title: EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens.
abstract: BACKGROUND
  Mutational analysis of the Epidermal Growth Factor Receptor (EGFR) and K-ras genes to select non-small cell lung cancer (NSCLC) patients for treatment with novel EGFR tyrosine kinase inhibitors is an appealing possibility currently under investigation. Although frozen tumor tissue would probably be the optimal source for analysis, the most common source of tumor material is fixed and paraffin embedded (FPE) archival specimens. Here, we evaluate how different procedures of tissue sample processing and preservation may affect the outcome of EGFR and K-ras mutation analysis. Furthermore, we compare the sensitivity of the analysis using genomic DNA (gDNA) versus RNA.
  
  
  METHODS
  We used PCR amplification and direct sequencing to analyze EGFR and K-ras genes in paired FPE and frozen tumor samples corresponding to 47 NSCLC patients. In frozen samples, the analysis was carried out using both gDNA and RNA extracted in parallel.
  
  
  RESULTS
  Whereas 100% of frozen samples were successfully amplified, the rate of successful PCR amplification in FPE samples was approximately 50%. We detected three previously described EGFR point mutations in 2 samples. In ten other samples, a K-ras mutation was observed. These mutations were detected in DNA extracted from frozen samples as well as in DNA obtained from FPE tissue. In addition, 10 nucleotide changes, were detected in FPE samples that were not detected in the frozen specimens. Upon re-analysis, these nucleotide changes could not be confirmed and were most likely the result of paraffin embedding and fixation procedures. All mutations found in gDNA were also detected in the corresponding RNA and, in two cases, the presence of the mutant allele was easier to identify by using RNA.
  
  
  CONCLUSIONS
  Our results indicate that RNA extracted from frozen tissue is the preferred source for EGFR and K-ras mutation testing. When analyzing FPE samples, reducing the size of the amplified fragments would increase PCR success rate, and care should be taken to control for false-positive results.
authors: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors: Mariëlle I
authors: MI
authors: Gallegos Ruiz
authors: Karijn
authors: K
authors: Floor
authors: Frank
authors: F
authors: Rijmen
authors: Katrien
authors: K
authors: Grünberg
authors: José A
authors: JA
authors: Rodriguez
authors: Giuseppe
authors: G
authors: Giaccone
mesh_terms: D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D066246:ErbB Receptors / Q000235:genetics*; D005404:Fixatives; D005629:Frozen Sections; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; D005404:Fixatives; D012334:RNA, Neoplasm; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras)