Comparison of MET gene amplification analysis by next-generation sequencing and fluorescence in situ hybridization

Area of Science:

• Oncology
• Molecular Diagnostics
• Genetics

Background:

• MET gene alterations are implicated in acquired resistance to EGFR inhibitors.
• MET amplifications are a therapeutic target in non-small cell lung cancer (NSCLC).
• Current clinical cut-off values for NGS-based MET amplification assessment are lacking, with FISH as the gold standard.

Purpose of the Study:

• To evaluate and propose clinically relevant cut-off values for assessing MET amplifications using next-generation sequencing (NGS).
• To compare NGS results with fluorescence in situ hybridization (FISH) for MET amplification detection in NSCLC.
• To establish reliable NGS parameters for identifying MET alterations in lung cancer.

Main Methods:

• Analysis of 20 formalin-fixed paraffin-embedded NSCLC tissue samples.
• Assessment of MET gene copy number variations (CNV) and mutations using QIAGEN GeneReader NGS.
• Confirmation and comparison using FISH (MET/CEN7) as the gold standard.

Main Results:

• NGS identified 17 MET-amplified and 1 rearranged samples; FISH confirmed only one highly amplified case.
• The single highly amplified case by FISH showed a fold change (FC) of 3.18 and mean copy number (CN) of 20.5 by NGS.
• A significant discrepancy was observed between NGS and FISH results for MET amplification detection.

Conclusions:

• NGS is capable of detecting MET gene amplifications, point mutations, and rearrangements using DNA and RNA.
• Proposed NGS cut-off values for MET amplification are FC ≥ 3.0 and CN ≥ 20.0, correlating with FISH results.
• Establishing standardized NGS cut-offs is crucial for accurate therapeutic target identification in NSCLC.