Ming-Tseh Lin1, Li-Hui Tseng1,2, Jonathan C Dudley1,3, Stacey Riel1, Harrison Tsai1, Gang Zheng1, Keith W Pratz4, Mark J Levis4, Christopher D Gocke5,6. 1. Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA. 2. Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan. 3. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. 4. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 5. Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA. cgocke1@jhmi.edu. 6. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. cgocke1@jhmi.edu.
Abstract
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.
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 AMLpatients. 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.
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