Studies on the core binding factor complex in patients with acute myeloid leukaemia.

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 a subunit AMLl (CBFA2/RUNX1) and the p subunit CBFB, which interacts with AMLl to increase DNA binding. The AMLl 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) (pl3q22) result in the creation of AMLl-ETO and CBFB-MYHll 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-M YHll fusion transcripts. AMLl-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 AMLl-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 AMLl-ETO transcripts and TMA/HPA methodology was subsequently applied to samples from AML patients with t(8;21). TMA/HPA was able to identify AMLl-ETO positive samples at a sensitivity of <1 in 10"̂ cells at diagnosis and retrospectively demonstrate changes in the AMLl-ETO/ABL ratio which reflected patient status or treatment, including prediction of relapse. Mutations in the DNA binding runt domain of AM Ll have been reported in AML patients with the minimally differentiated FAB type MO 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 MO 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 AMLl conformation. The incidence of mutations in MO AML patients (12%) was lower than that previously reported in 2 other similar series (24% and 33%). In addition, a germline mutation of AMLl was identified in two members of a family with FPD/AML. These studies illustrate the importance of AMLl 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.