A 48-year-old man with metastatic non-small-cell lung cancer (NSCLC) is suspected to have an EGFR activating mutation. Which investigation is the most specific and appropriate to confirm EGFR mutation status and guide tyrosine kinase inhibitor (TKI) therapy?

## Molecular Testing for EGFR Mutations in NSCLC **Key Point:** EGFR activating mutations (exon 19 deletions and exon 21 L858R point mutation) are predictive biomarkers for response to EGFR-TKIs (erlotinib, gefitinib, afatinib). Identification of these mutations is essential for personalized therapy and is a standard-of-care test in NSCLC. ### Why DNA Sequencing is the Gold Standard **High-Yield:** DNA sequencing (ARMS-PCR or next-generation sequencing [NGS]) directly detects EGFR mutations at the nucleotide level and is the most sensitive and specific method. NGS is increasingly preferred as it can simultaneously detect multiple mutations and other actionable alterations (KRAS, TP53, ALK, ROS1) in a single assay. **Clinical Pearl:** EGFR mutations are found in ~40–50% of NSCLC patients in East Asia and ~10–15% in Western populations. Patients with activating mutations have significantly improved response rates (70–80%) and progression-free survival with EGFR-TKIs compared to chemotherapy. ### Comparison of EGFR Testing Methods | Method | Detects | Sensitivity | Specificity | Clinical Use | |---|---|---|---|---| | **DNA Sequencing (ARMS-PCR/NGS)** | Activating mutations (exons 18–21) | 95–99% | 99% | **Gold standard; first-line test** | | IHC for EGFR protein | Protein expression level | Variable | Low | Prognostic only; NOT predictive of TKI response | | FISH | Gene amplification | High | High | Detects amplification, NOT mutations; different mechanism | | Western blot | Phosphorylated EGFR | N/A | N/A | Research tool; not used clinically | **Mnemonic: EGFR Mutation Testing = **SEQUENCE** (Specific, Exons 18–21, Quantitative, Ultrasensitive, Enables TKI selection, Nucleotide-level detection, Concurrent alterations)** ### Specimen Requirements 1. Formalin-fixed, paraffin-embedded (FFPE) tissue from primary tumor or metastasis 2. Cytology specimens (pleural fluid, bronchial brushings) if tissue unavailable 3. Circulating tumor DNA (ctDNA) from plasma as a non-invasive alternative when tissue is inaccessible **Warning:** IHC for EGFR protein expression does NOT predict TKI response. High EGFR protein expression can occur in wild-type EGFR tumors, and some mutation-positive tumors may have low protein expression. IHC is prognostic but not predictive. **Tip:** If initial tissue testing is negative but clinical suspicion is high, repeat testing with NGS (which has better sensitivity than older ARMS-PCR) or liquid biopsy (ctDNA) should be considered.