Minerva Elements Records
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ItemControl of the Intrinsic Pathway of Apoptosis( 2019)Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.
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ItemDefining functional drivers of oesophageal tumourigenesis( 2019)The incidence of oesophageal adenocarcinoma (OAC) has risen rapidly over the last four decades and has a high overall mortality rate that has shown only incremental improvements over the same duration. OAC develops from the precursor intestinal metaplasia of the oesophageal mucosa known as Barrett’s oesophagus. However, limited knowledge of the molecular mechanisms driving disease progression makes effective clinical management of OAC challenging. One of the common genetic events associated with the progression from Barrett’s oesophagus to OAC is loss of the tumour suppressor, SMAD4 (mutated in 13% or loss of function in 34% of OAC cases). Herein, this thesis firstly investigated the effect of SMAD4 inactivation in Barrett’s carcinogenesis. Genetic knockdown or knockout of SMAD4 was sufficient to initiate tumourigenesis of dysplastic Barrett’s oesophagus cell line, CP-B, in vivo, establishing SMAD4 loss as a crucial event driving progression to OAC. Further, low coverage whole genome sequencing (LC-WGS) analysis revealed that tumourigenic SMAD4 knockdown/knockout CP-B cell lines exhibited distinctive and consistent copy number alterations (CNAs) compared to non-tumourigenic SMAD4 wild-type parental cells. Amongst the alterations we observed were loss of chromosome arm 14q, while amplified regions include chromosome arms 6q and 12p, consistent with common CNAs found in patient tumours. This high genomic instability, characterized by structural chromosomal rearrangements within the tumours following SMAD4 loss, implicates SMAD4 as a protector of genome integrity in OAC development and progression. Moreover, initial in vitro data shows that SMAD4 knockout in CP-B cell line, results in differential expression of transforming growth factor-beta (TGF-beta) pathway target genes (such as ACTA2, CRYAB, PTK2B, ATF3 and CDC6) and loss of cell cycle arrest in response to TGF-beta1 cytokine compared to SMAD4 wild-type parental cells. Furthermore, SMAD4 knockout negatively regulated transcript expression of the multifunctional tumour suppressor INK4/ARF locus, demonstrating the novel and complex network of SMAD4 tumour suppressive activity. This thesis also focused on deciphering the functional role of growth factor receptor bound protein 7 (GRB7) amplification and overexpression in OAC and its potential targeting. GRB7 gene is positioned within known 17q12 amplicon, together with HER2 gene encoding for human epidermal growth factor receptor 2 (HER2). GRB7 is an adaptor molecule that mediates networking of multiple cell surface receptors with downstream signalling pathways. GRB7 high expression was found to be associated with worse survival in OAC patient cohort. Further, genetic GRB7 knockdown (siRNA) inhibited cell proliferation and clonogenic survival and induced apoptosis in GRB7 amplified and overexpressing OAC cell lines (OE19 and Eso26), without altering proliferation of the cells with normal GRB7 expression. Furthermore, whilst HER2 amplification and overexpression was also observed in OE19 and Eso26 cells, they were not uniquely sensitive to trastuzumab (HER2 inhibitor), with Eso26 cells exhibiting resistance in vitro. Taken together, initial findings raise the possibility that GRB7 may be a better molecular therapeutic target than HER2 in OAC with the 17q12 amplicon. To address this possibility, OE19 and Eso26 cell line xenograft models with inducible expression of shRNA targeting GRB7 were used. Consistent with in vitro findings, HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Of high importance, mimicking GRB7 inhibition with inducible-shRNA significantly inhibited tumour growth in both OE19 and Eso26 xenografts. Thus, this part of the thesis demonstrates the functional role of GRB7 overexpression as an oncogenic driver independent of HER2. In summary, the identification of functional genetic drivers and a deeper understanding of the mechanisms that underlie tumour progression in Barrett’s carcinogenesis will lead to improved strategies for the clinical management of OAC patients. To this end, SMAD4 loss was sufficient for progression from dysplasia to OAC. Tumours driven by SMAD4 loss exhibit distinctive CNAs consistent with OAC and metastatic potential. In addition, GRB7 high expression predicts poor outcome in patients with OAC and as such, GRB7 represents an oncogenic driver that could be used as a therapeutic target. Crucially, this thesis provides in vitro and in vivo preclinical and molecular biology evidence for the potential therapeutic benefit of targeting GRB7 in cancer.
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ItemGlobal meaning and its implications for emotional adjustment following a cancer diagnosis( 2007)This thesis examined the course of global meaning following diagnosis with cancer and explored its role in emotional adjustment using Janoff-Bulman’s (1992) assumptive world model. The study aimed to find evidence that there is a normative tendency to hold unrealistically positive global meanings which are contradicted by an adverse life event and the resultant emotional distress is resolved either through reinterpreting the event in a more positive light (assimilation) or revising meanings to be more realistic (accommodation). Assimilation is thought to be associated with favorable emotional functioning while accommodation has been linked to worse emotional outcomes (Janoff-Bulman, 1992). Using a longitudinal design, 86 adults with a hematologic cancer were assessed three times: following diagnosis; after primary treatment and: one year postdiagnosis (N=63). Data collected included six indices of emotional adjustment (depression, anxiety, traumatic stress, joviality, self-assurance and serenity); a measure of global meaning, the World Assumptions Scale (Janoff-Bulman, 1992) and, demographic and medical information. Three sets of analyses were conducted. The first set of analyses examined the course of emotional outcomes and global meaning over the year-long study period. Anxiety was the only emotional outcome showing overall change (decrease) while randomness was the only meaning displaying change (increase) over time. The study samples’ world assumptions were also compared with those of general community groups and motor vehicle accident survivors. At study entry, the sample displayed more positive bias in self and benevolence meanings than the other groups. By study exit, they showed less positive bias in contingency assumptions than the comparison groups, a pattern consistent with accommodation. The second set of analyses assessed whether global meaning predicted emotional outcomes when effects of demographic and medical factors had been accounted for. Meaning was implicated in all six emotional outcomes, with variability in their predictive power relative to the outcome measure and time of assessment. Self-worth and luck assumptions were the strongest predictors with justice and randomness also showing some predictive power. The final set of analyses used longitudinal data to focus on the association between global meaning and trajectories of emotional adjustment over time. The findings indicated that self-worth was associated with better outcomes for anxiety and TSS; luck was associated with depression while people benevolence was associated with anxiety. The directions of associations for all but people benevolence were consistent with the expectation that more positive bias in meanings is related to more favorable outcomes. The finding of a significant increase in randomness provides some support for the hypothesis that positively biased global meanings are challenged by the cancer experience and accommodate to become more realistic, although randomness did not predict worse emotional outcomes over time. Self-worth and luck, which were relatively high compared to other groups and stayed stable over the study period, were most strongly implicated in adjustment. The results also provide some impetus for a reconceptualization of the meaning in adversity models to take into account state-like variability in global meaning constructs.
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ItemExploration of novel regulators of mutant p53 in cancer cells: a role for NDFIP1( 2019)Mutations in the tumour suppressor gene, TP53 occur in more than 50% of human cancers. Mutant p53 proteins not only lose their tumour suppressive capacities, but also gain oncogenic functions, broadly referred to as gain of function (GOF). Cancer cells frequently accumulate mutant p53 and may become addicted to this protein for their survival. During development, mutant p53, like its wild-type counterpart, is inherently labile, however in cancer cells mutant p53 frequently accumulates. In part, this is due to the interrupted auto-regulatory loop with MDM2. However, MDM2 alone cannot explain the stability of mutant p53 in many cancer contexts. We therefore argued that additional factors are responsible for its stability in cancer cells. In order to identify the major players in the regulation of mutant p53, we performed a high-throughput RNAi screen through which we evaluated 18,120 genes for their effects on mutant p53 levels in two different mutant p53 expressing cell lines. Based on network analyses, pathway analyses and extensive literature mining, we selected 37 candidate genes to be validated through p53 immunoblotting. From the validated genes, we have selected one candidate for further studies. The results described in this thesis highlight a previously unknown mode of mutant p53 regulation in cancer cells. Future studies will explore in greater depth the functional consequences of this new interaction with mutant p53. Excitingly, these studies expose new vulnerabilities for therapeutic intervention and these opportunities for targeting aggressive cancers with mutant p53 will also be the focus of ongoing research.
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ItemHormonally induced defects of DNA damage repair genes: an oncogenic driver of prostate cancer( 2018)Patients with defects in the mismatch repair pathway, driven by either MSH2 or MSH6 loss, experience a significant increase in the incidence of prostate cancer, while germline mismatch repair defects in either MLH1 or PMS2, exhibit no such increase. This PhD project demonstrates that androgen-receptor activation, a known driver of prostate cancer, can disrupt the MSH2 gene in prostate cancer model systems through the induction of structural variations. Prostate tumours from two contrasting risk cohorts were screened to confirm loss of MSH2 protein expression in a small number of patients. Surprisingly, it was also found that a small but significant fraction of high-risk cases exhibited reduced expression of MSH2 without complete loss. Stratifying a large independent TCGA prostate cancer cohort for MSH2 expression levels revealed that patients whose tumours exhibited either complete loss or aberrant levels of MSH2 had significantly worse survival outcomes and accelerated clinical progression. In contrast, aberrant MSH2 levels had no impact on clinical survival in colorectal cancer. This PhD project also demonstrates that reduced expression of MSH2 can be explained by androgen-induced microRNA regulatory mechanisms. Here, it is demonstrated that miR-21 and miR-141 may both target the MSH2 gene leading to reduced MSH2 protein staining and both microRNAs were seen to be upregulated in prostate cancer patients with reduced MSH2 levels. Interestingly, this thesis also shows that miR-21 and miR-141 are both regulated by androgens, implicating this mechanism as a second androgen driven method of MSH2 downregulation. Importantly this PhD project also found that aberrant MSH2 expression in prostate tumours does not induce the same enhanced immune cell mobilisation seen in colorectal tumours suggesting that the prostate is an immune privileged site. This is contrary to the findings of other studies and may warrant a re-evaluation of whether MSH2 deficient prostate cancers are likely to benefit from immunotherapies. To further investigate if the prostate tumour microenvironment is indeed in an immunosuppressive state, a detailed investigation of the transcriptomic profile of the cells of the tumour microenvironment was also conducted. This resulted in not only the discovery of immunosuppressive signatures in tumour infiltrating T-cells but also significant transcriptomic alterations in other cancer associated pathways such as osteogenesis, cell migration, epithelial mesenchymal transition (EMT), hormone signalling and angiogenesis throughout the tumour microenvironment compared to the cellular make-up of benign prostatic tissue. The data presented in this thesis constitute a significant contribution to the current understanding of how defects in the mismatch repair gene MSH2 may affect prostate cancer severity. Additionally, these studies demonstrate multiple mechanisms through which the prostate tumour microenvironment may enhance prostate cancer progression.
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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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ItemMOF-mediated destruction of cancer using Fenton reaction( 2018)Cancer which is the second greatest cause of death worldwide has reached critical levels. In the past various therapies including photodynamic, photothermal and chemo-therapy are utilized for selective tumor treatment. Unfortunately, these methods suffer from various problems which limit their efficiency and performance. For this reason, novel strategies are being explored which improve the efficiency of these traditional therapeutic methods or treat the tumor cells directly. One such strategy utilizing the Fenton reaction has been investigated by many groups for the possible treatment of cancer cells. This therapy involves the utilisation of existing high levels of H2O2 in cancer cells to react with iron nanoparticles following the Fenton reaction to produce hydroxyl radicals capable of killing the cells. However, studies which attempted to use classical Fenton reaction alone to destroy the tumor cells, requires high concentrations of nanoparticles in order to be toxic to cancer cells. For this reason, there has not seen a successful nanoparticle which can treat cancer cells using the Fenton reaction without the need for external H2O2 sources. The aim of my work was to synthesize and develop novel metal organic frameworks (MOFs) for cancer treatment using the Fenton reaction. These specific nanoparticles can be utilized directly to destroy the cancer cells via the Fenton reaction or indirectly to deliver the Fenton reagent into cancer cells. In the first approach, a novel reduced iron metal-organic framework nanoparticle with cytotoxicity specific to cancer cells was fabricated. Iron present on the MOF can react with high levels of hydrogen peroxide found specifically in cancer cells to increase the hydroxyl radical concentration. The hydroxyl radicals oxidize proteins, lipids and/or DNA within the biological system to decrease cell viability. In vitro experiments demonstrate that this novel nanoparticle is cytotoxic to cancer cells through generation of hydroxyl radical using the cell’s own hydrogen peroxide. However, this emerging method is largely restricted due to the poor selectivity of reported nanoparticles. Subsequent improvements in nanoparticle size were facilitated by PEGylation on the particles through surface-initiated atom transfer radical polymerization, thus improving the stability, reducing the size and increasing the selectivity. In vitro experiments show that the selectivity index increased from 2.45 to 4.48 for HeLa cells, which is significantly higher than those reported in the literature for similar strategies. Finally, in an alternative approach, pH-responsive MOFs have been utilized for hemoglobin (Fenton reagent) and glucose oxidase (starvation reagent) delivery into the cancer cells. In a slightly acidic environment of cancer cells, GOx is released and consumes glucose and molecular oxygen that are essential survival nutrients in cancer cells and produces gluconic acid and hydrogen peroxide, respectively. The produced gluconic acid increases the acidity of the tumor microenvironment so completes MOFs destruction and enhances hemoglobin and GOx release. Fe ion from the heme groups of hemoglobin also releases in the presence of both endogenous and produced H2O2 and generate hydroxyl radical. In vitro experiments demonstrate that this novel nanoparticle is cytotoxic to both cancer (HeLa and MCF-7) cells at very low concentration (>2 µg/mL). Due to the great potential of the reported metal-organic frameworks in this thesis, these interesting particles may function as a new type of agents for controlled delivery and hydroxyl radical generation to treat cancer cells
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ItemUsing transcriptomics to understand cancer progression and predict response to therapy( 2018)Transcriptomics data provide useful information to better understand molecular phenotypes in cancer. Epithelial-to-mesenchymal transition (EMT) is one of these molecular phenotypes that is hijacked by cancer cells to obtain mobile mesenchymal characteristics which may assist cells to intravasate into blood stream, generate circulating tumour cells (CTCs) and metastasize to distant organs. CTCs also have heterogeneity in their molecular phenotypes and it is of utmost importance to understand these variations to be able to understand differences in their therapy response and use them to monitor treatment outcome. Using transcriptomics, we can also explore and predict molecular phenotypes associated with sensitivity to different therapeutic regimen. Although EMT is a single molecular phenotype, it can be regulated through different underlying molecular mechanisms, leading to differences in response to therapies. To identify samples with TGFβ-driven EMT, I derive a gene expression signature of EMT induced by TGFβ using metaanalysis and transcriptomics data integration. This signature is able to identify transcriptional profiles arising during TGFβ-driven EMT, and yields highly consistent results in multiple independent pan-cancer cell lines and patients data. Samples fitting this signature show lower number of mutations in elements of TGFβ signalling, poorer overall survival outcome and preferential response to certain drugs. Meta-analysis and data integration such as the above require careful attention to batch effects in datasets. I apply different batch correction methods in order to perform general normalisation or obtain differentially expressed genes (DEGs) in integrated transcriptomics data sets. Further, to classify the fit of individual samples to a gene signature, I apply existing single-sample scoring methods. However, these methods all use information borrowed from the whole set of samples, meaning they are not truly single sample scores. To address this, I developed a rank-based scoring method, called singscore, which generates more stable scores that are independent from sample size and composition in a dataset. CTCs are integral to cancer progression, but while these cells are extremely rare in blood, they have great potential to provide a real-time representation of cancer progression and treatment efficacy. I perform an assessment of current markers for enrichment and/or detection of CTCs, and then, introduce new CTC markers, including general, epithelial and mesenchymal markers obtained by analysing multiple breast cancer and blood data sets. I then assess their expression in publically available CTC data and a number of in-house patient samples. Finally, I use pharmacogenomics data in breast cancer cell lines and the singscore method to predict drug response outcome for 90 drugs based on gene expression data, which have been shown to be the most predictive molecular feature in breast cancer. I derive drug sensitivity signatures by quantifying associations between gene expression and drug response and evaluate the utility of these gene signatures using cell lines, PDX models and patient data and show consistent pattern of response across independent data sets. Further associations between drug sensitivity scores and EMT phenotype are assessed.
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ItemThe evolutionary history of genes and transcriptional networks reveals fundamental properties of cancer associated with the breakdown of multicellularity( 2018)All biological systems follow the rules and constraints imposed during their evolution. Current-day gene phenotypes such as gene expression, gene essentiality, gene function and protein localization are linked with the time of evolutionary emergence of genes. In cancer, tumours rely on cellular processes that date back to unicellular ancestors (e.g., cell replication, glycolysis), while dysregulating key pathways linked to the emergence of multicellularity, suggesting that the transition from unicellularity to multicellularity left vulnerabilities in cells that act as guiding principles during cancer development. Therefore, in this thesis I integrate genomics, systems biology and evolutionary biology to investigate fundamental principles of tumourigenesis related to the evolutionary history of genes using gene expression and somatic mutation information across multiple tumour types. First, I coupled the evolutionary age of genes and cellular processes with their expression levels in tumour and normal samples, and found that tumours consistently activate genes from unicellular ancestors while switching off genes related to multicellularity. These consistent patterns were supported by a mutual exclusivity between the activity of genes and transcriptional networks of unicellular and multicellular ancestors, which promoted convergent evolution towards a state of loss of multicellularity. Second, I investigated how somatic mutations disrupted gene regulatory networks. Genes that emerged together with early metazoans were enriched in point mutations and copy- number alterations, indicating that gene innovations that took place at the onset of multicellularity play a fundamental role in cancer development. Importantly, the uncoupling of regulatory networks of unicellular and multicellular ancestors was mostly due to point mutations in gene regulators linking these networks. On the other hand, copy-number aberrations were directly involved in the activation and inactivation of unicellular and multicellular genes, suggesting point mutations and copy-number aberrations play complementary roles in the loss of regulation between unicellular and multicellular transcriptional networks in cancer. Third, I focused on novel transcriptional associations formed during tumourigenesis using gene co-expression module analysis. Significant levels of rewiring between unicellular and multicellular genes were found across tumours. This rewiring was mostly driven by gene amplifications, which promoted the formation of tumour-specific modules composed of novel transcriptional associations between unicellular and multicellular genes, once more linking the genes and regulatory associations evolved at the onset of multicellularity to cancer development. The findings of this work reveal fundamental principles driving cancer development associated with genes and transcriptional networks evolved during the transition from unicellularity to multicellularity. I propose a model whereby activation of programs that date back to unicellular ancestors and the deactivation of multicellular programs is driven by an inherent mutual exclusivity of these genes together with the breakage of regulation between unicellular and multicellular genes by point mutations, whereas the formation of novel transcriptional associations between these genes in tumours is driven by copy-number changes. Finally, I identify potential novel drivers based on their key role in uncoupling unicellular and multicellular transcriptional networks across tumours and suggest novel treatment strategies derived from this evolutionary approach. The results presented in this thesis contribute to our understanding of how past evolutionary events led to vulnerabilities in transcriptional networks that influence cancer development, and highlight the benefits of the integration of evolutionary concepts with genomics and network biology to identify fundamental principles of cancer.
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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.