PS1038 FLT3 TESTING IN THE RYDAPT ERA
Authors: S. Scott, A. Cartwright, D. Travis, A. Tapley, H. Khan, D. Barnett, L. Whitby
Journal: HemaSphere (2019)

Background: The approval of Rydapt (midostaurin) heralds the first targeted therapy in acute myeloid leukaemia (AML). Accurate detection of both FLT3 internal tandem duplications (ITD) and tyrosine kinase domain (TKD) variants is required to ensure that patients correctly receive the therapy. Additionally, recent European LeukaemiaNet AML guidelines now require the testing of a number of genetic markers including FLT3 to risk stratify patients. Aims: With the increased importance of FLT3 testing for the diagnosis, prognosis and treatment of AML, the aims of this study were to assess methodological variation and the quality of laboratory testing of FLT3 ITD variants. Methods: The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI), has been providing external quality assessment (EQA) to laboratories undertaking FLT3 ITD testing by molecular genetic methods since 2011, and became IS0 17043 accredited in 2014. The programme currently has 130 active participants in 36 countries globally. Two lyophilised patient donated or cell line based samples are shipped to laboratories 3 times per annum and participants are asked to test the samples with their in‐house techniques, reporting both qualitative (ITD present/absent) and quantitative (mutation load/allelic ratio) data along with a number of methodological details. Results: Forty samples (including samples both positive and negative for the FLT3 ITD) have been issued to laboratories, in 20 separate distributions, since 2011. Positive samples have featured ITD sizes ranging from 30–126 bp, with mutation loads ranging from 10–90%. The most popular analysis methods used by participants were capillary electrophoresis (70.8%) and agarose gel electrophoresis (20.0%), with a small number of participant using next generation sequencing (NGS), melt curve analysis, acrylamide gel electrophoresis, microfluidic electrophoresis and Sanger sequencing. The majority of laboratories use laboratory developed (in‐house) tests (85.8%), with the remaining participants using commercially available assays. Concordance rates ranged from 83.3% to 100% (average 97.5%). Higher error rates were associated with low mutation load samples and larger ITDs (126 bp). High error rates were associated with gel electrophoresis and NGS based methods. Quantitatively, all laboratories were able to distinguish a high (median mutation load = 92%) and low (median mutation load = 11.7%) mutation load sample with a high degree of concordance (interquartile range = 2.5% and 3.7%, respectively), but variation in calculation methods used were identified. Summary/Conclusion: The quality of FLT3 testing was generally high, although a higher error rate was associated with low mutation load samples and large ITDs when participants used agarose gel or NGS based techniques. Gel electrophoresis is an inherently variable, outdated method and its use should not be encouraged. Standard NGS analysis software has difficulty in detecting large ITDs, as large insertions do not retain sufficient homology in the regions flanking the insertion to permit accurate alignment. Pair‐end based analysis approaches have been developed that can reliably detect larger insertions. The high degree of concordance seen in quantitative data was reassuring but could be improved further by standardising calculation methods.