# Comprehensive Molecular Diagnostic Tests in Non-Small Cell Lung Cancer: Frequency of ALK, ROS1, RET, and Other Gene Fusions/Rearrangements in a Romanian Cohort

## TL;DR

This study examines the frequency of gene fusions in lung cancer patients in Romania, highlighting the importance of comprehensive genetic testing for personalized treatment.

## Contribution

The study provides new insights into the prevalence of gene fusions in a Romanian NSCLC cohort, emphasizing the need for broad genetic profiling.

## Key findings

- ALK fusions were the most common, followed by ROS1 and RET fusions.
- Rare fusions like ETV6::NTRK3 and FGFR3::TACC3 were detected, showing the value of comprehensive testing.

## Abstract

Lung cancer remains one of the leading causes of cancer-related death worldwide. Molecular testing plays an essential role in detecting specific genetic alterations that can guide targeted therapy, especially in non-small cell lung cancer (NSCLC). In this study, we analyzed a cohort of newly diagnosed, treatment-naïve, non-squamous and non-smoking squamous NSCLC patients from Romania, particularly to assess the presence of gene fusions involving ALK, ROS1, RET, and NTRK, and, additionally, less common genes such as FGFR3 and MET. These gene fusions were present in a relatively small percentage of cases, supporting the need for comprehensive molecular profiling in both non-squamous and squamous NSCLC, particularly among non-smokers. Overall, this study emphasizes the value of broad genetic testing for improving diagnosis and opening new directions for personalized therapy.

Background/Objectives: Lung cancer remains among the most frequently diagnosed malignancies in Romania, with a high mortality rate. Beyond EGFR mutations, clinically relevant genetic alterations in non-small cell lung cancer (NSCLC) include fusions involving ALK, ROS1, RET, and NTRK1/2/3. This study aimed to determine the prevalence of these mutations in a Romanian cohort and evaluate their associations with clinicopathological features. Methods: DNA and RNA were simultaneously extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections using the Genexus Purification System (ThermoFisher Scientific). Concentrations were quantified fluorometrically, and gene fusions were analyzed with Ion Torrent NGS (Ion GeneStudio S5) with the Oncomine Focus Assay (ThermoFisher Scientific). Library preparation was automated with the Ion Chef System, and data interpretation was conducted using Ion Reporter. Results: Among 721 newly diagnosed NSCLC patients, 28 (3.88%) harbored gene fusions. Adenocarcinoma prevailed among fusion-positive cases (85.7%). The subgroup included 15 males and 13 females, with a mean age of 63.25 years (range 43–83). ALK fusions were most frequent (1.66% of the cohort; 42.86% of positives), predominantly EML4::ALK. ROS1 fusions were detected in five patients (0.7%), most frequently CD74::ROS1. RET fusions occurred in 1.11%. Rare fusions included one ETV6::NTRK3, one PTPRZ1::MET, and one FGFR3::TACC3 co-occurring with EGFR L858R. Conclusions: Gene fusions were present in a minority of NSCLC cases, with ALK, ROS1, and RET being the most clinically relevant. These alterations were mutually exclusive with common drivers such as EGFR or KRAS. Detection of rare fusions highlights the therapeutic potential of comprehensive NGS profiling in Romanian NSCLC patients.