Reflex testing in non-small cell lung carcinoma using DNA- and RNA-based next-generation sequencing-a single-center experience.

Background: Targeted treatment modalities for non-small cell lung carcinoma (NSCLC) patients are expanding rapidly and demand a constant adaptation of molecular testing strategies. In this regard, broad reflex testing via next-generation sequencing (NGS) might have several advantages. However, real-world data regarding practical feasibility and clinical relevance are scarce, especially for RNA-based NGS.

Methods: We performed a retrospective study comparing NGS use in two consecutive years (2019 and 2020). In 2019, reflex testing mainly consisted of DNA-based NGS for mutations and immunohistochemistry (IHC) for , , and fusion products. At the beginning of 2020, our approach has changed, with DNA- and RNA-based NGS panels now being simultaneously performed. This change in protocol allowed us to retrospectively evaluate if broad molecular reflex testing brings additional value to lung cancer patients.

Results: Within the whole cohort (n=432), both DNA- and RNA-based NGS yielded almost always evaluable results. Only in 6 cases, the RNA content was too little for an appropriate analysis. After integrating RNA-based NGS in the reflex testing approach, the number of detected fusions increased significantly (2.6% . 8.2%; P=0.0021), but also more patients received targeted therapies. Furthermore, exceedingly rare alterations were more likely to be detected, including the so far undescribed - fusion.

Conclusions: Our study demonstrates that a comprehensive approach to reflex NGS testing is practically feasible and clinically relevant. Including RNA-based panels in the reflex testing approach results in more detected fusions and more patients receiving targeted therapies. Additionally, this broad molecular profiling strategy identifies patients with emerging biomarkers, underscoring its usefulness in the rapidly evolving landscape of targeted therapies.