Screening and characterisation of BCR::ABL1 kinase domain mutations in chronic myeloid leukaemia participants at Tygerberg Hospital, South Africa

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dc.contributor.advisorSwanepoel, Carmenen_ZA
dc.contributor.advisorChapanduka, Zivanaien_ZA
dc.contributor.authorShareefa, Isaacsen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Science. Dept. of Pathology. Haematological Pathology.en_ZA
dc.date.accessioned2023-03-06T10:29:21Z
dc.date.accessioned2023-05-18T07:11:23Z
dc.date.available2023-03-06T10:29:21Z
dc.date.available2023-05-18T07:11:23Z
dc.date.issued2023-03
dc.descriptionThesis (MMed)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH SUMMARY: To date, an increased number of drug resistant cancers have been observed. The underlying mechanisms are not well understood; therefore, we use Chronic Myeloid Leukaemia (CML) as a disease model to explore the type and frequencies of potential drug resistant causing mutations and the effect on survival at Tygerberg Hospital (TBH). The underlying genetic abnormality is a t(9::22) chromosomal translocation, and consequent fusion between the Breakpoint Cluster Region and the Non-Receptor Tyrosine Kinase Abelson genes (BCR::ABL1). Although Tyrosine Kinase Inhibitors (TKD), such as Imatinib (Gleevec), effectively decrease BCR::ABL1 mRNA transcript levels whereby the drug acts on the protein and inhibits its mode of action, point mutations within the kinase domain (KD) have shown to confer resistance to treatments. A retrospective audit was conducted on a CML cohort from 2013 to 2020, and 20 of these participants were recruited for detection of KD mutations within the ABL1 oncogene. Peripheral blood from routine diagnostic screening by the NHLS Molecular Haematology diagnostic laboratory were obtained for the mutation detection assay via bi-directional Sanger Sequencing. Identified sequence variants were then subjected to various bioinformatics tools to investigate variant, protein, and consequent effect on pathways. For the audit, a total of 165 patients with confirmed CML treated at TBH was captured. Descriptive statistics showed 46.1% of CML patients were female, and 53.9% were male. The patients were from 69 different areas in the Northern part of the Cape Metropole and surrounding Boland areas in Western Cape, South Africa. Quantitative analysis showed the youngest patient to be two years old and the oldest 88 years old, x̄ = 45-46 years. We found a weak linear correlation between age and BCR::ABL1 mRNA transcript levels. A significant difference in BCR::ABL1 mRNA transcript levels (p < 0.000) was seen in CML patients on treatment after 1 year. Of interest’s sake, 104 of the initial 165 patients who were diagnosed were still undergoing treatment. Moreover, 57 out of the 165 patients presented with possible resistance; 75% were categorized as failure and 25%at warning stage according to European Leukaemia Net guidelines. In the second part of the study, 20 participants’ samples were screened for variants. The screening sample group had a mortality rate of 20% who had a relative survival rate of less than five years. Sanger sequence analysis showed potential variants of interest in Exon 4 and of the ABL1 gene, although bioinformatics analysis alludes to the fact that these variants are not likely to play a role in resistance. In conclusion, the TBH CML cohort has a younger age at diagnosis which puts a greater strain on the public sector, and a significant number of patients are lost to follow up. More so, the age relative to CML deaths, as well as relative survival greatly differs from literature. Additionally, although synonymous mutations have recently shown to be pathological, the two variants found within our cohort has not been published to do so. CML could be a poster child for personalised medicine as the survival rate of CML patients is nearing that of the general population and treatment-free remission is attainable. Unfortunately, the paucity of CML data, drug availability and monitoring limitations are some of the obstacles that hinders South Africa from achieving these goals.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Tot op datum is 'n verhoogde aantal middel weerstandige kankers waargeneem. Die onderliggende meganismes word nie goed verstaan nie; daarom gebruik ons Chroniese Myeloiede Leukemie (CML) as 'n siektemodel om die tipe en frekwensies van potensiele geneesmiddel weerstandige veroorsakende mutasies en die effek op oorlewing te ondersoek. Die onderliggende genetiese abnormaliteit is 'n translokasie, en gevolglike samesmelting tussen die Breakpoint Cluster Region geen en die Nie-reseptor Tyrosien Kinase Abelson geen (BCR::ABL1), t(9::22). Alhoewel Tyrosien kinase-inhibeerders, soos Imatinib (Glivec), transkripsievlakke effektief verlaag duur inhibisie, het punt mutasies binne die kinase domein (KD) getoon dat dit weerstand teen behandelings verleen. 'n Terugwerkende oudit is uitgevoer op 'n CML-kohort vanaf 2013 tot 2020, en 20 van hierdie deelnemers is gewerf vir die opsporing van KD-mutasies binne ABL1-onkogeen. Perifere bloed van roetine-diagnostiese toets deur die NHLS Molekulere Hematologie-diagnostiese laboratorium is verkry vir die mutasie-opsporingstoets via tweerigting Sanger-volgordebepaling. Geidentifiseerde variante is aan verskeie bioinformatika-instrumente onderwerp om proteien en gevolglike effekte te ondersoek. Vir die oudit is 'n totaal van 165 pasiente met bevestigde CML wat by TBH behandel is, opgeneem. Beskrywende statistieke het getoon 46,1% van CML-pasiente was vroulik, en 53,9% was manlik. Die pasiente was van 69 verskillende gebiede in die Noord-distrik van Wes-Kaap. Kwantitatiewe analise het getoon die jongste pasient was twee jaar oud en die oudste was 88 jaar oud, x̄ = 45-46 jaar. Ons het 'n swak lineere korrelasie tussen ouderdom en BCR::ABL1-transkripsievlakke gevind. 'n Beduidende verskil in BCR::ABL1-transkripsievlakke (p < .000) is gesien by pasiente op behandeling na 1 jaar. Interessant is dat 104 van die aanvanklike 164 pasiente wat gediagnoseer is, steeds behandeling ondergaan. Altesaam 57 uit die 165 pasiente het moontlike weerstand getoon; 75% is gekategoriseer as mislukking en 25% op waarskuwing stadium volgens ELN-riglyne. In die tweede deel van die studie is 20 deelnemers monsters vir variante gekeur en die steekproefgroep het 'n sterftesyfer van 20% gehad, met 'n relatiewe oorlewingsyfer van minder as vyf jaar. Volgorde-analise het potensiele interessante variante in Ekson 4 en 9 van die ABL1-geen getoon. Bioinformatiese analise sinspeel egter op die feit dat hierdie variante waarskynlik nie 'n rol in weerstand sal speel nie. Ten slotte, die TBH CML-kohort kom voor by jonger ouderdom groepe, wat 'n groter druk op die openbare sektor plaas, en 'n groot aantal pasiente is verlies om op te volg. Die ouderdom tot CML- sterftes, sowel as relatiewe oorlewing verskil grootliks van literatuur. Alhoewel synonymous mutasies onlangs as patologies getoon het, is die twee variante wat binne ons kohort gevind word nie gepubliseer om dit te doen nie. Selfs al kan CML 'n plakkaatkind vir persoonlike medisyne wees met die oorlewingsyfers van CML-pasiente wat die van die algemene bevolking nader en behandelingsvrye remissie wat nou bereikbaar is, is min CML-data, dwelmbeskikbaarheid en moniterings beperkings van die dinge wat Suid-Afrika verhinder om hierdie doelwitte te bereik.af_ZA
dc.description.versionMasters
dc.format.extentxviii, 100 pages : illustrations, includes annexures
dc.identifier.urihttp://hdl.handle.net/10019.1/127238
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch University
dc.rights.holderStellenbosch University
dc.subject.lcshCancer -- Treatment -- South Africaen_ZA
dc.subject.lcshDrug resistance -- South Africaen_ZA
dc.subject.lcshChronic myeloid leukemia -- Treatment -- South Africaen_ZA
dc.subject.nameUCTD
dc.titleScreening and characterisation of BCR::ABL1 kinase domain mutations in chronic myeloid leukaemia participants at Tygerberg Hospital, South Africaen_ZA
dc.typeThesisen_ZA
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