A CLINICOPATHOLOGICAL AND MOLECULAR GENETIC ANALYSIS OF LOW-GRADE GLIOMA IN ADULTS Presented by ANUSHREE SINGH MSc A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy Brain Tumour Research Centre Research Institute in Healthcare Sciences Faculty of Science and Engineering University of Wolverhampton November 2014 i DECLARATION This work or any part thereof has not previously been presented in any form to the University or to any other body whether for the purposes of assessment, publication or for any other purpose (unless otherwise indicated). Save for any express acknowledgments, references and/or bibliographies cited in the work, I confirm that the intellectual content of the work is the result of my own efforts and of no other person. The right of Anushree Singh to be identified as author of this work is asserted in accordance with ss.77 and 78 of the Copyright, Designs and Patents Act 1988. At this date copyright is owned by the author. Signature: Anushree Date: 30th November 2014 ii ABSTRACT The aim of the study was to identify molecular markers that can determine progression of low grade glioma. This was done using various approaches such as IDH1 and IDH2 mutation analysis, MGMT methylation analysis, copy number analysis using array comparative genomic hybridisation and identification of differentially expressed miRNAs using miRNA microarray analysis. IDH1 mutation was present at a frequency of 71% in low grade glioma and was identified as an independent marker for improved OS in a multivariate analysis, which confirms the previous findings in low grade glioma studies. IDH1 mutation was associated with MGMT promoter methylation when partially methylated tumours were grouped with methylated tumours. Grade II and grade III tumour comparison analysis revealed 14 novel significant miRNAs with differential expression. A miRNA signature was shown for histological subtypes, oligoastrocytoma and anaplastic oligoastrocytoma, following the miRNA expression analysis in grade II and grade III tumors based on histology. Oligoastrocytoma presented a more similar profile to oligodendroglioma, but anaplastic oligoastrocytoma was more similar to anaplastic astrocytoma. Five novel miRNAs were identified in grade III tumours, when comparing IDH1 mutant and IDH1 wild type tumours. Analysis of paired samples of primary/recurrent tumours revealed that additional genomic changes may promote tumour progression. For each of the pair, the two samples were genomically different and in each case, the reccurent tumours had more copy number aberrations than the corresponding primary tumours. iii Cell cultures derived from the tumour biopsies were not representative of the low grade glioma in vivo, which was evident from the differences identified in the miRNA expression and copy number changes in the paired samples. IDH1 mutation present in tumour biopsies was not maintained in their respective cell cultures. These findings give an insight into the molecular mechanisms involved in the tumourigenesis of low grade glioma and also tumour progression. iv TABLE OF CONTENTS Abstract ........................................................................................................................... iii Table of Contents ............................................................................................................. v List of Figures ................................................................................................................ xiii List of Tables ................................................................................................................. xvi List of Abbreviations ……………………………………………………………….....xix Acknowledgements ..................................................................................................... xxiii Chapter 1. Introduction .................................................................................................... 1 1.1 Incidence of glioma .................................................................................................. 1 1.2 Glioma histology ...................................................................................................... 3 1.2.1 Astrocytoma ....................................................................................................... 3 1.2.2 Oligoastrocytoma ............................................................................................... 3 1.2.3 Oligodendroglioma ............................................................................................ 4 1.2.4 Glioblastoma multiforme (GBM) ...................................................................... 4 1.3 Clinical presentation and diagnosis of LGG ............................................................ 6 1.4 Prognostic factors for LGG ...................................................................................... 6 1.5 Management and treatment of LGG ........................................................................ 8 1.5.1 Surgery ............................................................................................................... 9 1.5.2 Radiotherapy ...................................................................................................... 9 1.5.3 Chemotherapy .................................................................................................. 10 1.6 Genetic alterations in LGG .................................................................................... 12 1.6.1 Copy number aberrations in LGG .................................................................... 12 1.6.2 TP53 abnormalities in LGG ............................................................................. 13 1.6.3 Altered epression of platelet derived growth factor receptor in low grade glioma ............................................................................................................... 14 1.7 Endogenous role of IDH1 and IDH2 ...................................................................... 15 v 1.8 Frequency and type of IDH mutations in glioma .................................................... 17 1.9 Functional consequences of IDH1 mutation ........................................................... 18 1.9.1 HIF-1 pathway .................................................................................................. 18 1.9.2 2-Hydroxyglutarate ........................................................................................... 19 1.9.3 G-CIMP ............................................................................................................. 20 1.9.4 PI3K-Akt signaling pathway ............................................................................. 21 1.10 Prognostic value of genetic alterations ................................................................. 21 1.10.1 1p/19q .............................................................................................................. 21 1.10.2 MGMT ............................................................................................................. 22 1.11 Clinical correlation of IDH1 mutation .................................................................. 23 1.12 Molecular changes associated with IDH1 mutation .............................................. 24 1.12.1 EMP3 .............................................................................................................. 25 1.13 MicroRNA (miRNA) ............................................................................................ 26 1.13.1 miRNA in glioma ............................................................................................ 27 1.13.2 Role of miRNA in malignant progression ...................................................... 29 1.14 Cell culture model ................................................................................................. 30 1.15 Aims and objectives .............................................................................................. 31 Chapter 2. Materials and Methods ............................................................................. 33 2.1 Tumour samples .................................................................................................... 33 2.2 Tissue culture ........................................................................................................ 34 2.2.1 Primary cultures .............................................................................................. 33 2.2.2 Maintaining cells in culture ............................................................................. 35 2.2.3 Passaging cells ................................................................................................ 35 2.2.4 Reviving cells from storage in liquid nitrogen ................................................ 35 2.2.5 Preparation of cells for storage in liquid nitrogen ........................................... 36 2.3 Calculation of doubling time ................................................................................. 36 2.4 Assessment of tumour cell growth on feeder layers ............................................. 37 vi 2.5 Immunostaining ................................................................................................... 37 2.6 Isolation of DNA .................................................................................................. 38 2.6.1 Frozen tissues ................................................................................................