Usefulness of peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) for clinical diagnosis of KRAS codon 12 mutations in lung adenocarcinoma: comparison of PNA-clamp SmartAmp2 and PCR-related methods

Abstract

KRAS is an oncogene that can be activated by mutations. Patients with non-small cell lung cancer who have KRAS mutations do not respond to tyrosine kinase inhibitors; therefore, accurate detection of KRAS mutations is important for deciding therapeutic strategies. Although sequencing-related techniques have been frequently used, they are usually too complex, have low sensitivity, and are time-consuming for routine screening in clinical situations. We evaluated peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) as a detection method for KRAS codon 12 mutations in patient specimens compared with traditional sequencing and polymerase chain reaction-related methods. Among 172 lung adenocarcinoma samples, direct sequencing, enzyme-enriched sequencing, and PNA-enriched sequencing showed that 16 (9.3%), 26 (15.7%), and 28 (16.3%) tumors, respectively, contained KRAS mutations in codon 12. Using PNA-clamp SmartAmp2, we could identify 31 (18.0%) tumors that had KRAS mutations in codon 12 within 60 minutes, three of which were undetected by polymerase chain reaction-related methods. On the other hand, we examined 30 nonmalignant peripheral lung tissue specimens and found no mutations in any of the samples using PNA-clamp SmartAmp2. In this study, we confirmed that PNA-clamp SmartAmp2 has high sensitivity and accuracy and is suitable for the clinical diagnosis of KRAS codon 12 mutations.

Figure 1

Evaluation of the sensitivity of each detection method for KRAS codon 12 mutations. A: sensitivity of enzyme-enriched sequencing (anti-sense strand). B: sensitivity of PNA-enriched sequencing (sense strand). C: sensitivity of PNA-clamp SmartAmp2. The amplification curve shows the presence of the KRAS mutation. The mutant and mismatched sequences are shown with boxed and underlined text, respectively.

Figure 2

Results using different methods of detection for KRAS mutations (case 6). The results of PNA-clamp SmartAmp2 (A), PCR-based direct sequencing (sense strand sequence) (B), PNA-enriched sequencing (sense strand sequence) (C), enzyme-enriched sequencing (anti-sense strand sequence) (D), and enzyme-enriched PCR-RFLP (E) are shown. The sample contains KRAS mutation (G35C). The mutant sequences are shown with boxed text.

Figure 3

Results using different methods of detection for KRAS mutation (case 8). The results of PNA-clamp SmartAmp2 (A), PCR-based direct sequencing (sense strand sequence) (B), PNA-enriched sequencing (sense strand sequence) (C), enzyme-enriched sequencing (anti-sense strand sequence) (D), and enzyme-enriched PCR-RFLP (E) are shown. The sample contains KRAS mutation (G35A).