The genomic profile of low frequency chromosome 17p deletion sub-clones in Chronic Lymphocytic Leukaemia: detection and characterisation

Author: Cuc Do

Do, Cuc, 2018 The genomic profile of low frequency chromosome 17p deletion sub-clones in Chronic Lymphocytic Leukaemia: detection and characterisation, Flinders University, School of Medicine

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Abstract

Chronic lymphocytic leukaemia (CLL) is a disease predominantly of elderly people and therefore is becoming increasingly important given Australia's ageing population. One of the most important prognostic markers in CLL is a deletion of the short arm of chromosome 17 (del17p), which harbors a tumour suppressor gene (TP53). Loss of function of the encoded p53 protein, either by deletion or mutation, results in aggressive disease. However, it has been hypothesised that low frequency loss of TP53 genes (<25% of cells carrying del17p) may not carry the same clinical significance, particularly early in the disease course. To further confound this issue, it has not previously been possible to identify the additional genetic mutations which may be contained within these small del17p sub-clones in CLL. Clinicians are faced with these fundamental issues when selecting best therapy, which is usually guided by risk calculations based on the genetics of the disease. Therefore, to address this gap in knowledge, this study investigated primary CLL samples containing del17p low frequency clones, including their baseline characteristics, genomic profiling and relationships with other genetic aberrations.

With respect to the intra-clonal heterogeneity of low frequency del17p CLLs, this study has demonstrated a sub-clonal architecture and hierarchy that is different to current assumptions. From the data presented, it is apparent that del13q associates with low frequency del17p sub-clones; this association is not evident in the patients where del17p forms the dominant or high-risk clone. Additionally, through the application of high-throughput laser scanning FISH analysis, it is evident that del17p sub-clones arise independently or at least simultaneously with del13q clones, which is currently thought to represent a founder event. Whilst determining the clinical impact of this finding requires larger studies, this finding demonstrates a clear genetic difference between dominant and non-dominant del17p clones, and may help to explain the differences in their clinical course.

A direct comparison of the clinical application of three current FISH techniques was also undertaken, incorporating analysis of trisomy 12 CLL samples with the chromosome 12 centromeric probe. This work clearly demonstrates the clinical applicability of automated approaches to FISH analysis.

In order to identify the underlying driver mutations in low frequency del17p CLL sub-clones, a reliable technique is required to identify and analyse these small sub-clones. This study has demonstrated the ability to utilise fluorescence in situ hybridisation in suspension (FISH-IS), a method which combines a state of the art flow cytometer with image analysis of individual cells, to capture and record thousands of cells by ImageStreamX. This study has provided the first demonstration of the sensitivity/accuracy of FISH-IS in distinguishing aneuploidy subgroups at 1% with various centromere enumerate probes (CEP) on CLL samples. This is also the first report of a locus-specific probe set being used to identify low frequency del17p in CLL by FISH-IS, with the fluorescence signal made analysable by the use of a 17p BAC probe contig (consisting of labelled bacterial artificial chromosome DNA from the RP11 library). This method enables a prognostic test to reliably detect very low frequency del17p sub-clones with a reliable number of cells monitored at 3%, with the potential for these cells to be genetically analysed as a separate sub-clone.

Hence, the ultimate aim of this work was to flow sort low frequency del17p sub-clones and genetically analyse these cells specifically. This has been accomplished as a proof of principle experiment, which is the first time the genomic landscape of del17p sub-clones has been able to be interrogated in an unbiased manner. Taking this work forward, this technique will enable, for the first time, a specific and in depth genetic analysis of the untreated low frequency del17p sub-clones, with a view to being able to identify the early genetic abnormalities that accompany this event. This method will thereby provide information regarding the differences between early and late events that may lead to the chemo-refractoriness and aggressive CLL phenotype accompanying higher frequency TP53 genes loss. Critical to further understanding of the relevance of early minor sub-clones is the determination of the genetic profiles of these sub-clones and the identification of potentially druggable driver mutations.

Keywords: Chronic lymphocytic leukaemia, deletion of the short arm of chromosome 17 (del17p), sub-clones, FISH, laser scanning cytometry, ImageStreamX, FISH-IS.

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2018
School: School of Medicine
Supervisor: Associate Professor Bryone Kuss