Tandem duplication PCR: An ultrasensitive assay for the detection of internal tandem duplications of the FLT3 Gene

Ming Tseh Lin, Li Hui Tseng, Katie Beierl, Antony Hsieh, Michele Thiess, Nadine Chase, Amanda Stafford, Mark J. Levis, James R. Eshleman, Christopher D. Gocke

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)149-155
Number of pages7
JournalDiagnostic Molecular Pathology
Volume22
Issue number3
DOIs
StatePublished - Sep 1 2013

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Keywords

  • FLT3
  • acute myeloid leukemia
  • internal tandem duplication
  • minimal residual disease
  • tandem duplication PCR

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

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