Browsing by Author "Shires, Karen"

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    Cancer/Testis Antigen Expression Panel Incorporating MAGEC1 and BAGE2 Predicts Multiple Myeloma Disease Stage and Severity
    (OMICS publishing group, 2016-04-07) Shires, Karen; Teuchert, Andrea; Wienand, Kirsty; Shankland, Iva; Novitzky, Nicolas
    To provide a single molecular assay that could be used to easily stage Multiple Myeloma patients at diagnosis, we investigated the association between the simultaneous expression of 7 Multiple Myeloma-associated Cancer/Testis Antigens and biochemical parameters that are currently used for disease staging. We analysed the mRNA expression of MAGEC1, MAGEA3/A6, BAGE2, PRAME, NYESO1, SSX2 and PAGE by qualitative reverse transcription PCR using RNA extracted from diagnostic bone marrow samples from 39 patients covering the Multiple Myeloma disease continuum and compared this to levels of key biochemical parameters at diagnosis. We found that the Cancer/Testis Antigen panel was expressed in a specific order that was specifically associated with the severity of disease. This allowed the Cancer/Testis Antigens expression profile to successfully place the patient clearly into either stage I or stage III of the disease, with further sub-stratification in the stage III grouping. In addition, we putatively identified MAGEC1 expression as a confirmatory diagnostic marker for symptomatic Multiple Myeloma and clearly associated BAGE2 expression exclusively with stage III disease. We also demonstrated the novel finding of PAGE expression in Multiple Myeloma, with an association with more advanced disease. We suggest that this particular molecular Cancer/Testis Antigen panel can be used at diagnosis as a single test to clearly stage patients
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    Cytogenetically normal acute myeloid leukaemia at a single centre in South Africa
    (2022) Jenkins, Nicholas; Shires, Karen
    Introduction The heterogeneous molecular landscape of cytogenetically normal acute myeloid leukaemia (CN-AML) renders it an ongoing therapeutic challenge worldwide. The latest European LeukaemiaNet (ELN) 2017 guidelines attempt to address this by guiding post-remission therapy according to six prognostically informative mutations. However, its applicability in a South African setting remains elusive due to limited local data. This retrospective study aimed to describe a South African CN-AML cohort according to clinicopathological, molecular and treatment outcomes and consequently investigate the local applicability of a triple mutation testing approach for nucleophosmin (NPM1), fms-like tyrosine kinase internal tandem duplication (FLT3-ITD) and CCAAT/enhancer binding protein alpha (CEBPα) mutations in accordance with the ELN 2017 guidelines. Methods A review of cytogenetic results for all adult de novo AML cases diagnosed at Groote Schuur Hospital between 2005 and 2018 was performed. CN-AML cases were further characterized via molecular testing and review of clinical and laboratory data. Results In total, 218 patients with AML were identified of which fifty-six (33%) were cytogenetically normal. NPM1, FLT3-ITD and CEBPα mutations were found in 39%, 34% and 9% of CN-AML cases respectively, and allowed for definitive prognostication of 50% of cases. The 2-year overall survival rate for the entire CN-AML cohort was 16%. Conclusion Local rates of CN-AML and associated NPM1 and FLT3-ITD mutations were comparable to European cohorts. In contrast, local survival outcomes were notably inferior. Triple testing proved a resource effective prognostication approach for CN-AML. High throughput sequencing for adverse risk mutations should be considered for CN-AML patients inconclusively stratified via triple testing.
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    Defining the functional role of MAGE-C1 in Multiple Myeloma
    (2024) Stone, Marian; Shires, Karen
    The expression of Cancer/Testis antigens (CTAs) in various cancers, including multiple myeloma, makes them attractive targets for therapy and biomarker development. Despite intensive studies, their potential has not been fully realized due to lack of knowledge regarding their pathogenic role. To start elucidating the function of melanoma antigen (MAGE) protein-C1 (MAGE-C1) in multiple myeloma, this project aimed to firstly establish the suitability of culture cell line RPMI-8226 as an in vitro multiple myeloma model. Additionally, the project aimed to establish relevant reagents and techniques to study MAGE proteins by comparing MAGE-C1 to better described CTAs: MAGE-C2 and MAGE-A1. Firstly, the expression of MAGE-C1, MAGE-A1, MAGE-C2 and their respective binding partners, Ski interacting protein (SKIP) and Ring-box 1 (RBX1), was confirmed in RPMI-8662. Next, functional similarity was investigated through co-immunoprecipitation, to determine MAGE-C1 interaction with RBX1 or SKIP, and immunofluorescence microscopy, to assess co-localisation of these proteins. Lastly, cellular pathways influenced by MAGE-C2 were evaluated using heat shock to induce a stress response and activate the pathways of interest. The expression of MAGE-C1, MAGE-A1, MAGE-C2 and their binding partners were confirmed at the mRNA level. Unfortunately, interaction with their binding partners could not be demonstrated using co-immunoprecipitation, although immunofluorescence microscopy showed co-localisation of MAGE-C1 with both SKIP and RBX1. An abnormal stress response, via the E3 ligases known to interact with MAGE-C2 was observed in RPMI-8226. An array of reagents and techniques for future MAGE investigations were established during this study. However, the findings of this project indicated that RPMI-8226 may not be an ideal model for further MAGE investigations as initially hypothesized
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    The development of a flow cytometric method to detect the presence of mutated nucleophosmin in Acute Myeloid Leukaemia
    (2014) Du Pisani, Louis Almero; Shires, Karen
    Nucleophosmin (NPM1) is an acidic, nucleo-cytoplasmic, shuttling protein with predominant nucleolar localisation that plays multiple roles in cell growth and proliferation. Deletion insertion mutations of NPM1 (NPM1 DIM) seem to disrupt it normal physiologic role as a molecular chaperone, which likely leads to its oncogenic potential.NPM1 if present alone (not associated with FLT3 internal tandem duplications (FLT3-ITD)) is associated with significantly better overall survival and disease free survival in AML and has been entered as a provisional category in the World Health Organisation (2008) classification of Acute Myeloid Leukaemia with recurrent genetic abnormalities. Current methodology uses reverse transcriptase polymerase chain reaction (RT-PCR) and genomic deoxyribonucleic acid (DNA) PCR techniques to detect NPM1 DIM. Although these methods are robust and relatively easy to perform they can be expensive, labour intensive and not universally available. Six major variants of NPM1 DIM (Types A-F) have been described all leading to frame shift. All six types share the same last five amino acids in the C-terminal.The aim of this study was to develop a robust flow cytometry methodology that could be used in the routine assessment of AML samples to determine the mutational state of NPM, using a commercially available polyclonal antibody against the mutated NPM1.
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    Development of a rapid diagnostic screen for telomerase mutations associated with immunosuppressive therapy failure in patients with aplastic anaemia
    (2013) Xulu, Khethelo Richman; Shires, Karen; Mowla, Shaheen
    Includes abstract. Includes bibliographical references.
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    Efficiency of Buccal DNA sampling device in the mortuary
    (Avens Publishing Group, 2015-08-31) Tredoux, Simone; Mfolozi, Sipho; Shires, Karen
    Identification of forensic DNA samples by short tandem repeat (STR) profiling is currently an essential component of criminal investigations and can aid in linking perpetrators to crimes as well as identifying missing individuals or unidentified remains. In South Africa, recent amendments to legislation have allowed for the mandatory acquisition of reference DNA samples from certain offenders in order to populate the new National Forensic DNA Database. A novel method for the collection of buccal samples, the EasiCollect device, has been proposed to facilitate the collection of these DNA samples, replacing blood collecting devices as the standard method of DNA collection. Subsequently, this device has been introduced into South African state mortuaries to assist in the identification of deceased individuals. In order to ascertain if this device is suitable for use in the postmortem setting, an investigation was performed to compare the main methodology currently utilised within South African mortuaries, namely femoral blood transferred to ‘Fast Technology for Analysis of nucleic acids’ (FTA) cards, and buccal cells obtained using the EasiCollect device. DNA yields and STR genotyping results were compared between the two collection methods in thirty deceased individuals. Buccal samples provided a significantly greater DNA yield than blood samples, while no significant difference was observed between the qualities of the sample types as measured by the 260/280 nm ratio. Full STR profiles were obtained from all blood and buccal samples, with amplification efficiency showing limited DNA degradation and PCR inhibition in these samples, and only 3% of samples giving potentially disputable results. Numerous issues surrounding the collection of blood samples, however, indicated that this method is not optimal for use in the mortuary, with the EasiCollect device providing a more practical and robust method for the collection of DNA samples in the mortuary
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    HIV alters the expression of miRNA hsa-miR-200c-3p in B-cells, leading to enhanced migration of lymphoma cells
    (2018) Ramorola, Beatrice Relebogile; Mowla, Shaheen; Shires, Karen
    Background: The sub-Saharan African region is one that is affected most by the HIV/AIDS pandemic, with South Africa being the country with the highest number of infected individuals at 7.06 million. Infection with HIV is often associated with co-morbidities, including HIV-associated Non-Hodgkin’s Lymphomas (HIV-NHLs). Burkitt’s lymphoma (BL), a highly aggressive cancer, is one of the most common NHLs associated with HIV infection. Despite receiving highly active anti-retroviral therapy, the prognosis for this HIV-associated lymphoma remains poor and the incidence keeps on increasing in this group of patients. Recent studies have shown that microRNA (miRNA) dysregulation play essential roles in the pathogenesis of many cancers, including NHLs. While several human pathogenic viruses have been shown to deregulate cellular miRNAs, to date, no comprehensive studies have been carried out to determine whether HIV infection can lead to miRNA dysregulation in B-cells, which may contribute to the development of HIV-associated lymphomas. Objective: This research project aimed to validate the differential expression of selected miRNAs which were identified as potentially important in a PCR array, and characterise their roles in Burkitt’s lymphoma cells exposed to an attenuated strain of HIV-1, compared to control cells. Methods: Single-tube TaqMan miRNA assays were used to validate the previously observed differential expression of four selected miRNAs in Burkitt’s lymphoma cell lines (Ramos and BL41) exposed to HIV-1 compared to matched-microvesicle treated (control) cells. Following validation, the role of miRNA hsa-miR-200c-3p in the development of HIV-associated BL was investigated. This was done by using online bioinformatic prediction tools, as well as literature searches, to identify gene targets. Thereafter, the differential expression of a selected gene target was investigated by qPCR and western blotting. The functional significance of the observed changes in miRNA and gene expression was investigated by performing cell viability and migration assays. Results: Three upregulated (hsa-miR-575, hsa-miR-363-3p and hsa-miR-222-3p) and one downregulated (hsa-miR-200c-3p) miRNAs that were significantly deregulated by 2-fold or more (p< 0.05) in the PCR array were selected for validation. Thereafter, the miRNA hsa-miR200c-3p was selected for further analysis. Upon exposure to attenuated HIV-1, hsa-miR-200c3p was downregulated in the BL cell line Ramos, and this was reproducible in a second BL cell line BL41. The transcription factors ZEB1 and ZEB2, which are involved in cancer cell migration, were identified as targets of hsa-miR-200c-3p. Contrary to what is expected, the mRNA expression of both genes was found to be significantly downregulated in Ramos and BL41 exposed to attenuated HIV-1. At the protein level, in the Ramos cells, ZEB1 and ZEB2 matched what was observed for the mRNA. In contrast, both ZEB1 and ZEB2 protein were upregulated in BL41 cells under the same treatment conditions. At the functional level, the migration of both cell lines was enhanced when exposed to attenuated HIV-1, compared to control cells. Conclusions: The present study has demonstrated that HIV-1 has the ability to modulate cellular miRNA expression in Burkitt’s lymphoma cells. Of these miRNAs, hsa-miR-200c-3p is consistently downregulated when two BL cell lines were exposed to HIV. The ZEB transcription factors ZEB1 and ZEB2, which promote Epithelial-to-Mesenchymal Transition (EMT) through enhancing cellular migration, were investigated as hsa-miR-200c-3p targets. The mRNA levels of ZEB1 and ZEB2 were downregulated in both cell lines under the same experimental conditions. This is contrary to what is expected, since miRNAs lead to the attenuation of transcription or translation of their target genes and a downregulation of a miRNA should lead to an upregulation of its target. However, protein expression rather than mRNA expression has been described as a more accurate indication of target validation for miRNAs. The protein expression levels for ZEB1 and ZEB2 correlated with the mRNA expression results observed in the Ramos cells. In the BL41 cells, ZEB1 and ZEB2 protein levels were upregulated. Furthermore, in both cell lines, an increase in migratory ability was observed when cells were exposed to attenuated HIV-1. These results demonstrate that exposure to HIV enhances the cancer phenotype and that this is potentially due to changes in cellular miRNA expression brought about by the virus or viral components. Future studies should focus on gain-offunction and loss-of-function studies to determine whether the increase in cell migration is specifically due to a decrease in hsa-miR-200c-3p.
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    The Use of MAGE C1 and Flow Cytometry to Determine the Malignant Cell Type in Multiple Myelom
    (PLoS One, 2015-03-20) Wienand, Kirsty; Shires, Karen
    The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/−/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34+/MAGE C1+ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells. Introduction