Biomarkers and Risk Stratification in Colorectal Neoplasia
A thesis submitted to the University of Manchester for the degree of Doctor of Medicine in the Faculty of Biology, Medicine and Health.
2019
Lyndsay E Pearce BMedSci BMBS FRCS
School of Medical Sciences
Table of Contents
1 Introduction ...... 18 1.1 Pre symptomatic diagnosis and screening for Colorectal Cancer ...... 21 1.1.1 Background to Colorectal Cancer Screening ...... 21 1.1.2 The UK National Bowel Cancer Screening Programme ...... 23 1.1.3 Faecal Occult Blood Testing ...... 23 1.1.4 Faecal immunohistochemical testing ...... 24 1.1.5 Flexible sigmoidoscopy ...... 25 1.1.6 Individualising risk and colorectal cancer screening ...... 26 1.1.7 Moderate Familial Risk ...... 26 1.1.8 Screening in familial colorectal cancer syndromes ...... 27 1.2 Single Gene disorders ...... 29 1.2.1 Familial Adenomatous Polyposis ...... 29 1.2.1.1 Introduction and History ...... 29 1.2.1.2 Variation in phenotypic features ...... 30 1.2.1.3 Desmoid Tumours ...... 31 1.2.1.4 Polyp surveillance in FAP and AFAP ...... 32 1.2.2 Lynch Syndrome ...... 33 1.2.1.1 Introduction and History ...... 33 1.2.1.2 Diagnosis of Lynch Syndrome ...... 34 1.2.1.3 Amsterdam/Bethesda Criteria ...... 37 1.2.1.4 Other predictive models ...... 40 1.2.1.5 Genetic modifiers ...... 41 1.2.1.6 Surveillance and screening in Lynch Syndrome ...... 42 1.2.3 Polyposis Phenotype ...... 45 1.2.3.1 Hyperplastic Polyposis ...... 45 1.2.3.2 Multiple Adenoma Phenotype ...... 46 1.2.3.2.1 Oligopolyposis ...... 46 1.2.3.2.2 MUTYH associate polyposis ...... 47 1.2.3.2.3 NTHL1-associated polyposis ...... 48 1.2.3.3 Peutz Jeghers Syndrome ...... 48 1.2.3.4 Juvenile Polyposis Syndrome ...... 48 1.2.3.5 Cowden’s Syndrome ...... 49 1.3 Diagnosis and Staging of Colorectal Cancer ...... 50 1.3.1 Imaging and diagnosis of CRC ...... 50 1.3.2. Pathology of Colorectal Cancer ...... 53 1.3.2.1 Adenocarcinoma ...... 53 1.3.2.2 Precursor lesions (polyps) ...... 53 1.3.2.2.1 Adenomas ...... 53 1.3.2.2.2 Serrated Adenomas ...... 54 1.3.2.2.3 Hamartomas ...... 55 1.3.2.2.4 Inflammatory Polyps ...... 56 1.3.3 Staging of colorectal cancer ...... 56 1.3.3.1 Radiological staging ...... 57 1.3.3.1.1. PET CT ...... 58 1.4 Treatment of Colorectal Cancer ...... 58 1.4.1. Early colorectal cancer ...... 58 1.4.2 Stage II , III and IV Colorectal cancer ...... 58 1.4.3 Treatment considerations in Lynch Syndrome ...... 59 1.4.3.1 Prophylactic Surgery ...... 60 1.4.3.2 Adjuvant Therapy ...... 61 1.4.3.2.1 Fluorouracil-based adjuvant chemotherapy ...... 62
2 1.5 Follow-up after resection for Colorectal Cancer ...... 64 1.5.1 Carcinoembryonic Antigen ...... 64 1.5.2 CT scanning ...... 65 1.5.3 Positron Emission Tomography (PET CT) ...... 65 1.5.4 Surveillance programmes ...... 66 1.5.5 Colorectal liver metastases ...... 68 1.6 Colorectal Cancer Molecular Genetics ...... 69 1.6.1 Fearon and Vogelstein’s genetic model for tumourigenesis .... 70 1.6.2 Jass’ molecular classification of colorectal cancer ...... 70 1.6.3 Genetic Instability ...... 71 1.6.3.1 Epigenetics ...... 71 1.6.3.2 Jass’ Classification ...... 72 1.6.3.2.1 The Microsatellite Instability Pathway ...... 73 1.6.3.2.2 The Chromosomal Instability Pathway ...... 73 1.7 Principles of Biomarkers ...... 74 1.7.1 Categorisation of Biomarkers ...... 75 1.7.2 Biomarker Discovery ...... 76 1.7.3 Biomarker Validation ...... 77 1.7.4 Biomarker discovery roadmaps ...... 77 1.7.5 Biomarkers in Colorectal Cancer ...... 77 1.7.5.1 Risk stratification (predisposition) biomarkers ...... 78 1.7.5.2 Screening and early detection biomarkers ...... 78 1.7.5.3 Diagnostic Biomarkers ...... 79 1.7.5.4 Pharmacological Biomarkers ...... 79 1.7.5.5 Predictive Biomarkers ...... 79 1.7.5.5.1 KRAS ...... 80 1.7.5.6 Population based screening biomarkers ...... 81 1.7.5.7 Predictive value of MSI in chemotherapy ...... 81 1.7.5.8 Circulating Tumour Cells ...... 82 1.7.5.9 Tumour biology ...... 83 1.7.5.9.1 Beta-2 microglobulin as a biomarker in MMR-deficient CRC 84 1.7.5.9.2 Immune response to tumour cells ...... 85 1.7.5.9.3 Natural Killer Cells ...... 87 1.8 Common genetic variants and Colorectal Cancer ...... 88 1.8.1 Genome Wide Association Studies ...... 88 1.8.2 Single Nucleotide Polymorphisms ...... 89 1.8.3 Polygenic risk ...... 89 1.8.4 Genetic Linkage analysis ...... 90 1.8.4.1 Common Disease/Common Variant Hypothesis ...... 90 1.8.4.2 Linkage disequilibrium ...... 91 1.8.4.3 Direct and Indirect Association ...... 91 1.8.5 GWAS and colorectal cancer ...... 92 1.8.5.1 History and Discovery of SNPs in colorectal cancer ...... 94 1.8.6 Functions of CRC association single nucleotide polymorphisms ..... 95 1.8.6.1 BMP ...... 95 1.8.6.2 TERC ...... 96 1.8.7 Population specific risk ...... 96 1.8.7.1 CRC risk and SNPs in high risk populations ...... 97 1.8.7.2 Lynch syndrome and susceptibility loci ...... 98 1.8.8 SNPs and adenoma ...... 99 1.8.8.1 Polyposis Phenotype ...... 100 1.8.8.1.1 FAP ...... 100 1.8.8.1.2 Oligopolyposis ...... 101 1.8.9 Patterns of disease and types of disease ...... 102
3 1.8.9.1 Protective effects ...... 103 1.8.9.2 Long term survival and early onset colorectal cancer ...... 104 STUDY ONE ...... 105 The effect of intensive CT scanning in surveillance to detect the recurrence of colorectal cancer ...... 105 2.1 ABSTRACT ...... 106 2.2 Introduction ...... 108 2.2.1 Background ...... 108 2.2.1.1 Purpose of colorectal cancer follow-up ...... 109 2.2.1.2 Follow up regimens ...... 109 2.3 Methods ...... 111 2.3.1 Statistical Analysis ...... 111 2.4 Results ...... 112 2.4.1 Demographics ...... 112 2.4.2 Compliance with follow up ...... 112 2.4.3 Recurrent disease ...... 112 2.4.5 Limitations of the study ...... 113 2.5 Discussion ...... 114 STUDY TWO ...... 122 Analysis of the effect of single nucleotide polypmorphisms on age of onset of colorectal cancer in patients with Lynch Syndrome (hereditary nonpolyposis colorectal cancer) ... 122 3.1 ABSTRACT ...... 123 3.2 Introduction ...... 125 3.2.1 Current screening guidelines for Lynch Syndrome Patients ...... 125 3.2.2 Single Nucleotide Polymorphisms and risk prediction ...... 126 3.2.2.1 Single Nucleotide Polymorphisms and Colorectal Cancer ...... 127 3.2.2.2 Single Nucleotide Polymorphisms in other high-risk cancer patients 127 3.2.2.3 Single nucleotide polymorphisms in Lynch Syndrome ...... 127 3.2.3 Polymerase chain reaction (PCR) ...... 128 3.2.4 Genotyping Techniques ...... 129 3.2.4.1 Sanger Sequencing ...... 129 3.2.4.2 Sequenom ...... 130 3.2.5 Polygenic Risk Scores ...... 131 3.2.6 Hypothesis ...... 131 3.2.7 Ethical Approval ...... 131 3.3 Methodology ...... 132 3.3.1 Participants ...... 132 3.3.2 SNP Panel design ...... 133 3.3.3 Sequenom PCR Primers ...... 135 3.3.4 Coriell Cell Line Sanger Sequencing ...... 137 3.3.4.1 Purification of PCR products prior to Sanger sequencing ...... 137 3.3.5 Sequenom Assay Development ...... 139 3.3.5.1 Samples ...... 139 3.3.5.2 Amplification of target loci by PCR ...... 139 3.3.5.3 Post PCR; SAP reaction clean-up ...... 139 3.3.5.4 Primer Extension (iPLEX Pro extend reaction) ...... 139 3.3.5.5 Primer extension reaction resin clean up ...... 140
4 3.3.5.6 Spotting primer extension products on SpectroCHIPs ...... 140 3.3.5.7 Detection of primer extension products by mass spectrometry 140 3.3.6 Data analysis ...... 141 3.3.7 DNA Samples ...... 141 3.3.8 PCR Primer multiplex optimisation ...... 142 3.3.9 Extension Primer Optimisation ...... 143 3.3.10 Redesigning of SNP Panel ...... 143 3.3.11 Clinical Data ...... 145 3.3.12 Genotyping ...... 145 3.3.13 Overall Colorectal Cancer Risk Score (OCRS) ...... 148 3.4 Results ...... 150 3.4.1 Control Group ...... 153 3.4.2 Colorectal cancer cases ...... 154 3.4.3 Overall colorectal cancer risk scores ...... 155 3.4.4 Age of Onset of Disease ...... 155 3.4.4.1 Sporadic Colorectal cancer cases ...... 155 3.4.4.2 MLH1 Mutation carriers ...... 156 3.4.4.2.1 Female carriers ...... 158 3.4.4.2.2 Male Carriers ...... 160 3.4.4.3 MSH2 Carriers ...... 161 3.4.4.3.1 Female carriers ...... 163 3.4.4.3.2 Male carriers ...... 165 3.5 Discussion ...... 166 3.5.1 Sporadic Colorectal Cancers ...... 167 3.5.2 High-risk mutation carriers ...... 167 3.6 Limitations ...... 169 3.7 Conclusions ...... 169 3.7.1 Further work and recommendations ...... 170 STUDY THREE ...... 171 Beta-2 microglobulin as a prognostic biomarker in mismatch repair deficient colorectal cancer ...... 171 4.1 Abstract ...... 172 4.2 Introduction ...... 173 4.2.1 Mismatch Repair deficient colorectal cancer (dMMR) ...... 173 4.2.2 Beta-2 microglobulin ...... 174 4.2.3 HLA class I complex ...... 175 4.2.4 Immune-Escape Phenotype ...... 176 4.2.5 Tumours and Natural Killer cells ...... 176 4.2.6 The B2M gene ...... 177 4.2.6.1 B2M mutations in CRC ...... 178 4.3 Hypothesis and aims ...... 181 4.4 METHODOLOGY ...... 181 4.4.1 Ethical approval ...... 181 4.4.2 Participants ...... 182 4.4.3 Tissue specimens and DNA Samples ...... 182 4.4.4 Tissue specimen preparation ...... 183 4.4.5 Sanger sequencing ...... 184 4.4.5.1 Primer design ...... 184 4.4.5.2 Polymerase Chain Reaction (PCR) ...... 185 4.4.5.3 PCR conditions for B2M ...... 186
5 4.4.5.4 Gel electrophoresis of PCR product ...... 186 4.4.5.4.1 Running samples on the gel ...... 186 4.4.5.4.2 Analysis of the gel ...... 187 4.4.5.5 Agencourt AMPure XP Purification protocol ...... 187 4.4.5.6 Sequencing PCR products ...... 188 4.4.5.7 Agencourt CleanSEQ 384 Protocol ...... 188 4.4.6 Genotyping ...... 189 4.4.7 Immunohistochemistry ...... 189 4.4.7.1 Development of the IHC Scoring System ...... 190 4.4.7.2 Clinical Data ...... 192 4.4.8 Statistical Considerations ...... 192 4.5 Results ...... 192 4.5.1 B2M mutation frequency ...... 192 4.5.2 Effect of B2M mutation on recurrence ...... 193 4.5.3 Immunohistochemistry results ...... 194 4.6 Discussion ...... 194 STUDY FOUR ...... 197 Oligopolyposis within the bowel cancer screening programme; implications for regional genetics centres ... 197 5.1 Abstract ...... 198 5.2 Introduction ...... 200 5.2.1 The NHS Bowel Screening Programme ...... 200 5.2.2 Adenoma Surveillance ...... 201 5.2.3 Faecal Immunohistochemical Testing ...... 201 5.3 Hypothesis and aims ...... 203 5.4 Methods ...... 204 5.5 Results ...... 205 5.5.1 Endoscopic findings ...... 205 5.5.2 Histological findings ...... 206 5.5.3 Referral to regional genetics service ...... 206 5.5.4 National use of guidelines for referral of patients with oligopolyposis to regional genetic service ...... 207 5.6 Discussion ...... 208 6 Overall Conclusion ...... 212 References ...... 215
6 List of Figures Figure 1. Tubulovillous Adenoma (H&E stain – tubular component left of image, villous component right of image) ...... 54 Figure 2. Polyp of Sigmoid Colon ...... 54 Figure 3. Sessile Serrated adenoma ...... 55 Figure 4. Beta-2-Microglobulin ...... 85 Figure 5; Distribution of colonic resections ...... 117 Figure 6. Number of recurrences of colorectal cancer detected by CT during follow up by Dukes’ Stage ...... 117 Figure 7. Proportion (%) of colorectal cancer recurrence detected by CT scanning during follow up ...... 118 Figure 8. Site of distant recurrence of colorectal cancer by Dukes’ Stage ...... 119 Figure 9. Incidences of recurrence of colorectal cancer by Dukes’ stage of disease ...... 119 Figure 10. Cumulative overall survival by Dukes’ stage ...... 120 Figure 11. Purification of PCR products – Agencourt AMPure ...... 138 Figure 12. Gel Electrophoresis for rs1169552 ...... 144 Figure 13; Example of suboptimal sequenom analysis of the spectrochip indicated by traffic light colour coded plate map ...... 146 Figure 14; Example of optimal sequenom analysis of the spectrochip indicated by traffic light colour coded plate map ...... 146 Figure 15; Example of unextended primer ...... 147 Figure 16; Example of poor amplification ...... 147 Figure 17; Example of Typer 4.0 Analyser output including cluster plots, spectra and peak information ...... 148 Figure 18. Study consort diagram ...... 151 Figure 19; Age distribution across risk quintile for control population ...... 153 Figure 20. Age distribution across risk quintile for Sporadic ...... 156 Figure 21; Age distribution across risk quintile for MLH1 mutation carriers colorectal cancer population ...... 157 Figure 22. Cumulative hazard of developing colorectal cancer in 162 MLH1 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs...... 158 Figure 23; Cumulative hazard of developing colorectal cancer in 84 Female MLH1 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs...... 159 Figure 24; Cumulative hazard of developing colorectal cancer in 78 Male MLH1 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs...... 161 Figure 25. Age distribution across risk quintiles for MSH2 mutation carriers ... 162 Figure 26; Cumulative hazard of developing colorectal cancer in MSH 2 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs ...... 163 Figure 27; Cumulative hazard of developing colorectal cancer in 133 Female MSH 2 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs ...... 164 Figure 28; Cumulative hazard of developing colorectal cancer in 75 male MSH 2 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs ...... 166 Figure 29; B2m Gene (chr 15q21) (from Alamut v2.2) ...... 178 Figure 30; Cancer Research UK Prognostic / Predictive Biomarker Roadmap .. 180 Figure 31; Plate WS74764; Example of electrophoresis gel (11 samples and 1 coriell cell line control) ...... 187 Figure 32; B2M expressivity scoring scale ...... 191 Figure 33; British Society of Gastroenterology Guidelines on Surveillance after colorectal adenoma removal...... 202
7 List of Tables Table 1. Wilson and Jungner Screening Criteria ...... 22 Table 2. Risk of developing Colorectal Cancer based on family history ...... 27 Table 3. BSG Guidelines for colorectal cancer screening in high-risk groups .... 28 Table 4. Manchester Colorectal Cancer Pathway Group Guidelines for groups to be assessed for MMR status ...... 37 Table 5. Amsterdam II criteria developed to identify patients with Lynch Syndrome ...... 38 Table 6. Revised Bethesda Criteria ...... 39 Table 7. Risk prediction models of dMMR CRC ...... 41 Table 8. Colorectal cancer staging and survival ...... 52 Table 9. Nice Guidelines CG131. Section 1.4.1. Staging of Colorectal Cancer. December 2014 ...... 56 Table 10. Nice Guidelines CG131. Section 1.4.1. Follow up after apparently curative resection. December 2014 ...... 65 Table 11. Indications for the use of PET-CT in colorectal cancer ...... 66 Table 12. SNPs with statistically significant association with CRC risk. Lubbe et al 2012...... 103 Table 13. Rates of detectable recurrent disease in randomised studies prior to and after the introduction of CT scanning as routine follow up for colorectal cancer. * Pelvic CT for patients who underwent Abdominoperineal resection only. ** Liver CT in combination with CXR and colonoscopy ...... 121 Table 14. SNP panel design ...... 134 Table 15; Rehydration of SNP primers ...... 136 Table 16. Standard PCR mix ...... 137 Table 17; Optimisation SAP Mix ...... 142 Table 18; Optimisation of assay ...... 142 Table 19; Optimisation iPLEX Mix ...... 143 Table 20. Patients demographics of MLH1, MSH2, Sporadic CRC and Control groups. Ages are shown as mean values and ranges...... 151 Table 21. Polygenic Risk scores calculations for each SNP tested and Control risk allele frequencies. *Case and Control MAF from Lubbe et al. Relationship between 16 susceptibility loci and colorectal cancer phenotype in 3146 patients...... 152 Table 22; Number of colorectal cancers per population per quintile ...... 154 Table 23. Comparison of overall colorectal cancer score (polygenic risk score) between population groups ...... 155 Table 24. Hazard ratios from the cox model for age at the development of colorectal cancer in MLH1 mutation carriers by overall colorectal cancer risk score (split into quintiles) ...... 157 Table 25. Hazard ratios from the cox model for age at the development of colorectal cancer in female MLH1 mutation carriers by overall colorectal cancer risk score (split into quintiles) ...... 159 Table 26. Hazard ratios from the cox model for age at the development of colorectal cancer in Male MLH1 mutation carriers by overall colorectal cancer risk score (split into quintiles) ...... 160 Table 27. Hazard ratios from the cox model for age at the development of colorectal cancer in MSH2 mutation carriers by overall colorectal cancer risk score (split into quintiles) ...... 162 Table 28. Hazard ratios from the cox model for age at the development of colorectal cancer in Female MSH2 mutation carriers by overall colorectal cancer risk score (split into quintiles) ...... 164 Table 29. Hazard ratios from the cox model for age at the development of colorectal cancer in Male MSH2 mutation carriers by overall colorectal cancer risk score (split into quintiles) ...... 165 Table 30; Primers of all three exons of the B2M gene (Sigma-Aldrich) ...... 185 Table 31; Thermal cycling conditions for PCR...... 186
8 Table 32; Thermal cycling conditions for sequencing PCR...... 188 Table 33; Description and frequency of B2M mutations identified in the MSH2 dMMR CRC samples ...... 193 Table 34; Summary of studies comparing outcome of dMMR CRC based on B2M mutation status ...... 196 Table 35; Application criteria for accreditation as BSCP screening colonoscopist through Screening Assessment and Accreditation System (SAAS)...... 201 Table 36; Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups16 ; Guidance on Gastrointestinal Surveillance for High Risk Genetic Disorders ...... 210
Word Count 51,542
9 List of Appendices
Appendix 1; Manchester Cancer Guidelines for the assessment of mismatch repair (MMR) status in Colorectal Cancer. May 2015, Version 3………..……….….231
Appendix 2; Genotype frequencies; Wild type, Heterozygous and Homozygous for control group, Sporadic colorectal cancer group and MLH1 and MSH2 mutation carriers………………………………………..………………………………………………..……232
Appendix 3; Freedom of Information Request; Trust guidelines for referral to genetics services after colonoscopy undertaken as part of the UK Bowel Cancer Screening Programme………………….………………………………………………………….………..233
Appendix 4; BCSP centres sent Freedom of Information requests………………….234
10
Abstract
Over the last decade there have been considerable advances in the concept of personalising and individualising risk across a number of different cancer groups to identify more effective strategies for disease prevention, screening, treatment and prevention of recurrence.
Colorectal cancer (CRC) is a heterogenous disease and the second most common cause of cancer death in the UK. Early detection of disease through the NHS Bowel Cancer Screening Programme (NHS BCSP) enables identification of cancers at an earlier stage with wider ranging treatment options and potentially less risk of recurrent disease.
Hereditary colorectal cancer syndromes account for approximately 5% of all colorectal cancers. Lynch Syndrome patients exhibit DNA mismatch repair deficiency and have up to an 80% lifetime risk of colorectal cancer (compared to 5% in the background population) with wide ranging age of onset of disease.
This thesis consists of four related studies. The purpose of the research was to conduct a thorough literature review and investigate different aspects relevant to individualisation of risk in patients with or at risk of colorectal cancer. These include; single nucleotide polymorphisms (SNPs) and their role in predicting age of onset of colorectal cancer in patients with Lynch Syndrome (LS); follow up after curative resection for sporadic colorectal cancer; Beta-2 Microglobulin (B2M) mutation status and the impact on prevalence of recurrent disease in patients with MSH2 loss and finally oligopolyposis detection through the NHS BCSP and role of regional genetic centres in the management of these patients.
Incidence of oligopolyposis within the bowel cancer screening programme is small (0.79%) but there is a high incidence (50%) of synchronous or high grade dysplasia in these patients. There are no local, regional or national guidelines for the management of patients with oligopolyposis detected through the NHS BCSP. SNPs associated with sporadic CRC were not demonstrated to have a multiplicative effect on age of onset and risk of developing colorectal cancer in patients with MLH1 and MSH2 loss.
11 In sporadic colorectal cancer, a high proportion of patients who develop recurrent colorectal cancer do so in the first three years after surgery (95%) and have surgically treatable disease (36.6%). Intensive CT imaging is demonstrated to be pragmatic in clinical practice. There was no disease recurrence in patients with colorectal cancer, MSH2 loss and B2M mutation.
CRC is a heterogenous disease with implications for familial risk and, in some patients, other syndrome specific malignancies. Individualising and stratifying risk is appropriate in high risk and sporadic populations, throughout screening, management and surveillance of the disease within to optimise clinical outcomes for patients.
12 Declaration
No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning
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13 Acknowledgements
The molecular genetics work conducted as part of this thesis was undertaken in the Molecular Genetics laboratories at St Mary’s hospital, Central Manchester Foundation Trust. I would like to acknowledge Dr. Andrew Wallace (Consultant Clinical Scientist), Kerry Bean and Salqa Pervez (Genetic Technologists) for their assistance and guidance with SNP panel design, PCR optimization and in development of sequenom assay.
The histopathology work conducted as part of this thesis was performed in the pathology laboratory at Manchester Royal Infirmary, Central Manchester Foundation Trust. I would like to acknowledge Dr. Lucy Foster (Consultant Histopathologist) and Catherine Keeling (Advanced Biomedical Scientist) for their assistance.
Thank you to Dr. Steve Roberts (Applied Biostatistician, University of Manchester) and Dr. Elaine Harkness (Research Fellow, University of Manchester) for their advice and support statistics.
Thank you also to my supervisors, Prof. James Hill (Consultant Colorectal Surgeon, Manchester Royal Infirmary) and Prof. D. Gareth Evans (Consultant in Genetic Medicine, St Mary’s Hospital Manchester) for their guidance, understanding and support throughout my thesis. Thank you to Kate Green (Database Manager, St Mary’s Hospital) for her patience and for helping me access the data.
Finally, thank you to my family for encouraging me. To my mum for her time and constant support, to my partner Tom and children Theo and Lucia for understanding and always making me smile, and to our dog Winston for his hours of companionship in writing this thesis.
14 Preface
The author of this thesis graduated from the University of Nottingham Medical School in 2004 with BMBS and BMedSci and intention to pursue a career in surgery. She was first appointed as pre-registration house officer (PRHO) at Derby City General Hospital. She underwent basic surgical training at University Hospitals Leicester before relocating to Manchester in August 2007 following introduction of “Modernisation of Medical Careers’ (MMC).
In 2008, she entered higher surgical training in the North West Deanery working at a number of hospitals across the region. She had her first child in 2009 and returned to surgical training. She declared Colorectal Surgery as her chosen subspecialty in 2012. During her training she developed a strong interest in clinical research and with the North West Research collaborative developed an NIHR funded national clinical observational study, and subsequent randomised controlled trial.
In 2013 she took 2 years out of higher surgical training to conduct research into risk stratification in patients with, or at risk of, colorectal cancer. During this period of research she gained experience and techniques in molecular genetics and skills including sequencing using Sequenom and immunohistochemistry. She also developed research skills in data handling and statistical analysis relevant to other areas of her ongoing clinical research. She had her second child in 2015, and returned to full time clinical practice prior to completion of the research, to finish her Higher Surgical Training. She was awarded the Lady Estelle Wolfson Emerging Leaders Fellowship by the Royal College of Surgeons in 2016 for outstanding leadership in various aspects of surgery. She was then awarded Young Coloproctologist of the Year 2017 by the Association of Coloproctology of Great Britain and Ireland for her performance throughout her training and contributions and excellence in the areas of research and academia, leadership or education and training.
She was appointed as Consultant Colorectal and General Surgeon at Salford Royal Hospital in 2018 and completed her writing for this thesis. She continues to develop her clinical research interests in Frailty, Acute Care and Colorectal Surgery.
15
Presentations relating to this research
Oral Presentations
International: Analysis of the effect of single nucleotide polymorphisms on age of onset of colorectal cancer in patients with lynch syndrome (hereditary non-polyposis colorectal cancer). L Pearce, K Bean, A Wallace, J Hill, DG Evans. Presented by L Pearce. Presented at International Society for Gastrointestinal Hereditary Tumours (InSiGHT) Biennial Conference, Palazzo dei Congressi, Florence, Italy, 8th July 2017.
Regional: Is multiple SNP testing in MLH1 and MSH2 carriers ready for use in clinical practice? Results from a large genetic centre in the UK. L Pearce, K Bean, A Wallace J Hill, DG Evans. Presented by L Pearce. Presented at Manchester Medical Society, Section of Surgery meeting, Chancellors Conference Centre, Manchester, March 2016.
• Awarded Manchester Medical Society, Section of Surgery, Trainee Prize March 2016
Poster Presentations
International:
The Value of CT Scanning following Curative Resection for Colorectal Cancer. L Pearce, J Law, S Lee, J Hill. American Society of Colon and Rectal Surgeons, Seattle, WA June 2017
Analysis of the effect of single nucleotide polymorphisms on age of onset of colorectal cancer in patients with Lynch Syndrome (hereditary non-polyposis colorectal cancer). L Pearce, K Bean, A Wallace, J Hill, DG Evans.
16 American Society of Colon and Rectal Surgeons, Seattle, WA June 2017
National:
The Value of CT Scanning following Curative Resection for Colorectal Cancer. L Pearce, J Law, S Lee, J Hill. Association of Coloproctology of Great Britain and Ireland Annual Conference Bournemouth July 2017
Analysis of the effect of single nucleotide polymorphisms on age of onset of colorectal cancer in patients with Lynch Syndrome (hereditary non-polyposis colorectal cancer). Association of Coloproctology of Great Britain and Ireland Annual Conference, Bournemouth July 2017
Multiple SNP testing in MLH1 and MSH2 carriers. Results from a large genetic centre in the UK. L Pearce, A Wallace, J Hill, DG Evans. Association of Coloproctology of Great Britain and Ireland Annual Conference, Edinburgh July 2016.
Oligopolyposis within the bowel cancer screening programme; implications for regional genetics centres. L Pearce, C Chan, J Hill. Association of Coloproctology of Great Britain and Ireland Annual Conference, Edinburgh July 2016.
17
1 Introduction
One million people worldwide develop colorectal cancer each year and the disease affects 5% of the UK population. In 2010 40,695 new cases were diagnosed in the UK. In 2011 there were 15,695 deaths from bowel cancer in the UK (http://www.cancerresearchuk.org/cancer- info/cancerstats/types/bowel/ Accessed 2014). Fifty five per cent of patients newly diagnosed with colorectal cancer will die within five years of systemic or locally advanced disease 1. After lung and breast cancer, colorectal cancer is the most common cause of death from malignancy in the western world. Over the last thirty five years there have been great advances in the detection, diagnosis, treatment and on-going management of patients with colorectal cancer.
At the start of the 1980’s there were no reliable means of detecting asymptomatic pre-cancerous polyps or early colorectal cancers. Patients presented to clinicians symptomatically depending on site of the tumour, predominantly with advanced disease, reporting a change in bowel habit, rectal bleeding, palpable abdominal or rectal mass, or presenting acutely with large bowel obstruction.
The mainstay of investigations at this time was a barium enema, a radiological contrast study to introduce radiopaque contrast with gas into the rectum and colon in an attempt to identify areas narrowed by colorectal cancer. Laparoscopic surgery was not widely performed and advanced local or metastatic disease was identified at laparotomy. Treatments for patients with colorectal cancer were largely generic and there was negligible acknowledgment of risk stratification or opportunity to individualise risk and tailor management plans on a case-by-case basis. The only risk stratification symptoms commonly in clinical usage at that time were the Dukes’’ staging system and phenotypic identification (through generic investigations) of patients with familial adenomatous polyposis coli (FAP).
Since the 1980’s through clinical and laboratory based research, colorectal cancer has been recognised as widely heterogenetic disease and there have been outstanding advances in understanding the genetics of colorectal cancer.
18 In parallel, there have been other major advances in the detection and role of biomarkers in the management of colorectal disease. These biomarkers include faecal occult blood testing (FOBT), CEA testing, endoscopic screening and surveillance, computerised tomography (CT) and magnetic resonance (MR) cross sectional imaging, KRAS mutation and other polyposis syndromes.
Single gene disorders are either autosomal dominant or recessive and therefore inherited in a Mendelian fashion. All the currently identified disorders have high penetrance. Single gene disorders recognising a predisposition to developing colorectal cancer include; Familial Adenomatous Polyposis (FAP), Lynch Syndrome (or Hereditary Non-Polyposis Colorectal Cancer: HNPCC), Peutz-Jeghers Syndrome (PJS), Juvenile Polyposis Syndrome (JPS), NTHL1 associated polyposis and MUTYH (MutY Homolog (E. Coli)) associated polyposis syndrome (MAP). A small proportion of cases, around 5%, can be attributed to the high penetrance single gene mutations such as Adenomatous polyposis coli (APC)2 or Lynch Syndrome (MLH1, MSH2, MSH6, PMS1, PMS2) but most CRCs occur in patients who are not carriers of germline gene mutations.
Those individuals at increased risk of developing colorectal cancer are identifiable by family history, phenotypic recognition of a polyposis or hamartomatous syndrome, molecular features of the colorectal cancer of the index patient, or disease in relatives suggesting a familial syndrome or detection of a germline mutation.
Many studies have shown that colorectal cancers (CRCs) develop in genetically susceptible individuals. A study of Swedish, Danish and Finnish twins found a relatively large effect of heritability in colorectal cancer. In nearly 45,000 (monozygotic and dizygotic) twin pairs it was suggested that genetic susceptibility might play a role in up to 35% (95% confidence interval 10- 48%) 3.
First-degree relatives of those diagnosed with a colorectal cancer have a two- fold risk of the disease compared with the background population. This risk is further increased if the first degree relative is under the age of 45 years at the time of diagnosis or if more than one first degree relative is affected. Many of these cases are not attributable to single gene disorders and are unaccounted for. Much of this unaccounted heritable risk is thought to be as a consequence
19 of co-inheritance of common, low-penetrance variants each exerting a small influence on risk and following a polygenic model of inheritance 4 5. It is possible that whilst these are associated with relatively small effects on the risk in the individual, they contribute substantially to overall risk in the population. It is possible that as yet unidentified high penetrance genes are responsible but a polygenic mechanism now appears more plausible. Low penetrance polymorphisms (single base changes in the non-coding and coding regions of the DNA sequence) could be responsible. Some polymorphisms may have a direct functional effect but a SNP may be associated with susceptibility through linkage disequilibrium with a functional variant.
Pre-symptomatic diagnosis and screening of patients (including those with high risk gene defects), diagnosis and staging of disease, follow up, molecular genetics and utility of biomarkers have all evolved with the modern management of colorectal cancer. Pharmacological and technological advances in these fields have increased scope for personalising and individualising risk for patients and subsequently tailoring treatments. This work aims to investigate ways of personalising risk for patients’ with or at risk of developing CRC in the current era and how this may impact on their clinical care.
20 1.1 Pre symptomatic diagnosis and screening for Colorectal Cancer
1.1.1 Background to Colorectal Cancer Screening
In the UK, Colorectal Cancer (CRC) is the second most common cause of cancer death. The condition is slightly more predominant in men than women (lifetime incidence of 1:15 and 1:20 respectively). It remains a predominantly western disease with relatively low rates in Asia and Africa. As with other malignancies, there are specific patient groups at increased risk of developing CRC but increasing age is a significant risk factor. Other risk factors for the disease include smoking, alcohol intake and dietary factors (diets high in animal fat and red meat and low in fibre). These factors may account for some of the regional variation in incidence seen throughout the UK, particularly in areas with high levels of socioeconomic deprivation. Most colorectal cancers arise in the left colon (60%) presenting with symptoms of rectal bleeding, altered bowel habit and abdominal discomfort whereas right sided tumours tend to present more insidiously with weight loss and iron deficiency anaemia6. In recent years, the advent of greater awareness of CRC through media campaigns and social networking has increased public awareness of the disease. The apparent increase seen in diagnosis in patients 60- 69 years of age is likely due to this combined with the introduction of the National Bowel Cancer Screening programme in 2006.
The purpose of screening a population for a disease is early detection of that disease or risk assessment in order to improve outcomes. Screening principles were described by Wilson and Junger’s 1968 World Health Organisation criteria. Preventive screening programmes are distinct from early detection programmes. Preventive screening programmes aim to target the disease before it becomes malignant. Most colorectal cancers are believed to have arisen from benign precursor lesions (adenomatous polyps) in ‘average risk’ individuals i.e. no known CRC risk factors e.g. genetic susceptibility.
The Wilson and Jungner Screening Criteria are shown in Table 1. There has been no international consensus on the best strategy for screening in colorectal cancer.
21
Table 1. Wilson and Jungner Screening Criteria
Wilson & Jungner Screening criteria 1968 1. The condition screened for should be an important health problem 2. The natural history of the condition should be well understood 3. There should be a detectable early stage 4. Treatment at an early stage should of more benefit than at a later stage 5. A suitable test should be devised for early stage disease 6. The test should be acceptable 7. Intervals for repeating the test should be determined 8. Adequate health service provision should be made for extra clinical workload resulting from screening 9. The risks, both physical and psychological, should be less than the benefits 10. The costs should be balanced against the benefits.
Colorectal cancer survival is markedly affected by the stage of the disease at the time of diagnosis and therefore there is a survival advantage associated with early detection and treatment. Several staging systems are used clinically and these are discussed further in section 1.3.3. Detection of precancerous lesions is key to improving outcomes in susceptible patients at risk of developing colorectal cancer. It is widely accepted that the identification of colorectal cancer at an earlier stage improves outcome. Five-year survival of CRC exceeds 90% if the disease in confined to the bowel wall and falls to 60% in those patients with lymph node involvement and less than 10% if metastases are present at the time of diagnosis. Treatments for early stage cancers are less aggressive than for advanced stage cancers and as such are associated with less morbidity7.
There are several modalities available for CRC screening including guaiac FOBT, faecal immunohistochemical testing (FIT), flexible sigmoidoscopy and colonoscopy. Faecal occult blood testing (FOBT) has been extensively studied. This test relies upon detecting the peroxidase activity of the haeme moiety of haemoglobin and has been a widely used clinical test for many years. A systematic review by Burch et al in 2007 provided a summary of the evidence for FOBT and
22 informed the decision to utilise the test as the first part of the UK National Bowel Cancer Screening Programme (UK BCSP).
1.1.2 The UK National Bowel Cancer Screening Programme
In England, the NHS Bowel Cancer Screening Programme commenced in July 2006. The programme is non-specific and invitation is based upon age criteria alone. Men and women aged 60-69, registered with a GP, are sent an invitation for non-invasive screening by FOBT ages recently changed. Patients aged over 70 years are not routinely screened as part of the programme but can elect to be screened. The programme is currently being extended in England to include screening patients up to 75 years of age. Patients are sent a faecal occult blood testing kit by post every two years. For those patients in whom the test result is ‘unclear’, up to two further testing kits are issued before inviting the patient to attend for screening colonoscopy. A screening colonoscopy is offered to all patients who test positive for faecal occult blood.
1.1.3 Faecal Occult Blood Testing
The NHS bowel cancer screening programme uses guaiac faecal occult blood testing (g-FOBT) which was first proposed for use in the detection of colorectal cancer in the 1970s8. The first national screening programme with FOBT was introduced in Germany in 1977. Detection of occult blood using guaiac testing has been shown to reduce mortality and incidence of colorectal cancer. Overall three long term randomised trials reported a 15% - 33% reduction in CRC related death in users of the test. The test is non-invasive, cheap and relatively simple to perform at home. Faeces is smeared onto the test paper, which is impregnated with alpha-guaiaconic acid. Hydrogen peroxidase is then dropped onto the test spot, which oxidises the alpha-guaiaconic acid to a blue coloured quinone. This reaction is catalysed by the haem component of blood. As such, a positive test rapidly produces an intense blue colour. Unfortunately, g-FOBT is not specific for colorectal cancer and false positive results occur in patients with gastrointestinal bleeding due to another cause and in patients who on certain medication or who have ingested blood in their diet e.g. black pudding (commonly eaten in the North
23 West of England), raw meat. Test samples become dehydrated during processing and rehydration has shown to increase sensitivity, but decrease specificity leading to more false positive results. Low specificity is the main and significant problem with g-FOBT.
1.1.4 Faecal immunohistochemical testing
More recently the use of gFOBT is being replaced by faecal immunohistochemical testing (FIT). FIT is specific for the human-globin protein in stool. If globin is lost proximal to the colon e.g. oesophagitis, gastritis, peptic ulcer disease, the protein is degraded in the gastrointestinal tract thus making FIT specific for colonic blood loss 9. FIT is superior to gFOBT for other reasons, including greater sensitivity for advanced adenomas, quantitative results and improved cost effectiveness. Therefore, the European Group on Tumour Markers have recommended that all new centres embarking on FOBT screening utilise FIT and also welcome work to improve the standardisation of FIT assays and future research into DNA based tests.
The NHS bowel cancer-screening programme (BCSP) is moving towards replacing gFOBt with FIT. In Scotland FIT replaced gFOBT as the test for bowel cancer screening (cut-off threshold 80ug/g) in November 2017. Wales are conducting a phased roll out of FIT testing from January 2019 (150ug/g) and England are expected to implement FIT testing in Spring/Summer 2019 with cut off threshold 120ug/g. Utilisation of a ‘cut off threshold’ provides a binary outcome to FIT testing but here is variability in the cut off thresholds within these UK populations and between different countries across Europe. European countries are investigating using FIT testing to stratify risk through personalised risk prediction, combining FIT results with age and gender10. As haemoglobin concentrations and lifestyle data are largely not transferable between populations, it is important to understand the distribution within our own populations to ascertain how best to utilise this new screening test.
The role of FIT for UK symptomatic patients is not yet clear. 10-15% of all patients referred on a two-week wait basis with suspected colorectal cancer have precancerous polyps or a bowel cancer. In 2015 NICE expanded the criteria for colorectal two week wait referral and as such this has place unprecedented
24 demand of diagnostic services. The NICE FIT Study (which is currently open to recruitment) will identify if there is a role for FIT testing as a triage tool in the symptomatic population.
1.1.5 Flexible sigmoidoscopy
Only flexible sigmoidoscopy and FOBT have been tested as screening modalities in randomised trials but despite this colonoscopy is still considered to be the gold standard screening test. The obvious benefit of colonoscopy is direct visualisation of the entire colon. However, flexible sigmoidoscopy allows examination of the rectum and sigmoid colon in which two-thirds of colorectal cancers arise. Flexible sigmoidoscopy will miss approximately one third of colorectal neoplasia which are located in the proximal colon11. However, several studies have shown that although sigmoidoscopy does not allow visualisation of the entire colon, the risk of more proximal cancers can be predicted from characteristics of polyps found in the rectum and sigmoid colon. Therefore, patients who are risk stratified at flexible sigmoidoscopy and where appropriate proceed to full colonoscopy. Both colonoscopy and flexible sigmoidoscopy are invasive procedures with associated risks of complications. A UK multicentre study randomised study of one-off flexible sigmoidoscopy reported a 43% reduction in colorectal cancer related death after eleven years of follow up. The number of patients needed to be screened to prevent one colorectal cancer death in this study based on one off flexible sigmoidoscopy was 489. Which is considered to be favourable when 1250 people need to be screening using gFOBT to prevent one colorectal cancer death.
From March 2013 the NHS Bowel Cancer Screening Programme has piloted a new screening test whereby men and women in certain predetermined geographical areas are invited for a ‘one-off’ flexible sigmoidoscopy at the age of 55 years. The need for this pilot study is supported by a 2013 Cochrane review of FOBT and flexible sigmoidoscopy in colorectal cancer screening which reported that whilst there is high quality evidence that FOBT and flexible sigmoidoscopy reduce colorectal cancer deaths, there is insufficient evidence to judge the superiority of one over the other12.
25 1.1.6 Individualising risk and colorectal cancer screening
Traditionally, most clinical population screening programmes e.g. cervical screening, breast cancer screening, abdominal aortic aneurysm screening, use age to identify the target population to be screened. A significant criticism of evidence based population screening guidelines are that they apply to an ‘average’ individual within the population and do not take into consideration population subgroups with differing levels of risk i.e. genetic and environmental factors. False positive results, over diagnosis and overtreatment are all potential problems with screening programmes. Recent advances in cancer genomics are providing more information about an individual’s potential risk of developing a malignant disease and as such are enabling clinicians to contemplate a move away from homogenous ‘blanket’ screening of an entire target population.
Several studies have attempted to identify a robust risk prediction model within the screening population. Such a risk prediction model could potentially stratify risk and allow screening on the basis of risk e.g. age, sex, smoking, obesity, family history etc. rather than generic screening based upon age alone. A recent systematic review of these case-control, cohort and cross sectional studies by Ma and Ladabaum identified that most risk prediction models have weak discriminatory power between low, intermediate and high-risk individuals13. Currently, age is the only reliable discriminator14 and further work is needed in the development of risk prediction models to inform colorectal cancer screening strategies.
1.1.7 Moderate Familial Risk
Most colorectal cancer cases occur by chance but it is estimated that one in ten of the population have a relative who has had a diagnosis of colorectal cancer. Approximately forty thousand new cases of colorectal cancer are registered each year but the majority of these occur by chance and not through inherited germline susceptibility. The population lifetime risk for developing the disease in the UK is around 1:20 due to a combination of genetic and environmental factors.
26 Meta-analyses have been performed twice over the last fifteen years examining the effect of family history on risk of colorectal cancer. Johns and Houlston collated risk estimates for developing colorectal cancer dependent upon family history15 (see table 2). Butterworth et al reported a personal increased risk of colorectal cancer dependent upon the age of the affected relative resulting in a greater increased life time risk the younger the patient16.
1.1.8 Screening in familial colorectal cancer syndromes
Inherited susceptibility to colorectal cancer is thought to play a role in up to 35% of disease cases5 and accounts for a two to three fold increased risk in relatives of colorectal cancer cases15. It is well recognised that identification and surveillance of these susceptible patient populations can improve morbidity and mortality in this group. In 2010, the British Society of Gastroenterology published updated guidelines for colorectal cancer screening and surveillance of moderate to high-risk groups17. This document highlighted the importance of considering the basis upon which an individual is identified to have an increased risk of CRC. This includes a heterogenous composite of high-penetrance single gene disorders, multiple low-penetrance genetic factors (polygenic inheritance) and shared familial environment exposure’17.
Table 2. Risk of developing Colorectal Cancer based on family history
Family History Relative risk of CRC Absolute Risk by age 79 years None 1 4% 1 FDR* with CRC 2.25 9% >1 FDR with CRC 4.25 17% >1 FDR with CRC below 3.87 15.5% age of 45 *FDR First Degree Relative
The British Society of Gastroenterology guidelines (for colorectal cancer screening in high risk groups) define two groups of patients at moderate risk of developing colorectal cancer due to their family history; high-moderate and low-moderate
27 (table 3). These are patients whose family history is significant enough to warrant colonoscopic surveillance but not suggestive of one of the single gene disorders17.
Table 3. BSG Guidelines for colorectal cancer screening in high-risk groups Family History Lifetime risk Risk Recommended of Colorectal Category Surveillance Cancer CRC in 3 or more FDRs* (in first degree kinship with each other), none <50 years old 1:6-10 High 5-yearly CRC in 2 affected moderate colonoscopy relatives <60 years old or recommended with a mean age of < 60 from age 50 to 75 years old (in first degree years kinship with each other)
CRC in 2 FDRs > 60 years old Low Once only 1:12 moderate Colonoscopy CRC in 1 FDR < 50 years recommended age old 55 years
*FDR: First degree relative
A more personalised and ‘risk-stratified’ approach to screening at risk populations could result in tailoring screening regimes to each population stratum and potentially improve the efficiency of a screening programme whilst addressing the balance of harm and benefit of screening. Potential strategies targeting screening programmes based upon polygenic risk have been reported in breast cancer and prostate cancer18. However, the implementation of such a screening program would have ethical, legal and social implications. In breast cancer and colorectal cancer genetic variants have been identified, through GWAS and validation studies, to have weak additive or multiplicative effects associated with an increased risk of cancer. Therefore there is a possibility that some population stratification risk for CRC on the basis of polygenic inheritance could be undertaken with age and family history to improve the uptake and efficiency of current screening programmes.
28 1.2 Single Gene disorders
There are five high risk single gene disorders increasing an individual’s risk of colorectal cancer; Familial Adenomatous Polyposis (FAP), Hereditary Non- Polyposis Colorectal Cancer (HNPCC or Lynch Syndrome), MUTYH-Associated Polyposis, Juvenile Polyposis and Peutz-Jeghers Syndrome. These conditions account for 3-5% of all cases of colorectal cancer17. Other colorectal cancer syndromes and associated genes include oligopolyposis (POLE, POLD1), NTHL1 associated recessive polyposis, NTHL1, Juvenile polyposis syndrome (BMPR1A, SMAD4), and Cowden syndrome (PTEN) Patients with these hereditary conditions predisposing them to developing colorectal cancer are identified through any or all of, history of affected family members, family history of CRC, phenotypic features identified (e.g. multiple colonic polyps in young patient), genetic mutation detection through genetic sequencing. This highlights the importance of regional genetic centres in, identification, counselling and the management of those patents with a strong family history of CRC.
1.2.1 Familial Adenomatous Polyposis
1.2.1.1 Introduction and History
Familial Adenomatous Polyposis (FAP) accounts for only a small proportion (<1%) of all colorectal cancers but is the second most common hereditary colorectal cancer syndrome, affecting approximately 1 in 8,619 live births19. There is equal incidence in men and women. FAP is an autosomal dominant inherited condition mostly due to pathogenic variants of the APC gene on chromosome 5q encoding a protein consisting of 2843 amino acids (310kDa) and has 15 exons. Phenotypic features are a hundred to thousands of colorectal adenomas. As such, lifetime risk of CRC is almost 100% in the absence of any colonoscopic screening or surgical intervention. The average age of CRC diagnosis without intervention is 39 years, and 95% will develop CRC by age 50 years20. There is however significant variation in the clinical course even between family members with the same mutation and full explanation and reasoning for this is as yet unknown.
Attenuated FAP (AFAP) is described in those patients with less than 100 adenomatous polyps, later onset polyposis and mutations in specific region of the
29 APC gene (5’ region, exon 9 and 3’ region). Attenuated FAP is often considered a ‘less severe’ form of the syndrome, however there is still a 70% lifetime risk of developing CRC21.
AFAP and FAP are a result of germline mutations in the APC gene, which encodes the tumour suppressor gene, which is part of the WNT signalling pathway and has a particular role in the degradation of beta-catenin within the cell cytoplasm. Within the cell, the APC protein is present predominantly in the cytoplasm.
The protein encoded by the APC gene functions as a tumour suppressor gene22. Around 85% of mutations (both germline and somatic) occur in the mutation cluster region between codons 1250 and 144023,24. More than 1000 germline APC mutations are reported in the APC mutation database25. Within FAP families, germline pathogenic variants are detected in between 30 and 85% depending upon patient group and genetic technique. The risk of developing other clinical conditions and disease manifestations associated with FAP is often correlated with the position of the inherited APC mutation.
1.2.1.2 Variation in phenotypic features
Genotype/Phenotype correlations vary according to the position of the germline mutation within the APC gene. Whilst most are fully penetrant there is variation in severity of the condition and extra-intestinal manifestations. Severe polyposis (>5000 colorectal polyps) is associated with mutations between codons 1250 and 144023,24. Mutations in codon 1309 and immediate 3’ end of it tend to cause a severe phenotype with earlier age of onset of disease. In contrast, mutations at the extreme 5’ or 3’ ends of the gene are associated with attenuated FAP26. The development of desmoid tumours is discussed further in section 1.2.1.327.
Approximately 30% of cases of FAP are not hereditary and are due to a sporadic or de novo APC mutation. These patients often present as index cases of colorectal cancer and are identified at endoscopy for symptomatic disease. The detection rates of APC mutation are significantly associated with polyp burden28. Clinically, a polyp burden of >100 polyps is typically used as criteria to determine “classic FAP” whilst <100 polyps is often considered to be AFAP. MUTYH associated polyposis and homozygous loss of function germline mutation in NTHL1
30 gene (a base excision repair gene) can also have similar phenotypic features to FAP and AFAP and predispose to developing colorectal cancer. New techniques for sequencing the genome have made it possible to identify approximately 95% of all mutations.
In addition to polyps in the lower gastrointestinal tract, there are a number of FAP associated phenotypic features. Upper gastrointestinal polyps can also develop, particularly in the duodenum and periampullary region and these can undergo malignant change (adenocarcinoma) in approximately 10% of those affected. For this reason, patients with FAP undergo upper GI endoscopic surveillance (see section 1.2.1.4). Gastric fundic polyposis also occurs but there is no discernible increased risk of gastric malignancy. Other sites with increased risk of malignancy associated with the syndrome include ampulla of Vater (adenocarcinoma), brain (glioblastoma), thyroid (papillary carcinoma), liver (hepatoblastoma). Other features include retinal lesions in 60-90% of FAP patients (congenital hypertrophy of the retinal pigment; CHRPE), epidermoid cysts, osteomas (benign bone tumours). One third of patients also have dental anomalies including supernumerary and unerupted teeth. Desmoids tumours occur in 5-10% of patients with FAP.
1.2.1.3 Desmoid Tumours
Desmoid tumours are benign tumours of connective tissue (myofibroblasts). They are non-metastatic but infiltrate locally and therefore cause symptoms and signs related to their direct compressive effects and impingement on surrounding structures. Desmoid tumours often result in small bowel obstruction, ischaemia or hydronephrosis29. They are an important cause of morbidity and mortality in patients with FAP. They occur most commonly in women affected by the syndrome. APC gene mutations in the desmoids region of 3’-1399 are associated with attenuated FAP and a tendency to develop desmoids tumours27. There are few effective treatment strategies for the management of desmoids tumours and due to their proximity to surrounding structures treatment options can be difficult and clinical course challenging. There are high recurrence rates (up to 80%) in surgical excision, and mesenteric desmoids radical excision is associated with a high mortality (up to 60%)30. Non-operative management includes non-steroidal anti-inflammatory drugs, anti-oestrogens and cytotoxic chemotherapy. There have also been reports of radiofrequency ablation successfully treating abdominal wall desmoids tumours31 but this technique has not been used in the treatment of
31 intrabdominal desmoid tumours as the heat generated by the treatment can affect surrounding structures.
1.2.1.4 Polyp surveillance in FAP and AFAP
Early screening and diagnosis in patients with FAP or AFAP is essential. It has been shown in several studies that screening reduces the incidence of CRC in this high-risk group. Colonoscopic surveillance enables the identification of those patients most at risk of developing CRC and to offer prophylactic surgical intervention. Colonoscopy is considered to be superior to flexible sigmoidoscopy as it affords assessment of proximal disease, which is particularly important in patients with AFAP who may have distal colon and rectal sparing. However, surveillance through colonoscopic screening is an invasive and uncomfortable procedure and studies have demonstrated poor compliance. Poor compliance with screening regimes in this young patient population can result in affected patients presenting late with advanced symptomatic disease and limited treatment options.
Patients with documented APC gene mutations are offered annual flexible sigmoidoscopy and alternating colonoscopy in accordance with UK guidelines until polyp load indicates a need for surgery17. Patients with FAP are also at risk of gastroduodenal polyposis, duodenal and periampullary malignancies. Three- yearly upper GI endoscopy is recommended from age 30 years. Those patients with a large number of duodenal polyps are recommended to undertake yearly screening32.
By participation in regular screening, affected individuals can reduce their risk and make informed decisions about prophylactic surgery. Regular screening also allows the detection of colorectal carcinomas at an earlier stage than in an unscreened population affording treatments with less associated morbidity.
1.2.1.5 Prophylactic surgery
Prophylactic surgery is strongly recommended in patients with FAP before the age of 25 years. Patients may be offered colectomy and ileorectal anastomosis or proctocolectomy with ileoanal pouch. Any remaining colonic mucosa in either the
32 rectum or the anorectal cuff in these patients should be kept under annual endoscopic review for life32.
1.2.2 Lynch Syndrome
1.2.1.1 Introduction and History
Lynch Syndrome is also known as Hereditary non-polyposis colorectal cancer; HNPCC. It is the most prevalent form of inherited colorectal cancer. It is caused by an autosomal dominant inherited mutation in one of the mismatch repair genes (MLH1: mutL Homolog, MSH2: mutS Homolog 2, MSH3: mutS Homolog 3, MSH6: mutS Homolog 6, PSM2: post meiotic segregation increased 2). The condition is the most prevalent form of inherited CRC and thought to account for around 4% of colorectal cancer burden. There is variable disease penetrance, pattern of disease and age of onset between and within each single gene defects but the condition results in up to 80% lifetime risk of CRC if unscreened and undetected. Multiple generations are affected at an early age (mean approximately 45 years).
Henry Lynch first described Lynch Syndrome in 1966. Lynch described two large American families with familial colorectal and endometrial carcinoma whose phenotype did not fit into the already discovered genetic colorectal cancer syndrome i.e. Familial Adenomatous Polyposis.
In 1913 Aldred Scott Warthin, Chairman of the Department of Pathology at University of Michigan in Ann Arbor reported the first family with the hereditary disease we now know to be Lynch Syndrome33. He reported malignancies in half of the ten siblings of a seamstress with endometrial cancer who had uterine, stomach and ‘abdominal cancer’ and that the descendants of those affect also had multiple cancers. The family had emigrated from Germany to Michigan before the Civil War and Warthin named them “Family G”34. In 1925 Warthin further reported the families susceptibility to uterine and gastrointestinal cancers35. He also noted early age of onset of the cancers and postulated that the cancers may be occurring “at an earlier age in successive generations”. Three young members of the family diagnosed with appendicitis were found at surgery to have advanced colonic malignancy and we now know there is a tendency towards proximal colonic malignancy in some of those affected.
33
After his death in 1931, Warthin’s colleagues made a further report of the family in 193636 and concluded the family displayed an “inheritable organ-specific predisposition to cancer”.
In the 1960’s Henry Lynch noted similar clustering of cancers in other families and went on to published reports on these families. He also revisited “Family G” in 1971 and used the term ‘Cancer Family Syndrome’, noting the autosomal dominant inheritance pattern and again that the descendants of affected individuals were at risk of early onset cancers. He reported data on more than six hundred and fifty family members, ninety five of whom had by this time developed cancers37.
Ten years later, Richard Boland reported on two further families with “Lynch Syndrome”. It was noted at this time that two distinct phenotypes existed; those with only CRC and those with previously well-described extra-colonic cancers. As such, the terms Lynch Syndrome I (site specific colorectal cancer) and Lynch Syndrome II (familial cancer syndrome) were used to distinguish the two syndromes. The genetic basis for the disease remained unknown and in 1985 Lynch first used to term “Hereditary Non Polyposis Colorectal Cancer” or HNPCC.
We now know that extracolonic manifestations associated with Lynch Syndrome include endometrial, gastric, urothelial, ovarian, and brain tumours. Endometrial cancer is the most common extracolonic manifestation of Lynch syndrome and occurs as the index cancer in around 35% of women. It has been reported that patients with MSH6 mutations have an increased risk of endometrial cancer and a later onset of colorectal cancer than those patients with MLH1 and MSH2 mutations. However, extracolonic tumours are more commonly associated with MSH2 mutations38,39.
1.2.1.2 Diagnosis of Lynch Syndrome
Despite significant advances in the understanding of Lynch Syndrome over the last fifty years, and since the 1980’s, identification of patients with Lynch Syndrome remains difficult. There is significant phenotypic overlap between Lynch Syndrome patients and those patients who present with sporadic colorectal cancers. Additionally, many families will display a susceptibility to develop colorectal cancer without an identifiable underlying genetic basis for increased
34 risk. Some cases of teenagers with Lynch syndrome developing colorectal cancers have been described but the average age of onset of colorectal cancer in patients with Lynch Syndrome is 40 years. It is now known that colorectal cancer risk and type of cancer is directly related to within which gene the pathogenic mutation is identified. It has been reported that cumulative colorectal cancer risk by age 70yrs is around 40% for MLH1 and MSH2 patients whilst only 12% for MSH6 patients40. However, even within single gene defects there is much variability in age of onset of adenoma detection and colorectal cancer formation. The reasons for wide ranging age of onset remain unclear.
It is important to identify Lynch syndrome patients, as there are implications for both the index case and relatives in terms of genetic mutation testing, colonoscopic surveillance and timing of surgical intervention.
Currently in the UK, patients are referred based on their family history by their hospital clinician (and in some cases general practitioner) to regional clinical genetics services. At consultation with a clinical geneticist or counsellor a family history is taken and assessed against family history criteria i.e. the Amsterdam II (see table 5) or Revised Bethesda Criteria (see table 6). If a family fulfils the Amsterdam II criteria, if available, a living affected relative’s DNA is tested for germline mismatch repair mutations. If an individual’s personal or family history fulfils Bethesda Criteria archived tumour tissue (where available) undergoes tumour biomarker analysis (either MSI or MMR protein IHC). If MMR deficiency is demonstrated mutation analysis is then performed on germline DNA. A more effective strategy would be to test all patients presenting with colorectal cancer for possible Lynch Syndrome by means of tumour testing or even germline DNA testing for MMR mutations.
Immunohistochemistry testing detects the presence or absence of the protein products of the mismatch repair genes. Absence of the protein suggests a genetic mutation in the encoding gene. Currently tumour testing for assessment of MMR by IHC follows local and regional guidelines. National NICE guidelines were also published in February 201741 which now advocate molecular testing to all patients with colorectal cancer using immunohistochemistry for mismatch repair proteins or microsatellite instability testing to identify dMMR tumours and guide further testing for Lynch Syndrome. Expansion of MMR IHC testing for all colorectal cancers would result in a significant increase in the number of histopathological tests performed annually across the UK. IHC testing is reported to have 83%
35 sensitivity and 89% specificity regardless of which MMR gene is involved39. Yield of cases may be small but detection of germline MMR mutations through tumour testing would have significant implications for screening for Lynch Syndrome families and may be of some health economic benefit. The Manchester Colorectal Cancer Pathway group has published guidelines for the assessment of MMR status in colorectal cancer. The hierarchy used for assessment of MMR by IHC is shown in table 4.
36
Table 4. Manchester Colorectal Cancer Pathway Group Guidelines for groups to be assessed for MMR status
1 All cases of colorectal cancer occurring in patients aged under 50 years old
2 Cases by case following discussion at local Colorectal Cancer MDT meetings;
a: Additional cases aged between 50 and 60 years old with histopathological and clinical features suspicious of Lynch Syndrome and a family history of LS related cancers i. Features suspicious of LS in the patients may include: 1. Synchronous colorectal cancers 2. High grade / poor differentiation 3. Signet ring cancers 4. Mucinous tumours 5. Previous or current adenomas 6. Previous or current LS tumours (endometrial, ovarian, biliary tree, stomach, upper urinary tract, pancreas) ii. Features in family history 1. First degree relative with colorectal, small bowel, endometrial, ovarian, biliary tree, stomach, upper urinary tract, pancreas diagnosed <60 years of age 2. Two close relatives (first / second degree) with any of above <70 years of age
b: At oncologist’s request – in cases where prognostic information will aid decision making re chemotherapy e.g. TNM stage 2 cancers aged 50-70 years.
1.2.1.3 Amsterdam/Bethesda Criteria
In 1990 the first formal meeting of the “International Collaborative Group on Hereditary Non Polyposis Colorectal Cancer” (ICG-HNPCC) was held in Amsterdam
37 and the first clinical criteria was developed in an attempt to identify Lynch Syndrome patients for collaborative studies42; The Amsterdam I Criteria. This included only CRC and as such the criteria were revised in 1999 to the Amsterdam II Criteria to include all malignancies within Lynch Syndrome43. If a patient fulfils all criteria (listed in table 5) they should be referred to a regional genetic centre for screening and gene testing.
Table 5. Amsterdam II criteria developed to identify patients with Lynch Syndrome
Amsterdam II Criteria 1 At least three relatives with a Lynch Spectrum cancer (CRC, endometrial, upper urothelial, small bowel, ovarian) 2 At least one of these relatives is a first degree relative of the other two 3 At least two generations are affected 4 At least one of the affected patients was under the age of 50 years at the time of diagnosis 5 Familial Adenomatous Polyposis is excluded 6 Malignancies are verified from pathological records
In 1997, the US National Cancer Institute sponsored a workshop in Bethesda. The workshop manuscript reported a standard diagnosis panel of microsatellite markers, and criteria to identify CRC tissue for targeted analysis, either MSI or abnormal immunohistochemistry. Only one criterion needs to be fulfilled to recommend tumour testing. The Amsterdam II criteria were designed to select those patients who should be referred directly for mutation testing whilst the Bethesda Criteria were to be used in combination with MSI or IHC as pre-screening biomarkers. Much as the Amsterdam criteria were revised in 1999, the Bethesda Criteria were revised in 2004 (see Table 6).
38 Table 6. Revised Bethesda Criteria
Revised Bethesda Criteria 1 Any CRC diagnosed under the age of 50 years 2 *Synchronous or *aMetachronous CRC or any other Lynch spectrum cancer diagnosed at any age 3 CRC with typical MSI-H histology*b in a patient under the age of 60 years 4 CRC diagnosed in one or more first degree relatives with a Lynch spectrum cancer, with one of the cancers being diagnosed under the age of 60 years 5 CRC diagnosed in two or more first degree relatives or second-degree relatives with a Lynch spectrum cancer regardless of age
* Synchronous cancer is a second CRC which is histologically distinct from but occurs at the same time as, the primary CRC
*a Metachronous CRC is a second CRC which occurs more than 12 months after the primary CRC, is located in a different part of the colon or rectum, and was not present at the time of the primary CRC
*b “Presence of tumour-infiltrating lymphocytes, Crohn’s like lymphocytic reaction, mucinous/signet ring differentiation or medullary like growth pattern”
Generally, patients with early age onset CRC (<50 years) without family history are at very low risk for Lynch Syndrome. Neither the Amsterdam II Criteria, nor the Bethesda Criteria, are ideal screening tools for the identification of patients with probable Lynch Syndrome. A study by Barnetson et al in 2006 prospectively recruited 870 patients under the age of 55 years with a new diagnosis of colorectal cancer. The study found the Amsterdam Criteria to be superior to the Bethesda Criteria in terms of specificity (98% versus 38%) but that the opposite was true for sensitivity (42% versus 95%). The study group also reported their own risk prediction model; MMRpredict44.
A more recent German study evaluated the performance of Amsterdam and Bethesda criteria and the result of tumour tissue analysis in predicting MMR. They reported that the highest frequency of MMR gene mutations were found in families fulfilling the Amsterdam Criteria (46.4%) and that MMR mutations were found
39 significantly45 more often in families with at least one MSI-H small-bowel cancer (p<0.001).
1.2.1.4 Other predictive models
Mutation analysis to identify carriers of mismatch repair gene mutations is time- consuming and not cost effective. As such clinical features and tumour analysis are often utilised as pre-screening methods. Several risk prediction models have been developed to help refine calculation of the probability that an individual carries an MMR mutation, notably, the MMRPredict44, PREMM46, MMRPro47, Wijnen38 and Myriad Genetics Prevalence Model48. The MMRPredict model devised by Barnetson et al is more suited to larger families and older patients, as a detailed family history is fundamental for its application. The two-stage model uses location of tumour, metachronous or synchronous CRC, family history, age of youngest affected relative and presence of endometrial cancer to calculate probability of identifying a mutation carrier.
The PREMM model devised by Balmana et al is restricted to probability of identifying MLH1 or MSH2 mutation. The first developmental stage of the model involved mutation testing in 1618 serum samples for germline MLH1 and MSH2 mutation. This model was then validated in a separate cohort of 1016 patients using age of onset, type of tumour and number of primary cancers in first and second-degree relatives. The authors reported variable sensitivity and specificity rates depending upon predicted probability returned by the model. (These are shown in Table 7).
Chen et al designed a two-tier model, MMPro, in 2006. This model was validated using a much smaller US cohort. The model was designed using available published data for specificity and sensitivity of IHC and MSI tumour testing and germline mutation analysis. The first tier of the model assesses probability of the individual testing positive for MMR mutation based upon personal and family history of CRC and endometrial cancer. Tumour testing then affords probability modification in the second tier47.
40
Table 7. Risk prediction models of dMMR CRC
Study Model n Sensitivity Specificity Barnetson et al MMRPredict 870 62% 99% 2006 Amsterdam Criteria 42% 98% Bethesda Criteria 95% 38%
Balmana et al PREMM (5% likelihood) 1016 94% 29% 2006 (10% 85% 60% likelihood) Chen et al 2006 MMR Pro 279 83% 94%
Balmana et al were unable to demonstrate that either the PREMM or MMRPredict models were superior to the revised Bethesda guidelines in predicting MMR mutation carrier status49. Green et al compared these models, MMPro and Wijnen logistic regression model in 700 patients with unknown MMR gene mutation status. Wijnen found that younger age at diagnosis of colorectal cancer within a family (P<0.001), fulfilment of the Amsterdam criteria (P<0.001), and the presence of endometrial cancer (P<0.001) were independent risk factors but did not publish sensitivity or specificity rates for the model38. When compared with other models the MMRPredict model has been shown to be superior at predicting MMR gene mutation status in low risk populations49. In high risk (family history of colorectal cancer) populations the PREMM model has been shown to have slightly better predictive capability than MMPro and MMRPredict50. A validation study of all these models in moderate-high risk families found that the PREMM model demonstrated best performance status in predicting carrier status above 10% probability threshold51. None of these prediction models have replaced Amsterdam or Bethesda Criteria in prediction mutation status and warrant further research and validation.
1.2.1.5 Genetic modifiers
Whilst genetic factors are most amenable to retrospective studies, environmental factors are most suited to prospective studies. It is most likely that it is a combination of these factors that result in variable disease expression both within and between Lynch Syndrome families harbouring the same genetic mutation. The original spectrum of malignant disease reported in the first Lynch Syndrome family in 1913 included mainly gastric cancers33. Now, colorectal cancer is the most common tumour in patients with Lynch Syndrome, which parallels the
41 reduction of gastric malignancy, and increase in colonic malignancy that is now seen in western populations. Furthermore, there is a higher reported incidence of gastric cancer in LS families from eastern Asia. Studies have also reported that smoking and a high body mass index (BMI) were associated with an increase in risk of colorectal cancer52-57.
1.2.1.6 Surveillance and screening in Lynch Syndrome
Pre-symptomatic genetic testing allows identification and surveillance of known mutation carriers. Despite the condition having variable penetrance, cumulative lifetime risk of developing colorectal cancer is 60-80% (higher in men than women) without an appropriate screening regime.
For patients with Lynch Syndrome, prospective studies comparing outcomes for mutation carriers participating and non-participating in colonoscopic surveillance programmes have reported a 60% reduction in the incidence of CRC. However, some Lynch Syndrome family members who are known to have 50% chance of harbouring a mutation do not wish to undergo mutation analysis. In this situation it is recommended patients be screened according to mutation protocols with accepted potential 50% over investigation and unnecessary risk exposure.
For the background population, the UK National Bowel Cancer Screening programme has been identified to reduce the cumulative incidence of CRC in mutation positive individuals up to the age of 70 years58. However, current UK guidelines recommend at least biennial colonoscopic surveillance in Lynch Syndrome patients from the age of 25 years17. It has been suggested that MSH6 mutation carriers may not require surveillance until the age of 30-35 years but there has not been any prospective study data to confirm this. Studies have reported that regular screening reduces the incidence of colorectal cancer and improves mortality related to colorectal cancer by identifying early stage tumours to a larger extent that in a background general population. Three-year interval between surveillance colonoscopies has been reported to be effective but there have been no studies that have prospectively compared the effectiveness between intervals. However, advanced stage cancers have been reported between two and three years after surveillance and as such the recommended interval between surveillance colonoscopies remains one to two years. In one retrospective and three prospective studies reporting the effectiveness of colonoscopic surveillance,
42 the proportion of stage I and II interval cancers varied from 78% to 95%. Most of these tumours (57-62%) were right-sided and therefore this highlights the importance of colonoscopy rather than flexible sigmoidoscopy in Lynch Syndrome patients.
It has been reported that up to fifty per cent of adenomas are missed with standard white-light colonoscopy and it is therefore prudent to optimise adenoma detection in high-risk groups such as patients with Lynch Syndrome. Screening high-risk populations has led to the development of advanced endoscopic techniques (narrow-band imaging, auto fluorescence endoscopy, chromoendoscopy). Colonoscopy in at-risk patients affords direct visualisation of the entire colon with the opportunity to remove adenomas safely using endoscopic techniques. In those patients in whom a malignancy is detected, the CRC is often identified at an earlier and therefore more treatable stage and without the associated complications of more aggressive therapies. Advances in interventional endoscopic techniques (endoscopic mucosal resection, wide field endoscopic mucosal resection) have also pushed the boundaries of adenomas and early malignancies that, in a background population, can be managed endoscopically59,60. Lynch Syndrome patients with an early malignancy should undergo segmental resection or colectomy depending on a number of primary tumour and patient factors (these are discussed in section 1.4.3).
For women with Lynch Syndrome it is also important to offer patients screening for endometrial and ovarian cancer. Cumulative lifetime incidence of gynaecological cancers has been reported to be 32.5% (95% CI 29.1 – 35.9)61. Studies have reported risk to be as high as 40-60% for endometrial cancer and 7-12% lifetime risk for ovarian cancer62-64. As with colonic cancer, the age of onset of endometrial cancer and ovarian cancer is variable but average age at diagnosis is younger than the background population.
Published data suggests that large deletions affecting MSH2 can also affect the 3’ end of the EPCAM gene, lying in close proximity (15kb upstream) to the MSH2 gene. Similarly, deletions of the 3’end of EPCAM gene can lead to inactivation of the MSH2 promoter and therefore can be considered causative for Lynch Syndrome. Colorectal cancer risk in patients with EPCAM mutations was similar to that of EPCAM-MSH2 and MSH2 mutation carriers. In contrast the mosaic inactivation of MSH2, rather than constitutive lack of EPCAM, resulted in risk of endometrial cancers was lower65. Despite this low rate of endometrial cancer this
43 is the second most common Lynch syndrome associated malignancy. However, the relatively low risk of endometrial cancer in EPCAM deletion carriers, particularly those with a deletion at the 3’end of EPCAM and not extending in proximity to the MSH2 promoter region argues against prophylactic surgical intervention and questions the role of hysteroscopic surveillance and biopsies in this patient group66. Discoveries such as this may challenge current screening protocols in Lynch Syndrome patients to achieve more individualised risk-adjusted screening.
There are no current recommendations for endometrial or ovarian screening in the UK general population. A number of surveillance strategies have been suggested including, transvaginal ultrasound, outpatient hysteroscopy and endometrial biopsy. Current guidelines recommend annual endometrial biopsy from the age of 30-35 years or if family pedigree is known, 5-10 years prior to the earliest diagnosis of endometrial cancer in the family67. This is often done using hysteroscopy in an outpatient setting.
Urinary tract cancers (UTC) are rare but the highest occurrence is in the male MSH2 mutation carriers age 50-70 years68. There is limited evidence in the published literature for efficacy of screening modalities and their potential use in screening programmes. It is therefore suggested to educate known mutation carriers as to the potential symptoms of a urinary tract cancer rather than implement a screening programme of poor sensitivity and specificity.
As with other screening programmes, a major problem with screening for colorectal cancer in Lynch Syndrome patients and the development of interval cancers is lack of engagement and compliance with the screening programme. Reasons for non-compliance have been cited as anticipation or perception of discomfort, embarrassment at the time of the procedure and human error58. It is therefore recommended that patients are registered regionally or nationally with a hereditary cancer registry, facilitating an effective coordinated screening programme and reminder systems for screening patients69. Screening programmes in at risk groups warrant the same high standards seen in the UK Bowel Cancer Screening Programme.
44 1.2.3 Polyposis Phenotype
1.2.3.1 Hyperplastic Polyposis
Hyperplastic Polyposis syndrome (HPS), also called serrated polyposis, has been described as a rare condition affecting 1:3000 people70. However, this estimate was based upon detection rates with flexible sigmoidoscopy and as such there was no assessment of the right colon. More recent reports as a result of the NHS bowel cancer screening programme (BCSP) have reported that the prevalence of hyperplastic polyposis may be as much as 1:1818 (colonoscopy BCSP patients)71. Biswas et al reported higher prevalence this in the general population and affecting around 1:151 guaiac faecal occult blood test (gFOBT) BCSP patients72. Numerous serrated type polyps (hyperplastic, sessile serrated, traditional serrated and mixed polyps) are typically found proximal to the sigmoid colon. An increased risk of colorectal cancer has been reported, particularly of large, atypical, dysplastic or proximal polyps73. There is strong association with smoking, serrated polyps, and MSI-H colorectal cancer.
Hyperplastic Polyposis (serrated polyposis) is characterised by at least one of the following diagnostic criteria defined by WHO 2010:
1. More than five hyperplastic polyps proximal to the sigmoid colon, of which two or more are larger than 10mm. 2. Any number of hyperplastic polyps proximal to the sigmoid colon in a patient with a first-degree relative with a diagnosis of hyperplastic polyposis. 3. More than 20 polyps of any size distributed throughout the colon.
Hyperplastic Polyposis (now called serrated polyposis syndrome) is a heterogenous disease and is not associated with a single gene defect but does appear to be inherited in a simple Mendelian fashion. Surveillance is recommended for any patient with a serrated polyp >10mm or with dysplasia or a traditional serrated adenoma (3 year surveillance interval) or for those patients with multiple serrated polyps meeting the criteria for serrated polyposis syndrome (surveillance colonoscopy every one to two years until the colon is cleared)74.
45 1.2.3.2 Multiple Adenoma Phenotype
1.2.3.2.1 Oligopolyposis
Many cases of classical familial adenomatous polyposis coli present with more than 100 polyps at colonoscopy and are accounted for by APC gene mutation. However, there are a large number of patients with high polyp burden (5-100) for which there is no detectable APC gene mutation. These patients are considered to have oligopolyposis.
Oligopolyposis is a term used to describe a synchronous adenomatous polyp burden that is higher than would normally be expected in a screening population but that does not fit the criteria for classical FAP. The name ‘oligo’ is derived from the Greek term meaning few. Within the oligopolyposis spectrum there is variation in number of polyps and there is difficulty in determining the minimum polyp burden required for the term to be applied to patients. Within the literature some refer to polyp counts of 10-100 whilst others describe 5-100 polyps. These patients may present symptomatically or asymptomatically and are often detected as part of national screening programmes. The NHS bowel cancer-screening programme is discussed in section 1.13.
Whilst many patients do not have an identifiable genetic cause for their oligopolyposis, some patients with oligopolyposis are subsequently identified to have a detectable germline variant. A minority of patients with five to twenty colorectal adenomas test positive for any pathogenic germline variant28. Identifying a germline variant in patients with oligopolyposis detected at screening or colonoscopy for symptomatic disease relies upon the responsible clinician referring the patient to a regional genetics centre. There are currently no UK guidelines for the referral of patients with Oligopolyposis and there is no published data as to the yield of detectable germline variants from those patients referred.
Lynch syndrome is a potential diagnosis in patients with polyp counts at the lower end of the oligopolyposis spectrum. Lynch syndrome is rarely considered in trying to identify causation for an oligopolyposis phenotype. However, detection of one to ten adenomas could still be suggestive of Lynch syndrome, particularly in a symptomatic young patient or older screening patient.
MUTYH associated polyposis (MAP) accounts for around 1% of cases of adenomatous polyposis but for up to 30% of cases of oligopolyposis (less than
46 100 adenomas at presentation). The most common MUTYH gene pathogenic mutations in the Caucasian population are Y165C and G382D. The incidence of biallelic mutations is highest in patients with 15-100 colorectal adenomas75. The polyp burden is very similar to patients with attenuated FAP, likewise the age of diagnosis (45-55 years) is also similar for both these syndromes. Most cases of attenuated FAP will have less than 100 polyps by their fourth decade and age at CRC diagnosis delayed by 15 years when compared with classic FAP76. For both AFAP and MAP there is correlation between reduced polyp count and decreased likelihood of APC gene and MUTYH gene mutation respectively. If there is family history suggestive of autosomal recessive inheritance, then a diagnosis of MAP should be considered.
In up to two thirds of these patients with oligopolyposis, germline genetic variants can be uncovered for a number of less common hamartomatous polyposis conditions e.g. Peutz-Jeghers syndrome, Juvenile polyposis syndrome (see section 1.6.2).
Patients with five or more colorectal adenomas are considered to have a phenotype that is suggestive of an inherited genetic predisposition. It is therefore important to try and determine the genetic basis for these patients’ multiple adenoma phenotype in order to ascertain the likely natural history of their disease and thus optimise screening and surveillance programs for both the index patient and also any relatives that may also be affected. However, only a minority of individuals with 5-20 colorectal adenomas test positive for any pathogenic germline variant28. The numbers of adenomas in each of the Mendelian polyposis syndromes (FAP, AFAP, MAP) are widely variable and reasons for this, including variable phenotype in familial adenomatous polyposis, have been discussed in section 1.2.
1.2.3.2.2 MUTYH associated polyposis
MUTYH associated polyposis (MAP) is caused by bialleic recessively inherited mutations in the base excision repair (BER) gene MUTYH. The gene is located on chromosome 1p and functions to protect DNA from oxidative damage. The syndrome is an adenomatous polyposis syndrome with high penetrance that confers nearly a 100% lifetime risk of developing colorectal cancer. MAP can
47 mimic attenuated FAP77 as adenomas typically develop at around the age of 40 years but can occur at any age78.
1.2.3.2.3 NTHL1-associated polyposis
The introduction of readily available high throughput sequencing techniques has led to reporting of many novel germline mutations associated with increased risk of CRC. Through exome sequencing, a recent study of 51 patients with a suspected diagnosis of polyposis has identified a novel colorectal cancer syndrome79. The authors reported a homozygous nonsense mutation in the base excision repair gene NTHL1 in three unrelated families and a novel recessive adenomatous polyposis and CRC predisposition syndrome; NTHL1-associated polyposis (NAP). NAP, like LS also appears to present with an extended spectrum of extracolonic malignancies include endometrium, duodenal and skin. The association of NTHL1 with increased risk of colorectal cancer has recently been validated80.
1.2.3.3 Peutz Jeghers Syndrome
Peutz-Jeghers syndrome (PJS) is dominantly inherited with high penetrance. Germline mutation in STK11/LKB1 gene is causal in 20-63% of cases. There is also a further possible locus on chromosome 19q (PTEN)81,82. The condition gives rise to gastrointestinal hamartomatous polyposis in combination with cutaneous and mucosal macular melanin deposits. Hamartomas of the small bowel are characteristic, which lead to bleeding, anaemia, intussusception and obstruction within the first three decades of life83. Gastric (25%) and colonic (30%) hamartomas are also common84 and may present similarly. There is also a 50% lifetime risk of breast cancer83. Periorbital and perioral cutaneous pigmentation are typically noted in childhood and may raise suspicion of diagnosis77. Screening colonoscopy is recommended at 1-2 yearly intervals from 15-18 years of age17. Upper gastrointestinal screening and MRI enteroclysis is recommended from the age of 25 years at similar intervals17.
1.2.3.4 Juvenile Polyposis Syndrome
48 Juvenile Polyposis Syndrome (JPS) is characterised by multiple juvenile type hamartomatous polyps in the large and small bowel. Twenty per cent of cases are thought to be due to germline mutations in SMAD4 gene and 20% due to BMPR1A mutation. DPC4 gene and a further locus on chromosome 6 has also been implicated in some studies. Diagnosis is made on the basis of more than five juvenile polyps in the colon or rectum, juvenile polyps throughout the gastrointestinal tract, or any number of juvenile polyps in a person known to have a positive family history. Patients may present with symptoms reflective of polyposis including abdominal pain, rectal bleeding and change in bowel habit. In patients with multiple gastric polyps the lifetime risk of gastric malignancy is 21%. The lifetime risk of colorectal cancer is higher (39%). Small bowel and pancreatic cancers have also been documented. Screening colonoscopy and duodenoscopy is recommended at 1-2 yearly intervals from the age of 25 years17.
1.2.3.5 Cowden’s Syndrome
Cowden’s syndrome is a rare autosomal dominantly inherited condition characterised by multiple hamartomas of the gastrointestinal tract, skin, thyroid, breast and brain. The condition has a prevalence of 1:200,000. Caused by mutations in PTEN85. Cutaneous manifestations are particularly important in diagnosing the condition. The syndrome is a mixed polyp syndrome with hyperplastic polyps most prevalent and a lifetime risk of colorectal cancer is estimated at around 10%.
49 1.3 Diagnosis and Staging of Colorectal Cancer
Since the introduction of the National Bowel Cancer Screening Programme (NBCSP) there has been an increase in the numbers of patients presenting for colonic investigations. Radiological imaging plays a key role in both the screening for, and diagnosis of, colorectal cancer in the modern era. Computerised tomography (CT) scanning is utilised in the staging of colorectal cancer and magnetic resonance imaging in combination with CT scanning in the staging of rectal cancers.
1.3.1 Imaging and diagnosis of CRC
CT Colonography (CTC) was first described in 1994. Patients are given pre procedural bowel preparation and peri-procedural colonic insufflation with 4-6 litres of carbon dioxide to facilitate colonic distension. Intravenous contrast is only used in symptomatic patients. Patients undergo high resolution CT scan in a combination of prone and supine positions.
Although colonoscopy is still the “gold standard” for evaluating the colon, CT colonoscopy has in recent years replaced barium enema (BE) as a safer, more accurate and better tolerated radiological investigation in patients deemed unfit or unsuitable for colonoscopy. The SIGGAR trial compared the diagnostic accuracy of CTC with colonoscopy or BE in symptomatic UK patients. CRC detection rates were similar between the colonoscopy and CTC groups (11.4% versus 10.7% respectively, p=0.69) but greater in the CTC group than BE group (7.3% versus 5.6%, p=0.039)86,87. CTC is less invasive than colonoscopy, does not have the same associated risks, uses reduced bowel preparation and allows for review of intra-abdominal organs. The numbers of patients unsuitable for colonoscopy is increasing as patients are living longer (in part due to the improvement in medical therapies), are often frail and present symptomatically with CRC at a more advanced age than seen in previous years.
The numbers of CTC are increasing with time along with numbers of referral for colonoscopy. CTC uses low dose ionising X-radiation, which is unlikely to decrease life expectancy in the older patient population in which CTC is most commonly
50 used. If used in younger patients, such as in the screening of patients with Lynch Syndrome, radiation associated exposure could increase risk of malignancy.
Guidelines for the use of CTC in the setting of the NHS BCSP state that BE should not be performed as a first line alternative to colonoscopy and where high-quality CTC is not available locally, patients should be referred to other centres for examination. CTC should be performed and interpreted by radiographers and radiologists who satisfy the professional standards required by the NHS BCSP.
1.3.2 Staging of Colorectal Cancer
CRC develops from the colonic mucosa invading the muscular and serosal layers of the bowel wall as it proliferates. This can result in direct invasion to adjacent structures or organs, or via lymphatic or haematogenous spread. Lymphovascular invasion results in lymphatic spread to the mesenteric and para-aortic lymph nodes and haematogenous spread to the liver via the portal venous system6 and lungs.
Staging of colorectal disease is extremely important in the diagnosis and management of the condition to determine appropriate methods of surgical and chemotherapeutic treatment. Radiological staging investigations routinely used in the diagnosis of colorectal cancer include CT, MRI and PET scanning. Pathological staging can only be performed after surgical intervention and review of the resected specimen by a histopathologist. This pathological staging is essential in order to plan adjuvant treatments for patients. Colorectal staging systems take into consideration the extent of local invasion, degree of lymph node involvement and presence or absence of distant metastases.
1.3.2.1 Staging and Prognosis
In 1932 a British pathologist, Cuthbert Dukes’ (1890-1977) devised the Dukes’ Classification for colorectal cancer (Stages A-C). In 1935, the Dukes’ system was modified by Gabriel, Dukes’ and Bussey, to further subdivide stage C (lymph node involvement). This classification was widely adopted with the subsequent addition of Stage D, by Turnbull, to denote presence or absence of distant metastatic spread.
51 In 1998, the Union for International Cancer Control System (UICC) and the American Joint Committee on Cancer (AJCC) collaborated to establish an internationally consistent staging system based on the primary tumour, nodal involvement and distant metastases but provides more detailed information about tumour depth and lymph node status. A median of 12 lymph nodes should be retrieved and examined per specimen for adequate staging88. Over the last half- century we have seen significant improvement in the survival rates in colorectal cancer sufferers. Five-year survival rates have doubled and more than half of all patients with CRC will survive for more than ten years. This improvement in survival is multifactorial and can be attributed to improvement in detection, diagnostic, surgical and chemotherapeutic techniques. Staging systems used in colorectal cancer and the associated 5-year survival rates are shown in table 8.
Colorectal cancer has a well-described natural history and pattern of disease progression. Improving understanding of the interaction of environment and genetic factors in the development of colorectal cancer and variability in tumour biology and behaviour, thus creating a more individualised approach to screening, surveillance and treatment has the potential to further improve morbidity and mortality outcomes.
Table 8. Colorectal cancer staging and survival
Dukes’’ Stage TNM Stage UICC 5yr Survival Stage (CRUK 2012) A Limited by muscularis T1-2, N0, I 93.2% propria M0 B Serosal or extra rectal T3-4. N0, II 77.0% tissue involvement M0 C Nodal Involvement T1-4, N1, III 47.7% C1: Apical node M0 negative C2: Apical node positive D Distant Metastases T1-4, N0-1, IV 6.6% M1
52
1.3.2. Pathology of Colorectal Cancer
1.3.2.1 Adenocarcinoma
The majority (90%) of colorectal cancer is adenocarcinoma. Adenocarcinoma is an epithelial malignancy that originates within glandular epithelium of the bowel mucosa. Colorectal adenocarcinoma is an invasive carcinoma developing from dysplastic cells within a precursor lesion before invading the muscularis mucosae into the submucosa, muscularis propria and serosa. Malignant cells invade lymphatic and vascular plexuses which results in transcoelomic spread, lymph node and distant metastases.
1.3.2.2 Precursor lesions (polyps)
Colorectal polyps project into the lumen of the rectum or colon and arise from the mucosal layer. They are classified according to their histological morphology or macroscopic appearance i.e. flat (sessile) or pedunculated (on a stalk). There are four types of colorectal polyps including adenomas, serrated polyps (including hyperplastic polyps and sessile serrated adenomas), hamartomas and inflammatory polyps.
1.3.2.2.1 Adenomas
Adenomas are benign tumours arising from glandular epithelium and are the most common type of polyp resected. Proliferation within the adenoma is described as degrees of atypia or dysplasia. Dysplasia is characterised by four major pathological microscopic changes; Anisocytosis (cells of unequal size), Poikilocytosis (nuclear elongation resulting in abnormally shaped cells), Hyperchromatism (excessive pigmentation resulting from increased nuclear density) and presence of mitotic figures (an unusual number of cells which are currently dividing).
The architecture of adenomas may be tubular, villous or tubulovillous. Tubular adenomas are distinguished on the percentage of adenoma surface which displays tubular and villous formation. Tubular adenomas have more than 75% of their epithelium arranged in a tube like fashion and when transected in a horizontal
53 plane (“en face sections”) appear like transacted gun barrels. Villous adenomas have more than 50% of their epithelium arranged in the finger-like projections (villous length at least twice the thickness of the normal mucosa) seen in the epithelium of the small intestine. Tubulovillous adenomas have both tubular and villous components. The villous components make up 25-50% of the polyp and the remainder is tubular.
Adenomas are the precursor lesions in the Adenoma-Carcinoma sequence described by Fearon and Volgenstein 89. It is generally accepted that in the general population the time for transition from adenoma to carcinoma is around ten years. Risk of progression can be stratified according to the size and number of adenomas and this has been utilised to develop UK Polyp Screening Guidelines 17. These guidelines recommend colonic endoscopic examination and resection of adenomatous polyps. The size and number of adenomatous lesions determine the interval of screening endoscopies.
Figure 1. Tubulovillous Adenoma (H&E stain – Figure 2. Polyp of Sigmoid Colon tubular component left of image, villous component right of image)
1.3.2.2.2 Serrated Adenomas
Serrated adenomas can be hyperplastic polyps or sessile serrated adenomas (SSAs). Hyperplastic polyps (formerly metaplastic polyps) are benign non- dysplastic polyps and have a saw-like i.e. serrated epithelium, architectural atypia (non-uniformity of crypt structure and orientation) and crypt dilatation. They increase in frequency with age and most of these lesions are innocuous and occur in the distal colon with no malignant potential. These lesions represent a failure of anoikis (shedding of mature cells) from the gastrointestinal mucosa. In recent years there has been evidence of serrated variants similar in morphology with malignant potential. These lesions are termed sessile serrated adenomas. SSAs
54 are serrated polyps with unusual architecture with horizontal orientation of deep crypts with serration down to the crypt base. Whilst there is no conventional dysplasia, they may have nuclear atypia (ovoid nuclei with preserved polarity and prominent nuclear membrane due to chromatin condensation) or ‘hypermucinous’ change (reduced cytoplasm:mucin ratio due to mucin production) with crypt dilatation and abnormal proliferation. These lesions tend to be larger and are more commonly found in the right colon 90.
The progression of tubulovillous and tubular adenomas has long been recognised, but there is also evidence that serrated adenomas have the potential for malignant transformation. Serrated adenomas are thought to develop into serrated carcinomas which are morphologically different, but not completely distinct, from more typical colorectal carcinomas 91,92.
Figure 3. Sessile Serrated adenoma
1.3.2.2.3 Hamartomas
Hamartomas are almost entirely benign overgrowths of mesenchymal elements e.g. vascular tissue and fibrous stroma normally found at that site but grows in a disorganised mass. These lesions commonly demonstrate glandular proliferation. The autosomal dominant conditions of Peutz-Jeghers syndrome (PJS) and Juvenile Polyposis Syndrome (JPS) cause gastrointestinal hamartomatous polyposis (amongst other phenotypic features). Hamartomatous polyps in these patient populations have a high malignant potential and result in colorectal cancer in 70% of PJS patients 83 and 39% of JPS patients 93.
55 1.3.2.2.4 Inflammatory Polyps
Inflammatory polypoid lesions are associated with inflammatory bowel disease including Crohn’s Disease and Ulcerative Colitis. They are also found in infectious gastrointestinal disease processes resulting in mucosal inflammation such as amoebiasis, shistosomiasis and bacillary dysentery. Regenerative response to mucosal inflammation results in polyp formation. These lesions are stable and may not resolve after the inflammatory process subsides. These polyps have no malignant potential as they do not exhibit dysplasia. They are usually asymptomatic.
1.3.3 Staging of colorectal cancer
Clinical and radiological staging in newly diagnosed colorectal cancer aims to identify and accurately assess the extent of local disease and presence or absence of distant metastases. CT scanning and digital rectal examination was historically used to stage disease but the introduction of new imaging techniques such as endoscopic ultrasound (EUS), magnetic resonance imaging (MRI) and PET-CT has increased the armamentarium of available modalities. This has facilitated a more robust pre- operative assessment of disease and tailored treatments through a more individualised risk of disease recurrence. Current NICE recommendations for staging in colorectal cancer are shown in Table 9.
Table 9. Nice Guidelines CG131. Section 1.4.1. Staging of Colorectal Cancer. December 2014
NICE Guidelines; Staging of Colorectal Cancer
1. Offer CT Chest, Abdomen and Pelvis unless contraindicated. 2. Offer MRI to all patients with rectal cancer to assess risk of local recurrence determines by anticipated resection margin, tumour and lymph node staging 3. Offer Endoanal ultrasound to all patients with rectal cancer if MRI imaging show disease is amenable to local excision or if MRI is contraindicated. 4. Do not use the findings of a digital rectal examination as part of the staging assessment.
56
1.3.3.1 Radiological staging
CT scanning with intravenous contrast is used to stage local and distant disease in patients with colorectal cancer. Although the Dukes’ staging system is still widely used in clinical practice, radiologically it has been superseded by the TNM classification system (Table 8). It can be difficult to distinguish between T1 and T2 lesions on CT as both conform to the contours of with bowel wall without causing any distortion. Advanced T3 tumours may be detected by distortion of the bowel wall or adjacent fat stranding or the presence of satellite nodules. Staging of nodal disease is difficult on CT as micrometastases in small nodes cannot be seen but features suggestive of nodal involvement include nodes more than 1cm in size, enhancing nodes and any clusters of nodes (>3). The sensitivity and specificity of CT scanning in the staging of colorectal cancer is much greater for tumour invasion than for nodal disease (86% and 76% versus 70% and 78% respectively)94.
Accurate staging, with particular reference to involvement of the circumferential resection margin (CRM) is essential in planning operative approach (anterior resection or abdominoperineal resection) and neoadjuvant therapy in patients with rectal cancer. MRI scanning is more accurate than CT in determining involvement of the CRM (margin <2mm). Patients therefore undergo CT scanning of Thorax, abdomen and pelvis and Pelvic MRI scanning. The role of short course radiotherapy preoperatively in the management of T1 or T2 disease varies across the UK. Short course treatment has been shown to reduce risk of local recurrence in operable disease95. Some centres advocate the use of short course radiotherapy to reduce risk in all T1 and T2 tumours whilst others reserve treatment for operable T2 disease with adverse features. Patients with T3 or T4 tumours that have involvement of CRM are typically treated with neoadjuvant long course chemoradiotherapy in an attempt to downstage the tumour to an operable stage. This has been shown to reduce the risk of local recurrence and improve survival. Some patients (~15%) who undergo long course chemoradiotherapy are left with no detectable residual disease. Studies have not yet clearly determined the risk of recurrence in this patient group, if a watch and wait policy is followed.
57 1.3.3.1.1. PET CT
PET-CT was introduced into clinical practice in the UK in 1990 and has become a key investigation in the assessment of malignant conditions. Tumour cells rely on glucose metabolism and concentrate the FDG (fluorodeoxyglucose) tracer. PET- CT has more of a role in patients with metastatic colorectal cancer. PET-CT is discussed further in section 1.5.3.
1.4 Treatment of Colorectal Cancer
1.4.1. Early colorectal cancer
Stage I colorectal cancer describes those tumours, which are either confined to the submucosa (T1) or extend into but not beyond the muscularis propria (T2) without evidence of lymph node involvement. These tumours have five-year cancer free survival rates in excess of 95% after segmental resection with clear resection margins. Segmental resection involves excision of the primary tumour and its draining lymph nodes. If there are no involved lymph nodes the patient has undergone a more radical resection than required with the associated procedural and post procedural risks. Risk of lymph node metastasis can be predicted based upon tumour size, poor differentiation, depth of invasion in to the submucosa and the presence of lymphovascular invasion in the submucosa. For some patients, segmental resection may not be the most appropriate means of treatment for these tumours and advances in specialised endoscopic techniques such as endoscopic submucosal dissection (ESD) or transanal endoscopic microsurgery (TEMS) provide means of local resection with reduced morbidity and mortality risks. The risk with local excisional techniques is that if the disease is ‘undertreated’ oncological outcomes will be poor.
1.4.2 Stage II , III and IV Colorectal cancer
Stage II and III CRC includes T3 and T4 tumours or those with nodal involvement. These patients generally undergo segmental resection. Post-operative adjuvant chemotherapy is recommended in patients with stage III disease or pathological features that have been shown to be associated with poor prognosis in stage II
58 disease e.g. extramural invasion, pT4 disease, poor differentiation, obstructed tumours, perineural invasion and low lymph node recovery from the resection specimen. Current NICE guidelines96 recommend either capcitabine as a monotherapy or oxaliplatin in combination with 5-fluorouracil (5-FU) and folinic acid for the treatment of Stage II (with adverse features) and stage III colorectal cancer. There is a move towards more personalised medicine and a better understanding of cancer molecular biology. it is hoped that biomarkers will assist the continued development of novel pharmacotherapeutic agents (with improved side effect profiles) and may even be able to risk predict with greater accuracy those individuals at risk of recurrence and metastatic disease. The role of 5-FU is discussed further in section 1.4.3.2.1.
Stage IV describes those patients with distant metastases from a primary colorectal cancer. More than a third of patients with colorectal cancer will have liver metastases at the time of index presentation (synchronous metastases) or will go on to develop the disease in the liver during follow up (metachronous metastases). Patients may be offered resection of hepatic metastases either at the time of surgery for colorectal cancer (synchronous resection) or as a stand- alone resection, most commonly in the follow up period. Suitability for hepatic resection depends upon a number of factors. These include, local disease extent, metastatic burden, patient co-morbidities and future remnant liver (FRL). Techniques such as portal vein embolization to increase the volume of the FRL and neoadjuvant chemotherapy to reduce the volume of liver disease are now commonly used in clinical practice.
1.4.3 Treatment considerations in Lynch Syndrome
There is continued debate as to the true cumulative cancer risk in MMR mutation carriers. This is largely due to population studies utilising cancer registry groups with highly penetrant alleles resulting in an over-estimation of cumulative cancer risk. General population based mutation analysis irrespective of family history or disease state would be impractical and not cost effective. It is estimated that within a Lynch Syndrome population, the risk of CRC up to the age of 70 years is between 35 and 55%. The cumulative risk of endometrial cancer in females with Lynch Syndrome is between 10% and 45%.
59 There are 30,000 new Lynch Syndrome diagnoses worldwide every year. It is therefore important for clinicians to be familiar with the methods of diagnosis, the ongoing management of the affected patients and the surveillance and management of other potentially affected family members. Surgery for Lynch Syndrome, as for sporadic CRC, can be curative or palliative but it is also important to consider prophylactic or preventative surgery in affected individuals. Decision making processes in the management of mutation positive affected individuals who have not developed colorectal cancer is challenging. Individualising a patient risk through risk stratification is not yet considered an ‘exact science’ and is largely based on previous ages of disease onset family pedigrees. However, this is naturally not possible in those individuals with a sporadic rather than inherited mutation.
In a newly diagnosed Lynch Syndrome patient with colorectal cancer management will depend on tumour related factors i.e. the site and stage of the primary tumour, any other adenomas or metachronous cancers, evidence of advanced local or distant disease and patient related factors. The mainstay of operative interventional decision-making is whether to offer segmental resection and continued colonoscopic surveillance or whether to proceed with more extensive subtotal colectomy with ileorectal anastomosis.
1.4.3.1 Prophylactic Surgery
Historically, prophylactic colectomy was considered for patients with Lynch syndrome but provided patients participate in well co-ordinated colonoscopic surveillance programmes, there is no indication for total colectomy as a prophylactic measure. However, it is known that patients with Lynch Syndrome are at higher risk of metachronous tumours. Some have reported this risk to be as high as 40% ten years after diagnosis97. However, in a study by Parry et al 2011 cumulative risk of metachronous CRC inpatient undergoing segmental colectomy for primary colorectal cancer was 16% at ten years, 41% at twenty years and 62% at thirty years after the primary procedure. This study reported the risk of CRC was reduced by 31% for every 10cm of bowel removed98. However, there is a paucity of published long term survival data and despite a relative lack of evidence to support survival benefit98,99, guidelines recommend total colectomy and ileorectal anastomosis in young mutation carriers who have developed primary colorectal cancer100. Functional outcomes and quality of life
60 after subtotal colectomy remains a cause for concern. These patients can be troubled by stool frequency. However, increased metachronous cancer rates in those undergoing segmental rather than total colectomy coupled with reduced CRC specific survival rates partially offsets the reduced functional outcomes after total colectomy100. There has been recent evidence suggesting that although cumulative incidence of colorectal cancer detected by age 70 years, through colonoscopic surveillance programmes is high101 (MLH1; 46%, MSH2; 35%, MSH6; 20% and PMS2; 10%), this results in few deaths. Five and ten-year survival rates were reported as 94% and 91% respectively for colorectal cancer101. This evidence supports a programme of colonoscopic surveillance for these patients rather than prophylactic major surgical intervention.
There is little debate surrounding the surgical removal of the uterus and ovaries in identified female mutation carriers past childbearing age. This is in part due to the suboptimal surveillance for ovarian disease and the reduction in risk of endometrial and ovarian malignancy following surgery102. Ideally prophylactic surgery should be discussed with premenopausal women who have completed their families or in postmenopausal women who are undergoing abdominal surgery. Current guidelines recommend prophylactic total abdominal hysterectomy and bilateral salpingo-oophorectomy at age 50 years99,103.
1.4.3.2 Adjuvant Therapy
Fifteen per cent of CRC is a result of somatic DNA MMR deficiency leading to high frequency microsatellite instability (MSI-H). Microsatellite instability has been previously explained in section 1.3.3.
MSI-H is a hallmark of Lynch Syndrome resulting from germline mutations in MMR Genes (MLH1, MSH2, MSH6 and PSM2). These gene defects have high penetrance but only account for around 5% of all CRC and the majority of MSI-H CRC are sporadic non-Lynch Syndrome cases and arise in the setting of DNA hypermethylation (see section 1.3.2.2).
MSI-H tumours have distinct features including proximal colonic distribution, poor differentiation, mucinous tumours with high number of tumour infiltrating lymphocytes. There have been some studies reporting outcomes for patients with MSI-H tumours and tumour MSI has been shown to have significant survival
61 advantage independent of standard prognostic factors and be less likely to metastasise to locoregional lymph nodes or result in distant metastases104 e.g. Liver or Lung. It therefore stands to reason why the detection of microsatellite instability and mismatch repair deficiency is of high importance in colorectal cancer, not only when predicting or determining prognosis but also when planning adjuvant chemotherapy. MSI and MMR status are important in considering the risk/benefit of adjuvant treatment versus risk and also in choice of chemotherapeutic agents.
1.4.3.2.1 Fluorouracil-based adjuvant chemotherapy
5-fluorouracil is the cornerstone of chemotherapy treatment for colorectal cancer. For many years, adjuvant chemotherapy with fluorouracil (5-FU) and levamisole and subsequently with leucovorin had been the standard of care for patients with stage III and selected stage II colorectal cancer. A large study published by Ribic et al 2003 investigated the usefulness of microsatellite-instability status as a predictor of the benefit of adjuvant chemotherapy with fluorouracil in stage II and III colorectal cancer. Tumour specimens were collected form CRC patients who had been enrolled in randomised trials of 5-FU based chemotherapy. Ribic reported that 16.7% of colorectal cancers in the study demonstrated MSI-H and that there was significant association with proximal colonic site, histological tumour grade and improved survival in patients randomised to no chemotherapy. Tumour MSI-H was not correlated with an increased overall survival in the group receiving adjuvant chemotherapy. Benefit of treatment differed significantly according to tumour MSI status (p=0.01). Adjuvant chemotherapy with 5-FU improved survival only in patients with non MSI-H tumours (p=0.04)105. Previous and subsequent studies have contradicted these findings but the methodology of these studies106-108 has been suboptimal i.e. non-randomised trials, therefore introduced an element of selection bias. The most recent study published by Sargent et al109 using patients from previous randomised trials, confirmed the findings of Ribic et al. Microsatellite instability negatively predicts response to 5FU. This recognition of molecular subtypes of colorectal cancer has begun to revolutionise adjuvant treatment. This ‘individualised’ approach of personalising oncological treatment will help guide the development of new chemotherapeutic agents and regimes and reduce unnecessary treatments and improve patient outcomes. It is therefore important that when diagnosing new colorectal cancer as much information as possible is gathered about the primary tumour subtype and molecular heterogeneity. This information will help guide the timing and
62 approach of surgical intervention, follow up regimen and ongoing surveillance where appropriate.
In recent years there has been the development of online tools to aid healthcare professionals in the management of breast cancer. Adjuvant! Online (www.adjuvantonline.com) and Predict (www.predict.nhs.uk/predict.html) estimate the risk of negative outcomes (cancer related mortality or recurrence) without systemic adjuvant therapy and forecasts the reduction of these risks with various therapy options. Whilst there are surgical postoperative complication risk calculators (www.riskcalculator.facs.org/riskcalculator/) no such tools currently exist for the utility and benefit of adjuvant therapies for colorectal cancer.
63 1.5 Follow-up after resection for Colorectal Cancer
Traditionally, after treatment for colorectal cancer, patients undergo follow up for at least five years. Current follow up regimens promote the detection of locoregional and metastatic disease and two systematic reviews have suggested an overall survival benefit associated with more intensive follow up. Two recognised modalities of follow-up, which are accessible and affordable, are CT scanning of the chest, abdomen and pelvis and carcinoembryonic antigen (CEA) measurement.
1.5.1 Carcinoembryonic Antigen
The only biomarker approved for diagnostic use in colorectal disease is carcinoembryonic antigen110 (CEA). CEA is an oncofetal antigen produced by gastrointestinal tissue during foetal development normally present in low levels in healthy adults111. It is a complex glycoprotein, playing a role in intercellular adhesion, antigen presentation and as an antigen receptor. Gold and Freedman first identified CEA in 1965 in colorectal cancer specimens. Around 90% of colorectal cancers will produce CEA and as such it has become the most widely used and recognised biomarker in CRC. CEA serum levels may also be elevated in pancreatic, gastric and lung carcinoma111. However, CEA is not specific to malignant conditions and increased circulating levels are found in a number of inflammatory and benign conditions including inflammatory bowel disease, cirrhotic liver disease, cholelithiasis and bowel obstruction112. Due to low specificity and widely variable sensitivity (30-80%) CEA is not used currently in the screening and diagnosis of CRC. However, in newly diagnosed patients, preoperative CEA levels have been shown to be an independent prognostic factor for poor outcome and reduced 5-year survival in patients undergoing potentially curative resections. It is important to measure CEA prior to resection of the primary tumour as some primary tumours do not secrete CEA and therefore, are unlikely to demonstrate high CEA levels in the presence of metastatic disease. Some tumours may also undergo de-differentiation when they metastasise and stop secreting CEA111. However, in the most part, CEA has been demonstrated to have an important role for the surveillance of patients in detection of recurrent disease and may be the first indicator of recurrence. Serial CEA testing has a sensitivity of around 80% and a specificity of 70% in the detection of recurrent colorectal cancer. This should be used with caution as it has been recognised that
64 patients with normal serum CEA levels prior to colorectal resection maintain these normal levels through disease recurrence, especially in cases of local recurrence versus cases of metastasis113.
Table 10. Nice Guidelines CG131. Section 1.4.1. Follow up after apparently curative resection. December 2014
Clinical Off follow up to all patients from 4-6 weeks post potentially curative resection
CT scanning Minimum of 2 CT Chest, Abdomen and Pelvis in first 3 years
Serum CEA At least every 6 months for first 3 years
Colonoscopy Offer at 1 year and 5 years post resection
1.5.2 CT scanning
CT scanning was first introduced in the 1970s. Since then it has become one of the most important radiological modalities in the detection and screening for disease. Computer processed x-rays are used to generate tomographic images from a large series of two dimensional radiographic images taken around a single axis of rotation. The newer technique of PET-CT combines such three dimensional CT images with positron emission tomography (PET). An analogue of glucose in combination with a radionuclide tracer is used to indicate areas of metabolic activity (which take up the glucose analogue).
Computed tomography (CT) scanning is commonly used as a follow up modality to detect recurrence in patients following resection of a primary colonic malignancy. However, CT scanning has been reported to have high false-positive rates for pulmonary lesions and high false-negative rates for extrahepatic intra- abdominal lesions (e.g. paraortic nodes)114,115.
1.5.3 Positron Emission Tomography (PET CT)
18F-flurodeoxyglucose (FDG) positron emission tomography (PET) and FDG PET/CT is less widely available than conventional CT scanning. In current clinical
65 practice PET or PET/CT is typically performed when CT findings are equivocal. Some studies have reported that PET/CT may be a more appropriate radiological investigation for patients with suspected local or distant disease based upon clinical findings or elevated serum CEA levels116.
Indications for the use of PET-CT in colorectal carcinomas are shown in Table 11. Table 11. Indications for the use of PET-CT in colorectal cancer
Colorectal Carcinoma – Use of PET-CT 1. Staging of patients with synchronous metastases at presentation suitable for resection or patients with equivocal findings on other imaging; for example, pulmonary or liver lesions. 2. Restaging of patients with recurrence being considered for radical treatment and/or invasive techniques (e.g. metastectomy/selective internal radiation therapy (SIRT)) 3. Assessment of treatment response in patients with rectal carcinoma post (chemo) radiotherapy with indeterminate findings on other imaging 4. Assessment of treatment response following targeted therapy (ablative techniques for liver or lung metastases, selective internal radiotherapy for liver metastases) in metastatic colorectal carcinoma when findings on other imaging are inconclusive 5. Detection of recurrence in patients with rising tumour markers and/or clinical suspicion of recurrence with normal or equivocal findings on other imaging. 6. Evaluation of indeterminate pre-sacral masses post-treatment.
1.5.4 Surveillance programmes
Most surveillance programmes for colorectal cancer now favour a multimodal approach utilising endoscopy, CT scanning and biomarkers. There is limited evidence to support the intensive clinical, radiological and biochemical follow up of colorectal cancer patients that has become commonplace in the UK. The Greater Manchester and Cheshire Cancer Network Guidelines for post resection follow up include six monthly CT scans for two years followed by annual CT for three years and colonoscopy at two and five years post resection.
66 Meta-analyses have suggested that CEA and CT imaging are the only modalities of follow up with significant potential to detect curatively treatable recurrence in patients with colorectal cancer117,118. Recently, the FACS randomised clinical trial reported the effect of scheduled serum measurement of carcinoembryonic antigen (CEA) and CT as follow-up to detect recurrent colorectal cancer treatable with curative intent. 1202 patients who had undergone curative surgery for colorectal cancer were recruited over a six-year period. Equal proportions of patients were randomised to CEA measurement only, CT only, CEA and CT or minimum follow- up. CEA was measured every three months for two years and then every six months for 3 years and CT scans were performed every six months for two years and then annually for three years. The minimum follow-up group received follow up only if symptoms occurred. The authors found that 16.6% of patients developed recurrent disease and 35.6% of these were treated for recurrence with curative intent. There was little difference according to Dukes’ staging (Stage A, 5.1%; Stage B, 6.1%; Stage C, 6.2%). Both CEA and CT scanning were associated with increase rate of curative surgery for recurrence when compared with minimum follow up (p=0.02). Odds ratios for detection of recurrence were similar between CEA and CT only with little difference in the combined CEA and CT group providing evidence that there is no additive effect of using CT and CEA combined. One third of all recurrences of colorectal cancer were identified incidentally, not as part of the follow up regime, or between interval follow up. No mode of follow up demonstrated a statistically significant survival advantage and the authors concluded that if there was a survival advantage to any strategy it was likely to be small. A further recent prospective cohort study by Jones et al has reported low sensitivity (23%) and specificity (27.2%) of clinical review in detecting disease recurrence in stage I-III colorectal cancer. Clinical review in this study did not identify any disease recurrence that was not detected by scheduled CT119.
It is worth considering also that intensive CT scanning in young patients is not without risk of consequences of repeated large doses of radiation, the long-term effects of which are not entirely understood. Ideally frequency of follow up would be tailored to a patient’s individual risk, utilising biochemical, radiological and clinical modalities best identified to detect disease recurrence based upon features of both the patient and primary tumour.
67 1.5.5 Colorectal liver metastases
Colorectal cancer metastasises to the liver by lymphatic (mesenteric lymph nodes), haematogenous (portal venous drainage of the liver) or by direct invasion (e.g. tumours at the hepatic flexure). Twenty five per cent of patients with a new diagnosis of colorectal cancer will have liver metastases at the time of diagnosis120,121 (synchronous metastases), and up to 50% of patients undergoing curative resection for colorectal cancer will develop liver metastases after resection of the primary tumour (metachronous metastases)122,123. Hepatectomy for Colorectal Liver Metastases (CLM) has developed on the basis that metastatic disease may remain confined to the liver for a variable period of time and that clearing the liver of disease can afford cure. Over the last fifty years, surgery for colorectal liver metastases has developed from segmental resection for isolated metastases to the utilisation of pre-resection strategies such as neoadjuvant chemotherapy and advanced radiological techniques to modify the future remaining remnant liver (portal vein embolisation) to maximise resectability.
Detection of CLM is reliant upon CT scanning follow up after resection of the primary colonic tumour. Similarly, three monthly CT scanning in the early period after liver resection allows early detection of recurrent disease. This may in some patients permit further surgical resection and improve overall survival.
Of those patients who are identified to have metastatic disease limited to the liver, up to 30% can be treated with surgical intervention with curative intent124-128. Hepatectomy is the standard of care for these patients and overall median survival after colorectal liver metastases resection in these patients has been reported as 3.6 years with a 40% 5 year survival129. As a result of development of improved systemic chemotherapeutic neoadjuvant treatments hepatobiliary surgeons are increasingly encountering patients who have had apparent complete pathological response to systemic therapy. However, residual microscopic disease is found in up to 45% of patients at the time of surgery and long term remission occurs in only 50% of patients130. Therefore, all patients with CLM should undergo liver resection of all disease sites where possible even in the setting of apparent complete response.
68 1.6 Colorectal Cancer Molecular Genetics
The mainstay of treatment for non-metastatic colorectal cancer is surgical resection. The oncological surgical approach and timing of surgery is generic and whilst may be guided by radiological imaging and staging, it is independent of any histopathological features eventually identified within the resection specimen. Histopathology reporting provides information regarding tumour differentiation, grade of tumour and lymphovascular invasion, which are all widely accepted to be indicators of risk of tumour recurrence. However, there remains a lack of understanding of the patient and tumour specific molecular factors, which may affect risk of metastasis and recurrence.
The cell cycle is a highly integrated process whereby cell division and differentiation are normally tightly regulated through a series of intra and extracellular signalling pathways. Malignant cells escape this normal physiological control of the cell cycle.
Many genes that influence the passage from one phase of the cell cycle to another have been identified. Altered expression of these genes are termed oncogenes. Similarly, oncoproteins are proteins that are encoded by an oncogene which play an important role in the synthesis of proteins linked to tumour cell proliferation. Some proteins act to hold the cell at a distinct point in the cell cycle i.e. a checkpoint, the genes that encode these proteins are known as tumour suppressor genes.
Epigenetic changes are heritable changes in DNA that alter gene expression but do not alter the sequence of DNA. Colorectal cancer arises as a result of accumulation of genetic and epigenetic mutations, which transform normal glandular epithelial cells into benign neoplasms (adenomas) and subsequently into invasive carcinomas 131,132. The majority of these genetic alterations are somatic (mutations occurring in the somatic cell line i.e. cells that are not gametes and therefore diploid containing two copies of every chromosome) but some may be inherited. Genomic and epigenomic instability results in increased likelihood of accelerating “the neoplastic evolutionary process” 133 through mutations driving carcinogenesis to occur with a clonal lifespan. The majority of current molecular classification models are based on Microsatellite Instability (MSI), Chromosomal Instability (CIN), and CpG Methylator Phenotype (CIMP).
69 1.6.1 Fearon and Vogelstein’s genetic model for tumourigenesis
It was presumed for many years that CRC was an entirely homogenous disease. A single linear pathway was thought to be responsible for tumour development with mutations occurring in a predictable linear fashion. This is now known not to be true. It was thought that Adenomatous Polyposis Coli (APC) gene mutation was an early event triggering adenoma and neoplasia formation before Kirsten Rat Sarcoma Virus (KRAS) and tumour protein 53 (TP53) mutations created more aggressive subclones. In 1990 Fearon and Vogelstein challenged this belief and proposed that colorectal neoplasia developed through multistep carcinogenesis, and that the sequential loss of specific fragments of DNA was fundamental to this process; a genetic model of colorectal tumourigenesis. Loss of DNA in a tumour was termed “loss of heterozygosity” and these became presumptive loci for tumour suppressor genes. They described a sequential change from normal epithelium to hyperproliferative epithelium, early then late adenoma, and finally carcinoma and metastasis. It was suggested that the majority of colorectal cancers develop within pre-existing benign adenomas due to progressive acquisition of genetic defects within a monoclonal cell line, on the background of polyclonal field changes 89. Fearon and Vogelstein’s model included chromosomal instability, APC gene loss or mutation, DNA global hypomethylation, KRAS gene mutation, long arm of chromosome 18(q) loss resulting in deletion of the DCC gene (deleted in colon cancer) and TP53 gene mutation.
1.6.2 Jass’ molecular classification of colorectal cancer
Since Fearon and Vogelstein’s model for tumourigenesis, it has become widely accepted that colorectal cancer is in fact a heterogenous disease. It is clear that the development of colorectal carcinoma occurs through a variety of pathways that may or may not be mutually exclusive. Identification of different molecular phenotypes of tumours has been possible through advances in genomics, proteomics, transcriptomics and metabolomics. There is significant overlap between the molecular pathways that cause colorectal cancer in hereditary and sporadic cancers. In 2007 Jass, a British pathologist, proposed five possible pathways. These pathways were defined on the basis of two molecular features: DNA microsatellite instability and presence or absence of DNA methylation.
70 1.6.3 Genetic Instability
Genetic instability may be chromosomal or microsatellite. Chromosomal instability (CIN) predominates and is the defining characteristic of most human cancers. Mutation of CIN genes increases the probability that whole chromosomes or large fractions of chromosomes are lost or gained during cell division. This results in aneuploidy (imbalance in the number of chromosomes per cell) 134.
Microsatellites are simple repeating sequences of nucleotides. The repeating base pair sequence is usually no more than ten nucleotides long and often no more than two to five base pairs. Microsatellites are often found in non-coding areas of DNA. The total length of microsatellite repeats varies between individuals but should remain the same within the DNA of every cell of an individual. These repeating nucleotide sequences are susceptible to copy errors on the background of poorly functioning DNA mismatch repair systems. Microsatellite instability (MSI) is a change in the length of microsatellite alleles due to deletion or insertion of repeating units during DNA replication. Microsatellites frequently occur in promoter regions of tumour suppressor genes e.g. BAX and IGF2R92. It is therefore logical that the promotor function will be altered in the presence of variants in region and thus resulting in altered or reduced gene expression in a similar way to if the DNA sequence of the gene contained a mutation.
DNA methylation is an enzymatic process that adds a methyl group to the 5- position of cytosine by DNA methyltransferases (DNMT) to produce 5- methlycytosine. CpG (cytosine preceding guanine) island methylator phenotype (CIMP) is a subtype of colorectal carcinoma that arises through an epigenetic instability pathway and is characterised by vast hypermethylation of promoter CpG island sites. Some CpG dinucleotides that are methylated in normal cells are unmethylated or hypermethylated in cancer cells. Hypermethylation results in the inactivation of several tumour suppressor genes or other tumour-related genes. It has been noted that most sporadic MSI tumours have methylated MLH1 (this is rare in Lynch Syndrome tumours), a biomarker used when considering genetic testing for Lynch syndrome135.
1.6.3.1 Epigenetics
Some genes are at times inactive. There are a variety of mechanisms which cells use to control functionally relevant genetic changes without altering the nucleotide
71 sequence. DNA methylation is one of the epigenetic mechanisms that cells use to control gene expression. DNA methylation is necessary for normal cellular function and cellular differentiation and is the result of the addition of a methyl group to the fifth nitrogen of a cytosine base.
1.6.3.2 Jass’ Classification
Five subtypes of colorectal cancer were described by Jass on the basis of molecular genetics.
• Type 1: MSIhigh, chromosomal stable, methylation of MLH1 promotor region, CIMP high, BRAF (V-RAF Murine Sarcoma viral oncogene homolog B1) gene mutation. Originates from serrated polyps not adenomas. This pathway is believed to account for 13% of colorectal cancers.
• Type 2: MSI-stable or low, chromosomal stable, CIMP-high, partial methylation of MLH1 promoter region, BRAF gene mutation. Originates from serrated polyps not adenomas. This pathway is believed to account for 8% of colorectal cancers.
• Type 3: MSI-stable or low, chromosomal instability, CIMP-low, KRAS mutation, MGMT (O-6 methylguanine-DNA methyltransferase) promoter region methylation. Originates in adenomas or serrated polyps. This pathway is thought to account for 20% of colorectal cancers.
• Type 4: MSI-stable, chromosomal instability, CIMP-negative. Originates in adenomas. This pathway is the commonest and thought to account for 57& of all CRC which includes those of Familial adenomatous polyposis (FAP) and MUTYH associated polyposis (MAP).
• Type 5: MSI-high, chromosome stable, CIMP-negative, BRAF mutation negative. Originates in adenomas. This pathway is thought to account for all the colorectal cancers in Lynch syndrome, so is thought to be roughly 3% of all CRC.
72 Currently there are thought to be three main molecular pathways to colorectal carcinogenesis that exist. The theory of three pathways is a progression from Fearon and Vogelstein’s proposed model but a simplification of Jass’ classification. The three pathways outlined here include all the fundamental epigenetic and genetic elements. The first pathway is the chromosomal instability pathway (CIN) (Type 4 from Jass classification). The second is that which causes colorectal cancer in Lynch Syndrome. Progression from adenomas, or flat adenomas, to colorectal cancer (Type 5 from Jass classification) appears to be expedited compared to the CIN pathway. The third pathway is the CpG Island methylator pathway. CRC develops within either serrated adenomas if MSI-high is present or adenomas if not. Methylation is the hallmark of this heterogenous pathway and includes features of Jass’ Type 1, 2 and 3.
1.6.3.2.1 The Microsatellite Instability Pathway
Microsatellite instability occurs in around 22% of cases of colorectal cancer136. Microsatellites are short repetitive DNA sequences, which are susceptible to frame shift mutations and base pair substitutions. They are involved in the DNA repair system and mutations in these microsatellites were initially associated with Lynch Syndrome. Mismatch Repair (MMR) Genes in Lynch Syndrome are mutS homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6) and post meiotic segregation increased 2 (PMS2)92. Loss of these genes results in defective mismatch repair and microsatellite instability.
Microsatellite instability status is clinically relevant but the predictive value of MSI status is still debated. MSI-H tumours tend to be proximally located, poorly differentiated and occur at an earlier age. MSI-H is a strong prognostic factor for recurrence free survival in patents with stage II and stage III colorectal cancer137.
1.6.3.2.2 The Chromosomal Instability Pathway
The chromosomal instability pathway is thought to account for around 60% of sporadic colorectal cancer cases and all cancers cause by Familial Adenomatous Polyposis (FAP)138,139. This pathway represents the typical adenoma-carcinoma sequence of carcinogenesis89 with structural chromosomal losses (aneuploidy) or gains (polyploidy).
73 The APC (adenomatous polyposis coli gene) is located on the long arm of chromosome 5 (5q) and mutation of loss of this gene is an early event in the CIN pathway. The gene product, the APC protein, is involved with Wnt-signalling pathway. This signalling pathway is crucial to cell survival, growth and differentiation. During the normal cell cycle, proteins including APC, modulate the degradation of stimulatory transcription factors. APC gene mutations prevent normal binding to proteins e.g. ß-catenin. Without this complex the normal Wnt- signalling pathway is impaired conferring an abnormal survival advantage to that cell133,140. APC mutations are found in up to 80% of all colorectal adenomas, 60% of colonic cancer and 82% of rectal cancers133.
KRAS (Kirsten rat sarcoma virus) is also involved in the CIN pathway. It is a proto-oncogene involved in cellular response to extracellular stimuli137. The gene is located on the short arm of chromosome 12 (12p). Mutation results in structural activation of downstream signalling pathways e.g. mitogen activated protein kinase (MAPK). When mutated growth and differentiation is unchecked, tumour cells are more resistant to inhibition of surface receptors of tyrosine kinase such as epidermal growth factor receptor. The MAPK pathway has been reported as a fundamental mechanism involved in tumourigenesis136. It has been established that wild-type KRAS is associated with a better response to EGFR inhibitors such as cetuximab136,137,141. It is of significant clinical relevance when planning adjuvant treatment for patients with colorectal cancer and routine testing is performed prior to treatment with cetuximab.
1.7 Principles of Biomarkers
A biomarker (BM) is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes or responses (pharmacological or otherwise) to a therapeutic intervention. Biomarkers can be either prognostic or predictive137,138 and when considering a disease process they can be used to detect disease, predict prognosis or predict response to a pharmacological agent. In order to be of use clinically a biomarker must facilitate an improvement in life expectancy or in quality of life112. Biomarkers are essential in the management of malignant disease and underpin the development of personalised treatments and disease specific targeted therapy.
74
1.7.1 Categorisation of Biomarkers
The Cancer Research UK Biomarker Discovery and Development committee (BIDD) have defined the following categories of biomarkers.
The cancer research UK biomarker roadmap initiative has identified several categories of biomarker; 1. Risk assessment/predisposition biomarkers: these identify individuals at increased risk of developing cancer and can be identified in large population based studies, clinical trials or family studies. These may measure exposure to a carcinogen or be markers of genetic predisposition. For example the Adenomatous Polyposis Coli (APC) gene and Familial Adenomatous Polyposis (FAP). 2. Screening/early detection biomarkers: These can be used to aid early diagnosis of a condition this allowing detection at a more treatable stage. These biomarkers can be developed in large population based studies and/or clinical trials. For example faecal occult blood testing (FOBT) or flexible sigmoidoscopy in colorectal cancer. 3. Diagnostic Biomarkers: These biomarkers are used to define the type of cancer or disease that a patient has. These are often used in conjunction with standard imaging techniques. E.g. PSA, CEA and Ca125. Diagnostic biomarkers can also be used to detect and define recurrent disease after primary therapy. 4. Pharmacological Biomarkers: These biomarkers measure the effects of a drug treatment on a specific target (Proof of Mechanism (POM); e.g. enzyme inhibition or receptor blockade) or on a feature of tumour biology (Proof of Concept (POC); induction of cell death (apoptosis) or inhibition of blood vessel formation or function (angiogenesis)). Pharmacological biomarkers are particularly important in early phase drug development where failure to demonstrate POM or POC at tolerated doses can result in termination of drug development programme. 5. Predictive biomarkers: These identify subgroups of populations that are most likely to respond to a given therapy e.g. wild-type KRAS and response to anti-EGFR therapy in metastatic colorectal cancer 6. Prognostic biomarkers: These indicate the likely course of the disease. Prognostic biomarkers can reflect the metastatic state or potential and the
75 likely growth rate of the tumour and are used to estimate patient outcome without consideration of the treatment given. These biomarkers may also be used to guide treatment and surveillance decisions.
1.7.2 Biomarker Discovery
Biomarker discovery is the process through which putative biomarkers are investigated. This is either done in random hypothesis free “fishing exercises” or hypothesis driven through the understanding of the molecular pathophysiology of a condition.
Advances in genomics have allowed progression from hypothesis driven candidate gene research and family linkage analysis, to hypothesis-free genome-wide association studies142 Genome-wide scanning is a relatively new genetic technology for finding biomarkers associated with disease. It is a method of scanning the entire genome to identify single nucleotide polymorphisms (SNPs) that are correlated with disease. Whilst the vast majority of SNPs are innocuous, SNPs associated with disease are identifiable in combination with other data, including epidemiology studies that compare large groups of individuals with the disease against other groups without the disease. Candidate gene studies (based on knowledge of a disease or positional association based on previous family- based linkage studies) are population based association studies involving a small number of single nucleotide polymorphisms (SNPs). Unlike candidate gene studies, Genome wide association studies (GWAS) “allow massive investigations not based on hypotheses”143 search for genetic variants across the entire genome, which display differences in frequencies between cases and controls. The disadvantage associated with this design is that the large number of SNPs being tested (around 300,000) mean highly stringent significance criteria must be set in order to control for associations occurring purely by chance i.e. false positives.
Researchers are now utilising the findings of these hypothesis generating GWAS studies to investigate gene and environment interaction. Observational studies have consistently reported an association between the use of regular aspirin and other non-steroidal agents and reduced colorectal cancer risk144. The functionality of SNPs associated with colorectal cancer remains largely unclear and there is little published evidence of altered phenotype through SNP interaction
76 with pharmacological agents such as Aspirin. Sheth et al 2018 reported that variant alleles in genes involved in aspirin pathways (CYP2C9, ODC1, UGT1A6) may be responsible for modification of colorectal cancer or colorectal cancer risk. This study also reported SNPs previously reported to be associated with an increased risk of colorectal adenoma (rs11694911) conveyed a 16% reduction in colorectal cancer risk with use of regular aspirin145.
1.7.3 Biomarker Validation
All biomarkers must be validated before being introduced into clinical practice and assay techniques must be quality assured and standardised. Generally, biomarker validation may be “analytical” or “clinical” 146. To a certain extent the type of validation process depends upon the category of biomarker being tested.
1.7.4 Biomarker discovery roadmaps
In order to minimise uncoordinated research programmes, Cancer Research UK have developed a series of ‘roadmaps’ defining a robust research pathway. Roadmaps for screening, diagnostic, prognostic/predictive and pharmacological biomarkers have been developed along with dedicated CR UK funding streams for biomarker research and collaborative initiatives.
These roadmaps have been divided into four sequential phases:
1. Rationale 2. BM assay development 3. BM discovery 4. BM qualification
1.7.5 Biomarkers in Colorectal Cancer
Numerous established and putative biomarkers have been reported112. These can be helpful clinically as predisposition biomarkers (utilised in risk stratification),
77 molecular, screening, diagnostic, pharmacological and predictive. The biomarkers relevant to this thesis are discussed below.
1.7.5.1 Risk stratification (predisposition) biomarkers
In order for a biomarker to be useful clinically in a risk stratification setting it must meet certain criteria. Firstly, the biomarker must be readily obtainable by a method acceptable to the patient i.e. be as minimally invasive as possible. Secondly the biomarker must be shown to have high specificity within the population in which it is being tested. Thirdly, the biomarker must be able to diagnose the disease earlier than the ‘normal’ expected clinical presentation. A large prospective study must demonstrate a correlation between biomarker and incidence of disease and finally a prospective randomised trial must support benefit to long-term outcomes through reduction in morbidity and mortality. Apart from engaging in colorectal cancer screening programmes patients can take preventative measures through lifestyle changes (increased exercise, maintaining a healthy weight, stopping smoking) and dietary modification (increased fibre intake, reduction in intake of red, cured and processed meat.
In those patients affected by single gene disorders, germ line mutations can be considered to be predisposition biomarkers. Increased risk of colorectal cancer in these patients’ groups and appropriate surveillance programmes for these patients reduces the incidence of colorectal cancer and prolongs life expectancy32. Single nucleotide polymorphisms identified through GWASs to date only confer modest risk of colorectal cancer and as such these are currently not utilised clinically to stratify risk as predisposition markers currently. However, the accumulative and multiplicative effects of these variants have not been extensively investigated. As more is understood about risk conferred by these common variants they may have more clinical significance particularly within patient specific populations identified to be at higher risk than the background population but in whom clinical course of disease is variable e.g. hereditary non- polyposis colorectal cancer.
1.7.5.2 Screening and early detection biomarkers
Screening biomarkers have some properties similar to predisposition biomarkers. However, any screening test including screening biomarkers should comply with
78 screening criteria described by Wilson and Jungner in 1968147 that should be considered prior to starting a screening programme. The Wilson and Jungner Screening Criteria are shown in table 1. There is no international consensus on the best strategy for screening in colorectal cancer. The importance of colorectal cancer screening and currently available screening modalities are described in section 1.1.
Advances in genomics and proteomics have enabled the development of innovative markers on a diagnostic and therapeutic basis with potential application to the screening of patients at risk of developing colorectal cancer.
1.7.5.3 Diagnostic Biomarkers
The distribution of the biomarker value must be defined using samples from a representative population and the distribution must indicate a clinical relevance. The biomarker must significantly improve diagnostic accuracy in a prospective study.
1.7.5.4 Pharmacological Biomarkers
It is now widely accepted that an individual’s germline DNA can affect clinical response to chemotherapeutic agents through alteration in drug metabolism. Two examples of such mechanisms include dihydropyrimidine dehydrogenase and the anti-metabolite 5- fluorouracil (5FU) and the gene UGT1 and irinotecan, which is a prodrug topoisomerase inhibitor. Pyrimidine antagonists incorporate as false precursors in DNA or RNA through inhibition of proteins involved in nucleotide metabolism. All pyrimidine antagonists are prodrugs and in order to produce cytotoxic metabolites require intracellular conversion. Deficiency of dihydropyrimidine dehydrogenase (DPD) impairs the catabolism of 5FU and this can cause severe toxicity. Failure of glucoronidation in UGT1 deficiency similarly leads to toxicity with irinotecan148.
1.7.5.5 Predictive Biomarkers
The distribution of the biomarker marker value must be defined using samples from a representative population. There must be statistically robust correlation
79 between the biomarker and clinical outcome. This should be validated and clinical outcomes should be improved by use of the biomarker.
1.7.5.5.1 KRAS
KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is a protein coded by the KRAS gene. The gene is involved in several signal transduction pathways within the cell. Activation of the KRAS gene results in cell proliferation. KRAS was identified as playing a key role in the conversion of intermediate adenomas to carcinomas by Fearon and Vogelstein89. KRAS mutations are present in up to 50% of all colorectal cancers and confer a worse prognosis than a wild type copy of the gene149.
Activation of epidermal growth factor receptor (EGFR) by epidermal growth factor (EGF) results in cell proliferation, differentiation and promotes cell survival. Monoclonal antibodies (e.g. panitumumab and cetuximab) target EGFR and block binding of EGF to the receptor. In cancer cells this halts proproliferation signals. These agents are used as second line treatment of metastastic disease150. Several clinical studies have reported that the benefit of cetuximab is limited to patients with no mutations (wild type) of the KRAS gene151-153.
The KRAS wild type genotype has become an important predictive biomarker for treatments with anti-EGFR drugs. Mutant KRAS genotype is consistent with resistance to this group of drugs and therefore KRAS genotype is utilised clinically in patients with metastatic colorectal cancer in deciding chemotherapy regimens. However, half of patients with KRAS wild type genotype also do not respond to cetuximab154. This may be due to intratumoural heterogeneity155 and genotyping from a single block will wrongly assign wild type status in approximately ten per cent of cases.
1.7.5.5.2 BRAF
BRAF (BRaf murine sarcoma viral oncogenes B1) encodes a protein kinase. Mutated BRAF is present in 5% to 10% and has also been found to reduce the effectiveness of anti-eGFR agents. BRAF and KRAS mutations are thought to be mutually exclusive. It may be that BRAF mutations are responsible for non- response to treatment with anti-EGFR chemotherapeutic agents in patients with wild type KRAS (WTKRAS). Resistance to anti-EGFR agents has been reported in patients with a WT-KRAS but with a downstream mutation in BRAF112,153. Similar
80 to KRAS mutations, patients with BRAF mutations appear to have a worse prognosis irrespective of the treatment given156,157 however, there have been conflicting studies suggesting that risk of recurrent disease does not differ significantly between BRAF mutant and wild type tumours150.
1.7.5.6 Population based screening biomarkers
The national bowel cancer screening programme (BCSP) was commenced in England in 2006 and is discussed in detail in section 1.1. This programme has improved the cancer stage at diagnosis of colorectal cancer resulting in a greater proportion of the population presenting with disease amenable to surgery and less aggressive neoadjuvant and adjuvant treatments158.
1.7.5.7 Predictive value of MSI in chemotherapy
Patients with colorectal cancer exhibiting high level microsatellite instability (MSI- H) or tumours with a deficient mismatch repair system (dMMR) have been reported to have an improved overall survival and no benefit from adjuvant treatment with 5-FU based therapy, when compared to those with MSI stable tumours or those with a proficient mismatch repair system. However, there still remains significant uncertainty. A large prospective study by Sargent et al in 2010 reported no improvement in disease free survival in patients with Stage II colorectal cancer with dMMR tumours receiving adjuvant 5-FU compared to those assigned surgery alone (HR 1.10; 95% CI 0.42-2.91, p=0.85). It is unclear whether this is attributable to the already favourable prognosis with dMMR tumours or whether it is a resistance to treatment with 5FU109. More recently, the QUASAR study has reported evidence to suggest that 5FU based chemotherapy is beneficial in preventing disease recurrence in patients with stage II dMMR colorectal cancer (20.8% (183/880) of adjuvant-treated patients had recurrences at 10 years as compared with 27.6% (254/920) of untreated patients (RR 0.71; 95% CI 0.59–0.85; P = 0.0003)159.
Mismatch repair deficient colorectal cancers and microsatellite instability are discussed in section 1.4.3.
81 Clinically, selected use of MMR testing by IHC would therefore be useful to identify patients who primarily have a good prognosis based upon tumour biology but also those in whom chemotherapy may not be beneficial and would unnecessarily expose patients to harmful side effects. In the North West Region, the colorectal pathway group have developed guidelines for the assessment of MMR status in all patients under the age of 50 years with colorectal cancer (Appendix 1).
Other criteria are considered on a case-by-case basis following discussion at local CRC MDT meetings. This move toward individualisation of risk based upon tumour biology represents the beginning of more personalized management of colorectal cancer through prognostic biomarkers.
1.7.5.8 Circulating Tumour Cells
Despite decades of research and development in systemic colorectal cancer treatment, metastatic spread of the disease to distant organs is still a major focus of concern. Circulating tumour cells (CTCs) can arise from both the primary tumour and any pre formed metastases. Circulating tumour cells can be isolated using immune-magnetic techniques. However, general functional characterisation of CTCs represent a major challenge as the systemic number of CTCs in colorectal cancer is particularly low when compared with other malignancies. Despite this, there have been some studies, which have supported the clinical utility of identifying circulating tumour cells in the management of colorectal cancer. A systematic review by Peach et al in 2010 reported that six of the nine studies included which took blood samples 24 hours or more postoperatively found detection of postoperative CTCs to be an independent predictor of cancer recurrence. A prospective study of 430 patients with metastatic colorectal cancer reported that CTCs can provide prognostic and predictive information. Patients with more circulating tumour cells were found to have a worse prognosis than those with less CTCs (Overall survival 9.4 versus 18.5 months; p=0.0001). The role of circulating tumour cells in colorectal cancer continues to be explored. More recently, an ex vivo study by Grillet et al culturing CTCs it was reported that cancer stem cells (CSCs) are present within CTCs and that these CSCs express high levels of CSC markers160 and have multilineage differentiation. It was suggested that culturing CTCs could be utilised clinically to facilitate access to personalized medicine by predicting tumour behaviour and targeting treatments. In the future
82 it is hoped that “virtual biopsies” through circulating tumour cells may aid in the diagnosis of colorectal cancer and also in developing pharmacotherapies.
The discovery of circulating cell free DNA (cfDNA) has also attracted much attention in the field of cancer research. Colorectal cancer tumours are constantly in evolution. Obtaining a “liquid biopsy”, into which the tumour is constantly releasing biomolecules such as DNA (ctDNA), is considered to be more reflective of tumour heterogeneity (and potentially less invasive) than a standard tissue biopsy. The role of ctDNA in the detection of precancerous lesions or early colorectal cancer, or to guide treatments for patients with advanced disease has not yet been accurately defined to change clinical practice161. Common themes reported in this area of research are high proportions (up to 90%) of detectable serum mutations matching that of tumour DNA, particularly in patients with metastatic disease162-164. Similar concordance has not been reported in patients with early stage disease and as such little clinical validity has been described in this patient population165. In the post-operative setting, ctDNA detection has been demonstrated to be predictive of recurrence irrespective of adjuvant chemotherapy use (chemotherapy: HR 10.0; P<0.001; without chemotherapy: HR 22.0; P<0.001) in patients with locally advanced rectal cancer166. It is clear that further studies are required to both validate the role of ctDNA in different patient populations and there is need for clinical trials to ascertain the potential benefits of utilising ctDNA to guide clinical decision making and treatments161.
1.7.5.9 Tumour biology
The mismatch repair system (MMR) is responsible for recognising and repairing errors that arise during DNA replication. Colorectal cancers can be classified as to whether this system is proficient or deficient167. Mismatch deficient tumours account for 15% of all colorectal cancers and these may be sporadic or hereditary. Accumulation of multiple genetic errors, occurring within tumour suppressor genes or oncogenes, result in malignant change. Mutation in one of the four main MMR genes (MLH1, MSH2, MSH6 and PMS2) may result in deficient mismatch repair (MMR-deficient). These mutations lead to gene silencing and high-level microsatellite instability (MSI-H). Alternatively, sporadic change may occur through the epigenetic silencing of the MLH1 gene. It is this mechanism that is responsible for up to 15% of sporadic colorectal cancer.
83 Immunohistochemistry (IHC) is a histological process in which tissue antigens can be identified through using antibodies that bind to specific antigens. Immunohistochemical analysis is used to evaluate the expression of MLH1, MSH2, MSH6, PMS2 proteins and MSI-H in CRCs or lynch syndrome related cancers. It is not however possible to detect which of these CRCs are hereditary and which are sporadic based upon IHC alone.
Mismatch repair deficient CRCs (dMMR) are biologically different from proficient mismatch repair (MMR-proficient) CRCs arising through the chromosomal instability pathway. A quarter of all right sided cancers are MMR-deficient168. These tumours exhibit MSI-H phenotype169 (poor differentiation, mucinous tumours with tumour infiltrating lymphocytes and intraepithelial lymphocytes) and have improved long term outcomes and survival despite lack of benefit from standard 5-fluorouracil-based chemotherapy150,170,171 (see section 1.5.4). In stage II disease colorectal cancer it has been shown that MMR-deficient CRCs have half the recurrence rate for MMR-proficient CRC150. The true causality of this reduction in recurrence rates remains unclear, however, it has been proposed that the frame shift peptides seen in MMR-deficient CRC leads to numerous aberrant protein formation and tumours display a high immunogenicity. Immune surveillance may control the local and systemic spread of MMR-deficient tumours especially in patients with germline mutations resulting in Lynch Syndrome172,173.
1.7.5.9.1 Beta-2 microglobulin as a biomarker in MMR-deficient CRC
Beta-2 microglobulin (B2M) is a protein component of the major histocompatability complex (MHC) class I molecules. These molecules are present on all nucleated cells apart from red blood cells. These molecules have a trimeric complex consisting of a variable heavy chain, encoded by Human Leucocyte Antigen-A (HLA-A), HLA-B and HLA-C genes, the processed antigen peptide which binds to the heavy chain groove and the constant B2M light chain. B2M has no transmembrane region and lies adjacent to the alpha-3 molecule on the cell surface (Figure 4. Beta-2-Microglobulin). The B2M gene (chromosome 15q21) encodes beta-2 microglobulin. In deficient mismatch repair colorectal cancers accumulate somatic mutations in the gene that are usually preserved, such as the beta-2 microglobulin gene (B2M).
84
Figure 4. Beta-2-Microglobulin
The B2M gene spans 10,673 bps, contains several coding microsatellites and encodes for the beta-2 microglobulin protein, which is 119 amino acids in length. Four repetitive nucleotide sequences within exon 1 and 2 are vulnerable to somatic mutations and in the presence of mismatch repair deficiency are mutagenic
174 targets. Exon 1 contains a [CT]4 sequence (codon 13-15) and exon 2 contains two A5 repeats (codon 67-68, 94-95). B2M mutations are seen more commonly in patients with germline mismatch repair mutations compare with sporadic MSI- H tumours175.
1.7.5.9.2 Immune response to tumour cells
There is increasing evidence supporting the theory that an individual’s immune system plays an important role in tumorigenesis. The concept of cancer immune- editing and therefore resulting in escape of the host defence immunity is now widely accepted.
Immunoediting is a process of elimination, equilibrium and escape, whereby an individual is protected from developing malignancy and tumour progression is altered through immunogenic processes176. These phases may function independently or in sequence177. Tumour infiltrating T Lymphocytes (TILs) are often found within tumours, supporting the theory that malignant tumours trigger an immune response in the host. These cells are characterised by their cluster of differentiation 3 (CD3) surface proteins. Two subgroups of T cells have been identified; CD8+ (cytotoxic) and CD4+ (helper) cells.
85 CD4 and CD8 are transmembrane glycoproteins that are found on the surface of immune cells. CD8+ cells are specific for the Class I HLA complex. CD4 cells are specific for Class II HLA complex. CD4+ T cells are further subdivided in to T1 helper cells or T2 helper cells. Cytotoxic lymphocytes (CTLs) rely on T1 helper cells for their proliferation through the production of interleukin-2. CD4+ T cells only respond to HLA class II proteins expressed by antigen presenting cells. Therefore, CD8+ T cells are required for effective tumour elimination as most tumour cells express HLA class I molecules. Within the Class I complex there are six sub-groups of the HLA heavy chain (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G).
For CD8+ T cells (cytotoxic T Lymphocytes; CTLs) to recognise antigens, the antigens need to be exposed on the tumour cells with the HLA class I proteins. Therefore, this HLA class I complex has an important role in recognition by the cytotoxic (CD8+) T lymphocytes (CTL) through tumour cell adhesion and presentation of antigenic material to the CTLs as part of the elimination phase and cellular immune response to malignancy. On recognition of the tumour cell antigen/HLA I complex, CTLs clonally expand and differentiate. Differentiation into killer cells results in large numbers of lysosomes. Activated CTLs release cytotoxic mediators (e.g. perforin, granzymes, granulysin) in case of cell-to-cell interaction mediating destruction of the tumour cell. Cell death ensues through disruption of the cell membrane and activation of apoptosis (programmed cell death). Immediate response to tumour cells comes from Natural Killer (NK) cells. NK cells migrating from the bloodstream into a solid tumour only account for a minor fraction of the total tumour infiltrating leucocyte population178,179. However, NK have a major role in eliminating malignant cells through their ability to clear tumour cells lacking HLA class I expression.
Loss of HLA class I complex expression on tumour cells, allows the cell to avoid recognition by CTLs and tumour growth is permitted unchallenged174,180. This is termed ‘immune escape phenotype’ and has been demonstrated in several types of malignancy including colorectal cancer181, lymphoma, melanoma182 and prostate cancer183. It has been demonstrated in colorectal cancer that in addition to tumour stage, infiltration of CD8+ and CD57+ cells in the tumour margin are independent prognostic factors, and that there is an inverse correlation between these markers and some types of HLA expression (HLAB/C)178.
86 Seventy to ninety per cent of colorectal cancers have aberrant MHC class I expression. Mismatch repair deficient colorectal cancers are particularly immunogenic with prominent infiltration of TILs180,184 and upregulation of cytotoxic mediators185. HLA class I expression is lost in approximately 60% of mismatch repair deficient colorectal cancers usually due to B2M inactivation186. The incidence of HLA class I loss is less common in mismatch repair proficient colorectal cancers (16.7%)186-188.
B2M mutations lead to complete loss of HLA class I mediated antigen presentation and represent the most important mechanism of immune evasion in MSI-H colon cancers181,187,188. Loss of HLA class I expression has been implicated in local tumour progression and distant metastases. Metastases occur when malignant cells are transported from one organ or tissue to another e.g. liver or lungs usually by lymphatic or haematogenous spread. The formation of metastasis is complex. Tumour cells must leave the original tumour and migrate through lympho-vascular system and migrate from the circulation to tissue thereby forming micro- metastases. When these micro-metastases have developed their own blood supply they are able to form macroscopic metastases. It is reported that less than 2% of cells that migrate from the initial tumour site are able to initiate growth at distant sites, and less than 1% of these cells are able to maintain growth at that site189. It is therefore likely that the cells that are successful in forming distant metastases are a subset of highly specialised cells. There have been increasing numbers of reports suggesting that tumour cells are less likely to establish distant metastasis which are deficient in expressing HLA class I complex. T cell mediated immunity, by CTLs, cannot be activated in the absence of HLA class I. Natural Killer cells are inhibited by CTLs.
1.7.5.9.3 Natural Killer Cells
Loss of HLA class I results in activation of NK cells in the circulation. Natural killer cells recognise tumour cells by the activating receptors (like natural cytotoxicity receptors) which detect altered expression of their ligands on the tumour cell surface. They respond rapidly to the presence of tumour cells and thereby play a significant role in antitumour responses.
This activation may explain how whilst favouring local tumour growth, loss of HLA class I is protective against distant metastases. B2M mutations have been shown
87 to be more frequent in colorectal cancer than other malignancies including breast, ovary and lymphoma181 and to be positively related to advancing tumour stage175. However, in the study by Kloor et al, no B2M mutations were identified in stage IV tumours suggesting that B2M may play a protective role in the development of distant metastases.
As the immune system plays such an important role in the pathological course of malignancy it has been logical to attempt to develop new therapies in the treatment of colorectal cancer based upon manipulation of an individual’s immune response. These include checkpoint inhibitors and immune modulators, monoclonal antibodies, therapeutic vaccines, adoptive cell therapy, oncolytic virus therapy, adjuvant immunotherapies and cytokines. These treatments are being evaluated in early phase (phase I and II) clinical trials of patients with stage IV or recurrent colorectal cancer or those with stage IIa or III colorectal cancer in the adjuvant setting.
Once colorectal cancer becomes metastatic, the treatment options available to patients are more aggressive with high treatment related morbidity and mortality and are aimed at more palliative than curative outcomes. It is therefore beneficial, through risk stratification, to target adjuvant treatments to those most at risk of disease recurrence or metastasis following surgical resection and immunological properties inherent to the tumour may have a role in clinical decision-making.
1.8 Common genetic variants and Colorectal Cancer
1.8.1 Genome Wide Association Studies
It is proposed that much inherited risk in colorectal cancer is due to combination of low risk penetrance variants. It is this hypothesis that underpins the use of Genome Wide Association studies in colorectal cancer.
A Genome Wide Association Study or GWAS aims to identify genetic risk factors for disease that are common in the population through measurement and analysis of large numbers of DNA. Ultimately these genetic risk factors can be utilised through understanding the biology of disease to develop prevention and treatment strategies for the population at risk through screening and development of pharmacological therapies. Such studies have been successful in identifying risk factors for diseases such as age-related macular degeneration (AMD) and also
88 generally in developing pharmacological therapies through the identification of DNA sequence variations in several genes affecting drug metabolism, efficacy and adverse events. These developments have realised the possibility of future management strategies that are tailored to individual patients on the basis of their genetic profile.
1.8.2 Single Nucleotide Polymorphisms
A well-described unit of genetic variation is a SNP. Single nucleotide polymorphisms are single base pair changes in the DNA sequence that occur frequently in the human genome. SNPs are the most common genetic variant in the human genome and have minimal effect on cellular and biological mechanisms. However, these changes in a single base pair can have functional consequences through changes in amino acids, mRNA transcript stability and transcription factor binding ability190.
The term SNP is generally applied to common single base-pair changes. Single Nucleotide Polymorphisms have two commonly occurring base pair possibilities for a single SNP location i.e. two alleles. The frequency of a SNP in a given population is described as the minor allele frequency (MAF; the less common allele). The major allele is the more common allele. Rare genetic variants that result in detrimental change in protein function and rare genetic disorders such as cystic fibrosis (CF) are referred to as mutations rather than SNPs.
1.8.3 Polygenic risk
The genetic testing for monogenic disorders and hereditary forms of colorectal cancer e.g. HNPCC and FAP are well established. However, much debate surrounds the clinical relevance of polygenic profiling for patients at risk of other complex diseases including type 2 diabetes, breast cancer and prostate cancer. Multiple genetic loci contribute to the increased risk of common cancers and have been increasingly identified in Genome Wide Association Studies over the last decade. At any locus an individual may carry zero (homozygous wild type), one (heterozygous) or two (homozygous) risk alleles. These alleles usually confer a slight increased risk of developing the disease individually but appear to have a multiplicative effect on relative risk in combination.
89
A more personalised and ‘risk-stratified’ approach to screening at risk populations could result in tailoring screening regimes to each population risk stratum and potentially improve the efficiency of a screening programme whilst addressing the balance of harm and benefit of screening. Potential strategies targeting screening programmes based upon polygenic risk have been reported in breast cancer and prostate cancer18. However, the implementation of such a screening program would have ethical, legal and social implications. In breast cancer and colorectal cancer genetic variants have been identified, through GWAS and validation studies, to have weak additive or multiplicative effects associated with an increased risk of cancer. Therefore there is a possibility that some population stratification risk for CRC on the basis of polygenic inheritance could be undertaken with age and family history to improve the uptake and efficiency of current screening programmes191.
1.8.4 Genetic Linkage analysis
1.8.4.1 Common Disease/Common Variant Hypothesis
Common variants by definition do not have high penetrance. If the minor allele frequency of a SNP is 20% resulting in a disease phenotype, then 20% of the population would have that disease. However, only slight correlation between a SNP and disease prevalence will exist if the SNP has a small effect on risk of disease. Furthermore, where common alleles have low penetrance and common disorders display heritability, multiple common alleles must influence an individual’s susceptibility of a diseased state.
Alleles with high penetrance for Mendelian disorders are rare but result in a disease state i.e. large effect, whereas most GWAS findings are common SNPs that often occur in healthy individuals and do not result in a disease state (small effect).
The National Human Genome Institute GWAS catalogue (http://www.genome.gov/gwastudies) lists over 3600 SNPs that have been identified for common diseases or traits. This catalogue (as of August 2014) includes 1961 publications and 14012 SNPs. There were twenty GWAS studies identifying variants associated with colorectal cancer. This online resource,
90 together with bioinformatic predictions of the underlying functionality at trait/disease-associated loci, is well-suited to guide future investigations of the role of common variants in complex disease aetiology.
In order to capture common variants, the location and frequency of commonly occurring SNPs must be identified to allow further interrogation of these sites whilst taking consideration of population specific differences. These population differences must therefore be recorded. The international HapMap project was designed to first identify variation, and secondly characterise correlation amongst these variants through sequencing techniques in a number of populations. These populations included those of European descent, Yoruba population of African origin, Han Chinese from Beijing and Tokyo Japanese192. This data allowed the examination of linkage disequilibrium.
1.8.4.2 Linkage disequilibrium
Linkage disequilibrium refers to the changes in genetic variation over time in a population and infers a degree of dependence. It describes the extent to which a SNP is inherited or correlated with an allele of another SNP within a population and is directly related to the concept of chromosomal linkage. In chromosomal linkage two markers on a chromosome remain physically joined on that chromosome for several generations within a specific family. Recombination events during meiosis break apart chromosomal segments thus breaking apart these chromosomal markers. This effect is amplified over generations and in a population undergoing random mating these segments of contiguous chromosome become disassociated until eventually all alleles in the population are in linkage equilibrium i.e. independent. It is therefore logical that the rate of linkage equilibrium within a population is related to population size.
1.8.4.3 Direct and Indirect Association
Single nucleotide Polymorphisms that directly influence a phenotype, which is genotyped in a GWAS study, and found to be statistically significant is referred to as a SNP with direct association. GWAS studies have enabled thousands of SNPs to be associated with large numbers of phenotypes. These SNPs are known as ‘tag SNPs’. Indirect associations occur when a ‘tag SNP’ is identified in high linkage disequilibrium with the influential SNP. Areas of linkage disequilibrium are
91 population specific and as such tag SNPs for one population may not work well for an alternative population. It is therefore important to try to identify the association between genetic variant and disease at a molecular level to understand the functional consequences of these common variants. SNPs can also be described, as ‘functional SNPs’ if they directly influence a biological system resulting in a specific phenotype, however, the number of SNPs shown to be functional is small.
It is for the reason of indirect and direct association that significant associations identified in GWAS studies should not be presumed to have a direct causal effect. Therefore, further studies are needed to identify the precise location of the influential SNP or causative variant that may be rarer and have a larger effect size.
To conduct a GWAS study for common SNPs with common phenotypes, the alleles of half a million to a million SNPs for each individual must be genotyped in a time and cost-efficient manner. Recent developments in genotyping technologies have made this possible through chip-based microarray technology.
Genome wide association studies have made great impact upon human genetics over the last few years. Future development in next-generation sequencing techniques will provide further armamentarium to the geneticist leading to whole genome sequencing.
1.8.5 GWAS and colorectal cancer
Genome wide association studies have identified single nucleotide polymorphisms (SNPs) associated with colorectal cancer in the general population. Some of these SNPs are located close to loci functionally related to the Mendelian polyposis syndrome genes (e.g. GREM1, BMP2, BMP4, POLD3 and MYC). SNPs individually have small effect sizes but in combination are thought to have a multiplicative effect on the development of malignant disease. This has been shown in BRCA2 mutation positive patients and breast cancer risk193. A study by Carvajal-Carmona et al identified eight SNPs associated with adenoma development predisposition in the absence of colorectal cancer and ten other SNPs (which have previously been identified as associated with colorectal cancer) to not be associated with the development of adenomatous polyp formation. The authors of this study
92 concluded that colorectal cancer risk in the general population is likely in part to be mediated by predisposition to adenomas194. A more recent study has examined whether the multiple adenoma phenotype can be explained in some cases by common risk alleles of individually modest effect. One hundred and seventy-eight cases were identified from the St Mark’s Polyposis registry, Harrow, UK with between five and one hundred adenomas. To avoid overlap with published CRC GWAS cancer-free individuals were identified from the Colorectal Cancer Family Registry (CFR) as controls. Significantly higher SNP risk scores were seen in the adenoma group (mean=2.44, SD=0.40) than in the controls (mean=2.27, SD=0.42). The 18 CRC SNPs explained 4.3 % of the variance in the risk, however, three SNPs alone accounted for 3% of the variance in multiple adenoma risk (rs6983267, rs10795668, rs3802842). However, this study did not support the findings of previous other studies194,195.
There have been two phases in the identification of common genetic variants, or polymorphisms, associated with CRC risk. Early studies investigated pre identified candidate polymorphisms but due to these studies being underpowered it is now thought that levels of significance reached were not high enough for the associations to be regarded as proven69,196,197. More recently, GWASs have utilised genome wide hypothesis free approaches in which haplotype tagging SNPs (tagSNPs) are used to screen for associations based on linkage disequilibrium. Linkage disequilibrium has been previously discussed in section 1.12.2.
Genome wide association studies have now identified several common risk variants associated with colorectal cancer risk, at stringent levels of significance (p<5x10-8). Generally, discovery phases of GWASs are based on hundreds of thousands of tagSNPs in around 10,000 CRC cases and controls. Multiple subsequent validation stages involving tens of thousands of cases and controls (e.g. derived from the UK and COGENT collaborators throughout Europe) have been used to confirm CRC risk modulation with these SNPs196. Several meta- analyses of GWASs have also been used to identify further high-risk loci associated with colorectal cancer. More recent GWAS studies in European descendants, East Asian, African Americans and Hispanics have identified many more new variants. The total of known independent signals for colorectal cancer is now more than 100198,199.
Through the series of validation and association studies it has been concluded that some of the inheritable risk of colorectal cancer is now thought to be attributable
93 to multiple low risk variants. Several SNPs have been identified to be associated with CRC susceptibility, with variable effect sizes both within and between ethnic groups200, in a number of studies. However, for the majority of SNPs these associations are not reported universally across studies and it is not uncommon to identify conflicting reports of strengths of associations or even lack of association at all. Lubbe et al 2012 evaluated the impact of fourteen colorectal cancer variants that had been identified in 8,878 cases and 6,051 controls. The authors were able to identify that some SNPs clearly have stronger effects than others on colorectal cancer risk. They also described the SNPs to confer a moderate association with CRC but a strong cumulative association with CRC risk201. Cumulative effects models (additive or multiplicative) for these low risk markers have been used in many studies.
The functionality of the SNPs identified to be associated with colorectal cancer remains unclear and this is discussed further in section 1.12.4.2. Some studies have also demonstrated that these colorectal cancer susceptibility SNPs are not strongly modified by sex, BMI, alcohol, smoking, aspirin and dietary factors202. It is not known if the SNPs are responsible for increasing the risk of premalignant change i.e. adenoma formation or at the carcinoma stage of tumorigenesis. As most CRC arise from adenomas it stands to reason that SNPs associated with CRC may be partially responsible for the development of adenomas. Several studies have evaluated the association between SNP and polyposis phenotype, and this is discussed further in section 1.12.5.2.
1.8.5.1 History and Discovery of SNPs in colorectal cancer
The first SNP identified through GWAS to be susceptibility variant for CRC was identified in 2007 (rs6983267, loci 8q24.21) and has been validated in a number of independent studies203-205. Over the next year, five further susceptibility loci were identified with strongly associated SNPs (rs4939827, rs4779584, rs16092766, rs10795669, rs3802842)206-210. Meta-analysis of GWASs have been performed to enhance the power of GWASs to detect additional CRC risk loci. Houlston et al in 2008 identified an additional four common genetic variants, rs4444235, rs9929218, rs10411210 and rs961253 through meta-analysis of previous studies. These associations were replicated in more than 20,000 cases and controls5. A further meta-analysis of three genome wide association studies in 2010 was able to identify six more SNPs associated with colorectal cancer
94 (rs669170, rs6687758, rs10936599, rs11169552, rs7136702, and rs4925386)211. There have been around 30 SNPs validated at the time of writing.
1.8.6 Functions of CRC association single nucleotide polymorphisms
The function role of the associated SNPs is largely unknown. It is thought that these common low risk variants may impact on morphogenetic pathways, in the presence of different risk factors, thereby influencing not only predisposition to colorectal cancer but also tumour subtype (tumour site, stage, degree of differentiation and microsatellite instability status)212.
A study by Tuupanen et al has provided some evidence that the first CRC SNP identified, rs6983267, is in itself functional213. This SNP lies a few hundred kilobases form the c-Myc oncogene and alters the TCF4 binding site. The SNP rs10411210 is a known CRC predisposition SNP that lies within an intron of rhophilin2 (RHPHN2) but also within on hundred kilobases of the DNA repair gene FAAP24. However, studies have reported that RHPHN2 is actually the target of functional variation tagged by rs1041120 rather than FAAP24214. Candidate gene analyses have been unable to identify convincingly that DNA repair SNPs are directly involved in CRC predisposition. This finding is also true in studies of other cancer susceptibility. However, it has been demonstrated in SNP set analysis that variation around DNA repair loci does contribute to CRC risk196. It is therefore possible, that DNA repair SNPs have a small multiplicative effect on CRC risk and that many loci with individually small effects alter DNA repair capacity i.e. polygenic model.
1.8.6.1 BMP
Of the 14 loci identified in the meta-analysis by Houlston et al, 3 loci were identified to be close to the bone morphogenic protein (BMP) signalling pathway; GREM1 (rs4779584); BMP4 (rs4444235) and BMP2 (rs961253). Misregulation of the pathway is an important factor in the development of colon cancer.
95 1.8.6.2 TERC
Chromosome telomeres consist of multiple short nucleotide repeats that protect against large-scale genetic rearrangements. Long telomeres have been associated with long life span. Ordinarily telomere length reduces with advancing age and has been shown to be associated with the development of CRC. Telomerase enzyme activity is provided by telomerase reverse transcriptase (TERT) that uses telomerase RNA component (TERC) as a template for the addition of nucleotide repeats to existing telomeres. The major allele at polymorphism rs10936599 is associated with an increased risk of colorectal carcinomas, adenomas and telomere length215. Jones et al investigated both telomere length and TERC SNP and identified that TERC SNP (rs10936599) is associated with susceptibility to colorectal cancer. However, contrary to previous studies, the SNP data identified that those with longer telomeres were at increased risk of CRC. They concluded that previous studies between cancer risk and telomeres had wrongly emphasised the chromosomal instability aspects of the cancer risk. They proposed that shorter telomeres actually arise as a result of the disease and as such have been identified in retrospective collection of CRC data.
1.8.7 Population specific risk
How much a risk allele contributes to an individual’s risk of colorectal cancer may vary between populations to allele frequencies or other genetic and environmental backgrounds of the population. These factors may result in a modification of an effect of a variation. Understanding the differential effects in different populations is important for the translation of results to accurate and relevant risk predication in different populations216. Kirac et al reported 111% increased risk in those patients homozygous for the mutant allele (TT) in rs4939827 than those homozygous for wild type allele. This is a much stronger association between SMAD7 variant rs4939827 and colorectal cancer than had been reported previously in other populations. Other SNPs tested in the same study showed less strong associations that had been reported previously in other studies, which may be due to the study being underpowered, or true variations between populations. Understanding differential effects between populations is key in providing more risk adjusted screening, treatment and surveillance approaches for patients. Some studies have explored the association of colorectal cancer susceptibility loci in inherited colorectal cancer disorders. There have been no studies to date which
96 have explored the effects of susceptibility loci upon outcomes in patients with inherited gene disorders that increase an individual’ s risk of developing colorectal cancer.
Picelli et al conducted a meta-analysis of mismatch repair polymorphisms within the Cogent Consortium for Colorectal Cancer following a preliminary case-control study for SNPs in four colorectal cancer genes (APC, MLH1, MSH6 and MUTYH) in a Swedish population. In this follow up case control association study, six SNPs were investigated (rs459552, rs1799977, rs1800932, rs1800935, rs3219484 and rs3219489) in more than 8000 cases and 6000 controls, all of which showed no association with CRC risk. These SNPs have not been the subject of many validation studies and therefore their association with CRC risk are less well documented. However, this study again raises the possibility that phenotypic heterogeneity, differing environmental exposure and population linkage disequilibrium patterns may explain the observed difference of genetic effects between Swedish and other European cohorts217.
There is clearly value in the development of population-based risk stratification for the screening, treatment and surveillance of colorectal cancer patients. Increased risk of developing colorectal cancer has been demonstrated with increased number of risk alleles with a dose response relationship in patients with no known high risk mutation, stratified by family history (first degree relative, second degree relative and no family history)218. Unnecessary risk and morbidity may be avoided by tailoring management programmes according to population specific risk. However, more studies and meta-analyses are needed to better define the association with increased colorectal cancer risk, phenotype and particularly long-term outcomes for patients’ common genetic variants.
1.8.7.1 CRC risk and SNPs in high risk populations
Several SNPs have now been identified and associated with colorectal cancer susceptibility. Each of these variants confer a modest risk which is suggestive that a significant proportion of familial risk remains unexplained and other susceptibility variants have not yet been identified. Each of these variants acting together has the potential to confer a more profound effect on CRC risk. Whilst several studies have investigated the effect of CRC associated SNPs within a background population, relatively few studies have determined their effect on higher risk population e.g. single gene defects predisposing to early development
97 of colorectal cancer. Within the mismatch repair single gene defect populations there is wide variation in both lifetime risk of colorectal cancer (28-75% in men and 24-52% in women)100 and age at first adenoma detection or development of colorectal cancer.
1.8.7.2 Lynch syndrome and susceptibility loci
Cancer risks for MLH1 and MSH2 mutation carriers vary widely from family to family and this supports a role for polygenic modifiers in age of onset of carcinoma. If SNPs are responsible for some of the variation in phenotype between single gene defect carriers this may have some clinical applications in terms of risk-adjusted surveillance and management of these patients.
In 2009, a study of 675 Lynch syndrome patients by Wijnen et al, two SNPs (rs16897266 and rs3802842) were identified to be associated with increased risk of developing colorectal cancer219. Interestingly, for rs16892766 possession of the C-allele resulted in a dose dependent increase in risk of CRC. Homozygosity for the C-allele was associated with a 2.16-fold increased risk of developing CRC. An additive model, as used in most association studies, identified that the risk of CRC was significantly associated with the number of risk alleles with effects being more predominant in female than male carriers. Talseth-Palmer et al went on to further genotype nine SNPs (rs16892766, rs7014346, rs6983267, rs10795668, rs3802842, rs10318, rs4779584) at six colorectal cancer susceptibility loci in 684 mutation positive Polish and Australian patients with Lynch syndrome220. Again, an increased risk of CRC was demonstrated with rs3802842 and rs16892766 in female MLH1 carriers. Female carriers homozygous for rs3802842 were at highest risk of developing CRC. By combining the number of risk alleles a difference of 24 years was detected between individuals carrying three risk alleles to those carrying no risk alleles. The authors went on to further confirm the association in a subsequent study of over 1300 Lynch syndrome patients. In this study a pair wise combination of SNPs rs3802842 and rs16892766 revealed a significant difference in median age of diagnosis of CRC with patients carrying three risk alleles being 28 years younger compared to individuals carrying no risk alleles221. A limitation of this study is lack of genotyping a control group for comparison, however, there is some evidence to suggest that genotyping of rs3802842 and rs16892766 may be beneficial in patients with MLH1 mutations. This would provide personalised risk assessment and consequently a tailored risk-adjusted surveillance program to be offered to patients harbouring high numbers of risk
98 alleles. This also could have potential implications for targeting prophylactic surgical intervention for this high-risk group at risk of early onset CRC. However, such associations have not been reported universally.
A study by Houlle et al in 2011 genotyped 748 French patients with known MMR mutation carriers (without a control group) for five common genetic variants and did not identify any association with age at first CRC occurrence222. Only 44% (n= 329) of subjects had developed colorectal cancer at the time of analysis and given the relatively small sample sizes within each single gene defect group the authors recommended caution in interpreting their findings.
A more recent study found no association with any of the eleven colorectal cancer associated SNPs genotyped and has supported the findings of the French study223. However, this study also did not genotype a control population and only 46% of those tested had colorectal cancer. Both this and the French study evaluated frequency of SNPs in MMR gene mutation carriers with and without colorectal cancer. Broeke et al also reported that twenty four previously identified SNPs do not modify colorectal cancer risk in a study of PMS2 mutation carriers with colorectal cancer, using a control population of PMS2 carriers with no colorectal cancer224.
No studies have evaluated the frequency of SNPs in MMR gene mutation carriers compared to a background population with colorectal cancer or a control group without colorectal cancer. Further studies are needed in this population group before there is conclusive evidence that knowledge of SNP frequencies is not beneficial in accurately predicting individual risk estimates and tailoring surveillance and treatment programs for patients with Lynch Syndrome.
1.8.8 SNPs and adenoma
As the majority of colorectal carcinomas arise from adenomas it is reasonable to hypothesise that SNPs associated with colorectal cancer may have an effect upon this part of the tumorigenesis pathway. This hypothesis that SNPs associated with CRC are associated with adenoma formation has been tested by Carvajal-Carmona et al. Eight SNPs (rs10936599, rs6983267, rs10795668, rs3802842, rs444235,
99 rs1957636, rs4939827 and rs961253), known to be associated with colorectal cancer risk were over-represented in colorectal cancer free patients with adenomas, compared with controls194. However, two of these SNPs (rs380842 and rs6983267) have also been shown in other studies to be associated with non- polypoid disease i.e. HNPCC (Lynch Syndrome) which contradicts their contributory role to the development of adenomatous change. Additionally, one of two SNPs (rs9929218) that had been previously reported by Abuli et al to be associated with colorectal adenomas when present in combination with rs6983267 was not identified in the study by Carvajal-Carmona. However, there was no control group used in the study by Abuli. It is prudent to genotype a control group in studies of variant association and validation to be able to draw meaningful results from data if findings are to be used to inform, and potentially change, clinical practice through risk adjustment and stratification.
1.8.8.1 Polyposis Phenotype
1.8.8.1.1 FAP
Patients with adenomatous polyposis are at increased risk of developing colorectal cancer. Those most at risk are usually considered to be those with mutation in the APC gene resulting in familial adenomatous polyposis. Number of polyps is variable, but FAP patients usually develop hundreds to thousands of polyps. Few studies have evaluated the role of SNPs in onset of colorectal cancer in this patient group and although position of the APC germline variant has been shown to be associated with severity of polyposis, no other variable has shown any association225. Familial adenomatous polyposis coli is discussed further in section 1.8 and phenotypic variability between attenuated FAP and FAP is well described. As severity of disease has been stratified in this patient group to those with FAP and attenuated FAP the clinical importance of further trying to risk stratify this high-risk patient group is of questionable clinical relevance as most patients with FAP will undergo prophylactic colectomy in their second decade. It is the attenuated FAP group who may benefit more from further risk stratification to inform surveillance programs.
100 1.8.8.1.2 Oligopolyposis
Oligopolyposis is a term commonly used to describe a phenotype of polyp count, which is greater than anticipated in a screening population, but is not sufficient to correlate with expected endoscopic findings of an individual with familial adenomatous polyposis. The presence of five to one hundred colorectal adenomas suggests an inherited predisposition. Whilst the majority of these are explained by mutation in the APC or MUTYH gene, an estimated 20-30% remain unexplained by Mendelian polyposis syndromes of familial adenomatous polyposis (FAP) or MUTYH-associated polyposis (MAP). It has been proposed that some susceptibility of these unexplained cases might be attributable to the common genetic variants associated with CRC.
A recent study published in 2014 attempted to identify whether sixteen common genetic variants are associated with disease in 252 patients, without APC or MUTYH mutation, with or without CRC195. All patients had more than ten colorectal adenomas. A statistically significant association was identified for patients with SNPs rs3802842 (p=0.0037) and rs4779584 (p=0.023) and polyposis only phenotype (i.e. no CRC). It would appear that colorectal cancer risk alleles contribute to the development of multiple adenomas in patients without pathogenic germline mutations in APC or MUTYH genes.
Findings for rs3802842 have been supported in a subsequent study which calculated an individual SNP risk score for eighteen SNPs included in the study225. Multiple adenoma phenotype was associated with a significantly higher number of risk alleles than the population controls (p=5.7 x 10-7) with strongest association seen in those patients with more than ten adenomas. The eighteen CRC SNPs studied accounted for 4.3% of the variance in multiple adenoma risk, but 3.0% of this variance was accounted for by three SNPs, the afore mentioned rs3802842 and in addition rs6983267 and rs10795668.
Additionally, phenotype in first degree relatives was also evaluated. Forty-one percent of index patients had one or more relatives with CRC. This over- representation of CRC patients in FDR of polyposis patients suggests that the relatives of patients identified with more than ten adenomas may benefit from surveillance.
101 1.8.9 Patterns of disease and types of disease
It is important to not only evaluate the role of common genetic variants in colorectal risk but also their potential influence of polyp and carcinoma phenotype. The identification of an association between some SNPs (either in isolation or in combination) and colorectal cancer phenotype may have implications for CRC management and surveillance programs. For example, a SNP with a strong association to rectal carcinoma may reduce the need for colonoscopic surveillance as flexible sigmoidoscopy would visualise the rectum adequately without exposing the patient the risks of full colonoscopy. rs10936599, rs10796558, rs16892766, rs9929218226, have been associated with advanced adenomas and rs16892766 is also associated with advance stage tumours. rs961253 is associated with non-advanced adenomas and serrated polyps. Familial history of colorectal cancer and adenoma development has also been associated with rs6983267227,228.
A study by Lubbe et al in 2012 aimed to determine if any of the now 16 CRC susceptibility variants differentially impact on CRC phenotype to confirm them as distinct and aetiological different risk factors in carcinogenesis. Only one (14q22.2 rs1957636) of the sixteen SNPs studied did not show a statistically significant association with colorectal carcinoma. The fifteen SNPs that did show a statistically significant association are shown in table 12. Interestingly, this study did not show statistically different associations with either tumour grade or stage, but differences were shown between SNP genotype and tumour site (i.e. colon or rectum) and microsatellite stable (MSS) and MSI cancers. Microsatellite stable rectal disease was associated with rs6691170 and rs3802842; rs4779584, rs961253 and rs4813802 were associated with microsatellite stable colonic disease and rs4444235 and rs4925386 with microsatellite instability colonic disease212. Thereby partially supporting findings of a smaller study that had suggesting that rs4939827 and rs6983267 was associated with rectal disease and rs3802842 with a lower risk of rectal disease229.
102 Table 12. SNPs with statistically significant association with CRC risk. Lubbe et al 2012.
SNP Loci Gene Major Minor Allele Allele rs6691170 1q41 G T rs10936599 3q26.2 MYNN C T rs16892766 8q23.2 (EIF3H) A C rs6983267 8q24.21 (c-MYC) G T rs10795668 10p14 G A rs3802842 11q23.1 C11orf93 A C rs11169552 12q13.13 C T rs4444235 14q22.2 (BMP4) T C rs4779584 15q13.3 (GREM1/SCG5) C T rs9929218 16q22.1 CDH1 G A rs4939827 18q21.1 (SMAD7) T C rs10411210 19q13.11 RHPN2 C T rs961253 20p12.3 C A rs4813802 20p12.3 T G rs4925386 20q13.33 LAMA5 C T
1.8.9.1 Protective effects
Most studies have identified association between common genetic variants and colorectal cancer risk. A less reported association is the protective effects conferred by some variants. The single nucleotides rs10795668 (homozygous) and rs4939827 have been reported to have a protective effect on colorectal cancer risk in two studies206,208. However, other studies have also reported these common genetic variants to both be associated with the development of adenomas. It may be that these SNPs have protective effects in certain patient groups, which have not yet been clearly identified by the published literature. The potential protective effects of some SNPs could have as much, if not more, clinical significance for those patients in high-risk groups under regular surveillance programs.
103 1.8.9.2 Long term survival and early onset colorectal cancer
Despite increasing numbers of susceptibility loci for CRC being identified, much smaller numbers of studies have investigated the effect of low risk variants on long-term survival and outcome209,230-232. These studies have reported varying results. Marginal significance has been reported with SNPs rs7013278 and rs7014246 but these did not show study wide significance after correction for multiple testing233. More studies are needed in this area. Similarly, early onset colorectal cancer and influence of CRC susceptibility loci is also under investigated. Studies of genetic variants in early onset colorectal cancer in patients without identifiable single gene defects have also reported associations with rs1079668, rs3802842 and rs4779584234. Further work is needed with large-scale well- designed studies to further define associations and individualise risk.
104
STUDY ONE
The effect of intensive CT scanning in surveillance to detect the recurrence of colorectal cancer
105 2.1 ABSTRACT
Background Colorectal cancer is a major cause of morbidity and mortality worldwide and is the second most common cause of cancer death in the UK. 15.6% of all patients who undergo apparently curative resection for colorectal cancer will go on to develop recurrent or metastatic disease in 5 years235. The aim of follow up after curative resection is to identify surgically treatable recurrence. There is widespread heterogeneity in follow up regimens used in clinical practice. Intensive follow up is commonplace but is based upon limited evidence and the role of each follow up modality (clinical, serum carcinoembryonic antigen testing, intensive imaging, and endoscopic surveillance) remains unclear.
Methods All patients presenting to the department of colorectal surgery at Manchester Royal Infirmary up to 1st September 2013 were identified through electronic patient records and clinical case notes. All patients who underwent segmental colonic resection by a single surgeon with curative intent were included in the study. Recurrence rates (locoregional and distant), median time to recurrence and overall survival patterns were compared by Dukes’ Stage. National Hospital Episode Statistics (HES) Data were used to determine deaths related to colorectal cancer. Statistical analysis was undertaken using SPSS version 22. Chi-squared testing was performed for binary or categorical data. Time to recurrence was analysed using the Kaplan Meier method and time to recurrence were compared using the log rank mantel cox statistic.
Results 210 patients were included in the study. Mean age 64.4 years. 126 patients (60%) were male. CT scans were performed at 6,12,18,24, 36, 48 and 60 months following resection. Overall, 4.5% of all CT scans performed detected disease recurrence in 60 patients (29.4%) patients. Greatest incidence of disease recurrence was detected at 6 months and 3 years post op. Only 5% of all patients developed disease recurrence during the fourth and fifth years post resection. 3.8% of patients developed local recurrence. Patients who received postoperative chemotherapy were less likely to develop recurrent disease (p=0.008). There was
106 no correlation between Dukes’ Stage and local or regional recurrence (p=0.66). 10.5% of all patients (22/210) underwent surgery with curative intent for recurrence.
Conclusions This is the first study to validate the findings of a UK multicentre national randomised control trial of follow up in patients with colorectal cancer. Intensive CT imaging is pragmatic and a readily available follow up modality in clinical practice. Of the 60 patients with recurrent disease, a high proportion (22/60, 36.6%), had surgically treatable recurrence.
As patient expectations increase, colorectal cancer specific outcomes are also improving. It seems likely that further improvements in outcomes will result from aggregation of marginal gains. This study demonstrates the value of intensive CT follow up as an incremental gain in this high-risk population.
107 2.2 Introduction
CT scanning is a commonly used radiological surveillance modality for patients who have undergone resection of a primary colorectal cancer. However, the benefit of CT scanning in detecting potentially curable disease recurrence over other means of follow up including serum carcinoembryonic antigen, clinical and endoscopic assessment remains unclear. Heterogeneity of patient and environmental factors are thought to play a role in why despite several randomised controlled trials and meta-analyses studies have been unable to identify a single follow up regime most appropriate to identify curable disease recurrence. This study evaluates the role of intensive CT scanning in disease surveillance after segmental colonic resection with curative intent.
2.2.1 Background
Colorectal cancer is a major cause of morbidity and mortality worldwide and is the second most common cause of cancer death in the UK. Clinical assessment, endoscopic and radiological investigations are utilised in the detection, diagnosis and staging of colorectal cancer. Treatment options and planning will depend upon the stage of the disease and other patient factors. Patients with potentially curable disease will go on to have surgical resection of the disease, sometimes before or after treatment with chemotherapeutic agents. Traditionally patients undergo regular radiological (CT scanning) and clinical follow up by specialist nurses or clinicians (oncologists and colorectal surgeons) for a period of five years. The goal of this intensive follow up is to detect disease recurrence, which is potentially amenable to cure or further treatment to prolong survival.
CT scanning of the chest, abdomen and pelvis, regular blood carcinoembryonic antigen measurement (CEA) and surveillance colonoscopy are well-recognised widely available means of identifying early and surgically treatable recurrence of colorectal cancer. The role of each of these follow up modalities in detecting recurrent colorectal cancer remains unclear. Follow up regimes worldwide are highly heterogeneous with respect to all modalities of follow up despite extensive research programmes. Meta-analyses have evaluated randomised controlled studies with inherently heterogeneous follow up strategies236.
108 2.2.1.1 Purpose of colorectal cancer follow-up
The aim of follow up surveillance programmes in patients treated for colorectal cancer is to detect local, regional or distant recurrence of disease which is amenable to intervention with curative intent. This is not possible in all patients and some patients may develop incurable disease recurrence between surveillance tests. As no single investigation is best suited to detecting all sites of disease recurrence a combination of clinical, serum, endoscopic and radiological tests are used.
Locoregional recurrence colorectal cancer is associated with poor prognosis. Operative salvage in these patients is aimed at reducing the morbidity of local tumour involvement and prolonging overall survival. Some specialist centres have reported improved outcomes in selected patients with locoregional recurrence after curative intent surgery when treated with combined mode treatment.
2.2.1.2 Follow up regimens
There is no clear consensus on the most appropriate follow up regimen in terms of clinical review or investigations in order to maximise detection of treatable disease recurrence and improve survival. A 2015 Cochrane review recommended the use of CEA in detecting recurrent colorectal cancer suggested using a cut off of 10µg/L in surveillance for the detection of recurrent colorectal cancer but also adding another diagnostic modality (e.g. a single CT scan of the chest abdomen and pelvis at 12 to 18 months to detect the missed cases).
Current NICE guidelines237 recommend that patients are offered regular surveillance with a minimum of two CT’s of the chest, abdomen and pelvis in the first three years, and regular serum carcinoembryonic antigen tests (at least every 6 months in the first 3 years) after apparently curative resection.
Whilst it has been recognised in studies that increasing frequency of clinical review appointments has not been associated with any survival benefit it is important to remember that these visits are important for other reasons. They are important to maintain the clinician and patient relationship, to arrange, co-ordinate and discuss results of surveillance investigations and an opportunity to monitor the long-term effects of treatments and therapies.
109
As clinical studies have reported improved outcomes with more intensive follow up regimens, the Greater Manchester and Cheshire Cancer Network guidelines have adopted an intensive follow up approach. Patients post resection should undergo six monthly CT scans for two years followed by annual CT for three years and colonoscopy at two and five years. The British Society of Gastroenterology suggest much less intensive follow up is appropriate. Current guidelines238 (but pre dating systematic review publications) state that it is reasonable to offer liver imaging to asymptomatic patients under the age of seventy years in order to detect operable liver metastases once during the first two years after resection. On the basis that colonoscopy produces a yield of treatable tumours it is recommended that the colon is examined endoscopically five years after surgery and at five yearly intervals up to the age of seventy years. These guidelines also pre-date the introduction of the NHS Bowel Cancer Screening programme. Therefore, in the current era patients would be discharged on completion of follow up regimen to return to normal screening, or if older than the screening population.
Prior to 2014, seven published clinical trials had compared different levels of follow up for patients diagnosed with and treated for colorectal cancer239-245 (see table 13). A recently published clinical trial randomised 1202 patients in 39 NHS hospitals to four different follow up regimens; CEA only (serum testing every 3 months for 2 years and then every 6 months for 3 years), CT only (CT chest abdomen and pelvis every six months for 2 years and then annually for 3 years), CEA and CT or minimum follow up (only given follow-up if symptoms occurred). The authors reported disease recurrence in 16.6% of patients (n=199). Of those patients who developed disease recurrence 35.7% were treated with curative intent (with little difference according to Dukes’ staging). Compared to the minimum follow up group the absolute difference in the percentage of patients treated with curative intent in the CEA group was 4.4% (95%CI, 1.0-7.9%; OR 3.00, 95%CI 1.23-7.33) in the CT group 5.7% (95%CI, 2.2-9.5%; OR 3.63, 95%CI 1.51-8.69) in the CEA and CT group 4.3% (95%CI,1.0-7.9%; OR 3.10, 95%CI 1.10-8.71). The number of deaths was not significantly different between the intensive monitoring and minimum follow up groups (absolute difference 2.3%, 95%CI, -2.6%-7.1%). The statistical power of this study to assess mortality advantage of intensive follow up is limited. However, it does demonstrate that monitoring blood CEA is effective in detecting treatable recurrent bowel cancer, the impact of earlier detection on survival time and quality
110 of life is still not known. This study is the largest and highest quality clinical trial available in the current literature.
This study reports the effect of intensive CT surveillance follow up regimen (as per Greater Manchester and Cheshire Cancer Network guidelines) by a single surgeon in a single NHS hospital site, on the detection of recurrence of colorectal cancer.
2.3 Methods
Central Manchester Foundation Trust is a university teaching hospital serving an urban population of approximately 500,000 people. It is also a tertiary referral centre for colorectal and hepatobiliary disease. All patients presenting to the department of colorectal surgery (through primary or secondary care referrals) with confirmed diagnoses of colorectal malignancy were identified through prospectively maintained electronic patient records (Somerset cancer data system) and clinical case notes. All patients who underwent segmental colonic resection, by a single surgeon, with curative intent were included in the study.
Recurrence rates (locoregional, distant), median time to recurrence, overall survival, patterns were compared between Dukes’ stage.
2.3.1 Statistical Analysis
Statistical analysis was undertaken using SPSS version 22. Chi squared testing was performed for binary or categorical data. Time to recurrence was analysed using the Kaplan Meier method and plots of time to recurrence were compared using the log rank mantel cox statistic.
111 2.4 Results
2.4.1 Demographics
210 patients were included in the study. The mean age was 64.4 years (29.25 to 88.51 years, SD 11.57 years), 126 (60%) were male, 84 (40%) were female. All patients underwent segmental colonic resection with curative intent. Distribution of colonic resection is shown in Figure 5. Fifteen patients (7.1%) underwent resection of another viscus at the time of surgery, these included en bloc small bowel resection, hysterectomy, partial gastrectomy and nephrectomy for synchronous disease). All 101 patients (48.1%) undergoing anterior resection received pre-operative radiotherapy. 76 patients (36.2%) received postoperative chemotherapy.
2.4.2 Compliance with follow up
During the period of observation for recurrence (mean 8.35 years, range 2.92 – 20.33 years, SD 3.01 years) sixty patients (29.4%, 95% CI 23.0% - 35.0%) developed recurrence. 84.2% of patients in the study cohort completed 5 year follow up and 99.5% of patients completed 3 year follow up. There was 69.1% compliance with Great Manchester Cancer Network Guidelines at 3-year follow up. Forty-one patients (19.5%) died within the first three years of follow up compared with sixty-seven (31.2%) patients at 5 years.
2.4.3 Recurrent disease
Overall, sixty-seven (4.5%) CT scans performed detected recurrent colorectal cancer. Forty-five scans needed to be undertaken to detect one surgically treatable recurrence and one surgically treatable recurrence was detected for every ten patients scanned (22/210, 10.4%). Sixty-one patients (29%) developed locoregional or distant disease recurrence. The greatest incidence of recurrent disease detected by CT scanning was identified at six months post segmental resection and 3 years post resection. Only 5% of all patients developed disease recurrence during the fourth and fifth years post resection. Incidence of radiologically detectable disease recurrence over the follow up period is shown in Figure 6. Percentage of all patients who developed recurrent disease during the 5 year follow up period after curative resection for a Dukes’ A, Dukes’ B and Dukes’
112 C colorectal cancer is shown in Figure 7. The only patients with a Dukes’ D colorectal cancer underwent a synchronous right hemihepatectomy and en-bloc Right hemicolectomy with posterior sleeve gastrectomy. This patient developed recurrent disease at 12 months.
Of those patients who developed local recurrence (3.8%, 8/210), 81.8% also developed distant disease. Two patients underwent salvage surgery for local recurrence, one of whom underwent salvage surgery. Overall, 27.6% (58/210) of patients developed distant disease recurrence. 21.5% of all patients developed distant disease limited to the liver and/or lung. Site of distant recurrence by Dukes’ stage is shown in Figure 8. Patients who received postoperative chemotherapy were less likely to develop recurrent disease (p=0.008) including local or regional recurrence (p=0.01).
Four patients (0.27%) had detectable new primary malignancies on surveillance CT (Metachronous colorectal disease, lung cancer and two oesophageal cancers).
Dukes’ staging was not available for six (2.9%) of patients
There was no correlation between Dukes’ stage and local or regional recurrence (p=0.66). The proportion of patients with recurrence surgically treated with curative intent was 10.5% (22/210; 95% CI 0.6% - 14.7%). 16.7% of patients were treated for disease recurrence with chemotherapy and 3.3% with radiotherapy. There was a statistically significant difference (p=0.025) in those treated surgically with curative intent for disease recurrence and Dukes’ stage (Dukes’ A 7.9% (3/38); Dukes’ B 8.5% (6/71); Dukes’ C 12.8% (12/94); Dukes’ D 100% (1/1)). However, sample size is small and therefore may not be clinically significant.
Incidences of recurrence by stage of disease and cumulative overall survival by Dukes’ stage is shown in Figure 9.
2.4.5 Limitations of the study
Luminal recurrence rates during the study were not assessed, however, incidence of luminal recurrent disease is low and reported to be around 2%246,247. This is particularly true for cancers proximal to the rectum where luminal recurrence is
113 usually associated with extraluminal disease not amenable to curative resection248. Patient co-morbidities were not recorded and there was imperfect compliance with the intensive follow-up regime over the 5-year surveillance period during the pragmatic study.
2.5 Discussion
This study supports the findings of the study by Tsikitis et al in 2009249, that of patients who undergo segmental resection for colorectal cancer one third will develop recurrence that is amenable to treatment by surgery with curative intent. Although not part of routine follow-up, 18F-fluoro-deoxyglucose emission tomography/computed tomography (FDG-PET/CT) often has a role in investigating disease recurrence identified by serum biomarkers in the absence of disease on conventional imaging studies. More recently application of this imaging modality in routine follow up has been explored250,251, particularly in the detection of peritoneal disease and port site metastases252,253, but it has not yet has been validated as part of routine follow up. The diagnosis of treatable recurrent disease is robust upon available evidence and made by regional specialist multidisciplinary meetings (MDTs).
A recent Cochrane review (updated from previous review in 2007)118,254, reported that the average effect on intensive follow-up on colorectal cancer specific survival was ten fewer colorectal cancer specific survival deaths per 1000. The authors also reported an appreciable increase in surgery for recurrent disease in patients undergoing intensive follow up (RR 1.98,95% CI 1.53-2.56). This review reported significant heterogeneity between the studies but that there was evidence of an overall survival benefit at five years for patients undergoing more intensive follow up regimens (OR 0.73, 95%CI 0.59-0.91; RD 0.06, 95%CI -0.11 to -0.02). It was also reported that significantly more curative procedures were attempted in the intensively followed up patients (OR 2.41, 95%CI 1.63-3.54; RD 0.06, 95%CI 0.04 to 0.09). However, due to a wide variation in the follow up regimens in the included studies the authors were not able to suggest an optimal combination of clinical consultations, serum tests, endoscopic procedures or radiological investigation to maximally improve outcomes in this patient group.
For some patients, follow-up after surgery for non-metastatic colorectal cancer results in untold anxiety and worry amongst patients and their families. Some
114 studies have suggested that increased intensity of follow up has a beneficial effect on quality of life239. Patient experience and satisfaction with follow up have rarely been included in study outcomes which tend to focus on objectively measurable outcomes e.g. recurrence, survival and mortality. A study in 2006 by Wattchow et al255 investigated the effect of setting of follow up (in general practice) on quality of life, depression, anxiety and patient satisfaction. This study did not support the hypothesis that there would be improvement in depression and anxiety scores with GP led follow up and patient remained in ‘normal range’ at all times. However, the scores were only reported at 12 and 24 months post segmental resection. Our study reports that 95% of all recurrent disease occurs within the first three years postoperatively. The proportion of patients who develop recurrent disease in years four and five of surveillance after curative resection is not readily available in the published literature. However, there is some evidence to suggest that this number is so small a five-year surveillance period is not necessary256.
Current NICE guidelines recommend two surveillance CTs TAP in the first three years for all patients. There is a paucity of evidence in the literature to support follow-up tailored to stage of disease. Most randomised studies to date have disregarded stage of disease in allocation to follow up regimens239,240,242. Based on the results of this study performing surveillance CT scanning in patients with Dukes’ A colorectal cancer at six months and beyond three years post resection is questionable as no patients were identified as having recurrent disease. Nearly ten percent of all surveillance CT scans performed at three years post resection detected disease recurrence. The recent FACS trial reported no statistically significant difference in overall recurrence treated with curative intent according to Dukes’ Stage257 but did not report treatable recurrence by Dukes’ stage at timed intervals during follow-up. No studies have reported clinically significant or statistically significant results with respect to a risk stratified approach to follow up. Secco et al 2006 randomised patients to risk adapted follow up or minimum follow up, stratified by high or low risk prognostic factors (high risk features included low rectal cancer, T3 tumours of the left colon, CEA >7.5ng.ml, Dukes’ stage C, poor differentiation, mucinous features or signet ring cells). Low risk tumours had no high-risk characteristics. The authors reported a statistically significant difference in the incidence of recurrence treated by surgical resection in high-risk patients between the subgroups undergoing intensive follow-up and those undergoing minimal surveillance (p<0.05).
115 This study validates the use of intensive CT scanning in the follow-up of patients with colorectal cancer and demonstrates that this approach is practicable in routine clinical practice outwith a clinical trial. It provides useful information to patients regarding likelihood of cure after three years follow-up post resection (95%) and further supports the use of intensive follow up regimens for patients resulting in aggregation of marginal gains in the detection of surgically treatable recurrent disease and ultimately disease-free survival.
116
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Anterior Resection Left HemicolectomySigmoid Colectomy
Abdominoperineal resection
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Figure 5; Distribution of colonic resections
25
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15 DUKES A DUKES B 10 DUKES C DUKES D 5 ALL
0
2 years 3 years 4 years 5 years 6 months 12 months 18 months
Figure 6. Number of recurrences of colorectal cancer detected by CT during follow up by Dukes’ Stage
117
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Figure 7. Proportion (%) of colorectal cancer recurrence detected by CT scanning during follow up
118 18 16 14 12 Dukes Stage A 10 Dukes Stage B 8 Dukes Stage C 6 Dukes Stage D 4 2 0 Liver Lung Both Other
Figure 8. Incidence and site of distant recurrence of colorectal cancer by Dukes’ Stage
50 45 40 35 30 25 20 15 10 5 0 DUKES A DUKES B DUKES C DUKES D
Figure 9. Incidences of recurrence of colorectal cancer by Dukes’ stage of disease
119
Figure 10. Cumulative overall survival by Dukes’ stage
120
Study Title % Recurrence overall % of all patients % of recurrence surgically Surgically treated for treated recurrence PRIOR TO CT Makela, J. et al (1995) SCANNING Conventional Follow up (n=54) 39.0% 5.5% 14% Intensified Follow-up (n=52) 42.0% 9.6% 22% Ohlsson, B. et al (1995) No Follow-up (n=54) 33.0% 5.5% 16.6% Intensive Follow-up* (n=53) 32.0% 9.4% 29.4% Kjeldsen, B. J. et al (1997) Minimal Clinical Follow-up (n=307) 22.8% 1.6% 7.1% Intense Clinical Follow-up (n=290) 26.2% 5.2% 19.7% Pietra, N. et al (1998) Conventional Clinical Follow-up (n=103) 19.4% 1.9% 10.0% Intense Clinical Follow-up (n=104) 25% 16.3% 65.4% Schoemaker, D. et al (1998) Standard Follow-up (n=158) 8.2% - - Intensive Follow-up** (n=167) 5.9% - - Secco, G.B. et al (2002) Minimum surveillance (n=145) 57.2% 9.0% 15.7% “Risk adapted” Intensive (n=108) 23.1% 33.8% 52.6% Low intensity (n=84) 32.1% 22.2% AFTER CT Rodriguez-Moranta, F. et al (2006) SCANNING Minimum Follow up (n=132) 26.0% 7.6% 29.0% Intensive CT (n=127) 27.0% 14.1% 51.0% Primrose, J. et al (2014) Minimum Follow-up (n=301) 12.3% 6.7% 35.1% CEA & CT (n=302) 15.9% 8.0% 42.1% CT Only (n=302) 19.1% 6.6% 41.6% CEA Only (n=300) 19.0% 2.3% 18.9% Pearce, L et al (2016) Intensive follow up (n=210) 29.4% 10.5% 36.7% Table 13. Rates of detectable recurrent disease in randomised studies prior to and after the introduction of CT scanning as routine follow up for colorectal cancer. * Pelvic CT for patients who underwent Abdominoperineal resection only. ** Liver CT in combination with CXR and colonoscopy 121
STUDY TWO
Analysis of the effect of single nucleotide polymorphisms on age of onset of colorectal cancer in patients with Lynch Syndrome (hereditary nonpolyposis colorectal cancer)
122 3.1 ABSTRACT
Background
Lynch Syndrome (LS) in an inherited cancer predisposition syndrome. Patients develop colorectal cancer at a young age but age of onset of disease can be wide ranging. Single nucleotide polymorphisms (SNPS) have been shown to predict age of onset of malignancy in patients with high risk mutations for breast cancer. The ability to predict risk of age of onset of disease in patients with Lynch Syndrome could potentially reduce the repeated risk exposure and inconvenience that comes with screening colonoscopy, potentially improving compliance with current screening regimes and targeting prophylactic surgery to individual patients where appropriate.
Methods
MLH1 and MSH2 mutation carriers, patients with sporadic colorectal cancer but no genetic mutation and a control population were identified from the Manchester Genetics Database. A SNP panel of 16 SNPs previously identified in validation studies to be associated with colorectal cancer was developed. Lymphocyte DNA was sequenced and genotyped using Sequenom MassArray technology. Polygenic risk scores for each individual were stratified into quintiles. Cox proportional hazards model was used to assess the relationship between colorectal cancer and risk score.
Results
943 patients were included in the analysis (162 MLH1, 207 MSH2 mutation carriers, 251 sporadic colorectal cancer cases, and 323 controls). 67.1% MLH1 and 47.8% of MSH2 mutation carriers had developed colorectal cancer. Overall colorectal cancer risk score (OCRS) was higher in the sporadic group than any other group (MLH1 p=0.02, MSH2 p<0.001, Control p<0.001). There was no association with increasing OCRS and earlier age of onset of disease in MLH1, MSH2 or sporadic groups (p>0.05).
Conclusion
123 This study confirms that SNPs previously reported to be associated with colorectal cancer are more prevalent in patients with sporadic colorectal cancer than in high-risk mutation carriers or a control population. These SNPs do not appear to have current value in predicting age of onset of disease in any of the populations included in the study.
124 3.2 Introduction
Hereditary colorectal cancer syndromes account for around 4% of all incident colorectal cancers44,258. Lynch Syndrome (previously Hereditary Non polyposis Colorectal cancer) is caused by pathogenic germline mutations in one of the mismatch repair genes, namely MLH1, MSH2. MSH6 and PMS2, with lifetime risks of colorectal cancer of approximately 40-80%100. The majority (nearly 90%) of germline mutations in Lynch Syndrome have been identified in MLH1 and MSH2259,260 and approximately 10% in MSH6 and PMS2261. These patients frequently develop colorectal cancer at a young age and have a propensity to develop synchronous and metachronous tumours168. Colorectal polyposis precedes the development of malignancy in these patients and therefore affords a window of opportunity for colonic screening and early identification and endoscopic resection of polyps with ongoing endoscopic surveillance. Lynch syndrome is inherited in an autosomal dominant fashion and therefore first-degree relatives (FDR) have a 50% chance of inheriting the condition and are encouraged to undergo predictive genetic testing and colorectal screening in line with current screening recommendations.
3.2.1 Current screening guidelines for Lynch Syndrome Patients
Whilst the mean age of onset of colorectal cancer in MLH1 and MSH2 mutation carriers is around 45 years, there is a wide-ranging age of disease onset between 18 and 80 years of age. This raises several challenges for screening and surveillance programmes in this high-risk group of patients. Current guidelines recommend biennial screening commencing from age 25 years or five years earlier than the first detected cancer in families with known mutations and that this should continue until screening becomes unfeasible due to age or co-morbidity. Colonoscopic surveillance has been shown to be effective at 3 yearly intervals but reported advanced colorectal cancers between two and three years after surveillance have supported continuing 1- 2 yearly screening. For a patient who ceases screening at 75 years of age, biennial screening means that patients could potentially undergo twenty-five colonoscopies in their lifetime. Screening colonoscopy is a largely considered to be a safe procedure but nearly one third of patients will report transient
125 gastrointestinal symptoms after colonoscopy262. A systematic review in 2008 of twelve studies with over fifty thousand colonoscopies reported the serious adverse event rate to be 2.8 per 1000263. However, eighty five per cent of these adverse events occurred in patients undergoing polypectomy which has also previously been reported to be associated with a seven fold increase in colonic perforation or bleeding264. Current colorectal cancer screening programmes for Lynch Syndrome patients are not without risk.
Many studies have described a cumulative risk of colorectal cancer in patients with MLH1 and MSH2 mutations. A study by Barrow et al reported cumulative risk across the decades illustrating not only the wide-ranging age of onset of disease but also the increase in risk per decade (nearly 20% increase in cumulative risk in male MLH1 mutation over 30 years of age). This study highlighted that the same genetic mutation in patients of the same gender can still have a widely disparate ages of onset of disease. The reasons for this have not yet been identified at a clinical or molecular level. The ability to accurately estimate colorectal cancer risk is essential for counselling patients and tailoring effective and appropriate screening programmes. The ability to predict risk of age of onset of disease in patients with Lynch Syndrome could potentially reduce the repeated risk exposure and inconvenience that comes with screening colonoscopy, potentially improving compliance with current screening regimes and targeting prophylactic surgery where appropriate. It is therefore clinically relevant to investigate, at the molecular level, the effects of other genetic variations within a population with an increased genetic risk of developing colorectal cancer at a young age.
3.2.2 Single Nucleotide Polymorphisms and risk prediction
A single nucleotide polymorphism (SNP) is defined as a single base change in a DNA sequence that is replaced by any of the other three bases. They occur with relatively high frequency in the population. They predominantly reside in the non-coding regions of the genome. The common variant common disease hypothesis states that when the risk of large gene defects is excluded the remaining risk results from the summation of numerous low penetrant variants or SNPs. SNPs are the most common type of genetic variation amongst
126 people. Whilst most SNPs are thought to have no effect on an individual’s health, some studies have identified associations between SNPs and oncological disease such as colorectal cancer.
3.2.2.1 Single Nucleotide Polymorphisms and Colorectal Cancer
Genome wide association studies since 2007 have identified several SNPs associated with colorectal cancer and some studies have demonstrated that increased number of the SNPs increased an individual’s risk of developing colorectal cancer. Lubbe et al genotyped patients for 14 SNPs and demonstrated that whilst each risk allele conferred a relatively small risk, SNPs are common and in combination can contribute significantly to the overall risk of colorectal cancer201. However, little is known about the impact of SNPs in high risk mutation carriers and understanding their impact may translate into a more personalized and individualised screening programme for these high- risk patients with wide ranging age of onset of disease.
3.2.2.2 Single Nucleotide Polymorphisms in other high-risk cancer patients
Studies of large numbers of SNPs in breast cancer have used polygenic risk scores rather than the number of risk alleles to group patients into quintiles. Polygenic risk scores are generated based on the number of risk alleles and by the published odds ratios and minor allele frequencies. The more risk alleles you have the greater your risk of developing cancer. In BRCA2 patients Ingham et al demonstrated clear separation of the quintiles of polygenic risk scores for the SNPs and the authors reported a ten-year difference in median age of onset of breast cancer between the highest and lowest risk quintiles193.
3.2.2.3 Single nucleotide polymorphisms in Lynch Syndrome
Only one previous study has been undertaken to explore the relationship between SNPs and Lynch syndrome. Talseth-Palmar et al221 combined four datasets to study over one thousand patients with a molecular diagnosis of Lynch syndrome. Patients were grouped according to the number of risk alleles. They reported an association between two SNPs and increased
127 colorectal cancer risk in MLH1 mutations carriers. They reported a significant difference in age of diagnosis of colorectal cancer of twenty-eight years when comparing individuals with three risk alleles compared with no risk alleles.
3.2.3 Polymerase chain reaction (PCR)
A polymerase chain reaction selectively amplifies small amounts of DNA and is used in molecular biology prior to sequencing e.g. Sanger sequencing. It is an in vitro method for the enzymatic amplification of the genomic DNA and is based on the hybridisation of two synthetic oligonucleotide primers flanking the region of interest and amplification in the specified region. Thousands of copies of a particular DNA sequence are produced through an exponential DNA cloning chain reaction. Primers (short sequences of bases which are complimentary to the 3’ ends of the sense and antisense DNA) are specifically designed to initiate the reaction. DNA polymerase enzymes and thermal cycling facilitate and catalyse the reaction. DNA template, heat stable DNA polymerase (e.g. Taq DNA polymerase), deoxynucleotide diphosphates (dNTPs – which allow the polymerase to synthesis the new complimentary strand of DNA), buffer solution to optimise enzyme function, divalent Mg2+ cations and monovalent K+ cations.
A PCR reaction typically requires 20-45 cycles of amplification. The exact cycling conditions will depend on a variety of parameters. The steps within the thermal cycling conditions for a Sequenom PCR are as follows;
1. Initial denaturation: As a single step at the beginning of the PCR the reaction is heated to 95˚C to heat activate the DNA polymerase for two minutes. This step also disrupts the hydrogen bonds between the DNA bases of complementary strands in the genomic DNA thereby denaturing to single strand DNA.
2. Denaturation: The reaction is heated to 94˚C for a further thirty seconds. This disrupts the hydrogen bonds between the DNA bases thereby denaturing the DNA to single strand DNA.
128 3. Annealing: The temperature is lowered to 56˚C for thirty seconds to allow the primers to anneal to their complementary sequence of DNA. The DNA polymerase binds to the primer-DNA template complex and DNA synthesis commences.
4. Extension: This step occurs at 72˚C for a further one minute. The DNA polymerase adds dNTPs to the template DNA in the 5’ to 3’ direction. Steps 2 and 4 are repeated for a further 44 cycles.
5. Final Extension: This is the final step and occurs at 72˚C for five minutes. This ensures that all single stranded DNA is fully extended.
6. The sample is then cooled to 4˚C for five minutes before being held at 15˚C.
An Applied Biosystems Gene Amp PCR System 9700 was used for PCR amplification of both patient samples and Coriell cell line controls.
3.2.4 Genotyping Techniques
3.2.4.1 Sanger Sequencing
Frederick Sanger first described ‘Sanger sequencing’ in 1997 as a new method for determining nucleotide sequences in DNA by using chain terminating inhibitors265. The method relies upon the incorporation of inhibitory dideoxynucleotides, which prevent extension of the nucleotide chain265. Whilst having been surpassed by next generation sequencing for large-scale automated genome analyses, Sanger sequencing is still the gold standard of sequencing short nucleotide sequences (15-500 base pairs).
129 3.2.4.2 Sequenom
Sequenom MassARRAY system is a DNA analysis platform that is used to genotype DNA based on multiplex PCRs and mass spectrometric analysis of primer extension products. It has other applications in addition to genotyping including somatic mutation profiling, methylation analysis, molecular typing and quantitative gene expression (QGE).
The Assay has 5 main steps…
1. DNA extraction 2. DNA amplification (PCR) 3. PCR purification using Shrimp Alkaline Phosphatase (SAP) treatment 4. iPLEX extension followed by sample condition and nanodispensing (onto 96 well SpectroChip microarrays) 5. MALDI-ToF (matrix assisted laser desorption/ionization-time of flight)
Sequenom systems use Matrix-Assisted Laser Desorption/Ionisation-Time of Flight mass spectrometry (MALDI-ToF). A ‘SpectroChip’ is coated with a matrix, which allows crystallisation of the PCR product on its surface. MALDI is a two-step process. Firstly, desorption of matrix is triggered by a UV laser beam and secondly ionisation of analytes (energy is transferred via the matrix). Time of flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion’s mass-to-charge ratio is determined via a time measurement between desorption and detection. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will also depend on the mass-to-charge ratio of the particle. Time of flight measures the difference in time that different molecules hit the detector and software then calculates the mass of fragments. MALDI-ToF can resolve mass differences as little as 16 Da (daltons or unified atomic mass unit). There is a difference in mass between DNA species differing in nucleotide sequences, hence existence of a SNP within a sequence results in a predictable difference in mass and hence can be identified.
130 3.2.5 Polygenic Risk Scores
Polygenic scores are increasingly being used to summarise the genetic effect of individual markers which when considered in isolation in large-scale association studies do not achieve significance. Each allele within each subject is weighted, forming different scores for homozygous, heterozygous and wild type. The majority of studies use polygenic risk scores to report individual risk by summing all of an individual’s risk alleles. This implies that all risk alleles are weighted equally. Alternatively, previously observed effect sizes can be used to weight each allele risk differently. The ‘population attribution risk’ method integrates population minor allele frequency (MAF) and odds ratio (OR) and form a multiplicative overall polygenic risk score. This method has been validated in a study by Ingham et al193 in BRCA2 and BRCA1 female carriers. The authors used published odds ratios and risk allele frequencies to calculate an overall risk score for each woman. These scores were then split into quintiles (Lowest quintile represents highest overall risk scores). The author’s reported a significant difference in age at the development of breast cancer between the quintiles (p>0.001) and clear trend for reducing hazard with reducing overall risk score (increasing quintile). Similar results were also found when assessing breast cancer risk in familial non-BRCA women in a familial risk clinic266.
3.2.6 Hypothesis
SNPs have a multiplicative effect on age of onset of colorectal cancer in patients with MLH1 or MSH2 mutations.
3.2.7 Ethical Approval
Ethical approval was not required for this study. All patients had given prior consent to be registered onto the Manchester Genetic Register. Part of this consent process included consent for DNA to be extracted and stored from lymphocyte samples. Following identification, all samples were anonymised
131 and information was not disseminated to patients. There were no future management implications for these patients.
3.3 Methodology
3.3.1 Participants
Lynch syndrome patients with MLH1 or MSH2 pathogenic mutations, sporadic colorectal cancer cases with no pathogenic mutation and non-colorectal cancer patients (control group) were identified from the Manchester Genetic Register. Mutation heterozygotes were defined as those who had tested positive for a pathogenic germline mutation. For the cases, only patients with a pathogenic mutation and DNA available for analysis were used for the study. Some patients at initial identification were found to have insufficient DNA available for analysis due to their samples having been exhausted in previous studies or transfer of samples to another centre for diagnostic or research purposes. Any patient with insufficient DNA or incomplete genotyping and insufficient DNA to retest was excluded from the study.
Patients with a personal or family history suggestive of Lynch syndrome are referred to the Department of Genetics at St Mary’s Hospital from primary and secondary care services across the North West of England. At consultation in addition to patient demographics and clinical history, full pedigree information was taken including dates or birth, dates of death of immediate and extended family members. These were confirmed with the North West Cancer Intelligence Service (NWCIS; cancer registration data) and NHS summary care records. Since 1996 all families who fulfil Amsterdam or Bethesda Criteria (where there was evidence of mismatch repair in tumour)267 were tested for germline mutations in mismatch repair genes (if there was an available living affected relative) by a range of technologies but most recently by Next Generation Sequence analysis for point mutations and small insertions/deletions and Multiplex Ligation Probe Dependence Amplification (MLPA) for large genomic rearrangements in MLH1, MSH2 and MSH6. Tumours from patients suspected of having Lynch syndrome were tested for evidence of mismatch repair deficiency. Mismatch repair testing was performed by microsatellite instability of DNA up until 2008 but after concerns about high false negative rates (particularly for MSH6) and poor economic
132 viability mismatch repair immunohistochemistry has been used from 2008 to date as the pre-screening method of choice. If tumours tested demonstrated mismatch repair deficiency germline mutation testing was performed providing there was an available affected living relative. A range of technologies are used for mutation screening in MLH1, MSH2 and MSH6 but most recently Next Generation Sequence analysis has been used to screen for point mutations and small insertion/deletions across the coding sequences and immediate intron/exon boundaries plus Multiplex Ligation Probe Dependence Amplification (MLPA) for large genomic rearrangements.
SNPs identified in previous genome wide and validation studies found to be associated with sporadic cases of colorectal cancer were identified through a literature search. Sixteen SNPs were identified for testing in this study (including two previously reported to be associated with Lynch Syndrome). All SNPs had published minor allele frequencies for control groups and these were utilised to calculate polygenic risk score (see section 3.2.5). Some SNPs were reported to be risk reducing and others risk producing. SNPs for testing were ranked in order of strength of association with colorectal cancer based upon size of validation studies, use of control group and width of published confidence intervals.
3.3.2 SNP Panel design
The Sequenom online assay design suite v1.0 was used to create a new genotype assay design. The assay design suite is a comprehensive efficient online tool (www.mysequenom.co.uk) that utilises human, bovine and mouse genomes for sequence searches and comparisons. The system enables complex manipulation of SNP genotyping assay designs.
All ‘rs’ identification numbers for sixteen identified and validated SNPs were entered into the design programme. High multiplexing panel settings and the minimal multiplex level of sixteen were chosen as a preset in an effort to maximise the number of SNPs in a single panel. The organism was set as human and the reference sequence as (GRCh37/hg19) (the most up to date reference sequence on NCBI at the time of design). This retrieved and formatted sequences for each SNPs and one hundred base pairs either side of
133 the SNP. During the assay design phase it is important to identify other SNPs in the sequence to avoid designing primers over them. At the stage of identifying proximal SNPs one was identified at position 94 (rs114226621) near rs4813802 at position 100. The minor allele frequency/minor allele count of this proximal SNP was identified to be 0.001/2 in a study of 1000 genomes (dbSNP www.ncbi.nlm.nih.gov). As this SNP is of a low minor allele frequency the probability of finding this SNP in our patient population was low. Using inosine in place of this proximal SNP could control for this SNP. However, as the allele frequency for this SNP was judged as low (i.e. <1%) it was not felt necessary to control for this proximal SNP in the initial assay design.
By re-entering the rs numbers and excluding step 2 (find proximal SNPs) it was possible to develop a single multiplex of fifteen chosen SNPs (W1) and another monoplex with the remaining SNP (rs4925386) (W2) (see table 14).
SNP Multiplex rs4939827 W1 rs4444235 W1 rs3802842 W1 rs1892766 W1 rs4813802 W1 rs11169552 W1 rs7014346 W1 rs6983267 W1 rs691170 W1 rs10936599 W1 rs4779584 W1 rs10411210 W1 rs9929218 W1 rs961253 W1 rs10795668 W1 rs4925386 W2 Table 14. SNP panel design
134 3.3.3 Sequenom PCR Primers
SNP extensions and forward and reverse primers were obtained from Metabion International AG® via their online ordering system and received at stock concentration of 50µMol in an unhydrated form. These were diluted to a target concentration of 0.5µMol according to manufacturer’s instructions (see
135 Table 15). Four microlitres were required at a concentration of 0.5µMol for each PCR primer (2µl forward primer and 2µl reverse primer). For the first multiplex of fifteen SNPs this gave a total primer volume of 60µl to which 140µl of water was added to achieve a total W1 multiplex primer volume of 200µl. For the second multiplex of a single SNP 196µl water was added to the SNP primer to achieve the same total volume of W2 multiplex primer.
136 Table 15; Rehydration of SNP primers
SNP Forward Multiplex Volume of water added / (µl) Reverse rs4939827 F W1 286 R 466 rs4444235 F W1 442 R 466 rs3802842 F W1 454 R 452 rs1892766 F W1 408 R 454 rs4813802 F W1 456 R 450 rs11169552 F W1 452 R 432 rs7014346 F W1 412 R 478 rs6983267 F W1 430 R 462 rs691170 F W1 424 R 462 rs10936599 F W1 436 R 476 rs4779584 F W1 440 R 430 rs10411210 F W1 420 R 412 rs9929218 F W1 428 R 416 rs961253 F W1 420 R 414 rs10795668 F W1 370 R 400 rs4925386 F W2 362 R 426
137 3.3.4 Coriell Cell Line Sanger Sequencing
Coriell cell lines are maintained by the Coriell Institute for Medical research Human Genetic Cell Repository. These cell lines are well characterised and can be a source of high quality DNA with a defined set of variants. They are accepted to have an error rate of around 1-2%. Six Coriell Cell line samples held by Department of Genetics, St Mary’s Hospital were Sanger sequenced (3730 DNA analyser, Applied Biosystems, Hitachi) for SNPs prior to Sequenom testing in order to aid Sequenom assay development for the W1 and W2 multiplex and optimise diagnostic validity of subsequent Sequenom analysis. These cell lines were known not to carry MLH1 or MSH2 mutations. Standard Sequenom PCR mix was used (see Table 16Table 16).
Table 16. Standard PCR mix
Agent Vol (µl)
H2O 1.9 1Ox buffer 0.5
MgCl2 0.4 dNTP mix 0.1 Taq Roche 0.1 Primer mix 1.0 DNA 1.0 PCR reaction total volume 5.0
3.3.4.1 Purification of PCR products prior to Sanger sequencing
For the Coriell cell line samples only the Agencourt® AMPure® Kit which provides automated purification was used. Purification was performed according to the manufacturer’s recommended procedure. A summary of this process is shown in Figure 2. Firstly the AMPure® kit was gently mixed until the beads were homogenised with the solution. The volume of Agencourt® AMPure® to be used for a given PCR reaction volume was calculated by using the following equation:
Volume of Agencourt® AMPure® per reaction = 1.8 x PCR reaction volume
138
By using this formula the required volume of AMPure kit for 6µl of PCR products was calculated as 10.8µl and was added to the PCR products. This step provides the basics for separation of PCR products bound to magnetic beads from contaminants. After binding of the PCR amplicons to magnetic beads, PCR products were separated from the excess dNTPs, salts and unincorporated primers and were washed in 70% ethanol. The purified PCR products were eluted from the magnetic beads and transferred into a clean plate away from the beads resulting in effective clean up (Fig 2. www.beckmancoulter.com). All technical work was carried out under the supervision of a qualified genetic technologist and Sanger sequencing results were verified by Dr. Andrew Wallace, Consultant Clinical Scientist.
Figure 11. Purification of PCR products – Agencourt AMPure
139 3.3.5 Sequenom Assay Development
3.3.5.1 Samples
Six Coriell cell line DNA samples whose genotype had been established by Sanger sequencing were used for validating the Sequenom assay design.
3.3.5.2 Amplification of target loci by PCR
PCR Reactions were set up using 10ng/uL of DNA with a total volume of 5uL. Amplification included one cycle of 94˚C for two minutes, followed by forty- five cycles of 94˚C for thirty seconds, 56˚C for thirty seconds and 72˚C for one minutes and one final cycle of 72˚C for five minutes.
3.3.5.3 Post PCR; SAP reaction clean-up
Unincorporated nucleotides are inactivated prior to Sequenom analysis by the addition of shrimp alkaline phosphatase (see Table 17) in the ‘SAP clean up’ stage. Following PCR amplification, any remaining free deoxynucleotide triphosphates (dNTPs) in the amplification reaction mixture must be dephosphorylated in order to prevent interference with the iPLEX extension reaction. Shrimp alkaline phosphatase (SAP) dephosphorylates unincorporated dNTPs by cleaving the phosphate groups from the 5’ termini and converts them to deoxynucleotide diphosphates (dNDPs), making them unavailable for future reactions. The mixture is incubated at 37°C for forty minutes, the SAP is then heat inactivated by heating to 85°C for five minutes. Failure of the SAP PCR clean up would result in excessive quantities of dNTP present in the iPLEX reaction which would interfere with incorporation of the dNTPs. Long extension of the primer beyond the position of the SNP would be seen as few or no analyte peaks in the output.
3.3.5.4 Primer Extension (iPLEX Pro extend reaction)
The primer extension or iPLEX Pro extend reaction is a method for detecting single nucleotide variants or small insertion/deletion/substitution variants in PCR amplified DNA. After PCR cleanup, a primer extension reaction mixture (containing extend primer, buffer, enzyme, and mass-modified ddNTPs) is
140 added to the SAP treated and heat inactivated PCR amplification products. The amplification products and reaction are subject to thermal cycling as follows; one cycle of 94˚C for thirty seconds, forty cycles of 94˚C for five seconds, five cycles of 52˚C for five seconds and 80˚C for five seconds followed by one cycle of 72˚C for three minutes. This allows the enzymatic addition of a nucleotide at the ‘diagnostic’ position. The primer is extended by one nucleotide, terminating the primer extension. Failure of the iPLEX reaction would be observed as unextended primer peaks and no analyte peaks.
3.3.5.5 Primer extension reaction resin clean up
SpectroCLEAN (Sequenom) is a cationic resin pretreated with acid reagents used to remove salts such as Na+, K+, and Mg2+ ions. If not removed, these ions can result in high background noise in the mass spectra and as such this step is important in optimizing the mass spectrometry analysis. SpectroCLEAN resin was added directly to primer extension reaction products in a 96-well plate. Extension products were then purified by incubation for 30 minutes at room temperature on a Scientific Industries Rota-Shake Genie with an ion exchange resin (SpectroCLEAN, Sequenom).
3.3.5.6 Spotting primer extension products on SpectroCHIPs
A spectroCHIP is a silica chip overlayed with a proprietary matrix crystal. A nanodispenser was used to dispense reaction products onto the chip. This adds the extended oligonucleotides onto an appropriate matrix forMALDI-TOF (3- hydroxypicolinic acid), 10nL of purified product was spotted onto the matrix of a small silica SpectroCHIP. Failure during nanodispensing is often seen as a complete lack of peaks in columns of six, which corresponds to the pins on the SpectroPOINT. This can be corrected by ‘respotting’ the chip. Incorrect loading of the chip can also results in unaligned peaks in the output.
3.3.5.7 Detection of primer extension products by mass spectrometry
A Matrix assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometer (Sequenom MassARRAY Analyzer 4) was used to resolve extension products. The SpectrocCHIP was placed into the mass spectrometer
141 and each matrix spot was then targeted with a laser under vacuum by the matrix-assisted laser desorption ionisation–time-of-flight (MALDITOF) method. The laser beam is the desorption and ionisation source in MALDI mass spectrometry. The matrix absorbs the laser light energy and causes part of the illuminated substrate to vaporise. The quickly expanding matrix plume carries analyte into the vacuum with it and aids the sample ionization process. The matrix molecules absorb most of the laser energy, limiting sample damage and ion fragmentation. Once the sample molecules are vaporised and ionised, they are transferred electrostatically into a time-of-flight mass spectrometer (TOF-MS), they are separated from the matrix ions, individually detected based on their mass-to-charge (m/z) ratios and analysed. Detection of an ion at the end of the tube is based on its flight time, which is proportional to the square root of its m/z.
3.3.6 Data analysis
A Sequenom Typer 4.0 Analyzer was used for automated data analysis. Genotype calls are made in real time during chip detection. Traces are available for viewing immediately after detection. Automated data analysis was not used as per departmental clinical protocol. Visual analysis of trace outputs was performed by myself and a genetic technologist for all SNPs in all samples.
3.3.7 DNA Samples
Previously extracted and archived DNA samples were used for the study. DNA had been extracted from samples with EDTA as the anticoagulant using standard procedures. DNA was stored frozen at -40oC in a DNA archive post extraction. The concentration of DNA was determined using a Nanodrop ND 8000 spectrophotometer (Labtech international). The Nanodrop was blanked first using elution buffer (Chemagen). 2 µL of each DNA sample were then pipetted onto the pedestal and the lid replaced. Concentrations were recorded and samples diluted to achieve a DNA concentration of 10ng/µL. Any archived samples with concentrations recorded as less than 10ng/µL were used undiluted.
142 3.3.8 PCR Primer multiplex optimisation
In the first instance, standard Sequenom sequencing reaction volumes were used. However, due to poor DNA amplification seen in W1 and W2 (in both standard Sanger sequencing and Sequenom analyses) varying quantities of primer were used to identify an optimum volume for DNA amplification without resulting in large quantities of residual unextended primer seen at results analysis. Volumes for SAP and iPLEX mix remained unchanged. (See Table 17, Table 18 and
Table 19). It was determined that 1.5uL of PCR primer yielded sufficient DNA amplification for Sequenom analysis. However, there were large residual amounts of unextended extension primer noted during analysis and therefore results did not meet quality standards.
Table 17; Optimisation SAP Mix SAP Mix Vol (µl) Agent
H2O 1.53 SAP buffer 0.17 SAP 0.30 Total 2.0
Table 18; Optimisation of assay
Agent Vol (µl) Vol (µl) Vol (µl) Vol (µl)
H2O 1.9 1.4 1.15 0.9 IOx buffer 0.5 0.5 0.5 0.5
MgCl2 0.4 0.4 0.4 0.4 dNTP mix 0.1 0.1 0.1 0.1 Taq Roche 0.1 0.1 0.1 0.1 Primer mix 1.0 1.5 1.75 2.0 DNA 1.0 1.0 1.0 1.0
143 Total 5.0 5.0 5.0 5.0
Table 19; Optimisation iPLEX Mix
iPLEX Mix Vol (µl) Agent
H2O 0.739 iPLEX buffer 0.200 iPLEX Termination Mix 0.100 Primer Mix 0.940 iPLEX Enzyme 0.021 Total 2.0
3.3.9 Extension Primer Optimisation
To address the problem of unextended extension primer, extension primer volumes were reduced in the mix from 0.95uL (standard) to 0.8uL, increasing PCR primer to 1.75uL, DNA to 2uL, PCR enzyme to 0.5uL and dNTPs to 0.15uL. These changes appeared to resolve the problem of unextended extension primer however, two of the SNPs in W1 panel were not amplified sufficiently for analysis. The reason for this was unclear. The SNP rs11169552 also failed Sanger sequencing in the W1 panel.
3.3.10 Redesigning of SNP Panel
Samples were repeated with a new W1 primer panel dilution to confirm primers had not been omitted in the original mix. Additionally, rs1169552 was Sanger sequenced in isolation to confirm primer function. Agarose gel electrophoresis confirmed the presence of a PCR product (See Figure 13 row A1 to G1), which underwent successful Sanger Sequencing and confirmed the presence of the expected sequence.
144
Figure 12. Gel Electrophoresis for rs1169552
In order to achieve amplification of rs11169552 and rs6991170 primers for these SNPs were duplicated in the W2 panel. The primers for these SNPs were not removed from the W1 panel as all other SNPs in the W1 panel were achieving optimal automated and visual Sequenom analysis and it was felt that removing the primers might have a deleterious effect on the results for other W1 panel SNPs. Primers for rs11169552 and rs6991170 in the W2 plex gave good results, which reached diagnostic quality in Sequenom results analysis.
As previously discussed the PCR primer for rs4813802 lies over the SNP rs11422621. This therefore results in the potential of the PCR Primer for rs4813802 not to anneal if a SNP for rs11422621 is present in the sequence. Inosine will bind to any base and therefore a primer was designed and ordered with inosine at the site of the SNP for rs11422621 to ensure binding even in the presence of a base change. This SNP is rare and therefore unlikely to be identified frequently in our patient population, however, it was thought potentially advantageous to add an inosine primer for this SNP into the W2 panel in order to compare results with the standard primer in the W1 panel.
Two SNPs rs961253 and rs4925386 did not meet diagnostic quality standards without further testing. Therefore, ten random anonymised samples were identified from the DNA database at St Mary’s Hospital. These patients were identified as not having Lynch Syndrome. Sequenom testing and Sanger sequencing was carried out in parallel on these samples. Genotyping was achieved, and therefore acceptable diagnostic validity of the Sequenom assay design confirmed.
145 All samples were analysed at the Genomic Diagnostics Laboratory (Manchester Centre for Genomic Medicine, St. Mary’s Hospital, Central Manchester University Hospitals NHS Trust, Manchester) according to Good Laboratory Practice (GLP) Standards. The technical work was carried out under the supervision of a competent Genetic Technologist (KB and SP) and results were verified by Dr Andrew Wallace, Consultant Clinical Scientist.
3.3.11 Clinical Data
All data were handled according to the Data Protection Act (1998) in a linked- anonymised fashion using the genetic laboratory number. Data collected included demographic information, diagnosis of Lynch Syndrome, previous molecular/genetic tests (germline MMR mutation). The minimum clinical inclusion criteria were date of death or date of last follow up (clinical or radiological). All analytical data files were stored on a password protected NHS computer in a locked office on NHS property.
3.3.12 Genotyping
On completion of processing of the spectrochip the traffic light colour coded plate map (see Figure 13, Figure 14) of the active chip was reviewed to evaluate the overall performance of the multiplex in each well. This provided an immediate opportunity for ‘respotting’ of the spectro chip if the output quality was low and it was suspected that insufficient DNA had been delivered via the nanodispenser onto the chip.
Competence based training in genotyping using Typer 4.0 Analyser, was undertaken by LP (primary researcher), SP and KB (genetic technologists) by NH, a Clinical Scientist. Genotyping was performed for all SNPS in all patient samples by two independent researchers (LP and KB or SP) using Typer 4.0 Analyzer cluster plots, spectra and peak information. Exampled of spectra for unextended primer, poor amplification and full Typer 4.0 output are demonstrated in Figure 15, Figure 16 and Figure 17.
If mutant allele peaks were present in the water sample none of the mutation calls were deemed reliable for that assay and the multiplex was repeated. Genotype results were inputted directly into a pre designed and formatted
146 Excel spreadsheet. An Excel calculation was used to check concordance between the Excel spreadsheets of the two independent researchers. The degree of concordance was calculated and a measure of inter-rater agreement was made using the Kappa statistic. Where the genotype obtained by the different analysers did not correlate both analysers reviewed the data. If a consensus could not be reached together the patient sample underwent repeat PCR and analysis (Sequenom) and both researchers again reviewed the output. If a consensus could still not be reached after two repeats of processing the serum sample or if there was insufficient DNA to re-run the sample the patient was excluded from the study. This only occurred with seven patient samples.
Figure 13; Example of suboptimal sequenom analysis of the spectrochip indicated by traffic light colour coded plate map
Figure 14; Example of optimal sequenom analysis of the spectrochip indicated by traffic light colour coded plate map
147
Figure 15; Example of unextended primer
Figure 16; Example of poor amplification
148
Figure 17; Example of Typer 4.0 Analyser output including cluster plots, spectra and peak information
3.3.13 Overall Colorectal Cancer Risk Score (OCRS)
Polygenic risk scores were calculated for each patient depending on the number of risk alleles and minor allele frequencies (MAF) described in previous validation studies. The minor allele frequencies for each SNP are reported in the validation studies and this enables identification of the minor and major alleles for the SNP. In the instance whereby the control minor allele frequency (MAF) is greater than the case minor allele frequency (e.g. control MAF = 0.32 versus case MAF = 0.30) it is evident that the minor allele (e.g. A) is in fact the protective allele and not the risk allele (e.g. G). The inverse is true if the case minor allele frequency is greater than the control allele frequency. If the minor allele is protective, the risk allele frequency (RAF) is calculated by subtracting the control minor allele frequency from one (RAF = 1 - 0.32 = 0.68).
A population of 1000 people has a total of 2000 alleles. Multiplying the percentage of minor alleles (0.32) by the total number of alleles in the population (2000) will give the total number of minor alleles in that population
149 (640 alleles). The percentage of wild type alleles in the population is calculated by multiplying the protective allele frequency by itself (e.g. 0.32 x 0.32 = 10.24%). The percentage of heterozygotes can be calculated by the number of minor alleles in the population that come from heterozygotes ((640 – (0.32 x 640)) x 100 = 43.5%). The percentage of homozygotes can then be calculated (100 - (10.24 + 43.5)) = 46.26%.
Weighted risk scores were then calculated for each SNP, using excel, for the presence of zero, one or two risk alleles (wild type, heterozygous, homozygous respectively). Relative risks published in validation studies were utilised such that the weighting for each genotype when multiplied by the population frequencies of the genotypes equals 100 (see Table 21). As such the odds ratios were normalized around a population average risk of 1.0.
These weightings were applied to each specific genotype for each SNP tested in each patient. The individual risk scores were multiplied, again in excel, to give an overall polygenic colorectal cancer risk score (OCRS) for each patient.
Lubbe et al genotyped for 16 SNPs in the 16 chromosomal regions reported to be associated with CRC risk (see Table 12). They reported that all SNPs showed an association with CRC risk, apart from rs1957636, which did not attain statistical significance and therefore this SNP was not incorporated into the SNP panel used in this study.
Clinical and genotyping data were compiled into a single excel spreadsheet. Advice regarding statistical analysis was sought from Dr Steve Roberts (Applied biostatistician and Senior Lecturer in biostatistics at the university of Manchester. Statistical tests were performed using Statistical Package for the Social Sciences (SPSS) version 22 (SPSS Inc, Chicago, IL, USA). Using the published per SNP odds ratio (OR) and minor allele frequencies (MAF) in previous published studies212,268, OR were calculated for each of the three SNP genotypes (no risk alleles, 1 risk allele, and 2 risk alleles), assuming independence. To obtain an overall colorectal cancer risk score (OCRS) for each patient genotype specific OR were multiplied together (Table 21). These OCRS were then utilized to separate the patients into quintiles of risk, using a formula within Excel. Age at the development of colorectal cancer (time from date of birth to date of diagnosis in cases) or from date of birth to the date of last follow up (if not a case) was analysed using the sensor variable set at 1
150 for colorectal cancer cases and 0 otherwise.
Mann Whitney U and Krushkal Wallis tests were used to compare the difference in OCRS between the groups. Cumulative hazard curves were calculated using Cox proportional hazards model to assess the relationship between colorectal cancer risk and OCRS (which was split into quintiles and entered into the model as an ordinal categorical variable) and calculate the hazard ratios. The proportional hazards assumption was assessed graphically by plotting –ln(- ln(survival)) versus ln(time) for each of the five risk groups and checking to see that the curves are parallel. The type I error (α) was set at 5%. This analysis assessed the impact of the multiplicative SNP genotypes on penetrance of colorectal cancer.
3.4 Results
In total, 1215 patients were identified from the Manchester regional genetics database for inclusion in the study. Of these, 459 were patients with confirmed MLH1 (213) or MSH2 (246) germline mutation carrier status, 336 had been diagnosed with colorectal cancer but with no genetic mutation (sporadic group) and 420 age and sex matched controls known not to have developed colorectal cancer and not to have any genetic mutation predisposing them to developing colorectal cancer (control group). Any patient with insufficient lymphocyte DNA for analysis (44 MLH1, 24 MSH2, 39 Sporadic CRC, 45 Control Group) or incomplete genotyping (7 MLH1, 15 MSH2, 46 Sporadic CRC, 52 Control Group) was excluded from the study. A total of 943 patients were included in the final analysis (162 MLH1, 207 MSH2, 251 Sporadic CRC, 323 Control Group). This is represented in Figure 18.
Patient demographics for each group are shown in Table 20.
151 MLH1% MSH2% Sporadic% No%CRC% CRC% (controls)%
Iden%fied'Pa%ents'' Iden%fied'Pa%ents'' Iden%fied'Pa%ents'' Iden%fied'Pa%ents'' n=213' n=246' n=336' n=420'
No'available' No'available' No'available' No'available' serum'DNA' serum'DNA' serum'DNA' serum'DNA' n'='44' n'='24' n'='39' n'='45'
Genotyped'pa%ents' Genotyped'pa%ents' Genotyped'pa%ents' Genotyped'pa%ents' n=169' n=222' n=297' n=375'
Incomplete' Incomplete' Incomplete' Incomplete' genotype'' genotype'' genotype'' genotype'' n'='7' n'='15' n'='46' n'='52'
Complete'Genotype' Complete'Genotype' Complete'Genotype' Complete'Genotype' n=162' n=207' n=251' n=323'
Figure 18. Study consort diagram
Table 20. Patients demographics of MLH1, MSH2, Sporadic CRC and Control groups. Ages are shown as mean values and ranges.
MLH1 MSH2 Sporadic CRC Control Male (n) 78 75 136 113 Female (n) 84 133 115 210
Age (yrs) 44.90 (21.16-79.03) 46.64 (20.74-89.64) 65.98 (27.55-90.00) 44.87 (15.97-84.33)
CRC (n) 100 99 251 0 No CRC (n) 62 108 0 323
Total patients (n) 162 207 251 323
152
Table 21. Polygenic Risk scores calculations for each SNP tested and Control risk allele frequencies. *Case and Control MAF from Lubbe et al. Relationship between 16 susceptibility loci and colorectal cancer phenotype in 3146 patients.
Wild type Heterozygous Homozygous Wild Type Heterozygous Homozygous Wild Type Heterozygous Homozygous Control Gene Risk Allele Control MAF* Case MAF* RAF Weight 0 Weight 1 Weight 2 0 freq 1 freq 2 freq RR W*F Control Population Total Alleles Population RAF rs10411210 19q13.11 RHPN2 C 0.1 0.09 0.9 0.86 0.96 1.07 1.0 62.0 37.0 1.12 100 6 59 258 646 0.89 rs10795668 10p14 G 0.32 0.30 0.68 0.83 0.95 1.08 10.2 43.5 46.2 1.14 100 36 151 136 646 0.65 rs10936599 3q26.2 TERC C 0.24 0.23 0.76 0.90 0.96 1.03 5.8 36.5 58.1 1.07 100 25 122 176 646 0.73 rs16892766 8q23.2 EIF3H C 0.08 0.1 0.08 0.96 1.23 1.57 84.6 14.7 0.6 1.28 100 276 44 3 646 0.08 rs3802842 11q23.1 POU2AF1 C 0.29 0.32 0.29 0.91 1.06 1.24 50.4 41.2 8.4 1.17 100 161 124 38 646 0.31 rs4444235 14q22.2 BMP4 C 0.47 0.49 0.47 0.92 1.00 1.09 28.2 49.8 22.0 1.09 100 86 163 74 646 0.48 rs4779584 15q13.3 GREM1 T 0.19 0.22 0.19 0.93 1.11 1.34 65.5 30.8 3.6 1.2 100 221 90 12 646 0.18 rs4939827 18q21.1 SMAD7 T 0.48 0.44 0.52 0.85 0.99 1.15 23.0 49.9 27.0 1.16 100 60 164 99 646 0.56 rs6983267 8q24.21 MYC G 0.48 0.45 0.52 0.87 0.99 1.13 23.0 49.9 27.0 1.14 100 60 158 105 646 0.57 rs7014346 A 0.37 0.41 0.37 0.87 1.04 1.24 39.7 46.6 13.7 1.19 100 112 161 50 646 0.40 rs961253 20p12.3 BMP2 A 0.35 0.38 0.35 0.91 1.03 1.17 42.3 45.5 12.3 1.13 100 137 143 43 646 0.35 rs9929218 16q22.1 CDH1 G 0.3 0.27 0.7 0.82 0.94 1.08 9.0 42.0 49.0 1.15 100 28 114 181 646 0.74 rs11169552 12q13.13 C 0.28 0.25 0.72 0.84 0.94 1.07 7.8 40.3 51.8 1.13 100 39 118 166 646 0.70 rs4813802i 20p12.3 G 0.36 0.38 0.36 0.94 1.02 1.12 41.0 46.1 12.9 1.09 100 185 138 0 646 0.21 rs4925386 20q13.33 LAMA5 C 0.32 0.3 0.68 0.88 0.96 1.06 10.2 43.5 46.2 1.1 100 72 192 59 646 0.48 rs6691170 1q41 DUSP10 T 0.35 0.38 0.35 0.93 1.03 1.14 42.3 45.5 12.2 1.11 100 141 144 38 646 0.34
153 Genotype frequencies for each SNP are shown in Appendix 2 for MLH1, MSH2, Sporadic CRC and Control Groups respectively. Polygenic risk scores were calculated for each patient (Table 23). Of the 213 MLH1 mutation carriers identified, 162 were included in the analyses (44 insufficient lymphocyte DNA available for genotyping, 7 incomplete genotyping). 246 MSH2 mutation carriers were identified and 207 were included in the final analyses (24 insufficient lymphocyte DNA available for genotyping, 15 incomplete genotyping).
3.4.1 Control Group
Risk allele frequencies were calculated for all SNPs tested in the plex for the control population and compared to published risk allele frequencies (Table 21). These were largely comparable to those published212. 323 patients were genotyped for 16 SNPS. There was no difference in age across the risk groups based on polygenic risk score (p=0.90) Figure 19. Median age of the control population was 43.81 years (SD 12.69 years).
Figure 19; Age distribution across risk quintile for control population
154
3.4.2 Colorectal cancer cases
61.7% (n=100) of MLH1 mutation carriers and 47.8% (n=99) of MSH2 mutation carriers had developed colorectal cancer by the cut-off date of 01/02/2014 for follow up data and data extraction.
There was a significant difference in median age of onset of nearly twenty years between the MLH1 mutation carriers and sporadic cases (49.36 v’s 68.04 years, p<0.001). Median age of onset of colorectal cancer was earlier in the MLH1 mutation carriers than the MSH2 mutation carriers (49.36 v’s 54.31 years). Numbers of colorectal cancer per group per quintile per group are shown in Table 22.
Table 22; Number of colorectal cancers per population per quintile
MLH1 MSH2 Sporadic Control
CRC (n) CRC (n) CRC (n) CRC (n) Quintile 1 (Highest Risk) 20 24 50 0 Quintile 2 21 16 50 0 Quintile 3 18 24 50 0 Quintile 4 16 17 50 0 Quintile 5 (Lowest Risk) 25 18 51 0
Total (n) 100 99 251 0
Similar numbers of colorectal cancer cases were identified in the MLH1 and MSH2 groups. There were a higher proportion of colorectal cancer cases in the MLH1 group. After the cases were separated into quintiles of risk based upon their polygenic risk scores, comparable numbers were seen in the highest and lowest risk quintiles in the MLH1 And MSH2 mutations carriers (Quintile 1 n=44, Quintile 5, n=43).
155 3.4.3 Overall colorectal cancer risk scores
Overall colorectal cancer risk scores did not have the same distribution between groups (p<0.001). OCRS in the sporadic population was significantly higher than in all other groups (MLH1 p=0.02; MSH2 p<0.001; Control p<0.001) see Table 23
No statistically significant difference was demonstrated between the overall colorectal cancer risk scores of the MLH1 and MSH2 mutation carriers (p=0.162). There was no statistically significant difference in OCRS in the MSH2 and the control group (p=0.86).
Table 23. Comparison of overall colorectal cancer score (polygenic risk score) between population groups
Mean n OCRS Range SD MLH1 162 1.06 0.5 - 2.32 0.35 MSH2 207 1.02 0.37 - 2.31 0.37 p<0.001 Sporadic CRC 251 1.17 0.46 - 3.15 0.43 Control population 323 1.02 0.37 - 3.28 0.4
3.4.4 Age of Onset of Disease
3.4.4.1 Sporadic Colorectal cancer cases
Mean age of onset in the sporadic colorectal cancer cases was 65.98 years (range 27.55 – 90.00, SD 12.20) with no difference in age of onset between the groups based on overall colorectal cancer risk score (p=0.68) Figure 20 . The same was true for female (mean 65.69 years, range 27.5 – 88.7 years, SD 12.24; p=0.93) and male sporadic colorectal cancer cases (mean 66.22 years, range 28.48 – 90.00 years, SD 12.25, p=0.68).
156
Figure 20. Age distribution across risk quintile for Sporadic
3.4.4.2 MLH1 Mutation carriers
Of the 162 MLH1 mutation carriers included in the analyses, 61% to date have developed colorectal cancer. Mean age of onset of colorectal cancer was 43.94 years (range 21.16 – 75.86, SD 11.70). There was a statistically significant difference in age of onset of colorectal cancer in MLH1 mutation carriers who had developed colorectal cancer across the risk groups based on overall colorectal cancer risk score (p=0.044) Figure 21.
Estimates of the hazard ratios (each quintile relative to the highest risk group, quintile 1) and the corresponding 95% confidence intervals are shown (Table 24). There was no difference in age at development of colorectal cancer between the risk groups (OCRS split into quintiles) based on the 16 SNPS (p=0.58, Table 24). There was little separation of the hazard curves (Figure 22). No statistically significant difference was shown between groups basing the risk scores on the five most validated SNPs in colorectal cancer (p=0.681), the remaining 11 SNPS in the plex (p=0.354) or the two SNPS previously described to be associated with Lynch Syndrome (p=0.20).
157
Figure 21; Age distribution across risk quintile for MLH1 mutation carriers colorectal cancer population
Table 24. Hazard ratios from the cox model for age at the development of colorectal cancer in MLH1 mutation carriers by overall colorectal cancer risk score (split into quintiles)
Median Age at development of CRC or Overall colorectal cancer date of last follow up Hazard risk score (95% CI) Ratio 95% CI p value 16 SNPS Quintile 1 (Highest Risk) 50.00 (48.83-51.16) 1.00 1.00 0.58 Quintile 2 54.84 (44.30 - 65.37) 0.74 0.41-1.42 Quintile 3 52.84 (42.19 - 63.48) 0.93 0.94 - 1.74 Quintile 4 43.73 (38.38 - 49.07) 1.13 0.61 - 2.09 Quintile 5 (Lowest Risk) 47.10 (37.80 - 56.39) 1.26 0.68 - 2.34
11SNPS Quintile 1 (Highest Risk) 50.13 (41.60 - 58.66) 1.00 1.00 0.35 Quintile 2 52.84 (45.67 - 60.01) 1.11 0.59-2.07 Quintile 3 50.86 (45.46 - 56.26) 0.94 0.50 - 1.79 Quintile 4 47.34 (45.08 - 49.60) 1.20 0.64 - 2.26 Quintile 5 (Lowest Risk) 42.40 (39.44 - 45.36) 1.70 0.92 - 3.13
5 SNPS Quintile 1 (Highest Risk) 49.36 (46.94 - 51.78) 1.00 1.00 0.68 Quintile 2 59.25 (44.26 - 74.24) 0.71 0.37 - 1.36 Quintile 3 48.71 (36.20 - 61.33) 0.85 0.47 - 1.54 Quintile 4 44.46 (37.92 - 51.00) 1.15 0.62 - 2.15 Quintile 5 (Lowest Risk) 48.82 (38.16 - 59.48) 0.86 0.47 - 1.59
2 SNPS Quintile 1 (Highest Risk) 62.40 (51.10-73.70) 1.00 1.00 0.20 Quintile 2 54.15 (40.24 - 68.05) 1.32 0.61-2.86 Quintile 3 48.16 (40.92 - 55.40) 1.91 0.96 - 3.81 Quintile 4 - - - Quintile 5 (Lowest Risk) 49.36 (42.44 - 54.33) 1.84 0.94 - 3.61
158
Figure 22. Cumulative hazard of developing colorectal cancer in 162 MLH1 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs.
3.4.4.2.1 Female carriers
Of the 162 MLH1 mutation carriers, 84 (51.9%) were female. 51 female mutation carriers (60.7%) had developed colorectal cancer. Estimates of the hazard ratios and 95% confidence intervals are shown. There was a statistically significant difference in the age of development of colorectal cancer between the groups (p=0.02, Table 25). Basing the risk score on the 5 most validated SNPs in colorectal cancer there was no statistically significant difference, but the difference persisted when testing the other 11 SNPs (p=0.96 and p=0.03 respectively). There was no statistically difference in age of development of colorectal cancer between the group when the risk score was based on two SNPS previously shown to be associated with Lynch Syndrome related colorectal cancer (p=0.71)(Table 25).
159 Table 25. Hazard ratios from the cox model for age at the development of colorectal cancer in female MLH1 mutation carriers by overall colorectal cancer risk score (split into quintiles)
Median Age at development of CRC or Overall colorectal cancer date of last follow up Hazard risk score (95% CI) Ratio 95% CI p value 16 SNPS Quintile 1 (Highest Risk) 50.13 (34.52-65.73) 1 1 0.024 Quintile 2 58.97 (47.43 - 70.51) 0.21 0.22-1.38 Quintile 3 55.69 (33.93 - 77.45) 0.88 0.39 - 2.24 Quintile 4 50.47 (36.47 - 64.47) 0.37 0.28 - 1.62 Quintile 5 (Lowest Risk) 41.89 (36.81 - 46.97) 2.36 1.03 - 5.4
11SNPS Quintile 1 (Highest Risk) 55.69 (45.80 - 65.58) 1 1 0.037 Quintile 2 62.58 (51.53 - 73.62) 0.58 0.23-1.42 Quintile 3 50.86 (45.28 - 56.43) 0.94 0.41 - 2.16 Quintile 4 58.96 (42.89 - 75.03) 0.86 0.35 - 2.13 Quintile 5 (Lowest Risk) 41.89 (39.88 - 43.89) 2.42 1.05 - 5.60
5 SNPS Quintile 1 (Highest Risk) 50.13 (36.38 - 63.88) 1 1 0.963 Quintile 2 50.86 (41.64 - 60.88) 1.4 0.55 - 3.57 Quintile 3 58.96 (37.12 - 80.80) 1.33 0.53 - 3.34 Quintile 4 50.47 (34.25 - 66.69) 1.39 0.48 - 3.99 Quintile 5 (Lowest Risk) 51.16 (34.95 - 67.37) 1.36 0.53 - 3.53
2 SNPS Quintile 1 (Highest Risk) 62.40 (50.81 - 74.0) 1 1 0.71 Quintile 2 60.30 (44.23 - 76.36) 1.44 0.56 -3.71 Quintile 3 58.96 (46.73 - 71.18) 1.21 0.53 - 2.74 Quintile 4 - - - Quintile 5 (Lowest Risk) 47.73 (39.72 - 55.74) 1.54 0.71 - 3.33
Figure 23; Cumulative hazard of developing colorectal cancer in 84 Female MLH1 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs.
160
3.4.4.2.2 Male Carriers
Seventy-eight MLH1 mutation carriers were male and 48 (29.6%) of these had developed colorectal cancer. Estimates of the hazard ratios and 95% confidence intervals are shown (Table 26). There was no statistically significant difference in the age of development of colorectal cancer between the groups for risk scores based on all 16 SNPS, 11SNPS, 5SNPS or 2SNPS (p=0.19, p=0.56, p=0.62 and p=0.07 respectively).
Table 26. Hazard ratios from the cox model for age at the development of colorectal cancer in Male MLH1 mutation carriers by overall colorectal cancer risk score (split into quintiles)
Median Age at development of CRC or Overall colorectal cancer date of last follow up Hazard risk score (95% CI) Ratio 95% CI p value 16 SNPS Quintile 1 (Highest Risk) 46.84 (35.40 - 58.28) 1 1 0.19 Quintile 2 54.15 (32.71 - 75.59) 1.13 0.42 - 3.01 Quintile 3 48.82 (47.86 - 49.76) 1.11 0.43 - 2.84 Quintile 4 39.63 (36.69 - 42.57) 1.46 0.57 - 3.74 Quintile 5 (Lowest Risk) 50.38 (41.38 - 59.38) 2.71 1.07 - 6.85
11SNPS Quintile 1 (Highest Risk) 48.82 (44.20 - 53.44) 1 1 0.56 Quintile 2 49.94 (34.74 - 65.14) 0.55 0.22 - 1.37 Quintile 3 49.36 (46.02 - 52.70) 0.53 0.22 - 1.27 Quintile 4 43.47 (33.82 - 53.12) 0.56 0.23 - 1.40 Quintile 5 (Lowest Risk) 40.01 (35.41 - 44.61) 0.76 0.33 -1.77
5 SNPS Quintile 1 (Highest Risk) 42.40 (32.71 - 52.09) 1 1 0.62 Quintile 2 55.35 (33.49 - 77.21) 0.52 0.18 - 1.43 Quintile 3 51.95 (39.82 - 64.08) 0.59 0.25 - 1.37 Quintile 4 49.36 (38.94 - 59.78) 0.9 0.38 - 2.15 Quintile 5 (Lowest Risk) 46.28 (41.96 - 50.60) 0.67 0.29 - 1.52
2 SNPS Quintile 1 (Highest Risk) 50.76 (42.97 - 58.55) 1 1 0.07 Quintile 2 55.50 (46.18 - 64.81) 1.7 0.2 - 14 Quintile 3 42.35 (38.13 - 46.58) 4.8 0.64 - 35.99 Quintile 4 46.68 (42.32 - 51.84) 3.02 0.40 - 22.56 Quintile 5 (Lowest Risk) - - -
161
Figure 24; Cumulative hazard of developing colorectal cancer in 78 Male MLH1 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs.
3.4.4.3 MSH2 Carriers
Of the 207 MSH2 carriers included in the analysis, 99 (47.8%) of them have developed colorectal cancer to date. Mean age of onset of colorectal cancer was 44.08 years (range 20.74 – 89.64 years, SD 10.91). There was no statistically significant difference in the age of colorectal cancer in MSH2 Mutation carriers who had developed colorectal cancer across the risk groups based on overall colorectal cancer risk score (p=0.67).
Estimates of the hazard ratios (each quintile relative to the highest risk group, quintile 1) and the corresponding 95% confidence intervals are shown (Table 27). There was no difference in age at development of colorectal cancer between the risk groups (OCRS split into quintiles) based on the 16 SNPS (p=0.96, Table 27). No statistically significant difference was shown between groups basing the risk scores on the five most validated SNPs in colorectal cancer (p=0.42), the remaining 11 SNPS in the plex (p=0.87) or the two SNPS previously described to be associated with Lynch Syndrome (p=0.52).
162 Table 27. Hazard ratios from the cox model for age at the development of colorectal cancer in MSH2 mutation carriers by overall colorectal cancer risk score (split into quintiles)
Median Age at development of CRC or Overall colorectal cancer date of last follow up Hazard risk score (95% CI) Ratio 95% CI p value 16 SNPS Quintile 1 (Highest Risk) 51.76 (35.90 - 67.61) 1.00 1.00 0.96 Quintile 2 52.18 (47.98 - 56.37) 0.66 0.63 - 2.09 Quintile 3 56.15 (47.96 - 64.33) 0.80 0.57 - 2.06 Quintile 4 53.89 (45.38 - 62.40) 0.98 0.54 - 1.83 Quintile 5 (Lowest Risk) 57.56 (52.16 - 62.95) 0.77 0.48 - 1.74
11SNPS Quintile 1 (Highest Risk) 60.64 (48.83 - 72.45) 1.00 1.00 0.87 Quintile 2 53.82 (45.42 - 62.22) 1.34 0.70 - 2.56 Quintile 3 54.69 (46.90 - 62.48) 1.36 0.73 - 2.54 Quintile 4 53.10 (46.08 - 60.12) 1.35 0.71 - 2.56 Quintile 5 (Lowest Risk) 57.35 (44.23 - 70.47) 1.35 0.69 - 2.62
5 SNPS Quintile 1 (Highest Risk) 51.21 (47.25 - 55.17) 1.00 1.00 0.42 Quintile 2 48.41 (40.23 - 56.59) 1.14 0.64 - 2.03 Quintile 3 60.51 (48.84 - 72.18) 0.69 0.36 -1.30 Quintile 4 57.35 (49.76 - 64.94) 0.69 0.36 - 1.32 Quintile 5 (Lowest Risk) 54.69 (42.97 - 66.41) 0.94 0.52 - 1.70
2 SNPS Quintile 1 (Highest Risk) 64.35 (48.39 - 80.30) 1.00 1.00 0.52 Quintile 2 53.59 (47.08 - 60.10) 1.39 0.79 - 2.46 Quintile 3 - - - Quintile 4 53.89 (46.20 - 61.58) 1.23 0.70 - 2.18 Quintile 5 (Lowest Risk) -
Figure 25. Age distribution across risk quintiles for MSH2 mutation carriers
163
Figure 26; Cumulative hazard of developing colorectal cancer in MSH 2 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs
3.4.4.3.1 Female carriers
Of the 207 MSH2 mutation carriers, 130 (82.8%) were female. 54 female mutation carriers (41.5%) had developed colorectal cancer. Estimates of the hazard ratios and 95% confidence intervals are shown (Table 28). There was no difference in the age of development of colorectal cancer between the groups (p=0.41, Table 28). Basing the risk score on the 5 most validated SNPs and the remaining 11 in the SNP plex there was no difference between the groups (p=0.87 and p=0.72 respectively).
164 Table 28. Hazard ratios from the cox model for age at the development of colorectal cancer in Female MSH2 mutation carriers by overall colorectal cancer risk score (split into quintiles)
Median Age at development of CRC or Overall colorectal cancer date of last follow up Hazard risk score (95% CI) Ratio 95% CI p value 16 SNPS Quintile 1 (Highest Risk) 67.66 (49.64 - 85.68) 1.00 1.00 0.93 Quintile 2 58.33 (48.72 - 67.95) 0.60 0.56 - 2.75 Quintile 3 59.45 (47.83 - 71.07) 0.50 0.55 - 3.36 Quintile 4 59.27 (48.92 - 69.92) 0.92 0.42 - 2.18 Quintile 5 (Lowest Risk) 60.00 (56.45 - 63.56) 0.82 0.44 - 2.81
11SNPS Quintile 1 (Highest Risk) 77.34 (56.95 - 73.09) 1.00 1.00 0.73 Quintile 2 58.33 (43.77 - 72.88) 1.45 0.58 - 3.62 Quintile 3 59.27 (51.71 - 66.78) 1.57 0.66 - 3.74 Quintile 4 53.89 (52.57 - 55.21) 1.46 0.58 - 3.64 Quintile 5 (Lowest Risk) 37.56 (30.71 - 84.41) 1.96 0.76 - 5.00
5 SNPS Quintile 1 (Highest Risk) 57.85 (44.70 - 71.00) 1.00 1.00 0.44 Quintile 2 53.10 (43.51 - 62.69) 1.50 0.70 - 3.19 Quintile 3 66.27 (53.36 - 79/17) 0.76 0.32 - 1.81 Quintile 4 69.12 (57.47 - 80.77) 0.74 0.29 - 1.91 Quintile 5 (Lowest Risk) 59.45 (55.36 - 63.53) 1.03 0.44 - 2.41
2 SNPS Quintile 1 (Highest Risk) 67.66 (48.61 - 86.71) 1.00 1.00 0.69 Quintile 2 59.27 (52.15 - 66.39) 1.33 0.61 - 2.93 Quintile 3 - Quintile 4 - Quintile 5 (Lowest Risk) 58.33 (51.42 - 65.24) 1.39 0.64 - 3.02
Figure 27; Cumulative hazard of developing colorectal cancer in 133 Female MSH 2 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs
165
3.4.4.3.2 Male carriers
Seventy-seven (37.2%) MSH2 mutation carriers were male and of these forty- five (58.4%) had developed colorectal cancer. There was no difference between the groups based on polygenic risk scores for all 16 SNPS (p=0.41), 11 SNPS (p=0.87), 5 SNPS (p=0.72) or 2 SNPs previously associated with Lynch Syndrome (p=0.30). All results are shown in Table 29.
Table 29. Hazard ratios from the cox model for age at the development of colorectal cancer in Male MSH2 mutation carriers by overall colorectal cancer risk score (split into quintiles)
Median Age at development of CRC or Overall colorectal date of last follow up Hazard cancer risk score (95% CI) Ratio 95% CI p value 16 SNPS Quintile 1 (Highest Risk) 47.96 (37.35 - 58.56) 1.00 1.00 0.41 Quintile 2 45.49 (37.03 - 53.95) 1.70 0.68 - 4.26 Quintile 3 51.16 (40.91 - 61.41) 1.17 0.44 - 3.13 Quintile 4 45.57 (31.90 - 59.24) 1.80 0.71 - 4.59 Quintile 5 (Lowest Risk) 54.69 (34.47 - 79.91) 0.82 0.31 - 2.21
11SNPS Quintile 1 (Highest Risk) 47.96 (33.86 - 62.06) 1.00 1.00 0.87 Quintile 2 49.65 (43.71 - 55.59) 1.34 0.70 - 2.56 Quintile 3 40.73 (39.09 - 42.37) 1.36 0.73 - 2.54 Quintile 4 43.29 (35.49 - 51.09) 1.35 0.71 - 2.56 Quintile 5 (Lowest Risk) 50.55 (30.30 - 70.79) 1.35 0.69 - 2.61
5 SNPS Quintile 1 (Highest Risk) 49.65 (34.41 - 64.89) 1.00 1.00 0.72 Quintile 2 40.73 (33.74 - 47.72) 1.59 0.62 - 4.05 Quintile 3 47.96 (37.36 - 58.56) 0.87 0.32 - 2.33 Quintile 4 51.16 (32.63 - 69.70) 1.00 0.39 - 2.57 Quintile 5 (Lowest Risk) 45.57 (34.34 - 56.80) 0.95 0.36 - 2.47
2 SNPS Quintile 1 (Highest Risk) 54.69 (44.23 - 65.15) 1.00 1.00 0.30 Quintile 2 40.43 (34.43 - 46.44) 1.76 0.76 - 4.04 Quintile 3 - - - Quintile 4 48.91 (38.41 - 59.41) 1.16 0.50 -2.66 Quintile 5 (Lowest Risk) - - -
166
Figure 28; Cumulative hazard of developing colorectal cancer in 75 male MSH 2 mutation carriers by risk group (overall colorectal cancer risk score split into quintiles), based on 16 SNPs
3.5 Discussion
The results from this study confirms that SNP risk alleles previously found to be associated with colorectal cancer in GWAS studies and validation studies are more prevalent in sporadic colorectal cancer cases than in MLH1 and MSH2 mutation carriers or a control population. The suggestion that SNPs previously associated with colorectal cancer have a multiplicative effect on risk in mutation carriers was not substantiated, with no evidence of earlier age of onset of colorectal cancer.
167 3.5.1 Sporadic Colorectal Cancers
Overall colorectal cancer risk scores were highest in the sporadic colorectal cancer group and this difference was statistically significant when compared with high-risk mutation carriers and the control population. This supports the previous literature in that these SNPs and risk alleles are associated with colorectal cancer in a background population. However, there was no separation of the quintiles of risk on the hazard curves in terms of age of onset of disease. Whilst SNPs have been validated in studies to play important role in the risk of colorectal cancer in a background population there has been little evidence in the published literature of significant heterogeneity by age at diagnosis of colorectal cancer269. These study findings support those of previous research, however, age of onset of colorectal cancer in a background population and SNP profile have not been the focus of many studies.
3.5.2 High-risk mutation carriers
Unexpectedly the frequency of risk alleles in the MLH1 and MSH2 mutation carriers is lower than in the sporadic colorectal cancer population despite mutation carriers having an earlier age of onset of colorectal cancer, which was statistically and clinically significant.
There have been conflicting studies in the published literature with regard to SNPs associated with colorectal cancer and their modifying effect on age of onset of disease in MLH1 and MSH2 mutation carriers. Some have reported strong associations between risk allele frequency and earlier age of onset of disease (up to 28 years earlier age in onset of disease221) whilst others have found no modification of risk with these SNPs270,271. There is limited available data in the published literature for risk allele frequencies in the control populations used in some studies and also the risk allele frequencies found by authors for the tested SNPs in the mutation carrier populations. Therefore, the
168 data upon which polygenic risk scores were based in this study were limited212,268.
In MLH1 mutation carriers the median age at development of colorectal cancer varies with risk group, but there is no trend for later age of diagnosis with lower risk score. In fact, in this study population, the converse is true. In MLH1 mutation carriers the age of onset in the quintile 5 ‘lower risk’ group is lower than in quintile 1 ‘higher risk’ (47.10 versus 50.00 years of age). There were no clinically significant differences found in age of onset of disease in MLH1 and MSH2 mutation carriers based upon risk group, according to overall colorectal cancer risk score. Unlike in other malignancies, e.g. breast cancer, SNPs associated with colorectal cancer in previous studies do not appear to have a multiplicative effect on age of onset of the disease in high-risk mutation carriers and therefore their clinical utility in this population is currently questionable. It might be that the odds ratios are not as well calibrated based on smaller case control cohort numbers than was the case for breast cancer, which used the iCOGs consortium data193,266,272. Alternatively, the effect sizes may be different in sporadic CRC to MLH1 and MSH2 as is the case for BRCA1 and BRCA2 compared to sporadic and non-BRCA familial breast cancer where different weightings are required266. Either way unlike in breast cancer substantial further work is required before an OCRS can be used in CRC.
There is considerable overlap of the 95% confidence intervals for the respective risk groups. This may be explained by the SNPs used in this study having been largely validated in sporadic colorectal cancer cases, therefore is possible that the risk allele frequencies used to calculate the weightings for the polygenic risk scores are inappropriate as they relate to a background population and not high-risk mutation carriers. It also may be explained by the relatively small sample size and small number of patients who have developed colorectal cancer (199 MLH1 and MSH2 mutation carriers). Finally, it may be attributable
169 to the modifying effect of these SNPs being much less in mutation carriers than in the background population.
Some studies have reported strong associations between the number of risk alleles for some SNPs (e.g. rs16892766, rs4779584, rs4939827) and age of onset of colorectal cancer in high-risk mutation carriers or those individuals with a history suggestive of Lynch Syndrome. However, these studies, as with many genetic studies, have been undertaken using several individuals from the same family and little has been reported about the inheritability of SNPs. It is therefore possible that other inherited factors are acting to cause earlier age of onset of disease in individuals with inherited common variants that in isolation have no effect on age of onset of disease. This may explain why other studies223, similarly to this one, report that SNPs associated with colorectal cancer in the general population are not modifiers of risk for mismatch repair mutation carriers and are therefore of no current clinical utility in Lynch syndrome.
3.6 Limitations
The main limitation of the study is the small sample size, particularly the number of mutation carriers who had developed colorectal cancer. Also, some patients may have undergone endoscopic interventions that had prevented them from developing colorectal cancer and therefore delaying the onset of disease. Time to first adenoma would have been a more appropriate event status but this was impossible to collect from the existing database.
3.7 Conclusions
This study confirms an association between SNPs previously reported to be associated with colorectal cancer and a diagnosis of colorectal cancer in a background population. No association was found between
170 these SNPs and age of onset of colorectal cancer in patients with confirmed pathogenic MLH1 and MSH2 mutations.
3.7.1 Further work and recommendations
More validation studies are required to refine the associations between SNPs identified in GWAS to be associated with colorectal cancer and true associations with the disease. Researchers should report and publish negative findings. There must be more widespread availability of the case and control risk allele frequencies identified in these studies to aid the design of future studies. The accuracy of frequencies of risk alleles versus a weighted and multiplicative polygenic risk score should also be investigated.
When considering inclusion criteria, it is important to include sporadic cases of mismatch repair gene deficiency and not just those with inherited Lynch syndrome to limit cofounding of other inherited factors.
Before SNPs are of any clinical utility in colorectal cancer many larger, well-designed studies are required. There have been no reports of single nucleotides polymorphisms being ‘protective’ in delaying age of onset of disease in either a sporadic population or those with inherited mismatch repair deficiency. Large-scale studies of well-validated SNPs associated with colorectal cancer are required with multivariate analysis of each individual SNP in order to determine strength risk producing or protecting effect.
171
STUDY THREE
Beta-2 microglobulin as a prognostic biomarker in mismatch repair deficient colorectal cancer
172 4.1 Abstract
Background; Beta-2-Microglobulin (B2M) mutations occur frequently in mismatch repair deficient colorectal cancer. Mutations in the coding microsatellites of the B2M gene have been identified as the primary mechanism affecting HLA Class I antigen presentation. Limited data suggests this may have a role in protection against disease recurrence.
Methods; Lynch Syndrome MSH2 mutation carriers who had undergone colonic resection for colorectal cancer were identified from the Manchester Familial Colorectal Cancer Registry. Sanger sequencing was performed for the three coding exons of B2M on 54 dMMR (MSH2 loss) tumours. B2M protein expression was also assessed by immunohistochemistry. Mutation status was correlated with disease recurrence.
Results; 50 dMMR CRCs were included in the final analysis. Deleterious B2M mutations were identified in 14 (36%). With median follow up of 9.6 years, none of the tumours with B2M mutations recurred compared to 5(13.8%) of the tumours with wild type B2M (p=0.142). Sensitivity and specificity of IHC in detecting B2M mutations was 78% and 46% respectively.
Conclusion; B2M mutations were identified in more than one third of dMMR cancers in patients with MSH2 loss, none of which recurred. There is increasing evidence that B2M mutation status has a potentially advantageous clinical utility in colorectal cancer.
173 4.2 Introduction
4.2.1 Mismatch Repair deficient colorectal cancer (dMMR)
During DNA replication, the recognition and repair of coding errors occurs through the mismatch repair system (MMR). When this system fails multiple genetic errors result. This can predispose to malignant change, particularly if these errors occur in tumour suppressor genes or oncogenes. Deficiencies in the mismatch repair system occur due to mutation in one of the four main MMR genes (MLH1, MSH2, MSH6 and PMS2), resulting in Lynch Syndrome (formerly Hereditary Nonpolyposis Colorectal Cancer; HNPCC). Sporadic cases of mismatch repair deficient colorectal cancer (dMMR) occur through the epigenetic silencing of the MLH1 gene (by hypermethylation in the MLH1 promoter region) and account for approximately 15% of all sporadic cases of colorectal cancer. The phenotype and tumour biology of dMMR colorectal cancers is different to those tumours that arise through the chromosomal instability pathway (pMMR – proficient mismatch repair). Phenotypically, dMMR tumours occur on the right side of the colon and account for 25% of all right sided colorectal cancers168. Additionally, dMMR is extremely rare in rectal cancers and as such should raise the possibility of Lynch Syndrome150. Histologically dMMR tumours have microsatellite instability phenotype (MSI- H). These features include poor differentiation, mucinous tumours and numerous tumour infiltrating lymphocytes and intraepithelial lymphocytes169. It has been proposed that the numerous aberrant proteins resulting from microsatellite instability, and subsequent high tumour immunogenicity, may play a role in why dMMR CRCs are associated with favourable outcomes when compared with pMMR tumours150 even though there is lack of benefit from standard 5-fluorouracil based chemotherapy regimens109,273,274. Histologically dMMR tumours appear to be expansile rather than infiltrative, lack heterogeneity and have peritumoural lymphoid nodules (crohn’s like inflammatory reaction) and an abundance of tumour infiltrating lymphocytes. They are less likely to metastasise than pMMR tumours. Hutchins in 2011 reported that the recurrence rate for dMMR tumours was half that for MMR- proficient tumours (11% vs 26%). High tumour immunogenicity of dMMR tumours (thus eliciting specific T-cell responses) due to numerous aberrant proteins (frame shift peptides (FSP)) produced as a result of MSI
174 (microsatellite instability) has been proposed as a potential explanation for these findings. The development of new immunotherapies has presented the potential possibility to exploit dMMR tumours intrinsic immunogenicity and target therapies to improve outcomes.
Mutations in the coding microsatellites of the Beta-2 Microglobulin (B2M) gene have been identified as the primary mechanism affecting HLA Class I antigen presentation, leading to a generation of immune escape variants188. It would therefore seem logical that these immune escape variants of dMMR CRC have a more aggressive clinical course with more frequent distant metastases, as there is loss of immune control. However, there is conclusive evidence in the literature that B2M mutation status may be utilised to identify CRC patients where B2M mutation confers a more favourable prognosis175,275.
4.2.2 Beta-2 microglobulin
The B2M gene consists of 4 exons. There are four repetitive nucleotide sequences within exon 1 and 2 that are vulnerable to somatic mutation in dMMR cases. The DNA sequence of the B2M gene can be altered in a number of ways. Types of mutation include missense, nonsense, insertion or deletion (splice site), duplication, frameshift and repeat expansion. A missense mutation changes base pairs and results in the substitution of one amino acid for another in the protein made by the gene (HLA Class I in the case of B2M). Nonsense mutations, rather than substituting amino acids, alter the DNA sequence to prematurely stop building a protein, the shortened protein may not function properly, if at all. Insertion and deletions alter the number of DNA bases. Large deletions may remove an entire protein or several neighbouring genes. A reading frame consists of groups of three bases that each code for one amino acid. Frameshift mutations alter the grouping of these bases changing the code for the amino acids. The resulting protein, different from the original sequenced protein, is usually non-functioning. Repeat expansions are short DNA sequences that are repeated e.g. trinucleotide or tetranucleotide. These types of mutations all result in non-functioning proteins.
It is known that dMMR colorectal cancers accumulate somatic mutations in genes, which are normally conserved, such as the B2M gene. The gene is a mutagenic target due to the number of coding microsatellites it contains.
175
B2M mutation positive dMMR colorectal cancers are characterized by a lack of distant metastasis and disease recurrence175,275. B2M mutations (confirmed by exon-wise sequencing) in microsatellite instability high (MSI-H) colorectal cancers have been reported to be associated with a complete absence of disease relapse or tumour related death events. A recent study by Barrow et al explored the relationship between B2M mutation status and recurrence in patients with stage II colorectal cancer. B2M mutations were detected in nearly one third of dMMR cancers. None of the B2M mutant tumours recurred compared with 18% of the B2M wildtype tumours (p=0005) 276.
4.2.3 HLA class I complex
HLA class I complex is present on the surface of all nucleated cells and serves to present cellular peptides to the immune system. Molecular changes to the HLA class I heavy or light chains directly affect the HLA class I complexes ability to present these peptides. The HLA class I complex is comprised of the HLA class I heavy chain, the light chain (B2M) and a peptide fragment. B2M is an essential component of the HLA class I complex. Around 30% of Lynch syndrome associated colorectal cancers display loss of HLA class I antigen expression as a results of B2M mutations175,277.
Alpha subunits of the HLA class I complex are encoded by genes HLA-A, HLA- B and HLA-C on chromosome 6. HLA class 1 has an important role in the presentation of antigenic material to cytotoxic lymphocytes (CTLs) as part of the immunological response to malignancy. It is this response that is impaired in patients who are immunosuppressed resulting in increased rates of malignancy in some high-risk groups e.g. transplant patients. Lack of recognition by CTLs facilitates local tumour growth (immune-escape phenotype) and loss of HLA class I expression enables malignant cells to circulate without immune attack and facilitates distant spread. However, there is growing evidence to suggest that HLA class I deficient tumour cells are less likely to establish distant metastases than HLA class I proficient cells through activation of Natural Killer cells (NK) to destroy circulating tumour cells175,181,186,188. The mechanism by which B2M mutation affords protection from distant metastases is yet to be fully elucidated. High levels of
176 lymphocytic tumour infiltration have been described in some tumour types and have shown in colorectal cancer to be associated with a favourable prognosis184. High tumour immunogenicity and prominent lymphocytic infiltration seen in dMMR CRCs, together with up regulation of cytotoxic mediators (Granzyme A, Granulysin), results in high levels of lymphocytes into the tumour microenvironment.
4.2.4 Immune-Escape Phenotype
The Immune-Escape Phenotype has been described in a number of malignant tumours including melanoma, lymphoma and prostate cancer. Essentially, loss of HLA class I expression allow tumour cells to escape recognition by CTLs and results in unopposed local tumour growth. MSI-H CRCs evade immune surveillance through loss of HLA class I antigen presentation because of mutation in the encoding B2M gene. B2M mutations, and therefore loss of HLA class I expressions, is identified in around 30% of dMMR CRCs and only 2% of pMMR CRCs175,180,181,187. Furthermore, the degree of lymphocytic infiltration in B2M mutant dMMR CRCs has been reported to be significantly less than in dMMR CRCs with wild type B2M180. It stands to reason that mutation in the B2M gene may interfere with the presentation of antigenic material to CTLs permitting local tumour growth and enabling unopposed circulation of malignant cells. However, there is increasing evidence that loss of HLA class I expression, through mutation of B2M, is associated with a decrease in the likelihood of developing distant metastases.
4.2.5 Tumours and Natural Killer cells
HLA class I is essential for T cell mediated immunity. Additionally, normal HLA class I plays a pivotal role in tumour cell adhesion and inhibition of natural killer cell activity. In the absence of HLA class I, the activity of natural killer cells is less regulated. Tumour cells are recognised by NK cells and apoptosis is initiated. This mechanism may explain why HLA class I expression favours local growth of tumour cells but prevents the formation of distant metastases.
Several mouse model and in-vitro studies in uveal melanoma and fibrosarcoma have investigated the role of natural killer cells in the prevention of metastases
177 and led to the development of novel immunotherapies278,279. Uveal melanoma has many similarities with dMMR CRC (haematogenous spread to the liver and similar B2M mutation patter) and improved survival with loss of HLA class I expression.
The prognostic significance of HLA Class I down regulation has been reported in a large cohort of CRC cases by Watson et al280. Cases with low expression of HLA class I were associated with a significant shorter mean disease specific survival period compared with tumours with high expression of HLA class I (41 months v’s 68 months). Interestingly patients with a complete loss of HLA class I in the primary tumour had a similar prognosis to those with high expression (mean disease specific survival 60 months). Low expression of HLA class I but not complete loss could avoid both T cell mediated, and Natural Killer Cell mediated immune surveillance, and this may be responsible for the association with a poor prognosis.
4.2.6 The B2M gene
The B2M gene, located on chromosome 15q21, codes for a protein 119 amino- acids in length and has four exons spanning 10,673 base pairs. It has four coding microsatellites in its coding region; 1 dinucleotide (CT)4 repeat in exon
1 (codon 13-15) and 3 mononucleotide repeats (A5, C5 and A5) repeats in exon 2 (codon 67-68, codon 91-92, codon 94-95 respectively). All of these are vulnerable to somatic mutations in the case of dMMR, particularly the CT4 sequence in exon 1174. Several mutations in the B2M gene have been reported in the published literature (the majority of which were heterozygous), particularly in MSI-H CRC and melanoma. In some tumours, two different mutations have been described174, one in each copy of the B2M gene resulting in complete loss of HLA class I expressions. The extent to which loss of heterozygosity (LOH) occurs in dMMR CRC is difficult to quantify due to microsatellite instability interacting with the markers used174 but has been reported in 35% of MSS CRC, 44% of bladder cancers and 53% of breast cancer.
178
Figure 29; B2M Gene (chr 15q21) (from Alamut v2.2)
Mutations affecting the coding microsatellite regions of the B2M gene have been reported as instrumental in mediating impairment of the HLA class I antigen presentation in MSI-H CRC, affecting about 30-60% of lesions 180,187.
4.2.6.1 B2M mutations in CRC
B2M mutations have been reported to occur at early stages of MSI-H tumorigenesis. B2M mutation frequency in MSI-H adenomas is around 16%, which suggests an early onset of immunoselective pressure. B2M mutation frequency increases with tumour progression and it is estimated that B2M mutation exists in around 40% of lymph node positive MSI-H CRC175.
Bicknell et al181 twenty years ago compared the frequency of B2M mutations in sporadic colorectal cancer along with other tumours and found that mutations were more frequent among CRC’s with microsatellite instability indicative of a defect in DNA mismatch repair181. Over the last twenty years studies have reported that somatic B2M mutations are rare in pMMR CRC but are found in around 30% of dMMR CRCs. Kloor et al 2007 analysed the prevalence of B2M mutations in MSI-H colorectal adenomas and carcinomas of different stages175. They found a higher frequency of B2M mutations in patients with germline mutations of MMR genes MLH1 or MSH2 compared to sporadic CRC. B2M mutations were positively related to stage in CRC without distant metastases. No B2M mutations were observed in metastasized CRC’s. These findings therefore suggest that functional B2M may be necessary for the formation of distant metastases in patients with colorectal cancer. If CRC patients with B2M mutations do not form distant metastases, and therefore B2M mutations are in fact protective of distant disease, there are important clinical (therapeutic and surveillance) and prognostic implications particularly in dMMR CRC.
179 More recently the role of circulating B2M has been investigated as a marker for cancer risk. Serum B2M may be secreted from the cell surfaces or as a result of intracellular release and is filtered predominantly by the kidneys. Elevated serum B2M is a marker of renal impairment or increased cell turnover281. In the absence of renal impairment, an elevated B2M has been identified as a marker of cell proliferation in a number of solid and haematological malignancies282-287. Prizment et al (2016) hypothesized that as colorectal cancer had been associated with immune response and inflammation, an elevated serum B2M would be a marker for increased risk of development of future colorectal cancer. More than twelve thousand patients were followed up for over ten years for incident cancers. Colorectal cancers were identified in 255 patients. B2M risk was separated into quartiles and there were significant positive associations with B2M and the risk of colorectal cancer (HR, 1.55; 95% CI, 1.04-2.30, p=0.02). This study has provided the first evidence that elevated serum B2M is associated with increased colorectal cancer risk and further studies are warranted to determine the role of serum B2M as a biomarker288.
B2M mutation status is not currently utilised in clinical practice. The CRUK prognostic/predictive biomarker (BM) road map (Figure 30) states the optimum method for establishing the clinical utility of a biomarker is to conduct a clinical trial where B2M status defines randomisation. If clinical outcome is improved by the prospective use of B2M, B2M status would be transferable to clinical practice and used in the clinical decision making about adjuvant therapies for patients with colorectal cancer.
180
Figure 30; Cancer Research UK Prognostic / Predictive Biomarker Roadmap
181 4.3 Hypothesis and aims
B2M plays an important role in disease progression in dMMR CRC and in particular the development of distant metastases. B2M mutations and disruption of the HLA class I molecule is thought to result in tumour cells avoiding recognition by cytotoxic T lymphocytes (immune escape phenotype). However, circulating tumour cells are attacked by natural killer cells preventing the formation of distant metastases in those patients with B2M mutation.
The aim of this study was to determine the proportion of B2M mutations (through immunohistochemistry for B2M protein expression and DNA sequencing) in a cohort of colorectal cancer patients known to have MSH2 mutations and having undergone surgical resection for colorectal cancer and the effect of the mutation on cancer outcome. Most of the previous published work has been undertaken in MLH1 mutation carriers69,175,275,289. This study is the largest dataset (and only dataset to focus solely) on MSH2 mutation carriers.
4.4 METHODOLOGY
4.4.1 Ethical approval
This study received a favourable review from the South Manchester Research Ethics Committee (no10/H31003/11 – substantial amendment 2). No pathological specimens were collected. As per the human tissue act 2004, residual archived tissue may be stored and archived for future research without prior consent if the study is ethically approved and the tissue anonymised. In this study, patient consent was not sought specifically as the results of this study have no clinical implication for their ongoing management. Additionally, many patients included in the study would have completed their treatment and some may have died. No data were recorded in the patient notes and no identifiable information was disseminated outside of the research team. Supplementary clinical data were obtained from the clinical case notes and pathology reports and other forms of electronic patient records (e.g. PACS radiology system). Clinical data were collected by an independent researcher and handled in accordance with the data protection act (1998) using a patient’s
182 identification key. Information was stored on an NHS computer which was password protected and in a locked office on NHS property.
4.4.2 Participants
Lynch Syndrome MSH2 mutation carriers who had undergone colonic resection for colorectal cancer were identified from the Manchester Familial Colorectal Cancer Registry. Mutation carriers were defined as those patients who had tested positive for a pathogenic germline mutation.
Regional clinicians refer patients with a family or personal history suggestive of Lynch Syndrome to the department of Genetic Medicine at Central Manchester Foundation Trust (now Manchester Foundation Trust). Full pedigree information is taken at consultation by a genetic counsellor. Since 1996, if families fulfilled either Amsterdam or Bethesda criteria, living affected relatives were offered testing for germline mutations in the mismatch repair genes. Multiplex Ligation Probe Dependant Amplification (MLPA) was used to screen all exons of MSH1, MSH2, and MSH6 genes. For all other patients, tumour samples were tested for evidence of mismatch repair deficiency. If tumours demonstrated mismatch repair deficiency germline mutation testing was then performed.
4.4.3 Tissue specimens and DNA Samples
Tumour blocks were obtained retrospectively for resected colorectal cancers from regional hospitals. Patients had previously been identified as having loss of MSH2 protein expression by serum testing or tumour immunohistochemistry and germline MMR mutation testing at Manchester Regional Genetics Service. Matched Lymphocyte DNA samples were also tested.
Case archived formalin-fixed paraffin-embedded (FFPE) tissue specimens were requested from the pathology departments at which the colonic cancer had been assessed histologically. Diagnosis of colorectal cancer was confirmed using the histopathology reports included with the requested and returned specimen blocks for each patient. Two blocks were requested for each case;
183 one block of tumour tissue and one block of normal tissue. On receipt of these blocks they were anonymised by an administrator in the histopathology department at Central Manchester Foundation Trust. All data were anonymised using the specimen laboratory ID number and handled in accordance with the data protection act (1998). No patient identifiable information was disseminated outside of the research team and results were not recorded in the patient’s notes.
4.4.4 Tissue specimen preparation
The BioRobot EZ1 system was used to purify DNA from paraffin embedded tissue using magnetic particle technology which was then used for genotyping. The BioRobot Ez1 performs all steps of the DNA isolation procedure.
DNA was isolated from formalin-fixed paraffin-embedded tissue (FFPE) using the EZ1 DNA tissue kit. Sections were taken from FFPE tumour and normal blocks using a microtome and a minimum of 5 sections were transferred into a 1.5ml eppendorf tube using a clean pipette tip. 190µL of G2 digestion buffer (QIAGEN) were added to each tube. The specimens were briefly centrifuged to ensure the sections were submerged and collected at the bottom of the tube. The tubes were incubated at 76°C for 5 minutes with vigorous mixing at ~ 1200rpm in a ThermoMixer (Eppendorf, UK). The temperature was lowered to 56°C and the samples were allowed to cool. 10µL of Proteinase K solution were added to each tube and mixed by gently tapping the tube. The samples were then incubated overnight at 56°C with continuous shaking at 1200rpm to ensure complete digestion of the sample. The next morning the tubes were centrifuges briefly and the samples transferred to a 2ml skirted (ribbed) tube leaving any wax behind.
DNA isolation was performed using BioRobot EZ1 workstation (QIAGEN). Tissue cartridges were inverted twice to mix the magnetic particles and then tapped to deposit the reagents at the bottom of their wells and loaded into the cartridge rack. The opened (lids off) 1.5ml tapered tubes were loaded into the elution tube rack (row 1). Tip holders containing filter tips were loaded into the front row of the tip rack (row 2) and the digested paraffin embedded tissue samples were then loaded into the back row of the tip rack (row 4). The automated purification procedure took 15-20minutes.
184
Any residual magnetic beads that may have been present in the eluted DNA samples were removed using the EZ1 magnetic rack and then each sample transferred into a new 1.5ml tapered tube. The concentration of DNA was determined using a Nanodrop ND 8000 spectrophotometer (Labtech Internation). The machine was ‘blanked’ using an elution buffer (chemagen). 2µL of each DNA sample were pipetted onto each pedestal and the lid replaced. Concentrations were recorded. All samples were diluted to a DNA concentration of 30ng/µL using standard techniques. Any samples with a DNA concentration of less than 30ng/µL were used neat. All samples were stored in a refrigerator. The concentration of DNA samples that subsequently failed repeated genotyping were confirmed using Qubit rather than Nanodrop spectrophotometer. Qubit fluorometric quantitation is based on target specific fluorescence and is more sensitive that UV absorbance-based quantification
4.4.5 Sanger sequencing
All samples were analysed at the Regional Molecular Genetics Laboratory (Department of Genomic Medicine, Central Manchester Foundation Trust) according to Good Laboratory Practice (GLP) standards. The technical work was carried out under the supervision of a qualified genetic technologist and results were verified by Dr Andrew Wallace, Consultant Clinical Scientist.
Standard polymerase chain reactions (PCR) techniques were used to amplify the DNA covering the B2M gene using N13-tailed forwards and reverse primers. Bi-directional sanger sequencing was performed, and mutation analysis carried out using Staden package. All samples were analysed in duplicate to exclude artifact from poor quality DNA and confirm mutations. Positive and negative controls were used in the analysis and a water blank included to identify possible contamination of the PCR product.
4.4.5.1 Primer design
The sequence for exon 1, 2 and 3 of the B2M gene was obtained. Primers were designed for all three exons of the gene. The largest exon (exon 2) was split into two parts (2A and 2B). Forward and reverse primers were designed
185 for each amplicon using an online design tool (http://primer3.ut.ee). Primers were chosen to ensure they were not larger than 250 base pairs long and checked using www.snpcheck.net/. Cluster reports provided information on the frequency data for population diversity for any SNPs found within the primer sequence. Common SNPs within the primer sequence would result in failure of the primer in binding to the sequence.
Primers were obtained from Sigma-Aldrich (Table 30) and tagged with an N13 tail: Forward: 5’-GTAGCGCGACGGCCAGT……primer Reverse: 5’-CAGGGCGCAGCGATGAC……primer
Table 30; Primers of all three exons of the B2M gene (Sigma-Aldrich)
Exons Primers Size
Forward Primer: 5’- CGTCGCTGGCTTGGAGAC-3’ Exon 1 Reverse primer: 5’- CAGAGCGGGAGAGGAAGGA-3’ 240bp Forward primer: 5’-ACACCAAGTTAGCCCCAAGT-3’ Exon 2A Reverse primer: 5’-AGATAGAAAGACCAGTCCTTGCT-3’ 250bp Forward primer: 5’- TGGGTTTCATCCATCCGACA-3’ Exon 2B Reverse primer: 5’- ACACAACTTTCAGCAGCTTACA-3’ 243bp Forward primer: 5’- CAGCCTATTCTGCCAGCCT-3’ Exon 3 Reverse primer: 5’- TCCTCAGGACAGTGAAACAA-3’ 188bp
4.4.5.2 Polymerase Chain Reaction (PCR)
Polymerase chain reaction was carried out using a 20µL reaction (10µL GoTaq, 2µL of forward and reverse primers at a concentration of 5µM, 6µL water and 2µL DNA). Each sample was analysed in four separated reactions (exon 1, exon 2A, exon 2B and exon 3) and each patient’s sample was analysed in duplicate. Samples were ordered in a 96 well plate with a normal control sample (coriell cell line) and water blank per exon.
A master mix of 18µL containing PCR buffer, forward and reverse primers and water was pipetted into each well of the plate. To this 2µL of sample DNA (concentration 30ng/µL) was added. The plate was covered with a peelable adhesive seal (Rainin). The mixture was agitated and centrifuged using a pulse vortex to ensure all reagents collected at the bottom of the plate wells
186 and the plate was centrifuged to ensure all reagents collected at the bottom of the wells.
4.4.5.3 PCR conditions for B2M
Amplification of tumour and normal DNA was undertaken with polymerase chain reaction (PCR) using verity 96 well thermal cycler (Applied Biosystems, USA). The conditions for thermal cycling are shown in Table 31.
Table 31; Thermal cycling conditions for PCR.
Temp (°C ) Duration Stage 1 Initial Denaturation 95°C 15 minutes 1 cycle Denaturation 95°C 10 seconds Stage 2 Annealing 60°C 1 minute 40 cycles Extension 72°C 1 minute Final Extension 72°C 5 minutes 1 cycle Stage 3 Hold 4°C Hold
4.4.5.4 Gel electrophoresis of PCR product
A 2% agarose gel was produced for each 96 well plate. 2 grams of agarose gel was mixed with 100ml of 1xTBE buffer in a conical flask. Cling film was placed over the flask and the solution warmed in a microwave for around one minute. The flask was agitated and warmed until the agarose dissolved and the mixture became clear. The mixture was cooled by running the bottom of the flask under cold water. 10µL of safe view (10% volume of the 1%TBE volume) was added into the flask and gentle mixed. The mixture was carefully poured onto the gel container to avoid any bubbles and combs were added to make wells within the gel. The gel was allowed to set for 45-60 minutes.
4.4.5.4.1 Running samples on the gel
Once set the gel was submerged in 1xTBE buffer covering all wells. The 96 well plate was removed from the PCR machine and centrifuged. The Rainin seal was removed from the plate. 3µL of PCR product from the 96 well plate containing the PCR product was transferred into the corresponding wells of the
187 new 96 well plate containing 2µL of 5 x TBE (glycerol loading buffer to keep the sample in the wells). The plate was sealed and briefly centrifuged. 3µL of 100bp marker was pipetted into the marker well (like a control). 5µL of the PCR product and the 5xtBE mixed together was dispense into appropriate corresponding wells. The tank was closed and a 200v charge was applied for approximately 10 minutes until the PCR product had passed an adequate distance through the gel. The gel was imaged in gel trans illuminator using UV light, a hard copy and electronic copy were saved.
4.4.5.4.2 Analysis of the gel
The image was analysed to ensure adequate DNA amplification and confirmation the water well was clear and thus no contamination had occurred (Figure 31). If primer dimer was observed the sample continued with standard processing.
Figure 31; Plate WS74764; Example of electrophoresis gel (11 samples and 1 coriell cell line control)
4.4.5.5 Agencourt AMPure XP Purification protocol
188 The Agencourt AMPure XP protocol was used to purify the PCR product in an automated process using a BiomekNX Laboratory Automation Workstation (Beckman Coulter). The system used magnetic bead technology and a specifically optimized buffer to selectively bind to the PCR amplicons to paramagnetic beads whilst reaction contaminants such as excess primers, primer dimers, dNTPs, slats and enzymes are removed by a simple washing procedure using 70% ethanol. The purified DNA was eluted from the beads using water as an elution buffer and the purified product transferred to an output plate.
4.4.5.6 Sequencing PCR products
Separate forward and reverse sequencing reactions were set up for each sample in a total volume of 10µL. A master mix was made in a 2ml eppendorf tube, to give a total of 9µL per reaction (3µL water, 3.75µL big dye diluent, 0.25µL big dye V3.1 and 2µL Forward or Reverse N13 primer). Forward primer master mix was made in one eppendorf and reverse in another. A multichannel dispenser was used to aliquot 9µL of the master mix to each well in 96 well plate. To this, 1µL of the amplified, purified DNA was added to each well using the NX robot. Plates were sealed and again centrifuged to spin down the contents. DNA fragments were further amplified by polymerase chain reaction (PCR) using the verity 96-well thermal cycler (Applied Biosystems). Thermal cycling conditions are show in Table 32.
Table 32; Thermal cycling conditions for sequencing PCR.
Temp%(°C) Duration Stage 1 Initial Denaturation 96°C 2%minutes 1%cycle Denaturation 96°C 10%seconds Stage 2 Annealing 55°C 20%seconds 30%cycles Extension 60°C 4%minutes Hold 15°C
4.4.5.7 Agencourt CleanSEQ 384 Protocol
The Agencourt CleanSEQ 384 protocol was used to ‘clean up’ the sequencing reaction form unincorporated dNTP’s and other contaminants.
189 4.4.6 Genotyping
Genotyping for mutation status was performed using trace subtraction software (STaden package; www.sourceforge.net). Tumour sequence data were compared against sequence data from a normal control sample (coriell cell line). Mutations and variants were named according to Human Genome Variation Society nomenclature using the B2M reference sequence NM_004048.2 (www.hgvs.org/mutnomen). All mutations and potentially deleterious variants were confirmed through sequencing of a second independent PCR amplification. Normal tissue from the same patient was tested to confirm abnormalities as true mutations or deleterious variants as necessary Frameshift and nonsense mutations were considered to be significant if within the coding regions of B2M exons or mutations affecting the splice site (within 2 base pairs of the flanking intronic sequence). Missense mutations were considered to be variants of uncertain significance and were analysed separately. Alamut (interactive biosoftware) was used to predict the likely effect upon protein expression and function. Almost all B2M mutations occur within exon 1 or exon 2 in the portion covered by the 2a amplicon174.
4.4.7 Immunohistochemistry
The automated Ventana BenchMark ULTRA IHC ⁄ ISH Staining Module (Ventana Co., Tucson, AZ, USA) was used together with the Ultraview 3, 3’ diaminobenzidine (DAB) version 3 detection system (Ventana Co.). It is this DAB enzyme (clinical standard use) which produced the brown colouring on the slides.
Tissue sections (4µm) from tumour and normal tissue blocks were loaded onto slides and baked at 70oC for 30 min. These baked slides were loaded onto the ULTRA platform and deparaffinized in EZPrep Volume Adjust (Ventana Co.). This was accepted as a more environmentally friendly method to prepare the slides rather than using xylene, alcohol and water to hydrate the sample. At intervals between steps the slides were washed with a TRIS-based reaction buffer, pH 7.6. A heat-induced antigen retrieval protocol (36 min) was carried out using a TRIS– ethylenediamine tetracetic acid (EDTA)–boric acid pH 8 buffer (Cell Conditioner 1). The sections were incubated with ultraviolet
190 inhibitor blocking solution for 4 min which removes endogenous peroxidase (found in blood cells and muscle) and electrical charge that could results in non-specific binding, the sections were then incubated with anti-B2M (Anti-B- 2-Microglobulin precursor polyclonal antibody, Sigma, HPA006361 – 100uL Lot b47616) at a dilution of 1:2000 for a set time of 32 min. This was optimized in order to balance out the intensity of the membrane stain versus the background stain. This was followed by incubation with horseradish peroxidase-linked secondary antibody (polymer) (8 min.), then DAB chromogen (8 min.), and copper for 4 min. Counterstain (haematoxylin II) was applied for 12 min before an incubation of 4 min with bluing reagent. Slides were then dehydrated through 99%1DA and Xylene and coverslipped with a xylene based mountant.
4.4.7.1 Development of the IHC Scoring System
The scoring system was developed by using images of varying B2M expressivity from the human protein atlas (www.proteinatlas.org) (
191 Figure 32). In addition to scoring for depth of stain, percentage of field stain was also reported (0%, <25%, 25-75% and >75%).
192 Figure 32; B2M expressivity scoring scale
0 No Stain Negative 1 Mild Staining Mildly Positive 2 Moderate/Strong Staining Positive
Normal Colonic Mucosa
Tumour Score 0 Tumour Score 1 Tumour Score 2
Staining of the stroma and cytoplasm was not reported and only membranous expression of B2M on tumour cells was reported as positive.
Tumours were scored by two independent researchers (LP,LF). Any slides with non-concordant scoring were reviewed and discussed. Any reason for exclusion of samples were recorded.
193
4.4.7.2 Clinical Data
All data were handled according to the data protection act (1998) in a link anonymised fashion using a pathology laboratory number. Data collected included patient demographic data, family history, previous molecular testing, anatomical site of the tumour, morphological characteristics, tumour stage at diagnosis, duration of follow up and locoregional recurrence or distant disease.
4.4.8 Statistical Considerations
I consulted Dr Stephen Roberts PhD (Applied Biostatistician and Senior Lecturer in Biostatistics at the University of Manchester) for advice regarding sample size. He advised that as B2M mutation analysis in Lynch syndrome patients remains exploratory a formal sample size calculation was not appropriate, and analysis should be undertaken on all identified samples
Statistical tests were performed using Statistical Package for the Social Sciences (SPSS) version 20 (SPSS Inc, Chicago, IL, USA). B2M mutation frequency was determined with binominal confidence intervals. Mutation frequency was correlated with UICC stage of cancer. Chi squared test was used to compare the difference in mutation frequency between groups. Mutation analysis was performed in a blinded fashion before correlating with clinical outcome.
4.5 Results
4.5.1 B2M mutation frequency
54 dMMR CRC (MSH loss) CRC samples, obtained from 16 NHS trust across the North West of England, were analysed (24 right sided tumours, 36 tumours beyond the hepatic flexure (including 3 rectal tumours); 4 samples did not amplify, or results could not be confirmed. 50 were verified and included in the final analysis. 14 samples contained a significant B2M mutation (28%). 5 of those samples contained more than one mutation. 7 samples contained mutations of unknown significance. None of these were in conjunction with
194 any significant B2M mutations. Overall 19 significant mutations were detected in 50 samples (Table 33).
Table 33; Description and frequency of B2M mutations identified in the MSH2 dMMR CRC samples
No significant mutations occurred in exon 3. Most mutations detected occurred in exon 2 (amplicon 2a or 2b) (13/19; 68.4%). 8 different mutations were identified. The majority of mutations were frameshift mutations (17/19; 89.5%). The most frequent mutation was c.204delAp.(Val69Trfp5*34) which was detected in 6 samples (and in both amplicon 2a and 2b in 2 samples).
Only 2 missense mutations were identified which cause altered expression of the B2M protein. No nonsense variants coding for stop codon mutations were identified.
4.5.2 Effect of B2M mutation on recurrence
14 (28%) samples had significant B2M mutation and 36 (72%) had B2M wild type. Half of all patients were male. Median age at resection was 38.9 years (range 20.8 – 64.6 years, Std. Dev 11.4 years). Median follow up period was 9.6 years (range 1.8-31.9 years, Std. Dev 7.01 years). 5 patients developed recurrent disease (10%). The majority of tumours tested were either Stage I (36%) or Stage 3 (34%). None of the 14 tumours with significant B2M mutations presented with Stage 4 disease. There was no correlation between
195 UICC stage and B2M mutation status (p=0.444). None of the tumours with significant B2M mutations recurred (either local or distant) compared to 5/36 (13.8%) of tumours with wild type B2M (p=0.142) during the follow up period (11.05 years (range 1.79 – 31.85 years).
4.5.3 Immunohistochemistry results
Six of the tumour samples were excluded from immunohistochemistry testing for B2M expression due to no tumour identifiable on the slide.
Inter-rater reliability was 42% (depth of stain and percentage stain). Of the 48% of samples where there was disagreement between raters, 75% (18/24) of samples were assessed differently for B2M mutation status by both raters as a binary outcome. Sensitivity and Specificity for B2M mutation testing on IHC compared with PCR was 78% and 46% respectively.
4.6 Discussion
In this study B2M mutation status was associated with an absence of recurrent disease (0/14), compared to 5/36 (13.8%) with wild type B2M with an average of 10 years follow up. This did not reach statistical significance (p=0.142) (likely due to a small sample size). This work has added further to previously published literature determining B2M mutation status and its influence on the recurrence of colorectal cancer in patients with MSH2 mutations (Table 34).
B2M mutations are reported to be infrequent in pMMR CRC as the coding satellites within the B2M gene are most susceptible to mutation in the presence of dMMR. B2M mutations occur in around 30% of all dMMR CRC’s but are rare in pMMR CRC (<3%). This study did not compare B2M mutation status in pMMR CRC.
Sensitivity of IHC for B2M mutation status was 78% and specificity 46%. Low sensitivity and poor specificity made it unfeasible to interpret B2M loss as via epigenetic mechanisms. Tumour heterogeneity is a consideration and
196 combining DNA from sections of multiple FFPE tumour blocks may be helpful if diminishing this variability.
After cost effectiveness analysis, NICE published guidelines for the molecular testing for Lynch Syndrome for patients with colorectal cancer in February 201741. This guideline recommended universal testing of all colorectal cancers at the time of diagnosis for MMR status with the purpose of detecting Lynch Syndrome. Recognition of the clinical effect and influence that immunological pathways have on colorectal cancer requires a multidisciplinary multifaceted but standardised approach. Following a Freedom of Information request by Bowel Cancer UK it is recognised that despite NICE guidance, there is still much variation in diagnostic testing for Lynch Syndrome. If all tumours were tested in accordance with NICE guidelines, the numbers of dMMR tumours, and diagnoses of Lynch Syndrome would increase. Identification of dMMR tumours and on available data, B2M mutation status, would have significant prognostic implications and influence decisions about chemotherapeutic agents.
197 Table 34; Summary of studies comparing outcome of dMMR CRC based on B2M mutation status
Recurrent Disease or Distant Metastases
UICC Tumour B2M mutation B2M Proficient Stage / loss
Kloor et al 58% Stage I / 0/23 9/54 (2007) II Case Control 30% Stage III Study 12% Stage IV
Tikidzhieva et al 24% Stage I / 0/10 6/24 (2012) II RCT 76% Stage III
Koelzer et al 53% Stage I / 0/19 14/79 (2012) II Case Control 47% Stage III Study Barrow et al 95% Stage II 0/39 14/77 (2018) 5% Stage III RCT *unpublished
Current Study 37% Stage I 0/14 5/36 Retrospective 20% Stage II Cohort 17% Stage III 4% Stage IV
198
STUDY FOUR
Oligopolyposis within the bowel cancer screening programme; implications for regional genetics centres
199 5.1 Abstract
Background; There is widespread recognition of the benefits of BCSP offering targeted screening to those patients aged 60-74 years. Those with large numbers of adenomas have a high likelihood of carrying an inheritable high- penetrance germline defect. Oligopolyposis is accepted to be a phenotype of 10 – 99 adenomatous colorectal polyps.
The aim of this study was to identify patients who have undergone screening colonoscopy at the Manchester Screening Centre and have been identified to have oligopolyposis. The secondary aim of the study was to identify local guidelines followed by each screening centre defining criteria for referral to local genetics services.
Methods; Patients were identified who underwent colonoscopic testing between December 2009 to February 2014. Patients with more than ten histologically confirmed adenomatous polyps detected at colonoscopic screening were included in the study. All patients aged over 60 years referred to the Manchester Regional Genetics Service during the same time period (December 2009 to February 2014) were identified from the registry. It was ascertained through the registry if any genetic testing had been completed. Freedom of Information (FOI) requests were sent to all NHS bowel cancer screening centres in the England.
Results; 1523 patients underwent colonoscopic screening at the Manchester Screening Centre as part of the National Bowel Cancer Screening Programme between December 2009 and February 2014. Twelve patients (0.79%) were confirmed histologically to have more than ten adenomatous polyps (oligopolyposis) removed at colonoscopy. At colonoscopy, and confirmed by histology, 25% of all patients with oligopolyposis had synchronous colorectal cancers. No detectable gene mutations were identified in the one patient referred to genetic services from the local screening centre. One of the four patients referred from other screening centres was confirmed to have PTEN germline mutation. No NHS trusts provided local trust guidelines for the management of patients with oligopolyposis detected through BCSP.
Conclusion; Numbers of patients with oligopolyposis detected through BCSP are small. However, 50% of patients with oligopolyposis had either
200 synchronous colorectal cancer or high-grade dysplasia. Awareness must be increased amongst screening centres that genetically related colorectal cancers can have an age of onset beyond 50 years. Although, numbers of patient with detectable mutations are small, implications for individual risk of colorectal cancer, other syndrome specific malignancies and familial risks could be considerable.
201 5.2 Introduction
5.2.1 The NHS Bowel Screening Programme
The Joint Advisory Group on Gastrointestinal Endoscopy (JAG) was established under the Academy of Medical Royal Colleges in 1994 with the aim of standardising training in endoscopy across all specialties. The NHS Bowel Cancer Screening Programme (NHS BCSP) in England commenced in July 2006.
Symptoms of colorectal cancer alone are poor predictors of underlying pathology290 and there is widespread recognition of the benefits of bowel cancer screening. Patients eligible for the BCSP are invited biennially to complete a six-window home-based guaiac Faecal Occult Blood test (gFOBt). The BCSP initially targeted screening at those patients aged 60-69 years but the upper age screening limit was extended to 74 years in 2010. Around 2% of patients will have an abnormal result and 4% an unclear result. Those patients with an unclear result will undergo repeat testing. If an abnormal result is returned, patients will be referred for further investigation, usually colonoscopic screening. In the first ten years of the BCSP, 30 million invitations were sent out for screening and 25,528 cancers were detected. Randomised trials have shown that screening for bowel cancer in this way (gFOBTs) can reduce mortality by 16% in people offered screening and by 25% in people who accept screening291,292. Colonoscopic screening allows for both early detection of malignant change and polypoid disease with malignant potential. Colonoscopy also affords the possibility of risk reducing intervention i.e. polypectomy.
In 2013, “bowelscope” screening was introduced offering a “one-off” flexible sigmoidoscopy to people aged 55 years. Flexible sigmoidoscopy affords the opportunity for adenoma detection and removal from the left colon (with self- administered enema bowel preparation at home). Patients who are identified to have left sided adenomas are offered a completion colonoscopy.
Variability in colonosopic skills of endoscopists was recognised prior to the commencement of NHS BCSP, and as such the Screening Assessment and Accreditation System (SAAS) was developed in order to quality assure
202 endoscopists within the BCSP. Diagnostic Colonoscopists may apply for accreditation if they meet the application criteria293 (Table 35).
Table 35; Application criteria for accreditation as BSCP screening colonoscopist through Screening Assessment and Accreditation System (SAAS).
Bowel Cancer Screening Colonoscopist Application Criteria
i. Candidates must be fully registered with the GMC or appropriate professional body and be in good standing ii. Candidates must be attached to a screening centre iii. Applicants must have a minimum lifetime experience of 1000 examinations iv. A minimum of 150 examinations is required in the 12 months prior to the submission of an application. v. Candidates should have a documented unadjusted completion rate on an intention-to-treat basis of 90% or greater over the preceding 12 months. vi. Candidates should have polyp detection rated of 20% or more and meet the current criteria with respect to sedation vii. All candidates must have an appropriately qualified BCSP mentor viii. Candidates must submit four completed DOPyS forms.
5.2.2 Adenoma Surveillance
Most colorectal carcinomas develop from adenomatous precursors and therefore patients who undergo polypectomy at colonoscopy are offered a clinic appointment to discuss results and are offered ongoing adenoma surveillance in line with British Society of Gastroenterology (BSG) guidelines17 (Figure 33; British Society of Gastroenterology Guidelines on
Surveillance after colorectal adenoma removal.). Patients on adenoma surveillance programmes are excluded from subsequent gFOBt screening until adenoma surveillance is complete.
5.2.3 Faecal Immunohistochemical Testing
An important consideration is the psychosocial impact of the relatively binary outcomes of gFOBt colorectal cancer screening. Anxiety is heightened by uncertainty and false positive results. False positive rates are much lower with Faecal Immunochemical Testing (FIT). FIT testing is being introduced as part
203 of the BCSP in England in late 2018. FIT testing is considered to be a superior method of colorectal cancer screening for a number of reasons. Only one stool sample on a single day is required rather than 3 samples on 3 days required for gFOBt. The FIT test is specific for human haemoglobin and undergoes automated analysis to provide a quantifiable result. Faecal immunochemical testing (FIT) can be applied at variable thresholds depending upon patient population, capacity of endoscopic services and desired detection rates. The recommended threshold level is 10 micrograms of haemoglobin per gram of faeces.
Risk stratification within colorectal cancer screening is a relatively new concept but a change to colorectal cancer screening in Scotland has prompted a clinical trial to evaluate the impact of personalised risk information after positive FIT test294. Personalised risk information would hopefully increase uptake of colonoscopy after positive screening tests and aid healthcare services to direct resources to those patients most likely to have colorectal cancer or high risk adenomas, including those with multiple polyps.
Figure 33; British Society of Gastroenterology Guidelines on Surveillance after colorectal adenoma removal.
204
5.2.4 Multiple colonic polyps
Mendelian colorectal cancer syndromes are typified by a predisposition to adenomatous disease e.g. familial adenomatous polyposis (FAP). As such, patients with large numbers of adenomas have a high likelihood of carrying an inheritable high-penetrance germline defect. Patients with FAP typically have hundreds to thousands of colorectal polyps. In most patients affected, a germline mutation or, inherited defect for polypoid disease is identified at a young age.
5.2.5 Oligopolyposis
There is a clinically important subset of patients who are found to have a smaller number of polyps consistent with a diagnosis of oligopolyposis. The definition of oligopolyposis varies somewhat but is generally accepted to be a phenotype of 10-99 adenomatous colorectal polyps. Whilst this is suggestive of an inherited genetic defect, an estimated 30% of patients with polyposis do not have an explained inheritable genetic cause. The 70% of patients with oligopolyposis that do have a detectable germline mutation would potentially benefit from tailored screening and surveillance programs or even prophylactic surgical intervention. It may also be important to detect pathogenic mutations in patients with oligopolyposis presenting with a colorectal cancer as this may influence clinical decision making in terms of surgery and treatment offered in the adjuvant setting. There are obviously also implications for relatives of those identified as having inheritable gene defects.
5.3 Hypothesis and aims
There is very little published literature and no national or international guidelines for the management of patients with oligopolyposis detected through bowel cancer screening programmes. The proportion of oligopolyposis patients detected in this way, the number referred or not referred to genetics services and proportion of these patients identified as having inheritable mutation defects, is unknown.
205
There are sixty four bowel screening centres in England. The Manchester Screening Centre provides BCSP services for around 1.2 million people in the South of Greater Manchester across two screening centres.
The aim of this study was to identify the proportion of patients who have undergone screening colonoscopy at the Manchester Screening centre (Withington Community Hospital or Manchester Royal Infirmary) that have been identified to have oligopolyposis. The secondary aim of the study was to identify local guidelines followed by each screening centre defining criteria for referral to local genetics services.
5.4 Methods
Patients were identified who underwent colonoscopic testing in the Manchester Screening Centre from commencement of the screening programme in December 2009 to February 2014 following positive gFOBt results. Patients with more than ten histologically confirmed adenomatous polyps detected at colonoscopic screening were included in the study. Those patients with less than ten adenomatous polyps were excluded from the study. Colonoscopy reports, histopathology reports and NHS electronic patients’ records were reviewed for each patient screened.
All patients aged over 60 years referred to the Manchester Regional Genetics Service during the same time period (December 2009 to February 2014) were identified from the registry. Database records were reviewed to ascertain if any genetic testing had been undertaken and subsequent outcome.
Lynch Syndrome MSH2 mutation carriers who had undergone colonic resection for colorectal cancer were identified from the Manchester Familial Colorectal Cancer Registry. Mutation carriers were defined as those patients who had tested positive for a pathogenic germline mutation.
Regional clinicians refer patients with a family or personal history suggestive of Lynch Syndrome to the department of Genetic Medicine at Central Manchester Foundation Trust (now Manchester Foundation Trust). Full
206 pedigree information is taken at consultation by a genetic counsellor. Since 1996, if families fulfilled either Amsterdam or Bethesda criteria, living affected relatives were offered testing for germline mutations in the mismatch repair genes. Multiplex Ligation Probe Dependant Amplification (MLPA) was used to screen all exons of MSH1, MSH2, and MSH6 genes. For all other patients, tumour samples were tested for evidence of mismatch repair deficiency. If tumours demonstrated mismatch repair deficiency germline mutation testing was then performed.
A Freedom of Information request was made via NHS digital to obtain a list of all NHS bowel cancer screening centres in England. Further Freedom of Information (FOI) requests were then sent to all NHS bowel cancer screening centres in the England. Requests were made either by letter (attached to email) (Appendix 3) or by electronic submission via trusts websites depending on local process. All screening centres were provided with the same information about the study and response was requested with 20 working days of submission in keeping with standards set out in NHS England Freedom of Information policy295.
5.5 Results
1523 patients underwent colonoscopic screening at the Manchester Screening centre as part of the National Bowel Cancer Screening Programme between December 2009 and February 2014.
5.5.1 Endoscopic findings
Thirty-one patients (2.04%) were identified as having ten or more polyps at colonoscopy. There was some discrepancy between number of polyps reportedly removed at screening colonoscopy and number of polyps reported by histopathology. This is likely due to the piecemeal nature and fragmentation of polyps during retrieval. Nineteen of these thirty-one patients were found to have hyperplastic polyps histologically contributing to their polyp burden and as such were removed from the analysis.
207 5.5.2 Histological findings
Twelve patients (0.79%) were confirmed histologically to have more than ten adenomatous polyps (oligopolyposis) removed at colonoscopy (aged 62 to 71 years). Of those twelve patients, 58.3% presented with symptoms of GI disturbance and 41.6% did not. Two thirds of all patients with oligopolyposis had a weakly positive gFOBt, and one third had an abnormal gFOBt. There was equal identification of these patients across the two hospital sites of the Manchester Screening Centre.
At colonoscopy, and confirmed by histology, 25% of all patients with oligopolyposis had synchronous colorectal cancers. A further 25% had high grade dysplasia within a retrieved polyp, ranging in size from 17-30mm. All these polyps were left sided.
Screening outcomes for all patients with oligopolyposis in the absence of a synchronous colorectal cancer was repeat colonoscopy in 12 months apart from one patient with a 30mm Sigmoid adenoma with high grade dysplasia which underwent repeat colonoscopy at 3 months. At repeat colonoscopy the patient was identified to have a further 15 identified polyps.
5.5.3 Referral to regional genetics service
No patients with oligopolyposis and synchronous colorectal cancer were referred to the regional genetics service. Only one patient with oligopolyposis and no synchronous colorectal cancer (3%) was referred to the regional genetics centre for consideration of mutation testing. Sequencing and MLPA testing revealed no detectable APC, MUTYH, SMAD4 and BMPR1A gene mutation in this patient.
Seventeen patients aged over 60 years were referred to the Manchester Regional Genetics service between December 2009 and February 2014. Four of these seventeen patients (23.5%) were referred due to findings at BCSP colonoscopy at other regional centres. Three of these patients underwent genetic testing for APC / MUTYH without identifiable mutation. One of these patients underwent additional testing for SMAD4, BMPR1A and PTEN. This patient was confirmed to have a PTEN germline mutation.
208
5.5.4 National use of guidelines for referral of patients with oligopolyposis to regional genetic service
Freedom of information requests were submitted to all sixty-four UK screening centres (Appendix 4). Fifty-three responses were received within the 20- working day standard set out by NHS England. Overall, responses were received from fifty-nine (response rate 92.2%). Five screening centres under three NHS trusts did not respond (United Lincolnshire Hospitals NHS Trust, Nottingham University Hospitals NHS Trust and St Georges University Hospital NHS Trust).
All fifty-nine responding screening centres reported that there were no local BCSP guidelines for the referral of patients to regional genetics services who were found at screening colonoscopy to have oligopolyposis.
Nine screening centres (15.3%) reported that they adhered to National Guidelines including BSG National Guidelines for Colorectal Cancer Screening and Surveillance17, BSG position statement on the management of serrated polyps74 and BSCP guidelines for adenoma surveillance296, in the absence of local guidelines.
Four screening centres provided local trust guidelines. Only one of these was specific to BCSP patients and advised referral to genetics for patients with ³ 20 adenomatous polyps. One centre reported that in the absence of BCSP specific guidelines they followed the guidelines of the local paediatric hospital guidelines which is to refer any patient with ³10 adenomatous polyps in the absences of family history of colorectal cancer or ³5 adenomatous polyps in the presence of a family history of colorectal cancer. One centre reported to follow local guidelines for ‘Familial Colorectal Cancer’ which stated to refer if ‘multiple gastrointestinal polyps’ but if not quantify a threshold for referral. The remaining centre provided local guidelines that stated that the identification of ‘multiple polyps’ would be decided on a ‘case by case basis if referral to a geneticist is required’.
209 Five centres indicated that patients with ‘polyposis’ would be discussed in their local colorectal MDT meetings but no numeric threshold for discussion was provided by any centre. One trust responsible for two screening centres indicated that they provided a ‘clear pathway for all patients with polyposis or a genetic cause for cancer’. This pathway included specific family history endoscopy lists providing a one stop service in conjunction with genetics. There was also an established polyposis MDT every 3 months. It was not clear from the information provided the criteria which would need to be met for a BCSP to be referred to this pathway.
Whilst many trusts indicated the absence of local or national guidelines for the referral of patients with oligopolyposis, one screening centre did not recognise that genetically related cancers may occur beyond the age of 50. The centre state that “the Bowel Cancer Screening Programme covers people from the age of 55 and 60-74 years, so by definition, these are not genetically related cancers (cancers that develop aged 50 or below)”.
None of the screening centres indicated that they would directly refer patients from BCSP to regional genetics services and twelve centres stated that this responsibility resided with the patient’s General Practitioner.
5.6 Discussion
In this study less than 1% of all patients who underwent screening as part of the NHS BCSP were identified to have >10 adenomatous colorectal polyps at colonoscopy, but 50% of those did have either synchronous colorectal cancer or high-grade dysplasia. This suggests that although patient numbers in this study were small, oligopolyposis with a threshold of >10 adenomatous polyps is a clinically important phenotype with published data reporting mutation in APC or MUTYH in 9% of these patients28.
None of the screening centres in England were able to demonstrate robust local guidelines for the management of patients identified at screening to have oligopolyposis and there are no current UK guidelines.
210 The American Society of Colon and Rectal Surgeons (ASCRS) published Clinical Practice Guidelines for the Management of Inherited Polyposis Syndrome in 2017297. These guidelines acknowledge that for patients with fewer than 100 adenomas there are diagnostic challenges. There is no consensus as to the number of cumulative lifetime polyps that should prompt referral to genetic services for genetic testing. However, based on findings by Grover et al in 2012 that APC and MUTYH mutations occur in 17% of individuals with 20-99 polyps and 9% of individuals with 10-19 polyps28, ASCRS recommend referral for genetic testing for patients with ³ 20 cumulative lifetime adenomas. In this study, no germline mutations were identified either in the patient referred from the Manchester Screening Centre or those patients (>60 years) referred from the North West Region to the regional genetics centre.
Compared to the high-risk colorectal cancer gene disorders (FAP, HNPCC, MUTYH associated polyposis, Juvenile Polyposis and Peutz-Jeghers Syndrome) relatively little is known about the importance of high polyp burden in patients with no detectable APC or gene mutation. ASCRS recognise that in patients in whom no mutation is found patients should undergo surveillance (including gastroscopy of the upper GI tract) in keeping with classic or attenuated polyposis based on their observed phenotype.
Relatively recently germline mutations in the exonuclease domains of POLE and POLD1 have been shown to be associated with polyposis and colorectal cancer predisposition298-301. Detecting germline variants in patients with oligopolyposis relies on a responsible clinician referring patients with multiple polyps to regional genetics centre. In the context of the current study and those patients identified through the bowel cancer screening programme it appears that the responsibility for this is placed with primary care physicians. However, in the absence of any national, regional or local guidelines for referral it stands to reason that referral of patients will depend on the recommendation of the screening colonoscopist and be ad hoc and unstandardized.
Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups17 are adhered to by NHS BCSP screening centres. The guidelines do not however advise on referral to genetic services for patients found to have multiple polyps at screening colonoscopy. The recommendation in these guidelines is simply for patients with ³ 5 small adenomas to undergo repeat surveillance colonoscopy at 1 year. However, the guidelines do
211 recommend that individuals with an elevated personal risk of gastrointestinal malignancy (see Table 36 for criteria) should be referred to a regional genetics centre for assessment. It appears through qualitative analysis of the responses provide by screening centre in England that ‘pathognomy features of a characterised polyposis syndrome’ does not equate to the lower polyp burden of the spectrum of oligopolyposis.
Table 36; Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups16 ; Guidance on Gastrointestinal Surveillance for High Risk Genetic Disorders
Guidance on Gastrointestinal Surveillance for High Risk Genetic Disorders: Hereditary non-polyposis colorectal cancer, Familial Adenomatous Polyposis, MUTYH-Associated Polyposis, Juvenile Polyposis and Peutz-Jeghers Syndrome
People with a greatly elevated personal risk of gastrointestinal malignancy can be identifies on the basis of one or more of the following criteria:
i. A family history consistent with an autosomal dominant cancer syndrome
ii. Pathognomic features of a polyposis syndrome personally or in close relative
iii. The presence of a germline pathogenic mutation in a colorectal cancer susceptibility gene
iv. Molecular features of a familial syndrome in a colorectal cancer arising in a first degree relative
Relatively recently specific guidelines have been published by the British Society of Gastroenterology (BSG) for the management of serrated polyps74. Serrated polyposis syndrome (previously called Hyperplastic polyposis) is diagnosed if at least one of the following criteria are met; 1) at least five serrated polyps proximal to the sigmoid colon, two of which are greater than 10mm in diameter 2) any number of serrated polyps occurring proximal to the sigmoid colon in an individual who has a first-degree relative with serrated polyposis; and 3) more than 20 serrated polyps of any size distributed throughout the colon. For the majority of cases, SPS has no identifiable genetic mutation302. However, genes that have been associated with an SPS phenotype include; MUTYH (MAP), BMPR1A (juvenile polyposis
212 syndrome)303,304, PTEN (Cowden syndrome)305,306, GREM1 (hereditary mixed polyposis syndrome)307, RNF43 (multiple serrated polyps)308. BSG guidelines recommend that all patients with SPS are referred to clinical genetics services or a polyposis registry, where local resources allow.
Within the adenoma surveillance guidelines for the NHS BCSP296 there is no mention of patients with a polyposis phenotype being referred to local genetics centres.
Existing guidelines for NHS BCSP patients need updating to include genetic testing and potentially more regular endoscopic surveillance after polypectomy if surveillance rather than surgical resection is considered to be the most favourable treatment plan. Adenomatous polyp numbers can increase quickly in these patients.
There is clearly a role to increase awareness amongst screening centres that genetically related colorectal cancers can have a later age of onset beyond 50 years. Although the potential numbers of patient with detectable genetic mutations are small, implications for individual risk of colorectal cancer, other syndrome specific malignancies and familial risks could be considerable.
213 6 Overall Conclusion
This research evaluates some aspects of colorectal cancer presentation and management which may be amenable to establishing a more individualised risk profile, treatment plan and follow up regimen for patients.
Study One explores the role of intense CT surveillance of patients after treatment of sporadic colorectal cancer. There is no correlation between Dukes’ Stage and local or regional recurrence. Despite widespread heterogeneity in follow up regimens used in clinical practice throughout the UK it is generally accepted that a follow up period of 5 years is appropriate for these patients, but forty-five CT scans needed to be undertaken to detect one surgically treatable recurrence. This exposes patients to significant amounts of ionising radiation and associated risks.
The majority of disease recurrence occurs in the first three years after curative resection and only 5% of recurrence is detected in years four and five. Further work is needed to identify if a shorter “standard” follow up regime is more suitable for early disease and if there is any scope for stratification of frequency of cross-sectional imaging during the first two years after resection based on phenotypical, histological and molecular features of the primary tumour.
Many centres have recognised the anxiety generated by colorectal cancer follow up. Reassuring patients that if their disease does not return within the first three years of follow up there is a 95% chance of being disease free at five years may go some way to allay this anxiety.
175,000 people are estimated to have Lynch Syndrome in the UK yet fewer than 5% are known. Lynch Syndrome patients develop colorectal cancer at a young age but age of onset of disease can be wide ranging. In order to reduce the incidence of colorectal cancer in patients with Lynch Syndrome it is important that patients enter into screening programmes at a time appropriate to their level of risk. Whilst risks of current screening modalities are small there is a significant accumulative life time risk of repeated colonoscopy.
In Breast Cancer, SNPs have been shown to predict age of onset in patients with high risk mutations. GWAS have identified increasing numbers of SNPs
214 to be associated with sporadic colorectal cancer. Study Two did not identify any association between these SNPs and age of onset of colorectal cancer in MLH1 and MSH2 mutation carriers. Further work is needed with larger patient numbers to identify if any of the SNPs identified to date have an association with age of onset of colorectal cancer in these patients, or indeed if there is any protective effect.
Patients with Lynch Syndrome exhibit disease mismatch repair and subsequently have high frequency of B2M mutations. The results of Study Three support those recently published that deleterious B2M mutations occur in more than 30% of dMMR tumours and that B2M mutation status seem to afford some protective effect for disease recurrence.
Recently published NICE guidelines (2017) now recommend mismatch repair testing be offered to all patients with colorectal cancer, irrespective of age. In January 2018, a freedom of information request, carried out by Bowel Cancer UK, found that only 17% of hospitals in the UK are testing all bowel cancer patients for features of Lynch Syndrome. More must be done to raise the profile of Lynch Syndrome nationally. NHS trusts must provide the resources needed to support the MMR testing of all colorectal cancers. If the mechanism of protection afforded by B2M mutations is established and increased number of dMMR tumours are identified, B2M mutation status may have clinical utility as a prognostic biomarker.
Lack of recognition of the importance in inheriting germline mutations in older patients has been highlighted in Study Four. There is common misconception that genetic related cancers only affect patients of younger age. Numbers of patients with oligopolyposis identified through the BCSP are small but there is an association between this phenotype and the identification of colorectal cancer or high-grade dysplasia at index colonoscopy. The absence of any national or local guidelines for the management of these patients is concerning and at present this seems to be on an ad hoc basis with considerable variability from institution to institution.
Development of national guidelines and a standardised approach for referral of patients to genetic services with high risk phenotypes (regardless of age) and further research into genetic and environmental factors which determine age of onset of colorectal cancer is now needed to improve outcomes for
215 patients. Mismatch repair testing of all colorectal cancers and stratification of follow up regimens based on individual risk is essential to personalise treatments and ensure appropriate utilisation of NHS resources.
216 References
1. Weitz, J. et al. Colorectal cancer. Lancet 365, 153-65 (2005). 2. Aaltonen, L., Johns, L., Jarvinen, H., Mecklin, J.P. & Houlston, R. Explaining the familial colorectal cancer risk associated with mismatch repair (MMR)-deficient and MMR-stable tumors. Clin Cancer Res 13, 356-61 (2007). 3. Lichtenstein, P. et al. Environmental and heritable factors in the causation of cancer-- analyses of cohorts of twins from Sweden, Denmark, and Finland. N Engl J Med 343, 78-85 (2000). 4. Balmain, A., Gray, J. & Ponder, B. The genetics and genomics of cancer. Nat Genet 33 Suppl, 238-44 (2003). 5. Houlston, R.S. et al. Meta-analysis of genome-wide association data identifies four new susceptibility loci for colorectal cancer. Nat Genet 40, 1426-35 (2008). 6. RJC, S. Colorectal Cancer. in Colorectal Surgery (ed. RKS, P.) 40-66 (2005). 7. Burch, J.A. et al. Diagnostic accuracy of faecal occult blood tests used in screening for colorectal cancer: a systematic review. J Med Screen 14, 132-7 (2007). 8. Greegor, D.H. A progress report. Detection of colorectal cancer using guaiac slides. CA Cancer J Clin 22, 360-3 (1972). 9. Young, G.P. & Cole, S. New stool screening tests for colorectal cancer. Digestion 76, 26-33 (2007). 10. van de Veerdonk, W. et al. Risk stratification for colorectal neoplasia detection in the Flemish colorectal cancer screening programme. Cancer Epidemiol 56, 90-96 (2018). 11. Anderson, J.C. et al. Predictors of proximal neoplasia in patients without distal adenomatous pathology. Am J Gastroenterol 99, 472-7 (2004). 12. Holme, O., Bretthauer, M., Fretheim, A., Odgaard-Jensen, J. & Hoff, G. Flexible sigmoidoscopy versus faecal occult blood testing for colorectal cancer screening in asymptomatic individuals. Cochrane Database Syst Rev 9, CD009259 (2013). 13. Ma, G.K. & Ladabaum, U. Personalizing Colorectal Cancer Screening: A Systematic Review of Models to Predict Risk of Colorectal Neoplasia. Clin Gastroenterol Hepatol (2014). 14. Usher-Smith, J.A. et al. External validation of risk prediction models for incident colorectal cancer using UK Biobank. Br J Cancer 118, 750-759 (2018). 15. Johns, L.E. & Houlston, R.S. A systematic review and meta-analysis of familial colorectal cancer risk. Am J Gastroenterol 96, 2992-3003 (2001). 16. Butterworth, A.S., Higgins, J.P. & Pharoah, P. Relative and absolute risk of colorectal cancer for individuals with a family history: a meta-analysis. Eur J Cancer 42, 216-27 (2006). 17. Cairns, S.R. et al. Guidelines for colorectal cancer screening and surveillance in moderate and high risk groups (update from 2002). Gut 59, 666-89 (2010). 18. Pharoah, P.D., Antoniou, A.C., Easton, D.F. & Ponder, B.A. Polygenes, risk prediction, and targeted prevention of breast cancer. N Engl J Med 358, 2796-803 (2008). 19. Evans, D.G. et al. Birth incidence and prevalence of tumor-prone syndromes: estimates from a UK family genetic register service. Am J Med Genet A 152A, 327-32 (2010). 20. Samadder, N.J., Jasperson, K. & Burt, R.W. Hereditary and Common Familial Colorectal Cancer: Evidence for Colorectal Screening. Dig Dis Sci (2014). 21. Burt, R.W. et al. Genetic testing and phenotype in a large kindred with attenuated familial adenomatous polyposis. Gastroenterology 127, 444-51 (2004).
217 22. Groden, J. et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66, 589-600 (1991). 23. Evans, D.G. et al. Molecular genetic tests in surgical management of familial adenomatous polyposis. Lancet 350, 1777 (1997). 24. Friedl, W. et al. Can APC mutation analysis contribute to therapeutic decisions in familial adenomatous polyposis? Experience from 680 FAP families. Gut 48, 515-21 (2001). 25. Laurent-Puig, P., Beroud, C. & Soussi, T. APC gene: database of germline and somatic mutations in human tumors and cell lines. Nucleic Acids Res 26, 269-70 (1998). 26. Fearnhead, N.S., Britton, M.P. & Bodmer, W.F. The ABC of APC. Hum Mol Genet 10, 721-33 (2001). 27. Davies, D.R. et al. Severe Gardner syndrome in families with mutations restricted to a specific region of the APC gene. Am J Hum Genet 57, 1151-8 (1995). 28. Grover, S. et al. Prevalence and phenotypes of APC and MUTYH mutations in patients with multiple colorectal adenomas. JAMA 308, 485-92 (2012). 29. Groen, E.J. et al. Extra-intestinal manifestations of familial adenomatous polyposis. Ann Surg Oncol 15, 2439-50 (2008). 30. Knudsen, A.L. & Bulow, S. Desmoid tumour in familial adenomatous polyposis. A review of literature. Fam Cancer 1, 111-9 (2001). 31. Barrow, E. et al. Successful radiofrequency ablation of an anterior abdominal wall desmoid in familial adenomatous polyposis. Colorectal Dis 15, e160-3 (2013). 32. Dunlop, M.G., British Society for, G., Association of Coloproctology for Great, B. & Ireland. Guidance on gastrointestinal surveillance for hereditary non-polyposis colorectal cancer, familial adenomatous polypolis, juvenile polyposis, and Peutz-Jeghers syndrome. Gut 51 Suppl 5, V21-7 (2002). 33. A, W. Hereditary wth reference to carcinoma as shown in the study of cases examined in the Pathological Laboratory of the University of Michigan, 1895-1912 . Archives for Internal Medicine 12, 546-555 (1913). 34. Boland, C.R. & Lynch, H.T. The history of Lynch syndrome. Fam Cancer 12, 145-57 (2013). 35. A, W. Further study of a cancer family. J Cancer Res 9, 279-286 (1925). 36. Hauser IJ., W.C. A further report on the cancer family of Warthin. Am J Cancer 27, 434-449 (1936). 37. Lynch, H.T. & Krush, A.J. Cancer family "G" revisited: 1895-1970. Cancer 27, 1505-11 (1971). 38. Wijnen, J.T. et al. Clinical findings with implications for genetic testing in families with clustering of colorectal cancer. N Engl J Med 339, 511-8 (1998). 39. Hendriks, Y.M. et al. Diagnostic approach and management of Lynch syndrome (hereditary nonpolyposis colorectal carcinoma): a guide for clinicians. CA Cancer J Clin 56, 213-25 (2006). 40. Bonadona, V. et al. Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA 305, 2304-10 (2011). 41. Excellence, N.I.f.H.a.C. Molecular testing strategies for Lynch syndrome in people with colorectal cancer (NICE Guideline DG27). ( https://www.nice.org.uk/guidance/dg27 [Accessed 10 February 2019], 2017). 42. Vasen, H.F., Mecklin, J.P., Khan, P.M. & Lynch, H.T. The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC). Dis Colon Rectum 34, 424-5 (1991).
218 43. Vasen, H.F., Watson, P., Mecklin, J.P. & Lynch, H.T. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 116, 1453-6 (1999). 44. Barnetson, R.A. et al. Identification and survival of carriers of mutations in DNA mismatch-repair genes in colon cancer. N Engl J Med 354, 2751-63 (2006). 45. Steinke, V. et al. Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: a comprehensive analysis of 3,671 families. Int J Cancer 135, 69-77 (2014). 46. Balmana, J. et al. Prediction of MLH1 and MSH2 mutations in Lynch syndrome. JAMA 296, 1469-78 (2006). 47. Chen, S. et al. Prediction of germline mutations and cancer risk in the Lynch syndrome. JAMA 296, 1479-87 (2006). 48. Laboratory, M.G. (http://www.myriadtests.com/provider/mutprevhnpcc.htm). 49. Balmana, J. et al. Comparison of predictive models, clinical criteria and molecular tumour screening for the identification of patients with Lynch syndrome in a population- based cohort of colorectal cancer patients. J Med Genet 45, 557-63 (2008). 50. Pouchet, C.J. et al. A comparison of models used to predict MLH1, MSH2 and MSH6 mutation carriers. Ann Oncol 20, 681-8 (2009). 51. Monzon, J.G. et al. Validation of predictive models for germline mutations in DNA mismatch repair genes in colorectal cancer. Int J Cancer 126, 930-9 (2010). 52. Winkels, R.M. et al. Smoking increases the risk for colorectal adenomas in patients with Lynch syndrome. Gastroenterology 142, 241-7 (2012). 53. Win, A.K. et al. Body mass index in early adulthood and colorectal cancer risk for carriers and non-carriers of germline mutations in DNA mismatch repair genes. Br J Cancer 105, 162-9 (2011). 54. Watson, P., Ashwathnarayan, R., Lynch, H.T. & Roy, H.K. Tobacco use and increased colorectal cancer risk in patients with hereditary nonpolyposis colorectal cancer (Lynch syndrome). Arch Intern Med 164, 2429-31 (2004). 55. Pande, M. et al. Smoking and colorectal cancer in Lynch syndrome: results from the Colon Cancer Family Registry and the University of Texas M.D. Anderson Cancer Center. Clin Cancer Res 16, 1331-9 (2010). 56. Diergaarde, B. et al. Environmental factors and colorectal tumor risk in individuals with hereditary nonpolyposis colorectal cancer. Clin Gastroenterol Hepatol 5, 736-42 (2007). 57. Botma, A. et al. Body mass index increases risk of colorectal adenomas in men with Lynch syndrome: the GEOLynch cohort study. J Clin Oncol 28, 4346-53 (2010). 58. Newton, K., Green, K., Lalloo, F., Evans, D. & Hill, J. Colonoscopy screening compliance and outcomes in patients with Lynch Syndrome. Colorectal Dis (2014). 59. Moss, A. et al. Long-term adenoma recurrence following wide-field endoscopic mucosal resection (WF-EMR) for advanced colonic mucosal neoplasia is infrequent: results and risk factors in 1000 cases from the Australian Colonic EMR (ACE) study. Gut 64, 57-65 (2015). 60. Kaltenbach, T. & Soetikno, R. Endoscopic mucosal resection of non-polypoid colorectal neoplasm. Gastrointest Endosc Clin N Am 20, 503-14 (2010). 61. Barrow, E. et al. Cumulative lifetime incidence of extracolonic cancers in Lynch syndrome: a report of 121 families with proven mutations. Clin Genet 75, 141-9 (2009). 62. Aarnio, M. et al. Cancer risk in mutation carriers of DNA-mismatch-repair genes. Int J Cancer 81, 214-8 (1999).
219 63. Grindedal, E.M. et al. Survival in women with MMR mutations and ovarian cancer: a multicentre study in Lynch syndrome kindreds. J Med Genet 47, 99-102 (2010). 64. Dunlop, M.G. et al. Cancer risk associated with germline DNA mismatch repair gene mutations. Hum Mol Genet 6, 105-10 (1997). 65. Kempers, M.J. et al. Risk of colorectal and endometrial cancers in EPCAM deletion- positive Lynch syndrome: a cohort study. Lancet Oncol 12, 49-55 (2011). 66. Ligtenberg, M.J., Kuiper, R.P., Geurts van Kessel, A. & Hoogerbrugge, N. EPCAM deletion carriers constitute a unique subgroup of Lynch syndrome patients. Fam Cancer 12, 169-74 (2013). 67. Lindor, N.M. et al. Recommendations for the care of individuals with an inherited predisposition to Lynch syndrome: a systematic review. JAMA 296, 1507-17 (2006). 68. Barrow, P.J. et al. The spectrum of urological malignancy in Lynch syndrome. Fam Cancer 12, 57-63 (2013). 69. de Jong, A.E. et al. Decrease in mortality in Lynch syndrome families because of surveillance. Gastroenterology 130, 665-71 (2006). 70. M Lockett, M.A.W.S. Hyperplastic Polyposis: prevalence and cancer risk. . Gut 49 (supp 1), A4 (2001). 71. Orlowska, J. Hyperplastic polyposis syndrome and the risk of colorectal cancer. Gut 61, 470-1; author reply 471-2 (2012). 72. Biswas, S. et al. High prevalence of hyperplastic polyposis syndrome (serrated polyposis) in the NHS bowel cancer screening programme. Gut 62, 475 (2013). 73. Hiraoka, S. et al. The presence of large serrated polyps increases risk for colorectal cancer. Gastroenterology 139, 1503-10, 1510 e1-3 (2010). 74. East, J.E. et al. British Society of Gastroenterology position statement on serrated polyps in the colon and rectum. Gut 66, 1181-1196 (2017). 75. Gearhart, S.A., N. Early Diagnosis and Treatment of Cancer; Colorectal, (Saunders, 2011). 76. Knudsen, A.L. et al. Attenuated familial adenomatous polyposis: results from an international collaborative study. Colorectal Dis 12, e243-9 (2010). 77. Jasperson, K.W., Tuohy, T.M., Neklason, D.W. & Burt, R.W. Hereditary and familial colon cancer. Gastroenterology 138, 2044-58 (2010). 78. Lubbe, S.J., Di Bernardo, M.C., Chandler, I.P. & Houlston, R.S. Clinical implications of the colorectal cancer risk associated with MUTYH mutation. J Clin Oncol 27, 3975-80 (2009). 79. Kuiper, R.P. & Hoogerbrugge, N. NTHL1 defines novel cancer syndrome. Oncotarget 6, 34069-70 (2015). 80. Broderick, P. et al. Validation of Recently Proposed Colorectal Cancer Susceptibility Gene Variants in an Analysis of Families and Patients-a Systematic Review. Gastroenterology (2016). 81. Mehenni, H. et al. Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity. Am J Hum Genet 63, 1641-50 (1998). 82. Trojan, J., Raedle, J. & Zeuzem, S. [Mutated serine-threonine kinase gene (STK11) is the cause of Peutz-Jeghers syndrome]. Z Gastroenterol 36, 871-3 (1998). 83. Gammon, A., Jasperson, K., Kohlmann, W. & Burt, R.W. Hamartomatous polyposis syndromes. Best Pract Res Clin Gastroenterol 23, 219-31 (2009). 84. McGarrity, T.J. & Amos, C. Peutz-Jeghers syndrome: clinicopathology and molecular alterations. Cell Mol Life Sci 63, 2135-44 (2006).
220 85. Kersseboom, R. et al. PTEN in colorectal cancer: a report on two Cowden syndrome patients. Clin Genet 81, 555-62 (2012). 86. Atkin, W. et al. Computed tomographic colonography versus colonoscopy for investigation of patients with symptoms suggestive of colorectal cancer (SIGGAR): a multicentre randomised trial. Lancet 381, 1194-202 (2013). 87. Halligan, S. et al. Computed tomographic colonography versus barium enema for diagnosis of colorectal cancer or large polyps in symptomatic patients (SIGGAR): a multicentre randomised trial. Lancet 381, 1185-93 (2013). 88. Loughrey MD., Q.P.a.S.N.e.a.C.S.W.G. Standard and datasets for reporting cancers; Dataset for colorectal cancer and histopathology reports. (Royal College of Pathologists, www.rcpath.org, July 2014). 89. Fearon, E.R. & Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 61, 759-67 (1990). 90. Torlakovic, E. & Snover, D.C. Serrated adenomatous polyposis in humans. Gastroenterology 110, 748-55 (1996). 91. Young, J. et al. Serrated pathway colorectal cancer in the population: genetic consideration. Gut 56, 1453-9 (2007). 92. Jass, J.R. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology 50, 113-30 (2007). 93. Brosens, L.A. et al. Risk of colorectal cancer in juvenile polyposis. Gut 56, 965-7 (2007). 94. Dighe, S. et al. Diagnostic precision of CT in local staging of colon cancers: a meta- analysis. Clin Radiol 65, 708-19 (2010). 95. Kapiteijn, E. et al. Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer. N Engl J Med 345, 638-46 (2001). 96. Excellence, N.I.f.H.a.C. Capecitabine and oxaliplatin in the adjuvant treatment of stage III (Dukes’ C) colon cancer. (https://www.nice.org.uk/guidance/ta100/resources/capecitabine-and- oxaliplatin-in-the-adjuvant-treatment-of-stage-iii-dukes-c-colon-cancer-pdf-373071709 [Accessed 10 February 2019], 2006). 97. Fitzgibbons, R.J., Jr. et al. Recognition and treatment of patients with hereditary nonpolyposis colon cancer (Lynch syndromes I and II). Ann Surg 206, 289-95 (1987). 98. Parry, S. et al. Metachronous colorectal cancer risk for mismatch repair gene mutation carriers: the advantage of more extensive colon surgery. Gut 60, 950-7 (2011). 99. Baucom, R.B. & Wise, P.E. Endoscopic and surgical management of hereditary nonpolyposis colorectal cancer. Clin Colon Rectal Surg 25, 90-6 (2012). 100. Vasen, H.F. et al. Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer). J Med Genet 44, 353-62 (2007). 101. Moller, P. et al. Cancer incidence and survival in Lynch syndrome patients receiving colonoscopic and gynaecological surveillance: first report from the prospective Lynch syndrome database. Gut (2015). 102. Schmeler, K.M. et al. Prophylactic surgery to reduce the risk of gynecologic cancers in the Lynch syndrome. N Engl J Med 354, 261-9 (2006). 103. Crosbie, E.J. et al. The Manchester International Consensus Group recommendations for the management of gynecological cancers in Lynch syndrome. Genet Med (2019). 104. Gryfe, R. et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 342, 69-77 (2000).
221 105. Ribic, C.M. et al. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 349, 247-57 (2003). 106. Elsaleh, H. et al. Association of tumour site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet 355, 1745-50 (2000). 107. Hemminki, A., Mecklin, J.P., Jarvinen, H., Aaltonen, L.A. & Joensuu, H. Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy. Gastroenterology 119, 921-8 (2000). 108. Kim, G.P. et al. Predictive value of microsatellite instability-high remains controversial. J Clin Oncol 25, 4857; author reply 4857-8 (2007). 109. Sargent, D.J. et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 28, 3219-26 (2010). 110. Wang, J.Y., Tang, R. & Chiang, J.M. Value of carcinoembryonic antigen in the management of colorectal cancer. Dis Colon Rectum 37, 272-7 (1994). 111. Young, P.E. et al. Early detection of colorectal cancer recurrence in patients undergoing surgery with curative intent: current status and challenges. J Cancer 5, 262-71 (2014). 112. Newton, K.F., Newman, W. & Hill, J. Review of biomarkers in colorectal cancer. Colorectal Dis 14, 3-17 (2012). 113. Su, B.B., Shi, H. & Wan, J. Role of serum carcinoembryonic antigen in the detection of colorectal cancer before and after surgical resection. World J Gastroenterol 18, 2121-6 (2012). 114. Pfannschmidt, J. et al. Diagnosis of pulmonary metastases with helical CT: the effect of imaging techniques. Thorac Cardiovasc Surg 56, 471-5 (2008). 115. Wiering, B., Ruers, T.J., Krabbe, P.F., Dekker, H.M. & Oyen, W.J. Comparison of multiphase CT, FDG-PET and intra-operative ultrasound in patients with colorectal liver metastases selected for surgery. Ann Surg Oncol 14, 818-26 (2007). 116. Maas, M. et al. What is the most accurate whole-body imaging modality for assessment of local and distant recurrent disease in colorectal cancer? A meta-analysis : imaging for recurrent colorectal cancer. Eur J Nucl Med Mol Imaging 38, 1560-71 (2011). 117. Renehan, A.G., Egger, M., Saunders, M.P. & O'Dwyer, S.T. Impact on survival of intensive follow up after curative resection for colorectal cancer: systematic review and meta-analysis of randomised trials. BMJ 324, 813 (2002). 118. Jeffery, M., Hickey, B.E. & Hider, P.N. Follow-up strategies for patients treated for non- metastatic colorectal cancer. Cochrane Database Syst Rev, CD002200 (2007). 119. Jones, R.P., McWhirter, D., Fretwell, V.L., McAvoy, A. & Hardman, J.G. Clinical follow- up does not improve survival after resection of stage I-III colorectal cancer: A cohort study. Int J Surg 17, 67-71 (2015). 120. Aloia, T.A. et al. Solitary colorectal liver metastasis: resection determines outcome. Arch Surg 141, 460-6; discussion 466-7 (2006). 121. Rees, M., Tekkis, P.P., Welsh, F.K., O'Rourke, T. & John, T.G. Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients. Ann Surg 247, 125-35 (2008). 122. Abdalla, E.K. et al. Improving resectability of hepatic colorectal metastases: expert consensus statement. Ann Surg Oncol 13, 1271-80 (2006). 123. Donadon, M., Ribero, D., Morris-Stiff, G., Abdalla, E.K. & Vauthey, J.N. New paradigm in the management of liver-only metastases from colorectal cancer. Gastrointest Cancer Res 1, 20-7 (2007).
222 124. Bismuth, H. et al. Resection of nonresectable liver metastases from colorectal cancer after neoadjuvant chemotherapy. Ann Surg 224, 509-20; discussion 520-2 (1996). 125. Folprecht, G., Grothey, A., Alberts, S., Raab, H.R. & Kohne, C.H. Neoadjuvant treatment of unresectable colorectal liver metastases: correlation between tumour response and resection rates. Ann Oncol 16, 1311-9 (2005). 126. Nordlinger, B. et al. Combination of surgery and chemotherapy and the role of targeted agents in the treatment of patients with colorectal liver metastases: recommendations from an expert panel. Ann Oncol 20, 985-92 (2009). 127. Stangl, R., Altendorf-Hofmann, A., Charnley, R.M. & Scheele, J. Factors influencing the natural history of colorectal liver metastases. Lancet 343, 1405-10 (1994). 128. Simmonds, P.C. et al. Surgical resection of hepatic metastases from colorectal cancer: a systematic review of published studies. Br J Cancer 94, 982-99 (2006). 129. Kanas, G.P. et al. Survival after liver resection in metastatic colorectal cancer: review and meta-analysis of prognostic factors. Clin Epidemiol 4, 283-301 (2012). 130. Bischof, D.A., Clary, B.M., Maithel, S.K. & Pawlik, T.M. Surgical management of disappearing colorectal liver metastases. Br J Surg 100, 1414-20 (2013). 131. Pritchard, C.C. & Grady, W.M. Colorectal cancer molecular biology moves into clinical practice. Gut 60, 116-29 (2011). 132. Markowitz, S.D. & Bertagnolli, M.M. Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med 361, 2449-60 (2009). 133. Worthley, D.L. & Leggett, B.A. Colorectal cancer: molecular features and clinical opportunities. Clin Biochem Rev 31, 31-8 (2010). 134. Nowak, M.A. et al. The role of chromosomal instability in tumor initiation. Proc Natl Acad Sci U S A 99, 16226-31 (2002). 135. Bouzourene, H., Hutter, P., Losi, L., Martin, P. & Benhattar, J. Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation. Fam Cancer 9, 167-72 (2010). 136. Corso G., P.V., Flauti G., Ferrara F., Marrelli D., Roviello F. Oncogenic mutations and microsatellite instability phenotype predict specific anatomical subsite in colorectal cancer patients. Eur J Hum Genet 21, 1383-1388 (2013). 137. Lee, J.K. & Chan, A.T. Molecular Prognostic and Predictive Markers in Colorectal Cancer: Current Status. Curr Colorectal Cancer Rep 7, 136-144 (2011). 138. Church, D., Midgley, R. & Kerr, D. Biomarkers in early-stage colorectal cancer: ready for prime time? Dig Dis 30 Suppl 2, 27-33 (2012). 139. Grady, W.M. Genomic instability and colon cancer. Cancer Metastasis Rev 23, 11-27 (2004). 140. Cadigan, K.M. & Liu, Y.I. Wnt signaling: complexity at the surface. J Cell Sci 119, 395- 402 (2006). 141. Ceelen, W.P. Progress in rectal cancer treatment. ISRN Gastroenterol 2012, 648183 (2012). 142. Van Limbergen, J., Russell, R.K., Nimmo, E.R. & Satsangi, J. The genetics of inflammatory bowel disease. Am J Gastroenterol 102, 2820-31 (2007). 143. Vineis, P. & Perera, F. Molecular epidemiology and biomarkers in etiologic cancer research: the new in light of the old. Cancer Epidemiol Biomarkers Prev 16, 1954-65 (2007). 144. Algra, A.M. & Rothwell, P.M. Effects of regular aspirin on long-term cancer incidence and metastasis: a systematic comparison of evidence from observational studies versus randomised trials. Lancet Oncol 13, 518-27 (2012).
223 145. Sheth, H. et al. Interaction between polymorphisms in aspirin metabolic pathways, regular aspirin use and colorectal cancer risk: A case-control study in unselected white European populations. PLoS One 13, e0192223 (2018). 146. Brenner, D.E. & Normolle, D.P. Biomarkers for cancer risk, early detection, and prognosis: the validation conundrum. Cancer Epidemiol Biomarkers Prev 16, 1918-20 (2007). 147. Wilson, J.M. & Jungner, Y.G. [Principles and practice of mass screening for disease]. Bol Oficina Sanit Panam 65, 281-393 (1968). 148. Brown, S., Hartley, J., Hill, J., Scott, N., Graham Williams, J. Contemporary Coloproctology, (Springer, 2012). 149. Esteller, M. et al. K-ras and p16 aberrations confer poor prognosis in human colorectal cancer. J Clin Oncol 19, 299-304 (2001). 150. Hutchins, G. et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol 29, 1261- 70 (2011). 151. Heinemann, V., Stintzing, S., Kirchner, T., Boeck, S. & Jung, A. Clinical relevance of EGFR- and KRAS-status in colorectal cancer patients treated with monoclonal antibodies directed against the EGFR. Cancer Treat Rev 35, 262-71 (2009). 152. Siddiqui, A.D. & Piperdi, B. KRAS mutation in colon cancer: a marker of resistance to EGFR-I therapy. Ann Surg Oncol 17, 1168-76 (2010). 153. Richman, S.D. et al. KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol 27, 5931-7 (2009). 154. Baldus, S.E. et al. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res 16, 790-9 (2010). 155. Richman, S.D. et al. Intra-tumoral heterogeneity of KRAS and BRAF mutation status in patients with advanced colorectal cancer (aCRC) and cost-effectiveness of multiple sample testing. Anal Cell Pathol (Amst) 34, 61-6 (2011). 156. Ogino, S. et al. Predictive and prognostic roles of BRAF mutation in stage III colon cancer: results from intergroup trial CALGB 89803. Clin Cancer Res 18, 890-900 (2012). 157. Di Nicolantonio, F. et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 26, 5705-12 (2008). 158. Logan, R.F. et al. Outcomes of the Bowel Cancer Screening Programme (BCSP) in England after the first 1 million tests. Gut 61, 1439-46 (2012). 159. Hutchins, G.G.A. et al. Intratumoral stromal morphometry predicts disease recurrence but not response to 5-fluorouracil-results from the QUASAR trial of colorectal cancer. Histopathology 72, 391-404 (2018). 160. Grillet, F. et al. Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture. Gut (2016). 161. Yamauchi, M. et al. Serial profiling of circulating tumor DNA for optimization of anti- VEGF chemotherapy in metastatic colorectal cancer patients. Int J Cancer 142, 1418- 1426 (2018). 162. Strickler, J.H. et al. Genomic Landscape of Cell-Free DNA in Patients with Colorectal Cancer. Cancer Discov 8, 164-173 (2018).
224 163. Schmiegel, W. et al. Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing. Mol Oncol 11, 208-219 (2017). 164. Bettegowda, C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6, 224ra24 (2014). 165. Merker, J.D. et al. Circulating Tumor DNA Analysis in Patients With Cancer: American Society of Clinical Oncology and College of American Pathologists Joint Review. J Clin Oncol 36, 1631-1641 (2018). 166. Tie, J. et al. Serial circulating tumour DNA analysis during multimodality treatment of locally advanced rectal cancer: a prospective biomarker study. Gut (2018). 167. Boland, C.R. & Goel, A. Microsatellite instability in colorectal cancer. Gastroenterology 138, 2073-2087 e3 (2010). 168. Barrow, E. et al. Colorectal cancer in HNPCC: cumulative lifetime incidence, survival and tumour distribution. A report of 121 families with proven mutations. Clin Genet 74, 233-42 (2008). 169. Umar, A. et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 96, 261-8 (2004). 170. Malesci, A. et al. Reduced likelihood of metastases in patients with microsatellite- unstable colorectal cancer. Clin Cancer Res 13, 3831-9 (2007). 171. Sinicrope, F.A. & Yang, Z.J. Prognostic and predictive impact of DNA mismatch repair in the management of colorectal cancer. Future Oncol 7, 467-74 (2011). 172. Schwitalle, Y. et al. Immune response against frameshift-induced neopeptides in HNPCC patients and healthy HNPCC mutation carriers. Gastroenterology 134, 988-97 (2008). 173. Speetjens, F.M. et al. Prediction of the immunogenic potential of frameshift-mutated antigens in microsatellite instable cancer. Int J Cancer 123, 838-45 (2008). 174. Bernal, M., Ruiz-Cabello, F., Concha, A., Paschen, A. & Garrido, F. Implication of the beta2-microglobulin gene in the generation of tumor escape phenotypes. Cancer Immunol Immunother 61, 1359-71 (2012). 175. Kloor, M. et al. Beta2-microglobulin mutations in microsatellite unstable colorectal tumors. Int J Cancer 121, 454-8 (2007). 176. DuPage, M., Mazumdar, C., Schmidt, L.M., Cheung, A.F. & Jacks, T. Expression of tumour-specific antigens underlies cancer immunoediting. Nature 482, 405-9 (2012). 177. Sengupta, N., MacFie, T.S., MacDonald, T.T., Pennington, D. & Silver, A.R. Cancer immunoediting and "spontaneous" tumor regression. Pathol Res Pract 206, 1-8 (2010). 178. Menon, A.G. et al. Immune system and prognosis in colorectal cancer: a detailed immunohistochemical analysis. Lab Invest 84, 493-501 (2004). 179. Sandel, M.H. et al. Natural killer cells infiltrating colorectal cancer and MHC class I expression. Mol Immunol 42, 541-6 (2005). 180. Yamamoto, H., Yamashita, K. & Perucho, M. Somatic mutation of the beta2- microglobulin gene associates with unfavorable prognosis in gastrointestinal cancer of the microsatellite mutator phenotype. Gastroenterology 120, 1565-7 (2001). 181. Bicknell, D.C., Kaklamanis, L., Hampson, R., Bodmer, W.F. & Karran, P. Selection for beta 2-microglobulin mutation in mismatch repair-defective colorectal carcinomas. Curr Biol 6, 1695-7 (1996).
225 182. Paschen, A. et al. The coincidence of chromosome 15 aberrations and beta2- microglobulin gene mutations is causative for the total loss of human leukocyte antigen class I expression in melanoma. Clin Cancer Res 12, 3297-305 (2006). 183. Kitamura, H. et al. Down-regulation of HLA class I antigens in prostate cancer tissues and up-regulation by histone deacetylase inhibition. J Urol 178, 692-6 (2007). 184. Nosho, K. et al. Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature review. J Pathol 222, 350-66 (2010). 185. Drescher, K.M., Sharma, P. & Lynch, H.T. Current hypotheses on how microsatellite instability leads to enhanced survival of Lynch Syndrome patients. Clin Dev Immunol 2010, 170432 (2010). 186. Cabrera, C.M. et al. Total loss of MHC class I in colorectal tumors can be explained by two molecular pathways: beta2-microglobulin inactivation in MSI-positive tumors and LMP7/TAP2 downregulation in MSI-negative tumors. Tissue Antigens 61, 211-9 (2003). 187. Kloor, M. et al. Immunoselective pressure and human leukocyte antigen class I antigen machinery defects in microsatellite unstable colorectal cancers. Cancer Res 65, 6418- 24 (2005). 188. Dierssen, J.W. et al. HNPCC versus sporadic microsatellite-unstable colon cancers follow different routes toward loss of HLA class I expression. BMC Cancer 7, 33 (2007). 189. Luzzi, K.J. et al. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and limited survival of early micrometastases. Am J Pathol 153, 865-73 (1998). 190. Johnson, A.D. et al. Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues. Pharmacogenet Genomics 18, 781-91 (2008). 191. Khoury, M.J., Janssens, A.C. & Ransohoff, D.F. How can polygenic inheritance be used in population screening for common diseases? Genet Med 15, 437-43 (2013). 192. Tenesa, A. et al. Ten common genetic variants associated with colorectal cancer risk are not associated with survival after diagnosis. Clin Cancer Res 16, 3754-9 (2010). 193. Ingham, S.L. et al. Is multiple SNP testing in BRCA2 and BRCA1 female carriers ready for use in clinical practice? Results from a large Genetic Centre in the UK. Clin Genet 84, 37-42 (2013). 194. Carvajal-Carmona, L.G. et al. Much of the genetic risk of colorectal cancer is likely to be mediated through susceptibility to adenomas. Gastroenterology 144, 53-5 (2013). 195. Hes, F.J. et al. Colorectal cancer risk variants on 11q23 and 15q13 are associated with unexplained adenomatous polyposis. J Med Genet 51, 55-60 (2014). 196. Tomlinson, I.P., Houlston, R.S., Montgomery, G.W., Sieber, O.M. & Dunlop, M.G. Investigation of the effects of DNA repair gene polymorphisms on the risk of colorectal cancer. Mutagenesis 27, 219-23 (2012). 197. Houlston, R.S. & Tomlinson, I.P.M. Polymorphisms and colorectal tumor risk. Gastroenterology 121, 282-301 (2001). 198. Schmit, S.L. et al. Novel Common Genetic Susceptibility Loci for Colorectal Cancer. J Natl Cancer Inst (2018). 199. Huyghe, J.R. et al. Discovery of common and rare genetic risk variants for colorectal cancer. Nat Genet 51, 76-87 (2019). 200. Kupfer, S.S. et al. Genetic heterogeneity in colorectal cancer associations between African and European americans. Gastroenterology 139, 1677-85, 1685 e1-8 (2010).
226 201. Lubbe, S.J., Di Bernardo, M.C., Broderick, P., Chandler, I. & Houlston, R.S. Comprehensive evaluation of the impact of 14 genetic variants on colorectal cancer phenotype and risk. Am J Epidemiol 175, 1-10 (2012). 202. Kantor, E.D. et al. Gene-environment interaction involving recently identified colorectal cancer susceptibility Loci. Cancer Epidemiol Biomarkers Prev 23, 1824-33 (2014). 203. Tomlinson, I. et al. A genome-wide association scan of tag SNPs identifies a susceptibility variant for colorectal cancer at 8q24.21. Nat Genet 39, 984-8 (2007). 204. Zanke, B.W. et al. Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24. Nat Genet 39, 989-94 (2007). 205. Haiman, C.A. et al. A common genetic risk factor for colorectal and prostate cancer. Nat Genet 39, 954-6 (2007). 206. Broderick, P. et al. A genome-wide association study shows that common alleles of SMAD7 influence colorectal cancer risk. Nat Genet 39, 1315-7 (2007). 207. Jaeger, E. et al. Common genetic variants at the CRAC1 (HMPS) locus on chromosome 15q13.3 influence colorectal cancer risk. Nat Genet 40, 26-8 (2008). 208. Tomlinson, I.P., Webb E., Carvajal-Carmona et al. A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3. Nature Genetics 40, 623-630 (2008). 209. Tenesa, A. et al. Genome-wide association scan identifies a colorectal cancer susceptibility locus on 11q23 and replicates risk loci at 8q24 and 18q21. Nat Genet 40, 631-7 (2008). 210. Pittman, A.M. et al. Refinement of the basis and impact of common 11q23.1 variation to the risk of developing colorectal cancer. Hum Mol Genet 17, 3720-7 (2008). 211. Houlston, R.S. et al. Meta-analysis of three genome-wide association studies identifies susceptibility loci for colorectal cancer at 1q41, 3q26.2, 12q13.13 and 20q13.33. Nat Genet 42, 973-7 (2010). 212. Lubbe, S.J., Whiffin, N., Chandler, I., Broderick, P. & Houlston, R.S. Relationship between 16 susceptibility loci and colorectal cancer phenotype in 3146 patients. Carcinogenesis 33, 108-12 (2012). 213. Tuupanen, S. et al. The common colorectal cancer predisposition SNP rs6983267 at chromosome 8q24 confers potential to enhanced Wnt signaling. Nat Genet 41, 885-90 (2009). 214. Carvajal-Carmona, L.G. et al. Fine-mapping of colorectal cancer susceptibility loci at 8q23.3, 16q22.1 and 19q13.11: refinement of association signals and use of in silico analysis to suggest functional variation and unexpected candidate target genes. Hum Mol Genet 20, 2879-88 (2011). 215. Jones, A.M. et al. TERC polymorphisms are associated both with susceptibility to colorectal cancer and with longer telomeres. Gut 61, 248-54 (2012). 216. Kirac, I. et al. SMAD7 variant rs4939827 is associated with colorectal cancer risk in Croatian population. PLoS One 8, e74042 (2013). 217. Picelli, S. et al. Meta-analysis of mismatch repair polymorphisms within the cogent consortium for colorectal cancer susceptibility. PLoS One 8, e72091 (2013). 218. Weigl, K. et al. Strongly enhanced colorectal cancer risk stratification by combining family history and genetic risk score. Clin Epidemiol 10, 143-152 (2018). 219. Wijnen, J.T. et al. Chromosome 8q23.3 and 11q23.1 variants modify colorectal cancer risk in Lynch syndrome. Gastroenterology 136, 131-7 (2009).
227 220. Talseth-Palmer, B.A. et al. Colorectal cancer susceptibility loci on chromosome 8q23.3 and 11q23.1 as modifiers for disease expression in Lynch syndrome. J Med Genet 48, 279-84 (2011). 221. Talseth-Palmer, B.A. et al. Combined analysis of three Lynch syndrome cohorts confirms the modifying effects of 8q23.3 and 11q23.1 in MLH1 mutation carriers. Int J Cancer 132, 1556-64 (2013). 222. Houlle, S. et al. Evaluation of Lynch syndrome modifier genes in 748 MMR mutation carriers. Eur J Hum Genet 19, 887-92 (2011). 223. Win, A.K. et al. Are the common genetic variants associated with colorectal cancer risk for DNA mismatch repair gene mutation carriers? Eur J Cancer 49, 1578-87 (2013). 224. Ten Broeke, S.W. et al. SNP association study in PMS2-associated Lynch syndrome. Fam Cancer 17, 507-515 (2018). 225. Cheng, T.H. et al. Common colorectal cancer risk alleles contribute to the multiple colorectal adenoma phenotype, but do not influence colonic polyposis in FAP. Eur J Hum Genet 23, 260-3 (2015). 226. Burnett-Hartman, A.N. et al. Variation in the association between colorectal cancer susceptibility loci and colorectal polyps by polyp type. Am J Epidemiol 180, 223-32 (2014). 227. Abuli, A. et al. Susceptibility genetic variants associated with colorectal cancer risk correlate with cancer phenotype. Gastroenterology 139, 788-96, 796 e1-6 (2010). 228. Ghazi, S. et al. Colorectal cancer susceptibility loci in a population-based study: Associations with morphological parameters. Am J Pathol 177, 2688-93 (2010). 229. Mates, I.N. et al. Single nucleotide polymorphisms in colorectal cancer: associations with tumor site and TNM stage. J Gastrointestin Liver Dis 21, 45-52 (2012). 230. Cicek, M.S. et al. Functional and clinical significance of variants localized to 8q24 in colon cancer. Cancer Epidemiol Biomarkers Prev 18, 2492-500 (2009). 231. Gruber, S.B. et al. Genetic variation in 8q24 associated with risk of colorectal cancer. Cancer Biol Ther 6, 1143-7 (2007). 232. Xing, J. et al. GWAS-identified colorectal cancer susceptibility locus associates with disease prognosis. Eur J Cancer 47, 1699-707 (2011). 233. Hoskins, J.M. et al. Association of eleven common, low-penetrance colorectal cancer susceptibility genetic variants at six risk loci with clinical outcome. PLoS One 7, e41954 (2012). 234. Giraldez, M.D. et al. Susceptibility genetic variants associated with early-onset colorectal cancer. Carcinogenesis 33, 613-9 (2012). 235. Augestad, K.M. et al. Metastatic spread pattern after curative colorectal cancer surgery. A retrospective, longitudinal analysis. Cancer Epidemiol 39, 734-44 (2015). 236. Mokhles, S. et al. Meta-analysis of colorectal cancer follow-up after potentially curative resection. Br J Surg 103, 1259-68 (2016). 237. Excellence, N.I.f.H.a.C. Colorectal Cancer: diagnosis and management (NICE Guideline CG131). (https://www.nice.org.uk/guidance/cg131 [Accessed 10 February 2019], 2017). 238. Scholefield, J.H., Steele, R.J., British Society For, G., Association of Coloproctology for Great, B. & Ireland. Guidelines for follow up after resection of colorectal cancer. Gut 51 Suppl 5, V3-5 (2002). 239. Kjeldsen, B.J., Kronborg, O., Fenger, C. & Jorgensen, O.D. A prospective randomized study of follow-up after radical surgery for colorectal cancer. Br J Surg 84, 666-9 (1997).
228 240. Makela, J.T., Laitinen, S.O. & Kairaluoma, M.I. Five-year follow-up after radical surgery for colorectal cancer. Results of a prospective randomized trial. Arch Surg 130, 1062-7 (1995). 241. Ohlsson, B., Breland, U., Ekberg, H., Graffner, H. & Tranberg, K.G. Follow-up after curative surgery for colorectal carcinoma. Randomized comparison with no follow-up. Dis Colon Rectum 38, 619-26 (1995). 242. Pietra, N. et al. Role of follow-up in management of local recurrences of colorectal cancer: a prospective, randomized study. Dis Colon Rectum 41, 1127-33 (1998). 243. Rodriguez-Moranta, F. et al. Postoperative surveillance in patients with colorectal cancer who have undergone curative resection: a prospective, multicenter, randomized, controlled trial. J Clin Oncol 24, 386-93 (2006). 244. Schoemaker, D., Black, R., Giles, L. & Toouli, J. Yearly colonoscopy, liver CT, and chest radiography do not influence 5-year survival of colorectal cancer patients. Gastroenterology 114, 7-14 (1998). 245. Secco, G.B. et al. Efficacy and cost of risk-adapted follow-up in patients after colorectal cancer surgery: a prospective, randomized and controlled trial. Eur J Surg Oncol 28, 418-23 (2002). 246. Couch, D.G., Bullen, N., Ward-Booth, S.E. & Adams, C. What interval between colorectal cancer resection and first surveillance colonoscopy? An audit of practice and yield. Colorectal Dis 15, 317-22 (2013). 247. Mathew, J., Saklani, A.K. & Borghol, M. Surveillance colonoscopy in patients with colorectal cancer: how often should we be doing it? Surgeon 4, 3-5, 62 (2006). 248. Kahi, C.J. et al. Colonoscopy surveillance after colorectal cancer resection: recommendations of the US multi-society task force on colorectal cancer. Gastrointest Endosc 83, 489-98 e10 (2016). 249. Tsikitis, V.L. et al. Postoperative surveillance recommendations for early stage colon cancer based on results from the clinical outcomes of surgical therapy trial. J Clin Oncol 27, 3671-6 (2009). 250. Sanli, Y. et al. The utility of FDG-PET/CT as an effective tool for detecting recurrent colorectal cancer regardless of serum CEA levels. Ann Nucl Med 26, 551-8 (2012). 251. Choi, E.K. et al. Value of Surveillance (18)F-FDG PET/CT in Colorectal Cancer: Comparison with Conventional Imaging Studies. Nucl Med Mol Imaging 46, 189-95 (2012). 252. Goshen, E., Davidson, T., Aderka, D. & Zwas, S.T. PET/CT detects abdominal wall and port site metastases of colorectal carcinoma. Br J Radiol 79, 572-7 (2006). 253. Panagiotidis, E. et al. High incidence of peritoneal implants in recurrence of intra- abdominal cancer revealed by 18F-FDG PET/CT in patients with increased tumor markers and negative findings on conventional imaging. Nucl Med Commun 33, 431-8 (2012). 254. Jeffery, M., Hickey, B.E., Hider, P.N. & See, A.M. Follow-up strategies for patients treated for non-metastatic colorectal cancer. Cochrane Database Syst Rev 11, CD002200 (2016). 255. Wattchow, D.A. et al. General practice vs surgical-based follow-up for patients with colon cancer: randomised controlled trial. Br J Cancer 94, 1116-21 (2006). 256. Adams, K., Higgins, L., Beazley, S. & Papagrigoriadis, S. Intensive surveillance following curative treatment of colorectal cancer allows effective treatment of recurrence even if limited to 4 years. Int J Colorectal Dis 30, 1677-84 (2015).
229 257. Primrose, J.N. et al. Effect of 3 to 5 years of scheduled CEA and CT follow-up to detect recurrence of colorectal cancer: the FACS randomized clinical trial. JAMA 311, 263-70 (2014). 258. Bulow, S. & Myrhoj, T. [Hereditary colorectal cancer]. Ugeskr Laeger 158, 2959 (1996). 259. Peltomaki, P. Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Hum Mol Genet 10, 735-40 (2001). 260. Liu, B. et al. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat Med 2, 169-74 (1996). 261. Lynch, H.T. & de la Chapelle, A. Hereditary colorectal cancer. N Engl J Med 348, 919- 32 (2003). 262. Ko, C.W. & Dominitz, J.A. Complications of colonoscopy: magnitude and management. Gastrointest Endosc Clin N Am 20, 659-71 (2010). 263. Whitlock, E.P., Lin, J.S., Liles, E., Beil, T.L. & Fu, R. Screening for colorectal cancer: a targeted, updated systematic review for the U.S. Preventive Services Task Force. Ann Intern Med 149, 638-58 (2008). 264. Rabeneck, L. et al. Bleeding and perforation after outpatient colonoscopy and their risk factors in usual clinical practice. Gastroenterology 135, 1899-1906, 1906 e1 (2008). 265. Sanger, F., Nicklen, S. & Coulson, A.R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463-7 (1977). 266. Evans, D.G. et al. The impact of a panel of 18 SNPs on breast cancer risk in women attending a UK familial screening clinic: a case-control study. J Med Genet (2016). 267. Evans, G.D., Lalloo, F., Mak, T., Speake, D. & Hill, J. Is it time to abandon microsatellite instability as a pre-screen for selecting families for mutation testing for mismatch repair genes? J Clin Oncol 24, 1960-2; author reply 1962-3 (2006). 268. Schafmayer, C. et al. Investigation of the colorectal cancer susceptibility region on chromosome 8q24.21 in a large German case-control sample. Int J Cancer 124, 75-80 (2009). 269. Poynter, J.N. et al. Variants on 9p24 and 8q24 are associated with risk of colorectal cancer: results from the Colon Cancer Family Registry. Cancer Res 67, 11128-32 (2007). 270. Talseth, B.A. et al. Lack of association between genetic polymorphisms in cytokine genes and disease expression in patients with hereditary non-polyposis colorectal cancer. Scand J Gastroenterol 42, 628-32 (2007). 271. Reeves, S.G. et al. DNA repair gene polymorphisms and risk of early onset colorectal cancer in Lynch syndrome. Cancer Epidemiol 36, 183-9 (2012). 272. Michailidou, K. et al. Large-scale genotyping identifies 41 new loci associated with breast cancer risk. Nat Genet 45, 353-61, 361e1-2 (2013). 273. Jover, R., Castells, A., Llor, X. & Andreu, M. Predictive value of microsatellite instability for benefit from adjuvant fluorouracil chemotherapy in colorectal cancer. Gut 55, 1819- 20 (2006). 274. Sargent, D. & Grothey, A. Adjuvant therapy for colon cancer: learning from the past to inform the future. Ann Surg Oncol 17, 947-9 (2010). 275. Tikidzhieva, A. et al. Microsatellite instability and Beta2-Microglobulin mutations as prognostic markers in colon cancer: results of the FOGT-4 trial. Br J Cancer 106, 1239- 45 (2012).
230 276. Barrow, P. et al. Confirmation that somatic mutations of beta-2 microglobulin correlate with a lack of recurrence in a subset of stage II mismatch repair deficient colorectal cancers from the QUASAR trial. Histopathology 75, 236-246 (2019). 277. Le, D.T. et al. A Blueprint to Advance Colorectal Cancer Immunotherapies. Cancer Immunol Res 5, 942-949 (2017). 278. Maat, W. et al. Evidence for natural killer cell-mediated protection from metastasis formation in uveal melanoma patients. Invest Ophthalmol Vis Sci 50, 2888-95 (2009). 279. Tong, A.A. et al. Adoptive natural killer cell therapy is effective in reducing pulmonary metastasis of Ewing sarcoma. Oncoimmunology 6, e1303586 (2017). 280. Watson, N.F. et al. Immunosurveillance is active in colorectal cancer as downregulation but not complete loss of MHC class I expression correlates with a poor prognosis. Int J Cancer 118, 6-10 (2006). 281. Bianchi, C. et al. Reappraisal of serum beta2-microglobulin as marker of GFR. Ren Fail 23, 419-29 (2001). 282. Huang, W.C. et al. beta2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis. Cancer Res 66, 9108-16 (2006). 283. Li, K. et al. Characterization of beta2-microglobulin expression in different types of breast cancer. BMC Cancer 14, 750 (2014). 284. Teasdale, C. et al. Serum beta2-microglobulin in controls and cancer patients. Clin Chim Acta 78, 135-43 (1977). 285. Zhang, Y.X. et al. Correlation of serum beta2-microglobulin levels with prostate-specific antigen, Gleason score, clinical stage, tumor metastasis and therapy efficacy in prostate cancer. Arch Med Res 44, 259-65 (2013). 286. Yang, J. et al. Targeting beta2-microglobulin for induction of tumor apoptosis in human hematological malignancies. Cancer Cell 10, 295-307 (2006). 287. Rossi, D. et al. Beta-2-microglobulin is an independent predictor of progression in asymptomatic multiple myeloma. Cancer 116, 2188-200 (2010). 288. Prizment, A.E. et al. Circulating Beta-2 Microglobulin and Risk of Cancer: The Atherosclerosis Risk in Communities Study (ARIC). Cancer Epidemiol Biomarkers Prev 25, 657-64 (2016). 289. Clendenning, M. et al. Somatic mutations of the coding microsatellites within the beta- 2-microglobulin gene in mismatch repair-deficient colorectal cancers and adenomas. Fam Cancer 17, 91-100 (2018). 290. Jellema, P. et al. Value of symptoms and additional diagnostic tests for colorectal cancer in primary care: systematic review and meta-analysis. BMJ 340, c1269 (2010). 291. Hewitson, P., Glasziou, P., Watson, E., Towler, B. & Irwig, L. Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update. Am J Gastroenterol 103, 1541-9 (2008). 292. Towler, B. et al. A systematic review of the effects of screening for colorectal cancer using the faecal occult blood test, hemoccult. BMJ 317, 559-65 (1998). 293. BSCP Guidelines - Accreditation of screening colonoscopists V1.5. (Royal College of Physicians, Joint Advisory Group on GI Endoscopy and NHS Bowel Cancer Screening Programme). 294. Steele, R.J.C., Digby, J., Chambers, J.A. & O'Carroll, R.E. The impact of personalised risk information compared to a positive/negative result on informed choice and intention to undergo colonoscopy following colorectal Cancer screening in Scotland (PERICCS) - a randomised controlled trial: study protocol. BMC Public Health 19, 411 (2019).
231 295. NHS England. Freedom of Information Policy. Version 1.02. (NHS England). 296. NHS Cancer Screening Programmes, N.B. Adenoma Surveillance. 297. Herzig, D. et al. The American Society of Colon and Rectal Surgeons Clinical Practice Guidelines for the Management of Inherited Polyposis Syndromes. Dis Colon Rectum 60, 881-894 (2017). 298. Valle, L. et al. New insights into POLE and POLD1 germline mutations in familial colorectal cancer and polyposis. Hum Mol Genet 23, 3506-12 (2014). 299. Rosner, G. et al. POLD1 and POLE Gene Mutations in Jewish Cohorts of Early-Onset Colorectal Cancer and of Multiple Colorectal Adenomas. Dis Colon Rectum 61, 1073- 1079 (2018). 300. Esteban-Jurado, C. et al. POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cancer. Oncotarget 8, 26732-26743 (2017). 301. Bellido, F. et al. POLE and POLD1 mutations in 529 kindred with familial colorectal cancer and/or polyposis: review of reported cases and recommendations for genetic testing and surveillance. Genet Med 18, 325-32 (2016). 302. Clendenning, M. et al. Germline Mutations in the Polyposis-Associated Genes BMPR1A, SMAD4, PTEN, MUTYH and GREM1 Are Not Common in Individuals with Serrated Polyposis Syndrome. PLoS One 8, e66705 (2013). 303. Cheah, P.Y. et al. Germline bone morphogenesis protein receptor 1A mutation causes colorectal tumorigenesis in hereditary mixed polyposis syndrome. Am J Gastroenterol 104, 3027-33 (2009). 304. Mongin, C. et al. Unexplained polyposis: a challenge for geneticists, pathologists and gastroenterologists. Clin Genet 81, 38-46 (2012). 305. Heald, B. et al. Frequent gastrointestinal polyps and colorectal adenocarcinomas in a prospective series of PTEN mutation carriers. Gastroenterology 139, 1927-33 (2010). 306. Sweet, K. et al. Molecular classification of patients with unexplained hamartomatous and hyperplastic polyposis. JAMA 294, 2465-73 (2005). 307. Jaeger, E. et al. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Nat Genet 44, 699-703 (2012). 308. Gala, M.K. et al. Germline mutations in oncogene-induced senescence pathways are associated with multiple sessile serrated adenomas. Gastroenterology 146, 520-9 (2014).
232 APPENDIX 1. Manchester Cancer Guidelines for the assessment of mismatch repair (MMR) status in Colorectal Cancer. May 2015, Version 3.
233
APPENDIX 2. Genotype frequencies; Wild type, Heterozygous and Homozygous for control group, Sporadic colorectal cancer group and MLH1 and MSH2 mutation carriers
CONTROL GROUP SPORADIC COLORECTAL CANCER (n=251) MLH1 MUTATION CARRIERS MSH2 MUTATION CARRIERS
SNP Risk Allele WILD TYPE (n) HETEROZYGOTE (n) HOMOZYGOTE (n) WILD TYPE (n) HETEROZYGOTE (n) HOMOZYGOTE (n) WILD TYPE (n) HETEROZYGOTE (n) HOMOZYGOTE (n) WILD TYPE (n) HETEROZYGOTE (n) HOMOZYGOTE (n) rs10411210 C 258 59 6 215 33 3 136 25 1 166 38 3 rs10795668 G 36 151 136 23 95 133 12 83 67 26 88 93 rs10936599 C 25 122 176 13 86 128 10 57 95 13 74 120 rs16892766 C 276 44 3 202 48 1 132 29 1 187 19 1 rs3802842 C 161 124 38 111 115 25 72 67 23 93 91 23 rs4444235 C 86 163 74 70 120 61 45 93 24 75 93 39 rs4779584 T 221 90 12 155 80 16 98 55 9 117 79 11 rs4939827 T 60 164 99 53 104 94 31 81 50 45 113 49 rs6983267 G 60 158 105 49 119 83 48 81 33 50 108 49 rs7014346 A 50 161 112 45 121 85 15 79 68 23 96 88 rs961253 A 137 143 43 96 119 36 63 75 24 79 92 36 rs9929218 G 28 114 181 21 104 126 15 62 85 21 96 90 rs11169552 C 39 118 166 17 96 138 20 64 78 19 85 103 rs4813802i G 185 138 0 86 129 36 72 74 16 82 90 35 rs4925386 C 72 192 59 21 121 109 16 63 83 25 92 90 rs6691170 T 141 144 38 108 110 33 57 85 20 86 101 20
234 APPENDIX 3. Salford Royal NHS Foundation Trust
Stott Lane
Salford
M6 8HD
18th January 2019
Dear Sir/Madam,
This is a request under the Freedom of Information Act.
I request that a copy of the following documents be provided to me: Trust Guidelines for Referral to Genetic Services after colonoscopy undertaken as part of the UK Bowel Cancer Screening Programme (BCSP). In particular I would like you to include any guidelines for referral of patients identified to have ten or more colonic polyps at colonoscopy (oligopolyposis).
In order to help to determine my status to assess fees, you should know that I am a postgraduate student of the University of Manchester Faculty of Medical and Health Science. This is an educational institution, and this request is made for a scholarly or scientific purpose and not for a commercial use.
I request a waiver of all fees for this request. Disclosure of the requested information to me is in the public interest because it is likely to contribute to standardising care for patients at high risk of colorectal cancer and improving patient outcomes for this patient population.
Thank you for your consideration of this request.
Sincerely,
Miss Lyndsay Pearce BMBS BMedSci FRCS
235 APPENDIX 4. BCSP centres sent Freedom of Information requests.
Wolverhampton Bowel Nottinghamshire Bowel Cancer Screening Cancer Screening Centre Centre East and North Merseyside And North Hertfordshire Bowel Cheshire Bowel Cancer Cancer Screening Screening Centre Centre Dorset Bowel Cancer Berkshire Bowel Cancer Screening Centre Screening Centre South of Tyne Bowel Sussex Bowel Cancer Cancer Screening Screening Centre Centre Kettering and St Georges Bowel Northamptonshire Cancer Screening Bowel Cancer Centre Screening Centre Calderdale, Kirklees West London Bowel And Wakefield Bowel Cancer Screening Cancer Screening Centre Centre South East London Cornwall Bowel Cancer Bowel Cancer Screening Centre Screening Centre Herefordshire & South Yorkshire And Worcestershire Bowel Bassetlaw Bowel Cancer Screening Cancer Screening Centre Centre Barking, Havering And Somerset Bowel Redbridge Bowel Cancer Screening Cancer Screening Centre Centre Buckinghamshire And Coventry And Milton Keynes Bowel Warwickshire Bowel Cancer Screening Cancer Screening Centre Centre County Durham And University Hospitals of Darlington Bowel Leicester Bowel Cancer Cancer Screening Screening Centre Centre North Derbyshire Bowel North Staffordshire Cancer Screening Bowel Cancer Centre Screening Centre Liverpool and Wirral Lincolnshire Bowel Bowel Cancer Cancer Screening Screening Centre Centre Western Sussex Bowel West Herts Bowel Cancer Screening Cancer Screening Centre Centre Cambridge Bowel Bedfordshire Bowel Cancer Screening Cancer Screening Centre Centre Cheshire Bowel Cancer Cumbria And Screening Centre Morecambe Bay Bowel
236 Cancer Screening Centre North Essex Bowel Bristol And Weston Cancer Screening Bowel Cancer Centre Screening Centre Norwich Bowel Cancer Solent Bowel Cancer Screening Centre Screening Centre North of Tyne Bowel Pennine Bowel Cancer Cancer Screening Screening Centre Centre Peterborough And Bolton Bowel Cancer Hinchingbrooke Bowel Screening Centre Cancer Screening Centre Bradford And Airedale Gloucestershire Bowel Bowel Cancer Cancer Screening Screening Centre Centre South Essex Bowel West Kent And Medway Cancer Screening Bowel Cancer Centre Screening Centre Lancashire Bowel East Kent Bowel Cancer Screening Cancer Screening Centre Centre Hull and East Yorkshire Harrogate, Leeds And Bowel Cancer York Bowel Cancer Screening Centre Screening Centre Hampshire Bowel Kings Bowel Cancer Cancer Screening Screening Centre Centre Withington Bowel North and East Devon Cancer Screening Bowel Cancer Centre Screening Centre Shropshire Bowel Tees Bowel Cancer Cancer Screening Screening Centre Centre Surrey Bowel Cancer Surrey Bowel Cancer Screening Centre Screening Centre Bath, Swindon And Bath, Swindon And Wiltshire Bowel Cancer Wiltshire Bowel Cancer Screening Centre Screening Centre Sandwell And West Sandwell And West Birmingham Bowel Birmingham Bowel Cancer Screening Cancer Screening Centre Centre University College University College London Bowel Cancer London Bowel Cancer Screening Centre Screening Centre South Derbyshire North East London Bowel Cancer Bowel Cancer Screening Centre Screening Centre
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