Characterising the Chronic Lymphocytic Leukaemia methylome; a focus on an epigenetically-determined mitotic clock

Abstract

Epigenetically determined cumulative mitoses (epiCMIT) is a mitotic clock that is constructed from DNA methylation data of normal B-cells to track the proliferative history of neoplastic B-cells. It is strongly associated with clinical behaviour in Chronic Lymphocytic Leukaemia (CLL) patients, where higher scores indicate more aggressive phenotypes within each epigenetic subgroup. However, its utility as a prognostic or predictive biomarker in clinical trials is untested and the mechanisms behind the aggressive nature of high epiCMIT subgroups are unclear. DNA methylation heterogeneity in CLL, in part driven by allele-specific methylation (ASM), may explain differential gene expression between the two major prognostic subsets of CLL, unmutated CLL (U-CLL) and mutated CLL (M-CLL), defined by IGHV gene mutational status.
The aims of this research are to assess epiCMIT as an independent prognostic marker in CLL specifically within a clinical-trial cohort, to evaluate the reliability of a novel sequencing technology, five-base seq, in quantifying DNA methylation, to identify differential methylation patterns in CLL patients with high epiCMIT scores and explore functional pathways potentially regulated by these differentially methylated CpG sites, and to identify IGHV-related genes associated with recurrent ASM in CLL to explore regulatory potential of recurrent ASM in CLL.
The clinical significance of epiCMIT was assessed in 241 treatment-naïve CLL patients randomised to clinical trials of chemoimmunotherapy, by conducting survival analysis (univariate and multivariate). The epiCMIT score was significantly associated with a prolonged progression-free survival (PFS). While epiCMIT did not show significant independent prognostic value for PFS, overall survival (OS) and time-to-first-treatment (TTT) in multivariate analysis, high epiCMIT scores were associated with worse prognosis in both n-CLL and m-CLL patients across all clinical endpoints, except for TTT in m-CLL. Further, genome-wide DNA methylation data was generated for 20 CLL patients using five-base seq. High concordance was demonstrated between five-base seq and gold standard Infinium HumanMethylation450 BeadChip array. Subsequent differential methylation analysis identified differentially methylated CpGs between IGHV subsets and epiCMIT subgroups in U-CLL and M-CLL with minimal overlap. Overrepresentation analysis was performed to explore potential pathway impacts, though further validation is needed to confirm these findings. Finally, using Oxford Nanopore Technologies platform, whole-genome methylation data were generated from three U-CLL and three M-CLL samples. This enabled the identification of recurrent ASM in promoter regions of genes whose expression is differentially expressed between the IGHV subsets, including ZAP-70 in U-CLL cases.
Overall, the thesis focused on investigating the clinical significance of the epiCMIT and reliability of the novel technology in capturing the DNA methylome of CLL. For the first time, differential DNA methylation profiles of high epiCMIT patients were characterised in U-CLL and M-CLL. Finally, ASM analysis showed that DNA methylation heterogeneity exhibited by CLL is characterised by the presence of IGHV-specific ASMs. Given the small sample sizes and limited sequencing read depth used in this study, further research with larger cohorts and deeper sequencing is needed to validate these findings.

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Identifiers

ORCID for Anna Nilsson-Takeuchi: orcid.org/0000-0002-6628-1680

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