Molecular prognostication of thyroid tumors : with special focus on TERT
Authors: Hellgren Samuel 
Published: October 2024

<p dir="ltr">Thyroid carcinoma is the most common endocrine malignancy, with its incidence steadily increasing worldwide. These tumors can be challenging to diagnose, and treatment decisions are typically based on tumor size and a limited number of histomorphological features. To improve diagnosis, risk assessment, and treatment outcomes, there has been a growing focus on identifying new markers. In other types of tumors, molecular and immunohistochemical markers have significantly enhanced the diagnostic accuracy and prognostic evaluation. However, research on thyroid tumors has not yet resulted in markers that affect the management guidelines for thyroid tumors. Several potentially important markers are yet to be examined in terms of their effect on patient outcomes. My thesis aimed to study the clinical implication of molecular and immunohistochemical markers in thyroid tumors, with a special focus on telomerase reverse transcriptase (TERT) promoter mutations.</p><p dir="ltr">In Paper I, we examined the Ki-67 labeling index and its diagnostic and prognostic value in follicular thyroid tumors. Ki-67 is a nuclear protein expressed during all phases of the cell cycle except the resting state. Immunohistochemical analysis of Ki-67 is a widely used tool for assessing cell proliferation in various tumor types. Although it is used in the assessment of thyroid tumors, it is not determined exactly when or how to use it. In our study, we found that a Ki-67 index >4% was the most optimal threshold for indicating malignancy. However, there was considerable overlap between benign and malignant tumors using this cut-off, limiting its utility as a purely diagnostic marker. On the other hand, a Ki- 67 labeling index >4% effectively identified patients with follicular thyroid carcinoma (FTC) at risk of recurrence with metastatic disease. This cut-off allowed us to predict recurrence in FTCs more accurately, both across the entire group and within individual tumor stage subgroups. Moreover, the Ki-67 labeling index was an independent predictor of both metastatic disease and disease- specific mortality, suggesting its potential as an important prognostic tool in thyroid tumors. Although these results need to be reproduced in additional studies, our research demonstrates the potential of Ki-67 as a valuable marker in the evaluation of follicular thyroid tumors. To date, this is the largest study examining Ki-67 data in follicular thyroid tumors.</p><p dir="ltr">In Paper II, we examined if TERT promoter mutational analysis, using digital droplet polymerase chain reaction (ddPCR), could be performed on preoperative fine-needle aspiration cytology (FNAC) samples. We compared the preoperative mutational status of tumors with their postoperative status from the same tumors. Our results demonstrated a perfect correlation between the preoperative FNAC and postoperative formalin-fixed paraffin-embedded (FFPE) samples, with both mutated and wild-type tumors retaining their status in both contexts. Notably, all tumors with TERT promoter mutations were malignant, highlighting the potential of preoperative mutational analysis as a diagnostic tool for malignancy. Additionally, these mutations were associated with more aggressive histological subtypes, further underscoring their prognostic significance in the preoperative setting. In conclusion, ddPCR proved highly effective in detecting TERT promoter mutations in FNAC samples, showing high specificity. If validated in larger studies, this approach could influence surgical decisions for a subset of indeterminate thyroid lesions.</p><p dir="ltr">In Paper III, we introduced a novel in situ hybridization (ISH) technique to analyze TERT mRNA expression. TERT expression is associated with poor prognosis in FTCs, often due to TERT promoter mutations, but can also result from gene amplification or promoter hypermethylation. This novel technique allows for the visualization of TERT mRNA expression directly within the tissue, regardless of the underlying cause. We tested this method on FTCs with known TERT mRNA expression and TERT promoter mutation status, using two separate probes targeting different regions of the mRNA sequence. Our findings revealed that eight out of ten cases with known expression were detected by at least one of the probes, with no signals observed in TERT mRNA-negative samples. Interestingly, the positive signals were confined to the nuclear compartment of tumor cells and appeared in only a small fraction of the cells. This nuclear localization suggests that TERT mRNA may have an unconventional role in thyroid cancer development, warranting further investigation. Given the accuracy of ISH in identifying TERT promoter-mutated cases with confirmed mRNA expression, it holds significant potential for clinical screening of TERT abnormalities.</p><p dir="ltr">In Paper IV, we evaluated a clinical screening program designed to detect TERT promoter mutations in minimally invasive follicular thyroid carcinoma (miFTC) and minimally invasive oncocytic thyroid carcinoma (miOTC) using ddPCR. We analyzed a cohort of patients diagnosed with miFTC or miOTC. TERT promoter mutations were detected in 11.9% of the cases, including a small pT1 tumor measuring less than 20 mm. The mutations were frequently subclonal, and patients with mutated tumors were generally older compared to those with wild-type tumors. However, other clinical variables, such as tumor size and metastatic rate, did not differ significantly between the mutated and wild-type groups. These findings suggest that TERT promoter mutations can occur in miFTCs and miOTCs regardless of tumor size, supporting the rationale for testing all such tumors, regardless of tumor stage. The short follow-up time, along with the fact that the mutations were often subclonal, additional studies with longer follow-up periods are needed to fully determine their clinical relevance.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Hellgren LS</b>, Stenman A, Paulsson JO, Höög A, Larsson C, Zedenius J, Juhlin CC. Prognostic utility of the Ki-67 labeling index in follicular thyroid tumors: a 20-year experience from a tertiary thyroid center. Endocr Pathol. 2022 Jun;33(2):231-42. <a href="https://doi.org/10.1007/s12022-022-09714-4">https://doi.org/10.1007/s12022-022-09714-4</a><br></p><p dir="ltr"><br></p><p dir="ltr">II. Hysek M, <b>Hellgren LS</b>, Stenman A, Darai-Ramqvist E, Ljung E, Schliemann I, Condello V, Larsson C, Zedenius J, Juhlin CC. Digital droplet PCR TERT promoter mutational screening in fine needle aspiration cytology of thyroid lesions: A highly specific technique for pre-operative identification of high-risk cases. Diagn Cytopathol. 2023 Jun;51(6):331-40. <a href="https://doi.org/10.1002/dc.25120">https://doi.org/10.1002/dc.25120</a><br></p><p dir="ltr"><br></p><p dir="ltr">III. <b>Hellgren LS</b>, Olsson A, Kaufeldt A, Paulsson JO, Hysek M, Stenman A, Zedenius J, Larsson C, Juhlin CC. Nuclear-specific accumulation of telomerase reverse transcriptase (TERT) mRNA in TERT promoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool? J Clin Pathol. 2021 May 19;75(10):658-62. <a href="https://doi.org/10.1136/jclinpath-2021-207631">https://doi.org/10.1136/jclinpath-2021-207631</a><br></p><p dir="ltr"><br></p><p dir="ltr">IV. <b>Hellgren LS</b>, Stenman A, Jatta K, Condello V, Larsson C, Zedenius J, Juhlin CC. Catching the silent culprits: TERT promoter mutation screening of minimally invasive follicular and oncocytic thyroid carcinoma in clinical practice. Endocr Pathol. 2024 Oct 4. Online ahead of print. <a href="https://doi.org/10.1007/s12022-024-09828-x" rel="noreferrer" target="_blank">https://doi.org/10.1007/s12022-024-09828-x</a></p>