSP) assays on the MyIQTM Single-Color Real-Time PCR machine (Bio-Rad). Primers and probes were listed in Tables 2.1 and 2.5. The MethyLight assay was carried out in 20 l reactions containing two l of bisulfite treated DNA or plasmid standard, 1X IQ Supermix (Bio-rad), 0.3 M of respective forward and reverse primers and 0.2 M of respective probes. The qMSP assay was performed in 20 l reactions containing two l of bisulfite treated DNA or plasmid standard, 1X IQ SybrGreen supermix (Bio-rad) and 0.3 M of respective forward and reverse primers. Cycling conditions for respective assays were as follows: 95°C for six minutes, 40 X 94°C for 30 seconds, respective annealing temperature for 30 seconds, and 72°C for 30 seconds. The fluorescence output signal was measured during the extension step at 72°C for MethyLight assays or in an extra incubation step (> 72°C) for 30 seconds following extension for qMSP assays. Quantitative PCR was considered valid if the standard curve resulted in efficiency (E) of 80-100% and a correlation of coefficient (R2) value of greater than 0.95. In all quantitative PCR reactions, a negative control containing no DNA template was used to exclude contamination.

74 The intensity of methylation was calculated as Percentage of Methylated Reference (PMR) values as described by Eads et al., 2000 (Eads et al., 2000). The following formula was applied:

Copy number of the gene Copy number of the gene of interest of interest (sample) (100% methylated DNA) / × 100% Copy number MyoD Copy number MyoD (sample) (100% methylated DNA)

2.2.13 Assessment of assay sensitivity and linearity using artificially mixed DNA with varying percentages of methylation

The completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) was mixed in unmethylated blood DNA to construct dilutions of DNA with varying percentages of methylation (Table 2.13). One g of each DNA mixture was bisulfite- treated as described in 2.2.10 and assayed in triplicate using the qMSP assay.

Mixture Source of Methylated DNA Ratio of Methylated mixture : Final % mixture for dilution unmethylated DNA 1 Completely methylated DNA 100:0 100% 2 Mixture 1 3:1 75% 3 Mixture 1 1:1 50% 4 Mixture 1 1:3 25% 5 Mixture 1 1:9 10% 6 Mixture 1 1:45 5% 7 Mixture 1 1:99 1% 8 Mixture 7 1: 1 0.5% 9 Mixture 8 1:4 0.1%

Table 2.13 DNA mixture of varying percentages of methylation

2.2.14 Agarose gel electrophoresis

Two grams of agarose powder was dissolved in 100 ml of 1X TBE by heating, and allowed to cool down at room temperature while remaining in liquid form. Once cooled, 20 ng/ml of ethidium bromide was added to the liquid agarose, which was then poured into a gel former with gel forming combs, and allowed to set at room temperature. Once set, the gel was submerged in 1X TBE buffer in a gel electrophoresis tank. PCR products and DNA marker pUC19/MspI were loaded into the wells, and subjected to gel

75 electrophoresis at 120V. During electrophoresis, negatively-charged DNA migrated towards the anode at varying speeds, according to their size and conformation and this was subsequently visualised under UV light using the Bio-Rad Gel Doc 2000 system.

2.2.15 Purification of PCR product

Purification of PCR products was performed using the QIAquick DNA Purification Kit (Qiagen) according to the manufacturer’s instructions.

2.2.16 Clonal sequencing

2.2.16.1 Ligation of PCR product

PCR products were ligated into the pGEM®-T Easy vector (Promega) according to the manufacturer’s instructions. In brief, each 10 l of ligation reaction containing five l 2X rapid ligation buffer, one l T4 DNA ligase, 0.5 l vector and 1-3 l PCR product was incubated at room temperature for one hour or overnight at 4°C. PCR products amplified by Taq DNA polymerase contained 3’ A overhangs that ligated easily into the pGEM®-T Easy vector containing the 5’ T overhangs.

2.2.16.2 Transformation One fifth of each ligation reaction described in section 2.3.14.1 was added to 50 l of competent E. coli DH5 cells (Invitrogen, USA, USA). Following an incubation of 20 minutes on ice, each tube of transformation reaction was subjected to heat shock at 37°C for 45 seconds, allowing the uptake of plasmid into DH5 cells. Nine hundred microlitres of pre-warmed (37°C) SOC media was added into each tube of transformed bacteria. The tubes were then incubated for one hour at 37°C with shaking at 225 rpm to allow recovery of bacteria to complete antibiotic resistance. After the incubation period, the transformed bacteria were plated onto an agar plate containing 100 g/ml carbenicillin, 0.08 mg/ml X-gal and 5M IPTG. The bacteria were allowed to grow overnight on the agar plate at 37°C.

76 2.2.16.3 Selection of colonies with desired insert From each plate, 8-12 white colonies containing the desired insert were picked and inoculated into LB broth containing 100ug/ml carbenicillin and incubated overnight at 37°C with shaking at 225 rpm. Following the incubation, plasmid DNA was extracted from these bacteria using the Plasmid Miniprep96 Kit (Millipore) according to the manufacturer’s instruction. Restriction digestion at 37°C was performed using EcoRI since its recognition sites are found in the sequence flanking the multiple cloning site of this vector. Gel electrophoresis was then performed on each sample to determine the presence of insert with correct sizes by reference to DNA size marker resolved on the same gel with the samples.

2.2.16.4 Sequencing of plasmid DNA Plasmid DNA containing the correct size insert was then sequenced using the BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems) as per the manufacturer’s instructions. The universal vector primer SP6 was used as the sequencing primer. Briefly, each sequence reaction contained 100-200 ng of plasmid DNA, one l Big Dye Sequencing Terminator, 3.5 l sequencing buffer, one l 3.2 mM primer, and water up to a total reaction volume of 20 l. Cycle sequencing was performed by incubation at 96°C for two minutes, 27 repeated cycles of 96°C for ten seconds, 55°C for ten seconds, and 60°C for four minutes, followed by 72°C for seven minutes. Following cycle sequencing, the sequenced products were precipitated using 100% ethanol, cleaned up using 70% ethanol and dried at ambient temperature as described in the ABI PRISM® dGTP BigDyeTM Terminator v 3.0 Ready Reaction Cycle Sequencing kit protocol. The sequenced products were then delivered to the Ramaciotti Centre at the University of New South Wales for analysis with the ABI PRISM® 3700 automated DNA sequencer.

2.2.16.5 Analysis of DNA sequence Electronic electrophoretograms generated were analysed for the presence of methylated CpG using the DNASTAR lasergene software (DNASTAR).

77 2.2.17 Combined bisulfite restriction analysis (COBRA)

2.2.17.1 PCR amplification of fragments with CpG islands within promoter regions of genes PCR amplification was carried out in 25 l reactions containing 2.5 l bisulfite treated

DNA, 1X PCR buffer, three mM MgCl2, 0.2 mM of each dNTP, 0.4 M of each PCR primer and one unit of FastStart Taq DNA polymerase (Roche). The following PCR conditions were used: an initial denaturation at 94°C for five minutes, then 35 repeated cycles of 94°C for 30 seconds, annealing temperature for 30 seconds, and 72°C for 30 seconds. This was followed by a final extension phase at 72°C for seven minutes. Each PCR product was shown to be successfully amplified on a 2% agarose gel prior to restriction enzyme digestion.

2.2.17.2 Restriction enzyme digestion Restriction enzyme digestion was carried out in 20 l reactions containing 5-10 l of PCR product, 1X restriction enzyme buffer and 1-2 units of restriction enzyme, depending on product intensity. Each reaction was mixed well and incubated for up to four hours at the designated optimal temperature for the activity of restriction enzyme as recommended by the manucfacturer. Each digested product was subjected to gel electrophoresis on 2% agarose gel and visualised under UV illumination to determine the fragmentation pattern.

2.2.18 BRAF mutation analysis

BRAF mutation analysis was performed using the real-time allele-specific amplification assay as described by Jarry and co-workers (2004) (Jarry et al., 2004). Primers used for this assay are listed in Table 2.3. For each sample, PCR amplification was performed in two different mixtures, one that will only amplify the BRAF wild-type (primers: BRAF forward wild-type and reverse) and the other that will only amplify the V600E mutant fragments (primers: BRAF forward mutant and reverse). PCR was performed in a 20 l reaction containing ten l of IQ sybergreen For Supermix (Bio-Rad), 0.5 uM each of forward and reverse primer, and ten ng of template DNA on a MyIQ Real-time PCR system (Bio-Rad). The cycling conditions were as follows: 95°C for five minutes, 35

78 repeated cycles of 95°C for 30 seconds, 66°C for ten seconds and 72°C for 30 seconds. Fluorescence signal was measured at an extra incubation step of 80°C for 30 seconds which followed each extension cycle. Samples were considered BRAF V600E mutant if fluorescence signal was detected using both the wild type and mutant specific primer sets in conjunction with a difference in Ct values (Ct) of 11 between the mutant and wild-type reactions (detailed in Chapter 5).

2.2.19 KRAS mutation analysis

KRAS codon 12 and 13 mutations were assessed by other members of the Molecular and Cellular Oncology Group at St. Vincent’s Hospital by pyrosequencing. In brief, PCR products generated using biotinylated primers spanning KRAS codon 12 and 13 were immobilised on streptavidin beads, denatured and subjected to pyrosequencing using the Pyromark ID instrument (Biotage) according to manufacturer’s instructions. Pyrosequencing results were analysed using PyroMark 1.0 software (Biotage).

2.2.20 Statistical analyses

Categorical variables were compared using the 2 test or Fisher’s exact test. Mann- Whitney U Test was used to compare continuous variables that were not normally distributed. Multivariate analysis was performed using multivariate logistic regression analysis. Associations were considered significant when p0.05. All analyses were performed using the SPSS statistical package, Version 15.0 (SPSS Inc, Chicago, IL).

CHAPTER 3

MLH1 methylation in individuals

with colorectal cancer

3.1 Introduction

As discussed earlier, CRCs can be divided into those demonstrating the microsatellite unstable (MSI) or microsatellite stable (MSS) phenotypes. Individuals with MSI CRC demonstrate a better outcome than those with MSS CRC of similar stage and grade (Colella et al., 1999; Elsaleh et al., 2000; Watanabe et al., 2001; Popat et al., 2005; Kim et al., 2007b). More importantly, there are indications that MSI CRC may be less responsive to adjuvant chemotherapy and such treatment may instead make the outcome worse (Ribic et al., 2003). The clinical management of individuals who have had an MSI cancer is potentially different from their counterparts who have had an MSS cancer and therefore, it is important to distinguish between the two. Microsatellite instability testing and immunohistochemical analysis of mismatch repair proteins serve this purpose well. However, these laboratory tests are often not able to distinguish between MSI cancers that occur in the HNPCC setting and those that occur sporadically. From a clinical perspective, the distinction between sporadic and HNPCC-associated MSI cancers is important since individuals with a hereditary MSI cancer and their at-risk family members would require more intensive clinical surveillance and removal of polyps that may otherwise develop into tumours. From a molecular viewpoint, this distinction has led to the understanding of the mechanistic differences that underlie the development of sporadic MSI cancers and their hereditary counterparts.

The methylation of CpG islands within the promoter region of MLH1 can lead to the silencing of MLH1, and this epigenetic event is known to underlie the development of over 80% of sporadic MSI CRCs. MLH1 methylation rarely occurs in the context of HNPCC. Therefore, determination of the methylation status of MLH1 complements MSI testing and immunohistochemical analysis of mismatch repair proteins in detecting sporadic MSI CRC. While MLH1 methylation is clearly an important event in the development of most sporadic MSI CRC, it is unknown whether low levels of MLH1 methylation is present in the peripheral blood or normal colonic mucosa of individuals who have been diagnosed with this type of CRC and if so, whether this represents a risk factor. It is also unclear whether MLH1 methylation is present in the normal colonic mucosa early in the development of sporadic MSI CRC. There are discrepant data pertaining to the incidence of MLH1 methylation in the normal colonic mucosa of individuals with sporadic CRC. One study reported dense methylation of the entire

81 MLH1 promoter region in the normal mucosa of 55% of individuals with MSI and 20% of those with MSS CRC (Nakagawa et al., 2001). This observation has not been confirmed by others who reported mosaic methylation of MLH1 in the normal mucosa of individuals with MSI CRCs (Miyakura et al., 2001; Furukawa et al., 2002; Miyakura et al., 2003). The finding of MLH1 methylation in the normal mucosa of individuals with MSS CRC by Nakagawa et al is somewhat surprising due to the lack of association between MLH1 methylation and MSS CRC. These discrepancies in data may be attributed to different methodologies applied by each investigator as these methods may vary in terms of sensitivity and specificity. Therefore, a sensitive and specific assay that is capable of detecting low levels of MLH1 methylation is required to address the controversial question of whether methylation of this gene is present in these normal tissues, and if this might represent a biomarker for individuals at risk of developing sporadic MSI CRC .

Soma-wide monoallelic methylation of MLH1 has been observed in individuals with germline epimutations of this gene (Gazzoli et al., 2002; Miyakura et al., 2004; Suter et al., 2004b; Hitchins et al., 2005; Valle et al., 2007; Hitchins et al., 2007a; Morak et al., 2008). Germline epimutations have demonstrated a non-Mendelian pattern of inheritance and therefore, the mode of inheritance remains uncertain (Suter et al., 2004b; Hitchins et al., 2005). Based on clonal bisulfite sequencing, individuals with a germline epimutation showed a mosaic pattern of MLH1 methylation in some of their epimutant alleles (Suter et al., 2004b; Hitchins et al., 2007a). It remains a possibility that some family members had low levels and mosaic patterns of allelic MLH1 methylation which may be due to incomplete erasure of an epimutation during embryogenesis, or a cis-acting effect that predisposes the affected allele to subsequent somatic methylation. Thus, it is important to determine whether low level mosaic patterns of MLH1 methylation may be found in the family members of individuals affected by a germline epimutation. Again, such an investigation requires a sensitive, accurate and quantitative method to detect mosaic patterns of MLH1 methylation.

To date, various methods with different sensitivities have been developed and used for the detection of CpG methylation at the MLH1 promoter. Of these, gel-based methods such as the combined bisulfite restriction analysis (COBRA) or restriction enzyme digestion method (digestion with methylation-sensitive and -insensitive restriction

82 enzymes such as HpaII and MspI respectively) (Kane et al., 1997; Rein et al., 1998) are limited in sensitivity when compared to quantitative assays such as MethyLight. Others, such as the methylation-specific PCR (MSP) and methylation-sensitive nucleotide primer extension (Ms-SNuPE) (Gonzalgo and Liang, 2007) are sensitive but not quantitative.

In addition, different studies have assessed methylation at different regions of the CpG island spanning the MLH1 promoter. As discussed earlier, the MLH1 promoter encompasses the 5’-most (distal) and 3’-most (proximal) regions, known also as MLH1- A and MLH1-C respectively (Figure 1.6). Methylation at the proximal (MLH1-C) but not the distal (MLH1-A) promoter of MLH1 correlates strongly with the loss of MLH1 expression (Deng et al., 1999). However, as reported by Capel et al, over 60% of the published studies on MLH1 promoter methylation have analysed regions that do not correlate with the expression of this gene (Capel et al., 2007). This includes 14/18 (78%) publications that have used the MethyLight assay, the most widely-used assay for the quantification of methylation levels (Capel et al., 2007). The variations in the methods used and the region within the CpG islands investigated in the detection of MLH1 methylation have no doubt contributed to the inconsistency between reports pertaining to the pattern of MLH1 methylation in biological samples. Therefore it is important to develop an assay that is not only sensitive in the detection of MLH1 methylation but accurately detects methylation in regions known to correlate with the functional loss of MLH1 protein.

The work in this chapter aims at providing a better understanding of the biology of MLH1 methylation and its potential utility as a biomarker for the detection of individuals at risk of sporadic MSI CRC. Firstly, two sensitive and accurate assays for the quantification of MLH1-C methylation were developed and compared. The more sensitive of these assays was then applied to biological samples. Foremost, the assay is validated on samples with known levels of methylation. These include a CRC cell line as well as completely methylated and unmethylated controls. Secondly, the chosen assay was applied to biological samples of individuals with sporadic CRC. In order to assess the efficiency of this assay in determining the functional loss of MLH1, a correlation was made between the detection of MLH1-C methylation and the loss of protein expression in CRCs (by immunohistochemistry). The presence of MLH1-C

83 methylation in the normal colonic mucosa, blood and lymph node of individuals with CRC was also assessed. Finally, the assay was used to quantify MLH1-C methylation in individuals with a germline epimutation and their family members.

3.2 Methods

The development of assays for the detection of MLH1 methylation is described in the results section. General methods are described in Chapter 2.

3.3 Results

3.3.1 Development and comparison of assays for the detection of methylation at the MLH1-C promoter region

Two quantitative PCR assays were developed for the detection of MLH1-C methylation, a probe-based MethyLight assay, and a SyberGreen-based quantitative methylation- specific PCR (qMSP) assay. Both assays utilised the same set of amplification primers designed to specifically amplify methylated templates following bisulfite conversion. The main difference between the two assays was that MethyLight uses a fluorescent- labelled probe that specifically detects and measures the levels of methylated sequences while the qMSP assay utilises the SybrGreen dye which intercalates into the amplicons to detect and measure methylated templates. The specificity, sensitivity and efficacy of these assays were compared and contrasted as described below.

3.3.1.1 Specificity of quantitative assays for methylation detection at the MLH1-C region Typically, the first step in the optimisation of a PCR reaction is to determine the specificity of amplicons generated using a given primers set. The specificity of the detection of MLH1-C methylation as described in this section is applicable to both the MethyLight and qMSP assays. The primers used in these assays amplified a large fraction of the MLH1-C region, generating PCR products of 214 bp in size (Table 2.1). Following a temperature gradient analysis (Figures 3.1 and 3.2), amplification of the MLH1-C fragments was found to be optimal within the annealing temperature range of 58.0-62.9°C. A temperature at the middle of this range (61.0°C) was selected as it was also suited for the amplification of the control gene, MyoD. Using the qMSP assay for the detection of MLH1-C methylation as an example, only amplicons of the methylated template present in the methylated sample (M) were detected while no PCR

85 amplification was observed in the unmethylated sample (U) (Figure 3.1 A and Figure 3.2, top). The reaction for MyoD was unbiased for methylation since the region amplified was devoid of CpG sites. Therefore, MyoD amplicons were detected equally in both methylated and unmethylated samples containing identical amounts of starting DNA template (approximately 100 ng) (Figure 3.1 B and Figure 3.2, bottom).

65.0 °C 64.2 °C 62.9 °C 61.0 °C 58.0°C 56.2°C 54.8 °C 54.0 °C MW M UMUUUU MU M M M M UUM A) MLH1-C

242bp 190bp

B) MyoD

190bp 146bp

Figure 3.1 Optimisation of the annealing temperatures for MLH1-C and MyoD amplifications The gel picture shows 214 bp PCR products generated by the qMSP amplification of A) MLH1-C and B) MyoD in temperature gradient reactions. DNA samples containing methylated (M) and unmethylated (U) templates were the M-SssI-treated peripheral blood mononuclear cells (PBMCs) from a healthy individual and the untreated PBMCs from the same individual, respectively. Each sample was added into the PCR mastermix and divided equally into respective wells for amplification. Specific amplification of the methylated MLH1-C fragments was detected at temperatures within 58.0-65.0°C. Unmethylated MLH1-C fragments which were identical in size to the methylated fragments were not amplified at 58.0°C and above. MyoD fragments were unbiased for methylation and in both methylated and unmethylated samples, were amplified equally within the temperature range of 56.0-63.0°C. MW: molecular weight marker, pUC19 DNA/MspI.

86 qMSP MethyLight

58.0 and 58.0 ºC 61.0 ºC 61.0 ºC 62.9 ºC 62.9 ºC 64.2 and 64.2 ºC 65.0 ºC 65.0 ºC -C

MLH1 Ct values

58.0 – 65.0 ºC 58.0 – 65.0 ºC

Cycle Cycle

58.0 – 65.0 ºC 58.0 – 65.0 ºC MyoD

Cycle Cycle

Figure 3.2 Real time PCR amplification plot of MLH1-C and MyoD by qMSP or MethyLight assays In each real-time PCR reaction (MethyLight or qMSP), a similar amount of DNA was used for both the methylated and unmethylated samples (approximately 100ng). Top, MLH1-C: Using either the qMSP or Methylight assay, cumulative fluorescence output was detected above the threshold (orange line) in a methylated sample, M, amplified at annealing temperatures that ranged from 58.0 to 65.0 °C (red plots) and absent in an unmethylated sample, U, amplified at the same range of annealing temperatures (blue plots). The optimal range of annealing temperatures was 58.0-62.9°C, whereby the threshold cycle (Ct) values were lowest for the methylation-positive reactions and thus indicating highest amplification efficiency while still demonstrating complete specificity for methylated templates. Bottom, MyoD: This PCR reaction was unbiased for methylation since the primers amplified a region of MyoD that contains no CpGs. Therefore, signals were detected equally above the threshold (orange line) in methylated (red plots) and unmethylated samples (blue plots). This PCR reaction was equally efficient across a broad range of annealing temperatures (56.2-62.9°C). The annealing temperature of 61°C, which is at the middle of the optimal range for both PCR amplifications of MLH1-C and MyoD, was selected for subsequent real-time PCR runs.

3.3.1.2 Sensitivity of quantitative assays for the detection of methylation at MLH1-C

3.3.1.2.1 MethyLight assay In order to determine the level of sensitivity of the MethyLight assay, a dilution series of 106 to 10 copies of plasmids containing the methylated MLH1-C, as well as one that contained MyoD were prepared to generate standard curves (detail in section 2.2.11.2). As shown in Figure 3.3, the probe-based MethyLight assay is capable of detecting methylation down to 100 copies of plasmid DNA harbouring the completely methylated template of the MLH1-C promoter sequence. Using a dilution series of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) in unmethylated peripheral blood mononuclear cell (PBMC) DNA, this assay detected MLH1 methylation down to a measurable proportion of 0.5% (approximately 100-250 copies) of the total input DNA (Figure 3.3, top and Figure 3.4) in the presence of at least 2x104 copies of the control gene MyoD (Figure 3.3, bottom). Diluted samples containing 0.1% methylated templates were not detected within the range, measurable by the standard curve. 0.1% 0.5% 1% 5% MLH1-C 10% 100%

MyoD > 2x104 copies MyoD

Figure 3.3 Limits of detection of MLH1-C methylation using the MethyLight assay

Standard curves generated from serial dilution of plasmids containing the target genes are shown as log10 copy number (blue circles). These were used for quantification of the methylated MLH1-C promoter and the internal reference gene MyoD in test samples (red squares). Test samples were generated using a serial dilution of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) in unmethylated PBMC DNA. MLH1 methylation was detectable in dilutions containing at least 0.5% methylated templates of the total input DNA in the presence of >2x104 starting copies of MyoD. Each sample was assayed in triplicate in a single run.

Figure 3.4 Real-time PCR amplification plot for MLH1-C methylation by MethyLight Multi-coloured real-time PCR amplification plots are shown for samples with known proportions of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) diluted in unmethylated PBMC DNA (0% to 100%). A sample was positive for MLH1-C methylation when the corresponding real-time PCR plot cut across the thresholed line (in orange). MLH1-C methylation was detected down to a proportion of 0.5% of the completely methylated DNA.

3.3.1.2.2 Quantitative Methylation-specific PCR (qMSP) Using the same serial dilution of plasmid DNA harbouring a completely methylated fragment of the MLH1-C promoter as tested using MethyLight, the qMSP assay detected methylation down to 10 copies of this plasmid. Furthermore, using the same dilution of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) in unmethylated PBMC DNA tested, this assay detected methylation down to a measurable proportion of 0.1% (approximately 15-25 copies) of the total input DNA (Figure 3.5, top and Figure 3.6) in the presence of at least 2x104 copies of the control gene MyoD (Figure 3.5, bottom). Therefore, the qMSP assay was at least five times more sensitive than the MethyLight assay.

MLH1-C

MyoD

Figure 3.5 Limits of detection of MLH1-C methylation using the quantitative methylation-specific PCR (qMSP) assay Standard curves generated from a serial dilution of plasmids containing the target genes are shown as log10 copy number (blue circles). These were used for quantification of the methylated MLH1-C promoter and the internal reference gene MyoD in test samples (red squares). Test samples were generated using a serial dilution of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) in unmethylated PBMC DNA. MLH1-C methylation was detectable in dilutions containing at least 0.1% methylated templates of the total input DNA in the presence of >2x104 starting copies of MyoD. Each sample was assayed in triplicate in a single run.

Figure 3.6 Real-time PCR amplification plot for MLH1-C methylation detection using the qMSP assay Multi-coloured real-time PCR amplification plots are shown for samples that contain known proportions of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) diluted in unmethylated PBMC DNA (0%-100%). A sample was positive for MLH1-C methylation when the corresponding real- time PCR plot cut across the threshold line (in orange). MLH1-C methylation was detected down to a proportion of 0.1% of the completely methylated DNA.

3.3.1.3 Intra- and inter-assay variations of the MethyLight and qMSP assays The intra-assay reproducibility for the detection of methylated MLH1-C and MyoD using the MethyLight and qMSP assays was examined using a DNA sample from the RKO CRC cell line which harbours biallelic MLH1 methylation, a sample of unmethylated PBMC DNA from a healthy donor, and samples containing either a proportion of 0.5% or 0.1% completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) diluted in the unmethylated PBMC DNA. A similar amount (1.5 g) of each of these samples was bisulfite-treated in the same batch. Within the same run, both the MethyLight and qMSP assays demonstrated highly reproducible threshold cycle (Ct) values. Standard deviations (SDs) were less than one cycle for six replicates of each sample assayed for either the methylated MLH1-C or the MyoD template (Table 3.1). For each sample, the coefficient of variation (CV) for Ct values of replicates was less than 0.1, indicating a good reproducibility of fluorescence signals detected within a single run.

Threshold cycle (Ct) Assay Template Sample R1 R2 R3 R4 R5 R6 Mean SD CV RKO 27.17 27.59 27.35 27.52 27.23 27.63 27.42 0.19 0.01 MLH1-C Unmeth PBMC NA NA NA NA NA NA NA NA NA Methy- 0.5% meth 34.14 33.43 33.38 33.12 33.69 34.05 33.64 0.40 0.01

Light RKO 25.72 25.46 26.14 26.03 26.12 26.22 25.95 0.30 0.01 MyoD Unmeth PBMC 25.73 25.71 25.96 25.55 26.48 26.16 25.93 0.34 0.01 0.5% meth 26.23 26.84 26.54 26.73 26.62 26.45 26.57 0.22 0.01

RKO 26.84 27.65 26.69 27.66 27.05 27.26 27.19 0.41 0.02 Unmeth PBMC NA NA NA NA NA NA NA NA NA MLH1-C 0.5% meth 32.15 32.29 32.61 31.68 32.57 32.03 32.22 0.35 0.01 0.1% meth 35.81 35.81 36.48 36.45 36.76 37.82 36.52 0.74 0.02 qMSP RKO 26.51 27.07 26.03 25.64 26.65 26.21 26.35 0.50 0.02 Unmeth PBMC 22.30 22.63 23.03 22.25 22.63 22.10 22.49 0.34 0.02 MyoD 0.5% meth 23.50 23.42 22.62 22.28 22.47 22.63 22.82 0.51 0.02 0.1% meth 22.99 22.49 22.14 23.40 23.45 23.59 23.01 0.58 0.03

Table 3.1 Intra-assay variation of threshold cycle (Ct) measured by either MethyLight or qMSP The Ct value of six replicates (R1-R6) for given samples and their mean and standard deviation (SD) as measured by either the MethyLight or qMSP assay within a single run are shown. 0.5% and 0.1% meth refer to samples containing either a proportion of 0.5% or 0.1% completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) diluted in unmethylated PBMC DNA. For each sample, the coefficient of variation (CV) for Ct values of replicates was less than 0.1, indicating a good reproducibility of fluorescence signals detected within a single run. NA, not assessable.

The inter-assay reproducibility for the MethyLight and qMSP assays was also tested using the samples stated above. Using either the MethyLight or qMSP assay, each sample was assayed in triplicate for both MLH1-C and MyoD templates on three separate occasions. A similar amount of each sample (1.5 g) was bisulfite-treated in three separate batches prior to each of the three real-time PCR runs (Run 1-3). Using either the MethyLight or qMSP assay, comparable values of the mean threshold cycle (Mean Ct) with standard deviations (s.d. mean) of one cycle or less was obtained for each sample between the three runs (Table 3.2). For each sample, the coefficient of variation (CV) for mean Ct values of three different runs was less than 0.1, indicating a good reproducibility of fluorescence signals detected between runs. Overall, there was a good reproducibility within and between runs in both bisulfite conversion and subsequent real-time PCR assays (MethyLight or qMSP) (Table 3.1 and 3.2).

92 Mean Threshold cycle (Mean Ct) Assay Template Sample Run 1 Run 2 Run 3 Mean SD CV (n=3) (n=3) (n=3) means RKO 28.45 27.67 28.93 28.35 0.64 0.02 MLH1-C Unmeth PBMC NA NA NA NA NA NA 0.5% meth 33.65 34.45 34.58 34.23 0.50 0.02 MethyLight RKO 28.26 27.98 27.87 28.04 0.20 0.01 MyoD Unmeth PBMC 26.59 27.46 27.15 27.07 0.44 0.02 0.5% meth 26.60 26.01 25.77 26.13 0.43 0.02 RKO 27.24 26.96 27.05 27.08 0.14 0.01 MLH1-C Unmeth PBMC NA NA NA NA NA NA 0.5% meth 32.09 33.66 32.22 32.66 0.87 0.03 0.1% meth 36.54 36.03 37.01 36.53 0.49 0.01 qMSP RKO 26.79 25.83 26.55 26.39 0.50 0.02 Unmeth PBMC 22.32 22.41 22.62 22.45 0.15 0.01 MyoD 0.5% meth 22.19 23.18 22.46 22.61 0.51 0.02 0.1% meth 23.33 23.48 22.54 23.12 0.51 0.02

Table 3.2 Inter-assay variation of threshold cycle (Ct) measured by either MethyLight or qMSP The mean Ct values of three replicates from three separate runs (Run 1 through Run 3) are listed for given samples, measured by either the MethyLight or qMSP assay. 0.5% and 0.1% meth refer to samples containing either a proportion of 0.5% or 0.1% completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) diluted in the unmethylated PBMC DNA. For each sample, the coefficient of variation (CV) for mean Ct values of three different runs was less than 0.1, indicating a good reproducibility of fluorescence signals detected between runs. NA, not assessable.

3.3.1.4 Linearity of the MethyLight and qMSP assay for MLH1-C methylation detection The linearity of response of the MethyLight assay in detecting deffering levels of MLH1-C methylation was studied by analysing the percentage of methylated reference (PMR) values on a series of artificially mixed DNA samples (as described in section 2.2.13) as determined by MethyLight. Experiments were repeated in three separate runs to ensure the reproducibility of PMR values detected using this assay. The correlation coefficient (R2) and P value for the test of linear association were 0.994, P=1.36 ×10-9 (Figure 3.8, upper panel). A similar linearity test for MLH1-C methylation detection as detected by the qMSP assay was also performed. In this case, the correlation coefficient (R2) and P value for the test of linear association were 0.995 and P=1.17 ×10-10 respectively (Figure 3.7, lower panel). On this basis, both assays showed a good linearity and reproducibility between runs in measuring the proportion of methylated MLH1-C template in a background of unmethylated templates across a broad range of dilutions.

93 level (PMR) values detected across this range of dilu of range this across detected values (PMR) level different runs. Both theMethyLi different occasions, each time using samples intripli MethyLight (upper panel)and qM unmethylated PBMCDNA was tested across a broad the finalmean PMRwhich

A serial dilution ofcomplet assaysfor thedetection of qu the Linearity oftheand Figure 3.7MethyLight MethyLight Quantitative methylation-specificPCR

Measured methylation (PMR) Measured methylation (PMR) 100 100 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 0 0 20406080100204060800 04 08 100 80 60 40 20 0 MLH1 meanrepresents the oftriplicate ely methylated CpGenome ght andqMSP assaysshowgoodlinearity inthedetection of methylation -C promoter methylation SP (lower panel) assays were performed onthese samples onthree ehltdDNAinTemplate % Methylated ehltdDNA in Template % Methylated tion points with known percentagestion pointswithknown ofmethylation. antitative methylatio cates. Error bars represent ±1 rangemethylation of percentagesThe (0-100%). TM Universal MethylatedDNA(Chemicon) in PMR values,each obtained from three R R 2 2 = 0.9955 = 0.9941 n-specific PCR (qMSP) standard deviation of

100

3.3.2 qMSP is superior to MethyLight in the detection of mosaic allelic methylation

In a pilot study using seven bisulfite-treated DNAs derived from CRC and normal colonic mucosa samples, PMR values as determined by both MethyLight and qMSP were comparable (Table 3.3). However, while these PMR values were similar, it is noted that qMSP consistently detected slightly higher PMR values when compared to MethyLight. One possible explanation for this is that the qMSP assay might have the ability to detect MLH1-C fragments with mosaic patterns of methylation. These heterogeneously-methylated fragments were less likely to be detected by the MethyLight assay since the methylation-specific oligonucleotide probe would be less efficient in binding to any internal region of the template that showed mosaic methylation patterns. This possibility was verified by clonal bisulfite sequencing of PCR products generated by both assays. Using sample 1 as an example, dense methylation of MLH1-C fragments was observed in all clones (Figure 3.8). However, a more heterogenous pattern of methylation was observed in the amplicons generated by the qMSP assay, suggesting that this assay could better detect DNA templates with a mosaic pattern of MLH1-C methylation than the MethyLight assay. Therefore, the qMSP assay is better suited to the detection of mosaic patterns of MLH1-C methylation which may be present in the normal colonic mucosa and blood of individuals with CRC. Mosaic patterns of MLH1-C methylation may represent an intermediate methylation pattern which may subsequently proceed to a dense or fully methylated allele resulting in MLH1 inactivation.

Mean intensity of MLH1-C methylation (PMR) DNA Sample source Methylight qMSP PMR Difference (qMSP- MethyLight) 1 NCM 2.4 3.7 1.3 2 MSI Cancer 55.5 60.2 4.7 3 NCM 0.5 1.2 0.7 4 MSI Cancer 83.2 85.8 2.6 5 NCM 0 0 0 6 MSS Cancer 0 0 0 7 NCM 0 0 0

Table 3.3 Intensity of MLH1-C methylation (PMR) detected by each quantitative PCR assay Mean intensity of MLH1-C methylation (PMR) detected using MethyLight and quantitative methylation- specific PCR (qMSP) assays are shown. The qMSP assay consistently detects higher PMR values in methylation-positive samples. NCM, normal colonic mucosa; MSI, microsatellite unstable; MSS, microsatellite stable.

MethyLight

Forward primer Probe Reverse primer

94 -100 % of CpGs were methylated in individual alleles

Total methylation = 99.5%

qMSP

Forward primer Reverse primer

81 -100 % of CpGs were methylated in individual alleles

Total methylation = 92.2%

Figure 3.8 Clonal bisulfite sequencing of MLH1-C fragments generated by either MethyLight or qMSP assays for one methylated sample Black and white circles denote methylated and unmethylated CpGs respectively. Patterns of methylation at individual CpG dinucleotides were determined by cloning and sequencing of PCR products generated by either the MethyLight or qMSP assays. The methylation status at individual CpG dinucleotides within the MLH1-C promoter, inclusive of those within the primer binding regions, is shown for 12 clones from a sample of normal colonic mucosa (Sample 1, Table 3.3). Each row represents a single clone. Arrows specify the positions of the PCR primers, whereas a horizontal bar indicates the probe binding region of the MethyLight assay. Circles below the arrows or the bar indicate CpG sites within the primer binding regions or the probe binding region respectively. There is more variable methylation in the qMSP products within the CpGs spanned by the probe in the MethyLight.

96 3.3.3 MLH1 methylation analyses in tissue samples by qMSP

3.3.3.1 Study population - Individuals with sporadic colorectal cancer and controls A total of 235 individuals from the St.Vincent’s hospital cohort (mean age 74.0 ± 9.1 years; range 51-99 years) were enrolled in this study. As described earlier, these individuals had undergone curative surgical resection at St.Vincent’s Hospital, Sydney and none were known to have a hereditary CRC. Of these individuals, 104 had MSI while 131 had MSS CRC. In order to determine the cutoff PMR for MLH1-C methylation (measured by qMSP) which correlated with the loss of MLH1 protein expression, 50 MSI CRCs showing loss of MLH1 protein expression as assessed by immunohistochemistry (detail in section 2.2.8) were selected and compared with 50 MSS CRCs that did not show MLH1 loss by immunohistochemistry. Following this, the qMSP assay was used to detect the presence of MLH1-C methylation in a normal colonic mucosa sample from each of the 235 individuals included in this study. As controls, biopsy samples of normal colonic or rectal mucosa were collected from 21 individuals without a history of CRC who had either undergone colorectal surgery or colonoscopy for a non-neoplastic disorder (mean age, 58.4±17.5; range, 33-82 years). The presence of MLH1-C methylation was also assessed in lymphoid samples from a subset of individuals with CRC. These samples included PBMC samples from 75 individuals and lymph node samples from 50 individuals for whom blood samples were not available. Following informed consent, PBMC samples from 25 healthy donors (mean age 65.4±8.3; range 50 to 71) were included as normal controls. The clinical and pathological details of individuals with sporadic CRC were procured as described in section 2.2.2 and 2.2.3. This study was approved by Human Research Ethics Committee, St. Vincent’s Hospital.

3.3.3.2 MLH1 methylation in sporadic colorectal cancers The mean PMR value detected in sporadic MSI CRCs showing MLH1 loss (Mean PMR=42.53) was significantly higher than that detected in sporadic MSS CRCs without MLH1 loss (Mean PMR=0.02) (P<0.0001, Mann-Whitney U test) (Figure 3.9).

100.00

d d

e e 75.00

m m r

r fi

fi n

o o

Mean C C

_ _ 50.00

PMR th th

PMR =42.53 e

e M

M 1

H H

L L

25.00 M M Mean PMR 0.00 =0.02 MSSMSS ( n=50(n=27)01) MSI ( n=50(n=60)) CancerTumourMSI_status microsatellite MSI status status

Figure 3.9 MLH1 methylation levels in sporadic colorectal cancers A comparison of the intensity of MLH1-C methylation (PMR) measured by the qMSP assay in 50 sporadic MSI colorectal cancers showing immunohistochemical loss of MLH1 and 50 sporadic MSS colorectal cancers expressing MLH1. Each circle represents the PMR level detected in an individual cancer sample from the MSS (left) or MSI (right) groups. Horizontal lines represent the mean PMR level for each group. MSI, microsatellite unstable; MSS, microsatellite stable; PMR, Percentage of Methylated Reference.

The PMR value that correlated best with the loss of MLH1 expression by immunohistochemistry was determined using a ROC curve. The area under the curve (AUC) was 0.938 showing that this test was highly accurate (Figure 3.10). The cutoff PMR value that corresponded to the loss of MLH1 expression was 4.4, consistent with a previous study using Methylight in which the PMR value of >4 was found to correlate with the loss of gene expression (Ogino et al., 2006). Using this cutoff PMR value of >4, methylation of MLH1-C detected by the qMSP assay demonstrated a high sensitivity (88.0%) and specificity (100%) with loss of expression of the MLH1 protein as assessed by immunohistochemistry (Table 3.4).

1.0 Best discrete classifier

0.8

0.6 AUC= 0.938

Sensitivity 0.4

0.2

0.0 0.0 0.2 0.4 0.6 0.8 1.0 1 - Specificity

Figure 3.10 Receiver Operating Characteristic (ROC) curve of MLH1-C methylation levels versus MLH1 expression by immunohistochemistry This figure shows the plot of the true positive rate (sensitivity) against the false positive rate (1- specificity) when PMR values detected by the qMSP assay were classified according to different cutoff values (0-100). Loss of MLH1 expression by immunohistochemistry in an MSI cancer was considered as the gold standard. Fifty sporadic MSI CRCs showing loss of MLH1 expression and 50 sporadic MSS CRCs expressing MLH1 were included in this analysis. The plot in blue is made up of discrete classifiers that represent a false positive/true positive pair corresponding to a single point on the ROC curve. The point indicated by a red circle represents the best discrete classifier corresponding to the best cutoff PMR of 4.4. The area under the curve (AUC) was 0.938 indicating that this test was highly accurate. The diagonal line in green indicates the point where random correlations occurred and therefore areas above and below this line specify areas of acceptable or worthless classification respectively.

MLH1 immunohistochemistry MLH1 – negative MLH1- positive MLH1-negative 44 MLH1-C 44 0 (PMR > 4) methylation MLH1-positive 56 status by qMSP 6 50 (PMR < 4) 50 50 Sensitivity= 44/50 (88.0%), specificity = 50/50 (100%), positive predictive value = 44/44 (100%), negative predictive value = 50/56 (89.2%), measure of agreement, Kappa (Area under the curve) = 0.938

Table 3.4 Comparison between the quantitative methylation specific PCR (qMSP) and MLH1 immunohistochemistry in the detection of the loss of MLH1 protein in colorectal cancer The number of samples that were interpreted as MLH1- negative and positive by either the qMSP assay or immunohistochemical analysis of MLH1 is shown. MLH1 immunohistochemistry was considered the standard method for the detection of MLH1 expression status.

3.3.3.3 MLH1 methylation in normal colonic mucosa The frequency and intensity of MLH1-C methylation in the normal colonic mucosa samples from 104 individuals with sporadic MSI CRCs was compared with that of 131 individuals with sporadic MSS CRC, as well as 21 individuals without neoplasia. MLH1-C methylation was detected in the normal colonic mucosa samples of 7/104 (approximately 7%) individuals with sporadic MSI CRC, albeit at low intensities (Median PMR =0.54; range 0.17-3.7). Higher levels of MLH1-C methylation were found in the corresponding cancers of these individuals (Figure 3.11). No methylation was detected in normal colonic mucosa from individuals with sporadic MSS CRC or those without neoplasia (PMR=0).

100 T4 60.0 T3

50.0

T1 40.0 T5

30.0

T2 T6

20.0 R R

R T7

M M M

P P P

4.0 N1 3.0

2.0 N2

N3 1.0 N4 N5 N6 N7 0 Normal colonic mucosa Matched cancers

Tissue sample

Figure 3.11 Intensity of MLH1 methylation in the normal colonic mucosa and matched cancers The percentage of methylated reference (PMR) value detected in each of the seven MLH1-C-methylated normal colonic mucosa samples, N1-N7 (in blue) was compared to that of the corresponding colorectal cancers, T1-T7 (in red). Multi-colour lines linked each normal colonic mucosa sample to its matched cancer. A high level of MLH1-C methylation (PMR>20.0) was found in the corresponding cancers of each normal colonic mucosa sample, N1-N7.

The presence of methylation at MLH1-C detected initially by the qMSP assay was verified by clonal bisulfite sequencing using PCR products generated by qMSP (described in section 2.2.16). Dense allelic methylation at this gene promoter was found in all seven normal colonic mucosa samples from individuals with MSI CRCs that tested positive for MLH1-C methylation using qMSP (Figure 3.12). This finding shows that alleles detected by qMSP were hypermethylated and therefore these alleles are most unlikely to express MLH1.

101 N1 1 2 3 4 5 6 7 8 9 10 11 N2 1 2 3 4 5 6 7 8 9 10 11

91-100% of CpG sites were methylated 64-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 94.7% Total methylation = 85.1% N4 N3 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

82-100% of CpG sites were methylated 73-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 95.5% Total methylation = 93.2% N5 N6 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

82-100% of CpG sites were methylated 73-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 97.5% Total methylation = 94.7% N7 1 2 3 4 5 6 7 8 9 10 11

73-100% of CpG sites were methylated in individual clones representing alleles Total methylation = 94.7%

Figure 3.12 Methylation at individual CpG dinucleotides of the MLH1-C promoter region studied Patterns of methylation at individual CpG dinucleotides were determined by cloning and sequencing of PCR products generated by the qMSP assay. Circles represent CpG sites (1-11) that were either methylated (black) or unmethylated (white), positioned between the binding regions of the qMSP primers (indicated by black arrows). Methylation patterns of 10-12 clones are shown for each of the normal mucosa samples from seven individuals (N1-N7) that showed methylation at this gene promoter by the qMSP assay. Total methylation, calculated as a percentage of methylated over total CpG sites in each sample, ranged from 85.1-97.5%. Individual clones representing alleles sequenced showed dense methylation of >60% CpG sites.

102 In order to investigate whether the low level of hypermethylated MLH1-C alleles detected resulted in the focal loss of MLH1 expression in apparently normal colonic crypts, immunohistochemistry was performed on sections of normal colonic mucosa from the colonic and/or rectal resection margin of the surgical resection specimens. Paraffin-embedded histological tissue blocks were available in six of these seven cases. On average, 1000 crypts were assessed in each of the available tissue sections. However, no focal loss of MLH1 staining was found in the histological sections of normal colonic mucosa from these individuals. Therefore, the present study was unable to correlate low level of hypermethylated alleles in the normal colonic mucosa samples with the loss of protein expression in available samples. An example of the MLH1 staining is shown in Figure 3.13.

100m

Figure 3.13 MLH1 expression in the normal colonic mucosa of a representative case with MLH1-C methylation Parrafin embedded normal colonic mucosa sections were assessed for MLH1 expression by immunohistochemistry using a monoclonal antibody against MLH1 (Pharmingen, San Diego CA, USA). Strong positivity of MLH1 immunostaining (brown staining) was found in the nuclei of proliferating cells located at the base of all colonic crypts. Lack of MLH1 protein expression (blue staining) was found in maturing cells towards the surface of the crypts but this represented normal staining. There was no evidence of focal MLH1 loss of expression within any of the normal colonic crypts examined.

103 3.3.3.3.1 Clinicopathological characteristics of individuals showing MLH1 methylation in normal colonic mucosa The clinicopathological characteristics of individuals showing low levels of MLH1-C methylation in their normal colonic mucosa samples are shown in Table 3.5. These characteristics were compared with those of other individuals included in the present study with no MLH1-C methylation found in their normal colonic mucosa samples (Table 3.6). While a significant association was found between the presence of MSI cancers and MLH1-C methylation in the normal colonic mucosa samples (P<0.005, Fisher’s exact test), no significant association was found with the other clinicopathological characteristics assessed. Likewise, no association was found between the methylation of MLH1-C in normal colonic mucosa samples and the frequency of neoplastic colorectal polyps in individuals with CRC (Table 3.7). These individuals do not have MLH1-C methylation in blood (as described in the next section) and no other types of somatic tissue were available for MLH1-C methylation screening.

Individual Age Sex Location Multiple Tumour Tumour Associated polyps (years) cancers MSI CIMP status status 1 51 F Right No MSI CIMP+ve 2 conventional adenomas, 3 HPs, one serrated adenoma

2 80 F Right No MSI CIMP+ve Multiple conventional adenomas and HPs 3 84 F Left No MSI CIMP+ve 3 HPs, 2 serrated adenomas 4 89 F Left No MSI CIMP+ve 4 HPs 5 67 M Left No MSI CIMP-ve None 6 79 M Right No MSI CIMP-ve None 7 80 M Right No MSI CIMP-ve None

Table 3.5 Clinicopathological details of individuals harbouring low levels of MLH1-C methylation in their normal colonic mucosa Conventional adenomas comprises of tubular, tubulovillus and villus adenomas. M, male; F, female; MSI, Microsatellite instability; HP, hyperplastic polyp; CIMP+ve, positive for CpG Island methylator phenotype; CIMP-ve, negative for CpG island methylator phenotype.

104 Normal mucosa showing MLH1-C methylation Yes No P Value (n = 7) (n =235) Mean age (± SD) 75.7±11.2 70.9±12.6 0.165

Sex Male 3 (43%) 116 (49%) 1.000 Female 4 (57%) 119 (51%)

Location 0.156 Right-sided 4 (57%) 106 (21%) Left-sided 3 (43%) 90 (79%) Not specified 0 39

Tumour microsatellite 0.005 status MSI 7 (100%) 97 (41%) MSS 0 (0%) 131 (59%)

Tumour CIMP status 0.102 CIMP+ve 5 (71%) 75 (35%) CIMP-ve 2 (29%) 138 (65%) Not assessed 0 20

Table 3.6 Clinicopathological characteristics of individuals with colorectal cancer stratified by the presence of MLH1-C methylation in their normal colonic mucosa The P value for the comparison of mean age between groups was obtained using a Mann-Whitney U test. P values for all other characteristics were determined using Fisher’s exact test. A significant association was found between the presence of MSI cancers and MLH1-C methylation in the normal colonic mucosa samples (P=0.005) SD, standard deviation; MSI, microsatellite unstable; MSS, microsatellite stable CIMP+ve, positive for CpG Island methylator phenotype; CIMP-ve, negative for CpG island methylator phenotype.

105 Normal mucosa showing MLH1-C methylation Yes No P value (n=7) (n=230) Conventional adenomas 0.253 Median 0 0 Range 0-3 0-12

Hyperplastic polyps 0.254 Median 2 0 Range 0-4 0-50

Serrated adenomas 0.269 Median 0 0 Range 0-1 0-3

Serrated polyps 0.251 (serrated adenomas and hypeplastic polyps) Median 2 0 Range 0-4 0-53

Total polyps 0.871 (conventional and serrated polyps) Median 1 0 Range 0-5 0-53

Table 3.7 The frequencies of different types of neoplastic polyps in individuals with sporadic colorectal cancer by the presence of MLH1-C methylation in their normal colonic mucosa The median frequency and range of each type of polyp are shown for the two groups. Conventional adenomas comprise tubular, villous and tubullovillous adenomas. Details of colorectal polyps were not available for five individuals who showed no MLH1-C methylation in their normal colonic mucosa samples. P values were determined using Mann-Whitney U tests. No association was found between the methylation of MLH1-C in normal colonic mucosa samples and the frequency of neoplastic colorectal polyps in individuals with colorectal cancer.

3.3.3.4 MLH1 methylation in peripheral blood mononuclear cells and lymph nodes Using the qMSP assay, the presence of MLH1-C promoter methylation was assessed in the peripheral blood mononuclear cells (PBMCs) from 75 individuals enrolled in the St.Vincent’s Hospital cohort, of whom 52 have had sporadic MSI and 23 have had sporadic MSS CRC. PBMCs from 25 individuals without cancer were included as controls. No methylation was detected in the PBMCs of any of the individuals screened, either with or without cancer (PMR=0).

The presence of MLH1-C methylation was also examined in the lymph nodes of 50 individuals with sporadic CRC in whom PBMC samples were not accessible. Of these

106 individuals, 34 and 16 have had MSI and MSS cancers respectively. Low levels of MLH1-C methylation (PMR= 0.5-5.0) were detected in the lymph nodes of 8/34 (23.5%) individuals with MSI CRC, seven of whom (L1-L7) had higher levels of methylation in corresponding CRCs (Figure 3.14). A matched cancer sample was not accessible for one of these individuals (L8). None of these individuals had MLH1-C methylation in their normal colonic mucosa samples. No methylation was detected in lymph nodes of individuals with MSS cancers (PMR=0).

T9 80.0 T8 70.0

T12 60.0 T11 T10 50.0

40.0 T12 30.0 T13

20.0

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M M

P P

5.0 L1

4.0

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1.0 L3 L4 L5, L6, L7 0.0 Lymph nodes Matched colorectal cancers TissueTissue samplesample

Figure 3.14 Intensity of MLH1 methylation in the lymph nodes and matched colorectal cancers The percentage of methylated reference (PMR) value detected in each of the MLH1-methylated lymph nodes (in yellow) from seven individuals (L1-L7) was compared to that of the corresponding colorectal cancers (T8-T11, in red). Multi-colour lines link each lymph node sample to its matched colorectal cancers.

Table 3.8 shows the results of histopathological assessment of lymph nodes resected from individuals who had detectable MLH1-C methylation within DNA extracted from one fresh lymph node (L1-L8). Involvement of the lymph nodes by metastatic

107 carcinoma was seen in only one case (L7). There is no clear evidence that MLH1-C methylation is associated with involved lymph nodes.

Individual Number of involved Number of lymph Lymph nodes tested include the lymph nodes nodes tested one assessed for MLH1-C methylation L1 0 28 No L2 0 30 No L3 0 6 No L4 0 11 No L5 0 20 No L6 0 14 No L7 1 11 No L8 0 11 No Table 3.8 Histopathological assessment of available lymph node samples in individuals in whom MLH1-C methylation was found in one of their lymph nodes The number of involved lymph nodes is shown against the number of lymph nodes tested for lymphatic spread of colorectal cancer (involvement status). Note: Histopathological assessment was not performed on any of the lymph node samples that demonstrate methylation of MLH1-C since none of these tissue samples remained.

3.3.3.5 MLH1 methylation in cases of germline epimutation

3.3.3.5.1 Study group Two individuals with a germline epimutation of MLH1 had previously been identified from a cohort of 24 patients referred to the Familial Cancer Clinics of St. Vincent’s Hospital, Sydney and the Women’s and Children’s Hospital, Adelaide (Hitchins et al., 2007a). Tissues samples such as blood, buccal mucosae and hair follicles were procured from probands and their family members following informed consent. Lymphoblastoid cell lines available for three individuals with germline epimutations, previously identified by our laboratory were also included in this study. This study was approved by the Human Research Ethics Committee of St. Vincent’s Hospital.

3.3.3.5.2 Assessment of MLH1 methylation An initial screen for MLH1 methylation was performed using combined bisulfite restriction analysis (COBRA) in 24 individuals with CRC demonstrating MSI and MLH1 loss but no MLH1 mutation. Two females were found to have an MLH1

108 epimutation. Using this same COBRA assay, one individual (proband A) was shown to have transmitted the epimutation to one son (individual II6-A) but not to two other sons (individuals II5-A and II7-A) who inherited the same maternal allele associated with the epimutation (Figure 3.15). A sister of proband A, individual I1-A, also had the allele associated with the epimutation but was nevertheless found to be negative for the epimutation by COBRA. In family B, no evidence of transmission of the germ-line epimutation was detectable by COBRA, although the mother (I1-B) and one son (III2- B) shared the same allele associated with the epimutation in proband B (Figure 3.15).

109

Figure 3.15 Pedigrees of patients A and B showing intergenerational transmission of an MLH1 epimutation and haplotypes Panel A shows a map of SNPs within the MLH1 and EPM2AIP genes, used to determine inheritance patterns of the epimutant alleles and to analyze allelic expression. Panels B and C show pedigrees for Patient A and Patient B, with large black circles denoting Patient A and Patient B and with the current age of each family member given. Generations are listed I to III, and patients are identified by number. In Panels B and C, combined bisulfite restriction analysis of the C region of MLH1 (digested with MluI) is shown, with the lanes corresponding to data for family members shown in the pedigrees directly above. Combined bisulfite restriction analysis was performed on DNA extracted from peripheral-blood leukocytes from all patients except family member II7-A and family member II8-A, from whom only hair-follicle DNA was available. Lane M indicates the pUC19/MspI DNA ladder, lane C indicates DNA extracted from peripheral-blood leukocytes from a control with an unmethylated MLH1 promoter, and lane C+ indicates DNA extracted from the biallelically methylated RKO colorectal-carcinoma cell line (ATCC). Band sizes (in base pairs) are shown at the right. At the bottom of Panels B and C, haplotypes generated from informative SNPs are listed according to the key for each pedigree. Alleles associated with the epimutation are highlighted in yellow, and the presence of methylation (Me) is indicated. Maternally inherited alleles are shown in red, and paternally derived alleles are shown in blue; black letters indicate unknown parental origin. Although the haplotypes associated with the epimutation were inherited by several children, only family member II6-A retained the epimutation. (Hitchins et al., 2007).

110 In order to overcome the limitations of the COBRA assay in detecting low levels or mosaic patterns of MLH1 methylation, the qMSP assay was used to measure the intensity of MLH1-C methylation in the constitutional DNA of individuals with germline epimutation and their family members. At the commencement of this study, it was hypothesised that family members of epimutation patients may harbour low levels and mosaic patterns of MLH1 methylation in their tissue samples due to incomplete erasure of an epimutation during embryogenesis, or a cis-acting effect that predisposes the allele associated with the epimutation to subsequent somatic methylation.

Intensities of methylation were calculated as PMR values. In the PBMCs of three individuals with germ-line epimutations (two from family A and one from family B), percentages of MLH1-C methylation detected were 42 ± 7.0%, 42 ± 4.6% and 38 ± 9.0% respectively (Table 3.9). Similar percentages of MLH1-C methylation were detected in the lymphoblastoid cell line of three individuals with germline epimutations, previously identified by our group (probands C, D and E, Table 3.9) (Suter et al., 2004b; Hitchins et al., 2005).

Individual Mean intensity MLH1-C methylation (PMR) ± SD PBMC LCL Buccal Mucosa Hair follicle Proband A 42 ± 7.0 ND 76 ± 16.1 66 ± 3.7 II6-A 42 ± 4.6 ND 57 ± 5.4 57 ± 1.9 Proband B 38 ± 9.0 ND 101 ± 13.4 80 ± 30.9 I1-A, I4-A, II5-A, II7-A, II8-A, I1-B, 0 ND 0 0 III2-B, III3-B Proband C NA 39.3 ± 5.4 NA NA Proband D NA 46.7 ± 3.7 NA NA Proband E NA 45.0 ± 3.1 NA NA

Table 3.9 Percentages of MLH1-C methylation in tissues of individuals with a germ-line epimutation and their family members Percentages of MLH1-C methylation (PMR value) detected in the peripheral blood mononuclear cells, lymphoblastoid cell line, buccal mucosa samples or hair follicles of individuals with germ-line epimutation and their family members. PMR, Percentage of methylated reference; SD, standard deviation; PBMC, Peripheral blood mononuclear cell; LCL, Lymphoblastoid cell line; ND, Not done; NA, not available.

111 In any given sample, MLH1-C methylation was detected in <50% of alleles and this indicated that individuals with germline epimutations harbour mosaic patterns of monoallelic MLH1-C methylation in their blood. This finding was consistent with allelic bisulfite sequencing that found mosaic or absence of methylation in a small proportion of alleles associated with the epimutation (although the majority of the affected alleles were hypermethylated) (Suter et al., 2004b; Hitchins et al., 2005). MLH1-C methylation was also readily detected in the buccal mucosa samples and hair follicles of proband A, individual II6-A, and proband B. However, no evidence of low level mosaic methylation was found in the PBMCs, buccal mucosa samples and hair follicles of other members of family A and B who carry a genetic allele associated with an MLH1 epimutation. This indicates a complete reversal of the epimutation of MLH1 in these individuals.

112

3.4 Discussion

3.4.1 Real-time PCR assay for the detection of MLH1 methylation

This study further extended the findings by others (Eads et al., 2000; Trinh et al., 2001; Chan et al., 2004) that the MethyLight and qMSP assays are useful for the detection of CpG methylation at the promoter region of a given gene, in this case, MLH1. Both assays demonstrated good reproducibility within and between runs, in that the standard deviations of threshold cycle (Ct) values were comparable to those that have been reported in established real-time PCR assays (Gruber et al., 2001; Wellmann et al., 2001; Ogino et al., 2006) In the presence of >2x104 copies of MyoD control (for the normalisation of DNA input), the calculated percentage of methylated reference (PMR) values were equivalent to the input methylated templates present in samples with a known percentage of methylation (Figure 3.7). This indicates the precision of the sodium bisulfite conversion and subsequent detection of methylation levels using both MethyLight and qMSP assays. Both assays demonstrated consistency in obtaining PMR values between 0-100 (or approximate) and therefore, they were not affected by such errors as the variation of standard curve in the real-time PCR measurement or the use of a reference sample that is not completely methylated. PMR values exceeding 100 had previously been observed in other studies due to these aforementioned errors (Trinh et al., 2001; Okuda et al., 2005).

While this study clearly showed the superiority of the qMSP assay compared to the MethyLight in terms of its lower limit of detection, it is noteworthy that the qMSP assay was also better able to detect mosaic patterns of methylation which could still be important in gene inactivation. Particularly for MLH1, methylation at a specific CpG site in the proximal region of the MLH1 promoter (MLH1-C) has been found to be sufficient to cause MLH1 silencing by preventing the binding of the transcription factor CBF (Deng et al., 2001). Mosaic patterns of MLH1-C methylation encompassing the aforementioned CpG site may be present in various biological samples of individuals with CRC, particularly in those demonstrating the MSI phenotype. In light of its ability to detect low levels and mosaic patterns of MLH1-C methylation, the qMSP assay was chosen as the method for all subsequent analyses of biological specimens performed in this study.

113 In this study, MyoD serves as the internal control that roughly represents the total amount of DNA input. The lower limit of detection by the qMSP assay is 15-25 copies of methylated templates in a background of over 2x104 copies of a given DNA sequence (0.1%) which is one of the lowest percentages of methylation detection that has been reported to date. However, it is important to recognise that this assay cannot quantify methylation at individual CpG sites; instead, it quantifies the level of CpG methylation within primers binding sites as a whole. Other methods like pyrosequencing and matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are better suited for the resolution of methylation levels at individual CpG dinucleotides (Colella et al., 2003; Ehrich et al., 2005). However, these methods are not always feasible as they require expensive instrumentations that are not always available in diagnostic laboratories. In contrast to a real-time PCR assay, these methods are also more likely to introduce contamination since they involve end-point analysis that requires downstream processing of the PCR products (Ogino et al., 2006).

3.4.2 MLH1 methylation in biological samples

3.4.2.1 MLH1 methylation in sporadic MSI colorectal cancers More than 160 articles to date have examined the methylation status of the mismatch repair gene MLH1 in tumours of individuals with colorectal, gastric and endometrial cancers. Surprisingly, some studies have reported the finding of tumours with MLH1 methylation despite having intact mismatch repair machinery. Much of this inconsistency can be explained by the observation by Capel et al. (2006), who concluded on the basis of an extensive survey that over 60% of the published literature did not study methylation of MLH1 promoter at the region that correlates with MLH1 expression (Capel et al., 2007). Indeed, it has now been established that methylation of the proximal region of the MLH1 promoter (MLH1-C) but not that of the distal region (MLH1-A) correlates with expression of the MLH1 protein (Deng et al., 1999). In view of this knowledge, the present study assessed the methylation status within the pertinent MLH1-C region.

In this present study, a PMR value of >4, as assessed by qMSP was found to correlate best with the loss of protein expression, consistent with previous reports using

114 MethyLight. Using this cutoff criterion, the qMSP assay for MLH1-C region methylation showed high sensitivity (88.0%) and specificity (100%) for the detection of apparently sporadic MLH1 negative MSI CRC, suggesting that this assay is a reliable indicator of the functional loss of MLH1 expression. This result confirmed and extended the findings of Deng et al. who found that MLHI methylation at the proximal promoter region, relative to the transcription start site, correlates invariably with the absence of MLH1 expression (Deng et al., 1999; Deng et al., 2002).

In a small number of cases included in this study, MLH1 protein expression was lost despite the absence of methylation (at PMR >4). This is possibly due to the involvement of a different mechanism of gene silencing such as gene mutation or deletion. While not found in the present study, rare cases have previously been detected using the MethyLight assay in which MLH1 protein expression (by immunohistochemistry) remained intact in the presence of MLH1-C methylation (PMR >4) (Ogino et al., 2006). This may indicate mono-allelic methylation or inconsistent results between the two methods.

3.4.2.2 MLH1 methylation in normal colonic mucosa as a field defect in colorectal cancer development As mentioned in section 1.7, there are discrepant data in the literature regarding the pattern of MLH1 methylation in the normal colonic mucosa of individuals with CRC. In light of the reliability and the high efficiency of the qMSP assay in detecting low levels of methylation, this assay was implemented to determine whether MLH1 methylation is present in the normal colonic mucosa of individuals with CRC. A finding of low level methylation in individuals with cancer would support the hypothesis that this epigenetic change is a precursor to tumour formation.

In the present study, low levels of methylation were found at the proximal promoter region of MLH1 in the normal colonic mucosa of seven (7%) individuals who had an MSI CRC. This finding supports a previous report by Nakagawa et al. (Nakagawa et al., 2001) that methylation of the MLH1 promoter can extend to the proximal (3’-most) promoter region; a pattern of methylation that is well known to correlate with the loss of MLH1 expression (Deng et al., 1999; Deng et al., 2002). The present study did not find CpG methylation at this proximal promoter region of MLH1 in the normal colonic

115 mucosa of those with MSS cancers or those without neoplasia. This second finding refutes the claim by some researchers that the presence of methylation at the proximal promoter region of MLH1 is not related specifically to the development of MSI cancers (Nakagawa et al., 2001; Minoo et al., 2006). It is conceivable that previous detection methods which lacked specificity have overestimated the presence of methylation at the proximal MLH1 promoter in the normal colonic mucosa of individuals with MSS cancers.

The significance of low level MLH1-C methylation in the normal colonic mucosa of approximately 7% of individuals with MSI CRC remains speculative. A possibility is that MLH1-C methylation affects only a small focus of colonic crypts in the normal colonic mucosa samples. In this present study, no single crypt from MLH1-methylated normal colonic mucosae was entirely deficient of MLH1 staining, suggesting that biallelic MLH1 methylation is not a clonal event originating from stem or progenitor cells at the basal of colonic crypts. However, it is important to note that the normal colonic mucosa samples included in the methylation study were taken from a different section of the colon than that used for making the paraffin-embedded section. Immunohistochemical analysis of normal mucosa samples was typically limited to distal or proximal resection margins, often long distances (>10cm) from the site of the tumours. Others have reported the concomitant loss of MLH1 protein in conjunction with hypermethylation of MLH1 in small foci of replicating cells towards the middle and surface of a given crypt (Nuovo et al., 2006). The significance of this pattern of methylation is not well-defined, yet it was postulated that MLH1 methylation-induced silencing can be inherited by the immediate progeny of these small foci of MLH1- methylated cells and is important in the early evolution of some colorectal neoplasms (Nuovo et al., 2006).

In the present study, seven individuals who had low levels of MLH1-C methylation in their normal colonic mucosa samples had MSI cancers with high levels of methylation at this gene promoter. Such low levels of MLH1-C methylation in the normal colonic mucosa may represent a field defect in the development of additional CRC in some individuals who have had an MSI cancer. Indeed, methylation of several genes including MyoD, N33, ER, and MGMT has also been found in the normal colonic mucosa of individuals with CRC in which these genes were methylated, possibly as a

116 field defect in the development of subsequent CRC (Ahuja et al., 1998; Issa, 2000a; Issa, 2000b; Shen et al., 2005). In the study by Shen et al (2005), normal mucosa located one cm from tumour margin was more likely to be methylated than those located 10 cm away (Shen et al., 2005). In the present study, normal mucosa samples were resected far from their matched tumours (>10 cm away). Thus, it is plausible that a higher frequency of MLH1-C methylation would be detected in normal colonic mucosa closer to their matched cancers. The present study has provided additional knowledge that the field defect in CRC development may also involve the promoter methylation of an important cancer related gene, MLH1, which accounts for the development of the vast majority of sporadic MSI CRC.

While the present study shows clearly that MLH1-C methylation was found only in the normal colonic mucosa samples of a small number (7%) of individuals who had MSI CRC, there was no other unique clinicopathological features that was associated with individuals who harbour MLH1-C methylation in their normal mucosa. These findings were consistent with that reported by Kawakami et al (Kawakami et al., 2006). However, it is interesting to note the presence of multiple serrated polyps in the resected colon or rectum from four out of seven of these individuals. Serrated polyps may be precursors to sporadic MSI CRC developing via the “serrated neoplasia pathway” (Hawkins and Ward, 2001; Young et al., 2001b; Hawkins et al., 2002a; Goldstein et al., 2003; Jass, 2003; Torlakovic et al., 2003) and MLH1 methylation has previously been detected in these precursor lesions (Kambara et al., 2004b; Minoo et al., 2006). Thus, it is possible that MLH1-C methylation begins in a small focus of crypts in the apparently normal colonic mucosa, early in the development of this type of cancer.

One limitation of the present study is that the normal colonic mucosa samples may be contaminated with a small number of tumour cells. However, this possibility is unlikely given the evidence in Chapter 5 that demonstrated a mosaic pattern of allelic methylation in normal colonic mucosa samples as opposed to the dense methylation in matched tumours. It remains entirely possible that these normal colonic mucosa samples contained aberrant crypt foci, which were macroscopically indistinguishable from apparently normal colonic epithelial cells. In the present study, no remaining tissues were available from any of the normal colonic mucosa samples for formal histological assessment. While the present study has been performed retrospectively, a well-

117 designed prospective study in the future that includes microscopic examination of fresh normal colonic mucosa samples at collection could ensure the accuracy of any molecular investigations performed on these samples.

3.4.2.3 MLH1 methylation in lymphocytes from peripheral blood or lymph nodes MLH1 methylation if detected in a readily assessable sample such as peripheral blood mononuclear cells would present a useful detection method for individuals at risk of CRC. While monoallelic methylation of MLH1 has previously been found in the PBMCs of individuals with a germline epimutation, the presence of MLH1 methylation in the PBMCs of individuals with sporadic CRC remained uncertain prior to this study.

Previous studies in plants and animals have shown a mosaic state of methylation patterns in different tissue types (Cubas et al., 1999; Chan et al., 2006). In the flowering plant, Linaria vulgaris, the methylation-induced silencing of Lcyc gene led to a defect in flower symmetry and this epimutation is stably inherited across generations (Cubas et al., 1999). However, this epimutation was less stable in somatic development, whereby spontaneous demethylation can occur in the branches of the mutant plant. A similar pattern of epimutation to that seen in Linaria vulgaris may also underlie the allele- specific epimutation of MSH2 which was recently found in a family with HNPCC (Chan et al., 2006). Members of this family who harboured the allele associated with epimutation showed low levels of MSH2 methylation in the blood (1-3%) while higher in buccal mucosa and colorectal tissues. Such a mosaic state of epimutation may lead to a reduced disease penetrance in which affected individuals would most likely appear as sporadic. At the commencement of the present study, it was postulated that a proportion of individuals, clinically diagnosed with sporadic CRC have MLH1-C methylation in 1- 5% of their somatic cells including blood. In this scenario, low levels of methylation might confer a risk to the development of MSI CRCs.

In this study, no methylation of MLH1-C was found in any of the PBMC samples from individuals with sporadic CRC, suggesting that this epigenetic marker in PBMC is of little utility and is unlikely to detect individuals at risk of the disease. In individuals for whom blood samples were not available, MLH1-C methylation was assessed in lymph nodes samples that generally contain monocytes and lymphocytes. While seven individuals harbouring MLH1-C methylated MSI cancers had low levels of methylation in their lymph node samples, there was no remaining tissue from these MLH1-

118 methylated lymph node samples for further histopathological assessment of the lymphatic spread of CRC. Histopathological assessment of other lymph nodes from these individuals found no involvement in all but one case. This result, together with the absence of MLH1-C methylation in the normal colonic mucosa samples from these individuals, suggests that the methylation observed in lymph nodes is unlikely to be a somatic event inherent to white blood cells in the lymph nodes. More plausibly, methylation of MLH1 in lymph nodes could indicate the presence of naked DNA which may have been transported into lymph nodes following macrophage invasion.

3.4.2.4 MLH1 methylation and germline epimutation

Somawide monoallelic methylation of MLH1 has been found in individuals with germline epimutation of MLH1, suggesting that this event could have been initiated before gastrulation (prior to the formation of the ectoderm, endoderm and mesoderm layers). However, germline epimutations appear to be inherited in a non-mendelian pattern and the mechanisms and mode of inheritance remain uncertain. While an error in epigenetic reprogramming in the primordial germ cells may lead to the intergenerational transmission of an epimutation, present evidence suggested that a reversal of an epimutation is likely to occur during gametogenesis following demethylation and transcriptional reactivation events (Suter et al., 2004b; Hitchins et al., 2007a). In previous reports, individuals with germline epimutation have been shown to harbour mosaic patterns of monoallelic MLH1 methylation through the use of allelic bisulfite sequencing (Suter et al., 2004b; Hitchins et al., 2007a). Based on these findings, an epimutation may not be fully penetrant but rather, it may be caused by an incomplete erasure or retention of an epigenetic memory during embryogenesis. It is conceivable that low levels and mosaic patterns of allelic MLH1 methylation can also occur due to an incomplete erasure of epimutation, or alternatively, an in cis defect that will promote subsequent methylation in somatic tissues (Chan et al., 2006; Raval et al., 2007). This pattern of methylation may be present in the constitutional DNA of individuals who harbour a genetic allele that may be susceptible to germline epimutation, including family members of epimutation patients.

Using the qMSP assay, levels of MLH1-C methylation below 50% were consistently detected in the PBMC or LCL samples of individuals with germline epimutation,

119 confirming the mosaic patterns of methylation in these samples. If the epimutation was monoallelic and fully penetrant, levels of MLH1-C methylation would have been expected to be 50% in these samples. While MLH1-C methylation was also detectable in the buccal mucosa and hair follicle samples available for some of these individuals, DNA extracted from such samples were of poor quantity (MyoD copy number <104 ), and thus the percentages of methylation calculated were unreliable.

The qMSP assay was then utilised to determine whether low level mosaic patterns of MLH1-C methylation could be found in the family members of individuals with germline MLH1 epimutation. Apart from individual II6-A who has inherited the epimutated MLH1 allele from his mother (proband A), no traces of MLH1-C methylation were found in the PBMCs, buccal mucosa and hair follicles of other members of this family across two generations, including three individuals who possess the same maternal allele associated with the epimutation. Likewise, low levels and mosaic patterns of MLH1-C methylation were not found in the constitutional DNA from family members of another individual with germline epimutation (proband B) who harboured the affected genetic allele. This evidence does not support the possibility that mosaic patterns of MLH1-C methylation may occur due to incomplete somatic erasure of an epimutation during embryogenesis. In addition, germline epimutation seems to be meiotically reversible through complete erasure of epigenetic memory during gametogenesis. However, as evident in the case of individual II6-A, it may be retained at an apparently low but uncertain frequency through an error in epigenetic reprogramming or action of a trans mutation in resetting the epimutation post fertilisation.

During the course of this thesis, a new study has reported the mosaic patterns of MLH1 methylation (total methylation = 8%) in constitutive DNA from a family member of an epimutation patient (Morak et al., 2008). Therefore it is always worthwhile to investigate in future cases whether mosaic patterns of germline epimutation have occurred at MLH1 in a manner analogous to that reported in the aforementioned study. This may allow the identification of those affected by germline epimutation within high risk families and may help in the improving management of associated neoplasia among affected members.

120 3.4.3 Summary

1. A highly sensitive quantitative methylation-specific PCR (qMSP) assay was successfully developed and implemented for the detection and quantification of the levels of MLH1-C methylation in biological samples, particularly from individuals with sporadic CRC as well as from individuals with germline epimutations of MLH1 and their family members.

2. Low levels of MLH1-C methylation were present in the normal colonic mucosa of 7% of individuals with MSI CRC, which may represent a field defect and may increase the risk of the development of additional CRC.

3. No MLH1-C methylation was detectable in the PBMC DNA of individuals with sporadic CRC and thus, the utility of this methylation marker in PBMC samples is limited as it is unlikely to identify individuals at risk of sporadic CRC in the general population.

4. In the present study, low levels and mosaic patterns of MLH1-C methylation were absent in the constitutional DNA from family members of individuals with germ-line epimutation, including those who possessed the same allele that was associated with the epimutation. However, mosaic patterns of germline MLH1 epimutation may be heritable within the family members of some epimutation patients as has been reported in a recent study.

121

CHAPTER 4

Clinicopathological and molecular significance of CpG island methylation of gene promoters within the 3p22 chromosomal region

122

4.1 Introduction

The association between MLH1 methylation and the development of sporadic MSI CRC has been well documented. However, as reported in a study by our laboratory, MLH1 is not the only gene within the 3p22 chromosomal domain (Figure 4.1) that is prone to hypermethylation in this type of cancer (Hitchins et al., 2007b). That study demonstrated tumour-specific hypermethylation at loci flanking MLHI within the 3p22 chromosomal domain, including AB002340, ITGA9, PLCD1 and DLEC1. While the role of proteins encoded by AB002340 and PLCD1 in the development of cancer remains unclear, the protein encoded by DLEC1 is a putative tumour suppressor. The - integrin gene, ITGA9 encodes an -integrin that heterodimerises with a ß- integrin to form a membrane receptor that mediates cell adhesion and migration during angiogenesis (Vlahakis et al., 2005).

Figure 4.1 Map of genes and CpG islands flanking MLH1 within the 3p22 chromosomal domain A, human genome map of chromosome 3p22.3-3p22.2 (UCSC genome browser). Gray boxes or connected vertical lines (exons) represents genes identified by the GeneID database. The distance of CpG islands (in kilobases) from MLH1 is indicated at the bottom. B, Map of genes (black boxes) and corresponding CpG island of several 3p22 genes studied by Hitchins et al. (2007). Genes transcribed from the sense and antisense strand are placed above and below the horizontal line respectively (Hitchins et al., 2007).

Several of the genes localised at the 3p22 chromosomal domain including ITGA9, CTDSPL, PLCD1 and DLEC1 are frequently inactivated through deletion in other

123 epithelial cancers (Protopopov et al., 2003; Senchenko et al., 2003; Senchenko et al., 2004). Hypermethylation of ITGA9, PLCD1 and DLEC1 individually is also common in various types of human cancers (Shu et al., 2006; Weisenberger et al., 2006).

In tumours of individuals with sporadic MSI CRC, hypermethylation-induced silencing occurs concomitantly at multiple genes within the 3p22 chromosomal domain alongside MLH1 methylation. Inactivation of these genes may be important for the development of this type of CRC. The expression of unmethylated genes residing between methylated 3p22 genes were also suppressed (Hitchins et al., 2007b). This finding is consistent with the concept of long-range epigenetic silencing that is said to mimic the loss of heterozygosity in cancer (Frigola et al., 2006).

Notably, the concomitant methylation of CpG islands encompassing multiple 3p22 genes was strongly associated with the activating V600E mutation of BRAF (Hitchins et al., 2007b). Consistent with this observation was the fact that this association was not observed in a small group of sporadic MSS CRCs with wild-type BRAF. However, the association between BRAF mutation, microsatellite status and methylation at multiple 3p22 genes was only studied in 48 CRCs and therefore, requires verification in a larger cohort. In light of the strong association between BRAF mutation and the CpG island methylator phenotype (CIMP), methylation at multiple 3p22 genes may be a reflection of the latter. It is well established that a small proportion of MSS CRCs harbouring BRAF V600E mutation is positive for CIMP status (Weisenberger et al., 2006; Ogino et al., 2007a). Yet, it is uncertain whether this subset of MSS cancer is also concomitantly methylated at gene promoters within the 3p22 chromosomal region.

The association between methylation of 3p22 genes individually or as a group, and other clinicopathological or molecular variables has not been reported. Similarly, the prognostic significance of methylation at these genes, together or individually, remains unknown. This warrants further investigation.

In addition, the mechanism by which DNA methylation spreads across a chromosomal domain remains an important question to be answered. While it was suggested that DNA methylation can spread from random methylation of CpG sites within a given gene promoter (Song et al., 2002), the pattern and mechanism by which DNA methylation spreads across a given chromosomal domain has not been investigated.

124 This chapter examines the frequency of promoter methylation at five 3p22 genes, namely AB002340, MLH1, ITGA9, PLCD1 and DLEC1 as assessed in 875 sporadic CRCs enrolled in the St.Vincent’s hospital CRC cohort. This included all cancers previously found to harbour the V600E mutation of BRAF (n=128) as well as 747 cancers that were BRAF wild-type. Methylation of the 3p22 genes individually and as a group was correlated with BRAF mutation and microsatellite status to investigate the possibility that a small proportion of BRAF V600E mutant MSS cancers shows concurrent methylation of 3p22 genes. Subsequently, methylation of the 3p22 genes individually, and as a group, was correlated with other clinicopathological and molecular features, as well as patient survival.

Findings from this chapter may provide a better characterisation of the clinicopathological and molecular features that underlie the development of cancers showing regional 3p22 methylation. In addition, it may also suggest the utility of regional 3p22 methylation as a surrogate marker for a subset of cancers demonstrating certain clinicopathological and molecular features. This work also interrogates the pattern and density of hypermethylation across the 3p22 chromosomal domain, which may provide insights into mechanisms of the spread of DNA methylation across chromosomal regions in cancer development.

125

4.2 Methods

4.2.1 Sporadic colorectal cancer cohort

A subset of CRC specimens were procured from a consecutive series of 959 individuals enrolled in the St. Vincent’s Hospital cohort as described in Section 2.2.1. DNA extraction was performed as described in section 2.2.6. A total of 875 cancers from 861 individuals were included in this study, selected by the availability of DNA for methylation and mutation analyses. Clinicopathological variables of all individuals were obtained as described in sections 2.2.2 and 2.2.3. Immunohistochemical analysis of p53 expression was performed as previously described (Ward et al., 1997). BRAF and KRAS mutation analyses were performed as described in sections 2.2.18 and 2.2.19 respectively.

4.2.2 Methylation analyses

The promoter methylation status of five 3p22 loci, namely AB002340, MLH1, ITGA9, PLCD1 and DLEC1 was assessed using a combination of combined bisulfite restriction analysis (COBRA) (Hitchins et al., 2007b) and quantitative methylation specific PCR (qMSP) (as described in section 2.2.12) using primers listed in Table 2.1. For COBRA, digestions were performed using respective restriction enzymes as previously described (Hitchins et al., 2007b).

Optimisation of qMSP assays for the detection of promoter methylation of AB002340, ITGA9, PLCD1, and DLEC1 was performed in a manner similar to that described for MLH1 (Section 3.3.1). Briefly, each assay was able to detect 25 copies of target DNA fragments in samples which contained at least 2x104 copies of a control gene, MyoD. Each assay displayed excellent linearity when tested using serial dilutions of methylated DNA in unmethylated DNA (Figure 4.2). For each assay, the correlation coefficient and the P value for the test of linear association were as follows: AB002340, R2= 0.999, P = 2.68 × 10-15; ITGA9, R2=0.996, P = 4.42 × 10-11; PLCD1, R2=0.997, P = 1.98 × 10-11; DLEC1, R2=0.994, P = 3.61 × 10-10.

126 AB002340 ITGA9

PLCD1 DLEC1

Figure 4.2 Linear regression analyses to assess the linearity of the percentages of methylation measured by quantitative methylation specific PCR (qMSP) assay against input percentage methylation Samples with known percentage methylation were generated using a serial dilution of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) in unmethylated blood DNA (section 2.2.13). For each gene, the qMSP assay was performed on three different occasions using samples in triplicates. Error bars represent ±1 standard deviation of the final mean PMR which represents the mean of triplicate PMR values, each obtained from three different runs.

127 CpG island methylator phenotype (CIMP) status of CRCs was assessed by staff of the Molecular and Cellular Oncology Group, St.Vincent’s Hospital, using MethyLight analysis as previously described by Weisenberger et al. (2006). For both MethyLight and qMSP assays, the percentage of methylated reference (PMR) (section 2.2.12) was determined at each locus. Methylation at a particular locus was defined as being present when PMR was 4. This PMR threshold was chosen because it has been shown to correlate best with loss of gene expression (Ogino et al., 2006).

4.2.3 Hierarchical clustering analysis

Hierarchical clustering analysis was performed using the Hierarchical Clustering Explorer version 3.5 (University of Maryland), available on-line at http://www.cs.umd.edu/hcil/hce. An average linkage method using Manhattan distance was used.

4.2.4 Statistical analyses

Univariate and multivariate analyses for comparisons between clinicopathological and molecular features were performed as described in section 2.2.20.

For the analysis of disease outcome, survival (months) was calculated from the date of resection to the date last seen, or the completion of five years of follow-up or the date of death. Only individuals who have undergone surgical resection of CRCs and in whom residual tumours (R2) did not exist (n=835) were included in this analysis (Hermanek and Sobin, 1998). For individuals with synchronous CRCs, the highest stage tumour was considered in the survival analysis. Survival was censored at the time of death for individuals who died of causes other than CRC. Univariate associations between risk factors and disease-specific survival were analysed using Kaplan-Meier estimates and Log-rank test. Multivariate survival analyses were performed using the Cox proportional hazards regression model.

128

4.3 Results

4.3.1 Clinicopathological and molecular characteristics of the study group

The study group consisted of 479 males and 382 females, with age ranging from 29 to 99 years (mean 68±12 years). Of 835 individuals included in the survival analysis, a total of 207 individuals had died of CRC at the census date, with a mean survival time of 22 months (range 1-60 months). Sixty-seven individuals had died of causes other than CRC, and 561 were alive at five years. Thirteen individuals were lost to follow-up. The median duration of follow-up time was 37 months (range 1-60 months).

Pathological characteristics Number of cases Percentage (%)

Tumour site Caecum 96 10.9 Right- Ascending colon 133 301 15.2 34.3 Transverse colon sided 72 8.2 Descending colon 52 5.9 Sigmoid Left- 184 568 21.0 64.8 Rectum sided 332 37.9 Not specified 6 0.9

Pathological Stage (TNM v.6) Stage I 170 19.4 Stage II 301 34.4 Stage III 277 31.7 Stage IV 127 14.5

Tumour grade High grade 120 13.7 Low grade 752 85.9 Not classified 3 0.4

Tumour type Mucinous 170 19.4 Non-mucinous 701 80.1 Not classified 4 0.5

Intraepithelial lymphocytes Prominent (>30 lymphocytes per 10 power field) 113 12.9 Scant (<30 lymphocytes per 10 power field) 539 61.6 Not assessed 223 25.5

Table 4.1 Summary of pathological characteristics of all colorectal cancers in the study group (n=875)

129 The pathological characteristics of CRCs including site, stage, grade, mucinous type, and the presence of intraepithelial lymphocyte are summarised in Table 4.1. The frequencies of various molecular features observed in the cancers from this cohort, including MSI, KRAS mutation, BRAF V600E mutation and p53 immunohistochemisty status are shown in Table 4.2. With the exception of one case, KRAS and BRAF mutations were mutually exclusive (data not shown).

Molecular characteristics Number of cases Percentage (%)

Microsatellite status MSI 98 11.2 MSS 777 88.8

BRAF V600E mutation Mutant 128 14.6 Wild-type 747 85.4

KRAS codon 12 or 13 mutations Mutant 285 32.6 Wild-type 590 67.4

P53 nuclear accumulation Present 327 37.4 Absent 267 30.5 Not assessed 281 32.1

CIMP Positive 139 15.9 Negative 736 84.1

Table 4.2 Summary of molecular features of all colorectal cancers in the study cohort (n=875) MSI, microsatellite unstable; MSS, microsatellite stable; CIMP, CpG island methylator phenotype.

Of all clinicopathological and molecular features considered, Kaplan-Meier estimates showed that higher pathological stage and higher tumour grade (poorly-differentiated) were significantly associated with poorer disease-specific survival (Figure 4.3, upper panel). Significant relationships were found between microsatellite instability (MSI) as well as the presence of intraepithelial lymphocytes and improved survival (Figure 4.3, lower panel). On multivariate analysis, only tumour stage was identified as a significant prognostic factor (Table 4.3).

130

Tumour stage Tumour grade 1.0 Stage I 1.0

Stage II 0.8 0.8 Low grade

0.6 Stage III 0.6 High grade

0.4 0.4 Proportion surviving Proportion surviving

0.2 0.2

P <0.0001(Log-rank) Stage IV P =0.007= 0.002 0.0 0.0 0 12 24 36 48 60 0 12 24 36 48 60 Survival time (months) Survival time (months)

MSI status Intraepithelial lymphocyte status

1.0 1.0

MSI ProminentIEL +ve IEL

0.8 0.8 MSS IELScantIEL -ve

0.6 0.6

0.4 0.4 rprinsurviving Proportion rprinsurviving Proportion

0.2 0.2

P=0.014 P =0.015 PP =0.001=0.006 0.0 0.0 0 12 24 36 48 60 0 12 24 36 48 60

Survival time (months) Survival time (months)

Figure 4.3 Survival analysis in SVH cohort Kaplan Meier plots showing proportion of surviving individuals against time (months) stratified by disease stage (TNM), tumour grade, MSI status and the status of intraepithelial lymphocytes (IEL). + specifies the survival (months), of individuals who were still alive at censor date or were censored because they were lost to follow-up or died of a cause other than colorectal cancer. MSI, microsatellite unstable; MSS, microsatellite stable; Prominent IEL, >30 lymphocytes per 10 power field; Scant IEL<30 lymphocytes per 10 power field.

131

Colorectal cancer survival Risk factor n HR 95% CI P Tumour stage 835 4.4 3.5-5.5 <0.001 High grade 835 1.4 0.9-2.2 0.091 MSI 835 1.2 0.5-2.6 0.699 Prominent IEL 623 1.2 0.6-2.1 0.589

Table 4.3 Multivariate Cox proportional hazards regression model showing significant prognostic factor in sporadic colorectal cancers This analysis evaluated all features that were significantly associated with survival in univariate analyses. Tumour stage was identified as an independent prognostic factor. HR, hazard ratio; CI, confidence interval; MSI, microsatellite instability; Prominent IEL, >30 lymphocytes per 10 power field.

4.3.2 Promoter methylation of 3p22 genes in the study group

The frequency with which sporadic CRCs were methylated at the promoter region of each of the 3p22 genes is presented in Table 4.4. Taking the entire cohort into account, CRCs were most frequently methylated at AB002340, followed by DLEC1, ITGA9, MLH1, and then PLCD1 as determined by a combination of COBRA and qMSP assays.

Gene Number of colorectal cancers % of colorectal cancers with with methylation methylation AB002340 160 18.3% MLH1 82 9.4% ITGA9 98 11.2% PLCD1 77 8.8% DLEC1 106 12.1%

Table 4.4 Frequency of colorectal cancers showing promoter methylation at each of the 3p22 gene Promoter methylation of a gene in a given sample was considered present when a COBRA reaction resulted in a digestion of PCR product into smaller fragments, or when qMSP assay measured a percentage of methylated reference (PMR) >4 . All samples were assessable at each loci (n=875).

Examples of gels illustrating the COBRA result from tumour DNA for each of the 3p22 genes are shown in Figure 4.4. CRC samples do not comprise a pure population of methylated cancer cells but are mixed with stromal and normal epithelial cells. Therefore, all methylated samples contained both methylated and unmethylated fragments. Amplification plots for the qMSP assays for each 3p22 genes were similar to that illustrated for MLH1 (detailed in Chapter 3) and are not reproduced here.

132 A B

Figure 4.4 COBRA results for the detection of promoter methylation of 3p22 genes A, Map of gene promoters within the 3p22 chromosomal regions as labelled. Individual CpG dinucleotides are indicated by vertical lines. Positions of nucleotides with reference to the translational start site (+1) are indicated below the map. Primers (represented by horizontal arrows) were unbiased for the amplification of methylated and unmethylated templates of bisulfite treated DNA. Red arrows represent the position of the translational start site, ATG, if within or near the amplified fragment. B, Agarose gels following electrophoresis showing examples of COBRA of 3p22 genes. Following sodium bisulfite conversion, PCR and restriction enzyme digestion, amplicons corresponding to methylated DNA (M) were cleaved into smaller fragments while those corresponding to unmethylated DNA (U) remained intact. MW, molecular weight marker puC19 DNA/MspI; T 1-9, CRC samples which are not the same from one gel to another; +ve, methylated control (M.SssI treated genomic DNA; -ve, unmethylated control (peripheral blood DNA from a healthy donor).

133

Significant association was found between methylation of each 3p22 gene individually and that of the four other genes (p <0.0001) (Table 4.5) showing that methylation occurred in a cluster in the 3p22 region. Based on hierarchical clustering analysis, no specific pattern of methylation spreading across the 3p22 chromosomal domain was observed (Figure 4.5). However, the dendrogram highlights that promoter methylation of MLH1 was more closely associated with that of ITGA9 compared to other genes.

U 688 78 MLH1 M 27 82* U 701 84 737 40 ITGA9 M 14 76* 29 69* U 708 90 743 55 753 45 PLCD1 M 7 70* 23 54* 24 53* U 684 85 720 49 733 36 744 25 DLEC1 M 31 75* 46 60* 44 62* 54 52* U M U M U M U M AB002340 MLH1 ITGA9 PLCD1

Table 4.5 The frequency of association between promoter methylation of each 3p22 gene and that of other 3p22 genes in colorectal cancers (n=875) Significant associations were observed between methylation of any one gene with that of all other genes. Methylation: U, unmethylated; M, methylated. * indicates p<0.0001 (2 test).

134

1.0 0.8 0.6 0.4 0.2 1- similarity MLH1 ITGA9 PLCD1 DLEC1 0 AB002340 CRC samples (n = 875)

Figure 4.5 Dendrogram and heat maps showing the relationship between concomitant methylation of genes the 3p22 cluster Hierarchical clustering analysis generates a dendrogram and heat map showing the association between methylation of one gene with another in colorectal cancer (CRC) tissues. Each horizontal line represents a single CRC sample. Red and green represent the presence and absence of methylation respectively. The 1-similarity scale represents a distance metric ranged from 0 to 1, which measured the strength of association between concomitant methylation at each gene and MLH1. The smaller the 1-similarity value, the stronger the association.

135 The frequency of CRCs showing methylation at various number of 3p22 genes is shown in Figure 4.6. Criteria previously used to charaterise CpG island methylator phenotype (CIMP) status have defined CIMP as methylation of 3 or more of 5 markers (Weisenberger et al., 2006). Consistent with this system, CRCs were dichotomised in this study into those with or without methylation at 3/5 3p22 loci. These groups were designated as positive and negative for regional 3p22 methylation respectively. Overall, 11% of CRCs in this study group were positive for regional 3p22 methylation (regional 3p22 methylation +ve) using this definition.

n=875

Figure 4.6 Frequency of colorectal cancers with promoter methylation at 0-5 genes within the 3p22 chromosomal domain Methylation at individual genes was defined as present if PMR4 by qMSP assays, or if DNA templates were digested using COBRA.

4.3.3 Association between promoter methylation of individual 3p22 genes, and BRAF V600E mutation and MSI status

As shown in Table 4.6, a significant association was observed between the promoter methylation of each 3p22 gene and the V600E mutation of BRAF (2, P <0.001). Methylation of 3p22 genes was also strongly associated with MSI in CRCs (2, P <0.001). A considerable number of CRCs showing methylation of AB002340, ITGA9, PLCD1 and DLEC1 but unmethylated at MLH1 were BRAF wild-type and MSS. Promoter methylation of MLH1 was not detected in MSS CRCs. When stratified by BRAF mutation and MSI status, methylation at each 3p22 gene with the exception of MLH1 was more frequently found in association with BRAF mutation (Figure 4.7).

136 AB002340 methylation MLH1 methylation ITGA9 methylation PLCD1 methylation DLEC1 methylation Present Absent P Value Present Absent P Value Present Absent P Value Present Absent P Value Present Absent P Value (n =160) (n =715) (n =82) (n =793) (n =99) (n =776) (n =77) (n =798) (n =106) (n =769)

BRAF <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 V600E Mutant 110 (69%) 18 (2%) 79 (96%) 49 (6%) 82 (83%) 46 (6%) 64 (83%) 63 (8%) 74 (68%) 54 (7%) WT 50 (31%) 697 (98%) 3 (4%) 744 (94%) 17 (17%) 730 (94%) 13 (17%) 735 (92%) 32 (30%) 715 (93%)

MSI # <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Status MSI 88 (55%) 10 (1%) 82 (100%) 16 (2%) 70 (71%) 28 (4%) 56 (73%) 42 (5%) 59 (56%) 39 (5%) MSS 72 (45%) 705 (99%) 0 (0%) 777 (98%) 29 (29%) 748 (96%) 21 (27%) 756 (95%) 47 (44%) 730 (95%)

Table 4.6 The associations between promoter methylation of individual 3p22 genes and BRAF V600E mutation, as well as MSI status in colorectal cancers # % was calculated by excluding samples in which respective clinicopathological features were unknown. P values are indicated for 2 or Fisher’s exact tests. WT, wild- type; MSI, microsatellite unstable; MSS, microsatellite stable.

137

100 90 80 70 60 50 40 30 20 Percentage of CRC (%) 10 0 CpG141 MLH1 ITGA9 PLCD1 DLEC1 (n=121) (n=82) (n=85) (n=71) (n=78) Methylation of 3p22 genes

BRAFMut MSI Mut MSI BRAFMut MSS Mut MSS BRAFWT MSI WT MSI BRAFWT MSS WT MSS

Figure 4.7 Distribution of colorectal cancers that were methylated at each 3p22 gene, stratified by BRAF mutation and MSI status Methylation at each 3p22 gene, with the exception of MLH1, was more frequently found in association with BRAF mutation (red and blue combined) than MSI (red and geen combined). BRAF Mut, BRAF V600E mutant; BRAF WT, BRAF wild-type; MSI, microsatellite unstable; MSS, microsatellite stable; CRC, colorectal cancer.

138 4.3.4 Association between simultaneous promoter methylation of multiple 3p22 genes, and BRAF V600E mutation and MSI status

The proportion of CRCs showing different numbers of methylated 3p22 genes is shown in Figure 4.8, stratified by BRAF V600E mutation status. In the BRAF mutant group, most cases showed heavy methylation at multiple loci (3 or more). In contrast, the percentage of BRAF wild-type tumours decreased consistently with increasing number of methylated 3p22 genes.

BRAF V600E Mutant BRAF V600E WT 100 n=128 100 n=747

90 90

80 80

70 70

60 60

50 50

40 40

30 30

20 20 Percentage of cases (%) Percentage of cases (%)

10 10

0 0 0 12345 012345 Number of methylated 3p22 loci Number of methylated 3p22 loci

Figure 4.8 Histogram showing the frequency of BRAF mutant and wild-type colorectal cancers with promoter methylation of 0, 1, 2, 3, 4 and 5 genes within 3p22 chromosomal domain

When CRCs were stratified by MSI status, a similar observation was made to that of BRAF mutants (Figure 4.9). The number of CRCs harbouring methylation at 3 of 5 3p22 genes (regional 3p22 methylation +ve) was significantly higher in BRAF mutant and MSI groups respectively (P<0.0001) (Table 4.7).

139 MSI MSS 100 n = 98 100 n = 777

90 90

80 80

70 70

60 60

50 50

40 40

30 30

20 20 Percentage of cases (%) Percentage of cases (%)

10 10

0 0 0 12345 0 12345 Number of methylated 3p22 loci Number of methylated 3p22 loci

Figure 4.9 Histogram showing the frequency of MSI and MSS colorectal cancers with promoter methylation at 0, 1, 2, 3, 4 and 5 genes within 3p22 chromosomal domain

Regional 3p22 methylation +ve -ve 2, P Value (n = 96) (n =779) BRAF V600E <0.0001 Mutant 91 (95%) 37 (5%) Wild-type 5 (5%) 742 (95%)

Microsatellite status <0.0001 MSI 80 (84%) 18 (2%) MSS 15 (17%) 762 (98%)

Table 4.7 The frequency of association between regional 3p22 methylation and BRAF V600E mutation, as well as MSI status in colorectal cancers (n=875) Positivity for regional 3p22 methylation is defined as the methylation at 3 of 5 genes within the 3p22 cluster.

While most regional 3p22 methylation +ve CRCs were BRAF mutant and MSI (83%), approximately 12% of these were BRAF mutant and MSS (Figure 4.10), in the absence of MLH1 methylation. No regional 3p22 methylation was found in CRCs that were BRAF wild-type and MSI, although MLH1 methylation was found in three of these cases (4%) (Figure 4.7). These findings show a strong correlation between regional

140 3p22 methylation and the V600E mutation of BRAF, and that regional 3p22 methylation was not entirely dependent upon MLH1 methylation and MSI status. P < 0.0001

V600EV600EV600E mutant V600EV600E wild-type wildtype 100 80 606 40 20 p2mtyain(%) methylation 3p22

Frequency of regional 0 MSI/BRAFMSI Mut MSS/BRAFMSS Mut MSI/BRAFMSI WT MMSS/BRAFSS WT mutant mutant wild-type wild-type (n=86) (n=42) (n=12) (n=735) (n=86) (n=42) (n=12) (n=735)

Groups Figure 4.10 Distribution of regional 3p22 methylation in colorectal cancers stratified by BRAF V600E mutation and MSI status into four distinct groups

4.3.5 Association between promoter methylation of individual 3p22 genes and other clinicopathological and molecular features of colorectal cancer

The association between methylation at each 3p22 gene and other clinicopathological as well as molecular features (apart from BRAF mutation and MSI status) in the study group is summarised in Table 4.8.

Significant correlations were observed between methylation at each of the five genes and female gender, right-sidedness of tumours, mucinous histology, low tumour grade, wild-type KRAS, absence of p53 nuclear accumulation by immunohistochemistry, prominent intraepithelial lymphocytes, and CIMP +ve phenotype (all P <0.05). Methylation of each of the five genes appeared to be associated with older age although this was not statistically significant in the case of DLEC1 (P=0.23). Importantly, methylation at MLH1 and ITGA9 was significantly associated with lower tumour stage.

141

Table 4.8 Clicopathological associations of individual 3p22 genes methylation in colorectal cancers P values are indicated for 2 or Fisher’s exact tests. Significant P values are highlighted in bold. SD, standard deviation; MSI, microsatellite unstable; MSS, microsatellite stable; CIMP, CpG island methylator phenotype. 142

Table 4.8 Continued.

143 4.3.6 Regional 3p22 methylation and the clinicopathological and molecular features of sporadic colorectal cancers

The association between regional 3p22 methylation and common clinicopathological as well as molecular features (apart from BRAF mutation and MSI status) in the study group is summarised in Table 4.9.

Significant correlations were observed between regional 3p22 methylation +ve CRCs and older age, female gender, right-sidedness of cancers, mucinous histology, low tumour grade, wild-type KRAS, negative p53 immunostaining, prominent intraepithelial lymphocytes, lower tumour stage, and CIMP +ve (all P <0.01).

Regional 3p22 methylation +ve -ve P Value (n = 95) (n =780) Mean age (± SD) 73.6±9.5 68.1±12.0 <0.0001

Sex <0.0001 Male 31 (33%) 456 (58%) Female 64 (67%) 324(42%)

Tumour site <0.0001 Right-sided 76 (81%) 229 (29%) Left-sided 18 (19%) 550 (71%) Not classified 1 0

Tumour type <0.0001 Mucinous 54 (57%) 116 (15%) Non-mucinous 41 (43%) 660 (85%) Not specified 0 4

Tumour grade <0.0001 Low grade 58 (62%) 694 (89%) High grade 36 (38%) 84 (11%) Not specified 1 2

Table 4.9 Clinicopathological associations of regional 3p22 methylation in colorectal cancers Positivity of regional 3p22 methylation is defined as the methylation of 3 of 5 3p22 loci. The P value for the comparison of mean age between groups was obtained using a Mann-Whitney U test. P values are indicated for 2 or Fisher’s exact tests for all other characteristics. Significant P-values are highlighted in bold. SD, standard deviation; CIMP, CpG island methylator phenotype.

144 Regional 3p22 methylation +ve -ve P Value (n = 95) (n =780) KRAS codon 12 or 13 <0.0001 Wild-type 93 (98%) 497 (64%) Mutant 2 (2%) 283 (36%)

P53 nuclear <0.0001 accumulation Present 13(23%) 314 (59%) Absent 44 (77%) 223 (41%) Not assessed 38 243

Intraepithelial <0.0001 lymphocytes Prominent 49 (73%) 64 (11%) Scant 18 (27%) 521 (89%) Not assessed 28 195

TNM stage 0.011 I 18 (19%) 152 (20%) II 46 (48%) 255 (33%) III 24 (26%) 253 (32%) IV 7 (7%) 120 (15%)

CIMP <0.0001 Positive 89 (94%) 50 (6%) Negative 6 (6%) 730 (94%)

Table 4.9 continued

Multivariate analysis of clinicopathological and molecular associations as shown in Tables 4.7 and 4.9 found CIMP+ve and MSI to be independent predictors of this regional 3p22 methylation (Table 4.10).

145 Clinicopathological/molecular feature OR (95%CI) P value Age 1.0 (0.9-1.0) 0.218 Sex (female) 2.6 (0.8-7.9) 0.097 Tumour site (right-sided) 0.7 (0.2-2.6) 0.643 Tumour type (mucinous) 2.8 (0.9-2.6) 0.072 Tumour grade (Low grade) 0.9 (2.5-3.3) 0.881 KRAS Codon 12 or 13 mutation (wild-type) 2.6 (0.3-20.3) 0.348 P53 nuclear accumulation (absence) 1.9 (0.4-8.5) 0.397 IEL (prominent) 2.4 (0.6-9.8) 0.215 TNM stage (lower tumour stage) 0.335 CIMP (positive) 46.8 (10.8-202.1) <0.0001 BRAF (V600E mutant) 5.3 (0.9-29.5) 0.056 Microsatellite status (MSI) 36.9 (7.6-178.7) <0.0001

Table 4.10 Multivariate logistic regression analysis showing independent predictors of regional 3p22 methylation Odds ratio (OR) and 95% confidence interval (95%CI) as well as P values obtained using the analysis are shown. IEL, intraepithelial lymphocytes; CIMP, CpG island methylator phenotype; MSI, microsatellite unstable.

4.3.7 Distribution of regional 3p22 methylation by BRAF mutation, CIMP and MSI status

The finding in multivariate analysis that BRAF mutation was not a predictor of regional 3p22 methylation was somewhat surprising. One explanation is that the majority of CIMP+ve and/or MSI CRCs were also BRAF mutant, this association acting to confound the relationship between BRAF mutation and regional 3p22 methylation. In order to confirm this possibility, the distribution of regional 3p22 methylation was examined in tumours stratified by BRAF mutation, CIMP and MSI status (Figure 4.11).

In this analysis, regional 3p22 methylation was seen predominantly in CRCs with BRAF V600E mutation, and occurred rarely in BRAF wild-type cancers. Amongst BRAF mutant tumours, regional 3p22 methylation was most frequently found in those that demonstrated concomitant CIMP+ve and MSI. Regional 3p22 methylation was found in a small number of BRAF mutant CRCs that showed either CIMP+ve/MSS or CIMP-ve/MSI phenotypes, and was entirely absent in CRCs that were both CIMP negative and microsatellite stable (CIMP-ve/MSS).

146 BRAF V600E Mut BRAF V600E WT 100 100 N=701 90 90

80 80

70 70 N=79 60 60

50 50 Frequency (%) Frequency (%) 40 40

30 N=29 30

20 20 N=13 10 N=7 10 N=31 N=0 N=12 0 0 CIMP+ve CIMP+ve CIMP-ve CIMP-ve CIMP+ve CIMP+ve CIMP-ve CIMP-ve MSI MSS MSI MSS MSI MSS MSI MSS

Yes No Regional 3p22 methylation

Figure 4.11 Distribution of regional 3p22 methylation in colorectal cancers stratified by BRAF V600E mutation, CIMP and MSI status Numbers for each category are expressed as a percentage of the total sample number in each group (BRAF mutant, n=128; or wild type, n=747).

4.3.8 Correlation between promoter methylation of 3p22 genes and survival of individuals in the entire study group

To determine the prognostic significance of methylation of 3p22 genes individually and as a group, survival analysis was performed using Kaplan-Meier analysis. The presence of MLH1 methylation was significantly associated with a better disease-specific survival of individuals with sporadic CRC (p=0.023, Log-rank test) (Figure 4.12). This association did not hold true when adjusted for tumour stage (data not shown), reflecting the fact that MLH1 methylated CRCs were much more likely to be at lower tumour stage. No relationships were observed between methylation of other 3p22 genes individually and survival, although individuals with promoter methylation of ITGA9, PLCD1 and DLEC1 showed a trend towards better survival. When methylation of 3p22 genes was considered as a group, individuals with CRCs showing regional 3p22 methylation demonstrated a propensity towards better survival, although this did not reach statistical significance.

147 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Proportion surviving Proportion surviving 0.2 0.2 n=835 n=835 AB002340 n=875 n=875 p=0.588p=0.576 MLH1 p=0.019p=0.023 0.0 0.0 0 12 24 36 48 60 0 12 24 36 48 60 Survival time (months) Survival time (months)

AB002340 methylation Methylated Unmethylated MLH1 methylation Methylated Unmethylated

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Proportion surviving Proportion surviving 0.2 0.2 n=835 n=835 ITGA9 n=875n=875 n=875 p=0.184p=0.259 PLCD1 p=0.389p=0.301 0.0 0.0 0 12 24 36 48 60 0 12 24 36 48 60 Survival time (months) Survival time (months)

ITGA9 methylation Methylated Unmethylated PLCD1 methylation Methylated Unmethylated

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Proportion surviving Proportion surviving 0.2 0.2 Regional 3p22 n=875n=835 n=875n=835 DLEC1 p=0.209p=0.243 methylation p=0.08p=0.110 0.0 0.0 0 12 24 36 48 60 0 12 24 36 48 60 Survival time (months) Survival time (months)

DLEC1 methylation Methylated Unmethylated Regional 3p22 methylation +ve -ve

Figure 4.12 Kaplan-Meier plots showing colorectal cancer survival by methylation status of 3p22 genes individually and as a group Regional 3p22 methylation was defined as methylation of 3 of 5 3p22 genes. MLH1 methylation was significantly associated with improved disease-specific survival that was not independent of tumour stage in this dataset (data not shown).

148 4.3.9 Regional 3p22 methylation, BRAF V600E mutation, CIMP and the survival of individuals with sporadic colorectal cancer

Individuals with CRCs demonstrating the MSI phenotype are known to have an improved survival. In order to eliminate the confounding effect of MSI on survival, the prognostic significance of regional 3p22 methylation, BRAF V600E mutation, and CIMP were assessed in patients within the MSI and MSS subgroups respectively. Furthermore, BRAF mutation and CIMP+ve phenotype were previously found to be associated with a poorer outcome amongst individuals with sporadic MSS cancers (Samowitz et al., 2005b). However, that study found no association between BRAF mutation or CIMP+ve phenotype and the survival of individuals with MSI cancers.

In the present study, 92 individuals had MSI CRC of which 13 died of CRC and 11 died of other causes. Using Kaplan-Meier survival estimates, no relationship was found between BRAF mutation, CIMP and regional 3p22 methylation status, and survival in this small number of individuals with MSI cancer (data not shown).

A total of 769 individuals had MSS CRC, of which 527 were still alive at census date. Of the 242 deceased individuals, 194 died of CRC. CRCs that showed either BRAF mutation or CIMP+ve phenotype were associated with poorer survival in individuals with sporadic MSS cancer (both p<0.001, Log-rank test) (Figure 4.13 A, and B). These associations remained after adjusting for tumour stage (Table 4.11). No relationship was found between regional 3p22 methylation+ve and survival of individuals with this type of cancer (Figure 4.13 C). Individuals harbouring CIMP+ve CRCs with concomitant BRAF mutation demonstrated the poorest survival amongst those with MSS cancers before (Figure 4.13 D) and after adjusting for tumour stage (Table 4.11). Mean survival time of individuals with this feature was 15-19 months less than individuals without this feature (Table 4.12). Interestingly, there was a trend demonstrating better survival of individuals with regional 3p22 methylation +ve amongst this group of individuals with the worst prognosis. Within this subset of 26 individuals, the presence of regional 3p22 methylation increased mean survival time from 26 to 45 months (Table 4.12), although this relationship did not reach statistical significance (P=0.099, Log-rank test).

149 A B

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Proportion surviving Proportion surviving 0.2 0.2 n=741n=777 n=741n=777 p=0.001P<0.0001 p=0.001p=0.001 0.0 0.0 0 12 24 36 48 60 0 12 24 36 48 60 Survival time (months) Survival time (months)

BRAF V600E status Mutant Wild-type CIMP status +ve -ve C D

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Proportion surviving Proportion surviving 0.2 0.2 n=741n=777 n=741n=777 p=p=0.468 0.373 P<0.0001 0.0 0.0 p<0.0001 0 12 24 36 48 60 0 12 24 36 48 60 Survival time (months) Survival time (months)

Regional 3p22 methylation +ve -ve BRAF mutation/CIMP status BRAF mutant/CIMP+ve BRAF mutant/CIMP-ve BRAF wild-type/CIMP+ve BRAF wild-type/CIMP-ve Figure 4.13 Kaplan-Meier plots showing the association between various molecular features and prognosis of individuals with MSS cancers A, BRAF mutation status. B, CIMP status. C, regional 3p22 methylation status. D, stratification by BRAF mutation and CIMP status.

150 Colorectal cancer survival Molecular features of MSS colorectal N HR (95% CI)* P value cancers BRAF wild-type 703 1.00 (reference) BRAF mutant 38 2.45 (1.46-4.13) 0.001 CIMP -ve 684 1.00 (reference) CIMP+ve 57 2.01 (1.27-3.18) 0.003 BRAF wild-type/CIMP-ve 672 1.00 (reference) BRAF wild-type/CIMP+ve 31 1.32 (0.65-2.70) 0.445 BRAF mutant/CIMP-ve 12 1.42 (0.45-4.48) 0.554 BRAF mutant/CIMP +ve 26 3.01 (1.70-5.35) <0.0001 * Adjusted for tumour stage Table 4.11 Multivariate Cox proportional hazards regression model comparing the disease-specific risk of mortality in individuals with microsatellite stable colorectal cancers These individuals were stratified by BRAF mutation and CIMP status of their cancers. Tumours that showed either BRAF mutation or CIMP+ve phenotype were associated with poorer survival in individuals with this type of colorectal cancer, after adjusting for tumour stage.

Molecular features of MSS n Cancer Death Mean survival P value colorectal cancers (months) (Log-rank) BRAF wild-type/ CIMP-ve 672 167 51 BRAF wild-type/ CIMP+ve 31 8 48 BRAF mutant/CIMP-ve 12 4 52 BRAF mutant/CIMP+ve 26 15 33 <0.0001 Regional 3p22 methylation+ve 10 3 45 0.099 Regional 3p22 methylation -ve 16 10 26

Table 4.12 Kaplan-Meier estimates of mean survival in relation to various stratifications of MSS colorectal cancers by combinations of BRAF mutation, CIMP and regional 3p22 methylation status Within the group of individuals with BRAF mutant/CIMP+ve/MSS colorectal cancers (the group with the worst prognosis), the presence of regional 3p22 methylation increased mean survival time from 26 to 45 months, even though this did not reach statistical significant (P=0.099, Log-rank test).

151 4.4 Discussion

4.4.1 Methylation of 3p22 genes in sporadic colorectal cancers

Promoter hypermethylation of individual 3p22 genes was found in 8.8-18.3% of sporadic colorectal cancers indicating that these events are not commonplace in the development of this type of cancer. This observation is not surprising since methylation of 3p22 genes was previously found to be a specific feature of sporadic colorectal cancers with concomitant MSI and BRAF mutation. Indeed, only 11.2 and 14.6% of sporadic cancers in this study group were MSI and BRAF mutant respectively (Table 4.2), and these percentages were also consistent with those found in other study groups (Weisenberger et al., 2006; Ogino et al., 2007a).

Importantly, methylation of each 3p22 gene individually was strongly associated with that of four other genes within the 3p22 chromosomal region, suggesting a concomitant occurrence of methylation at these genes. However, hierarchical clustering analysis did not demonstrate a consistent methylation pattern of 3p22 genes between individual cancers but rather, suggested that methylation of these genes occurred stochastically. This result is consistent with that previously observed within the 2q14 region, whereby the methylation pattern of genes within this region was inconsistent between different colorectal cancers (Frigola et al., 2006). In light of this, an ordered spread of methylation from one gene to another seems unlikely to be a mechanism that gives rise to concomitant methylation of genes within a given chromosomal region.

Colorectal cancers in this study group were dichotomised into two groups, negative and positive for regional 3p22 methylation that demonstrate methylation at 0-2 and 3 of 5 3p22 genes respectively. This dichotomy is consistent with the criteria used by others (Toyota et al., 1999; Weisenberger et al., 2006) for categorising tumours into CIMP-ve and CIMP+ve respectively and certainly it allows an easier correlation between regional 3p22 methylation status and CIMP, as well as other clinicopathological/molecular features that are reported in a categorical manner. It is also reasonable given the findings in this study of a significant increase in the frequency of MSI or BRAF V600E mutant cancers that harboured methylation at 3 of 5 3p22 genes.

152 4.4.2 Methylation of 3p22 genes, BRAF mutation and MSI status

A major aim of the work in this chapter was to validate the association between methylation of genes across the 3p22 chromosomal region and BRAF V600E mutation as well as the MSI phenotype, as previously reported by Hitchins et al (Hitchins et al., 2007b). In the BRAF mutant group, the number of individuals harbouring 3 of 5 methylated 3p22 genes increased considerably while in the BRAF wild-type group, only five of 747 individuals demonstrated this feature. This result suggested that the criterion for defining the positivity of regional 3p22 methylation was reasonable in indicating those individuals who are likely to have BRAF mutation. Overall, univariate analyses showed a remarkably close association of methylation of 3p22 genes individually and as a group (regional 3p22 methylation) with BRAF mutation (Table 4.6 and 4.7), consistent with that found by Hitchins et al ((Hitchins et al., 2007b).

Another aim of the work in this chapter was to determine whether 3p22 methylation would reflect general long range epigenetic silencing, or whether it was associated with MSI since MLH1 is a key gene in this region. The presence of methylation at individual 3p22 genes and regional 3p22 methylation was strongly associated with the MSI phenotype. However, this study shows that methylation of multiple 3p22 genes is not MSI-specific as previously reported in a smaller cohort (Hitchins et al., 2007b). Approximately 12% of CRCs methylated at 3 3p22 loci were MSS, in the absence of MLH1 methylation. This finding supports a possible concept by which methylation developed stochastically within the 3p22 chromosomal domain and gives rise to the MSI phenotype only when MLH1 methylation is involved.

Overall, methylation at individual 3p22 genes seems to be more closely related to the presence of the BRAF mutation than the MSI phenotype (Figure 4.7). In addition, regional 3p22 methylation was significantly more frequent in BRAF mutant CRCs regardless of MSI status. These findings suggest a similar observation to that previously demonstrated between BRAF mutation and methylation of multiple genes in conjunction with CpG island methylator phenotype (CIMP) (Kambara et al., 2004b; Samowitz et al., 2005a; Weisenberger et al., 2006; Ogino et al., 2007a). Like CIMP phenotype, regional 3p22 methylation was associated with the majority of MSI colorectal cancers and a small proportion of their MSS counterparts. While extensive analysis would be required to prove this, regional 3p22 methylation markers may serve

153 as an alternative set of markers to characterise CIMP status. On a broader perspective, the CIMP+ve phenotype may be more appropriately conceptualised as the presence of areas of long-range methylation of multiple chromosomal regions, including but not limited to regions where current markers for CIMP such as NEUROG1 and SOCS1 reside (Figure 4.14).

Figure 4.14. Conventional and alternate conceptions of CpG island methylator phenotype (CIMP) that incorporate the phenomenon of regional methylation. Upper panel: CpG island methylator phenotype as defined conventionally by the concomitant methylation of multiple and discrete loci throughout the entire genome. Lower panel: An alternate conception of CIMP by long range methylation of genes encompassing the chromosomal regions including but not limited to regions where the current markers for CIMP status reside. Red dots represent current CIMP markers. Blue dots represent other commonly methylated genes that either flank the current CIMP markers or are located at chromosomal domains other than that in which current markers for CIMP reside. D1-D7 indicates different chromosomal domains.

This possibility and its functional importance in terms of changes in gene expression and tumourigenesis remain to be investigated. Genome-wide profiling of methylated markers and their functional significance in colorectal cancer would be useful in understanding whether CIMP is indeed a phenomenon that encompassed long range epigenetic silencing of numerous chromosomal regions and not merely the methylation of multiple and discrete loci throughout the entire genome. Another concept worth

154 investigating is whether long range epigenetic silencing would occur in the absence of CIMP at different chromosomal regions.

4.4.3 Methylation of 3p22 genes and common clinicopathological and molecular features of colorectal cancers

Of all 3p22 genes included in this study, MLH1 is the only gene that is well-documented in terms of its silencing via promoter hypermethylation and relationship with clinicopathological, as well as molecular features. This study confirmed previous findings that MLH1 methylation was found in approximately 80% of sporadic MSI CRCs (Herman et al., 1998; Wheeler et al., 2000; Miyakura et al., 2001). Consistent with existing literature (Miyakura et al., 2001; Young et al., 2001b), MLH1 methylation in this study was significantly associated with characteristics of MSI cancers including older age, female gender, right-sideness of cancers, mucinous histology, high grade, and the prominence of intraepithelial lymphocytes (Table 4.8). In light of its strong association with BRAF mutation, it is not surprising that MLH1 methylation was inversely correlated with KRAS mutation. Indeed, BRAF and KRAS mutations were almost mutually exclusive in this and other study groups (Rajagopalan et al., 2002; Chan et al., 2003), supporting the concept that one of these is sufficient in itself to cause the cancer-related upregulation of the MAPK signalling pathway, and indeed that mutations of both may be disadvantageous to the tumour cell.

MLH1 methylation demonstrated a considerable sensitivity (93%) and specificity (92%) in detecting CRCs with CIMP+ve phenotype and this supports the suggestion that methylation of this gene should be included as one of the markers in the panel for the characterisation of CIMP (Ogino et al., 2007a). MLH1-methylated cancers were similar to CIMP+ve CRCs in general, which tend to lack p53 nuclear accumulation. However, they differ from CIMP+ve CRCs in their association with lower tumour stages. In this study (data not shown), no relationship was observed between CIMP status and tumour stages, although CIMP+ve has previously been associated with higher tumour stages in MSS CRCs (Samowitz et al., 2005a). The positive correlation between MLH1 methylation and lower tumour stage is likely to be associated with MSI phenotype, as previously suggested (Samowitz et al., 2001; Malesci et al., 2007).

155 Methylation of other 3p22 genes, AB002340, ITGA9, PLCD1 and DLEC1 individually also correlated with most clinicopathological and molecular features that were strongly associated with CRCs showing MLH1 methylation (Table 4.8), reinforcing the notion that methylation of 3p22 genes occurs concomitantly with MLH1 in a considerable subset of sporadic CRCs. Notably, methylation of ITGA9 but not other 3p22 genes was significantly associated with lower tumour stages. While this association may be of clinical interest, it is unlikely to be independent of the function of MLH1 methylation. Hierarchical clustering analysis showed a stronger association between methylation of MLH1 and ITGA9, suggesting that these two genes are more likely to be methylated simultaneously. MLH1 and ITGA9 are located approximately 400 kb apart and are separated by two other genes, LRRFIP and GOLGA2 that are also associated with CpG rich promoters. Surprisingly, hypermethylation of CpG dinucleotides within LRRFIP and GOLGA2 promoter regions are not common in the cancers tested. A simplistic linear model failed to explain the close association between methylation of MLH1 and ITGA9. At a higher degree of complexity, the coiling of DNA may have placed MLH1 and ITGA9 in close vicinity to one another and thus allowed them to be targeted simultaneously by DNA methyltransferases. Alternatively, promoter regions of these genes may have a similar binding site that can accommodate a trans-acting factor that potentiates DNA methylation. While these possibility remains to be elucidated, it is noteworthy that certain transcription factors can bind to gene promoters, and recruit DNA methyltransferases, thereby facilitating the methylation of CpG dinucleotides (Di Croce et al., 2002; Brenner et al., 2005).

Clinicopathological and molecular characteristics typical of colorectal cancers showing MLH1 methylation, including lower tumour stage were also found in cancers with concomitant methylation at three or more 3p22 genes (regional 3p22 methylation +ve) (Table 4.9). In order to determine the unique features that give rise to this phenomenon, multivariate analysis was performed. Notably, CIMP and MSI status were the only significant predictors of regional 3p22 methylation. Upon stratification of CRCs by BRAF mutation, MSI and CIMP status, it became clear that regional 3p22 methylation +ve was almost exclusive to BRAF mutant cancers with CIMP+ve and/or MSI phenotype. More than 50% of CIMP+ve/MSS CRCs did not display regional 3p22 methylation. A possible explanation for this is that 3p22 genes are not as frequently methylated in some CIMP+ve/MSS CRCs as compared to the panel of markers that

156 characterises CIMP status. If this is the case, regional methylation would represent a less efficient panel of CIMP markers. However, it is noteworthy that some MSI cancers were regional 3p22 methylation +ve but CIMP-ve. This observation suggests that methylation patterns in CRCs can be stochastic and therefore, the 3p22 methylation panel may be useful to identify CIMP+ve cancers when another panel of markers failed. Regional 3p22 methylation +ve may characterise a subgroup of colorectal cancers with overlapping features of BRAF mutation, CIMP+ve and/or MSI phenotype.

4.4.4 Methylation of 3p22 genes and patient survival

In order to determine the clinical importance of 3p22 gene methylation, the methylation status of individual 3p22 genes as well as regional 3p22 methylation was correlated with the survival of individuals with sporadic CRCs (Figure 4.12). As expected, MLH1 methylation was significantly associated with the improved disease-specific survival of individuals in the entire study group. This result is consistent with the fact that MLH1 methylation and the subsequent silencing of this gene underlie MSI cancers which are generally reported to have a better prognosis (Gafa et al., 2000). At least two suggestions have been proposed for this observation. Firstly, MSI may possibly inhibit progression of cells and their ability to metatasize. However, this is contrary to what might be expected (Tomlinson and Bodmer, 1999) since microsatellite instability is often thought as a mechanism that drives cancer progression. Secondly, MSI CRCs are strongly associated with increased CD8+ and CD103+ lymphocytes infiltration (Guidoboni et al., 2001; Quinn et al., 2003; Baker et al., 2007), which may provide a barrier for cancer progression (Naito et al., 1998; Pages et al., 2005; Zlobec et al., 2007).

The present study reports for the first time that the methylation of ITGA9, PLCD1, and DLEC1 genes individually, and regional 3p22 methylation, demonstrate a trend towards better survival in individuals with colorectal cancer. Given that methylation of these genes is closely related to that of MLH1, this observation may be associated with the function of MLH1 methylation and the resultant MSI phenotype.

The impact of 3p22 gene methylation and other molecular features on survival may be more meaningful if studied within the group of individuals with MSS CRC. Our laboratory and others have previously reported a poorer survival for individuals with

157 MSS cancers that are also CIMP+ve (Ward et al., 2003; Samowitz et al., 2005b; Ogino et al., 2007b) or BRAF V600E mutant (Samowitz et al., 2005b). Samowitz also used multivariate analysis to show that BRAF mutation rather than CIMP+ve was contributing to this greater risk of death (Samowitz et al., 2005b). The present study confirms the significant impacts of BRAF mutation and CIMP+ve on the poorer survival of individuals with MSS cancers (Figure 4.13 A and B). However, contrary to the study by Samowitz et al., 2005, this study has shown that BRAF mutation alone is not responsible for the poorer survival in individuals with MSS CRCs. Rather, it is the presence of both BRAF mutation and CIMP+ve that predicts a worse outcome for individuals with MSS cancers (Figure 4.13 D), and this association remained after adjusting for tumour stage, itself a powerful predictor of outcome.

Nevertheless, it is important to note that CIMP status in this study was characterised using the newly described panel of CIMP markers (Weisenberger et al., 2006) which differs from the conventional set of markers used by Samowitz et al, 2005. It is conceivable that the prognostic significance of CIMP may be dependent on the set of markers that is used to characterise this feature. While a consensus for CIMP markers remains to be elucidated, methylation of certain genes may be more important in terms of prognostic purposes and this factor should be taken into account, in the consideration of a practical set of CIMP markers.

Importantly, regional 3p22 methylation +ve was not significantly associated with the poorer survival of individuals with MSS cancers (Figure 4.13 C), suggesting that this phenomenon may have a biological effect distinct from those attributable to BRAF mutation and CIMP status. Indeed, regional 3p22 methylation +ve predicted better survival amongst individuals with MSS cancers with concomitant BRAF mutant and CIMP+ve phenotypes, a group that demonstrated the poorest prognosis overall (Table 4.12). However, there were only 26 individuals in this group with poorest survival, and the impact of regional 3p22 methylation on this subgroup needs to be verified in a larger sample set.

4.4.5 Summary

1. A stochastic pattern of methylation across the 3p22 chromosomal region was observed in sporadic CRCs, inconsistent with the concept that methylation

158 spreads from one gene promoter to another in a particular direction. This stochastic pattern of methylation also complicates the detection of any particular point which may be the site of initiation of the methylation process. Nevertheless, it is clear that methylation of each 3p22 gene is closely associated with the same event at the other four loci examined.

2. The concomitant methylation of multiple genes within the 3p22 chromosomal region is strongly associated with BRAF mutation and occurs more frequently in conjunction with CIMP+ve and MSI phenotype. This event is not MSI specific, suggesting it is more closely related to the CIMP+ve phenotype. Due to the stochastic pattern of DNA hypermethylation in colorectal cancers, this panel of 3p22 markers may serve as an additional marker for CIMP status.

3. Methylation of 3 of 5 3p22 genes (regional 3p22 methylation) shared overlapping clinicopathological and molecular features with cancers that harbour BRAF mutation or CIMP+ve or MSI phenotype. It is to be noted however that cancers with regional 3p22 methylation differed from BRAF and CIMP+ve cancers in that they tended to be of lower tumour stage (p<0.05).

4. Regional 3p22 methylation may be useful in predicting individuals with superior outcomes among those with the worst prognosis overall; those with BRAF V600E mutant and CIMP+ve MSS colorectal cancers. This study suggests that regional 3p22 methylation may negate the poorer outcome in individuals with cancers of the MSS, BRAF mutant, CIMP+ve phenotype. However, this result requires confirmation in larger studies.

5. This is the first large population-based study that has investigated the pattern of clustered DNA methylation within a large chromosomal domain in the development of sporadic colorectal cancer. Using the 3p22 chromosomal domain as an example, this study has provided insights into the similarity between the concomitant methylation of gene promoters at a given chromosomal domain, and genome-wide methylation as characterised by the CIMP+ve phenotype. Nevertheless, it is evident through this study that a given pattern of DNA methylation may be useful in predicting the rate of survival of individuals with colorectal cancer, within a group that share similar clinicopathological features.

159

CHAPTER 5

Methylation of 3p22 genes and BRAF V600E mutation in apparently normal colonic mucosa adjacent to

colorectal cancers

160

5.1 Introduction

In Chapter 4 of this thesis, it has been shown that the CpG island methylation within contiguous 3p22 gene promoters was strongly associated with the oncogenic V600E mutation of BRAF in the tumours of individuals with sporadic CRC. However, the sequence in which these events occur, if any, remains unknown. This knowledge may be important in better understanding the interaction between epigenetic and genetic events in the development of CRC. Colorectal cancers are known to progress via the clonal expansion of a single cell in the colonic or rectal epithelium. Thus, any genetic or epigenetic event that leads to the development of CRC may be detected early in apparently normal colorectal mucosa, or in other non-malignant lesions such as adenomas or other polyps.

Many studies have found aberrant hypermethylation of genes in normal colonic mucosa (Miyakura et al., 2001; Nakagawa et al., 2001; Deng et al., 2002; Furukawa et al., 2002; Shen et al., 2005; Minoo et al., 2006; Kawakami et al., 2006) and adenomas (Kambara et al., 2004b; Derks et al., 2006; Minoo et al., 2006) adjacent to tumours, suggesting that this epigenetic event may occur early in CRC development. In terms of regional methylation at 2q14, Frigola et al. (2006) found low-level methylation in normal colonic mucosa samples adjacent to CRCs with higher levels of regional methylation (Frigola et al., 2006). They suggested that DNA methylation at such low levels may either be attributable to minor cancer cell contamination or alternatively that it may represent “seeds” of methylation that promote subsequent hypermethylation. A previous study by our laboratory failed to identify methylation at the promoter of several genes across the 3p22 region including AB002340, ITGA9, MLH1, PLCD1 and DLEC1, in normal colonic mucosa adjacent to cancers that showed hypermethylation of these genes (Hitchins et al., 2007b). However, this data was based on COBRA, a relatively insensitive assay not well suited to detecting low levels of methylation.

Less information is available on the occurrence of BRAF mutation in normal colonic mucosa. To date, only one study has reported the presence of the BRAF V600E mutation in the normal colonic mucosa of an individual with sporadic CRC (Kadiyska et al., 2007). However, BRAF mutations have been identified in pre-malignant lesions such as aberrant crypt foci (Beach et al., 2005; Rosenberg et al., 2007 ) and serrated polyps in the colon or rectum (Chan et al., 2003; Yang et al., 2004; Kambara et al.,

161 2004b; Minoo et al., 2006) as well as in non-gastrointestinal lesions, most notably cutaneous melanocytic naevi (Pollock et al., 2003; Yazdi et al., 2003; Poynter et al., 2006).

Using sensitive real-time PCR assays, work in this chapter aims to determine the frequency of the BRAF V600E mutation as well as methylation at 3p22 genes in the normal colonic mucosa samples from individuals with sporadic CRC. The study group for this chapter included individuals in whom MLH1 methylation had been identified within their normal colonic mucosa (discussed in Chapter 3), and who were thus considered more likely to have concomitant methylation of other 3p22 genes. It also included a small group of individuals with sporadic CRC, in whom concomitant methylation of 3p22 genes and the V600E mutation of BRAF had been identified in their tumour samples (discussed in Chapter 4). The study group was used to identify clinicopathological correlates of BRAF mutation and extensive methylation of 3p22 genes within normal colonic mucosa samples.

Findings from this chapter will provide insights into the chronology of BRAF mutation and regional 3p22 methylation in the development of sporadic CRCs and better define the relationship between the two. This study may also facilitate the identification of clinicopathological features that are associated with the presence of BRAF mutation and regional 3p22 methylation in the normal colonic mucosa of individuals with sporadic CRC.

162

5.2 Methods

5.2.1 Normal colonic mucosa samples

Normal colonic mucosa samples were procured as described in section 2.2.1 from individuals undergoing curative surgical resection for colorectal cancer at St.Vincent’s Hospital, Sydney. The collection and subsequent DNA extraction of these normal colonic mucosa samples was detailed in section 2.2.1. Normal colonic mucosa samples included in the present study are shown in Table 5.1. In a pilot study, seven MLH1- methylated normal colonic mucosa samples were selected on the basis that they may be more susceptible to the methylation of other 3p22 genes (detailed in Chapter 3). For each of these seven cases, matched MLH1 methylation-negative normal colonic mucosa controls were selected from individuals with MSS CRCs who were matched for sex and age (within ±5 years). An additional study was later conducted on the normal colonic mucosa samples from 13 individuals (mean age 76.4±9.0; range 63-92 years), selected at random, who had CRCs with the V600E mutation of BRAF and concomitant methylation of 3 of 5 3p22 genes (regional 3p22 methylation), as determined in Chapter 4. These cases were compared to normal colonic mucosa samples from individuals matched for sex and age (±5 years), in whom no concomitant BRAF mutation or regional 3p22 methylation was found in their cancers (mean age, 75.2±10.3; range 58-90 years). As controls, biopsy samples of normal mucosa were collected from 21 individuals without cancer as described previously in Chapter 3 (mean age, 58.4±17.5; range, 33-82 years). The details of other colorectal polyps in individuals with CRCs were based on the histopathological report as described in section 2.2.3. This study was approved by Human Research Ethics Committee, St. Vincent’s Hospital, and informed consent was obtained from all participating individuals.

163 Pilot study Test samples Matched controls (MLH1-methylation +ve NCM) (MLH1 methylation –ve NCM) Sample Age Sex Sample Age Sex N1 80 M N8 75 M N2 51 F N9 46 F N3 89 F N10 85 F N4 68 M N11 68 M N5 84 F N12 82 F N6 79 F N13 79 F N7 79 M N14 75 M

Extended study Test samples Matched controls (NCM adjacent to BRAF V600E mutant and (NCM adjacent to BRAF wild-type and RM- RM+ve colorectal cancers) ve colorectal cancers) Sample Age Sex Sample Age Sex N15 81 F N28 82 F N16 74 M N29 74 M N17 88 M N30 90 M N18 79 F N31 83 F N19 92 F N32 87 F N20 63 M N33 58 M N21 64 M N34 59 M N22 85 F N35 84 F N23 68 M N36 65 M N24 75 M N37 71 M N25 82 F N38 81 F N26 72 F N39 72 F N27 71 M N40 72 M

Table 5.1 Normal colonic mucosa samples included in the present study Each test normal colonic mucosa sample (left) and its matched control (right) included in the respective pilot (upper panel) or extended studies (lower panel) are shown. In the pilot study, test samples (N1-N7) were selected on the basis that MLH1 methylation had previously been identified in these samples, with these samples subsequently considered more likely to have concomitant methylation of other 3p22 genes. For each of these seven cases, matched MLH1 methylation-negative normal colonic mucosa controls were selected from individuals with MSS colorectal cancers (N8-14). In the extended study, test normal colonic mucosa samples (N15-N27) were selected at random from 13 individuals who had colorectal cancers with the V600E mutation of BRAF and regional 3p22 methylation. These cases were compared to normal colonic mucosa samples from individuals in whom no concomitant BRAF mutation or regional 3p22 methylation was found in their cancers (N28-N40). Each test normal colonic mucosa sample was matched for sex and age (±5 years) with its normal mucosa control. NCM, normal colonic mucosa; +ve, positive; -ve, negative; RM, regional 3p22 methylation; F, female; M, male.

164 5.2.2 Methylation profiling of 3p22 genes and BRAF mutation analysis

The methylation profiles of 3p22 genes were assessed in normal colonic mucosa samples using the quantitative methylation specific PCR (qMSP) assay as described in Section 2.2.12. Confirmation of methylation status was carried out in selected samples using clonal bisulfite sequencing as described in Section 2.2.16. BRAF mutation analysis was performed by allele-specific real-time PCR as described in Section 2.2.18.

5.2.3 Statistical analyses

The values of percentage of methylated reference (PMR) detected using the qMSP assays were treated as a continuous variable and since the data were not normally distributed, the Mann-Whitney-U test was used to compare the differences in the levels of 3p22 gene methylation between groups of normal colonic mucosa samples, stratified by BRAF mutation status. The univariate analysis for comparisons between clinicopathological and molecular features was performed as described in section 2.2.20. All P values <0.05 were considered significant. All data were analysed using SPSS V15.0 (SPSS Inc., Chicago, IL)

165

5.3 Results

5.3.1 Methylation of 3p22 genes in normal colonic mucosa

The methylation profiles of 3p22 genes in the normal colonic mucosa samples included in the pilot study and matched colorectal cancers (as assessed in Chapter 4) are shown in Figure 5.1.

Individuals with MLH1 methylation-positive normal colonic mucosa samples

Control subjects with MLH1 methylation-negative normal colonic mucosa samples

Figure 5.1 The presence of regional 3p22 methylation in normal colonic mucosa samples Upper panel: In each row, the methylation status of five 3p22 genes is shown for either the colorectal cancer (C1-C7) or matched normal colonic mucosa sample (N1-N7) from individuals with sporadic colorectal cancer cases. MLH1-methylation had previously been found in the normal colonic mucosa samples (N1-N7). Lower panel: The methylation profile of cancers (C8-C14) and corresponding normal colonic mucosa samples (N8-N14) in cases matched for age and sex (±5 years) in which no MLH1- methylation had previously been detected in paired normal colonic mucosa samples (N8-N14). For each individual (1-14), the age at colorectal cancer resection, gender and BRAF V600E status of their tumour are also shown. Methylation: Black box, PMR >4.0-100.0; Grey box, PMR 0.5-4.0; Light grey box, PMR 0.1-0.5; White box, no detectable methylation ; F, female; M, male.

166 Overall, CpG island methylation at additional 3p22 genes was more common in the normal colonic mucosa samples of individuals who had low levels of concomitant MLH1 methylation (Figure 5.1). In the majority of cases, methylation was detected at low levels (PMR value between 0.1 and 4). When the methylation status of 3p22 genes was assessed as a group, low levels of methylation were detected at 3 of 5 3p22 genes in the normal colonic mucosa samples, in four of seven (57%) individuals who had low level MLH1 methylation in their normal mucosa samples (N1-N4) (Figure 5.1). Higher levels of methylation (PMR >4) had previously been detected at 3 of 5 3p22 genes (regional 3p22 methylation) in conjunction with V600E mutation of BRAF in the matched CRCs from each of these four individuals (C1-C4). No methylation of 3 of 5 3p22 genes was detected in the normal colonic mucosa samples (N5-N14) of individuals with CRCs in which regional 3p22 methylation and concomitant V600E mutation of BRAF were absent (C5-C14) (Figure 5.1). These data suggests that low level methylation of 3p22 genes as a group was more common in the normal colonic mucosa samples of individuals who had cancers showing concomitant V600E mutation of BRAF and extensive CpG island methylation of gene promoters at this chromosomal region.

The pilot study was extended to determine the frequency of low level methylation of 3p22 gene promoters in the normal colonic mucosa samples from 13 additional individuals who had cancers showing regional 3p22 methylation and concomitant V600E mutation of BRAF (as assessed in Chapter 4). Results of this extended study are shown in Figure 5.2. Only three of these 13 individuals showed methylation at one or two 3p22 genes, and in these cases (N15, N16 and N17), methylation was seen only at the ITGA9 and/or PLCD1 promoters. None of these 13 additional individuals had low level methylation of 3 of 5 3p22 genes in their normal mucosa (N15-N27) (Figure 5.2), suggesting that low levels of regional 3p22 methylation are uncommon in the normally appearing colonic mucosa, even when that mucosa is adjacent to a cancer showing high levels of regional 3p22 methylation. As expected, methylation of 3p22 genes individually or as a group was rare in the normal colonic mucosa samples from additional individuals whose cancer was concomitantly BRAF wild-type and devoid of regional 3p22 methylation (N28-N40) (Figure 5.2), or from individuals without neoplasia (N41-N61) (Figure 5.3).

167

Figure 5.2 The methylation profiles of 3p22 genes in normal colonic mucosa samples of individuals with colorectal cancer Each row shows the methylation profile of five 3p22 genes in the cancer (C) or matched normal mucosa sample (N) from individuals who showed (15-27) and did not show (28-40) concomitant regional 3p22 methylation (methylated at 3 of 5 3p22 genes) and V600E mutation of BRAF in their cancers. Each sample within the C/N15-27 group was matched for age (±5) and sex with a sample from the C/N28-40 group. The age at colorectal cancer resection, gender and cancer BRAF status are also shown for each individual (15-40). Methylation: Black box, PMR >4.0-100.0; Grey box, PMR 0.5-4.0; Light grey box, PMR 0.1-0.5; White box, no detectable methylation ; F, female; M, male.

168

Figure 5.3 The methylation profiles of 3p22 genes of normal colonic mucosa samples from control subjects without colorectal neoplasia Each row shows the methylation status of each of the five 3p22 genes in the normal colonic mucosa samples from individuals without neoplasia (N41-N61), as well as their age and sex. Methylation: Grey box, PMR 0.5-4.0; Light grey box, PMR 0.1-0.5; White box, no detectable methylation; F, female; M, male.

5.3.1.1 Confirmation of presence of methylation in normal colonic mucosa In order to confirm the presence of methylation in selected samples displaying PMR value 0.1, clonal bisulfite sequencing was performed on the PCR products obtained using the qMSP assays. Dense methylation of CpG sites was found within the majority of individual clones representing alleles sequenced (Figure 5.4-5.7). However, there was a mosaic pattern of methylation in some normal colonic mucosa samples, particularly within the promoter regions of ITGA9 and PLCD1 (Figure 5.5 and 5.6). In some alleles, less than 50% of CpG sites were methylated. This finding may indicate the occurrence of seeding methylation in some normal colonic mucosa samples, and this trace of methylation may not be sufficient to cause gene inactivation. This seed of methylation

169 may subsequently progress to a dense or fully methylated pattern, resulting in gene silencing.

N1 N2 1 2 3 4 5 1 2 3 4 5

80-100% of CpG sites were methylated 80-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 98.3% Total methylation = 96.7%

N3 N4 1 2 3 4 5 1 2 3 4 5

60-100% of CpG sites were methylated 80-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 93.3% Total methylation = 98.3%

Figure 5.4 Methylation at individual CpG dinucleotides of the AB002340 promoter region in normal colonic mucosa samples Patterns of methylation at individual CpG dinucletides were determined by cloning and sequencing of PCR products generated by the qMSP assay. The four normal colonic mucosa samples (N1-N4, labeled according to Figure 5.1) were positive for methylation using the qMSP assay. For each sample, methylation patterns for 10-12 clones representing alleles are shown. Each row represents an individual clone that was sequenced. Circles represent each of the five CpG sites (1-5), positioned between the binding regions of the qMSP primers (indicated by black arrows). Black and white circles represent methylated and unmethylated CpG sites respectively. This region corresponds to -36515 bp to -36370 bp relative to the transcriptional start site of the AB002340 gene. Total methylation was calculated as a percentage of methylated CpGs over total CpG sites. Over 90% of total methylation was observed in each of the four samples, in which 60-100% of CpG sites were methylated within individual clones.

170 N1 N2 1 2 34 5 6 7 8 9 10 11 12 1 23 4 5 6 7 98 121110

42-100% of CpG sites were methylated 8-92% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 77.1% Total methylation = 67.3%

N3 1 223 4 5 6 778891210 11 N4 1 3 4 5 6 91210 11

67-100% of CpG sites were methylated 75-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 88.2% Total methylation = 90.3% N15 N22 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

92-100% of CpG sites were methylated 92-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 95.8% Total methylation = 93.9%

Figure 5.5 Methylation at individual CpG dinucleotides of the ITGA9 promoter region in normal colonic mucosa samples Patterns of methylation were determined by cloning and sequencing of PCR products generated by the qMSP assay. The six samples (N1-N4, N15 and N22) represent normal colonic mucosa samples that were positive for methylation using the qMSP assay. For each sample, methylation patterns for 10-12 clones, as represented by individual rows, are shown. Circles represent all 12 CpG sites (1-12, positioned between the binding regions of the qMSP primers (indicated by black arrows). Black and white circles represent methylated and unmethylated CpG sites respectively. This region corresponds to -196 bp to-88 bp relative to the transcriptional start site of the ITGA9 gene. Total methylation in the six normal colonic mucosa samples varied from 67.3-95.8%, While the majority of individual clones sequenced showed dense methylation of >70% CpG sites, several clones were methylated at <50% CpG sites.

171 N1 1 2 3 4 5 6 7 8 9 1011 N2 1 2 3 4 5 6 7891011

55-100% of CpG sites were methylated 9-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 84.8% Total methylation = 80.9%

N3 1 2 3 4 5 6 7788991011 N4 1 2 3 4 5 6 1011

64-82% of CpG sites were methylated 18-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 75.7% Total methylation = 87.3% N20 N25 1 2 3 4 5 6 7 8 9 1011 1 2 3 4 5 6 7 8 9 1011

27-100% of CpG sites were methylated 27-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 69.0% Total methylation = 68.2%

Figure 5.6 Methylation at individual CpG dinucleotides of the PLCD1 promoter region in normal colonic mucosa samples Patterns of methylation were determined by cloning and sequencing of PCR products generated by the qMSP assay. The six samples (N1-N4, N20 and N25) represent normal colonic mucosa samples that were positive for methylation using the qMSP assay. For each sample, methylation patterns for 10-12 clones, as represented by individual rows, are shown. Circles represent all 11 CpG sites (1-11), positioned between the binding regions of the qMSP primers (indicated by black arrows). Black and white circles represent methylated and unmethylated CpG sites respectively. This region corresponds to -475 bp to - 341bp relative to the transcriptional start site of the PLCD1 gene. Total methylation in the six normal colonic mucosa samples varied from 68.2-87.3%, with the majority of individual clones sequenced showing methylation at >60% CpG sites. Several clones were mosaically methylated at <50% CpG sites.

172 N1 N2 1 2 3 4 5 6 1 2 3 4 5 6

83-100% of CpG sites were methylated 83-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 92.4% Total methylation = 97.0%

N3 1 2 3 4 5 6

67-100% of CpG sites were methylated in individual clones representing alleles Total methylation = 91.7%

Figure 5.7 Methylation at individual CpG dinucleotides of the DLEC1 promoter region in normal colonic mucosa samples PCR products generated by the qMSP assay for the three normal colonic mucosa samples positive for DLEC1 methylation (N1-N3, numbered according to Figure 5.1) were subjected to clonal bisulfite sequencing. For each sample, methylation patterns for 10-12 clones, as represented by individual rows, are shown. Circles represent each of the six CpG sites (1-6), positioned between the binding regions of the qMSP primers (indicated by black arrows). Black and white circles represent methylated and unmethylated CpG sites respectively. This region corresponds to +200 bp to +302 bp relative to the transcriptional start site of the DLEC1 gene. Over 90% of total methylation was observed in each of the three samples, in which 67-100% of CpG sites were methylated in individual clones.

5.3.2 BRAF mutation in the normal colonic mucosa

Using the allele-specific real-time PCR, BRAF mutation analysis was performed on each normal colonic mucosa sample included in the present study group. This method relies on the specificity of a set of PCR primers to detect BRAF V600E mutant in the background of BRAF wild-type alleles. As mentioned in section 2.2.18, samples were considered BRAF V600E mutant if fluorescent signal was detected using both the wild

173 type and mutant specific primer sets. Samples were designated as BRAF wild type if fluorescent signal was only detected using the wild type but not the mutant specific primers. At times, non-specific binding of primers for the mutant reaction may occur and lead to the detection of low false-positive signals. Given that this method had previously detected a cycle difference (Ct) of 11.24 to between the wild-type and mutant reactions in the peripheral blood of healthy individuals (data not shown), a sample was called positive for BRAF V600E mutation only when the Ct was <11. This cutoff is equivalent to the lower detection limit of one mutant in 2048 wild-type alleles (or 0.05%), as calculated using the formula 2Ct = fold difference (Sonia Gowan, personal communication).

Among the 18 individuals who had BRAF V600E mutation present in their cancers (C1- C7, C15-C27), four had the same mutation in their matched normal colonic mucosa (N1-N4). Ct values differences between the mutant and wild-type reactions (Ct) in samples N1-N4 ranged from 6.7–9.9 (mean Ct = 8.5), which were greater than that found in V600E mutant cancers in this study group (mean Ct = 5.6) (data not shown). Therefore, lower levels of BRAF V600E mutations were detected in samples N1-N4 than that in the corresponding cancers. No BRAF V600E mutations were found in the normal colonic mucosa samples of the 22 individuals who had wild type BRAF in their cancers (N5-N7, N28-N40) (range Ct = 12.7 - ). Likewise, BRAF mutations were absent in the normal colonic mucosa samples from the 21 individuals without neoplasia (N41-N61) (Ct = ). Examples of results from the BRAF mutation analysis of CRC and normal colonic mucosa samples are shown in Figure 5.8.

174 BRAF V600E mutant cancer (C3) BRAF V600E mutant NCM (N3)

Ct?Ct = 3.2 ?Ct = 6.5

Cycle Cycle

BRAF wild-type NCM (N18) BRAF wild-type NCM (N40)

?Ct Ct = 13.9 Ct?Ct =?

Cycle Cycle

Figure 5.8 Real-time PCR amplification plots for the detection of the BRAF V600E mutation in cancer and normal mucosa samples Typical real-time PCR plots for the BRAF mutant (red) and wild-type reactions (blue) are shown for four different scenarios observed using this assay. The upper panel shows typical results for a BRAF V600E mutant colorectal cancer (C3) (left) and normal colonic mucosa sample (N3) (right) respectively. The lower panel shows typical results for BRAF wild-type NCM samples as represented by samples N18 and N40. A sample was called positive for the mutation when the amplification plot for the BRAF mutant reaction cut across the threshold (orange line), in conjunction with a difference in Ct values (Ct) of 11 between the mutant and wild-type reactions. NCM, normal colonic mucosa.

175 5.3.3 BRAF mutation is associated with a higher frequency and intensity of methylation at 3p22 genes in normal colonic mucosa samples

The presence of BRAF V600E mutation was significantly correlated with the methylation of 3p22 genes individually or as a group in the normal colonic mucosa samples of individuals with sporadic CRCs (Table 5.2). Notably, each of the four individuals who had a low level of the BRAF V600E mutation in their normal colonic mucosa also showed low levels of methylation at three or more of the five 3p22 genes in these same samples. This finding suggests that BRAF V600E may be associated with an increase in the frequency of methylated 3p22 genes within normal colonic mucosa samples. Nevertheless, methylation at one or two individual 3p22 genes was present in some normal colonic mucosa samples in the absence of BRAF mutation (Table 5.2).

There was also a significantly higher intensity of methylation at each 3p22 locus in the normal colonic mucosa samples harbouring BRAF V600E mutations compared to normal colonic mucosa samples without the mutation (all P <0.001, Figure 5.9). The findings here indicated that V600E mutation of BRAF is strongly associated with both the frequency and intensity of methylation at 3p22 gene promoters.

176 BRAF V600E mutation status Methylation status Mutant Wild-type P Value (n = 4) (n =36) AB002340 <0.0001 Methylated 4 (100%) 0 (0%) Unmethylated 0 (0%) 36 (100%)

MLH1 <0.0001 Methylated 4 (100%) 3 (8%) Unmethylated 0 (0%) 33 (92%)

ITGA9 <0.0001 Methylated 4 (100%) 3 (8%) Unmethylated 0 (0%) 33 (92%)

PLCD1 0.001 Methylated 4 (100%) 4 (11%) Unmethylated 0 (0%) 32 (89%)

DLEC1 <0.0001 Methylated 3 (100%) 0 (11%) Unmethylated 1 (0%) 36 (89%)

Multiple loci <0.0001 3 3p22 genes methylated 4 (100%) 0 (0%) <3 3p22 genes methylated 0 (0%) 36 (100%)

0.002 1 3p22 genes methylated 4 (100%) 6 (17%) <1 3p22 genes methylated 0 (0%) 30 (83%)

Table 5.2 The associations between BRAF V600E mutation and the presence of methylation at 3p22 genes, either individually or as a group, in normal colonic mucosa samples of individuals with colorectal cancer P values were determined using Fisher’s exact tests. DNA methylation was considered present at individual 3p22 genes when PMR >0.1. The presence of BRAF V600E mutation was significantly associated with the presence of methylation at 3p22 genes individually or as a group in the normal colonic mucosa samples of individuals with colorectal cancer.

177

Figure 5.9 Comparison of the median and range of methylation level (PMR value) at 3p22 loci in normal colonic mucosa with or without BRAF V600E mutation The number beside a given circle (if present) indicates the total number of samples with a similar PMR value. Significantly higher median PMR was observed at each locus in colonic mucosa harbouring BRAF mutation than in those that were BRAF wild-type (P<0.001, Mann-Whitney U test). NCM, normal colonic mucosa.

178 5.3.4 Clinicopathological characteristics of individuals with BRAF V600E mutant and regional 3p22 methylation-positive normal colonic mucosa

The clinical characteristics of individuals and the pathological characteristics of cancers that were BRAF mutant and regional 3p22 methylation +ve, in whom normal colonic mucosa demonstrated concomitant BRAF mutation and low level methylation at 3 of 5 3p22 genes are shown in Table 5.3. These clinicopathological characteristics were compared with those of other individuals who had BRAF mutant and regional methylation +ve cancers but in whom no concomitant BRAF mutation and low level methylation at 3 of 5 3p22 genes were found in paired normal colonic mucosa samples. No significant difference was found in the clinicopathological characteristics assessed between the two groups (Table 5.4).

NCM Sex Age Location TNM Tumour Tumour Tumour Associated Stage Grade MSI CIMP polyps status status N1 M 80 Ascending II High grade MSI CIMP+ve 2 conventional colon adenomas, 3 HPs, one serrated adenoma N2 F 51 Ascending III Low grade MSI CIMP+ve Multiple colon conventional adenomas and HPs N3 F 89 Ascending III High grade MSI CIMP+ve 3 HPs, 2 colon serrated adenomas N4 M 68 Descending III Low grade MSI CIMP+ve 4 HPs colon

Table 5.3 Clinicopathological details of the four individuals harbouring concomitant BRAF mutation and methylation of 3 of 5 3p22 genes in their normal colonic mucosa samples Conventional adenomas comprise tubular, tubulovillus and villus adenomas. NCM, normal colonic mucosa; M, male; F, female; MSI, microsatellite instability; HP, hyperplastic polyp; CIMP+ve, positive for CpG Island methylator phenotype; CIMP-ve, negative for CpG island methylator phenotype.

179 Normal mucosa showing concomitant BRAF mutation and/or methylation of 3 of 5 3p22 genes Yes No P Value (n = 4) (n =14) Mean age (± SD) 72.0±16.4 77.0±8.8 0.633

Sex Male 2 (50%) 8 (57%) 1.000 Female 2 (50%) 6(43%)

Tumour site 1.000 Right-sided 3 (81%) 11 (21%) Left-sided 1 (19%) 3 (79%)

Tumour stage 0.119 I 0 (0%) 1 (7%) II 1 (25%) 9 (64%) III 3 (75%) 2 (14%) IV 0 (0%) 2 (14%)

Tumour grade 1.000 Low grade 2 (50%) 8 (57%) High grade 2 (50%) 6 (43%)

Tumour microsatellite status MSI 4 (100%) 14 (100%) NC MSS 0 (0%) 0 (0%)

Tumour CIMP status CIMP+ve 4 (100%) 14 (0%) NC CIMP-ve 0 (0%) 0 (0%)

Table 5.4 Clinical characteristics of individuals with BRAF V600E mutant and regional methylation-positive cancers as well as the pathological features of their cancers stratified by the presence of concomitant BRAF mutation and methylation of 3 of 5 3p22 genes in their normal mucosa. The P value for the comparison of mean age between groups was obtained using a Mann-Whitney U test. P values are shown for Fisher’s exact test for all other characteristics. No significant difference was found in the clinicopathological characteristics assessed between the two groups. SD, standard deviation; MSI, microsatellite stable; MSS, microsatellite unstable; CIMP+ve, positive for CpG island methylator phenotype; CIMP-ve, negative for CpG island methylator phenotype; NC, not computed because one of the variables assessed in the statistical test is a constant.

180 5.3.5 Concomitant BRAF V600E mutation and regional 3p22 methylation in normal colonic mucosa is associated with a higher frequency of neoplastic polyps in the colorectum

The frequency of neoplastic polyps in association with the presence of methylation of at least one 3p22 gene, or the presence of concomitant V600E mutation of BRAF and methylation of 3 of 5 3p22 genes was investigated in normal colonic mucosa samples. The number of serrated polyps and the total number of polyps overall (number of serrated and conventional polyps combined) were significantly increased in association with the presence of concomitant V600E mutation of BRAF and methylation of 3 of 5 3p22 genes in the normal colonic mucosa samples (Table 5.5).

Normal mucosa showing concomitant V600E mutation of BRAF and methylation of 3 of 5 3p22 genes Yes (n=4) No (n=36) P value Conventional adenomas 0.574 Median 1 0 Range 0-3 0-8

Serrated polyps (serrated 0.006 adenomas and hyperplastic polyps) Median 4 0 Range 3-5 0-20

Total polyps (conventional 0.029 and serrated polyps) Median 5 1 Range 4-6 0-20

Table 5.5 The frequencies of different types of neoplastic polyps in individuals with sporadic colorectal cancer stratified by the presence of concomitant V600E mutation of BRAF and regional 3p22 methylation in their normal mucosa samples The median frequency and range of each type of polyps are shown for the two groups. Statistical significance was defined using Mann-Whitney U test. Significant P values are highlighted in bold. The number of serrated polyps and the total number of polyps overall were significantly increased in association with the presence of concomitant V600E mutation of BRAF and methylation of 3 of 5 3p22 genes in the normal colonic mucosa samples.

Indeed, all of the four individuals harbouring concomitant BRAF mutation and methylation of 3 of 5 3p22 genes in their normal colonic mucosa samples had colorectal polyps with a serrated morphology (Table 5.3). No association was found between the methylation of at least one 3p22 gene in normal colonic mucosa samples

181 and the frequency of neoplastic colorectal polyps in individuals with CRC (data not shown).

Concomitant BRAF mutation and low level methylation at 3 of 5 3p22 genes may be a molecular feature that is more common in the colonic mucosa of individuals who have multiple serrated polyps contemporaneous with their cancers.

182 5.4 Discussion

Low level methylation of various human genes including APC, MLH1, and MGMT has previously been found in the normal mucosa adjacent to colorectal cancers (Miyakura et al., 2001; Nakagawa et al., 2001; Furukawa et al., 2002; Bai et al., 2004; Lee et al., 2004; Shen et al., 2005; Tominaga et al., 2005; Kawakami et al., 2006; Minoo et al., 2006; Ye et al., 2006; Iacopetta et al., 2007). In addition, low level methylation of contiguous genes within the same chromosomal region has been reported in the normal mucosa of individuals with CRCs (Frigola et al., 2006). The present study extends these findings by others and shows for the first time that genes flanking MLH1 including AB002340, ITGA9, PLCD1 and DLEC1 can also be methylated at low levels in the normal colonic mucosa. In a considerable number of cases, the methylation patterns at individual alleles were mosaic and this may represent an intermediate state that has yet to reach the hypermethylated state (methylated at >80% CpG sites within a given allele).

An explanation for the presence of low level or mosaic methylation of 3p22 genes in the normal colonic mucosa can only be speculative. Conceptually, such low level or mosaic methylation may represent a field defect that underlies the development of additional colorectal neoplasms in some patients. It has been suggested that the low level and mosaic patterns of methylation found in the biological samples can provide a nidus of methylation, around which subsequent hypermethylation can develop through stimulation of de novo methyltransferase activity (Lorincz et al., 2002). Furthermore, the present study and others (Shen et al., 2005; Frigola et al., 2006) have shown that a given gene methylated within the normal mucosa is usually methylated at lower levels than that seen in an adjacent cancer.

The present study found that extensive methylation (3 of 5) of 3p22 genes in normal colonic mucosa was seen predominantly in individuals with concomitant BRAF mutant cancers. This finding was consistent with a previous report (Minoo et al., 2006) that found extensive methylation of numerous genes, including a panel of CIMP markers (MINT1, MINT2, MINT31, p16), and proapoptotic genes (RASSF1, RASSF2, NORE1) in the normal colonic mucosa of individuals with hyperplastic polyposis in whom V600E mutations of BRAF were found in their serrated adenomas .

183 One novel finding from the present study is the presence of the BRAF V600E mutation at low levels in conjunction with methylation of three or more of five 3p22 genes in normal colonic mucosa. To date only one other study has reported the presence of BRAF V600E mutation in normal colonic mucosa from individuals with cancers harbouring the same mutation (Kadiyska et al., 2007). However, that study did not examine the DNA methylation status of the normal colonic mucosa harbouring the BRAF mutation. Several previous studies have failed to identify BRAF mutations in normal colonic mucosa adjacent to cancers (Koinuma et al., 2004; Lubomierski et al., 2005), and this failure could be due to the use of less sensitive assays for BRAF mutation detection. The normal colonic mucosa samples assessed in the present study and others in the literature were all macroscopically normal. It is conceivable that only a few crypts within these samples may actually be harbouring the genetic mutation. The method used in the present study can detect BRAF mutation down to a proportion of 0.35% mutated DNA in the total input DNA (Hitchins et al., unpublished data). Therefore, this method may be better able to detect the mutation events occurring at low frequency within an otherwise normal field of colorectal epithelial crypts.

In the present study, there is evidence that methylation of individual 3p22 genes can occur in the absence of the V600E mutation of BRAF in normal colonic mucosa (Table 5.2). Some of these normal colonic mucosa samples (N6, N7, and N31) were procured from individuals who had a BRAF wild-type cancer but others were from individuals who had a BRAF mutant cancer that showed regional 3p22 methylation (Figure 5.1 and 5.2). In these cases, methylation of individual 3p22 genes may precede BRAF mutation although this remains uncertain. This observation supported previous reports (Derks et al., 2006; Shin et al., 2007) that DNA methylation may occur earlier than a genetic mutation in the development of CRCs. On the contrary, it is noteworthy that the oncogenic V600E mutation of BRAF has previously been shown to potentiate methylation of MLH1 in a normal colonic mucosa cell line (Minoo et al., 2007).

The present study further suggests a strong association between BRAF mutation and a higher frequency and intensity of CpG island methylation at 3p22 gene promoters in the normal colonic mucosa. While it remains unclear whether there is a causal link between the two, there is some evidence that an oncogenic protein may promote DNA methylation through the recruitment of aberrant DNA methyltransferase activity

184 (Brenner et al., 2005). It is plausible that activating mutations of BRAF may enhance the aberrant hypermethylation of CpG islands within the promoter region of 3p22 genes. Further studies are required to determine this possibility.

Given the links to BRAF mutant cancers described earlier, it is not surprising that each of the four individuals in whom concomitant methylation of 3 of 5 3p22 genes was detected in normal colonic mucosa had CIMP+ve/MSI cancers. This is consistent with the observation detailed in Chapter 4 that CRCs with concomitant methylation of 3 of 5 3p22 genes were typically of the CIMP+ve/MSI phenotype. More interestingly, the present study shows that individuals with extensive methylation of 3p22 genes and concomitant V600E mutation of BRAF in their normal colonic mucosa are more likely to have multiple hyperplastic polyps and serrated adenomas. While polyps and adenomas from the aforementioned individuals were not available for any molecular analysis, previous reports have shown extensive methylation of multiple genes in conjunction with BRAF mutation in the serrated polyps of individuals with colorectal neoplasia (Kambara et al., 2004b; Minoo et al., 2006). These individuals were either diagnosed with hyperplastic polyposis or had multiple serrated polyps that did not meet the clinical diagnostic criteria for the hyperplastic polyposis syndrome. In one of these studies (Minoo et al., 2006), extensive methylation of multiple genes was also found in normal mucosa samples adjacent to serrated polyps. However, the same study did not assess the BRAF mutation status of the normal colonic mucosa samples that showed extensive DNA methylation. Given the findings of the present study, it is possible that the normal colonic mucosa samples reported by Minoo et al. may also have harboured low levels of BRAF mutation (Minoo et al., 2006).

Overall, the present study has extended observations made by other researchers and showed for the first time that BRAF mutation can occur concomitantly with extensive DNA methylation in normal colonic mucosa of individuals with multiple serrated polyps. BRAF mutation and DNA methylation may predispose to the development of neoplasms of the serrated pathway including hyperplastic polyps, serrated adenomas and sporadic MSI/CIMP+ve CRCs. While they had multiple serrated polyps, none of the individuals included in the present study had a clinical diagnosis of hyperplastic polyposis as outlined by the World Health Organisation (Burt and Jass, 2000). It remains possible that the concomitant low levels of BRAF mutation and the extensive

185 methylation of 3p22 genes will be found at a higher frequency in normal colonic mucosa samples of individuals diagnosed with this rare syndrome.

More interestingly, the present study has provided important molecular knowledge that may be useful to facilitate the development of specific chemopreventive agents that may reduce the risk of cancer recurrence within the group of individuals who are predisposed to the serrated neoplasia pathway.

As described in Chapter 3, one limitation of the present study is that the normal colonic mucosa samples studied may be contaminated with a small amount of cancer cells. However, this possibility is unlikely, given that normal colonic mucosa samples were taken as far as possible (>10cm) from the cancer tissue. Furthermore, the methylation pattern of 3p22 genes in some normal colonic mucosa samples (N5-N7, N15-N17 and N31) was different to their matched cancers (C5-C7, C15-C17 and C31). In that respect, the frequency of methylated 3p22 genes was generally lower in the normal colonic mucosa samples. Subsequent cloning and sequencing of methylated alleles has also shown a mosaic pattern of methylation in a considerable numbers of alleles, of which some showed methylation of 10-60% of the CpG sites present in a given allele. Such mosaic patterns of methylation were unlikely to be found in cancer cells that have lost the expression of a given 3p22 gene. In such cases, allelic methylation would be expected to be more dense (>80%) (Hitchins et al., 2007b). Finally, the present study was also limited by the small number of normal colonic mucosa samples with BRAF V600E mutation and/or methylation of 3p22 genes (n=4).

Nevertheless, it remains entirely possible that these apparently normal colonic mucosa samples contained aberrant crypt foci, which were macroscopically indistinguishable from the apparently normal colonic epithelial cells. Indeed, the V600E mutation of BRAF (Beach et al., 2005; Rosenberg et al., 2007) as well as the CpG island methylation of numerous genes including MLH1, RASSFIA, and P16 (Chan et al., 2002a; Li et al., 2003; Luo et al., 2005; Greenspan et al., 2006) have previously been identified in this type of benign lesion. As mentioned in Chapter 3, no remaining tissues were available from any of the normal colonic mucosa samples for formal histological assessment. Ideally, a carefully designed prospective study that involves the microscopic examination of fresh normal colonic mucosa samples prior to collection may facilitate the acquisition of a more reliable result regarding the presence of low

186 levels of BRAF V600E mutation and methylation of 3p22 genes in the normal colonic mucosa of CRC patients.

5.4.1 Summary

1. Low levels of the V600E mutation of BRAF and CpG island methylation of several 3p22 genes were present in the normal colonic mucosa of a proportion of individuals with CRCs.

2. When BRAF mutation is present in the normal colonic mucosa, as in the four cases reported in this study, it is associated with enhanced levels and frequencies of methylation of 3p22 gene promoters.

3. Both the BRAF V600E mutation and extensive methylation of 3p22 genes are more likely to be present in individuals with MSI/CIMP CRCs, particularly those who have an increased number of serrated polyps in their colon.

187

CHAPTER 6

The role of APC methylation in the development of familial and sporadic polyposis

188

6.1 Introduction

Familial adenomatous polyposis (FAP) is a hereditary syndrome which predisposes to the development of less than 1% of all cases of CRC. This autosomal dominant syndrome is characterised by the development of hundreds to thousands of adenomatous polyps in the colon and rectum of affected individuals. It is caused by truncating germline sequence mutations causing monoallelic silencing of the tumour suppressor gene, APC (Powell et al., 1992; Nagase and Nakamura, 1993; Laurent-Puig et al., 1998). Subsequent somatic mutation or deletion of the wild-type APC allele is the typical second hit that inactivates APC expression, and this event leads to the onset of adenomatous polyps. These polyps will lead to carcinoma within 10 to 15 years unless they are resected surgically. In addition, a small proportion of individuals with clinical features typical of FAP actually carry homozygous recessive or compound heterozygous mutations of the base excision repair gene, MYH (Varesco, 2004). However, approximately 20% of individuals with the clinical features of FAP show no evidence of germline mutation in either the APC or MYH genes suggesting that other as yet unrecognised processes may lead to this clinical appearance.

In some individuals with FAP, reduced expression from one allele of APC has been described in their lymphoblastoid cells in the absence of a germline sequence mutation in the coding region (Yan et al., 2002). The methylation status of the APC gene promoter was not examined in these individuals. It remains possible that the monoallelic silencing of APC has occurred through germline epimutations, similar to the somawide monoallelic methylation of the mismatch repair gene MLH1 observed in some cases of HNPCC (Gazzoli et al., 2002; Miyakura et al., 2004; Suter et al., 2004b; Hitchins et al., 2005; Valle et al., 2007; Hitchins et al., 2007a). In support of this theory, somatic methylation of the CpG island within the promoter 1A region of APC is a well- recognised phenomenon in colorectal carcinoma (Esteller et al., 2000a; Lind et al., 2004; Kim et al., 2005; Derks et al., 2006; Iacopetta et al., 2006; Judson et al., 2006; Wynter et al., 2006; Noda et al., 2007; Kamory et al., 2008, ), In one of these studies, hypermethylation of the APC promoter was shown to occur in conjunction with the loss of APC expression (Esteller et al., 2000a). These findings indicate that the APC-1A promoter is susceptible to epigenetic inactivation.

189 Like FAP, attenuated familal adenomatous polyposis syndrome (AFAP) is known to be associated with either the germline mutation of APC or biallelic MYH mutation in 30- 40% of cases (de la Chapelle, 2004; Varesco, 2004; Lipton and Tomlinson, 2006). The genetic basis of the majority of AFAP cases is yet to be determined (Lipton and Tomlinson, 2006). Again, it is possible that at least some of these cases may represent individuals with germline epimutations of APC.

Little is known about the genetic predisposition to the hyperplastic polyposis syndrome in which affected individuals develop numerous large serrated polyps (including hyperplastic polyps, sessile serrated adenomas, and serrated adenomas) in their colon or rectum. To date, there is no consensus regarding the diagnostic criteria for this condition, although the World Health Organization has preferred a clinical definition based on the presence of one of the following criteria: (i) more than 30 polyps proximal to the sigmoid colon, (ii) more than five polyps proximal to the sigmoid colon of which more than two are larger than 10 mm in diameter, (iii) any number of hyperplastic polyps proximal to the sigmoid colon in individuals who have a first-degree relative with hyperplastic polyposis (Burt and Jass, 2000). The hyperplastic polyposis syndrome (as defined by these suggested criteria) is associated with the serrated neoplasia pathway, in which a cancer may evolve from a serrated polyp (Young and Jass, 2006). While no functional APC mutations have been identified in serrated adenomas (Hiyama et al., 1998; Dehari, 2001), it remains possible that germline epimutations of APC underlie the development of some hyperplastic polyposis cases. Certainly, promoter methylation of a large number of genes is a common event in both serrated polyps (Kambara et al., 2004b; Minoo et al., 2006) and normal colonic mucosa (Minoo et al., 2006) from individuals with hyperplastic polyposis.

Finally, it is also possible that germline epimutations of APC may be found in individuals with multiple adenomatous and/or serrated polyps in their colon or rectum, but who fail to meet the diagnostic criteria for any formally recognised hyperplastic polyposis syndrome. For the purpose of this chapter, this group of individuals will be considered to have sporadic polyposis.

At the time of commencement of the work presented in this chapter, no study had assessed the prevalence of germline epimutations of APC in individuals with FAP, AFAP, hyperplastic or sporadic polyposis. A finding of such germline epimutations of

190 APC would improve our understanding of the aetiology of these syndromes, and allow better management of affected individuals and families. The aim of the work presented in this chapter was to determine if APC epimutations may underlie several phenotypes that are characterised by the development of multiple colorectal polyps. Using the combined bisulfite restriction analysis (COBRA) and/or the quantitative methylation specific PCR (qMSP) assays, this study screened for the presence of CpG island methylation at the APC-1A promoter region in the constitutional DNA of a group of individuals with the aforementioned conditions. It is important to recognise that the present study group consists of a heterogenous group of individuals with florid or mild polyposis, classified solely on the basis of clinical features. The clinical condition of these individuals may be biased by the subjective assessments by different clinicians. This is particularly true for cases of hyperplastic or sporadic polyposis in which there is limited consensus regarding clinical definitions.

In the present study, APC-1A methylation status was also examined in the matched normal and cancer samples from 40 individuals with a familial polyposis syndrome or sporadic polyposis. This investigation aimed at determining the frequency and extent of somatic APC methylation in matched normal colonic mucosa and cancers samples of individuals with polyposis and to relate this to the clinicopathological and molecular features in these cases.

191

6.2 Methods

6.2.1 Collection of samples

A total of 165 individuals recruited by family cancer clinics in the UK and Australia were enrolled in this study. Of these individuals, 140 were diagnosed with specific polyposis syndromes including 22 with hyperplastic polyposis, 29 with FAP and 89 with attenuated FAP. Clinical classification of these individuals was performed by clinical geneticists with experience in the management of hereditary colorectal neoplasia syndromes. These included Professor Ian Tomlinson (Cancer Research UK), Professor Rodney Scott (Hunter Research Institute, Newcastle, Australia), Professor Barbara Legget (School of Medicine, University of Queensland, Australia) and Professor Robyn Ward (St. Vincent’s Hospital, Sydney, Australia). Classification was based on standard clinical criteria for these syndromes. Based on information supplied by contributors, none of these 140 individuals harboured a germline sequence mutation of APC, and biallelic mutations of MYH had been ruled out in all but ten cases. The remaining 25 individuals were recruited as cases with multiple (4) colorectal polyps. This group of individuals was designated the sporadic polyposis group and none of these individuals had biallelic mutations of MYH. Of the 165 individuals with polyposis included in the present study, 40 had undergone curative surgical resection of adenocarcinoma at St.Vincent’s Hospital, Sydney (Table 6.1). For these cases, tissue and blood samples were collected, and DNA was extracted as described in section 2.2.6. The remaining 125 cases with polyposis were recruited from other hospitals and in these cases, only DNA derived from peripheral blood mononuclear cells (PBMCs) was available for analysis. As controls, blood and normal colonic mucosa samples were also collected from individuals without cancer. The population of healthy blood donors consisted of 40 males and 48 females with age ranging from 50 to 71 years. Control samples of normal mucosa were procured from 21 individuals without neoplasia as described previously in Chapter 3 (mean age, 58.4±17.5; range, 33-82 years). This study was approved by the Human Research and Ethics Committee of St. Vincent’s Hospital, Sydney. Informed consent was obtained from all participating individuals.

192 Patient ID Age at CRC resection Sex Disease Type

1 63 F FAP 2 89 F AFAP 3 70 M AFAP 4 66 F AFAP 5 67 M Hyperplastic polyposis 6 67 F Hyperplastic polyposis 7 62 M Hyperplastic polyposis 8 68 M Hyperplastic polyposis 9 72 F Hyperplastic polyposis 10 60 M Hyperplastic polyposis 11 70 F Hyperplastic polyposis 12 61 M Hyperplastic polyposis 13 76 M Hyperplastic polyposis 14 68 M Hyperplastic polyposis 15 62 M Hyperplastic polyposis 16 68 M Sporadic polyposis 17 79 F Sporadic polyposis 18 87 F Sporadic polyposis 19 80 M Sporadic polyposis 20 79 F Sporadic polyposis 21 60 F Sporadic polyposis 22 68 F Sporadic polyposis 23 67 F Sporadic polyposis 24 71 M Sporadic polyposis 25 68 M Sporadic polyposis 26 88 F Sporadic polyposis 27 79 F Sporadic polyposis 28 75 F Sporadic polyposis 29 48 F Sporadic polyposis 30 76 F Sporadic polyposis 31 42 F Sporadic polyposis 32 74 M Sporadic polyposis 33 76 M Sporadic polyposis 34 74 M Sporadic polyposis 35 80 M Sporadic polyposis 36 62 M Sporadic polyposis 37 80 M Sporadic polyposis 38 92 F Sporadic polyposis 39 68 F Sporadic polyposis 40 74 M Sporadic polyposis

Table 6.1 Individuals with a polyposis condition recruited from St. Vincent’s Hospital, Sydney and their clinical diagnosis The age at colorectal cancer (CRC) resection, gender, and disease type are shown for 40 individuals who underwent curative surgical resection for colorectal cancers at St. Vincent’s Hospital, Sydney. For these individuals, matched colorectal tumour and normal colonic mucosa, as well as PBMC samples were available for the assessment of APC-1A methylation. F, female; M, male; FAP, familial adenomatous polyposis; AFAP, attenuated familial adenomatous polyposis.

193 6.2.2 Analysis of APC-1A promoter methylation

Methylation of CpG dinucleotides at the APC-1A promoter region was analysed using combined bisulfite restriction analysis (COBRA) and quantitative methylation-specific PCR (qMSP) assays as described in sections 2.2.17 and 2.2.12 respectively. The primer sets and their annealing temperatures are listed in Table 2.1. For COBRA, the primer set amplified a DNA fragment that corresponds to a 229 bp region within the CpG island of the APC-1A promoter (Figure 6.1). The restriction enzymes RsaI and TaqI were used separately for subsequent digestion of the amplicons to detect CpG sites within this region that were methylated in the template DNA. A second set of primers that amplified a smaller 143 bp region within the APC-1A promoter was used in the qMSP assay (Fig. 6.1). MyoD was used as the internal control for the normalisation of DNA input. Optimisation of the qMSP assay for the detection of APC-1A promoter methylation was similar to that described in section 3.3.1 and 4.2.3. This assay demonstrated good linearity across a broad range of allelic methylation levels (R2= 0.997, P value for the test of linear association = 3.62 × 10-11) (Figure 6.2). It was also capable of detecting methylation down to a measurable proportion of 0.1% of the total input DNA. Results obtained from qMSP were verified using the clonal bisulfite sequencing method described in section 2.2.16.

-169 qMSP (143bp) -27

-209 COBRA (229 bp) +20

Figure 6.1 Regions of APC-1A promoter analysed by the COBRA or qMSP assay The regions of APC-1A promoter amplified by the COBRA (black line) or qMSP assays (grey line) are shown with reference to a CpG map of this gene promoter (bottom). The transcription start site for APC is at nucleotide +1. Individual CpG dinucleotides are denoted by vertical black bars. For COBRA, the primer set amplified a 229 bp PCR amplicon that correspond to the -209 bp to +20 bp region relative to the transcription start site of the APC gene. The primers for the qMSP assay amplified a smaller 143 bp region (corresponding to the -169 bp to -27 bp region relative to the transcription start site).

194 100

60 RR22= = 0.9966 0.997

Measured MethylationMeasured (PMR) 20

0 20406080100

% Methylated DNA in Template

Figure 6.2 APC-1A qMSP shows linearity in the detection of methylated DNA A serial dilution of completely methylated CpGenomeTM Universal Methylated DNA (Chemicon) in unmethylated blood DNA was tested across a broad range of percentages (0-100%, as described in section 2.2.13). The qMSP assay was performed on these samples on three different occasions, each time using samples in triplicates. Error bar represent ±1 standard deviation of the final mean PMR which represents the mean of triplicate PMR values, each obtained from three different assay runs. The qMSP assay shows good linearity in the detection of methylation level (PMR) values across this range of dilution points with known percentages of methylation.

6.2.3 Statistical analyses

Univariate analyses for comparisons between clinicopathological and molecular features were performed as described in section 2.2.20. The relationship between the levels of APC-1A methylation and age was assessed using Spearman’s Test. All P values <0.05 were considered significant. All data were analysed using SPSS V15.0 (SPSS Inc., Chicago, IL).

195

6.3 Results

6.3.1 Screening for germline epimutations of APC in polyposis cases using combined bisulfite restriction analysis (COBRA)

With the assistance of the staff of the Molecular and Cellular Oncology Group, St. Vincent’s Hospital, COBRA was performed to assess the methylation status of the APC-1A promoter in the PBMC DNA from a total of 140 individuals: 22 with HPS, 29 with FAP and 89 with AFAP. No methylation of the APC-1A promoter was detected in the constitutional DNA of any of the 140 individuals studied, suggesting that germline epimutations of APC do not account for cases of polyposis arising in individuals who have no detectable germline sequence mutation of the APC or MYH genes. A typical result for the COBRA assay is shown in Figure 6.3.

L C+ C- 1 2 3 4 5 6 7 8 9

229 bp U 162 bp M 67 bp Figure 6.3 Combined bisulfite restriction analysis of APC-1A promoter Upper panel: CpG Map of the APC-1A promoter .The transcription start site of APC exon 1 (grey box) is at nucleotide +1. The positions of the primers used to amplify a 229 bp fragment of the promoter upstream of exon 1 are shown by arrows. Individual CpG dinucleotides are denoted by vertical black bars. The diagnostic restriction enzyme sites that cleave specific CpG sites when methylated are indicated. Lower panel: A typical gel picture showing a batch of samples subjected to combined bisulfite and restriction analysis of the APC-1A promoter. L, pUC19/MspI DNA ladder; C+, in vitro methylated genomic DNA shows cleavage of the 229 bp amplicon when the methylated templates (M) were digested with RsaI to give a digestion pattern of 162 and 67 bp. C-, unmethylated genomic DNA (U) from a normal control individual remains undigested. Wells labelled 1–9 contain peripheral blood DNA from nine familial adenomatous polyposis subjects, each undigested and therefore negative for APC-1A methylation (Hitchins et al., 2006).

196 6.3.2 Assessment of low level methylation of the APC-1A promoter using quantitative methylation-specific PCR (qMSP) assay

6.3.2.1 APC-1A methylation in PBMC samples Recently, epimutations of the mismatch repair gene MSH2 have been characterised by a mosaic distribution of methylation levels in different somatic tissues of individuals who had the epimutated MSH2 allele (Chan et al., 2006). In these individuals, low levels of methylation were found in leucocytes, while higher levels were detected in buccal and colonic mucosa samples. Given that COBRA assays are not sensitive enough to detect low levels of methylation, it remains possible that despite the negative results described above, low levels of APC methylation could be found in the constitutional DNA of some individuals with sporadic or familial polyposis. In order to investigate this possibility, the quantitative methylation specific PCR (qMSP) assay was used to assess the levels of APC-1A methylation in the PBMC samples from each of the 165 individuals with a polyposis condition enrolled in the present study.

The extent of APC-1A methylation (PMR) detected in the PBMC of individuals with or without a polyposis condition is shown in Figure 6.4. Low levels of APC-1A methylation (PMR=0.11-0.32) were found in the PBMC from 7% (11/165) of individuals who had a polyposis. Of these 11 individuals, one was diagnosed as having FAP, eight were diagnosed as having AFAP while the remaining two had sporadic polyposis. Methylation of APC-1A was similarly detected at low levels (PMR=0.10- 0.44) in the PBMC of 5% (4/88) of healthy individuals. The median level of APC-1A methylation in the PBMC samples of individuals with polyposis was not significantly higher than that detected in healthy individuals.

197 P=0.410

0.50

0.40

R M

M 0.30 P P 0.20 Median 2 Median 0.10 methylation methylation (PMR) = 0.02 (PMR) = 0.00 141 84 0 PBMC of individual PBMC of healthy with polyposis control subjects

Figure 6.4 Distribution of APC-1A methylation levels in the peripheral blood mononuclear cell samples measured using the qMSP assay Circles represent the methylation level (PMR) of APC-1A in the peripheral blood mononuclear cell (PBMC) samples from individuals with a polyposis condition (left) and from healthy control subjects (right). The number superscripted beside a given circle (if present) indicates the total number of samples with a similar PMR value. Horizontal lines represent median PMR levels for each group. The median level of APC-1A methylation in the PBMC samples of individuals with polyposis was not significantly higher than that detected in healthy individuals (P=0.410, Mann-Whitney U Test). PMR, percentage of methylated reference; PBMC, peripheral blood mononuclear cell.

Clonal bisulfite sequencing was used to confirm the presence of methylation by the qMSP assay in five individuals with a polyposis condition (one with FAP, two with AFAP and two with sporadic polyposis) and four healthy donors. An example of this clonal bisulfite sequencing is shown in Figure 6.5. P1 and P2 represent individuals with FAP and AFAP respectively. W1 and W2 are two healthy blood donors. While the majority of the methylated APC-1A clones representing alleles in the PBMC samples showed patterns of dense methylation (80-100% of CpG sites were methylated), the overall methylation patterns were mosaic (Figure 6.5). In addition, as seen in samples P2 and W1, a considerable number of clones were methylated at less than 60% of CpG sites.

198

P1 P2 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

89-100% of CpG sites were methylated 33-100% of CpG sites were methylated in individual clones representing alleles in individual clones representing alleles Total methylation = 94.2% Total methylation = 64.8%

W2 W1 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

56-78 % of CpG sites were methylated in 78-100% of CpG sites were methylated in individual clones representing alleles individual clones representing alleles Total methylation = 67.6% Total methylation = 91.9%

Figure 6.5 Confirmation of qMSP result using clonal bisulfite sequencing Patterns of methylation at individual CpG dinucletides were determined by cloning and sequencing of PCR products generated by the qMSP assay. Examples of methylation patterns are shown for PBMC samples from individuals with polyposis (P1, an individual with FAP; P2, an individual with AFAP) and from individuals without neoplasia (W1 and W2). For each sample, methylation patterns for 10-12 clones, as represented by individual rows, are shown. Circles represent all 9 CpG sites (1-9), positioned between the binding regions of the qMSP primers (indicated by black arrows). This region corresponds to -169 bp to -27 bp relative to the transcription start site of the APC gene (shown in Figure 6.1). Black and white circles represent methylated and unmethylated CpG sites respectively. Total methylation calculated as a percentage of methylated over total CpG sites in each PBMC samples varied from 67.3-95.8%. While the majority of individual clones sequenced showed dense methylation of >70% CpG sites, several clones were methylated at <50% CpG sites.

199 The density of CpG methylation within methylated APC-1A clones representing alleles (Total methylation) in PBMC samples was not significantly different between individuals with a polyposis condition and healthy donors (Table 6.2).

Density of APC-1A methylation in PBMC samples N Median total Range (%) Mann- methylation (%) Whitney U test Individuals with 5 84.2 64.8-94.2 P = 1.000 polyposis

Healthy donors 4 80.9 61.6-91.9

Table 6.2 Density of allelic APC-1A methylation in PBMC samples Clonal bisulfite sequencing was performed on PCR products (generated by the qMSP assay) from several peripheral blood mononuclear cell (PBMC) samples that were positive for APC-1A methylation. These included PBMC samples from five individuals with a polyposis condition (one with FAP, two with AFAP and two with sporadic polyposis) and four healthy donors. For each PBMC sample, total methylation was calculated as a percentage of methylated over total CpG sites in 10-12 methylated APC-1A clones. No significant difference was observed in the density of CpG methylation within methylated APC-1A alleles (total methylation) between individuals with a polyposis condition and healthy donors.

6.3.2.2 APC-1A methylation in colorectal tumours and normal mucosa samples Of the 165 individuals enrolled in this study, matched colorectal tumour and normal colonic mucosa samples were available for 40 cases (Mean age = 71±10.1; range, 42-92 years). While all of these individuals underwent curative resection for presumed CRC, in three of these cases the final diagnosis was adenoma without evidence of invasive malignancy. The term tumour is used here to include either adenoma or carcinoma.

Of these 40 individuals, 16 had APC-1A methylation of PMR>4.0 (hypermethylation- positive) in their tumour samples (Median PMR = 29.8, range 6.9-69.8), a level which has been shown for MLH1 (detailed in chapter 3) and other genes including MGMT and p16 (Ogino et al., 2006) to correlate with the loss of protein expression. Lower levels of APC-1A methylation (Median PMR =0.31, range 0.12- 3.68) were found in the tumours of the remaining 24 individuals. In detail, high levels of APC-1A methylation (PMR>4.0) were found in each of the three individuals diagnosed with AFAP, in five of 11 (45%) individuals diagnosed with hyperplastic polyposis and in eight of 25 (32%)

200 individuals who had sporadic polyposis (Figure 6.6). Hypermethylation of the APC-1A promoter was not found in the tumour from the one individual with FAP in this study.

100

30 Frequency of APC-1A 20

hypermethylation in tumours in hypermethylation (%) 10

0 FAP AFAP Hyperplastic Sporadic (n=1) (n=3) Polyposis Polyposis (n=11) (n=25) Type of Polyposis condition

Figure 6.6 Frequency of APC-1A hypermethylation in tumours of individuals with a polyposis condition The frequency of APC-1A hypermethylation (%) in tumour samples is shown for each group of individuals with a given polyposis condition. The hypermethylation of APC-1A was defined as PMR>4.0. FAP, familial adenomatous polyposis; AFAP, attenuated familial adenomatous polyposis.

201 Table 6.3 shows the levels of APC-1A methylation in the matched normal colonic mucosa samples of individuals with a polyposis condition as well as individuals without neoplasia. Overall, the median levels of APC-1A methylation in the normal colonic mucosa samples was significantly higher in the group of individuals with polyposis than those without neoplasia (P<0.001, Mann-Whitney U test).

Levels of APC-1A methylation in normal colonic mucosa N Median (PMR) Range (PMR) P value Individuals with 40 0.59 0-1.75 <0.001 polyposis

Individuals without 21 0.23 0-0.75 neoplasia

Table 6.3 Comparison of the levels of APC-1A methylation in the normal mucosa samples of individuals with polyposis and those without neoplasia The median levels of APC-1A methylation (PMR) in the normal mucosa samples of individuals with a polyposis was higher than those without neoplasia (P<0.001, Mann-Whitney U test).

A more detailed analysis of this data in relation to the levels of APC-1A methylation in the paired tumour samples showed that methylation was highest in the normal colonic mucosa samples from the group of individuals who also showed higher levels of APC- 1A methylation (PMR>4.0) in their tumours (Figure 6.7). These findings not only indicate that the intensity of APC-1A methylation is likely to be higher in the normal colonic mucosa of individuals with polyposis, but suggest that this higher intensity of methylation may predispose to the development of a colorectal tumour characterised by hypermethylation of the APC promoter.

202 P<0.0001

2.00 P<0.031

1.50

1.00

0.50 P-A ehlto (PMR) methylation APC-1A 0.00 NCM adjacent to NCM adjacent to NCM of individuals hypermethylated non-hypermethylated without neoplasia tumour tumour (n=21) (n=16) (n=24) Figure 6.7 Distribution of APC-1A methylation in apparently normal colonic mucosa stratified by methylation status of paired tumour Each circle represents the APC-1A methylation level (PMR) in the normal-appearing colonic mucosa (NCM) of individuals with APC-1A hypermethylation-positive (as defined by PMR > 4.0) (left) and APC-1A hypermethylation-negative (centre) colorectal tumours, as well as from individuals without neoplasia (right). Horizontal lines represent median PMR levels for each group. Significantly higher levels of APC-1A methylation were found in the normal-appearing colorectal mucosa adjacent to hypermethylation-positive tumour when compared to NCM adjacent to hypermethylation-negative tumours or NCM in individuals without neoplasia (P, Mann-Whitney U-test = 0.031 and < 0.0001 respectively). PMR, Percentage of Methylated Reference.

6.3.3 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours in individuals with polyposis

Table 6.4 shows the clinicopathological and molecular characteristics of individuals with polyposis by the methylation status of APC-1A promoter in tumour samples. Hypermethylation of APC-1A (PMR >4.0) was found to correlate inversely with presence of the V600E mutation of BRAF (P=0.016), and the CIMP-positive phenotype (P=0.035). No significant correlation was observed between hypermethyation of APC- 1A and age, gender, tumour site, microsatellite status, or KRAS Codon 12 or13 mutations.

203 APC-1A hypermethylation- APC-1A hypermethylation- P Value Characteristics positive negative (n = 16) (n =24) Mean age (± SD) 73.3±10.1 69.5±10.1 0.562

Sex 0.093 Male 11 (69%) 10 (42%) Female 5 (31%) 14 (58%)

Tumour site 0.151 Right-sided 7 (44%) 16 (67%) Left-sided 9 (56%) 8 (33%)

Tumour stage 0.652 I 4 (25%) 5 (22%) II 9 (56%) 12 (55%) III 3 (18%) 3 (14%) IV 0 (0%) 2 (9%) Not assessed 0 2

Microsatellite status 0.329 MSI 4 (25%) 10 (42%) MSS 12 (75%) 14 (52%)

BRAF V600E mutation 0.016 Mutant 2 (15%) 13 (57%) Wild-type 13 (85%) 10 (43%) Not assessed 1 1

KRAS Codon 12 or 13 0.476 mutations Mutant 5 (33%) 5 (23%) Wild-type 10 (67%) 17 (77%) Not assessed 1 2

CIMP 0.035 Positive 2 (13%) 11 (50%) Negative 13 (87%) 11 (50%) Not assessed 1 2

Table 6.4 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours arising in individuals with polyposis Hypermethylation of APC-1A was defined by PMR >4.0. The P value for the comparison of mean age between groups was obtained using a Mann-Whitney U test. P values are indicated for 2 or Fisher Exact tests for all other characteristics. Significant P values are highlighted in bold. SD, standard deviation; MSI, microsatellite unstable; MSS, microsatellite stable; CIMP, CpG island methylator phenotype.

In a more detailed analysis within the group of individuals with hyperplastic polyposis, hypermethylation of APC-1A (PMR >4.0) in tumours was found to correlate inversely with the presence of BRAF V600E mutation (P=0.048, Table 6.5). Hypermethylation of APC-1A also appeared to be more frequent in tumours that demonstrated the MSS or

204 CIMP-ve phenotype in individuals with hyperplastic polyposis. However, this did not reach statistical significance (Table 6.5).

APC-1A APC-1A P Value hypermethylation- hypermethylation- Characteristics positive negative (n = 5) (n =6) Mean age (± SD) 66.2±67.24.2 6.4 ± 0.360

Sex 0.182 Male 5 (100%) 3 (50%) Female 0 (0%) 3 (50%)

Tumour site 0.567 Right-sided 2 (40%) 4 (66.7%) Left-sided 3 (60%) 2 (33.3%)

Tumour stage 0.323 I 0 (25%) 2 (22%) II 3 (56%) 3 (55%) III 2 (18%) 1 (14%) IV 0 (0%) 0 (0%)

Microsatellite status 0.061 MSI 0 (0%) 4 (66.7%) MSS 5 (100%) 2 (33.3%)

BRAF V600E mutation 0.048 Mutant 0 (0%) 4 (80%) Wild-type 5 (100%) 1 (20%) Not assessed 1 0

KRAS Codon 12 or 13 mutations 0.500 Mutant 2 (50%) 1 (16.7%) Wild-type 2 (50%) 5 (83.3%) Not assessed 1 0

CIMP 0.076 Positive 0 (0%) 4 (66.7%) Negative 4 (100%) 2 (33.3%) Not assessed 1 0

Table 6.5 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours arising in individuals with hyperplastic polyposis Hypermethylation of APC-1A was defined by PMR >4.0. The P value for the comparison of mean age between groups was obtained using a Mann-Whitney U test. P values are indicated for 2 or Fisher Exact tests for all other characteristics. Significant P values are highlighted in bold. SD, standard deviation; MSI, microsatellite unstable; MSS, microsatellite stable; CIMP, CpG island methylator phenotype.

In tumours of individuals with sporadic polyposis, hypermethylation of APC-1A was not associated with any common clinicopathological and molecular features (Table 6.6).

205 Given the small number of samples within the group of individuals with FAP or AFAP, no statistical analysis was performed for this group.

APC-1A APC-1A P Value hypermethylation- hypermethylation- Characteristics positive negative (n = 8) (n =17) Mean age (± SD) 76.9±10.0 70.7±11.6 0.294

Sex 0.411 Male 5 (63%) 7 (41%) Female 3 (37%) 10 (59%)

Tumour site 0.389 Right-sided 3 (37%) 11 (65%) Left-sided 5 (63%) 6 (35%)

Tumour stage 0.638 I 3 (38%) 3 (20%) II 4 (50%) 8 (54%) III 1 (22%) 2 (13%) IV 0 (0%) 2 (13%) Not assessed 0 2

Microsatellite status 0.667 MSI 4 (50%) 6 (35%) MSS 4 (50%) 11 (65%)

BRAF V600E mutation 0.338 Mutant 2 (25%) 8 (50%) Wild-type 6 (75%) 8 (50%) Not assessed 0 1

KRAS Codon 12 or 13 mutations 0.657 Mutant 3 (33%) 4 (23%) Wild-type 5 (67%) 11 (77%) Not assessed 0 2

CIMP 0.400 Positive 2 (25%) 7 (47%) Negative 6 (75%) 8 (53%) Not assessed 0 2

Table 6.6 APC-1A hypermethylation status and the clinicopathological and molecular characteristics of tumours arising in individuals with sporadic polyposis Hypermethylation of APC-1A was defined by PMR >4.0. The P value for the comparison of mean age between groups was obtained using a Mann-Whitney U test. P values are indicated for 2 or Fisher Exact tests for all other characteristics. SD, standard deviation; MSI, microsatellite unstable; MSS, microsatellite stable; CIMP, CpG island methylator phenotype.

206 6.3.4 The extent of APC-1A methylation in the normal colonic mucosa and clinicopathological features of individuals in the study group

The associations between the intensity of APC-1A methylation in the normal colonic mucosa and clinicopathological features of individuals in the study group was also investigated. Within the group of individuals who had a polyposis condition, levels of APC-1A methylation in the normal colonic mucosa samples were positively correlated with age (Spearman’s correlation: r = 0.21, P = 0.044, Figure 6.8).

2.0

1.5

1.0 Spearman’s rank, r =0.21 APC-1A methylationAPC-1A (PMR) 0.5

0 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Age (years)

Figure 6.8 Scatter plot of APC-1A methylation levels in the normal colonic mucosa samples against the age of individuals with a polyposis condition The APC-1A methylation level (PMR) in each normal colonic mucosa sample was plotted against the age of corresponding individuals who had a polyposis condition (blue triangle). APC-1A methylation levels were positively correlated with age in this group of samples (Spearman correlation: r =0.21, P = 0.044). PMR, percentage of methylated reference.

There was no association between the levels of APC-1A methylation in normal colonic mucosa samples and age within the group of individuals without neoplasia alone (Spearman’s correlation: r = -0.17, P = 0.451, Figure 6.9).

207

1.0

0.5

Spearman’s rank, r = -0.17 0

APC-1A methylation (PMR) 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Age (years)

Figure 6.9 Scatter plot of APC-1A methylation levels in the normal colonic mucosa samples against the age of individuals without a polyposis condition in this study cohort The APC-1A methylation level (PMR) in each normal colonic mucosa sample was plotted against the age of corresponding individuals without a polyposis condition (red triangle). APC-1A methylation levels were not related to age in this group of samples (Spearman correlation: r =-0.17, P = 0.451). PMR, percentage of methylated reference.

In summary, these data indicate that individuals with multiple polyps are more susceptible to APC-1A methylation as they age. However, this susceptibility is not seen in individuals without neoplasia.

208 No association was found between the levels of APC-1A methylation in normal colonic mucosa samples and other clinicopathological features examined in either the group of individuals who had a polyposis condition, or in the group of individuals without neoplasia (Table 6.7).

Gender Location Male Female Right Left Unknown Individuals N 21 19 24 16 with a Median PMR 0.71 0.38 0.65 0.56 polyposis Range 0.18-1.75 0-1.47 0-1.47 0.21-1.75 condition P value 0.055 0.989

Individuals N 11 10 0 2 19 without Median PMR 0.25 0.23 ND 0.2 neoplasia Range 0-0.67 0-0.33 ND 0.1-0.31 P value 0.647 NC

Table 6.7 Absence of correlation between the levels of APC-1A methylation in the normal colonic mucosa samples of individuals in the study group and clinicopathological features Statistical analyses were performed in the group with or without a polyposis condition (no neoplasia), or in both groups combined. P values are indicated for Mann-Whitney U tests. PMR, Percentage of methylated reference; ND, no data; NC, not computed since there is no data available for one of the variables.

6.3.5 APC-1A methylation in the normal colonic mucosa and the molecular features of matched tumours

Increased levels of APC-1A methylation in the normal colonic mucosa samples were significantly associated with hypermethylation of the APC-1A promoter (PMR>4) in the matched tumours (P=0.031, Mann- Whitney U test). No association was found between the levels of APC-1A in the normal colonic mucosa samples and other molecular characteristics of matched tumours assessed (Table 6.8).

209

Levels of APC-1A methylation in normal colonic mucosa N Median (PMR) Range (PMR) P value Microsatellite status 0.487 MSI 14 0.68 0-1.75 MSS 26 0.50 0.21-1.15

BRAF V600E mutation 0.492 Mutant 15 0.64 0.21-1.26 Wild-type 23 0.56 0-1.75 Not assessed 2

KRAS Codon 12 or 13 mutations 0.918 Mutant 10 0.47 0.16-1.75 Wild-type 27 0.64 0-1.26 Not assessed 3

APC-1A hypermethylation 0.031 (PMR>4) Present 16 0.68 0.24-1.75 Absent 24 0.40 0-1.26

CIMP 0.811 Positive 13 0.55 0.21-1.15 Negative 24 0.61 0-1.75 Not assessed 3

Table 6.8 Associations between the levels of APC-1A methylation in the normal colonic mucosa samples of individuals with a polyposis condition and various molecular characteristics of corresponding tumours P values are indicated for Mann-Whitney U tests. Significant P values are highlighted in bold. Increased levels of APC-1A methylation in the normal colonic mucosa samples were significantly correlated with hypermethylation of APC-1A in matched tumours (P=0.031) but not with other molecular features. PMR, percentage of methylated reference; MSI, microsatellite unstable; MSS, microsatellite stable; CIMP, CpG island methylator phenotype.

210

6.4 Discussion

6.4.1 Germline epimutation of APC is not associated with familial and sporadic colorectal polyposis

The present study using the COBRA assay is the first to consider germline epimitation as an alternate to the germline monoallelic loss of APC seen in individuals with FAP, AFAP or hyperplastic polyposis syndromes. This possibility was investigated in patients with one of these syndromes in whom no germline mutation of APC had been found (Hitchins et al., 2006). The study found no evidence that germline epimutations of APC contribute towards the development of FAP, AFAP or hyperplastic polyposis syndromes within the groups studied.

The present study investigates the presence of CpG methylation at APC-1A, but not at a second CpG island of APC promoter known as APC-1B. In part, this is due to the reported absence of methylation at the latter CpG island in colorectal cancer (Esteller et al., 2000a). It is however noteworthy that a recent study failed to identify methylation of either APC-1A or APC-1B in the constitutional DNA of 21 individuals with FAP and 39 individuals with AFAP (Romero-Gimenez et al., 2008). This study not only supports the findings presented in this chapter regarding the lack of germline APC-1A methylation, but has now clearly shown that APC-1B is not susceptible to germline epimutation.

While the mechanism underlying the development of some familial and sporadic polyposis remains uncertain, it is clear from the present study and that of Romero- Gimenez et al (Romero-Gimenez et al., 2008) that germline epimutations of APC are unlikely to be involved. One possible mechanism is a mosaic pattern of somatic APC mutations (somatic mutational mosaicisms) which has been found in the blood and colorectal adenomas of 20% of individuals diagnosed with FAP and AFAP (Aretz et al., 2007; Hes et al., 2008). The term somatic mosaicism refers to the presence of discrete populations of genetically distinct cells within a same individual (Tuohy and Burt, 2008). A mosaic distribution of a given somatic mutation may be present at levels below the limit of detection of common genetic screening techniques, such as DNA sequencing. Mosaicism for APC mutation can only be detected using sensitive assays such as pyrosequencing, denaturing high performance liquid chromatography, high

211 resolution melt analysis or pyrophosphorolysis-activated polymerisation (Aretz et al., 2007; Hes et al., 2008). Therefore, it remains possible that a mosaic form of APC mutation may underlie the development of a polyposis condition in some individuals in whom no APC mutation has been identified by standard genetic screening.

The essentially negative results presented in this chapter do not preclude the possibility that germline defects (genetic or epigenetic) in other genes may produce polyposis syndromes. For example, in the present study, the MYH mutation status of 10 individuals was not known. It is possible that a homozygous or compound heterozygous mutation of MYH may underlie the development of one or more of these 10 polyposis cases.

6.4.2 Somatic APC methylation in the tissues of individuals with a polyposis condition

The mosaic patterns of somatic CpG methylation at a given gene promoter can be predisposed by a cis-acting genetic defect. This has been reported in the case of FMR1 methylation in the fragile X mental retardation syndrome (Malter et al., 1997) and more recently in the case of DAPK1 methylation in chronic lymphocytic leukaemia (Raval et al., 2007). The recently reported MSH2 epimutation in a family with HNPCC (Chan et al., 2006) was suggested to be caused by a cis-acting genetic defect and therefore demonstrated a full penetrance within the affected family (Chong et al., 2007; Horsthemke, 2007; Suter and Martin, 2007). In a similar manner, it is plausible that somatic mosaicism in APC-1A methylation may underlie some cases of polyposis. The highly sensitive qMSP assay is well suited to the detection of mosaic patterns of methylation and this was therefore used to re-assess APC-1A methylation.

Low levels and mosaic patterns of APC-1A methylation were found at similar intensity in PBMC from either individuals with polyposis or healthy volunteers, suggesting that this event may be commonplace. Furthermore, APC-1A methylation has previously been found in non-cancerous gastric epithelial (Tsuchiya et al., 2000) and normal liver tissues (Harder et al., 2008; Nishida et al., 2008). Therefore, APC-1A methylation may occur in certain types of apparently normal tissues.

The qMSP assay was used to determine whether APC-1A methylation can be detected at a higher level in the colorectal mucosa of individuals with polyposis. One limitation

212 in the present study was the lack of consensus in defining hyperplastic polyposis. While the clinical diagnosis of a given patient was provided by experienced clinicians, it is in some ways inevitable that the present study consisted of a heterogenous group of individuals. Ideally, a tight case definition should be implemented, but the present study sought to determine whether APC-1A methylation might underlie the development of a broad range of phenotypes characterised by the development of multiple colorectal polyps. Thus, it was considered reasonable to perform the study in a heterogenous group of polyposis patients. Another limitation of this study was that normal colonic mucosa samples may be contaminated with a small amount of cancer cells. This possibility is considered unlikely, given the reasons described in Chapter 5, but cannot be fully discounted. Finally, the study was limited by the small number of normal mucosa samples and the lack of availability of fresh tissue of neoplastic polyps from individuals with polyposis.

The findings presented in this chapter suggest that a higher intensity of APC-1A methylation in the normal mucosa samples of individuals with polyposis may predispose to the development of a tumour harbouring a hypermethylated APC-1A promoter (Figure 6.7, Table 6.8). This result is consistent with a previous report of elevated levels of MGMT methylation in the normal colonic mucosa of individuals with sporadic colorectal carcinomas showing MGMT hypermethylation (Shen et al., 2005). Together, the present study and others show that increased DNA methylation at a given gene may be indicative of a field defect in the development of colorectal neoplasia. In light of a recent report that MGMT promoter methylation is strongly associated with the presence of a specific single nucleotide polymorphism (SNP) in the MGMT promoter (Ogino et al., 2007c), the propensity for APC promoter methylation may also be related to the presence of a given SNP in the APC promoter. Further studies are required to determine this possibility.

The present study found elevated levels (PMR>4) of APC-1A methylation in the tumours from individuals diagnosed with an AFAP, hyperplastic polyposis or sporadic polyposis. This finding suggests that somatic hypermethylation of APC promoter may serve as an alternative mechanism underlying the loss of the tumour suppressor protein, APC in at least some individuals with a polyposis condition, and in whom no germline APC mutation has been identified by standard genetic testing.

213 6.4.3 APC promoter methylation is inversely correlated with BRAF V600E mutation and CIMP+ve phenotype in colorectal tumours

Consistent with a previous study in an unselected group of colorectal cancers (Iacopetta et al., 2006), the present study demonstrated that hypermethylation of the APC-1A promoter is inversely correlated with BRAF V600E mutation and the CIMP+ve phenotype in colorectal tumours of individuals with polyposis. Similar to previous studies in colorectal cancer, the present study also found no association between APC hypermethylation in colorectal tumours and age, gender, tumour site, tumour stage, tumour grade, microsatellite status, and KRAS codon 12 and 13 mutations (Esteller et al., 2000a; Iacopetta et al., 2006; Hashimoto et al., 2008). It is apparent from these data that APC hypermethylation is not in itself responsible for a phenotypically distinct group of CRCs. APC methylation is unlikely to contribute to the development of CIMP- positive/BRAF mutant cancers, and as recommended by others, APC hypermethylation should not be included as a marker for CIMP (Iacopetta et al., 2006).

Given the heterogeneity of polyposis cases in the present study group, the correlations between hypermethylation of APC-1A and clinicopathological and molecular features of tumours were considered separately for the two major groups of individuals (hyperplastic or sporadic polyposis). While showing similar trends particularly in the hyperplastic polyposis group, the small number of individuals in this study group prevents meaningful subset analysis.

6.4.4 Age-related methylation of APC promoter in the normal colonic mucosa of individuals with polyposis

One interesting finding from the present study was the association between the increased levels of APC-1A methylation in normal colonic mucosa and the age of polyposis patients. This was notable in that no such association was found in individuals without neoplasia. One interpretation of these results is that individuals with polyposis may be more susceptible to hypermethylation of APC within their normal mucosa as they age.

Given the heterogeneity of polyposis cases included in the present study, two models may be proposed to fit with the observed facts regarding APC methylation and its role in the development of neoplastic polyps and subsequent colorectal cancers. Firstly, a

214 germline defect involving an as yet unknown cis-acting defect may predispose to APC methylation in the colonic mucosa. This model is likely to be applicable to the FAP and AFAP cases that exhibited strong familial inheritance. Secondly, APC methylation may have occurred in the colonic mucosa due to an environmental influence. This model may explain the presence of APC methylation in the vast majority of tissue samples included in the present study, including those from individuals diagnosed with either a hyperplastic or sporadic polyposis condition (Table 6.1). Unlike cases of FAP and AFAP that are typically associated with a strong genetic predisposition and therefore exhibit a relatively early onset, cases of hyperplastic or sporadic polyposis appear to lack a genetic basis. In these latter cases, the process of neoplastic development is likely to be similar to that seen in sporadic lesions, and is more susceptible to environmental factors.

To date, there is little evidence that aberrant hypermethylation of tumour suppressor genes is directly associated with an aging process (reviewed in Fraga and Esteller) (Fraga and Esteller, 2007). However, a progressive loss of DNMT1 expression has been reported in aging human fibroblasts, and this may lead to the global hypomethylation of DNA (Casillas et al., 2003). In contrast, expression of de novo DNA methyltransferase DNMT3B increased steadily in aging fibroblast cells, possibly as a natural response of cells to counter the loss of global DNA methylation (Casillas et al., 2003). While these observations need to be confirmed, a logical effect of increased DNMT3B expression would be the aberrant hypermethylation of otherwise unmethylated CpG islands of gene promoters. Hypermethylation at CpG islands which occurs in concert with global hypomethylation may be common in aging cells and these are also established epigenetic events in cancer development.

An age-related increase in the DNA methylation levels in normal colonic mucosa has previously been proposed as a manifestation of field defect in the development of sporadic colorectal cancers (Ahuja et al., 1998; Issa, 2000a; Issa, 2000b; Shen et al., 2005), and individuals with hyperplastic polyposis have been shown to have highly concordant patterns of DNA methylation in different tumours (Chan et al., 2002b). More recently, age-related increase in methylation has been reported for several genes including APC, HPP1, p16 and AXIN2 in sporadic colorectal cancers (Belshaw et al., 2008). The finding of a close correlation between age and APC-1A methylation in

215 normal colonic mucosa amongst polyposis patients, as found in the present study cohort, is consistent with these previous reports, and shows for the first time that age- related DNA methylation in the normal colonic mucosa may also predispose to the development of colorectal cancer in some individuals with polyposis, particularly in those with a hyperplastic or sporadic polyposis phenotype.

6.4.5 Summary

1. Germline epimutations of APC are not associated with the development of familial polyposis in APC mutation-negative cases.

2. Within cancers occurring in the setting of polyposis, hypermethylation of APC is inversely correlated with BRAF mutations and CIMP-positive phenotype.

3. Methylation of APC in individuals with a polyposis condition increases with age, and may represent a field defect that predisposes to the development of colorectal neoplasia.

216 Conclusions from this thesis The overall aim of the work undertaken in this thesis has been to better understand the role of DNA methylation in the development of colorectal neoplasia.

Methylation of the mismatch repair gene MLH1 underlies the development of over 80% of sporadic MSI colorectal cancers. However, there has been little knowledge regarding the patterns of MLH1 methylation in the tissues of individuals with this type of cancer. Furthermore, there has been some contention in the literature regarding the presence of MLH1 methylation in the cancer and normal colonic mucosa of individuals with MSS cancers. This reflects the lack of specificity of methods for the detection of DNA methylation, as well as assessment of CpG methylation at regions of the MLH1 promoter that did not correlate with MLH1 protein expression. The present study found that a sensitive method such as the qMSP assay is suited for the determination of MLH1 loss in sporadic MSI colorectal cancers, provided that it is used to assess MLH1 promoter methylation at appropriate regions (MLH1-C). In addition, work in this thesis refutes the claim by others that MLH1-C methylation in the normal colonic mucosa is not specifically associated with the development of MSI colorectal cancer (Nakagawa et al., 2001; Minoo et al., 2006). In fact, low levels of MLH1-C methylation were found only in the normal mucosa samples of individuals with MSI cancers. This may represent a field defect, and potentially may predispose to the development of additional MSI cancers in these individuals. No MLH1-C methylation was identified in peripheral blood mononuclear cells of individuals with colorectal cancer, indicating that analysis of this tissue would not be useful as a marker of risk for sporadic MSI colorectal cancer.

Another goal of this thesis was to assess the presence of low levels and mosaic patterns of MLH1 methylation in the constitutional DNA of family members of individuals (proband A, proband B and individual II6-A) with a germline epimutation of MLH1, particularly those who carry the same epimutant allele. These patterns of MLH1 methylation may indicate an incomplete reversal of an epimutation in individuals who harboured an MLH1 allele that is susceptible to an epimutation, or a cis-acting defect that promotes subsequent methylation of an epimutant allele. No traces of MLH1 methylation were found in the tissue samples from any of the family members of individuals with germline epimutations of MLH1 suggesting a complete reversal of epimutations in these individuals.

217 A recent study by our laboratory reported that MLH1 methylation occurs concomitantly with that of flanking genes within the 3p22 chromosomal domain in sporadic MSI and BRAF V600E mutant CRCs. Building on previous work from this laboratory on regional methylation (Hitchins et al., 2007b), work in this thesis assessed the relationship between regional methylation of 3p22 genes (methylation of three of five 3p22 genes) and common clinicopathological features of colorectal cancer. This work not only confirmed the strong association between regional 3p22 methylation, MSI and BRAF V600E mutations, but showed that this phenomenon is not MSI-specific. In fact, approximately 12% of cancers showing regional 3p22 methylation (in the absence of MLH1 methylation) demonstrated the MSS phenotype in conjunction with the BRAF V600E mutation. In addition, regional 3p22 methylation correlated strongly with the CIMP+ve phenotype, and its related clinicopathological and molecular features. It is clear from this work that 3p22 methylation and CIMP+ve are overlapping phenomena. On a broader perspective, the CIMP+ve phenotype may be more appropriately conceptualised as the presence of areas of long-range methylation of multiple chromosomal regions, including but not limited to regions where current markers for CIMP reside. Interestingly, the presence of regional 3p22 methylation may be a useful tool to predict individuals with better outcome among those individuals with MSS, BRAF V600E mutant cancers. This finding will require confirmation by analysis in separate colorectal cancer cohorts.

To further advance knowledge of regional 3p22 methylation in sporadic colorectal cancer, work in this thesis assessed this phenomenon in normal colorectal mucosa. Consistent with findings in cancer, regional 3p22 methylation in normal mucosa was strongly associated with the presence of the BRAF V600E mutation. This association was more likely in individuals with an increased number of serrated polyps in their large bowel. This finding suggests that regional 3p22 methylation and the BRAF V600E mutation may predispose to the development of neoplastic lesions within the serrated neoplasia pathway including hyperplastic polyps and serrated adenomas, as well as sporadic MSI and CIMP+ve cancers. From the point of view of future studies, the findings arising from this thesis may facilitate the development of specific cancer chemopreventive agents for use on those individuals who are predisposed to colorectal tumours of the serrated neoplasia pathway.

218 Work in this thesis has also investigated the role of APC methylation in the development of a broad range of polyposis conditions, including FAP, AFAP and hyperplastic polyposis. While germline sequence mutations of APC underlie the development of the majority of FAP and AFAP cases, the present study has shown that germline epimutations of APC do not contribute towards the development of these polyposis conditions. Further studies may be required to determine the molecular defect that underlie cases of polyposis in which no APC mutations have been identified by standard genetic screening.

Finally, work undertaken in this thesis has supported the finding that hypermethylation of APC in colorectal tumours is inversely correlated with the BRAF V600E mutation and CIMP-positive phenotype (Iacopetta et al., 2006). Methylation of APC may increase with age in the normal colonic mucosa of individuals with a polyposis condition, and this may predispose to the development of at least some of the colorectal neoplasms that demonstrate inverse correlations with the BRAF V600E mutation and CIMP-positive phenotype.

Taken together, the results presented in this thesis indicate the important role of hypermethylation of certain genes in the development of various subtypes of colorectal cancer. During the course of this thesis, several reports have been published by others, highlighting the importance of field defects characterised by either DNA methylation (Shen et al., 2005; Belshaw et al., 2008) or abnormal changes in protein expression (Polley et al., 2006) in the development of sporadic colorectal cancer. The work in this thesis that focused on well-established cancer-related genes (MLH1 and APC) clearly affirms that aberrant DNA methylation marks may indicate the emergence of focal lesions in the mucosa field adjacent to a pre-existing sporadic and possibly familial colorectal cancer as well.

The additional knowledge acquired from the work in this thesis may have important implications in understanding the pathogenesis of different ccolorectal ancer subtypes. Ultimately, it may help in the future assessment and management of individuals with colorectal cancer by identifying the risk of the development of metachronous neoplasms in these individuals, and by suggesting novel molecular targets for cancer therapy.

219 Future Research Directions

1. Methylation of MLH1 and other genes within the 3p22 chromosomal region may occur in the normal colonic mucosa as a field defect in the development of sporadic MSI colorectal cancer. A well-planned longitunal study would be important to determine whether individuals who had sporadic MSI colorectal cancer are at a high risk of developing additional colorectal neoplasia with a similar phenotype.

2. The work in this thesis proposed that CIMP may be a phenomenon that encompassed long range epigenetic silencing of numerous chromosomal regions. This requires confirmation through genome-wide profiling of methylated markers and the understanding of their functional significance in colorectal cancer. In addition, it may be worthwhile to investigate whether long range epigenetic silencing would occur at different chromosomal regions in cancers with CIMP-ve phenotype. This would provide better knowledge into different pathways that could lead to the development of colorectal cancer and potential targets for therapy.

3. In general, work presented in this thesis was limited by the small number of normal colonic mucosa samples or the unavailability of pre-cancerous lesions from individuals with colorectal neoplasia. Hence, it would be important to design a large population prospective study which would better address the role of field defects in the development of different types of colorectal neoplasia. Future studies are also needed to describe the mechanisms (such as environmental factors and their interaction with genotype or epigenetic changes) that can promote field defect in the development of colorectal neoplasia.

4. The work presented here suggested that the intensity of APC methylation may increase with age in the normal colonic mucosa of individuals with polyposis. Further studies using animal models may help in determining a better relationship between APC methylation, aging and the development of neoplastic lesions.

220 Appendix 1

Suppliers of Equipment and Reagents

Supplier Address Ajax Auburn, NSW, Australia Amresco Solon, Ohio, USA Applichem GmbH Darmstadt, Germany Applied biosystem Scoresby, VIC, Australia Bacto Liverpool, NSW, Australia Baxter Old Toongabbie, NSW, Australia BDH Dorset, United Kingdom Bio-rad Hercules, CA, USA Biotage Uppsala, Sweden Chemicon Temecula, CA, USA DNAStar Inc. Madison, WI, USA Epicentre Biotechnologies Madison, WI, USA Fermentas Glen Burnie, MD, USA Geneworks Adelaide, SA, Australia Gilson Middleton, WI, USA Invitrogen, USA Calsbard, CA, USA Milipore North Ryde, NSW, Australia NanoDrop Technologies Inc. Wilmington, DE, USA New England Biolabs Ipwich, MA, USA Probiogen Berlin, Germany Promega Annandale, NSW, Australia Qiagen GmbH Hilden, Germany Roche Basel, Switzerland Sigma Castle Hill, VIC, Australia SPSS Inc. Chicago, IL, USA Trilink Biotechnologies San Diego, CA, USA Whatman Clifton, NJ, USA Geneworks Adelaide, SA, Australia

221 Appendix 2

Publications arising from this thesis

1. Hitchins, M., Suter, C., Wong, J., Cheong, K., Hawkins, N., Leggett, B., Scott, R., Spigelman, A., Tomlinson, I., Martin, D. and Ward, R. (2006). Germline epimutations of APC are not associated with inherited colorectal polyposis. Gut. 55: 586-7.

2. Wong, J.J., Hawkins, N.J. and Ward, R.L. (2007). Colorectal cancer - a model for epigenetic tumourigenesis. Gut. 56: 140-148.

3. Hitchins, M.P., Wong, J.J., Suthers, G., Suter, C.M., Martin, D.I., Hawkins, N.J. and Ward, R.L. (2007). Inheritance of a Cancer-Associated MLH1 Germ-Line Epimutation. N Engl J Med. 356: 697-705.

4. Wong, J.J., Hawkins, N.J., Ward, R.L. and Hitchins, M.P. (2008). Regional methylation of genes within the 3p22 chromosomal region and the development of serrated colorectal neoplasia. Manuscript in preparation.

5. Wong, J.J., Hawkins, N.J., Tomlinson, I., Leggett, B., Scott, R., Ward, R.L. and Hitchins, M.P. (2008). Somatic methylation of APC as a field defect in the development of colorectal polyposis. Manuscript in preparation.

222 Appendix 3

List of poster presentations related to this thesis

1. Wong, J.J., Hitchins, M.P., Hawkins, N.J. and Ward, R.L. A sensitive and accurate real-time PCR assay for quantifying MLH1 promoter methylation in colorectal cancer (2005). The 15th St. Vincent’s & Mater Health Sydney Research Symposium 2005, The Garvan Institute, Sydney, Australia.

2. Wong, J.J., Hitchins, M.P., Hawkins, N.J. and Ward, R.L. A sensitive and accurate real-time PCR assay for quantifying MLH1 promoter methylation in colorectal cancer (2006). The Australian Society for Medical Research NSW Scientific Meeting, Sydney Australia.

3. Wong, J.J., Hitchins, M.P., Hawkins, N.J. and Ward, R.L. Hypermethylation of MLH1 promoter in the normal colonic mucosa of individuals with colorectal cancer (2006). The 15th St. Vincent’s & Mater Health Sydney Research Symposium 2006, The Garvan Institute, Sydney, Australia.

4. Wong, J.J., Hitchins, M.P., Ward, R.L. and Hawkins, N.J. Methylation of MLH1 and flanking genes in the normal colonic mucosa of individuals with sporadic colorectal cancer (2007). Lorne Genome Conference, Melbourne, Australia.

5. Wong, J.J., Hawkins, N.J., Ward, R.L. and Hitchins, M.P. Methylation of a cluster of 3p22 encompassing MLH1 occurs in conjunction with BRAF V600E mutation early in colorectal cancer development (2007). Epigenetic 2007 Australian Scientific Conference, Perth, Australia.

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