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Tandem Duplication PCR: An Ultrasensitive Assay for the Detection of Internal Tandem Duplications of the FLT3 Gene

Lin, Ming-Tseh MD, PhD*; Tseng, Li-Hui MD, PhD*,†; Beierl, Katie BS*; Hsieh, Antony BS*; Thiess, Michele BS*; Chase, Nadine BS*; Stafford, Amanda MS*; Levis, Mark J. MD; Eshleman, James R. MD, PhD*,‡; Gocke, Christopher D. MD*,‡

Diagnostic Molecular Pathology:
doi: 10.1097/PDM.0b013e31828308a1
Original Articles
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.

Author Information

Departments of *Pathology

Oncology, Johns Hopkins University School of Medicine, Baltimore, MD

Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan

Supported in part by R21HG004315 and R21HG005745 from National Institutes of Health.

The authors declare no conflict of interest.

Reprints: Christopher D. Gocke, MD, Department of Pathology, Johns Hopkins University School of Medicine, Park SB202, 600 North Wolfe St., Baltimore, MD 21287 (e-mail: cgocke1@jhmi.edu).

© 2013 by Lippincott Williams & Wilkins.