A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening

Pasi A. Jänne, Ana M. Borras, Yanan Kuang, Andrew M. Rogers, Victoria A. Joshi, Hema Liyanage, Neal Lindeman, Jeffrey C. Lee, Balazs Halmos, Elizabeth A. Maher, Robert J. Distel, Matthew Meyerson, Bruce E. Johnson

Research output: Contribution to journalArticle

183 Citations (Scopus)

Abstract

Purpose: 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. Experimental Design: 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.

Original languageEnglish (US)
Pages (from-to)751-758
Number of pages8
JournalClinical Cancer Research
Volume12
Issue number3 I
DOIs
StatePublished - Feb 1 2006
Externally publishedYes

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Epidermal Growth Factor Receptor
Paraffin
Formaldehyde
Mutation
Neoplasms
DNA Sequence Analysis
DNA
Deoxyribonuclease I
Non-Small Cell Lung Carcinoma
Protein-Tyrosine Kinases
Digestion
Exons
Research Design
High Pressure Liquid Chromatography
Sensitivity and Specificity
Population

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Jänne, P. A., Borras, A. M., Kuang, Y., Rogers, A. M., Joshi, V. A., Liyanage, H., ... Johnson, B. E. (2006). A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clinical Cancer Research, 12(3 I), 751-758. https://doi.org/10.1158/1078-0432.CCR-05-2047

A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. / Jänne, Pasi A.; Borras, Ana M.; Kuang, Yanan; Rogers, Andrew M.; Joshi, Victoria A.; Liyanage, Hema; Lindeman, Neal; Lee, Jeffrey C.; Halmos, Balazs; Maher, Elizabeth A.; Distel, Robert J.; Meyerson, Matthew; Johnson, Bruce E.

In: Clinical Cancer Research, Vol. 12, No. 3 I, 01.02.2006, p. 751-758.

Research output: Contribution to journalArticle

Jänne, PA, Borras, AM, Kuang, Y, Rogers, AM, Joshi, VA, Liyanage, H, Lindeman, N, Lee, JC, Halmos, B, Maher, EA, Distel, RJ, Meyerson, M & Johnson, BE 2006, 'A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening', Clinical Cancer Research, vol. 12, no. 3 I, pp. 751-758. https://doi.org/10.1158/1078-0432.CCR-05-2047
Jänne, Pasi A. ; Borras, Ana M. ; Kuang, Yanan ; Rogers, Andrew M. ; Joshi, Victoria A. ; Liyanage, Hema ; Lindeman, Neal ; Lee, Jeffrey C. ; Halmos, Balazs ; Maher, Elizabeth A. ; Distel, Robert J. ; Meyerson, Matthew ; Johnson, Bruce E. / A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. In: Clinical Cancer Research. 2006 ; Vol. 12, No. 3 I. pp. 751-758.
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abstract = "Purpose: 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. Experimental Design: 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.",
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AU - Borras, Ana M.

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AU - Joshi, Victoria A.

AU - Liyanage, Hema

AU - Lindeman, Neal

AU - Lee, Jeffrey C.

AU - Halmos, Balazs

AU - Maher, Elizabeth A.

AU - Distel, Robert J.

AU - Meyerson, Matthew

AU - Johnson, Bruce E.

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N2 - Purpose: 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. Experimental Design: 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.

AB - Purpose: 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. Experimental Design: 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.

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