Tese de Doutoramento Medicina 2024 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Thyroid nodular disease is very common and distinguishing benign from malignant nodules is still a major challenge in clinical practice. Thyroid cancer (TC) is diagnosed in 1% to 5% of nodules, with more than 90% being differentiated thyroid carcinoma (DTC). Ultrasound-guided fine needle aspiration cytology (US-FNAC) continues to be the gold standard method in the diagnosis of thyroid nodular disease, but up to 25% of nodules remain without a definitive diagnosis, particularly in indeterminate nodules. A significant understanding in molecular mechanisms of thyroid carcinogenesis has been achieved. The development of TC has been associated with the activation of oncogenes (e.g. TERTp, BRAF and RAS (NRAS, HRAS and KRAS)), that are implicated in the cell signaling pathway, interfering in cancer promotion and outcome. Moreover, studies of the microRNAs (miRNAs) expression profile in thyroid neoplasms allowed the identification of specific "signatures" associated with diagnosis, staging, prognosis, and response to treatment. The rising incidence of thyroid neoplasm and the low incidence of aggressive TC, urges the exploration of strategies to improve the diagnostic accuracy in a pre-surgical phase, particularly in indeterminate nodules, and to prevent unnecessary surgeries while effectively treating patients. Tests available are expensive and not adapted to different populations. The present Thesis compromises two studies aiming (1) to compare the cyto-histological genetic profile (TERTp, BRAF and RAS (NRAS, HRAS and KRAS)) in a series of differentiated thyroid carcinoma and (2) to determine miRNAs expression (miR-146b, miR-221, miR-222 and miR-15a) in cytology and histology samples, in order to evaluate the reliability of molecular tests in preoperative diagnoses, using US-FNAC, in accurately diagnosing differentiated thyroid carcinomas (DTCs) and predicting biologic behavior of papillary thyroid carcinomas (PTCs). This retrospective study was conducted in a series of patients with thyroid nodular disease suspected of malignancy that underwent surgery, in a single non oncologic hospital between 2013 and 2020. We studied 259 operated patients with paired cyto-histological samples, totalizing 518 samples. From the 259 patients included, histologies were 19.3% (50) benign controls and 80.7% (209) DTC cases, from which 182 were PTCs. Cytologies were 5.8% non-diagnostic, 18.2% benign, 39% indeterminate, and 37.1% malignant. The genetic alterations were examined by polymerase chain reaction (PCR), followed by DNA sequencing. The miRNA expression was detected using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The association of the genetic and molecular alterations with clinicopathologic features was evaluated in PTCs. Mutation frequencies in cytology and histology specimens were, respectively, TERTp: 3.7% vs. 7.9%; BRAF: 19.5% vs. 25.1%; and RAS: 11% vs. 17.5%. The overall cyto-histological agreement of the genetic mutations was 94.9%, with Cohen’s k = 0.67, and in indeterminate nodules was 95.7%, k = 0.64. The identified mutations exhibited a discriminative ability in diagnosing DTC with a specificity of 100% for TERTp and BRAF, and of 94% for RAS, albeit with low sensitivity. TERTp and BRAF mutations were associated with aggressive clinicopathological features and tumor progression in PTCs (p<0.001). The obtained good cyto-histological agreement suggests that molecular analysis via US-FNAC may anticipate the genetic profile and the behavior of thyroid tumors, confirming malignancy and contributing to referring patients to surgery. All the analyzed miRNAs showed a tendency to be over expressed in DTCs/PTCs when compared with benign lesions, both in cytology and histology samples. In cytology, miRNAs expression levels were higher in malignant than in benign tumors. In histology, corresponding discriminative abilities regarding PTC diagnosis were: miR-146b (AUC0.94, 95%CI 0.87-1), miR-221 (AUC0.79, 95%CI 0.68-0.9), miR-222 (AUC0.76, 95%CI 0.63-0.89) and miR-15a (AUC0.85, 95%CI 0.74-0.97). miRNAs were associated with aggressive clinicopathological features and tumor progression in PTCs (p<0.05), particularly for miR-222. Our data reveals a significant association of higher expression levels of miR-146b, miR-221 and miR-222, with the presence of BRAF mutation (p<0.001), and miR-146b (p=0.016) and miR-221 (p=0.010) with the presence of RAS mutation, suggesting an interplay of these mutations with miRNAs expression. The miRNAs showed a good discriminative ability in PTC diagnosis. The association between the miRNAs expression profile and genetic alterations results could be useful for an accurate diagnosis of DTCs/PTCs in US-FNAC. Our studies yielded compelling results, demonstrating a good cyto-histological agreement for genetic alterations, and a deregulation of the expression levels of selected miRNAs, confirmed malignancy in thyroid nodules. The molecular analysis via US-FNAC may contribute to refine the diagnosis of thyroid nodules, reducing repeated US-FNAC and optimizing the pre-surgical diagnosis. The biomarkers complexity associated with the lack of consensus on the best panels to use, the need of high technology and specialized laboratories, and the high costs not supported in the majority of countries for their national health care systems, determine that the implementation of biomarkers in clinical practice is not yet a reality. More studies are needed to optimize its use in routine practice, and artificial intelligence may be the next step for a better understanding of molecular biomarkers in thyroid nodular disease