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

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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*,‡

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Diagnostic Molecular Pathology 22(3):p 149-155, September 2013. | DOI: 10.1097/PDM.0b013e31828308a1


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.

© 2013 by Lippincott Williams & Wilkins.

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