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Title: The evolution of glioblastoma : a single case study
Author: Wendler, Astrid Svenja
ISNI:       0000 0004 7961 9294
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Glioblastoma multiforme (GBM) is the most malignant and common primary brain cancer in adults, with a 2.5-year survival of only ~8%. In this single case study we have analysed the development of a primary GBM over four years. We performed WGS and RNAseq on three, spatially distinct samples of the treatment naïve primary tumour, the first recurrence and the fatal second recurrence. We observed a massive increase in mutations from the first to the second recurrence, showing predominantly C > T and A > G transitions, which is a pattern typical of TMZ treatment. The fact that the hypermutation did not appear until the second recurrence raises questions about the mechanism of hypermutation. The therapeutic apoptotic action of TMZ relies on the function of the mismatch repair (MMR) pathway. In the second recurrence we observed disruptive mutations of MSH6, MSH2, PMS2 and MLH1 all of which are crucial to the MMR pathway. The pattern of mutations of these genes varied between the samples taken from the second recurrence, however, all three samples showed hypermutation. This suggests that the hypermutation observed after TMZ treatment only occurs if the MMR is damaged, but that this damage does not need to be at a specific locus. Using the CRISPR-Cas9 technology we explored the impact of MMR mutations on patient derived GBM cell lines. Results on MSH2 and MSH6 knockout cell lines treated with TMZ showed the reproduction of the hypermutation phenotype in vitro, but only in MGMT promoter methylated cell lines. Moreover, the DNA analysis of the patient showed one shared mutation in high copy number in all of the nine samples: a mutation in EGFR G598V. We hypothesise that subsequent tumours developed from an original clone bearing this mutation. This mutated oncogene is located on an extrachromosomal circular DNA structure known as a double minute (DM), which is found in an estimated 40% of GBM. We performed linked-read sequencing using the 10X Genomics Chromium technology to reconstruct the DM and shed light on its emergence.
Supervisor: Watts, Colin ; Tavaré, Simon Sponsor: Cancer Centre Cambridge
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Glioblastoma ; Genetics ; Evolution ; Hypermutation ; Recurrence