title: The "COLD-PCR approach" for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer.
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.
  
  
  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.
authors: *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.
authors: Fabian D
authors: FD
authors: Mairinger
authors: Claudia
authors: C
authors: Vollbrecht
authors: Anna
authors: A
authors: Streubel
authors: Andreas
authors: A
authors: Roth
authors: Olfert
authors: O
authors: Landt
authors: Henry F R
authors: HF
authors: Walter
authors: Jens
authors: J
authors: Kollmeier
authors: Thomas
authors: T
authors: Mairinger
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics; D003080:Cold Temperature; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D003955:Diagnostic Tests, Routine; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D055088:Early Detection of Cancer; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D058996:Quality Improvement; D015203:Reproducibility of Results
chemical_list: D047428:Protein Kinase Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors