THE PHARMACOGENOMICS OF VINCRISTINE-INDUCED NEUROTOXICITY IN PAEDIATRIC CANCER PATIENTS WITH WILMS TUMOR OR RHABDOMYOSARCOMA by Tenneille Loo A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2011 © Tenneille Loo, 2011 Abstract Vincristine is one of the most effective and widely utilized antineoplastic agents. However, the clinical utility of this drug is limited by severely debilitating vincristine- induced neurotoxicities (VIN). Previous studies have associated VIN with genetic polymorphisms in genes involved in the metabolism and transportation of vincristine, including CYP3A4, CYP3A5, and ABCB1. However, the findings of such studies have not been consistently reproduced. This study hypothesizes that there are specific variants in genes involved in general drug absorption, metabolism, distribution, excretion, and toxicity (ADME-Tox) that affect the individual susceptibility to VIN in patients with Wilms tumor and rhabdomyosarcoma. Detailed clinical data was collected from 140 patients with Wilms tumor and rhabdomyosarcoma by retrospective chart review. VIN cases were characterized by type of neurotoxicity, and severity was evaluated using a validated clinical grading system for adverse events (NCI-CTCAE v4.03). A customized Illumina GoldenGate Panel was used to genotype 4,536 single nucleotide polymorphisms (SNPs) in candidate genes involved in the metabolism and transportation pathway of vincristine, as well as in genes broadly involved in ADME-Tox. None of the SNPs that were previously reported to be associated with VIN were found to be significantly associated (p-value < 0.05). With similar effect sizes, six novel genetic variants in five genes (PON1, ABCA4, ABCG1, CY51A1, SLCO1C1) were significantly associated with VIN in both tumor types. Whereas none of these genes have been previously associated with VIN or the biotransformation of vincristine, interestingly, ii the biological functions of the encoded proteins have been indirectly linked to nerve function, neuropathy, or neurodegenerative diseases. Therefore, I hypothesize that the genetic basis of VIN is likely polygenic and that the six genes influence individual susceptibility to VIN by affecting: nerve regeneration (PON1, PPP1R9A), and cholesterol homeostasis and remyelination (ABCA4, ABCG1, CYP51A1), as well as the metabolism of vincristine (PON1, CYP51A1) and the transportation of lipids, vincristine, metabolites, or neuroprotectants (SLCO1C1, ABCA4, ABCG1). This study adds to the literature by identifying new potential biomarkers for VIN, providing novel hypotheses for the mechanisms underlying VIN susceptibility, and is a point of origin for replication studies. iii Preface The work contained in this thesis has been approved by the University of British Columbia and the Children’s & Women’s Health Centre of BC Research Ethics Board, the Ethics Certificate Numbers for this study are H10-01568 and H04-70358. iv Table of Contents Abstract ................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of Contents ..................................................................................................................... v List of Tables ............................................................................................................................ x List of Figures ......................................................................................................................... xi List of Symbols and Abbreviations ..................................................................................... xii Acknowledgements ............................................................................................................. xvii Chapter 1: Introduction ......................................................................................................... 1 1.1 Adverse Drug Reactions ............................................................................................... 1 1.1.1 Factors that Contribute to Adverse Drug Reactions ............................................... 2 1.1.2 Adverse Drug Reactions in Children ...................................................................... 2 1.1.3 Pharmacogenomics ................................................................................................. 4 1.2 Vincristine .................................................................................................................... 5 1.2.1 The Antineoplastic Mechanism of Action .............................................................. 6 1.2.2 Metabolism and Transportation .............................................................................. 6 1.2.3 Clinical Use of Vincristine in Children .................................................................. 9 1.3 Vincristine-Induced Neurotoxicity ............................................................................. 10 1.3.1 Signs and Symptoms ............................................................................................ 10 1.3.2 Clinical Diagnosis of Vincristine-Induced Neurotoxicity .................................... 11 1.3.3 Management ......................................................................................................... 13 1.3.4 Future Management: Neuroprotectants ................................................................ 14 v 1.3.5 Pathophysiology ................................................................................................... 15 1.4 Factors Affecting the Exposure to Vincristine ........................................................... 17 1.4.1 Dose Intensity, Frequency, and Cumulative Dose ................................................ 17 1.4.2 CYP3A Inhibitors ................................................................................................. 19 1.4.3 Genetic Variation in CYP3A4 and CYP3A5, and Ancestry ................................ 20 1.4.4 Vincristine Transporters ....................................................................................... 21 1.4.5 Previous Pharmacogenomic Studies ..................................................................... 22 1.5 Patient Cohort Selection: Wilms Tumor and Rhabdomyosarcoma ............................ 24 Chapter 2: Hypothesis, Project Goal and Objectives ........................................................ 28 2.1 Hypothesis .................................................................................................................. 28 2.2 Project Goal and Objectives ....................................................................................... 28 Chapter 3: Methodology ...................................................................................................... 29 3.1 Recruitment, Consent, and Establishing Causality ..................................................... 29 3.2 Defining Cases and Controls ...................................................................................... 31 3.3 Clinical Characterization of Vincristine-induced Neurotoxicity Cases ..................... 33 3.3.1 Classifying Each Vincristine-Induced Neurotoxicity Event by Type of Neuropathy ........................................................................................................... 33 3.3.2 Evaluating the Severity of Each Vincristine-Induced Neurotoxicity Event ......... 33 3.4 Study Inclusion and Exclusion Criteria ...................................................................... 40 3.5 Calculating Dose and Time to First Adverse Drug Reaction ..................................... 41 3.6 Association Studies ..................................................................................................... 42 3.6.1 General Drug Biotransformation Genotyping ...................................................... 42 3.6.2 Candidate Gene Analysis ...................................................................................... 43 vi 3.7 Genotyping: Illumina GoldenGate Assay ................................................................... 43 3.8 Quality Control and Assurance in Genotypic Data .................................................... 44 3.8.1 Quality of Patient Samples and SNPs ................................................................... 44 3.8.2 Data Cleaning ....................................................................................................... 45 3.9 Statistical Analyses: Logistic Regression Models and Manhattan Plot ..................... 46 3.10 Prediction Model ........................................................................................................ 48 3.11 Linkage Disequilibrium Studies ................................................................................. 49 3.12 Imputation Analyses ................................................................................................... 49 Chapter 4: Results ................................................................................................................ 51 4.1 Patient Cohort ............................................................................................................. 51 4.2 Patient Demographics and Clinical Factors ...............................................................