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Title: Clonal dynamics of TEL-AML1+ childhood ALL : an in vivo model
Author: Turati, Virginia
ISNI:       0000 0004 6061 0222
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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ETV6-RUNX1 gene fusion is an early or initiating genetic lesion of Childhood Acute Lymphoblastic Leukaemia, followed, in disease progression, by a modest number of recurrent or "driver" copy number alterations. Among patients diagnosed with this particular cytogenetic subtype of ALL cure rate reaches over 80% . However, treatment protocols are extremely demanding, and the molecular mechanisms driving drug-resistance and relapse in the remaining 10% of patient are still unknown. Recently a genetic signature of subclones arranged in a complex non-linear or branching architecture has been identified in this leukaemia, pioneering similar observations in many other cancer types. At present, most reports of this intratumor diversity represent static, in-depth snapshots of clonal diversity of tumours at a given time. "Real-time" longitudinal and spatial analysis of subclonal evolutionary dynamics is required to understand the functional characteristics of individual subclones. Such approach also promises to reveal the overall clinical relevance of this phenomenon. This project therefore aims to understand whether genetically distinct subclones are also functionally different, particularly with respect to chemoresistance. A mouse model that allows for the independent exposure of the same tumour to treatment multiple times, and the tracking of clonal dynamics by mean of a selected pool of genetic markers was established. While the analysis is still ongoing, preliminary data confirm that tumours generated from the same inoculum are highly similar in their genetic makeup across recipients, that multiple clones within a tumour can survive chemotherapy, but sensitivity of clones equally represented within the bulk can vary, and unexpectedly that spatially segregated subclones can be identified in this model of liquid cancer. The complete analysis of the collected samples by mFISH as well as by single cell whole genome sequencing will provide unprecedented insight into the impact of cytotoxic treatment on intratumour genetic heterogeneity, and might reveal novel mechanisms of chemoresistance and relapse. Additionally, a lentiviral overexpression system was designed to endogenously manipulate clonal interactions through the restored expression of TEL and PAX5 in primary cells from patients, and evaluate their impact on clonal relationships and hierarchies and to validate the results obtained from the first part of the study. Lentiviral vectors for TEL and PAX5 second hit overexpression were produced and tested by Western blot and qPCR. The TEL vectors were adopted in a preliminary in vivo experiment, showing no effect on overall engraftment ability.
Supervisor: Enver, Tariq ; Jacobsen, Sten Eirik Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available