Targeted RNA-sequencing assays: a step forward compared to FISH and IHC techniques?

ALK and ROS1 rearrangements are molecular targets of several tyrosine kinase inhibitors. RNA-sequencing approaches are regarded as the new standard for fusion gene detection, representing an alternative to standard immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) techniques.

We aimed to compare two recent amplicon-based RNA-sequencing techniques: FusionPlex^® Alk Ret Ros1 v2 Kit (Archer^® ) with FHS-003Z-12-Human Lung Cancer Panel (Qiagen^® ) and assessed the accuracy of the data for therapy management. Thirty-seven formalin-fixed paraffin-embedded non-small cell carcinoma (NSCC) lesions initially explored by IHC and FISH were selected for RNA-sequencing analysis.

Qiagen^® and Archer^® kits produced similar results and correctly identified 85.1% (23/27) and 81.5% (22/27) of IHC/FISH ALK- and ROS1-positive samples, respectively, and 100% (6/6) of the negative samples. With regard to the ambiguous IHC-positive/FISH-negative cases, RNA-sequencing confirmed 75% (3/4) of the FISH conclusion. Although not statistically significant, patients with common EML4-ALK variants presented shorter overall survival and progression-free survival compared with patients harboring rare variants.

Our findings assessed the implementation of RNA-sequencing approaches to explore ALK and ROS1 rearrangements from formalin-fixed paraffin-embedded samples. We highlighted the similarities between Qiagen^® and Archer^® kits in terms of handling time, cost, and outcomes. We confirmed the feasibility of molecular testing in routine organization and its possible use not only as an alternative for standard IHC and FISH techniques, but as a supplementary technique helping to classify discrepant cases.