Development of a Human Acute Myeloid Leukaemia Screening Panel and Identification of Novel Gene Mutations.

Mutation studies in Acute Myeloid Leukaemia (AML) are complicated by the existence of distinct morphological and cytogenetic subtypes; consequently although mutations in any one gene may occur in only 5% of AML, the frequency of mutation may differ both between the different FAB-types and cytogenetic risk groups studied. It is important therefore not only to validate the screening methodology used but also the suitability of the patient panel tested. A screening panel was developed allowing detection of novel recurring gene mutations within samples derived from patients with AML. Mutation analysis of 6 previously described genes (RUNX1, FLT3, KIT, CEBPA, PTPN11, NRAS) and 2 candidate genes (CCND3, FES) were carried out in a cohort of 175 AML samples representing all FAB types (except M3) and cytogenetic risk groups using a combination of SSCP, DHPLC and sequence analysis. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with 2 mutations, and 2 patients (1%) with 3 mutations. Fifty-five out of 88 (63%) patients with normal karyotype AML had at least one mutation. There was was a weak negative association between FLT3 ITD and loop mutation (p = 0.095), a positive association between KIT mutation and favourable risk cytogenetics (p = 0.001), CEBPA mutation and intermediate risk/normal cytogenetics (p = 0.045) and PTPN11 mutation and poor risk disease (p = 0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 (Y589D, D839G, Y842H) were detected in 4 patients that would have been overlooked by conventional gel electrophoresis techniques. A single in frame 51bp deletion of nucleotide 939 – 990, resulting in a deletion of 17 amino acids at the carboxyl-terminus of the cyclin D3 protein was identified in a single patient. Overall, both the pattern and mutation frequencies reported in this cohort are similar to those in the literature supporting its further use as an investigational tool in the evaluation of candidate genes in the genesis of myeloid malignancy.