A Novel Tandem Duplication Assay to Detect Minimal Residual Disease in FLT3/ITD AML
Ming-Tseh Lin1, Li-Hui Tseng1,2, Jonathan C Dudley1,3
1Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.
Background:
Internal tandem duplication (ITD) of the fms-related tyrosine kinase 3 (FLT3) gene is associated with a poor prognosis in acute myeloid leukemia (AML) patients with a normal karyotype. The current standard polymerase chain reaction (PCR) assay for FLT3/ITD detection is not sufficiently sensitive to monitor minimal residual disease (MRD). Clone-specific assays may have sufficient sensitivity but are not practical to implement, since each clone-specific primer/probe requires clinical validation.
Objective:
To develop an assay for clinical molecular diagnostics laboratories to monitor MRD in FLT3/ITD AMLs.
Methods:
We designed a simple novel assay, tandem duplication PCR (TD-PCR), and tested its sensitivity, specificity, and clinical utility in FLT3/ITD AML patients.
Results:
TD-PCR was capable of detecting a single ITD molecule and was applicable to 75 % of ITD mutants tested. TD-PCR detected MRD in bone marrow prior to patient relapse. TD-PCR also identified low-level ITD mutants not only in FLT3/ITD AMLs but also in initial diagnostic specimens that were reportedly negative by the standard assay in patients who progressed with the same ITDs detected by the TD-PCR assay.
Conclusion:
Detection of MRD by TD-PCR may guide patient selection for early clinical intervention. In contrast to clone-specific approaches, the TD-PCR assay can be more easily validated for MRD detection in clinical laboratories because it uses standardized primers and a universal positive control. In addition, our findings on multi-clonality and low-level ITDs suggest that further studies are warranted to elucidate their clinical/biological significance.
