title: Screening for EGFR and KRAS mutations in non-small cell lung carcinomas using DNA extraction by hydrothermal pressure coupled with PCR-based direct sequencing.
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.
authors.affiliation: Department of Pathology, Peking University Health Science Center Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Bing-Quan
authors.initials: BQ
authors.lastname: Wu
authors.forename: Hao-Hao
authors.initials: HH
authors.lastname: Zhong
authors.forename: Pei
authors.initials: P
authors.lastname: Hui
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Fang
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D000077192:Adenocarcinoma of Lung; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001706:Biopsy; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016612:Paraffin Embedding; D010641:Phenotype; D016133:Polymerase Chain Reaction; D057285:Precision Medicine; D011237:Predictive Value of Tests; D011312:Pressure; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D016707:Tissue Fixation; D055815:Young Adult; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: EGFR; FFPE; KRAS; hydrothermal pressure; lung cancer; mutation analysis
references.citation: Oncogene. 2008 Jul 17;27(31):4336-43
references.pmid: 18372921
references.citation: Clin Cancer Res. 2009 Dec 15;15(24):7502-7509
references.pmid: 20008850
references.citation: Lung Cancer. 2010 Sep;69(3):272-8
references.pmid: 20022659
references.citation: Acta Radiol. 2008 Nov;49(9):991-4
references.pmid: 18651255
references.citation: Lancet Oncol. 2009 May;10(5):432-3
references.pmid: 19410185
references.citation: JAMA. 2004 Apr 7;291(13):1644-5
references.pmid: 15069056
references.citation: J Thorac Oncol. 2011 Jan;6(1):203-6
references.pmid: 21178716
references.citation: Cancer Res. 2004 Dec 15;64(24):8919-23
references.pmid: 15604253
references.citation: Clin Cancer Res. 2006 Dec 15;12(24):7232-41
references.pmid: 17189394
references.citation: Cancer Biomark. 2011-2012;10(2):63-9
references.pmid: 22430133
references.citation: Chest. 2007 Aug;132(2):597-602
references.pmid: 17573511
references.citation: N Engl J Med. 2009 Sep 3;361(10):947-57
references.pmid: 19692680
references.citation: Nat Rev Cancer. 2005 May;5(5):341-54
references.pmid: 15864276
references.citation: Lung Cancer. 2004 Jun;44(3):295-301
references.pmid: 15140542
references.citation: Clin Cancer Res. 2007 Sep 1;13(17):4954-5
references.pmid: 17785543
references.citation: PLoS One. 2012;7(6):e40109
references.pmid: 22768234
references.citation: Clin Cancer Res. 2006 Mar 1;12(5):1647-53
references.pmid: 16533793
references.citation: Curr Drug Targets. 2010 Jul;11(7):851-64
references.pmid: 20388064
references.citation: PLoS Med. 2005 Jan;2(1):e17
references.pmid: 15696205
references.citation: Anticancer Res. 2010 May;30(5):1667-71
references.pmid: 20592359
references.citation: Adv Anat Pathol. 2008 Jul;15(4):241-7
references.pmid: 18580100
references.citation: Diagn Mol Pathol. 2013 Mar;22(1):52-8
references.pmid: 23370427
references.citation: J Exp Clin Cancer Res. 2010 Oct 06;29:132
references.pmid: 20925915
references.citation: Clin Cancer Res. 2008 Sep 15;14(18):5731-4
references.pmid: 18794081
references.citation: J Thorac Oncol. 2010 Oct;5(10):1664-7
references.pmid: 20871266
references.citation: Clin Cancer Res. 2011 Jun 1;17(11):3812-21
references.pmid: 21531810
references.citation: Acta Radiol. 2011 Dec 1;52(10):1083-7
references.pmid: 22006985
references.citation: Pathol Oncol Res. 2008 Dec;14(4):373-9
references.pmid: 18985444