Bowes, Amy; (2024) Profiling the genomic landscape of polyploid giant cancer cells in sarcomas. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: As tumours evolve, they accumulate somatic mutations that serve as an archaeological record of their evolutionary past. Undifferentiated pleomorphic sarcomas (UPSs) are malignant soft tissue tumours that frequently contain scattered polyploid giant cancer cells. Polyploid giant cancer cells often exhibit atypical nuclear features, including multinucleation and micronucleus formation, likely due to whole genome duplication (WGD) and chromothripsis events that have been observed through bulk WGS studies (Steele et al., 2019). Polyploid giant cancer cells are also a common feature of other high-grade sarcomas that can resemble UPS histologically, including dedifferentiated liposarcomas (DDLPS), pleomorphic liposarcomas and myxofibrosarcomas. Aims: This thesis aims to study the genomic landscape and evolutionary history of polyploid giant cancer cells in UPSs and their histological mimics using topographic single-cell DNA sequencing (scDNA-seq). Methods: The performance of two laser capture microdissection (LCM) systems and three commercially available single-cell whole genome amplification kits were assessed, as well as the sequencing quality of fluorescence-activated cell sorted nuclei versus LCM isolated cells. Following experimental optimisation, polyploid giant cancer cells identified in 10 high-grade sarcomas by histology (i.e. nuclear size and shape, as well as multinucleation) underwent topographic scDNA-seq thereby retaining important spatial and morphological information. DNA was also estimated at a single-cell level in one UPS by FACS followed by scDNA-seq. Allele-specific copy number aberrations (CNAs) were inferred in all single cells. Matching bulk tumour and normal WGS was also performed. Results: Topographic scDNA-seq is a reliable experimental method to study the genomic landscape of polyploid giant cancer cells. Polyploid giant cancer cells isolated from UPSs and their histological mimics exhibited extreme subclonal heterogeneity, characterised by additional copy number gains and losses compared to the bulk tumour population. Screening for chromothripsis-like events in single cells revealed that both clonal (identified in matching bulk) and subclonal de novo chromothripsis-like events were present. FACS analysis and copy number inference of single cells in one UPS revealed multiple aneuploid subpopulations, including a near-haploid, a near-diploid and a polyploid subpopulation. scDNA-seq and copy number inference in single cells showed that following near genome scale haploidisation, multiple haploid cells underwent successive rounds of WGD to produce a genetically diverse polyploid subpopulation. Conclusions: CNAs, chromothripsis and WGD emerge as major contributors to intra-tumour heterogeneity in polyploid giant cancer cells isolated from high-grade sarcomas in adults. The multiple subclonal WGD events seen in UPS raises the possibility that WGD is an ongoing process that contributes to continuing tumour evolution. This process may fuel further chromosomal instability and could be linked to the poor prognosis observed in these cancers.

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