Targeted sequencing with enrichment PCR

a novel diagnostic method for the detection of EGFR mutations

Suki Kang, Baek Gil Kim, Hyun Ho Han, Joo Hyun Lee, Ji Eun Kim, Hyo Sup Shim, Namhoon Cho

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01% and 0.05%, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692% in tissue samples. EGFR mutations with frequencies as low as 0.01% were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA.

Original languageEnglish
Pages (from-to)13742-13749
Number of pages8
JournalOncotarget
Volume6
Issue number15
DOIs
Publication statusPublished - 2015 May 30

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Epidermal Growth Factor Receptor
Polymerase Chain Reaction
Mutation
Mutation Rate
Tumor Cell Line
Constriction
Protein-Tyrosine Kinases
Neoplasms
Cell Survival
Cell Proliferation
DNA

All Science Journal Classification (ASJC) codes

  • Oncology

Cite this

Kang, Suki ; Kim, Baek Gil ; Han, Hyun Ho ; Lee, Joo Hyun ; Kim, Ji Eun ; Shim, Hyo Sup ; Cho, Namhoon. / Targeted sequencing with enrichment PCR : a novel diagnostic method for the detection of EGFR mutations. In: Oncotarget. 2015 ; Vol. 6, No. 15. pp. 13742-13749.
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abstract = "Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01{\%} and 0.05{\%}, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692{\%} in tissue samples. EGFR mutations with frequencies as low as 0.01{\%} were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA.",
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Targeted sequencing with enrichment PCR : a novel diagnostic method for the detection of EGFR mutations. / Kang, Suki; Kim, Baek Gil; Han, Hyun Ho; Lee, Joo Hyun; Kim, Ji Eun; Shim, Hyo Sup; Cho, Namhoon.

In: Oncotarget, Vol. 6, No. 15, 30.05.2015, p. 13742-13749.

Research output: Contribution to journalArticle

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