Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing

摘要: Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS associated TKI resistance. Accordingly, mutation status routinely examined specimens, but employed methods may have a dramatic impact on interpretation results and, consequently, therapeutic decisions. Specimens low tumour cell content are at particular risk false-negative reporting by sequencing Sanger chemistry. To improve reliability detecting clinically relevant variants and KRAS, we took full advantage 454 deep developed two-step amplification protocol analysis exons 18–21 2 3. We systematically addressed sensitivity, reproducibility specificity assay. Mutations could be reliably identified down to an allele frequency 0.2–1.5 %, as opposed 10–20 % detection limit sequencing. High (0–2.1 variant frequency) very background level (0.4–0.8 further complement this Notably, re-evaluation 16 samples ≤40 wild type according revealed frequencies 0.9–10 seven cases. summary, novel superior significantly increased enabling reliable independent content.