The most common single mutation observed in approximately one-third of AML cases, is an activating mutation of the tyrosine kinase receptor FLT3. The presence of the mutation is associated with adverse prognosis in terms of increased risk of disease relapse. The majority of mutations (25–35% of AML patients) occur as an internal tandem duplication (ITD) within the juxtamembrane region. A further 3–7% of patients have a mutation within the tyrosine kinase domain (TKD) of FLT3. The type of mutation, and its population size, may have prognostic implications. We have used the Agilent 2100 Bioanalyser to assay PCR reactions for the ITD and the TKD mutations. The “chips” used within the Bioanalyser separate RNA or DNA through glass etched micro channels filled with gel matrix, nucleic acid is stained and laser detected. The PCR primers and protocol for the ITD analysis were as previously published (
Kottaridis et al. Blood. 98, 2001
), but within our laboratory the reactions were analysed on the Bioanalyser using a DNA 500 chip. We detected the presence of an ITD in 19.9% of the 291 AML samples, with percent of mutant RNA varying from 2.9% to 97.5% (median 27%) and with the size of the ITD varying from 15–212 bp (median 41 bp). A separate PCR reaction using primers flanking the TKD domain was used, in combination with an Eco RV or Hinf I (Smith et al, MTAL Workshop, 2003) digestion, to detect and quantitate the different mutations. Distinctive digestion and/or heteroduplex patterns were observed that identified D835, D835del, I836del or D839G mutation. TKD mutations were observed in 12.9% (10.1% D835 mutations, 1.4% I836del, 1.4% D839G) of samples examined, with the mutant RNA ranging from 1.0% to 52.8%. The D835/6 mutation rate was higher than previously reported and may be due to the efficiency and sensitivity of the 2100 Bioanalyser for the analysis of FLT3 mutations. This gave an overall FLT3 mutation rate in the AML population studied of approximately 32.8%; females had a higher overall incidence of 39.1% compared to 27.2% for males. The samples were derived from two NCI trials, AML 14 for patients over 60 years old and AML15 for younger patients. The rate of ITD mutations was 15.3% in AML14 patients compared to 27.5% in AML15, although the incidence of TKD mutations was similar (13.5% v 14.3%). The incidence of ITD mutations was over 45% in patients in the 30–40 year age group at diagnosis. A large percentage of these patients have also been gene expression profiled and the molecular effect of the FLT3 ITD mutations across the age groups has also been examined.
