Contribution of tumour cell signalling and the microenvironment to the pathogenesis of EBV- associated B cell lymphoma and nasopharyngeal carcinoma BY MAHA IBRAHIM A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY Institute of Cancer and Genomic Sciences College of Medical and Dental Sciences University of Birmingham May 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract In this thesis I have explored different components of the pathogenesis of several related EBV associated cancers. In the first part of the thesis I focus on the microenvironment of two of these cancers, nasopharyngeal carcinoma (NPC) and diffuse large B cell lymphoma (DLBCL). Our group has developed a therapeutic vaccine against EBV which has already been shown to be safe in patients with NPC. Therefore, in the first results chapter (chapter 3), I present a description of the phenotyping of expression of the immune microenvironment including immune checkpoint (ICP) genes and MHC class I and class II genes in NPC tissues. I showed for the first time in NPC tissue samples, two types of PD-L1 expressing tumours: diffuse and marginal. In re-analysis of published data, I found co- expression of immune checkpoint genes and their receptors in EBV positive NPC samples; information which is likely to inform the design of combination immunotherapy in NPC patients. I have shown in my re-analysis of EBV positive NPC that PD-L1 is not up-regulated by LMP-1. In chapter 4, I show the results of studies of the expression of collagen and collagen receptors in DLBCL in which I have identified the over-expression of a potentially novel immune checkpoint receptor, LAIR-1, on the macrophages infiltrating this tumour. In the second part of the thesis I switch my line of inquiry to the tumour cells of EBV-associated cancers, this time focussing on Hodgkin lymphoma (HL), another EBV-associated lymphoma. During the course of the work presented in this chapter I was able to define a role for aberrant sphingosine-1-phosphate (S1P) signalling in driving PI3-K activation mediated through up-regulation of S1PR1 and downregulation of S1PR2 receptors in HL. I also showed that in turn, PI3-K signalling increases the expression of potentially oncogenic downstream transcription factors, such as BATF3 which I have shown to be overexpressed in HL. These data suggest that antagonists of S1P could be considered for the treatment of patients with HL. Acknowledgment I would like to thank the following establishments for generously funding my project while giving me the opportunity to pursue my postgraduate studies in such reputable University here in the UK: - The Egyptian Ministry of Higher Education - Southern Egypt Cancer Institute, Assiut University Also, I would like to recognize and thank all the staff members of the University of Birmingham for facilitating/assisting in my research. The specialized EBV Center is definitely a state of the art research center I would like to sincerely dedicate my special thanks and great gratitude to my principle supervisor Professor Paul Murray for all the sincere effort, his precious generous time he usually gave me and his sincere pieces of advices he has been providing me for the past four years. I would like to thank him for his continuous support, enthusiasm, positivity, patience, and willingness to share with me his great experience and vast knowledge in the field of oncology. He has such an amazing knowledge that is topped with his wonderful so helpful, kind, generous and thoughtful personality. His continuous enthusiasm and devotion have been the main drive for me throughout the entire time of my PHD studies. Professor Paul is not only a great resource for myself, members in PGM group but also has been a great resource for all groups in the Cancer Science Centre. Without him, this work would never succeeded. Also, I would like to extend my recognition as well as my gratitude to my co- supervisor Dr. Graham Taylor. Dr. Taylor's intellectual, valuable experience, patience was always available to guide me in my study. For certain his meticulousity for details had left a positive impact on my research and academic career. I am very grateful to Dr. Wenbin Wei as well as Dr. Robert Hollows for all the support they have provided me with through the bioinformatics work in my thesis. Also, I am greatly thankful to Dr.Abeer Shaaban. Her experience in pathology is remarkable. Thank you Dr. Abeer for sparing the time and effort to help me in my project. My special gratitude and thanks to all my lovely colleagues in the PGM group. - Tracey Perry and Lauren Lupino, Navta Massand, Grace Mitchel, thank you for teaching me all the necessary lab skills and technical support to master my PHD work here. - Ghada Mohamed, Sandra margielewska, Naheema Gordon, Regina Andrijes, Victoria Stavrou and Vera Novitskaia, thank you all for being there for me through all these years. Thank you for making me happy and enjoy work in a friendly smiley warm and fun environment. I can never forget all the assistance and support I have received from the following research groups: - Dr. Frank Mussai group; Dr. Caramla De Santo for the vast knowledge in the macrophage work that they shared with me. - Professor Ben's group; Dr. Neeraj Lal, Dr Galeb Gassous, Chris Bagnall for the Vectra help. - Professor Hisham Mehanna, Dr. Jill Brookes, Dr. Alex Dowel, Dr William Simmons, Dr Isla Humphreys, Dr. Nicholas Davies and Dr Christopher Dawson for all their enormous help, support and sincere advice. Also, I would like to show my appreciation and thanks to my family. Especially, my parents: Professor AbdelKalek and my mother Madeha El-Rafei who have been a source of inspiration to me throughout my life. I would like to let them know that their continuous encouragement and prayers have sustained me that far. Many thanks for all your sacrifices and love. You were behind every step of my work. If it wasn't for my parents, my brothers (Ahmed and Mahmoud) and my sister Shahnaz, I can never forget your support. Thank you all. Finally, although words can’t adequately express my feelings, I would like to express my deepest gratitude to my husband who showed me a lot of nice feelings and support. He has always been encouraging me to be enthusiastic. Thank you Ahmed El Amir Mohamed. Maha Ibrahim CONTENTS 1. INTRODUCTION 1 1.1 Epstein-Barr virus 3 1.1.1 EBV is a transforming B lymphotropic virus 3 1.1.2 Asymptomatic infection of B cells 6 1.2 Diffuse large B cell lymphoma 6 1.2.1 Incidence of Diffuse large B cell lymphoma 7 1.2.2 Histology of DLBCL 7 1.2.3 DLBCL in the WHO classifications 11 1.2.4 Cell of origin (COO) Classification of DLBCL 13 1.2.5 Immunophenotyping 16 1.2.6 Prognostic factors in DLBCL 17 1.3 Hodgkin lymphoma 18 1.3.1 Histology of HL 18 1.3.2 Origin of HRS cells of cHL 19 1.3.3 Suppression of the B cell phenotype in HRS cells 21 1.3.4 Deregulated cellular signalling in classical HL 22 1.4 Nasopharyngeal carcinoma 25 1.5 Contribution of EBV to B cell Lymphoma (DLBCL and HL) and 30 NPC 1.5.1 EBV-associated B cell lymphomas 30 1.5.2 Contribution of EBV latent genes to the pathogenesis of 30 lymphomas 1.5.3 Suppression of the EBV lytic cycle as a potential pathogenic 34 event in EBV-positive cHL 1.5.4 EBV and DLBCL; additional remarks 37 1.5.5 EBV association and contribution of EBV genes in NPC 38 1.5.5.1 Genetic factors in NPC development 38 1.5.5.2 Viral and cellular factors influencing tumour immunity 40 1.6 Tumour microenvironment 43 1.6.1 Collagen and collagen receptors 48 1.6.1.1 Collagen in haematolymphoid neoplasms 49 1. 6.2 Collagen receptors 50 1.6.2.1 Discoidin domain receptor-1 (DDR-1) 50 1.6.2.2 Leukocyte-associated Ig-like receptor-1 (LAIR-1) 51 1.6.3 PD-L1 52 1.6.4 Tumour microenvironment of NPC 56 1.6.5 MHC class I and class II 58 1.7 Sphingolipids 61 1.7.1 S1P synthesis and degradation 61 1.7.2 The sphingolipid rheostat 63 1.7.3 S1P signalling through its receptors 63 1.7.4 Over-expression of SPHK1 and cancer 66 1.7.5 Induction of aberrant cell signalling by S1P in cancer cells 67 1.7.6 Contribution of SPHK1-S1P signalling to altered cell migration in 68 cancer 1.7.7 Therapeutic targeting of S1P or its receptors 69 1.8 Aims of the Study 71 2. MATERIAL AND METHODS 73 2.1 Tissue samples 74 2.1.1 Nasopharyngeal carcinoma 74 2.1.2 DLBCL samples 74 2.1.2.1 Construction of tissue microarray of DLBCL 74 2.1.3 HL samples 76 2.2 Immunohistochemistry (IHC) 76 2.2.1 Positive and negative controls 76 2.2.2 Sectioning of tissues 76 2.2.3 Haematoxylin and eosin (H&E) protocol 76 2.3 Optimisation of antibodies 77 2.4 IHC 77 2.4.1 Citrate Antigen Retrieval 78 2.4.2 Cytospins preparation for IHC/IF 79 2.5 A20 syngeneic xenograft mouse model of DLBCL 80 2.6 Immunofluorescence (IF) 80 2.6.1 Detection of antigen by singleplex/multiplex immunofluorescence 80 (IF) using Opal staining method 2.6.2 Automated IHC and multiplex staining 89 2.6.3 Digital semi-automated quantitative scoring using Vectra scanner