AUTHOR=Kowalewski Adam , Siemanowski-Hrach Janna , Stehle Thomas , Rehker Jan , Siebolts Udo , Merkelbach-Bruse Sabine , Heydt Carina 

TITLE=NGS coverage accurately predicts MET and HER2 (ERBB2) gene amplifications in a real-world non-small cell lung cancer cohort

JOURNAL=Frontiers in Oncology

VOLUME=Volume 15 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2025.1618509

DOI=10.3389/fonc.2025.1618509

ISSN=2234-943X

ABSTRACT=BackgroundFluorescence in situ hybridization (FISH) is the current standard for detecting gene amplifications, yet its low throughput and practical constraints call for alternative methods. This study evaluates next-generation sequencing (NGS) as a potential tool for accurately predicting gene amplifications.MethodsWe analyzed 66 primary non-small cell lung cancer (NSCLC) samples, tested by both NGS and FISH. FISH was conducted to detect gene amplifications in MET in 26 samples, in HER2 (ERBB2) in 21 samples, in PIK3CA in 9 samples, and KRAS in 9 samples, with one tumor tested for both MET and ERBB2. NGS fold changes, reflected by gene coverage, were calculated as the ratio of the highest gene-specific coverage to the mean coverage across all genes.ResultsAmplification was detected in 46 (68.7%) samples. NGS fold changes correlated strongly with FISH Gene/CEN ratios (Spearman’s ρ = 0.720, p < 0.001) and gene copy number per cell (Spearman’s ρ = 0.847, p < 0.001). Among FISH-negative cases, NGS fold change ranged from 0.57 to 1.95, while in FISH-positive cases, it ranged from 2.11 to 25.08.ConclusionNGS fold changes demonstrate significant correlation with FISH metrics, supporting NGS as a promising marker for gene amplification. A fold change cutoff of 2.0 effectively distinguishes amplified from non-amplified cases, with NGS achieving a high degree of predictive reliability across the tested genes.