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Title: Investigation of methylation-based markers for relapse prediction in childhood acute lymphoblastic leukemia
Author: Naji, Fadhel Mohammed Lafta Alsaid
ISNI:       0000 0004 6424 7387
Awarding Body: University of Newcastle upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2015
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Acute lymphocytic leukemia (ALL) is the most common childhood cancer representing more than 80% of diagnosed childhood leukemia cases. More than half of these cases come from the high hyperdiploidy (HeH) and RUNX1-ETV6 (t (12; 21)) cytogenetic subgroups. Although HeH and ETV6-RUNX1 -XYX subgroups are considered to have a good prognosis, a significant number of relapses in these subgroups still occur. If molecular markers could be discovered which identify those at high risk of relapse within these groups, these patients could be treated with protocols used for high risk patients, with the hope of producing significant improvements in outcome. Alterations in the patterns of DNA methylation throughout the genome are one o f the hallmarks of cancer development. These alterations are also of potential clinical use as prognostic markers. This study aimed to investigate the utility of methylation- based markers for relapse prediction in childhood ALL. Two approaches have been used to investigate potential methylation markers that would predict outcomes using a panel of 189 (HeH, n=96 cases and RUNX1-ETV6, n=93 cases) diagnostic ALL patients' samples from the UK Medical Research Council ALL 97/99 randomized trial: 1) locus specific methylation analysis (using pyrosequencing) o f three candidate genes (TWIST2, IIOXA4 and TUSC3) previously found to be associated with survival in a small pilot study. 2) genome-wide analysis utilising Illumina450K BeadChip performed on 40 diagnostic samples from 20 patients who subsequently relapsed and 20 patients in continuing remission. This study also aimed to utilise the differentially methylated patterns identified from the above approaches for the identification of functionally relevant genes, mainly by using the lentiviral vector system to deliver/knock-down the expression of aberrantly methylated target genes. The study produced a number of findings: 1) neither the candidate methylation- based markers nor the genome-wide DNA methylation approach identified markers significantly associated with relapse prediction; 2) methylation patterns are associated with specific cytogenetic subtypes, and therefore could influence or drive different clinical behaviour; 3) KEGG pathway analysis of the ALL’S subtype-specific differentially methylated sites identified significant enrichment of genes related to immune function and infection in the ETV6-RUNX1 -ALL cytogenetic subtype. Additional analysis of the methylation patterns to identify potential novel leukaemia associated genes showed: 1) decreased TUSC3 methylation in the vast majority (79.5%) o f ETV6-RUNX1 cases, suggesting a selective pressure to maintain its expression and an oncogenic role in this type of childhood ALL. Incomplete and short duration knock­down of TUSC3 resulted in significant apoptosis induction and proliferation reduction (p=0.001) in the Reh cell line that carries ETV6-RUNX1 translocation. 2) HOXA4 aberrantly methylated Nalm6 cells were selected for when they treated with 15nM and 50nM of cytarabine compare to HOXA4 positive Nalm6 cells that showed significant induction of apoptosis (p=0.003).This result suggest that HOXA4 sensitizes Nalm6 for cyatrabine treatment and the methylation of its gene promoter could be used to monitor treatment responses. Overall the study did not find clear evidence for any methylation-based markers associated with relapse prediction in good prognosis ALL, indicating that patients who subsequently relapsed exhibit highly similar methylation patterns to those who did not relapse. The study confirmed the clear differences in methylation profiles between different ALL genetic subtypes, which may underlie their different clinical and biological behaviour. This study also identified a potential novel method for using methylation patterns for the identification of functionally relevant genes in leukaemia, which may be used to discover novel therapeutics targets in different types of cancers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available