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Title: Probing tumour origins and behaviour in b-cell lineage malignancies
Author: Moreno Rueda, Luz Yurany
ISNI:       0000 0004 8501 8786
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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The exquisite and complex pathways of normal B-cell differentiation present a challenge when deciphering the origins and mechanism that give rise to malignant B-cells. In this, advances in global genomic and proteome based analytical platforms are providing important tools to probe this challenge. In this Thesis, we evaluated specific questions with regard to tumour origins and behaviour in two contrasting mature B-cell neoplasia, in classical Hairy cell leukaemia (HCLc) and in multiple myeloma (MM) that differ markedly. HCLc is a disease of mature B-cells whereas MM is a disease of terminally differentiated malignant plasma cells. In HCLc, we explored three separate questions. In the first of these, we examined for the first time the spectrum of genome-wide acquired somatic lesions using whole genome sequencing (WGS). We probed the noncoding genome in HCLc in particular, and identified recurrent single nucleotide variants (SNVs) in common regulatory elements of 13 cancer-related genes. Interestingly, in 6 of these genes (AKAP9, BCL6, CD74, CIITA, MEF2C and MYC), the pattern of mutations was consistent with somatic hypermutation (SHM) activity catalysed by activation induced cytidine deaminase (AID). Both SHM and AID activity are localised during normal B-cell development to the germinal centre (GC). However, although mutations in these 6 genes implicated a role in tumourigenesis, they were not universal in HCLc disease, suggesting that multiple differing pathways operate that coalesce mutant drive with the signature BRAF p.V600E mutation to promote malignancy. Of coding mutations, nonsynonymous SNVs were identified often in Kruppel-like factor 2 (KLF2), whereas other gene mutations occurred sporadically. Somatic indels and genomic copy number variation were also derived by WGS. In the second HCLc question, we examined the molecular basis of its virtually unique phenotype where multiple surface immunoglobulin (mult-sIg) isotypes are expressed, in marked contrast to single sIg isotype generated by deletional class switch recombination (CSR) events in the GC in normal B-cells, also catalysed by AID. We interrogated the IGH locus in chromosome 14q32 ii by mining WGS data and by targeted sequencing. We observed that while some HCLc cases retain a germline IGH locus, in others there is evidence of slippage of CSR-like events and a role for AID that delete some isotype coding regions. However, in all cases the retention of at least one germline allele of IGH coding genes suggests that the mult-sIg isotypes are generated by differential transcriptional splicing mechanisms. Thirdly, we evaluated the impact of the signature BRAF p.V600E mutation on the miRNA epigenome in HCLc tumour cells, to model how a critical gene driver mutation dysregulates the epigenome in cancer. Using Vemurafenib to ablate mutant BRAF activity in HCLc cells, we utilised a small RNA sequencing approach to map global effects on the miRNome. From this, 15 miRNA species were found to be differentially regulated by BRAF p.V600E, and in silico analysis of potential targets indicated control of key oncogenic processes. These data now open future study of important miRNA species, notably mir-21, in HCLc pathogenesis and progression. In the final part of the Thesis, we address the question of when lethal MM arises during asymptomatic monoclonal gammopathy of undetermined significance (MGUS) that invariably precedes malignancy. Current therapy in MM is instituted when disease is diagnosed at a late stage with onset of organ failure, and early biomarkers that can guide therapy in the absence of morbidity associated with organ failure will have a profound effect on disease management. To pursue this, we utilised an ultra-precise proteomics TMT-3DLC-MS/MS approach to compare plasma proteins from healthy controls vs MGUS vs MM. We identified a panel of 28 plasma proteins that associated only with MM and not with MGUS or controls, presenting an opportunity for future validation work to cement which marker(s) are definitive in identifying onset of lethal MM, to then guide immediate therapeutic intervention.
Supervisor: Sahota, Surinder ; Weston-Bell, Nicola Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available