Follicular thyroid adenomas (FTAs) and carcinomas (FTCs), collectively the most common thyroid neoplasms, constitute a significant clinical challenge since histological evidence of invasive behaviour is required for a malignant diagnosis. Small subsets of FTAs relapse as manifest malignant FTCs, indicating that histology is not always adequate to predict malignant potential. Lately, recurrent mutations in the promoter of the Telomerase reverse transcriptase (TERT) gene have been coupled to FTCs, whereas FTAs usually lack this aberrancy. We describe three patients with follicular thyroid tumours in which TERT promoter mutational screening was employed as part of the clinical work-up to pinpoint malignant potential. In two retrospective analyses of seemingly benign lesions, the detected mutations predicted future skeletal metastases, and in one prospective case, the mutational screening led to a different clinical management of the afflicted patient. We therefore consider TERT promoter mutational screening an adjunct tool of value in equivocal cases. . . . Keywords TERT Thyroid cancer Mutation Clinical practice Introduction follicular thyroid adenoma (FTA) . TERT encodes the cat- alytic subunit of telomerase, an enzyme capable of extending Two point mutations of the Telomerase reverse transcriptase telomere length—a pivotal mechanism for cancer cells to (TERT) promoter (C228T and C250T, corresponding to posi- evade telomere shortening and subsequent programmed cell tions − 124 and − 146 base pairs from the ATG site) were death . Therefore, TERT promoter mutations and the ensu- recently shown to confer increased TERT expression, and ing TERT overexpression are expected to provide malignant these events have been demonstrated for various malignan- tumours with a selective advantage. In this study, three cases cies, including thyroid cancer [1–6]. More specifically, these from the clinical practice are highlighted for which TERT mutations have been intimately associated with follicular thy- promoter mutational screening conveyed an adjunct tool for roid cancer (FTC) and follicular tumours of uncertain malig- pinpointing malignant potential in follicular thyroid tumours nant potential (FT-UMP) as opposed to a near-total absence in with an equivocal histology. * C. Christofer Juhlin Case presentations email@example.com The first case is a 62-year-old male of Swedish ethnicity Department of Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden with a history of ankylosing spondylitis. In 2012, he pre- sented with a 30-mm nodule in his right thyroid lobe, and Department of Oncology-Pathology, Cancer Centre Karolinska (CCK) R8:04, Karolinska Institutet, 17176 Stockholm, Sweden a preoperative cytology fine-needle aspiration biopsy (FNAB) suggested a follicular tumour (Bethesda IV). Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden A diagnostic hemithyroidectomy was performed, and the histopathological examination revealed a 40-mm, Department of Breast, Endocrine Tumors and Sarcoma, Karolinska University Hospital, Stockholm, Sweden well-circumscribed FTA without signs of vascular or 640 Virchows Arch (2018) 473:639–643 capsular invasion, but with slightly elevated Ki-67 proliferation tumour and the FTC metastatic lesion using extraction of geno- index (4.6%) (Fig. 1(A)). In 2018, 6 years after the operation, the mic DNA from the formalin-fixated paraffin-embedded (FFPE) patient was diagnosed with a pathologic fracture of the humerus, material and bi-directional Sanger sequencing of the TERT pro- and radiology suggested a metastatic lesion. An intra-operative moter region covering upstream positions C228 and C250 using biopsy from the fractured area revealed metastatic thyroglobulin- the Genetic Analyser 3500 (Applied Biosystems, Foster City, positive adenocarcinoma with a follicular growth pattern, indic- USA). These analyses revealed a C228T TERT promoter muta- ative of metastatic FTC (Fig. 1(B)). Careful histopathological re- tion in both the original FT-UMP and the subsequent metastatic examination of the original thyroid tumour revealed small foci of lesion, providing molecular evidence that the original thyroid intra-capsular tumour cell deposits, but no evident malignant tumour exhibited malignant potential (Table 1). If the pathologist phenotype, suggesting a diagnosis of Bfollicular tumour of un- reviewing the original follicular tumour would have had access certain malignant potential^ as outlined by the novel 2017 WHO to TERT promoter mutational screening, the final histopatholog- criteria (Fig. 1(C)) . We then retrospectively performed TERT ical diagnosis would in theory remain unaltered—but the finding promoter mutational analyses from the patient’s original thyroid of such a mutation would certainly have intensified the follow-up a b c Case 1 d e f Case 2 g h i Case 3 Fig. 1 Photomicrographs illustrating the cases detailed in the study. All TERT promoter mutation. (E) Case 2: neck scar recurrence, initially cases are magnified × 100 unless otherwise specified. (A) Case 1: the interpreted as a tumoural implant. An asterisk marks the epidermal layer primary thyroid lesion diagnosed as FTA. Upon re-investigation 6 years of the skin. Magnification × 40. (F) Case 2: neck scar recurrence. Upon re- later, this tumour was found to harbour a C228T TERT promoter investigation, foci suggestive for vascular invasion were detected (arrows). mutation. (B) Case 1: scrapings from a pathological fracture of the A C228T TERT promoter mutation was demonstrated for this lesion. (G) humerus demonstrating metastatic FTC with strong immunoreactivity Case 3: pulmonary lesion demonstrating thyroid tissue arranged in a for thyroglobulin, magnified × 400. (C) Case 1: the originally macrofollicular growth pattern (left) encapsulated from the lung tissue diagnosed FTA was re-classified as an FT-UMP. Arrow indicates area (right). This lesion carried a C228T TERT promoter mutation, thereby with questionable capsular invasion. Insert (× 400) highlighting the favouring metastatic FTC over ectopic thyroid tissue as the final diagnosis. capsular engagement. Note the focal nuclear atypia within this part Magnification × 40. (H) Case 3: widely invasive FTC in the right thyroid of the tumour. (D) Case 2: the primary thyroid lesion originally classified lobe positive for the C228T TERT promoter mutation, macroscopic image. as multinodular goitre, and later re-classified as a follicular tumour, in Arrow indicates a focus of capsular invasion. (J) Case 3: histological which the distinction between FTA and FTC could not be ascertained representation of the capsular invasion. An asterisk marks the capsular based on the limited original sampling. This sample carried a C228T layer Virchows Arch (2018) 473:639–643 641 Table 1 Clinico-pathological characteristics of the three cases Case no. Gender Age at Tissue or site Original diagnosis Year diagnosed TERT promoter genotype Revised diagnosis Status diagnosis 1 M 62 Thyroid FTA 2012 C228T mutation FT-UMP – 1 – 68 Humerus Metastatic FTC 2018 C228T mutation – Alive 2 F 64 Thyroid Multinodular goitre 2001 C228T mutation Follicular tumour NOS – 2 – 71 Neck scar BImplanted FTA^ 2008 C228T mutation Metastatic FTC – 2 – 80 Pelvis Metastatic FTC 2017 No material available – Alive 3 F 74 Lung Metastatic FTC 2018 C228T mutation –– 3 – 74 Thyroid wiFTC 2018 C228T mutation – Alive FTA, follicular thyroid adenoma; FT-UMP, follicular tumour of uncertain malignant potential; FTC, follicular thyroid cancer; NOS, not otherwise specified; wiFTC, widely invasive FTC scheme of the patient. The patient underwent a completion the right thyroid lobe was indeed an FTC, and that the hemithyroidectomy in April 2018 and is currently awaiting subcutaneous manifestation was a scar recurrence of the radioiodine treatment. Thyroglobulin 1 week post-operatively same lesion. Thus, had the latter lesion been screened for was elevated (2122 microgram/L). He is clinically well. this genomic alteration, the diagnostic outcome might The second case is a 64-year-old female of Swedish have been different, and the patient would have been con- ethnicity who was diagnosed with multinodular goitre fol- sidered for adjuvant treatment. The patient received lowing a right-sided hemithyroidectomy (Fig. 1(D)) at an- radioiodine ablation with 7.3 gigabecquerel in February other hospital. Seven years later, the patient noted a sub- 2018. Further radiological examinations have revealed ad- cutaneous, nodular expansion underneath the neck scar. ditional metastases in the shoulder, ribs and chest. Latest FNAB suggested a follicular thyroid tumour (Bethesda thyroglobulin count was elevated (14,830 micrograms/L). IV), and the lesion was surgically removed. The histo- The third case is a 74-year-old female of Swedish ethnicity. pathological report was consistent with a subcutaneously In 2015, a small mass in her left superior lobe of the lung was located 24-mm encapsulated follicular thyroid tumour detected by conventional chest X-ray as a part of an investi- without overt signs of malignancy (Fig. 1(E)). The Ki- gation for intermittent chest pain. An ensuing FDG-PET scan 67 index was 2%. The lesion was therefore believed to was negative, and she was followed clinically. After 2 years, constitute an implantation remnant from a previous hypo- the slow growth rate raised suspicion that the lesion represent- thetical thyroid tissue seeding at the initial surgery. Nine ed a hamartoma or a neuroendocrine tumour, and since the years later, the patient complained of pain from the right lesion was also positive for a DOTATOC-positron emission pelvic area, and an MRI scan visualised a large destruc- tomography (DOTATOC-PET) CT scan, a diagnostic wedge tion from which an FNAB suggested metastatic FTC. A excision of the lesion was performed. The histopathological completion left-sided hemithyroidectomy was performed, diagnosis was surprisingly consistent with a 20-mm large in which two small (3 and 0.4 mm respectively) conven- macrofollicular lesion indicative of thyroid tissue (Fig. tional papillary thyroid cancer lesions were demonstrated. 1(G)). The cells demonstrated positivity for thyroglobulin No follicular tumour was visualised, and hence the ques- and TTF1, were without cellular atypia and endowed with a tion arose whether the original diagnoses of multinodular low Ki-67 proliferation index (<1%). The preliminary pathol- goitre as well as subcutaneous implantation tumour were ogy report listed two differential diagnoses: metastatic FTC or correct. Histopathological re-examination of the right thy- an inclusion of entrapped benign thyroid tissue. A subsequent roid lobe revealed a probable follicular thyroid tumour, in mutational screening revealed a C228T mutation of the TERT which the amount of tissue submitted for examination was promoter, strongly suggestive for metastatic FTC (Fig. 2a). too diminutive to characterise the relation to the surround- The patient was then referred to the endocrine surgery unit ing capsule. Moreover, the implantation tumour in the at our institution, and a 21-mm nodule was visualised in the neck was also re-investigated—in which small foci of right thyroid lobe by ultrasonography. An FNA biopsy was lymphovascular invasion were noted, and hence the no- consistent with a follicular tumour (Bethesda IV), and an en- menclature for this lesion was changed to metastatic FTC suing analysis of the cytology material could pinpoint the (Fig. 1(F)). Interestingly, tissues from both the original C228T TERT promoter mutation (Fig. 2b). With this informa- right thyroid lobectomy and the subsequent subcutaneous tion at hand, the patient underwent total thyroidectomy up- excision were positive for the C228T TERT promoter mu- front, and the histopathological examination was consistent tation (Table 1). This indicates that the original lesion in with a macrofollicular variant of FTC (Fig. 1(H–J)). This case 642 Virchows Arch (2018) 473:639–643 Fig. 2 Chromatograms a b illustrating the TERT promoter C228T C228T mutation C228T in case 3. a Chromatogram from the TERT promoter sequencing using genomic DNA from the excised pulmonary FTC metastasis. A heterozygous C>T mutation at position 228 corresponding to − 124 bp from the ATG site of the TERT gene was demonstrated (highlighted in light blue). b Chromatogram from the TERT promoter sequencing from the subsequent cytological examination of the primary tumour in the right thyroid lobe, in which the heterozygous C228T mutation was visualised (highlighted in light blue). The mutations were verified using reverse complement sequencing (data not shown) demonstrates the advantage of TERT promoter mutational employed for all cases of FT-UMP. If a mutation is detect- screening when determining whether extrathyroidal lesions ed, the patient is offered a contralateral lobectomy to allow of thyroid origin represent metastatic tissue or trapped embry- follow-up using thyroglobulin to detect eventual relapses. ological tissue . The patient is currently awaiting However, it should be noted that TERT promoter mutations radioiodine treatment, and she is clinically well. 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 . Moreover, a general pitfall when Discussion analysing thyroid tumours with a follicular growth pattern is the distinction between a follicular tumour and a follic- The introduction of TERT promoter mutational screening ular variant of PTC [13, 14]. In our case series, no sample in the clinical setting has given physicians an instrument to displayed PTC-like nuclear changes. Moreover, nuclear better estimate the true malignant potential of follicular atypia is sometimes associated to an underlying malignant thyroid tumours. Although the histopathological diagnosis potential of follicular thyroid tumours . Among our requires unequivocal signs of malignancy, such as capsular cases, mild atypia was seen in one case only, the FT- and/or vascular invasion , there is a disturbing gap be- UMP of case 1 (Fig. 1(C)). tween the current histopathological classification algo- We conclude that TERT promoter mutational screening en- rithm and the true clinical outcome—as subsets of cases tails several clinical benefits and constitutes a useful tool for a with worrisome histological features (denoted FT-UMP) proper detection of follicular thyroid tumours with potential do recur as full-blown malignant FTCs . This is not least for aggressive behaviour in histologically equivocal cases. We exemplifiedinthisstudy, inwhichtwoofthe caseswere further advocate that the finding of such a mutation should initially interpreted as benign, but later recurred. lead to an intensified follow-up scheme, as molecular attri- Moreover, TERT promoter mutational screening is cheap, butes of malignancy might precede the required histological fast, easy to perform and interpret, and the two mutational signs. sites are easily amplified by the same primer pair. Therefore, the test is also well suited for low-volume pa- Contributions MH and CCJ conceived and designed the study, thology centres without experience and/or economics to researched and analysed data and wrote, edited and reviewed the manu- accommodate more elaborate next-generation sequencing script. JOP and NW analysed data and edited and reviewed the manu- platforms, such as Thyroseq . In our institution, a post- script. KJ, CL, NFM, IS and JZ provided clinical details and edited and reviewed the manuscript. All authors gave final approval for publication. operative TERT promoter mutational screening is routinely Virchows Arch (2018) 473:639–643 643 CCJ takes full responsibility for the work as a whole, including the study Castro P, Castro L, Pardal F, Lopes JM, Santos LL, Reis RM, design, access to data and the decision to submit and publish the Cameselle-Teijeiro J, Sobrinho-Simões M, Lima J, Máximo V, manuscript. Soares P (2013) Frequency of TERT promoter mutations in human cancers. Nat Commun 4 2185 5. Cancer Genome Atlas Research Network (2014) Integrated geno- Funding This work was supported grants provided by the Swedish mic characterization of papillary thyroid carcinoma. Cell 159:676– Cancer Society. 6. Landa I, Ibrahimpasic T, Boucai L, Sinha R, Knauf JA, Shah RH, Compliance with ethical standards Dogan S, Ricarte-Filho JC, Krishnamoorthy GP, Xu B, Schultz N, Berger MF, Sander C, Taylor BS, Ghossein R, Ganly I, Fagin JA Informed consent Informed consent was collected from all patients (2016) Genomic and transcriptomic hallmarks of poorly differenti- enlisted in the study, and ethical permission has been granted by a local ated and anaplastic thyroid cancers. J Clin Invest 126:1052–1066 ethics committee (Regionala etikprövningsnämnden Stockholm). 7. Wang N, Liu T, Sofiadis A, Juhlin CC, Zedenius J, Höög A, Larsson C, Xu D (2014) TERT promoter mutation as an early Conflict of interest The authors declare that they have no conflict of genetic event activating telomerase in follicular thyroid adenoma interest. (FTA) and atypical FTA. Cancer 120:2965–2979 8. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Open Access This article is distributed under the terms of the Creative Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension Commons Attribution 4.0 International License (http:// of life-span by introduction of telomerase into normal human cells. creativecommons.org/licenses/by/4.0/), which permits unrestricted use, Science 279:349–352 distribution, and reproduction in any medium, provided you give appro- 9. Lloyd RV, Osamura RY, Klöppel G, Rosai J (2017) WHO priate credit to the original author(s) and the source, provide a link to the Classification of Tumours of Endocrine Organs WHO/IARC Creative Commons license, and indicate if changes were made. Classification of Tumours, 4th Edition, Volume 10 10. Cheng H, Yang L, Xiong J, Peng J, Ruan Q (2015) Multiple thyroid nodules in the lung: metastasis or ectopia? Diagn Pathol 10:61 11. 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Published: Jun 2, 2018
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