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Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa
Thesis (MMed)--Stellenbosch University, 2025.
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613968547184640 |
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| access_status_str | Open Access |
| author | Losper, Kaylee Chantel |
| author2 | Swanepoel, Carmen |
| author_browse | Losper, Kaylee Chantel Swanepoel, Carmen |
| author_facet | Swanepoel, Carmen Losper, Kaylee Chantel |
| author_sort | Losper, Kaylee Chantel |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MMed)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132631 |
| institution | Stellenbosch University (South Africa) |
| last_indexed | 2026-06-10T12:44:34.165Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/132631 Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa Losper, Kaylee Chantel Swanepoel, Carmen Musekwa, Ernest Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Haematological Pathology. Acute myeloid leukemia in children -- Molecular aspects Biochemical markers -- Diagnostic use Mutation (Biology) Genetic screening UCTD Thesis (MMed)--Stellenbosch University, 2025. Losper, K. C. 2025. Design and Validation of Molecular Diagnostic Assays for key Prognostic Biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in Cytogenetically Normal AML patients at Tygerberg Hospital, South Africa. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/97691985-032c-4530-bb7b-b8b92d941581 ENGLISH ABSTRACT: Acute myeloid leukaemia (AML) is a highly aggressive group of complex disorders with poor disease outcomes. Clinically and genetically, it is very heterogeneous and difficult to stratify, so it is subdivided based on clinical presentation and cytogenetic and molecular subtypes. The largest subgroup (40-50%) is classified as cytogenetically normal (CN-AML) and varying gene expression profiles, phenotype, prognosis and treatment responses are due to acquired variants in key "driver "genes FLT3, NPM1, CEBPA and DNMT3A. We therefore aim to design and validate molecular-based diagnostic assays to screen for sequence variants in these key biomarkers in a CN-AML subgroup. A 11-year (2012-2022) retrospective audit of the existing FLT3-ITD and NPM1 diagnostic tests requested and performed were done to aid case selection for subsequent mutational screening of 20 confirmed CN-AML patients. Test methodologies for FLT3-ITD and NPM1 were upgraded from Sanger Sequencing to Fragment analysis via capillary electrophoresis and concordance, estimated sensitivity and specificity were determined. For DNMT3A and CEBPA, new tests using PCR and Sanger Sequencing were designed and optimized. Sixty-one FLT3-ITD tests were performed from 2012-2022 compared to the 38 NPM1 tests that were performed. Ten percent (6/61) were positive for FLT3-ITD, while 13% (5/38), were positive for NPM1, with the c.860_863dupTCTG (Type A) sequence variant being the most common within our setting. A 4 base pair deletion, c.558_561del (p.Pro187fs) sequence variant that cause a frameshift was found in CEBPA while no sequence variants were found in DNMT3A. A concordance of 85% and 95% were respectively observed between the newly optimised FLT3-ITD and NPM1 compared to the existing assays while an estimated sensitivity (FLT3-ITD-90%; NPM1-100%) and specificity (FLT3-ITD-80%; NPM1-100%) were observed. Satisfactory results were also achieved via external quality control testing. In conclusion, we successfully developed, validated and implemented a diagnostic assay for FLT3-ITD and NPM1 sequence variants. Despite technical difficulties, once resolved, the implementation of DNTM3A and CEBPA diagnostic assays would expand our local molecular capabilities allowing for comprehensive profiling of AML. Our findings contribute to significantly improve treatment outcomes and aid to understand the genetic landscape of AML in South African populations. This in turn, may be essential for informing local clinical guidelines and protocols for AML management by supporting the development of region-specific treatment strategies that address the unique needs and challenges of South African patients. AFRIKAANSE OPSOMMING: Akute myeloïede leukemie (AML) is 'n hoogs aggressiewe groep komplekse afwykings met swak siekte-uitkomste. Klinies en geneties is dit baie heterogeen en moeilik om te klasifiseer, dus word dit onderverdeel op grond van kliniese aanbieding en sitogenetiese en molekulêre subtipes. Die grootste subgroep (40-50%) word as sitogeneties normaal (SN-AML) geklassifiseer en wisselende geenuitdrukkingsprofiele, fenotipe, prognose en behandelingsreaksies is as gevolg van verworwe variante in sleutel "drywer" gene FLT3, NPM1, CEBPA en DNMT3A. Ons beoog dus om molekulêr-gebaseerde diagnostiese toetse te ontwerp en te bekragtig om te ondersoek vir volgordevariante in hierdie sleutelbiomerkers in 'n SN-AML subgroep. 'n Retrospektiewe oudit van 11 jaar (2012-2022) van die bestaande FLT3-ITD en NPM1 diagnostiese toetse wat aangevra en uitgevoer was, was gedoen om met die keuse van gevalle vir daaropvolgende mutasie sifting van 20 bevestigde SN-AML pasiënte te help. Toetsmetodologieë vir FLT3-ITD en NPM1 was opgegradeer van volgordebepaling na Fragment analise via kapillêre elektroforese en konkordansie, beraamde sensitiwiteit en spesifisiteit was bepaal. Vir DNMT3A en CEBPA was nuwe toetse wat Amplifikase Refraktoriese Mutasie Amplifikase en volgordebepaling gebruik, ontwerp en geoptimaliseer. Een-en-sestig FLT3-ITD-toetse was vanaf 2012-2022 uitgevoer in vergelyking met 38 NPM1- toetse. Tien persent (6/61) was positief vir FLT3-ITD, terwyl 13% (5/38) positief was vir NPM1, met die c.860_863dupTCTG (Tipe A) volgordevariant wat die algemeenste in ons populasie was. 'n 4 basispaar delesie, c.558_561del (p.Pro187fs) variant wat 'n raamverskuiwing veroorsaak was gevind in CEBPA terwyl geen volgorde variante gevind is in DNMT3A. 'n Ooreenstemming van 85% en 95% is onderskeidelik waargeneem tussen die nuut geoptimaliseerde FLT3-ITD en NPM1 in vergelyking met die bestaande toetse terwyl 'n beraamde sensitiwiteit (FLT3-ITD-90%; NPM1-100%) en spesifisiteit (FLT3-ITD-80) % NPM1-100%) bepaal was. Bevredigende resultate was ook behaal deur middel van eksterne gehaltebeheertoetsing. Ten slotte het ons 'n diagnostiese toets vir FLT3-ITD en NPM1 variante suksesvol ontwikkel, bekragtig en geïmplementeer. Ten spyte van tegniese probleme, sodra dit opgelos is, sal die implementering van DNTM3A- en CEBPA-diagnostiese toetse ons plaaslike molekulêre vermoëns uitbrei wat 'n omvattende profilering van AML moontlik maak. Ons bevindinge dra by tot aansienlike verbetering van behandelingsuitkomste en help om die genetiese landskap van AML in Suid-Afrikaanse bevolkings te verstaan. Dit kan op sy beurt noodsaaklik wees om plaaslike kliniese riglyne en protokolle vir AML-bestuur in te lig deur die ontwikkeling van streekspesifieke behandelingstrategieë te ondersteun wat die unieke behoeftes en uitdagings van Suid-Afrikaanse pasiënte aanspreek. Masters 2025-06-12T06:57:24Z 2025-06-12T06:57:24Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132631 Stellenbosch University iii, 103 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Acute myeloid leukemia in children -- Molecular aspects Biochemical markers -- Diagnostic use Mutation (Biology) Genetic screening UCTD Losper, Kaylee Chantel Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa |
| title | Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa |
| title_full | Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa |
| title_fullStr | Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa |
| title_full_unstemmed | Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa |
| title_short | Design and validation of molecular diagnostic assays for key prognostic biomarkers, FLT3-ITD, NPM1, DNMT3A and CEBPA in cytogenetically normal aml patients at Tygerberg Hospital, South Africa |
| title_sort | design and validation of molecular diagnostic assays for key prognostic biomarkers flt3 itd npm1 dnmt3a and cebpa in cytogenetically normal aml patients at tygerberg hospital south africa |
| topic | Acute myeloid leukemia in children -- Molecular aspects Biochemical markers -- Diagnostic use Mutation (Biology) Genetic screening UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132631 |
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