Mutations of the Telomerase reverse transcriptase (TERT) gene promoter are recurrently found in follicular thyroid carcinoma (FTC) and follicular tumors of uncertain malignant potential (FT-UMP), but nearly never in follicular thyroid adenoma (FTA). We, therefore, believe these mutations could signify malignant potential. At our department, postoperative TERT promoter mutational testing of FT-UMPs was implemented in 2014, with a positive mutation screening leading to vigilant follow-up and sometimes adjuvant treatment. To date, we screened 51 FT-UMPs and compared outcomes to 40 minimally invasive FTCs (miFTCs) with known TERT genotypes. Eight FT-UMPs (16%) displayed TERT promoter mutations, of which four cases underwent a completion lobectomy at the discretion of the patient, and a single patient also opted in for radioiodine (RAI) treatment. Three mutation-positive patients developed distant metastases, registered in one patient receiving a completion lobectomy and in two patients with no additional treatment. Three out of four patients who received additional surgery, including the RAI-treated patient, are still without metastatic disease. We conclude that FT-UMPs with TERT promoter mutations harbor malignant potential and exhibit at least similar recurrence rates to TERT-promoter-mutated miFTCs. Mutational screening should constitute a cornerstone analysis in the histopathological work-up of FT-UMPs.

In thyroid carcinomas, telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T predict an unfavorable clinical outcome. The analysis is particularly valuable when assessing histologically equivocal follicular thyroid tumors of uncertain malignant potential (FT-UMPs). Given recent findings of TERT promoter mutational heterogeneity in thyroid cancer, we determined the frequency of this phenomenon in FT-UMPs and minimally invasive follicular thyroid carcinomas. DNA was extracted from several tissue blocks from 16 FT-UMPs as well as 10 minimally invasive follicular thyroid carcinomas, and interrogated using Sanger sequencing as well as digital droplet PCR (ddPCR). Mutational heterogeneity was observed by Sanger sequencing in four of seven (57%) FT-UMPs. In two FT-UMPs with C228T mutations, analyses of additional blocks gave wild-type results using Sanger sequencing in one or several blocks interrogated, whereas ddPCR found low-frequency C228T mutations in one of these fractions. In two additional FT-UMPs with the C228T and C250T mutation, respectively, sequencing of additional blocks revealed the opposite mutation. Moreover, in the C250T mutated area in one of these tumors, the ddPCR displayed a co-occurring C228T mutation that failed detection through Sanger sequencing. To conclude, most TERT promoter mutated FT-UMPs display mutational heterogeneity when analyzed by Sanger sequencing, thereby emphasizing the importance of the tissue sampling process. The ddPCR technique might overcome this phenomenon because of increased sensitivity and should be considered for clinical screening purposes.

Background: Despite the advent of comprehensive molecular testing in surgical pathology, most centers still rely on the morphological assessment of fine‐needle aspiration cytology (FNAC) to triage patients with thyroid nodules for surgery. Subsets of patients could benefit from the inclusion of molecular testing to increase the diagnostic and/or prognostic properties of the cytology analysis, including the assessment of TERT promoter mutations, an event coupled with thyroid malignancy, and poor prognosis.Methods: In this prospective study, preoperative FNAC material from 65 cases was assessed for TERT promoter hotspot mutations C228T and C250T using the digital droplet PCR (ddPCR) technique on frozen pellets and re‐evaluated postoperatively.Results: Our cohort consisted of 15 B‐III (23%), 26 B‐IV (40%), 1 B‐V (2%), and 23 (35%) B‐VI lesions according to the Bethesda System for Reporting Thyroid Cytopathology. TERT promoter mutations were detected in 7 cases; 4 papillary thyroid carcinomas (all with preoperative B‐VI status), two follicular thyroid carcinomas (one B‐IV and one B‐V status), and one poorly differentiated thyroid carcinoma (with B‐VI status). All mutated cases were verified by mutational analysis of tumor tissue derived from postoperative formalin‐fixed paraffin‐embedded tissue, while all cases identified as wild‐type on FNAC remained wild‐type postoperatively. Moreover, the occurrence of a TERT promoter mutation was significantly associated with malignant disease and higher Ki‐67 proliferation indices.Conclusion: In the present cohort, we found that ddPCR is a highly specific method for detecting high‐risk TERT promoter mutations on thyroid FNAC material that could guide different surgical approaches in subsets of indeterminate lesions if reproduced in larger materials.

Telomerase reverse transcriptase ( TERT) gene aberrancies correlate to adverse prognosis in follicular thyroid carcinoma (FTC). As loss of 5-hydroxymethylcytosine (5hmC) has been associated with TERT promoter mutations in papillary thyroid carcinoma, this study sought to analyze the levels of 5hmC in a cohort of follicular thyroid tumors with available TERT data. A total of 29 tumors (26 FTCs, 2 follicular thyroid tumors of uncertain malignant potential, and 1 oncocytic thyroid carcinoma) with known TERT promoter mutational status and TERT gene expression were assessed for 5hmC immunoreactivity using two antibodies (clones RM236 and 4D9.) Slides were analyzed using a semiquantitative scoring system. Of the 10 tumor cases with aberrant TERT, only 1 scored negative with both antibodies (1/10; 10%), whereas the remaining 9 cases (9/10; 90%) exhibited some positivity for at least one antibody. Of the 19 TERT wild-type tumors, no case was scored negative using RM236, and 2 cases (2/19; 11%) using 4D9. The differences between TERT promoter mutated and wild-type groups were non-significant. The sensitivity and specificity for 5hmC immunohistochemistry (IHC) to detect mutated cases were 10% and 100% (RM236) and 20% and 89% (4D9). Therefore, 5hmC IHC is not a sensitive marker for detecting TERT promoter mutations in follicular thyroid tumors.

Background and purpose: Distinguishing branchial cleft cysts (BrCCs) from cystic metastases of human papillomavirus (HPV) positive tonsillar or base of tongue squamous cell carcinoma and cancer of unknown primary (CUP) is challenging. Fine needle aspiration cytology (FNAC) from cystic metastasis can be nonrepresentative, while reactive squamous cells from BrCC can be atypical. It is unclear whether benign characteristics and the absence of HPV-DNA in FNAC can enhance distinguishing BrCC from a cystic metastasis; therefore, we investigated here. Patients/materials and methods: Morphology and HPV-DNA in FNAC were reevaluated preoperatively and correlated to final diagnosis of 304 BrCC and CUP patients at Karolinska University Hospital during 2016-2023. Results and interpretation: All 176 cases finally diagnosed as BrCC were HPV-DNA negative in the preoperative FNAC. HPV-DNA was present in 100/128 (78.1%) of the FNAC with a solitary cystic neck metastasis and in 3/3 CUPs separately investigated on surgical specimens, which is distributed in 40/58 (69.0%) CUP, 40/41 (97.6%) tonsillar cancer, 21/22 (95.5%) base of tongue cancer, 2/2 uterine cervical cancer, and 0/5 non-HPV-related cancers. Interpretation: All cases with final BrCC diagnosis were HPV-DNA negative in FNAC. HPV-DNA was only present in FNAC of malignant cystic neck masses of HPV-related tumors or CUP. The data suggest that HPV-DNA analysis in FNAC enhances the diagnostics of cystic masses of the neck. A FNAC with a benign morphology and the absence of HPV-DNA indicated a BrCC, while an HPV-DNA positive aspirate irrespective of morphology suggested an HPV-DNA positive cancer or CUP.

Objectives: Sentinel lymph node biopsy (SLNB) enhances staging and treatment of oral squamous cell carcinoma (OSCC) by enabling detection of micrometastases (Mi) and isolated tumour cells (ITCs). However, the prognostic significance of these microscopic tumour deposits remains unclear. This study aims to evaluate the impact of Mi, ITCs, and other nodal characteristics on survival and recurrence across all stages of OSCC. Methods: This retrospective cohort study included all patients with primary OSCC who underwent SLNB at Karolinska University Hospital between March 2019 and October 2024. Clinical and pathological TNM stages were recorded, including the presence of Mi, ITCs, and extranodal extension (ENE). Disease-free survival (DFS) and disease-specific survival (DSS) were analysed using Kaplan–Meier estimates and Cox proportional hazards models. Results: A total of 259 patients were included. 66.4% presented with early-stage disease (cT1-T2N0). Recurrence occurred in 15.8% of cases. Increasing tumour stage and nodal burden were associated with poorer DFS and DSS (p < 0.0001). Mi was identified in 11.0% of patients with early-stage OSCC and in 3.4% of cases with advanced disease. Three-year DFS was 88.0% in pN0 disease, 100% for pN0itc, 73.2% for pNmi, and 57.9% in patients with macrometastasis (pN +). Conclusions: Tumour stage, nodal burden and ENE are strong predictors of DFS and DSS in OSCC. Patients with Mi had outcomes comparable to those with pN1 disease, whereas ITCs did not negatively affect prognosis. These findings support classifying Mi as metastatic disease, and underscore the value of SLNB in management across all stages of OSCC.