Background/Objectives: Papillary thyroid carcinoma (PTC) is the most common malignancy of the endocrine system, characterized by various molecular alterations. This study evaluates the relationship between p16 promoter methylation status, BRAFV600E mutation presence, and ETS1 (E26 transformation-specific) expression, aiming to better understand their clinical significance and to enhance the risk stratification of PTC patients. Methods: p16 promoter methylation was analyzed by methylation-specific PCR (MSP), BRAFV600E by mutant allele-specific PCR amplification (MASA), ETS1 mRNA expression by quantitative PCR (qPCR), ETS1 protein expression by immunohistochemistry (IHC), and Western blot. All tested factors were further associated with the occurrence of unfavorable clinicopathological data of the patients. Results: While p16 methylation did not correlate with adverse clinical parameters or BRAFV600E mutation presence, it was significantly associated with the increased ETS1 mRNA levels. Combined p16 methylation with high ETS1 protein levels was significantly associated with advanced pT and pTNM stages. BRAFV600E-mutated PTC cases with p16 methylation showed increased mRNA and protein ETS1 expression. Conclusions: Therefore, although p16 methylation could not be used as a standalone prognostic marker, its association with elevated ETS1 levels points to its potential involvement in tumor progression and adverse clinical outcomes, particularly in BRAFV600E-mutated PTCs. Deeper insights into these interactions may enhance PTC prognosis and the selection of high-risk patients.

In order to enhance the risk stratification of papillary thyroid carcinoma (PTC) patients, we assessed the presence of the most common mutation in PTC (BRAFV600E) with the expression profiles of long non-coding RNA activated by BRAFV600E (BANCR) and microRNAs, which share complementarity with BANCR (miR-203a-3p and miR-204-3p), and thereafter correlated it with several clinicopathological features of PTC. BRAFV600E was detected by mutant allele-specific PCR amplification. BANCR and miRs levels were determined by quantitative RT-PCR. Bioinformatic analysis was applied to determine the miRs’ targets. The expression profile of miR-203a-3p/204-3p in PTC was not affected by BRAFV600E. In the BRAFV600E-positive PTC, high expression of miR-203a-3p correlated with extrathyroidal invasion (Ei), but the patients with both high miR-203a-3p and upregulated BANCR were not at risk of Ei. In the BRAFV600E-negative PTC, low expression of miR-204-3p correlated with Ei, intraglandular dissemination and pT status (p < 0.05), and the mutual presence of low miR-204-3p and upregulated BANCR increased the occurrence of Ei. Bioinformatic analysis predicted complementary binding between miR-203a-3p/204-3p and BANCR. The co-occurrence of tested factors might influence the spreading of PTC. These findings partially describe the complicated network of interactions that may occur during the development of PTC aggressiveness, potentially providing a new approach for high-risk PTC patient selection.

The expressions of ETS1, miR-203a-3p, and miR-204-3p in papillary thyroid carcinoma (PTC) are poorly described, and their clinical significance is unclear. To determine the prognostic value of ETS1 (E26 transformation-specific), its levels in divergent cell compartments were paired with miR-203a-3p/-204-3p levels and linked to the presence of unfavorable clinical characteristics of PTC patients. Immunohistochemistry and Western blot were performed to evaluate ETS1 protein expression in PTC and matched nonmalignant thyroid tissue (NMT). qPCR was utilized to quantify ETS1 mRNA, miR-203a-3p, and miR-204-3p expressions. Bioinformatic analysis was applied to predict biological interactions. Although there was a significant increase in ETS1 protein expression (p < 0.05), no difference was observed in ETS1 mRNA levels between PTC and matched NMT (p > 0.05). 98.7% of PTC samples exhibited positive staining for the ETS1 protein, detected in the nucleus, the cytoplasm, or both. In contrast, the ETS1 protein had positive staining in 70.9% of NMT samples, primarily localized in the nucleus. ETS1 cytoplasmic levels correlated with the pT status of PTC patients (p = 0.020, r = −0.267), while nuclear levels correlated with the occurrence of lymph node metastasis (p = 0.020, r = −0.271). According to the bioinformatic analysis, the 3′-untranslated region of ETS1 mRNA shares a seed sequence with miR-203a-3p/-204-3p. The mutual distribution of ETS1 and miR-203a-3p levels differs between aggressive and non-aggressive PTCs. ETS1 could be used in the identification of high-risk PTC patients; however, its subcellular localization should be considered. PTC aggression could be influenced by increased cytoplasmic ETS1 protein levels, which may be affected by reduced levels of miR-203a-3p or miR-204-3p.