Send to

Choose Destination
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

*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.



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.


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.


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.


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.

[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wolters Kluwer
Loading ...
Support Center