title: A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening.
abstract: OBJECTIVE
  Mutations in the epidermal growth factor receptor (EGFR) are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Currently available methods of EGFR mutation detection rely on direct DNA sequencing, which requires isolation of DNA from a relatively pure population of tumor cells, cannot be done on small diagnostic specimens, and lack sensitivity. Here we describe the use of a sensitive screening method that overcomes many of these limitations.
  
  
  METHODS
  We screened 178 non-small cell lung cancer specimens for mutations in exons 18 to 21 of EGFR using a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA. Samples were analyzed by high-performance liquid chromatography on the Transgenomic WAVE HS system. Selected specimens that produced digestion products using SURVEYOR were subsequently reanalyzed by size separation or under partially denaturing conditions, followed by fractionation and sequencing. The specimens included DNA isolated from frozen tumor specimens, dissected formalin-fixed, paraffin-embedded tumor specimens undergoing clinical sequencing, and undissected formalin-fixed, paraffin-embedded specimens. One hundred sixty specimens were independently analyzed using direct DNA sequencing in a blinded fashion.
  
  
  RESULTS
  EGFR mutations were detected in 16 of 61 fresh frozen tumor specimens, 24 of 91 dissected formalin-fixed, paraffin-embedded tumor specimens, and 11 of 26 undissected formalin-fixed, paraffin-embedded tumor specimens. Compared with sequencing, the sensitivity and specificity of the present method were 100% and 87%. The positive and negative predictive values were 74% and 100%, respectively. SURVEYOR analysis detected 7 (4%) mutations that were not previously detected by direct sequencing.
  
  
  CONCLUSIONS
  SURVEYOR analysis provides a rapid method for EGFR mutation screening with 100% sensitivity and negative predictive value. This unbiased scanning technique is superior to direct sequencing when used with undissected formalin-fixed, paraffin-embedded specimens.
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. pjanne@partners.org
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
authors.forename: Ana M
authors.initials: AM
authors.lastname: Borras
authors.forename: Yanan
authors.initials: Y
authors.lastname: Kuang
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Rogers
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Joshi
authors.forename: Hema
authors.initials: H
authors.lastname: Liyanage
authors.forename: Neal
authors.initials: N
authors.lastname: Lindeman
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Lee
authors.forename: Balazs
authors.initials: B
authors.lastname: Halmos
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Maher
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Distel
authors.forename: Matthew
authors.initials: M
authors.lastname: Meyerson
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Johnson
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000737:chemistry / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D009154:Mutation; D016612:Paraffin Embedding; D011237:Predictive Value of Tests; D011799:Quinazolines / Q000627:therapeutic use; D012680:Sensitivity and Specificity
chemical_list: D004273:DNA, Neoplasm; D011799:Quinazolines; D005557:Formaldehyde; D066246:ErbB Receptors; D003850:Deoxyribonuclease I; D000077156:Gefitinib