Clinical Pathology - Theses
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ItemImproving the diagnosis and treatment of cancer of unknown primary using genomic profiling and patient-derived organoids( 2023-04)Cancer of unknown primary (CUP) is a metastatic cancer for which a standard clinical investigation fails to identify a primary tissue of origin (TOO). Clinical decision-making is impaired without a primary TOO diagnosis since this is typically used to inform optimal patient care. Most CUP patients are treated with empirical chemotherapy leading to poor survival outcomes. Therefore, improved diagnostic and treatment options for CUP patients are needed. The diagnostic utility of targeted RNA and DNA profiling was investigated in a cohort of 215 CUP patients. A retrospective clinicopathology review was performed to assign a putative TOO diagnosis. Seventy-seven percent (166/215) of the cohort had insufficient clinicopathological evidence to support a TOO diagnosis, while the remaining 23% were putatively resolved and had a clinicopathology diagnosis assigned. A custom gene expression profiling (GEP) TOO classifier was applied. The clinicopathology-unresolved CUPs were more challenging to classify than the resolved CUPs, and only 56% had a high confidence TOO classification. Cancer-type specific DNA features, including mutations and mutational signatures, were investigated and provided diagnostic evidence to support a TOO in 31% of unresolved CUPs, while GEP classifications supported a diagnosis in 13%. A genomics-informed clinicopathology review could direct an unconfirmed TOO diagnosis in one-third of CUPs. The RNA and DNA profiled CUP cohort were also investigated for immune and genomic biomarkers that could direct immune checkpoint inhibitor (ICIs) treatment for CUPs. Sixteen percent of CUPs had a high tumour mutation burden, 33% had high expression of genes associated with immunotherapy response, and 47% of the TOO-resolved CUPs belonged to an ICI-responsive cancer type. Of a subset of 28 CUPs treated with ICIs, 8/28 (29%) responded to treatment, and all of the responders had a high gene expression score (7/8) and/or high tumour mutation burden (3/8), and most (5/8) belonged to ICI-responsive cancer types. Therefore, many CUP patients have molecular features indicative of ICI susceptibility. Whole genome sequencing (WGS) was explored in 30 CUPs with targeted DNA sequencing to identify additional diagnostic and therapeutic informative features. An 82% concordance of variants was detected between WGS and panel sequencing. WGS could detect additional genomic features, including mutational signatures. Actionable mutations and biomarkers were detected in 48% of cases by targeted DNA sequencing, while WGS increased this to 59%. Furthermore, 72% of CUPs could have a TOO assigned following genomics-informed iii histopathology assessment, of which a third resulted from WGS-identified features only, and all diagnostic features detected by targeted sequencing were supported by WGS. Although only assessed in a small subcohort, tumour DNA extracted from archival tissue and cell-free material could be sequenced to detect clinically relevant findings. Patient-derived organoids (PDOs) were attempted from fresh CUP tumours. PDOs were successfully derived from six of 20 tumour samples (30%). The PDOs were heterogenous, genomically and morphologically representative of the tumour and could be used for WGS. Anticancer compounds were selected for PDO drug screening based on molecular and clinically directed treatment. The PDO drug responses frequently concurred with genomic biomarkers, but multiple drug sensitivities were also unanticipated. By incorporating targeted DNA sequencing and GEP testing, many CUP patients had a putative TOO diagnosis assigned. The genomic tests were also used to identify molecular biomarkers for ICI treatment. When compared with WGS, there were specific diagnostic scenarios whereby WGS could improve the diagnosis and identify additional potentially useful therapies for patients. Additionally, CUP PDOs were used to test genomic-directed therapies through in vitro drug testing. Overall, the study provides insights into how genomic tests can improve the diagnosis and treatment of CUP.
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ItemImproving the Differentiation of Inherited from Sporadic Causes of DNA Mismatch Repair Deficient Cancers( 2022)People with Lynch syndrome (LS), the most common cancer predisposition syndrome, have an increased risk of developing colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumours, among other cancer types. LS is caused by germline (likely) pathogenic variants in the DNA mismatch repair (MMR) genes, leading to a characteristic MMR-deficient (dMMR) / microsatellite unstable (MSI) tumour phenotype. However, dMMR / MSI is not only caused by LS and can also result from somatic causes of MMR gene inactivation. Identification of the dMMR / MSI tumour phenotype is of clinical significance for cancer prevention, prognosis, and response to immune checkpoint inhibitor treatment. However, diagnostic challenges still remain for accurate dMMR identification using current clinical tools. Therefore, the aim of my thesis is to address these two clinically relevant challenges: 1) can we improve detection of dMMR / MSI status in CRCs, ECs, and SSTs using next generation sequencing (NGS) derived tumour features and bioinformatic tools, and 2) can we improve differentiation of inherited (high risk) from sporadic (low risk) dMMR in CRCs, ECs, and SSTs using NGS? With the increased adoption of NGS in clinical practice for precision oncology and cancer genetics, the opportunity exists to develop a tumour-focused approach to improve the identification of LS. In Chapter 3, I investigated whether routinely collected clinicopathological tumour features could differentiate LS (inherited) from MLH1 methylated (sporadic) and suspected LS (unknown) subtypes in over 631 dMMR CRCs from the Colon Cancer Family Registry. Although age at diagnosis, sex and other variables were different between these subtypes, their power to differentiate was limited. Significantly, MLH1me tumours presented with a higher mortality rate than LS and suspected LS (SLS) tumours. These findings confirmed the need to examine new NGS-based approaches for differentiation purposes. In Chapter 4, I developed a novel model using NGS data to accurately determine dMMR from MMR-proficient (pMMR) tumour status in CRC and tested this model on EC and SST tumours. In total, 104 tumour features derived from whole exome sequencing of 300 CRCs were tested for their ability to identify dMMR with 10 features displaying >80% prediction accuracies. I established a novel model which combines the best predictive features for improved dMMR detection in CRC, EC, and SST sequenced tumours. In Chapter 5, using a custom-designed panel sequencing assay that I developed, tumour and blood-derived DNA were assessed from 134 SLS dMMR tumours (79 CRCs, 32 ECs, and 23 SSTs) in one of the largest investigations of SLS in these tumour types to date. Using the model determined in Chapter 4, I found 8.2% were not dMMR as clinically reported. The predominant cause of dMMR in SLS was biallelic somatic MMR mutations (61.9%) while only 1.5% were identified as LS that were missed by clinical testing. The findings from my thesis provide an evidence base to implement NGS-based diagnostic approaches for accurately detecting dMMR status and to resolve an SLS diagnosis in CRCs, ECs, and SSTs using a single test, which will ultimately improve utilisation of limited clinical resources for improved clinical management and cancer prevention.
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ItemThe genetics of gene expression: from simulations to the early-life origins of immune diseases( 2019)Human complex traits and diseases are often highly polygenic. Genome-wide association studies (GWAS) have been successful in identifying the underlying genetic components. However, challenges still remain and one of them is the biological interpretation of these findings. Genetic variants that are associated with diseases or traits are enriched in regulatory regions of the genome, suggesting that they may have a role in the regulation of intermediate molecular phenotypes, such as mRNA gene expression. Studies investigating the genetic architecture of gene expression variation, or expression quantitative trait loci (eQTLs), have aided the interpretation of GWAS findings by providing potential mechanisms through which the genetic variants contribute to higher-order phenotypes. In addition, eQTLs identified in disease-relevant tissues, or those that are specific to certain cell types or conditions are more informative in disease pathogenesis. This thesis first explored eQTL study design and analysis choices using extensive, empirically driven simulations with varying sample sizes, true effect sizes, and allele frequencies of true eQTLs. False discovery rate (FDR) control applied to the entire collection of tests had inflated FDR of genes with eQTLs (eGenes) in most scenarios; in contrast, hierarchical correction procedures had well-calibrated FDR. Significant eQTLs with low allele frequencies identified using small sample sizes were enriched for false positives. Overestimation of eQTL effect sizes was common in scenarios with low statistical power, and a bootstrap method (BootstrapQTL) which can lead to more accurate effect size estimation was developed. Based on the insights of the eQTL simulation study, optimal strategies were selected for the following eQTL analysis in two types of neonatal immune cells (monocytes and T cells) under resting and stimulated conditions. A great proportion of cis-eQTLs were specific to a certain cell type or condition, and the majority of them were observed only upon stimulation. Response eQTLs (reQTLs), with effects on gene expression modified by immune responses, were identified for 31% of the eGenes in monocytes and 52% of the eGenes in T cells. Trans-eQTL effects that were mediated through expression of cis-eGenes were observed. Lastly, integrative analyses were performed, using the early-life eQTLs, as well as GWAS variants associated with immune-related diseases obtained from external large cohorts. Significant overlaps between neonatal eQTLs and postnatal disease-associated variants were observed. Some cell type- or condition-specific cis-eQTLs colocalised with disease associations, suggesting that the potential risk genes involved in disease pathogenesis are linked to the stimulation of certain immune cells. Causal effects of genes were evaluated using Mendelian randomisation, and changes in expression levels (e.g. BTN3A2) were identified to have causal associations with multiple immune-related diseases. Taken together, it demonstrates that the early-life genetic variants and gene expression might contribute to later disease development. In conclusion, this thesis provides a strong evidence base for eQTL study design and guidance for analysis strategies in future studies. The characterisation of genetic regulation of neonatal immune responses and the interaction between regulatory variants and stimulatory conditions is a useful resource, and generates insights on the early-life origins of immune-related diseases that develop later in life.