Assessment for the presence of fms-like tyrosine kinase 3 (FLT3) and nucleophosmin protein-1 (NPM1) mutations and their association with immunophenotypic markers in de novo Acute Myeloid Leukaemia (AML) diagnosed at a single academic centre

Abstract

Introduction: Acute Myeloid Leukaemia (AML) is known to be a heterogeneous clonal disorder of haemopoietic progenitor cells. Recently, the molecular pathogenesis of AML has become more apparent, with the description of two commonly found mutations, namely NPM1 (Nucleophosmin protein-1) gene mutation and the FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplication) mutation. These mutations are now routinely assessed and used in both prognostication and treatment decisions in the first world. The primary aim of this project was to assess for the presence of NPM1 and FLT3- ITD mutations in patients diagnosed with de novo AML at Charlotte Maxeke Johannesburg Academic Hospital. In order to achieve this aim certain objectives were completed. This included the establishment of a detection method for both NPM1 and FLT3-ITD mutations by means of PCR as well as determining the overall number of these mutations in the study population and assessing the frequency of these mutations alone and in combination. Correlation with previous immunophenotypic analysis, cytogenetics and other routine parameters was also assessed. Materials and Methods: A total of 164 cases of de novo AML were collected retrospectively over time period September 2004 to December 2009. A robust method for the detection of both NPM1 and FLT3-ITD mutations was established using genomic (g) DNA, extracted from blood or bone marrow aspirate samples. Two single PCR reactions were performed, with final analysis by means of capillary electrophoresis using the ABI 3130 Genetic Analyser. Results: Of the 164 cases analysed, NPM1 mutations were found in 26%, while FLT3-ITD was present in 21% of these cases. FLT3-ITD showed preferential targeting of the NPM1 mutation positive cases with 44% of these cases also found to be NPM1 mutation positive (P<0.04). Routine parameter assessment revealed some novel findings. The median age at diagnosis for the NPM1 mutation positive only cases was 30 years; this is younger than expected. The median WCC of the FLT3-ITD only cases was 101x109/l which was significantly increased compared to the NPM1 mutation only cases (P<0.03) and those with neither mutation present (P<0.01). Immunophenotypic analysis revealed a statistically significant increase in CD14, CD11b and HLA-DR (human leucocyte antigen DR) in NPM1-mut positive samples. When assessing CD34 expression and NPM1-mut. NPM1-wt cases were significantly more likely to be CD34 positive than NPM1-mut cases. Recurrent genetic translocations were found to co-occur with NPM1 mutations. This, in particular is a rare finding which requires further investigation. Conclusion: This much needed test has proven to be robust in its detection of these gene mutations. It will certainly prove clinically relevant, especially as the use of molecularly targeted therapy becomes part of routine treatment.