Epidermal growth factor receptor mutations in non-small cell lung cancer undetected by high-sensitivity allele-specific real-time PCR-based assays

BACKGROUND: Identifying epidermal growth factor receptor (EGFR) mutation status is critical for planning lung cancer treatment. Sanger sequencing detects both known and novel mutations but shows poor sensitivity. High-sensitivity allele-specific real-time PCR (ASRP) based assays offer quick and reliable results, but may overlook uncommon mutations. We aimed to define the rate at which high-sensitivity ASRP-based assays missed uncommon EGFR mutations. METHODS: Non-small cell lung cancer specimens that were diagnosed as EGFR wild-type (EGFR-WT) by high-sensitivity ASRP-based assays and had residual DNA samples were sent for Sanger sequencing. Patient characteristics and clinical features were evaluated by chart review, and outcomes of EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy were studied. RESULTS: Hundred DNA specimens diagnosed by high-sensitivity ASRP-based assays as EGFR-WT were rechecked by Sanger sequencing. Two samples which were re-biopsy specimens from patients with EGFR mutations were excluded from analysis. Sanger sequencing was failed in 24 samples. Among the remaining 74 samples, 6 (8.1%) had EGFR mutations-one exhibited exon 19 deletion (delT751_I759insS), two exhibited substitution mutations (S768I+V769L and L861Q), and three exhibited exon 20 insertions (N771_P772insN, P772_H773insHP, and H773_V774insAH). Only the patient with the exon 19 deletion had received EGFR-TKI therapy. Although the best tumor response was only stable disease, this was maintained for more than 10 months. CONCLUSION: High-sensitivity ASRP-based assays can overlook uncommon mutations. This detection failure rate is worth noting, especially when treating patients from regions known to have a high prevalence of EGFR mutation. Patients carrying uncommon mutations may still benefit from EGFR-TKI therapy.