Pathology - Theses
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ItemActivated platelets and antibody opsonization as tumor markers: novel avenues for cancer diagnosis, targeted therapy and monitoring therapeutic outcomes( 2018)With over 100 types of cancer known, the study presented in this thesis introduces novel means of targeting a wide range of cancers, rather than a specific cancer antigen. Here, unique cancer targets, such as components of the tumor microenvironment and monoclonal antibody opsonization are investigated as new approaches for cancer diagnosis and targeted therapy. The first part of the study investigates the possibility of targeting activated platelets in the tumor microenvironment as a novel cancer diagnostic target. I showed the feasibility of using a single-chain antibody, which targets the activated form of GPIIb/IIIa, the most abundant platelet-specific receptor on the platelet surface as a possible tool for cancer diagnosis using PET/CT, fluorescence imaging and ultrasound. The second part of the investigation aims to further expand the utility of the single-chain antibody as an antibody-drug conjugate for cancer therapy. Using a mouse metastasis model of triple negative breast cancer, I showed that the activated platelet targeting single-chain antibody, conjugated to Auristatin E, a clinically available chemotherapy agent, was successful in reducing tumor growth and preventing metastasis development. The final part of the thesis describes the development of an FcγRIIIa receptor dimer, which has been engineered to selectively bind avidly to multimeric Fc complexes. This binding mimics the engagement of FcγRIIIa on effector cells, such as on NK cells, with antibody-coated cells that leads to antibody-mediated target killing. Here, using a mouse xenograft model of B cell lymphoma treated with Rituximab and triple negative breast cancer adenocarcinoma, treated with an EGFR receptor antibody, I show that the FcγRIIIa receptor dimer, labeled with a near-infrared contrast agent, could be used to specifically image antibody opsonization of tumor cells in vivo.
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ItemThe role of next generation sequencing in the management of haematological malignancies( 2018)Next generation sequencing comprises a rapidly-evolving cohort of technologies which enable detection of genetic variants present in DNA or RNA. In the context of haematological malignancies, such variants may have diagnostic, prognostic or therapeutic relevance. The scope of diagnostic-grade genetic testing is increasing as the underlying molecular landscape of haematological malignancies is increasingly well-characterised and becomes further embedded in clinical management. Multiple conventional methods for the detection of somatic single-gene variants are already embedded in standard-of-care. The technical performance of next generation sequencing when compared to these existing methods is of interest, since this determines in which instances it can be used to supplement or replace the status quo. This work focuses on acute myeloid leukaemia and systemic mastocytosis, since detecting variants in these malignancies presents several technical challenges. Acute myeloid leukaemia demands detection of a broad spectrum of molecular lesions, whereas systemic mastocytosis requires high sensitivity testing. Chapter one reviews molecular testing for haematological malignancies, including aspects of next generation-based testing relevant to diagnostics. Molecular markers currently employed in the diagnosis of acute myeloid leukaemia and systemic mastocytosis are discussed. The second chapter details methods used in the generation of data for chapters three to five. In the third chapter the selection and use of a targeted sequencing panel is discussed, testing performance is compared to conventional molecular methods using data generated from 30 clinical samples. Limitations of panel-based testing are discussed, including sensitivity, detection of specific variant types, and relevant bioinformatic and analytical issues. In chapters four and five, proof of principle is demonstrated for two novel next generation sequencing library generation methods, addressing the issues of structural variant detection ‘HEPTAD’ and high sensitivity single nucleotide variant detection ‘LNA PCRbrary’, respectively. Theory, optimisation and testing results for a combination of 49 clinical samples and cell lines using HEPTAD are discussed. The technique is applied to detect chromosomal translocations involving RUNX1, BCR, ABL, RARA, MLL/KMT2A, a chromosomal inversion involving CFBF, and KMT2A partial tandem duplications. The method detects structural variants in diagnostic samples with 100% sensitivity but requires further optimisation for minimal residual disease testing. Successful prospective application in a clinical scenario where orthogonal FISH testing was performed is detailed. LNA PCRbrary’s application to the detection of KIT D816V single nucleotide variants in peripheral blood is discussed. Suboptimal results when testing RNA derived from clinical samples are detailed, but promising results when testing cell line DNA, and significant potential for further optimisation and testing. In conclusion this work shows that NGS does have a role in the management of haematological malignancies, but technical challenges remain, including high sensitivity detection of single nucleotide variants, minimal residual disease monitoring of structural variants and gene targets which are difficult to amplify. Some of these issues are in part addressed in this work, but careful validation of testing sensitivity and specificity is required when introducing such NGS assays into clinical diagnostics.
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ItemThe genomic landscape of phaeochromocytoma( 2015)Phaeochromocytomas (PCC) and paragangliomas (PGL) (collectively PPGL) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PPGL cases are associated with heritable syndromes involving 18 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PPGL, exome sequencing, high-density SNP-array analysis, and RNA sequencing was applied to 36 PCCs and four PGL tumours. All tumours displayed a low mutation frequency in combination with frequent large segmental copy-number alterations and aneuploidy, with evidence for chromothripsis seen in a single case. Thirty-one of forty (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PPGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation (e.g. TP53, STAG2, KMT2D). A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function (e.g. ASCL1, NCAM1, GOLGA1). In addition, the existing paradigm for gene-expression subtyping of PPGL was further refined by applying consensus clustering to a compendium of previously published microarray data, enabling the identification of six robust gene-expression subtypes and subsequent cross-platform classification of RNA-seq data. The majority of cases in the cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PPGL, suggesting there are yet unknown PPGL cancer genes that can phenocopy MAX mutations. The cross-platform classification model was then further refined to develop a 46-gene Nanostring-based diagnostic tool capable of classifying PPGL tumours into gene-expression subtypes. The strong genotype-to-subtype relationship in PPGL makes subtyping a powerful tool that can be used clinically to guide and interpret genetic testing, determine surveillance programs and aid in better elucidation of PPGL biology. In applying the diagnostic assay to a test set of 38 cases, correct classification into one of the six subtypes was achieved for 34 (90%) samples based on the known genotype to gene-expression subtype association. The observation that at least one of the six subtypes is likely defined by the presence of non-neoplastic cells led to further refinement into five, four, and three-class architectures, further improving classification accuracy. Increasingly tumour heterogeneity is being recognised as one of the most significant challenges facing modern oncology. Genomically diverse tumour regions create additional complexity in predicting treatment response and metastatic potential through biopsy. Multi-region sampling of multiple synchronous primaries from patients with a predisposing germline mutation was used to explore tumour evolution and heterogeneity in PPGL and concomitant medullary thyroid carcinoma. Evolutionary reconstruction of a single primary PPGL demonstrated periods of both branched and linear evolution resulting in a high degree of intratumoural heterogeneity. Comparison of multiple synchronous primaries provided strong evidence of convergent evolution through recurrent chromosomal aberrations, indicating these may be obligate events in tumourigenesis, and as such, may indicate potential novel therapeutic targets.
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ItemPerforin biochemistry: function and dysfunction( 2016)Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), collectively referred to as cytotoxic lymphocytes (CLs), are responsible for clearing virus infected and cancerous cells. The predominant manner by which CLs do this is through the delivery of the pore forming protein, perforin, and pro-apoptotic granzymes that synergise to induce apoptosis in a conjugated target cell. Humans who inherit bi-allelic inactivating perforin mutations develop the immunoregulatory disease familial haemophagocytic lymphohistiocytosis (FHL) and/or haematological malignancies, demonstrating the critical importance of expressing functional perforin for the maintenance of immune homeostasis and tumour immune surveillance. While most disease associated perforin mutations are rare, 8-9% of the Caucasian population are carriers of polymorphism A91V (rs35947132, 272C>T). It has been suggested that >50% of individuals homozygous for the A91V allele develop FHL and/or cancer and that individuals heterozygous for A91V have an increased susceptibility to ALL. Despite its frequency and disease association, it remained unknown whether heterozygous inheritance of the A91V allele impairs human CL cytotoxicity and, more broadly, whether perforin is rate limiting in CL cytotoxicity. Here, it has been demonstrated that NK cells from healthy humans heterozygous for the A91V allele show an almost 50% reduction in cytotoxicity compared to individuals homozygous for WT perforin. This reduction in function was due to A91V perforin protein being misfolded within human primary NK cells. Moreover, it was also observed that heterozygous perforin knockout mouse CTLs showed an ~50% reduction in cytotoxicity. Taken together, these data demonstrate that perforin is indeed rate limiting for CLs cytotoxicity and therefore, individuals heterozygous for defective perforin alleles have impaired CL function. Although FHL predominantly presents shortly after birth, a subset of patients present at an age greater than three years, owing to the expression of misfolded perforin variants. Previous studies have shown that when transiently expressed in CTLs, perforin variants associated with late onset disease failed to traffic within CTLs and the cells remained non-functional. Therefore, it was unknown how patient CTLs expressing these variants could avoid FHL in infancy, and maintain a level of immune homeostasis for many years, or even decades. Here, it is shown that perforin variants associated with late onset disease can fold correctly and traffic within CTLs, and thus provide a significant level of cytotoxic function. However, this function was found to be lost if CTLs were cultured at an increased temperature (39 ̊C). Taken together, these data suggest that the CTLs of late onset FHL patients may have sufficient cytotoxicity to delay FHL onset in infancy. However, prolonged fever and, potentially, a more rapid exhaustion of the limited pool of correctly folded perforin mutants may result in the loss of CTL function, leading to FHL and cancer later in life. Prior to its secretion from the CL, the evolutionarily conserved C-terminal residues of perforin are proteolytically cleaved. The functional significance of C- terminal processing has remained controversial. Here it is shown that perforin enriched from human NK cell with an intact and glycosylated C-terminus was not cytotoxic. However, removal of the C-terminal glycan from the protein was found to completely restore function. As full-length deglycosylated perforin has wild type activity, these data suggest that C-terminal cleavage of perforin is permissive for cytotoxic function due to removal of an inhibitory N-linked glycan moiety at the C-terminus of the protein. These findings position the protease(s) responsible for perforin cleavage as critical to CL function. In summary, the studies described in this thesis have added to the understanding to how perforin mutations affect CL cytotoxicity and described a critical final step in perforin maturation. Together, these advancements in perforin biology may contribute to the treatment of disease arising from perforin deficiency and also define new factors critical for CL function and human health.
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ItemMolecular profiling of ovarian cancer to guide targeted treatment( 2015)Ovarian cancer is a complex disease composed of multiple distinct molecular and clinical subtypes. The survival rate for ovarian cancer has remained largely unchanged over the past three decades, despite the rapid advancement of the knowledge of the molecular and genetic mechanisms underlying most of the subtypes of ovarian cancer. There is, therefore, an urgent need to rapidly translate this knowledge into improved clinical outcomes for patients with ovarian cancer. There have been significant clinical responses of certain types of cancer to targeted therapies that are designed to inhibit specific molecular defects that some tumours appear to be dependent upon. To assist in allocating patients with ovarian cancer to targeted therapies, two customised assays for mutation and copy number alteration detection were developed for molecular profiling. A panel of 29 genes, which are commonly mutated in ovarian cancer, and are potentially therapeutically targeted, was selected to be screened using an amplicon-based assay, designed for next generation sequencing. Seventy six ovarian cancer cases with matched formalin-fixed paraffin- embedded tumour tissue, snap-frozen tumour tissue and blood samples were used for the assay validation and estimation of the diagnostic yield. A panel of 11 commonly copy number altered genes in ovarian cancer was also selected for screening with a herein developed method for multiplex low-level copy number detection. Furthermore, a thorough assessment and optimisation of the available and developed analysis methods was performed to ensure accurate analysis and reporting of mutations and copy number alterations. Thirty five patients with advanced ovarian cancer were tested using the developed assays as part of the ALLOCATE study, with genetic changes detected in 90.9%, demonstrating a high diagnostic yield. Molecular profiling of these cases was not only useful in identification of possible targeted treatment strategies with the aim of improving clinical outcomes, but also assisted in determining the correct diagnosis. Moreover, a novel algorithm was proposed for the prediction of individual tumour response to PARP inhibitors, a promising targeted treatment in high-grade serous ovarian cancer.