TERT promoter mutational screening as a tool to predict malignant behaviour in follicular thyroid tumours—three examples from the clinical routine

Two point mutations of the Telomerase reverse transcriptase (TERT) promoter (C228T and C250T, corresponding to positions − 124 and − 146 base pairs from the ATG site) were recently shown to confer increased TERT expression, and these events have been demonstrated for various malignancies, including thyroid cancer [1‐6]. More specifically, these mutations have been intimately associated with follicular thyroid cancer (FTC) and follicular tumours of uncertain malignant potential (FT-UMP) as opposed to a near-total absence in follicular thyroid adenoma (FTA) [7]. TERT encodes the catalytic subunit of telomerase, an enzyme capable of extending telomere length—a pivotal mechanism for cancer cells to evade telomere shortening and subsequent programmed cell death [8]. Therefore, TERT promoter mutations and the ensuing TERT overexpression are expected to provide malignant tumours with a selective advantage. In this study, three cases from the clinical practice are highlighted for which TERT promoter mutational screening conveyed an adjunct tool for pinpointing malignant potential in follicular thyroid tumours with an equivocal histology.

The first case is a 62-year-old male of Swedish ethnicity with a history of ankylosing spondylitis. In 2012, he presented with a 30-mm nodule in his right thyroid lobe, and a preoperative cytology fine-needle aspiration biopsy (FNAB) suggested a follicular tumour (Bethesda IV). A diagnostic hemithyroidectomy was performed, and the histopathological examination revealed a 40-mm, well-circumscribed FTA without signs of vascular or capsular invasion, but with slightly elevated Ki-67 proliferation index (4.6%) (Fig. 1(A)). In 2018, 6 years after the operation, the patient was diagnosed with a pathologic fracture of the humerus, and radiology suggested a metastatic lesion. An intra-operative biopsy from the fractured area revealed metastatic thyroglobulin-positive adenocarcinoma with a follicular growth pattern, indicative of metastatic FTC (Fig. 1(B)). Careful histopathological re-examination of the original thyroid tumour revealed small foci of intra-capsular tumour cell deposits, but no evident malignant phenotype, suggesting a diagnosis of “follicular tumour of uncertain malignant potential” as outlined by the novel 2017 WHO criteria (Fig. 1(C)) [9]. We then retrospectively performed TERT promoter mutational analyses from the patient’s original thyroid tumour and the FTC metastatic lesion using extraction of genomic DNA from the formalin-fixated paraffin-embedded (FFPE) material and bi-directional Sanger sequencing of the TERT promoter region covering upstream positions C228 and C250 using the Genetic Analyser 3500 (Applied Biosystems, Foster City, USA). These analyses revealed a C228T TERT promoter mutation in both the original FT-UMP and the subsequent metastatic lesion, providing molecular evidence that the original thyroid tumour exhibited malignant potential (Table 1). If the pathologist reviewing the original follicular tumour would have had access to TERT promoter mutational screening, the final histopathological diagnosis would in theory remain unaltered—but the finding of such a mutation would certainly have intensified the follow-up scheme of the patient. The patient underwent a completion hemithyroidectomy in April 2018 and is currently awaiting radioiodine treatment. Thyroglobulin 1 week post-operatively was elevated (2122 microgram/L). He is clinically well.

The second case is a 64-year-old female of Swedish ethnicity who was diagnosed with multinodular goitre following a right-sided hemithyroidectomy (Fig. 1(D)) at another hospital. Seven years later, the patient noted a subcutaneous, nodular expansion underneath the neck scar. FNAB suggested a follicular thyroid tumour (Bethesda IV), and the lesion was surgically removed. The histopathological report was consistent with a subcutaneously located 24-mm encapsulated follicular thyroid tumour without overt signs of malignancy (Fig. 1(E)). The Ki-67 index was 2%. The lesion was therefore believed to constitute an implantation remnant from a previous hypothetical thyroid tissue seeding at the initial surgery. Nine years later, the patient complained of pain from the right pelvic area, and an MRI scan visualised a large destruction from which an FNAB suggested metastatic FTC. A completion left-sided hemithyroidectomy was performed, in which two small (3 and 0.4 mm respectively) conventional papillary thyroid cancer lesions were demonstrated. No follicular tumour was visualised, and hence the question arose whether the original diagnoses of multinodular goitre as well as subcutaneous implantation tumour were correct. Histopathological re-examination of the right thyroid lobe revealed a probable follicular thyroid tumour, in which the amount of tissue submitted for examination was too diminutive to characterise the relation to the surrounding capsule. Moreover, the implantation tumour in the neck was also re-investigated—in which small foci of lymphovascular invasion were noted, and hence the nomenclature for this lesion was changed to metastatic FTC (Fig. 1(F)). Interestingly, tissues from both the original right thyroid lobectomy and the subsequent subcutaneous excision were positive for the C228T TERT promoter mutation (Table 1). This indicates that the original lesion in the right thyroid lobe was indeed an FTC, and that the subcutaneous manifestation was a scar recurrence of the same lesion. Thus, had the latter lesion been screened for this genomic alteration, the diagnostic outcome might have been different, and the patient would have been considered for adjuvant treatment. The patient received radioiodine ablation with 7.3 gigabecquerel in February 2018. Further radiological examinations have revealed additional metastases in the shoulder, ribs and chest. Latest thyroglobulin count was elevated (14,830 micrograms/L).

The third case is a 74-year-old female of Swedish ethnicity. In 2015, a small mass in her left superior lobe of the lung was detected by conventional chest X-ray as a part of an investigation for intermittent chest pain. An ensuing FDG-PET scan was negative, and she was followed clinically. After 2 years, the slow growth rate raised suspicion that the lesion represented a hamartoma or a neuroendocrine tumour, and since the lesion was also positive for a DOTATOC-positron emission tomography (DOTATOC-PET) CT scan, a diagnostic wedge excision of the lesion was performed. The histopathological diagnosis was surprisingly consistent with a 20-mm large macrofollicular lesion indicative of thyroid tissue (Fig. 1(G)). The cells demonstrated positivity for thyroglobulin and TTF1, were without cellular atypia and endowed with a low Ki-67 proliferation index (<1%). The preliminary pathology report listed two differential diagnoses: metastatic FTC or an inclusion of entrapped benign thyroid tissue. A subsequent mutational screening revealed a C228T mutation of the TERT promoter, strongly suggestive for metastatic FTC (Fig. 2a). The patient was then referred to the endocrine surgery unit at our institution, and a 21-mm nodule was visualised in the right thyroid lobe by ultrasonography. An FNA biopsy was consistent with a follicular tumour (Bethesda IV), and an ensuing analysis of the cytology material could pinpoint the C228T TERT promoter mutation (Fig. 2b). With this information at hand, the patient underwent total thyroidectomy up-front, and the histopathological examination was consistent with a macrofollicular variant of FTC (Fig. 1(H–J)). This case demonstrates the advantage of TERT promoter mutational screening when determining whether extrathyroidal lesions of thyroid origin represent metastatic tissue or trapped embryological tissue [10]. The patient is currently awaiting radioiodine treatment, and she is clinically well.

The introduction of TERT promoter mutational screening in the clinical setting has given physicians an instrument to better estimate the true malignant potential of follicular thyroid tumours. Although the histopathological diagnosis requires unequivocal signs of malignancy, such as capsular and/or vascular invasion [9], there is a disturbing gap between the current histopathological classification algorithm and the true clinical outcome—as subsets of cases with worrisome histological features (denoted FT-UMP) do recur as full-blown malignant FTCs [7]. This is not least exemplified in this study, in which two of the cases were initially interpreted as benign, but later recurred. Moreover, TERT promoter mutational screening is cheap, fast, easy to perform and interpret, and the two mutational sites are easily amplified by the same primer pair. Therefore, the test is also well suited for low-volume pathology centres without experience and/or economics to accommodate more elaborate next-generation sequencing platforms, such as Thyroseq [11]. In our institution, a postoperative TERT promoter mutational screening is routinely employed for all cases of FT-UMP. If a mutation is detected, the patient is offered a contralateral lobectomy to allow follow-up using thyroglobulin to detect eventual relapses. However, it should be noted that TERT promoter mutations are coupled to older patient age, and as our three cases were all > 60 years at initial presentation, the true value of TERT promoter mutational analyses in younger patients is not established [12]. Moreover, a general pitfall when analysing thyroid tumours with a follicular growth pattern is the distinction between a follicular tumour and a follicular variant of PTC [13, 14]. In our case series, no sample displayed PTC-like nuclear changes. Moreover, nuclear atypia is sometimes associated to an underlying malignant potential of follicular thyroid tumours [14]. Among our cases, mild atypia was seen in one case only, the FT-UMP of case 1 (Fig. 1(C)).