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Appl Immunohistochem Mol Morphol. 2014;22(2):114-8. doi: 10.1097/PDM.0b013e31829a638d.

The "COLD-PCR approach" for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer.

Author information

1
*Institute of Pathology and Neuropathology, University Hospital Essen, University of Duisburg-Essen ∥Department of Interventional Pneumology, Ruhrlandklinik-University Hospital, Essen †Institute of Pathology, University Hospital Cologne, Cologne Departments of ‡Pathology ¶Pneumology, Helios Klinikum Emil von Behring §TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany.

Abstract

BACKGROUND:

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies.

MATERIALS AND METHODS:

Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing.

RESULTS:

COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible.

CONCLUSIONS:

COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

PMID:
24517913
DOI:
10.1097/PDM.0b013e31829a638d
[Indexed for MEDLINE]

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