Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

• Published in: British journal of cancer Vol. 100; no. 6; pp. 985 - 992
• Main Authors: Beau-Faller, M, Legrain, M, Voegeli, A-C, Guérin, E, Lavaux, T, Ruppert, A-M, Neuville, A, Massard, G, Wihlm, J-M, Quoix, E, Oudet, P, Gaub, M P
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24.03.2009; Nature Publishing Group; Cancer Research UK
• Subjects: Aged; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Cancer; Cancer Research; Cancer therapies; Carcinoma, Non-Small-Cell Lung; Carcinoma, Non-Small-Cell Lung - genetics; Chemotherapy; Drug Resistance; Epidemiology; ErbB Receptors - antagonists & inhibitors; Female; Genes, ras; Humans; Life Sciences; Lung cancer; Lung Neoplasms; Lung Neoplasms - genetics; Male; Medical research; Medical sciences; Middle Aged; Molecular Diagnostics; Molecular Medicine; Mutation; Nucleic Acid Hybridization; Oncology; Peptide Nucleic Acids; Peptide Nucleic Acids - genetics; Pneumology; Polymerase Chain Reaction; Polymerase Chain Reaction - methods; Proto-Oncogene Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins p21(ras); ras Proteins; ras Proteins - genetics; Receptor, Epidermal Growth Factor; Sensitivity and Specificity; Signal transduction; Tumors; Tumors of the respiratory system and mediastinum; France; non-small cell lung cancer; PNA; mutations; sensitivity; real-time PCR; Human; Lung disease; Respiratory disease; Lung cancer; Malignant tumor; non-small cell lung carcinoma; Real time; K ras Gene; Polymerase chain reaction; Sensitivity; Cancerology; C-Onc gene; Bronchus disease; Diagnosis; Mutation; Molecular biology; Ras gene; K-Ras mutations; Protooncogene; Cancer
• ISSN: 0007-0920; 1532-1827; 1532-1827
• DOI: 10.1038/sj.bjc.6604925

Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P <0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR ( P <0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.