Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

Abstract

The ability to identify the broadest range of targetable gene fusions is crucial to facilitate personalized therapy selection for advanced lung adenocarcinoma (LuADs) patients harboring targetable receptor tyrosine kinase (RTK) genomic alterations. In order to evaluate the most effective testing approach for LuAD targetable gene fusion detection, we analyzed 210 NSCLC selected clinical samples, comparing in situ (Fluorescence In Situ Hybridization, FISH, and ImmunoHistoChemistry, IHC) and molecular (targeted RNA Next-Generation Sequencing, NGS, and RealTime-PCR, RT-PCR) approaches. The overall concordance among these methods was high (>90%), and targeted RNA NGS was confirmed to be the most efficient technique for gene fusion identification in clinical practice, allowing the simultaneous analysis of a large set of genomic rearrangements at the RNA level. However, we observed that FISH was useful to detect targetable fusions in those samples with inadequate tissue material for molecular testing as well as in those few cases whose fusions were not identified by the RNA NGS panel. We conclude that the targeted RNA NGS analysis of LuADs allows accurate RTK fusion detection; nevertheless, standard methods such as FISH should not be dismissed, as they can crucially contribute to the completion of the molecular characterization of LuADs and, most importantly, the identification of patients as candidates for targeted therapies.

Unravelling the diagnostic pathology and molecular biomarkers in lung cancer

“…For single-gene assays, PCR-based testing can identify specific gene mutations with high sensitivity and specificity, for example, EGFR exon 19 deletions or L858R mutations [ 11 ], while FISH is generally used for the detection of gene fusion and amplification [ 12 ]. ALK rearrangements represent a notable exception, as IHC is an equivalent alternative for treatment decisions and predicting response to ALK inhibitors [ 13 ].…”

Fluorescent In Situ Hybridization for miRNA Combined with Staining of Proteins

“…They denatured DNA in situ and then formed DNA‐DNA hybrids with radioisotope‐labeled DNA probes that could be detected with autoradiography. Since then, in situ hybridization has been applied in numerous fields, including developmental biology, phylogenetics, molecular biology, pathology, microbiology, oncology, cardiology, and neurology (Cao et al., 2023; Carr, 1995; Cui et al., 2016; Fitzpatrick et al., 2023; Laurent‐Huck & Felix, 1991; Luo & Liu, 2019; Pecciarini et al., 2023; Zhou et al., 2023). Although FISH has been used to localize miRNAs in cells (Dixon‐McIver et al., 2008; Herzer et al., 2012; Silahtaroglu, 2010), it remains challenging to detect low‐abundant miRNAs in histological sections (Kasai et al., 2016; Nuovo et al., 2009; Sempere et al., 2010; Zaidi et al., 2000).…”

Novel insights into molecular patterns of ROS1 fusions in a large Chinese NSCLC cohort: a multicenter study

“…In comparison to DNA NGS, RNA-based NGS (RNA NGS) offers superior detection of functional fusions as it specifically sequences the coding regions rather than the introns, enabling more accurate identification of functional ROS1 fusions [10,19]. However, the high dependence on RNA quality and the substantial sample amount requirement have limited the widespread use of RNA NGS, particularly in formalin-fixed paraffin-embedded (FFPE) samples, which are predominate sample type of NSCLC in clinical practice [20]. Therefore, finding ways to optimize the utility of both DNA NGS and RNA NGS in detecting functional ROS1 fusions is essential.…”