Targeting the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways to enhance chemoradiotherapy responsiveness in colorectal cancer. Aoife Carr, BSc Department of Medical Oncology A thesis submitted to the School of Postgraduate Studies, Faculty of Medicine and Health Sciences, Royal College of Surgeons in Ireland, in fulfilment of the degree of Doctor of Philosophy May 2020 Supervisors: Prof. Bryan Hennessy, Dr. Sinead Toomey, Dr. Simon Furney, Prof. Elaine Kay, Dr. Brian O’Neill Candidate Thesis Declaration I declare that this thesis, which I submit to RCSI for examination in consideration of the award of a higher degree of Doctor of Philosophy, is my own personal effort. Where any of the content presented is the result of input or data from a related collaborative research programme this is duly acknowledged in the text such that it is possible to ascertain how much of the work is my own. I have not already obtained a degree in RCSI or elsewhere on the basis of this work. Furthermore, I took reasonable care to ensure that the work is original, and, to the best of my knowledge, does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text. Signed (Aoife Carr) . Student number 14130521 . Date 2020 . 2 Table of contents Chapter 1 Introduction .................................................................................................... 33 1.1. Colon and rectum: An overview. ........................................................................... 34 1.2. Histology ............................................................................................................... 34 1.2.1. Mucosa .......................................................................................................... 34 1.2.2. Submucosa .................................................................................................... 36 1.2.3. Muscularis externa / propria ........................................................................... 36 1.2.4. Serosa/adventitia ........................................................................................... 36 1.2.5. Recto-anal junction ........................................................................................ 36 1.3. Colorectal cancer .................................................................................................. 37 1.3.1. Colorectal polyps ........................................................................................... 37 1.3.2. Polyp formation .............................................................................................. 38 1.3.3. Symptoms of colorectal cancer ...................................................................... 38 1.3.4. Testing for colorectal cancer .......................................................................... 38 1.3.5. Staging and differentiation of colorectal cancer .............................................. 39 1.3.6. Treatment ....................................................................................................... 40 Chemotherapy ................................................................................................ 41 Radiation therapy............................................................................................ 41 Surgery: Total mesorectal excision (TME) ...................................................... 43 1.3.7. How radiation affects cellular survival ............................................................ 43 1.3.8. Radiation resistance ...................................................................................... 43 1.3.9. Radiation resistance and the hallmarks of cancer .......................................... 43 1.3.10. The role of the immune system in cancer ..................................................... 47 1.3.11. Recurrences ................................................................................................ 47 1.3.12. Survival ........................................................................................................ 48 1.4. Circulating tumour cells and their role in tumour metastasis. ................................. 49 1.5. Human genome .................................................................................................... 51 1.5.1 Structure of DNA ............................................................................................. 51 1.5.2. Transcription and translation of DNA ............................................................. 52 1.6. Mutations in the human genome ........................................................................... 54 3 1.6.1. Point mutations .............................................................................................. 54 1.6.2. Transition and Transversion point mutations .................................................. 55 1.6.3. Frameshift mutations ..................................................................................... 55 1.7. The role of genomic aberrations and the mutator phenotype in the development of CRC. ....................................................................................................................... 57 1.7.1. Chromosomal instability (CIN) ....................................................................... 58 1.7.2. Mismatch repair (MMR) ................................................................................. 58 1.7.3. CpG Island Methylator Phenotype (CIMP) pathway ....................................... 60 1.7.4. Causes of DNA damage ................................................................................ 61 1.8. Mutational signatures ............................................................................................ 65 1.9. The role of the PI3K and MAPK signalling pathways in cancer ............................. 69 1.9.1. PI3K signalling pathway ................................................................................. 69 PIK3CA ........................................................................................................... 70 1.9.2. Mutations in PI3K / MAPK pathways .............................................................. 73 1.9.3. PI3K pathway mutations ................................................................................ 73 1.9.4. The MAPK signalling pathway ........................................................................ 74 1.9.5. MAPK pathway mutations. ............................................................................. 75 1.9.6. Co-occurrence of PI3K/MAPK pathway mutations. ........................................ 77 1.10. Inhibition of the PI3K and MAPK pathways. .......................................................... 78 1.10 .1. Copanlisib / BAY80-6946 / Aliqopa ............................................................. 78 1.10.2. Refametinib / BAY86-9766 / RDEA119 ........................................................ 81 Aims ............................................................................................................................ 82 Chapter 2 Materials and methods ................................................................................... 83 2.1. Materials and methods utilised in aim 1 to determine the full spectrum of somatic genetic aberrations that activates the PI3K and related signalling pathways in LARC and how they are modulated by treatment. .............................................................. 84 2.1.1. Human rectal cancer tumour samples ............................................................ 84 2.1.2. DNA extraction from normal and tumour samples .......................................... 85 2.1.3. Whole exome sequencing .............................................................................. 86 2.1.4. DNA quantification ......................................................................................... 86 2.1.5. Determination of quality of DNA ..................................................................... 87 4 2.1.6. Whole-exome sequencing ............................................................................. 89 2.2.1. TRI-LARC (ICORG 12-38) clinical trial ............................................................... 90 Inclusion criteria ................................................................................................. 90 Exclusion criteria ............................................................................................... 91 Treatment regimens........................................................................................... 91 2.2.2. Blood collection.............................................................................................. 92 2.2.3. Processing of plasma samples ...................................................................... 94 2.2.4. Tumour tissue collection ................................................................................ 94 2.2.5. Whole blood for circulating tumour cell (CTC) analysis .................................. 95 Histological staining of ScreenCell filters ....................................................... 97 Identification of circulating tumour cells ......................................................... 97 Statistical analysis ......................................................................................... 97 2.3. Materials and methods utilised in aim 3 to determine if in-vitro inhibition of the PI3K and related kinase signalling pathways augments