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Int J Clin Exp Pathol 2013;6(9):1880-1889
Original Article
Screening for EGFR and KRAS mutations in non-small cell lung carcinomas using DNA
extraction by hydrothermal pressure coupled with PCR-based direct sequencing
Yan Liu, Bing-Quan Wu, Hao-Hao Zhong, Pei Hui, Wei-Gang Fang
Department of Pathology, Peking University Health Science Center, Beijing, China; Department of Pathology, Yale University School of
Medicine, New Haven, CT, USA
Received July 26, 2013; Accepted August 16, 2013; Epub August 15, 2013; Published September 1, 2013
Abstract: EGFR and KRAS mutations correlate with response to tyrosine kinase inhibitors in patients with non-small cell lung carcinoma
(NSCLC). We reported a hydrothermal pressure method of simultaneous deparaffinization and lysis of formalin-fixed paraffin embedded
(FFPE) tissue followed by conventional chaotropic salt column purification to obtain high quality DNA for mutation analysis using PCR-base
direct sequencing. This study assessed the feasibility of using this method to screen for exons 18-21 of EGFR and exon 2 of KRAS gene
mutations in surgical resection and core needle biopsy specimens from 251 NSCLC patients. EGFR mutations were identified in 140 (55.8%)
NSCLC patients (118 in adenocarcinoma, 11 in squamous cell carcinoma, 7 in adenocarcinoma and 4 in NSCLC-not otherwise specified),
including four novel substitutions (L718M, A743V, L815P, V819E). EGFR mutations were frequently present in female patients (72 of 113,
63.7%) and NSCLC with adenocarcinoma component (125/204, 61.3%) with statistical significance. Twenty-one patients had multiple
mutations at different exons of EGFR, in which seventeen patients had deletions in exon 19. KRAS mutations were found in 18 (7.2%) patients
(15 in adenocarcinoma, 2 in squamous cell carcinoma and one in NSCLC-not otherwise specified), including an uncommon substitution
G13C. Deparaffinization and lysis by hydrothermal pressure, coupled with purification and PCR-based sequencing, provides a robust
screening approach for EGFR and KRAS mutation analysis of FFPE tissues from either surgical resection or core needle biopsy in clinical
personalized management of lung cancer. (IJCEP1307047).
Keywords: EGFR, KRAS, FFPE, hydrothermal pressure, lung cancer, mutation analysis
Address correspondence to: Dr. Wei-Gang Fang, Department of Pathology, Peking University Health Science Center, Beijing 100191, China.
Tel: 86-10-82802599; Fax: 86-10-62015681; E-mail: wgfang@bjmu.edu.cn
Original Article
Screening for EGFR and KRAS mutations in non-small cell lung carcinomas using DNA
extraction by hydrothermal pressure coupled with PCR-based direct sequencing
Yan Liu, Bing-Quan Wu, Hao-Hao Zhong, Pei Hui, Wei-Gang Fang
Department of Pathology, Peking University Health Science Center, Beijing, China; Department of Pathology, Yale University School of
Medicine, New Haven, CT, USA
Received July 26, 2013; Accepted August 16, 2013; Epub August 15, 2013; Published September 1, 2013
Abstract: EGFR and KRAS mutations correlate with response to tyrosine kinase inhibitors in patients with non-small cell lung carcinoma
(NSCLC). We reported a hydrothermal pressure method of simultaneous deparaffinization and lysis of formalin-fixed paraffin embedded
(FFPE) tissue followed by conventional chaotropic salt column purification to obtain high quality DNA for mutation analysis using PCR-base
direct sequencing. This study assessed the feasibility of using this method to screen for exons 18-21 of EGFR and exon 2 of KRAS gene
mutations in surgical resection and core needle biopsy specimens from 251 NSCLC patients. EGFR mutations were identified in 140 (55.8%)
NSCLC patients (118 in adenocarcinoma, 11 in squamous cell carcinoma, 7 in adenocarcinoma and 4 in NSCLC-not otherwise specified),
including four novel substitutions (L718M, A743V, L815P, V819E). EGFR mutations were frequently present in female patients (72 of 113,
63.7%) and NSCLC with adenocarcinoma component (125/204, 61.3%) with statistical significance. Twenty-one patients had multiple
mutations at different exons of EGFR, in which seventeen patients had deletions in exon 19. KRAS mutations were found in 18 (7.2%) patients
(15 in adenocarcinoma, 2 in squamous cell carcinoma and one in NSCLC-not otherwise specified), including an uncommon substitution
G13C. Deparaffinization and lysis by hydrothermal pressure, coupled with purification and PCR-based sequencing, provides a robust
screening approach for EGFR and KRAS mutation analysis of FFPE tissues from either surgical resection or core needle biopsy in clinical
personalized management of lung cancer. (IJCEP1307047).
Keywords: EGFR, KRAS, FFPE, hydrothermal pressure, lung cancer, mutation analysis
Address correspondence to: Dr. Wei-Gang Fang, Department of Pathology, Peking University Health Science Center, Beijing 100191, China.
Tel: 86-10-82802599; Fax: 86-10-62015681; E-mail: wgfang@bjmu.edu.cn
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