PRIMARY LIVER CANCER: EPIDEMIOLOGICAL AND BIOMARKER DISCOVERY STUDIES Nimzing Gwamzhi Ladep Imperial College London Department of Medicine December 2013 Thesis submitted for Doctor of Philosophy 1 THESIS ABSTRACT With previous reports indicating changes in mortality, risk factors and management of primary liver cancer (PLC), evaluation of current trends in the incidence and mortality rates was indicated. Late diagnosis has been implicated to be a major contributor to the high fatality rates of PLC. This work aimed at: studying trends of PLC by subcategories globally in general, and in England and Wales, in particular; investigating liver-related morbidities of HIV infected patients in an African setting; and discovering urinary biomarkers of hepatocellular carcinoma. The World Health Organisation (WHO) and Small Area Health Statistics Unit (SAHSU) databases were interrogated respectively, in order to achieve the first aim. The second aim was achieved through utilisation of databases of an African-based HIV treatment programme- AIDS Prevention Initiative in Nigeria (APIN), located in Jos, Nigeria. The European Union-funded Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) case-control study in three West African countries was the platform through which urinary metabolic profiling was accomplished. Proton nuclear magnetic resonance spectroscopy (NMR) and parallel ultra-performance liquid chromatography mass spectrometry (UPLC-MS) were used for biomarker discovery studies. Mortality rates of intrahepatic bile duct carcinoma (IHBD) increased in all countries that were studied. Misclassification of hilar cholangiocarcinoma accounted for only a small increase in the rate of IHBD in England and Wales. With over 90% screening rate for viral hepatitides, the rates of hepatitis B (HBV), hepatitis C (HCV) and 2 HBV/HCV in HIV-infected patients in the APIN programme were 17.8%, 11.3% and 2.5% respectively. There was attenuated immune response as well as significantly lower survival observed in HBV/HIV co-infection, relative to HIV mono-infected patients (p=0.0097). Whereas single urinary metabolites, including acetylcarnitine, N- acetylglutamate, betaine aldehyde, 3’-sialyllactose, methionine among others possessed high discriminatory power to diagnose HCC, a combination of three metabolites: 3’-sialyllactose, methionine and 9-decenoylcarnitine significantly outperformed serum alpha-fetoprotein (AFP) in the diagnosis of HCC in a cirrhosis population (area under the receiver operating characteristic curve; [urinary panel= 0.96] compared to [AFP = 0.64]). This work informs a critical assessment of current control strategies in the prevention of HCC, and potentially assists in the development of more affordable means of early detection of PLC for most affected regions of the world. 3 DEDICATION To: Joyce, Jipon, Julbyen and Jembyen 4 DECLARATION OF ORIGINALITY I declare that the work presented in this thesis is my own and all else is appropriately referenced. Nimzing Gwamzhi Ladep London, UK December 2013. 5 COPYRIGHT DECLARATION The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use if for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 6 ACKNOWLEDGEMENT I would not have envisaged that my first trip to England in 2006, sponsored by the Royal College of Physicians International office, under the leadership of Professor Roger Williams would lead to significant research collaborations that ensued. That brilliant idea, aimed at exposing young physicians from developing countries to state- of-the-art conferences and interventional endoscopies led to meeting up with Professor Simon D. Taylor-Robinson. Simon is a knowledgeable man, keen to guide and not afraid to develop minds from the scratch. He was able to provide me all the support any PhD student would require in order to achieve research goals. Together with Dr Andrew Thillainayagam, Simon was able to source for and obtained a grant from The London Clinic that funded my work for the first 3 years of research. This effort will not easily be forgotten, especially during a time when funding for research is difficult to obtain. Both of them helped me to remain focussed and aim high on several occasions. Drs Shahid Khan and Mireille Toledano, two of my other primary supervisors painstakingly mentored my epidemiological skills. Dr Toledano ensured I attended Epidemiological and Biostatistics taught courses for 6 months, link up with other students in her department and gave me access to the primary liver cancer data being maintained by the Small Area Health Statistics Unit. Dr Khan took time out of his normal working hours to advise, read through and correct my epidemiological work. Through the advice of my supervisors, I was able to access primary liver cancer database from the World Health Organisation that formed the global primary liver cancer mortality chapter of this work. 7 Professor Phyllis Kanki of Harvard School of Public Health, Boston (MA, USA), the principal sponsor of AIDS Prevention Initiative in Nigeria deserves no less accolade from me. I had collaborated with Professor Kanki since when I worked in Nigeria. Her willingness and support led to my being able to access the HIV database of the programme that formed some chapters of this work. She has a profound mind and mentored two manuscripts that were published in peer reviewed journals. She is devoted to system developments and successfully established state-of-the-art laboratory in developing countries; one of which holds the samples that were used in the laboratory-based aspect of the work presented in this report. Perhaps, without the grant from the European Union FP-7 bid that ensured the sponsoring of liver cancer work in Gambia, Nigeria and Senegal, my research dream could have ended prematurely. I am grateful to my collaborators from institutions in the UK, France, Nigeria, Gambia and Senegal. Professor Mark Thursz, my Head of Section and principal investigator of PROLIFICA (Prevention of Liver Fibrosis and Carcinoma in Africa) has provided more than anticipated support to me and ensured that I maintained a keen research focus during the process of the current work. Other contributors to PROLIFICA and metabolic profiling include: Professors Tumani Corrah, Elaine Holmes, Edmund Banwat, Edith Okeke, Soulaymane Mboup, Mourtala Ka and Drs Maud Lemoine, Mohamed Shariff, Jeremy Cobbold, Matthew Lewis, Mark McPhail, Anisha Wijeyesekera, Debbie Garside and Ms Mary Crossey. I am a summary of their collective contributions to intellectual development. 8 TABLE OF CONTENTS THESIS ABSTRACT .................................................................................................. 2 DEDICATION ............................................................................................................. 4 DECLARATION OF ORIGINALITY ............................................................................ 5 COPYRIGHT DECLARATION ................................................................................... 6 ACKNOWLEDGEMENT ............................................................................................. 7 TABLE OF CONTENTS ............................................................................................. 9 LIST OF FIGURES ................................................................................................... 20 LIST OF TABLES ..................................................................................................... 25 LIST OF ABBREVIATIONS ...................................................................................... 27 1. INTRODUCTION ............................................................................................... 30 1.1 Definition ......................................................................................................... 30 1.2 Epidemiology .................................................................................................. 30 1.3 Aetiological Risk Factors for Hepatocellular Carcinoma.................................. 33 1.3.1 Cirrhosis.................................................................................................... 34 1.3.2 Non-cirrhotic hepatocellular carcinoma................................................. 34 1.3.3 Hepatitis C Virus ....................................................................................... 35 1.3.4 Hepatitis B Virus ....................................................................................... 36 1.3.5 Dietary aflatoxin ........................................................................................ 37 1.3.6 Alcohol ...................................................................................................... 38 1.3.7 Non-alcoholic liver disease and diabetes mellitus ..................................... 39 1.3.8 Hereditary Haemochromatosis and Iron Overload Syndromes ................. 40 9 1.3.9 Oral contraceptive pills ............................................................................. 40 1.3.10 Dietary factors ......................................................................................... 41 1.4 Risk factors and pathogenesis of intrahepatic cholangiocarcinoma