FLT3 TESTING IN THE RYDAPT ERA
Author(s): ,
Stuart Scott
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom;Department of Oncology and Metabolism,University of Sheffield,Sheffield,United Kingdom
,
Ashley Cartwright
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom
,
Debbie Travis
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom
,
Annie Tapley
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom
,
Habebah Khan
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom
,
David Barnett
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom;Department of Oncology and Metabolism,University of Sheffield,Sheffield,United Kingdom
Liam Whitby
Affiliations:
UK NEQAS for Leucocyte Immunophenotyping,Sheffield Teaching Hospitals,Sheffield,United Kingdom
EHA Library. Scott S. Jun 15, 2019; 266655; PS1038
Mr. Stuart Scott
Mr. Stuart Scott
Contributions
Abstract

Abstract: PS1038

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

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 30-126bp, 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 (126bp). 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.

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.

Session topic: 4. Acute myeloid leukemia - Clinical

Keyword(s): Acute myeloid leukemia, Genetic, Quantitative molecular analysis

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