Direct sequencing in cytological specimens as a useful strategy for detecting EGFR mutations in non-small cell lung cancer patients.

Background: New therapeutics targeting epidermal growth factor receptor (EGFR) have significantly improved tumor responses to therapy in non-small cell lung cancer (NSCLC) patients. Molecular testing for EGFR mutations informs important therapeutic decisions in clinical practice. In this study, we sought to validate the clinical relevance of sequencing-based EGFR mutation testing combined with cytological analysis using body fluid specimens.

Methods: Two NSCLC cell lines were used in sensitivity analyses. In addition, we performed cytological analyses and directly sequencing of exons 18-21, for 32 specimens. The absence of EGFR mutations determined by direct sequencing in 14 specimens was confirmed by real-time PCR. Changes made to patients' therapeutic strategies after reports of EGFR mutation status were investigated by querying electronic medical records.

Results: Sensitivity studies showed that detection of in-frame deletions in exon 19 and point mutations in exon 21 was possible in specimens containing 10% and 5% mutant DNA, respectively. In clinical practice, EGFR mutations were detected in 18 of 32 specimens (56.3%). Twelve patients with EGFR mutations detected by direct sequencing were started on treatment with EGFR tyrosine kinase inhibitor (TKI) after reports of EGFR mutation. EGFR-TKI therapy was discontinued for two patients with TKI-resistant T790M mutation. The results of real-time PCR were consistent with those of direct sequencing in 13 of 14 specimens (92.9%) in which no mutation was detected by direct sequencing.

Conclusions: Combined direct sequencing and cytological analysis of body fluid specimen might be clinically useful and sensitive test for the detection of EGFR mutations in NSCLC patients.