Implementation of NGS Analysis for the Detection of RET Rearrangements in Thyroid Cancer Patients. An Inter-Laboratory Validation of the Method

Background/Objectives: The clinical use of molecular biomarkers in thyroid cancer management requires standardized technical and analytical criteria, especially for gene fusion detection. Accurate identification of patients suitable for RET-selective inhibitors, a new treatment for advanced thyroid cancer, depends on effective molecular testing. This study aimed to validate molecular alterations in the RET gene and assess the diagnostic validity of the detection techniques through inter-institutional comparison. Methods: A two-site external inter-comparative study was conducted involving RNA and DNA analysis from sixty retrospective papillary thyroid carcinoma tumor samples that were wild-type for BRAF and TERT promoter mutations. Next-generation sequencing (NGS) was used to detect gene fusions and mutations, followed by confirmation using fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR). Concordance analysis between two independent institutions evaluated the reliability of diagnostic techniques. Results: The analysis revealed a high frequency of molecular alterations in thyroid cancer. RNA analysis identified RET gene fusions with various partners, as well as common fusions involving NTRK3 and ALK. DNA analysis found single nucleotide variations (SNVs) in NRAS, KRAS, ALK, MYC, and RET. The inter-hospital concordance analysis showed a high degree of agreement between the results of both institutions for RNA and DNA testing. Conclusions: The study confirms that NGS is a reliable technology for detecting genetic alterations in thyroid cancer. The high concordance between institutions emphasizes the importance of standardized protocols for molecular testing, supporting the use of molecular biomarkers in guiding patient management and treatment decisions.