title: Results of first proficiency test for KRAS testing with formalin-fixed, paraffin-embedded cell lines in China.
abstract: BACKGROUND
  Laboratory testing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutations in metastatic colorectal cancer (mCRC) is performed by various methods in China, but there is no standardized system for proficiency testing or assay performance evaluations. The aim of this study was to evaluate assay and laboratory performance with artificial samples derived from formalin-fixed, paraffin-embedded (FFPE) cell lines.
  
  
  METHODS
  Artificial FFPE samples were prepared from cultured cell lines to construct a proficiency panel of 10 samples covering eight KRAS mutations and two wild-type samples. The samples were validated by Sanger sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The panel was distributed to participating laboratories and their reported results were compared to the reference sequences.
  
  
  RESULTS
  The percentages of mutant KRAS alleles in each mutant sample were more than 50% by MALDI-TOF-MS. Sixty-three laboratories reported results, including 41 hospital laboratories and 22 commercial laboratories and reagent manufacturers. Only 55.6% (35/63) of the laboratories correctly identified the mutations in all samples and 33.3% (21/63) reported at least one false-positive result. The false-positive ratio was 7.1% (45/630) and the false-negative ratio was 3.0% (15/504).
  
  
  CONCLUSIONS
  KRAS mutations can be missed even by the most sensitive methods if the procedures are not performed correctly. False-positive results are a substantial problem in KRAS testing; laboratories must use sufficient negative controls to identify cross-contamination from PCR-amplified products or between samples during handling and DNA extraction.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Yanxi
authors.initials: Y
authors.lastname: Han
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.forename: Liang
authors.initials: L
authors.lastname: Ma
authors.forename: Yulong
authors.initials: Y
authors.lastname: Li
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
mesh_terms: D045744:Cell Line, Tumor; D002681:China; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology; D004252:DNA Mutational Analysis / Q000592:standards; D005189:False Positive Reactions; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D007753:Laboratories; D059021:Laboratory Proficiency Testing; D009154:Mutation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000592:standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000592:standards; D018631:ras Proteins / Q000032:analysis* / Q000235:genetics
chemical_list: D005557:Formaldehyde; D018631:ras Proteins