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Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa
Thesis (MMed)--Stellenbosch University, 2025.
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Stellenbosch: Stellenbosch University
2025
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| _version_ | 1867613733953470464 |
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| access_status_str | Open Access |
| author | Mhlongo, Zwelakhe Johan Michael |
| author2 | Swanepoel, Carmen |
| author_browse | Mhlongo, Zwelakhe Johan Michael Swanepoel, Carmen |
| author_facet | Swanepoel, Carmen Mhlongo, Zwelakhe Johan Michael |
| author_sort | Mhlongo, Zwelakhe Johan Michael |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MMed)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/132604 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:40:50.669Z |
| 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:10019.1/132604 Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa Mhlongo, Zwelakhe Johan Michael Swanepoel, Carmen Nell, Erica-Mari Chapanduka, Zivanai Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Haematological Pathology Leukemia, Hairy Cell -- Diagnosis Microbial mutation Biochemical markers -- Diagnostic use Molecular diagnosis Molecular diagnosis UCTD Thesis (MMed)--Stellenbosch University, 2025. Mhlongo, Z. J. M. 2025. Design, optimisation and validation of Molecular Diagnostic Assays for BRAF p.Val600Glu and associated genetic factors within Hairy Cell Leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/018d655f-4a34-4bad-8a15-cba0c0112431 ENGLISH ABSTRACT: The BRAF p.Val600Glu somatic sequence variant is a molecular distinctive feature of hairy cell leukaemia (HCL) and other cancers and an important diagnostic and therapeutic targeted biomarker. Its clonal, heterozygous and detectable via molecular based techniques, such as Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR). The present study sought to evaluate and validate a molecular diagnostic assay for the identification of the BRAF p.Val600Glu sequence variant for future implementation as a diagnostic tool within our setting. In addition, the design and optimisation of diagnostic assays for other biomarkers, Mitogen-activated protein kinase kinase 1 (MAP2K1) and Kru ppel-like Factor 2 (KLF2) are also evaluated as this would aid in distinguishing HCL from HCL-like patients which is challenging on a morphological level. ARMS-PCR was employed for the BRAF p.Val600Glu assay and compared to Sanger sequencing to determine concordance. Estimated sensitivity and specificity were also determined for the assay validation. For MAP2K1 and KLF2, both PCR and Sanger sequencing were employed to determine various sequence variants in targeted areas. Following successful validation (BRAF) and optimisation (MAP2K1 and KLF2), available HCL and HCL-like tissue samples were screened, comprised of 12 HCL cases and 20 HCL-like cases. The BRAF p.Val600Glu ARMS-PCR based diagnostic assay was successfully validated and a concordance rate of 100% was achieved between ARMS-PCR and Sanger Sequencing. Estimated sensitivity and specificity were also 100%. A satisfactory score via United Kingdom National External Quality Assessment Service (UKNEQAS) external quality assessment (EQA) performance testing was also achieved. Both KLF2 and MAP2K1 diagnostic assays were successfully optimised and a non-synonymous sequence (c311T>C; pLeu104Pro) variant was detected in 1 of 2 splenic marginal zone lymphoma (SMZL) cases, while no sequence variants were detected in MAP2K1. In conclusion, BRAF p.Val600Glu ARMS-PCR based assay was successfully validated and demonstrated to be “fit for use” and ready for clinical use implementation. It is ideal for our resource-limited settings and should be of value in other cancers as well. While KLF2 and MAP2K1 were successfully optimised, a larger sample size cohort of good quality samples are needed. Our study demonstrated the need for accurate sequence variants identification as they influence patient diagnosis and subsequently their treatment plans which is different in HCL and HCL-like patients. This in turn would fast track turnaround time and improve clinical care. AFRIKAANSE OPSOMMING: Die BRAF p.Val600Glu somatiese volgorde variant is 'n molekule re kenmerkende kenmerk van harige sel leukemie (HCL) en ander kankers en 'n belangrike diagnostiese en terapeutiese geteikende biomerker. Die volgorde variant is klonaal, heterosigoties en kan opgespoor word deur molekule re gebaseerde tegnieke, soos Amplifikasie RefraktorieseStelsel Mutasie Polimerase Ketting Reaksie (ARMS-PKR). Die huidige studie het gepoog om 'n molekule re diagnostiese toets te evalueer en te bekragtig vir die identifikasie van die BRAF p.Val600Glu volgorde variant vir toekomstige implementering as 'n diagnostiese hulpmiddel binne ons omliggende instellings. Daarbenewens word die ontwerp en optimalisering van diagnostiese toetse vir ander biomerkers, Mitogeengeaktiveerde proteï enkinase kinase 1 (MAP2K1) en Kru ppel-agtige Faktor 2 (KLF2) ook gee valueer aangesien dit sal help om HCL van HCL-soortgelyke pasie nte te onderskei. ARMS-PKR was gebruik vir die BRAF p.Val600Glu-toets en was vergelyk met Sangervolgordebepaling om konkordansie te bepaal. Geskatte sensitiwiteit en spesifisiteit was ook bepaal vir die toets validering. Vir MAP2K1 en KLF2 beide PCR en Sanger volgordebepaling was gebruik om verskeie volgorde variante in geteikende areas te bepaal. Na suksesvolle validering (BRAF) en optimalisering (MAP2K1 en KLF2), beskikbare HCL en HCL-soortgelyke weefselmonsters was ondersoek, bestaande uit 12 HCL gevalle en HCL-agtige gevalle. Die BRAF p.Val600Glu ARMS-PKR-gebaseerde diagnostiese toets was suksesvol bekragtig en 'n konkordansie koers van 100% was bereik tussen ARMS-PKR en volgordebepaling. Geskatte sensitiwiteit en spesifisiteit was ook 100%. 'n Bevredigende resultaant via United Kingdom National External Quality Assessment Service (UKNEQAS) eksterne kwaliteit assessering (EKA) prestasietoetsing was ook behaal. Beide KLF2 en MAP2K1 diagnostiese toetse was suksesvol geoptimaliseer en 'n nie-sinonieme (c311T>C; pLeu104Pro) variant was opgespoor in 1 van 2 milt marginale sone limfoon (SMZL) gevalle, terwyl geen volgorde variante in MAP2K1 opgespoor was nie. Ten slotte, BRAF p.Val600Glu ARMS-PKR-gebaseerde toets was suksesvol bekragtig en demonstreer dat dit "geskik vir gebruik" en gereed vir kliniese implementering is. Dit is ideaal vir ons hulpbron-beperkte instellings en behoort ook van waarde te wees in ander kankers. Terwyl KLF2 en MAP2K1 suksesvol geoptimaliseer was, is 'n groter steekproefgrootte van goeie gehalte monsters nodig. Ons studie het die behoefte aan akkurate volgordevariante-identifikasie getoon, aangesien dit pasie nt diagnose en gevolglik hul behandelingsplanne wat verskil in HCL en HCL-agtige pasie nte ook beï nvloed. Masters 2025-06-11T12:49:52Z 2025-06-11T12:49:52Z 2025-03 Thesis https://scholar.sun.ac.za/handle/10019.1/132604 en Stellenbosch University xiii, 99 pages : illustrations application/pdf Stellenbosch: Stellenbosch University |
| spellingShingle | Leukemia, Hairy Cell -- Diagnosis Microbial mutation Biochemical markers -- Diagnostic use Molecular diagnosis Molecular diagnosis UCTD Mhlongo, Zwelakhe Johan Michael Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa |
| title | Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa |
| title_full | Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa |
| title_fullStr | Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa |
| title_full_unstemmed | Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa |
| title_short | Design, optimisation and validation of molecular diagnostic assays for BRAF p.Val600Glu and associated genetic factors within hairy cell leukemia (HCL) and HCL-like entities at Tygerberg Hospital, South Africa |
| title_sort | design optimisation and validation of molecular diagnostic assays for braf p val600glu and associated genetic factors within hairy cell leukemia hcl and hcl like entities at tygerberg hospital south africa |
| topic | Leukemia, Hairy Cell -- Diagnosis Microbial mutation Biochemical markers -- Diagnostic use Molecular diagnosis Molecular diagnosis UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/132604 |
| work_keys_str_mv | AT mhlongozwelakhejohanmichael designoptimisationandvalidationofmoleculardiagnosticassaysforbrafpval600gluandassociatedgeneticfactorswithinhairycellleukemiahclandhcllikeentitiesattygerberghospitalsouthafrica |