Objectives: Molecular testing of fine-needle aspiration biopsy (FNAB) samples is increasingly used mainly for indeterminate categories of the Bethesda System for Reporting Thyroid Cytopathology. Our aim was to introduce a routine molecular analysis of the main genetic causes of thyroid cancer.
Methods: In total, 1354 FNAB samples of thyroid nodules were analyzed. Testing procedures mainly in samples evaluated as Bethesda categories III and above were gradually established. First, DNA for the most common mutation V600E in the BRAF gene using allele specific Real Time PCR (LC480, Roche) is analyzed. BRAF-positive samples are screened for TERT mutations using direct sequencing (CEQ 8000, Beckman Coulter). BRAF-negative samples are analyzed by next generation sequencing (MiSeq, Illumina) using the Thyro-ID panel (4bases) examining other 12 genes. The samples negative in the Thyro-ID panel are subjected to detection of 23 fusion genes including ALK, BRAF, GLIS3, NTRK1, NTRK3, PPARG, RET genes using Real Time PCR. Samples suspected of medullary thyroid carcinoma (MTC) for RET and RAS mutations are tested.
Results: BRAF mutations in 153 patients, RAS mutations in 87 patients, RET mutations in 4 patients, TERT mutations in 24 patients and fusion genes in 52 patients were detected. Genetic variants in other genes (TP53, PTEN, PIK3CA, KIT, TSHR) were detected in 25 patients. From our cohort, in 430 patients post-surgical histopathological evaluation has been known. Positive predictive values (PPV) of BRAF, TERT, KRAS, HRAS, NRAS mutations and fusion genes were 99.3%, 94.1%, 73%, 50%, 45.8% and 97.7%, respectively, if borderline tumors were not included in malignancy. PPV for BRAF, TERT and fusions were almost 100% except for a follicular adenoma with BRAF K601E mutation, one case of follicular tumor of uncertain malignant potential with TERT and NRAS mutations and one case of hyalinizing trabecular tumor (HTT) with PAX8/GLIS3 fusion that is pathognomonic for HTT.
Conclusions: We established molecular testing of thyroid nodules that significantly contributed to clinical management of patients in the Czech Republic. Mutations of BRAF, TERT and fusion genes are associated with almost 100% risk of malignancy or even worse prognosis, therefore according to ETA guidelines from 2017 and recent publications their carriers are recommended for the total thyroidectomy. The risk of malignancy of RAS mutations is lower, clinical interpretation is difficult and thus rather a lobectomy is recommended.
Supported by AZV NU21-01-00448 and MH CR RVO 00023761.
10 Sep 2022 - 13 Sep 2022