Ultra-Rapid EGFR Mutation Screening Followed by Comprehensive Next-Generation Sequencing: A Feasible, Informative Approach for Lung Carcinoma Cytology Specimens with a High Success Rate.

ABSTRACT Background For patients with advanced non-small cell lung carcinoma (NSCLC), cytologic samples may be the only diagnostic specimen available for molecular profiling. While both rapid and comprehensive assessment are essential in this setting, an integrated multi-test approach remains an important strategy in many laboratories, despite the risks and challenges when working with scant samples. In this study, we describe our experience and high success rate using a multi-test approach, focusing on the clinical validation and incorporation of ultra-rapid EGFR testing using the Idylla system, followed by comprehensive next generation sequencing (NGS). Methods Cytology samples received for routine molecular testing were included in this study. The performance characteristics of the EGFR Idylla assay were assessed; tissue suitability parameters and interpretation criteria to supplement automated mutation calling were established. Assay performance was monitored for one year, comparing results with concurrent NGS testing by MSK-IMPACT (primarily) and/or MSK-AmpliSeq and MSK-fusion solid panel in a subset of cases. Results In all, 301 samples were studied: 83 samples were included in the validation (60.2% (50/83) were positive for EGFR mutations). Concordance with the reference methods was 96.4% (80/83) with excellent reproducibility. The limit of detection was variable depending on the total tissue input and the specific mutation tested. Unextracted tissue inputs that maintained total EGFR Cq at ≤23 allowed all mutations to be detected if present at > 5% VAF. Mutations could be detected as low as 1% VAF with total EGFR Cq of ∼ 18. During the clinical implementation phase, 218 NSCLC were tested by Idylla (24.3% (53/218) were EGFR-mutation positive). Concurrent NGS testing was requested on 165 and successfully performed on 96.4% (159/165) of the samples. The Idylla automated results were concordant with those obtained by NGS in 96.2% (153/159) of cases and improved to 98.7 (157/159) after incorporation of manual review criteria to supplement automated calling, resulting in a diagnostic sensitivity of 95.6% (95% CI, 84.9% to 99.5%). In all, 9% (14/159) of the cases tested by NGS had EGFR mutations not covered by the Idylla assay, primarily insertions in exon 19 and 20 and minor mutations co-occurring with canonical sensitizing mutations. Conclusion Comprehensive molecular testing is feasible and has a high success rate in NSCLC cytology samples when using a multi-test approach. Testing with the Idylla system enables rapid and accurate determination of EGFR status without compromising subsequent NGS testing.