Design and analytical validation of Nexthyro 2.0, a custom next-generation sequencing panel integrating DNA mutation profiling and RNA fusion detection for routine thyroid pathology specimens

: The increasing role of molecular testing in thyroid pathology, from the pre-operative stratification of indeterminate fine-needle aspiration (FNA) categories to the management of advanced carcinomas, has prompted the development of institution-specific custom NGS solutions as a locally deployable alternative to centralized tests. The aim of the present study is to validate a new custom NGS panel dedicated to the molecular analysis of routine thyroid pathology specimens. The panel was evaluated on thyroid cancer cell lines, 124 FFPE thyroid tissue specimens, and 43 FNA samples. Nexthyro 2.0 achieved a high sequencing success rate on both histological (100%) and cytological (95.3%) material, with analytical sensitivity down to 0.5% dilution point. Mutational profiles across thyroid neoplasm histotypes and FNA categories were consistent with established molecular-morphological correlates. Nexthyro 2.0 represents a reliable local solution for thyroid molecular testing across cytological and histological specimens encountered in routine clinical practice.