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Development of a clinically applicable high throughput sequencing of immunoglobulin gene rearrangements in childhood precursor B-cell acute lymphoblastic leukaemia

Bartram, Jack; (2025) Development of a clinically applicable high throughput sequencing of immunoglobulin gene rearrangements in childhood precursor B-cell acute lymphoblastic leukaemia. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The overall survival of the latest childhood acute lymphoblastic leukaemia (ALL) trial published from the United Kingdom ALL group, UKALL was 92.3%. This excellent outcome was underpinned by accurate risk stratification according to minimal residual disease (MRD) assessment, using allele specific real time quantitative (ASO-RQ) PCR of immunoglobulin gene (IG) and T-cell receptor gene rearrangements, the current gold standard method in Europe. This method has limitations and requires the use of bone marrow (BM). It is only applicable to 92% of patients resulting in 8% of patients getting inferior risk stratification. The quantitative range of ASO-RQ PCR limits the ability to potentially identify patients with an even lower relapse risk who would benefit from protocols that further reduce treatment related mortality. Finally, many groups have demonstrated an unexpectedly complex clonal heterogeneity at diagnosis in patients with ALL and when disease relapse occurs, it often involves clones that were not identified or only viewed as minor clones at diagnosis. To overcome these issues a targeted ultrasensitive and accurate high throughput sequencing (HTS) approach for MRD (HTS-MRD) of immunoglobulin heavy chain (IGH) rearrangements has been developed. Novel bioinformatics pipelines enable interpretation and visualisation of millions of sequencing reads. The technique requires very small amounts of diagnostic DNA and allows quantitative tracking of multiple clones at different time points. As well as giving a more complete picture of clonality at diagnosis, the technique overcomes the secondary/ongoing rearrangements of IG genes during the disease course, which hampers MRD detection. HTS has the potential to increase sensitivity of detection, as sensitivity is limited by the number of cells input to the assay, as opposed to the background amplification in ASO-RQ PCR. This thesis describes the rigorous investigation into multiple sources of potential error during processing in an ultrasensitive assay and mitigations necessary to successfully implement HTS-MRD in clinical practice. The sensitivity is tested to one abnormal cell in one million normal cells threshold. A prospective feasibility study was then performed using peripheral blood instead of BM, concluding HTS-MRD can correctly stratify patients using blood, thereby reducing the need and inherent risks and pain associated with marrow sampling in children. The HTS technique was then applied to cerebrospinal fluid in patients with and without central nervous system (CNS) involvement of leukaemia. The increased sensitivity of detection allowed assessment of the dynamic response of CNS leukaemia throughout treatment and relapse. This, for the first time provided underpinning science to the long-held belief, from clinical observations, that CNS and BM relapses are “competing events” rather than distinct clinical entities. supporting the use of CNS-directed therapy for all children with ALL.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Development of a clinically applicable high throughput sequencing of immunoglobulin gene rearrangements in childhood precursor B-cell acute lymphoblastic leukaemia
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. - Some third party copyright material has been redacted from this e-thesis.
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health
UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10210984
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