Predicting and modifying response to neoadjuvant therapy in colorectal cancer This thesis is submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Paul Anthony Sutton September 2015 Dedication To my family. Without your love and support I would not be where I am today. i Declaration The work presented in this thesis was carried out in the Institute of Translational Medicine, University of Liverpool and was undertaken while working as a research fellow at the Liverpool Cancer Research (UK) Centre. The material contained within this thesis has not been, nor is currently being presented wholly, or in part, for any other degree or qualification. I declare that all the work presented in this thesis has been carried out by me except where indicated below: Cation exchange prior to iTRAQ analysis was performed by Mrs Jane Hamlett iTRAQ mass spectrometry was performed by Dr Roz Jenkins Assessment of radiological response was performed by Dr G Abbott Histopathological assessment of tumour response was performed by Dr M Terlizzo and Dr T Andrews Exome sequencing was performed at Genotypic Technology Paul Sutton ii Acknowledgments I am indebted to the many people who have helped me over the last few years. First and foremost my supervisors: Professor Dan Palmer, Dr Chris Goldring, Mr Hassan Malik and Mr Dale Vimalachandran. Thank you for your endless support, enthusiasm and patience as I attempted to make the transition from clinician to scientist. I’d also like to thank my ‘room-mate’, Dr Neil Kitteringham, who provided endless advice and guidance as well as acting as a sounding board and telling me when I was wrong. I feel extremely lucky to have been trained by you. One of the most challenging aspects of this work was the bioinformatics analysis, for which I am grateful for the time and assistance of Dr Puthen V Jithesh. I am also grateful to all the staff in the Medical Research Council Centre for Drug Safety Science who assisted me in the acquisition of laboratory technique, in particular Dr Cliff Rowe and Dr Jo Henry. The work presented in this thesis required the establishment of a regional network of colorectal surgeons to facilitate the recruitment of patients and collection of clinical samples. As such I am grateful to the following collaborators for their help and support: Trust Principal Investigator Royal Liverpool and Broadgreen Mr Paul Rooney Aintree University Hospital Mr Hassan Malik Countess of Chester Hospital Mr Dale Vimalachandran Arrowe Park Hospital Mr Ciaran Walsh Clatterbridge Centre for Oncology Professor Dan Palmer Mid Cheshire Hospital (Leighton) Mrs Caroline Bruce St Helens and Knowsley Hospital Mr Ajai Samad Liverpool Heart and Chest Hospital Mr Michael Shackcloth Southport and Ormskirk Mr Tarek Hany iii East Cheshire Hospital Mr Usman Khan Royal Bolton Hospital Mr David Smith North Wales Health Board Dr Simon Gollins Warrington and Halton Hospital Mr Mark Tighe My fellow surgical trainees have also made the research process much easier, and I wish to thank Mr Rob Jones, Mr Jonathan Evans, Mr Derek McWhirter, Mr David Bowden and Mr Declan Dunne. I am grateful to Professor John Neoptolemos both for his mentorship, and for affording me this opportunity. Finally I wish to thank Cancer Research (UK) who funded me and this work. iv Contents Dedication i Declaration ii Acknowledgements iii Contents v Abstract vii List of Tables viii List of Figures xv Abbreviations xxi Chapter 1 – General Introduction 1 1.1 Epidemiology of colorectal cancer 2 1.2 Risk factors for colorectal cancer 3 1.3 Genetics of colorectal cancer 3 1.4 Staging of colorectal cancer 9 1.5 Treatment of colorectal cancer with surgery 18 1.6 Treatment of colorectal cancer with radiotherapy 24 1.7 Chemotherapeutic agents used in colorectal cancer 29 1.8 Treatment of colorectal cancer with chemotherapy 35 1.9 Asssessment of response to chemoradiotherapy 43 1.10 Predictive and prognostic markers in colorectal cancer 49 1.11 Summary 58 1.12 Aims, hypotheses and study plan 59 Chapter 2 - Evaluation of a tissue stabilisation gel (AllprotectTM) to facilitate clinical sampling for translational research in surgical trials 63 2.1 Introduction 64 2.2 Methods 66 2.3 Results 71 2.4 Discussion 81 Chapter 3 - Exome sequencing of synchronously resected primary colorectal tumours and colorectal liver metastases to inform oncosurgical management 86 3.1 Introduction 87 3.2 Methods 91 3.3 Results 96 3.4 Discussion 117 v Chapter 4 - A global proteomic assessment of patient matched primary and metastatic colorectal tumours 126 4.1 Introduction 127 4.2 Methods 131 4.3 Results 137 4.4 Discussion 168 Chapter 5 - A global proteomic assessment of serial rectal tumour biopsies 177 5.1 Introduction 178 5.2 Methods 180 5.3 Results 184 5.4 Discussion 203 Chapter 6 – Targeted analysis of candidate biomarkers in the primary tumour for predicting response to neoadjuvant chemotherapy in colorectal liver metastases 208 6.1 Introduction 209 6.2 Methods 212 6.3 Results 226 6.4 Discussion 251 Chapter 7 – Concluding Discussion 261 7.1 Summary of aims and experimental design 262 7.2 Summary of results 263 7.3 Advances in the literature 267 7.4 Study limitations and further work 269 7.5 Review of hypotheses 270 7.6 Conclusions 271 Bibliography 273 Appendix 1 - Lists of single nucleotide variants from exome sequencing 299 Appendix 2 - Genetic variants in colorectal cancer identified by next generation sequencing studies 329 Appendix 3 - Proteomic comparison of metastatic tumour and normal adjacent liver parenchyma 337 Appendix 4 - Proteomic comparison of responders and non-responders in the primary tumour 353 Appendix 5 - Supporting Publications, Presentations, Prizes and Grants 361 vi Abstract Background Colorectal cancer is the fourth commonest cancer in the UK, and the second commonest cause of cancer-related death. A knowledge of the biology of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from the metastatic lesions is not feasible until after resection. We aimed to use proteomic and genomic analysis to establish the degree of biological similarity across disease sites and identify biomarkers in the primary tumour which predict response to neoadjuvant chemotherapy in liver metastases. An identical approach was also used to identify predictors of response to neoadjuvant chemoradiotherapy in rectal cancer. Methods Fresh tissue from both primary colorectal tumour and liver metastases from 16 patients was subjected to proteomic analysis using isobaric tagging for relative quantification (iTRAQ). Data were analysed with Protein Pilot (Ab Sciex, Framingham, MA, USA), with stratification of patients into those showing low or high response to chemotherapy permitting the identification of potential predictive biomarkers. These markers were subsequently investigated by immunohistochemistry on a tissue microarray of 56 patients, in parallel with a series of in vitro studies to investigate the concordance between primary and metastatic tumours of those proteins relevant to the activation and metabolism of 5-FU, irinotecan and oxaliplatin. The therapeutic potential of the identified biomarkers was also investigated by dosing SW480 cells with irinotecan/5FU with or without inhibition (using siRNA or a known competitive inhibitor) of the proteins of interest. Four of the 16 patients studied were resected synchronously, and tissue from these was also used for exome sequencing using the Ion Proton platform. Diagnostic, post- treatment and resection biopsies from 8 patients with rectal cancer were again subjected to iTRAQ with stratification into low or high response to neoadjuvant chemoradiotherapy to investigate potential response biomarkers. Findings We identified 5766 discrete proteins, of which 2.54% were differentially expressed between primary and metastatic tumours. There were 170 potential response biomarkers in the primary tumour and 27 in the metastases. Two proteins were common to both tissue types and showed consistent dysregulation, including NQO1. Immunostaining of NQ01 in metastases revealed lower expression in patients responding to chemotherapy (p=0.041), with a significant correlation between primary and metastatic disease sites (r=0.44, p=0.001). Knockdown of NQO1 followed by treatment with irinotecan and 5FU reduced the IC50 from 100.1µM to 49.8µM and from 200.1µM to 25.0µM respectively. Pre-treating cells with dicoumarol prior to incubation in irinotecan and 5FU reduced the IC50 from 100.0µM to 50.0µM and from 183.7µM to 49.9µM respectively. Exome sequencing identified 585 non-synonymous missense SNVs of which 215 (36.8%) were unique to the primary tumour, 226 (38.6%) unique to the metastasis and 81 (13.8%) present in patient matched pairs. Aberrations in the ErbB pathway were identified in paired samples (ratio 0.07, p=5.87x10-7), which were validated by Sanger sequencing along with a potential response biomarker in the CDAN1 gene. Changes to the phenotype of the rectal tumour with chemoradiotherapy were modest, although it is clear that base excision repair (and in particular PARP1) remains of interest, and that acid ceramidase is a potential response biomarker and novel radiosensitiser. Interpretation Proteomic sequencing of matched metastatic colorectal cancer samples is feasible with high coverage. The high degree of similarity between the primary and secondary tumours suggests that the primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in the liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity. vii List of Tables Table Legend Page 1.1 Percentage of cases and 5 year relative survival (%) by Dukes' stage at 2 diagnosis for colorectal cancer patients diagnosed 1996-2002 in England. (Reproduced without permission from Cancer Research (UK)). 1.2 Genetics of inherited colorectal tumour syndromes. (Reproduced without 4 permission from Fearon, 2011).