Endocrine Abstracts

Objectives: Molecular techniques are becoming increasingly important for the management of thyroid nodules. In particular, next-generation sequencing (NGS) can assess multiple predictive biomarkers simultaneously, enabling more accurate pre-operative risk stratification for patients with indeterminate fine-needle aspirates and identifying specific molecular alterations for targeted therapy in patients affected by advanced, radioactive iodine refractory carcinomas. To meet these needs, we developed and validated a custom NGS gene panel (Nexthyro) designed to identify clinically relevant mutations implicated in thyroid oncogenesis.

Methods: Our panel covers 265 mutations in 15 genes and 161 gene fusions, specifically targeting mutations in BRAF, EIF1AX, GNAS, HRAS, IDH1, KRAS, NF2, NRAS, PIK3CA, PPM1D, PTEN, RET, DICER1, CHEK2, TERT promoter, and fusions in RET, BRAF, NTRK, PAX8/PPARγ, ALK, and ROS1. The analytical sensitivity was assessed by extracting both DNA and RNA from two different cell lines: 1) gDNA OncoSpan (Horizon Diagnostics [HDx]), which harbours 386 variants in 152 genes, including those detected by our panel, and 2) LC-2/ad (Dr. Miguel Angel Molina-Vila, Laboratory of Oncology, Pangaea Oncology, Spain) which harbor RET gene fusions. The nucleic acids were mixed with wild-type cell lines reaching serially decreasing dilution points (Table 1). Each dilution point was tested twice to assess the reproducibility of the results.

Results: All samples were successfully analysed and the limit of detection (LOD) of the mutated allele was established at 2% for both DNA alterations and RNA fusions. These results were successfully replicated in the same NGS run (Table 1).

Conclusions: Our custom NGS gene panel demonstrated high reproducibility and analytical sensitivity. Further validation using cytological and histological routine samples, which is currently underway. is necessary to evaluate implementation in clinical practice. Table 1. Dilution point, mean fusions RNA read counts and allele frequency of DNA mutations. For each dilution point, the results from the two different set of NGS tests were compared in order to show their analytical reproducibility.