title: Tandem duplication PCR: an ultrasensitive assay for the detection of internal tandem duplications of the FLT3 gene.
abstract: Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with a poor prognosis in acute myeloid leukemia. Detection of ITD-positive minor clones at the initial diagnosis and during the minimal residual disease stage may be essential. We previously designed a delta-PCR strategy to improve the sensitivity to 0.1% ITD-positive leukemia cells and showed that minor mutants with an allele burden of <1% can be clinically significant. In this study, we report on tandem duplication PCR (TD-PCR), a modified inverse PCR assay, and demonstrate a limit of detection of a few molecules of ITD mutants. The TD-PCR was initially designed to confirm ITD mutation of an amplicon, which was undetectable by capillary electrophoresis and was incidentally isolated by a molecular fraction collecting tool. Subsequently, TD-PCR detected ITD mutation in 2 of 77 patients previously reported as negative for ITD mutation by a standard PCR assay. TD-PCR can also potentially be applied to monitor minimal residual disease with high analytic sensitivity in a portion of ITD-positive acute myeloid leukemia patients. Further studies using TD-PCR to detect ITD mutants at diagnosis may clarify the clinical significance of those ITD mutants with extremely low allele burden.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Tseng
authors.forename: Katie
authors.initials: K
authors.lastname: Beierl
authors.forename: Antony
authors.initials: A
authors.lastname: Hsieh
authors.forename: Michele
authors.initials: M
authors.lastname: Thiess
authors.forename: Nadine
authors.initials: N
authors.lastname: Chase
authors.forename: Amanda
authors.initials: A
authors.lastname: Stafford
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Levis
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Gocke
mesh_terms: D020440:Gene Duplication; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics*
chemical_list: C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3