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
Date
2013-03-05
Authors
Marshall, Robyn Cara
Journal Title
Journal ISSN
Volume Title
Publisher
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.