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Title: Studies on the core binding factor complex in patients with acute myeloid leukaemia
Author: Langabeer, Stephen Eirian
ISNI:       0000 0001 3604 8266
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2002
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The core binding factor (CBF) transcription factor complex is involved in the regulation of a number of genes essential for normal haemopoiesis. The complex is comprised of two subunits: the DNA-binding α subunit AML1 (CBFA2/RUNX1) and the β subunit CBFB, which interacts with AML1 to increase DNA binding. The AML1 and the CBFB genes are known to be disrupted in up to 25% of cases of acute myeloid leukaemia (AML). Therefore, in this thesis, several aspects of CBF leukaemia were investigated at the DNA and RNA level. The chromosomal abnormalities t(8;21)(q22;q22) and inv(16) (p13q22) result in the creation of AML1-ETO and CBFB-MYH11 transcriptionally active fusion genes, and are associated with the M2 and M4Eo AML FAB types respectively. Detection of these abnormalities at presentation is of clinical importance as these patients have a relatively favourable prognosis. Of 321 AML patients studied, 21 (6.5%) had inv(16) whereas 10.3% had reverse transcriptase (RT)-PCR evidence of CBFB-MYH11 fusion transcripts. AML1-ETO transcripts were detected in 51/396 (12.9%) of patients studied, although only 32 (8.1%) of the patients had cytogenetic evidence of t(8;21). Both fusion transcripts were demonstrated in AML patients with FAB types other than those with which they are most commonly associated. This suggests that RT-PCR analysis for these fusion transcripts in all new cases of AML would be of diagnostic relevance. Detection of AML1-ETO is not only of value in the classification of AML but affords the opportunity to study minimal residual disease which may aid in determining a patients' response to therapy or may indicate impending relapse. The novel, rapid, RNA based transcription mediated amplification and hybridisation protection assay (TMA/HPA) were evaluated for this purpose. Probes and primers optimised for the TMA/HPA analysis of BCR-ABL transcripts were initially available and were evaluated for quantification of these transcripts in chronic myeloid leukaemia patients. ABL transcripts were simultaneously quantified and used to control for template RNA quality and assay standardisation. Oligonucleotides were then developed for the quantification of AML1-ETO transcripts and TMA/HPA methodology was subsequently applied to samples from AML patients with t(8;21). TMA/HPA was able to identify AML1-ETO positive samples at a sensitivity of < 1 in 104 cells at diagnosis and retrospectively demonstrate changes in the AML1-ETO/ABL ratio which reflected patient status or treatment, including prediction of relapse. Mutations in the DNA binding runt domain of AML1 have been reported in AML patients with the minimally differentiated FAB type M0 and in the rare familial platelet disorder with predisposition to AML (FPD/AML). PCR-single stranded conformation polymorphism (SSCP) analysis was used to screen this region for mutations in DNA samples from 41 M0 and 20 FAB type M7 (megakaryoblastic) AML patients. Two polymorphisms and six mutations were identified. Three mutations would be predicted to result in a truncated protein and 3 would alter AML1 conformation. The incidence of mutations in M0 AML patients (12%) was lower than that previously reported in 2 other similar series (24% and 33%). In addition, a germline mutation of AML1 was identified in two members of a family with FPD/AML. These studies illustrate the importance of AML1 in both normal haemopoiesis and myeloid leukaemogenesis and identify several molecular abnormalities of the CBF complex which may have a potential impact on clinical management of AML patients.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medicine