Patients with thyroid cancer (TC) usually have an excellent prognosis; however, 5– 10% of them develop an advanced disease. The prognosis of this subgroup is still favourable if the lesions respond to radioactive iodine (RAI) treatment. Nearly two-thirds of advanced TC patients become RAI-refractory (RAI-R), and their management is challenging. A multidisciplinary approach in the context of a tumour board is essential to define a personalized strategy. Systemic therapy is not always the best option. In case of slow neoplastic growth and low tumour burden, active surveillance may represent a valuable choice. Local approaches might be considered if the disease progression is limited to a single or few lesions, also in combination and during systemic therapy. Antiresorptive treatment may be started in presence of bone metastases. In case of rapid and/or symptomatic progression involving multiple lesions and/or organs, systemic therapy has to be considered, in absence of contraindications. The multi-kinase inhibitors (MKIs) lenvatinib and sorafenib are currently available as first-line treatment for advanced progressive RAI-R TC. Among second-line options, cabozantinib has been recently approved in RAI-R TC who progressed during MKIs targeting the vascular endothelial growth factor receptor (VEGFR). In the last few years, next-generation sequencing (NGS) assays have been increasingly employed, permitting identification of the genetic alterations harboured by TC, with a significant impact on patients’ management. Novel selective targeted therapies have been introduced for the treatment of RAI-R TC in selected cases: REarranged during Transfection (RET) inhibitors (selpercatinib and pralsetinib) and Tropomyosin Receptor Kinase (TRK) inhibitors (larotrectinib and entrectinib) have recently expanded the panorama of the therapeutic options. Moreover, immune checkpoint inhibitors (ICIs) have shown promising results, and they are still under investigation.

Objective: Our objectives were to describe molecular profiling in a real-life cohort of patients with radioiodine-resistant (RAI-R) differentiated or poorly differentiated thyroid cancer (DTC or PDTC) treated with lenvatinib and to focus on factors potentially influencing the quality of tissue samples for molecular analysis, including the impact of storage time, defined as the interval between tissue collection and molecular testing. Design: We retrospectively included all lenvatinib-treated RAI-R DTC or PDTC patients tested with DNA- and/or RNA-based next-generation sequencing (NGS) in our center, also analyzing the results of fluorescence in situ hybridization (FISH) for RET fusions if the sample did not satisfy quality criteria for RNA-based NGS analysis. We investigated differences in terms of histotype, biopsy site, or storage time between adequate and inadequate samples for RNA-based NGS. Results: At least one gene alteration was detected in 50% of the cohort (18 out of 36 patients); RAS and BRAF were the most frequent mutations, while gene fusions accounted for 5.6% of cases. Tissue samples were more frequently adequate for DNA-based NGS compared to RNA-NGS analysis (93.9% vs. 58.3%, p < 0.001). The median storage time was significantly longer in the case of inadequate samples for RNA-based NGS compared with adequate specimens (41.5 vs. 9.5 months, p = 0.016); samples archived for ≥3 years led more frequently to an inadequate result. Conclusions: Advanced RAI-R TC candidates for systemic therapy often harbor gene alterations. An adequate result was less frequently achieved in cases of RNA-based NGS than in DNA-based NGS, especially if the interval between tissue collection and molecular analysis was longer; nevertheless, the limited cohort size precludes definitive conclusions.