title: A Novel Tandem Duplication Assay to Detect Minimal Residual Disease in FLT3/ITD AML.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
authors.affiliation: Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.|Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Tseng
authors.affiliation: Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.|Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Dudley
authors.affiliation: Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Riel
authors.affiliation: Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.
authors.forename: Harrison
authors.initials: H
authors.lastname: Tsai
authors.affiliation: Division of Molecular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe Street, Baltimore, MD, 21287, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Pratz
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Levis
authors.affiliation: 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.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. cgocke1@jhmi.edu.
authors.forename: Christopher D
authors.identifier: http://orcid.org/0000-0002-9541-2192
authors.initials: CD
authors.lastname: Gocke
mesh_terms: D004252:DNA Mutational Analysis; D020440:Gene Duplication; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D018365:Neoplasm, Residual; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3