The association of EGFR amplification with aberrant exon 20 insertion report using the cobas EGFR Mutation Test v2

Man-San Zhang ¹, Yi-Chen Yeh ^2,3, Hsien-Neng Huang ^4,5

⁰Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan.

Plos One +

|

April 30, 2024

View abstract on PubMed

Summary

The cobas EGFR test may inaccurately report epidermal growth factor receptor (EGFR) exon 20 insertions (ex20ins) in lung cancer. High EGFR amplification, not ex20ins, explains many discrepancies, necessitating validation by other methods.

Area Of Science

Oncology
Molecular Diagnostics
Genetics

Background

Accurate detection of epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations is crucial for lung cancer treatment.
The cobas EGFR test v2 has reported ex20ins mutations, but validation by Sanger sequencing has shown discrepancies, even with high tumor content.

Purpose Of The Study

To validate cobas-reported EGFR ex20ins mutations in lung cancer using Sanger sequencing and Idylla assay.
To identify clinicopathological factors associated with false-positive cobas ex20ins reports.
To investigate the role of EGFR gene copy number alterations in these discrepancies.

Main Methods

Retrospective collection of 123 cobas-reported ex20ins lung cancer cases.
Validation using Sanger sequencing and Idylla assay.
EGFR fluorescence in situ hybridization (FISH) for gene copy number analysis.
Comparison of clinicopathological features between validated and unvalidated groups.

Main Results

52 out of 123 (42%) cobas-reported ex20ins mutations could not be validated by Sanger sequencing.
The unvalidated group showed significantly higher tumor content, poor differentiation, and necrosis.
EGFR fluorescence in situ hybridization (FISH) revealed high EGFR copy number gain or amplification in 92% of unvalidated cases, suggesting these were the actual findings, not ex20ins mutations.
Cases with concomitant classic EGFR and ex20ins mutations reported by cobas also showed high EGFR amplification and lacked true ex20ins.

Conclusions

Aberrant cobas EGFR ex20ins reports are frequently associated with EGFR amplification, not true ex20ins mutations.
Independent validation of cobas-reported ex20ins, particularly those with co-occurring classic EGFR mutations, is essential.
EGFR FISH is critical for identifying high EGFR copy number alterations that may be misinterpreted as ex20ins.