TERT promoter mutations and additional molecular alterations in thyroid fine-needle aspiration specimens: A multi-institutional study with histopathologic follow-up

Abstract

Objectives

TERT promoter mutations are not infrequently encountered in thyroid carcinomas; however, it is unclear if additional molecular alterations may play a role in determining tumor behavior.

Methods

Fine-needle aspiration (FNA) specimens from 32 patients with TERT promoter mutations detected by ThyroSeq v3 from 4 institutions were included in the study. FNA diagnoses, molecular results, and surgical follow-up were retrospectively reviewed and analyzed.

Results

There were 5 benign and 27 malignant neoplasms, including 7 high-grade thyroid carcinomas (HGCs) on histopathologic follow-up. Of 4 cases with an isolated TERT mutation, 3 (75%) cases were malignant. Of 17 cases harboring a co-occurring TERT mutation with 1 additional molecular alteration, 13 (76%) displayed malignancy on histopathologic follow-up. All 11 cases with TERT mutations plus 2 or more additional molecular alterations were malignant on follow-up. Furthermore, HGC was not seen in cases with an isolated TERT mutation, while 80% of cases harboring TERT mutations plus 3 additional molecular alterations showed HGC.

Conclusions

TERT promoter mutations are commonly associated with malignancy, particularly HGCs, when multiple co-occurring molecular alterations are present. However, TERT promoter mutations may occasionally be detected in benign thyroid neoplasms when encountered in isolation or with fewer than 2 additional molecular alterations.

INTRODUCTION

Telomerase reverse transcriptase (TERT), an enzyme encoded by the hTERT gene, prevents telomere shortening in senescent cells, allowing for unlimited cellular proliferation. TERT is repressed in most normal tissues and benign tumors but can be highly expressed in malignant tumors with a TERT promoter mutation that upregulates TERT transcriptional activity.^1-5 Aggressive and advanced cancers such as metastatic melanomas and glioblastomas are further enriched with TERT promoter mutations.^6,7 Similarly, in the thyroid, TERT promoter mutations have been reported in tumors of different histologic grades, emerging as a prognostic biomarker for thyroid cancers with aggressive clinicopathologic features.⁸TERT promoter mutations have a similar prevalence in papillary thyroid carcinoma (PTC), oncocytic carcinoma (OCA), and follicular thyroid carcinoma (FCA), while the incidence rate increases in aggressive forms of thyroid cancers such as high-grade and anaplastic thyroid carcinomas.^8-10 Of note, TERT promoter mutation is one of the most recognized markers associated with aggressive thyroid cancer phenotypes. It has been hypothesized that a 2-step molecular alteration sequence may underlie the development of an aggressive thyroid tumor—the first step being early genetic events such as BRAF V600E and RAS mutations in the initial tumorigenesis of well-differentiated thyroid cancers, followed by a secondary molecular change such as TP53 or TERT promoter mutations leading to tumor progression or dedifferentiation.¹⁰ A high prevalence of those TERT promoter mutations and TP53 mutations has been confirmed by other studies.¹¹ The coexistence of TERT promoter mutations with other molecular alterations, such as BRAF V600E mutation, further augments tumor aggressiveness.^9,12-16TERT promoter mutations have been associated with aggressive clinicopathologic features such as a higher tumor stage and frequent disease recurrence.¹⁷ In addition, TERT promoter mutation is an independent molecular marker of poor prognosis, regardless of the clinicopathologic parameters.¹⁸ Studies that have investigated the incidence of TERT promoter mutations across thyroid lesions have largely examined histologic specimens.^9,18,19 In this study, we reported a multi-institutional experience with TERT promoter mutations identified in preoperative thyroid fine-needle aspiration (FNA) specimens with a predominantly indeterminate cytology diagnosis in correlation with molecular profiles and histopathologic outcomes.

MATERIALS AND METHODS

Study Design and Case Selection

After approval by each institution’s institutional review board, institutional pathology databases were searched for thyroid FNA cases in which a TERT promoter mutation was identified between January 2018 and December 2022 from 4 participating institutions. We only included cases in which molecular testing was performed with ThyroSeq, version 3 (v3), Genomic Classifier (GC) test and had histopathologic follow-up available. Information including patients’ demographics, FNA diagnosis, molecular results, and histopathologic follow-up was recorded.

Cytology Specimen Preparation

The FNA biopsies of thyroid nodules were performed under ultrasound guidance. Typically, direct smears were prepared, air-dried, and/or fixed immediately in 95% alcohol or using a commercial fixative spray. Air-dried smears were stained with a Diff-Quik stain (a modified Romanowsky stain) or a modified ultrafast Papanicolaou stain. In a subset of cases, needle rinse material was saved in a Cyto-Rich Red fixative (Epredia) or a ThinPrep PreservCyt Solution (Hologic) and processed for a ThinPrep slide. In selective cases, the entire FNA material was saved in ThinPrep PreservCyt Solution (Hologic) and processed for a ThinPrep slide. When available, ThinPrep slides and alcohol-fixed smears were processed with a Papanicolaou stain. Additionally, a sample was collected and saved in a ThyroSeqPreserve vial for a potential ThyroSeq molecular test (Sonic Healthcare USA Thyroseq Laboratory). Cytologic diagnosis/classification of thyroid FNAs was rendered according to the criteria outlined in the third edition of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC).²⁰

ThyroSeq Molecular Testing

TERT promoter mutation analysis was performed on FNA samples with an indeterminate cytology diagnosis, including atypia of undetermined significance (AUS, TBSRTC category III), follicular neoplasm/oncocytic follicular neoplasm (FN/FN-OFN, TBSRTC category IV), and suspicious for malignancy (TBSRTC category V) using the ThyroSeq v3, GC platform as a commercially available test. Molecular testing was extended to TBSRTC category VI: malignant (TBSRTC category VI), if requested by the clinician. The test analyzed a total of 112 genes for point mutations, fusions, DNA copy number variations (CNVs), and gene expression alterations. TERT promoter mutation was assessed as a part of the ThyroSeq v3 assay.²¹

Histopathologic Evaluation

Thyroid neoplasms were classified according to the fifth edition of the World Health Organization.²² Cases with a single totally encapsulated follicular-patterned nodule were classified as follicular adenoma (FA). Cases with a follicular-patterned nodule with oncocytic cells comprising more than 75% of the nodule were classified as oncocytic adenoma (OA). In FA and OA cases, the entire nodule and capsule were entirely submitted. Follicular thyroid carcinoma and OCA were defined as invasive malignant well-differentiated follicular neoplasm and invasive malignant well-differentiated follicular neoplasm composed of at least 75% of oncocytic cells, respectively, without nuclear features of PTC or high-grade features. Papillary thyroid microcarcinoma was defined as PTC less than 1 cm in size. Papillary thyroid carcinoma, FCA, and OCA were referred to as well-differentiated malignant thyroid neoplasms. High-grade follicular cell–derived nonanaplastic thyroid carcinoma (HGC) included poorly differentiated thyroid carcinoma (PDTC) and high-grade differentiated thyroid carcinoma (HGDTC). High-grade differentiated thyroid carcinoma was defined as differentiated carcinomas with 5 or more mitotic figures per 2 mm² and/or tumor necrosis using the Memorial Sloan Kettering Cancer Center (MSKCC) criteria.²³ Poorly differentiated thyroid carcinoma was defined as histologically poorly differentiated carcinoma diagnosed using the Turin proposal (solid, trabecular, or insular growth pattern; absence of nuclear features of papillary thyroid carcinoma; presence of at least 1 of the following 3 features: convoluted nuclei, ≥3 mitotic figures/2 mm², tumor necrosis).²⁴ Tumors were staged according to the eighth edition of the American Joint Committee on Cancer staging system.

RESULTS

The study cohort included 32 cases from 11 male and 21 female patients, with a median age of 68 years. Cytology diagnoses included AUS (n = 12), FN/FN-OFN (n = 16), and suspicious for malignancy or malignant, PTC (n = 4) TABLE 1. Histopathologic follow-up showed that 5 cases were benign neoplasms: FA (n = 2) and OA (n = 3). The remaining 27 cases were malignant, including HGDTC (n = 5), PDTC (n = 2), PTC (n = 7), OCA (n = 7), and FCA (n = 6) TABLE 1 and TABLE 2. Of the 12 cases classified as AUS, 9 (75%) were malignant and 3 (25%) were benign neoplasms on histopathologic follow-up TABLE 2. None of the 9 malignant cases showed high-grade features. Of the 16 cases cytologically classified as FN/FN-OFN, 14 (88%) were malignant and 2 (12%) were benign neoplasms on follow-up. Of the 14 malignant cases, 6 (38%) were high-grade carcinomas. All 4 cases cytologically classified as suspicious for malignancy or malignant were malignant (100%) on follow-up, including 1 case of HGDTC TABLE 2. It is worth noting that all HGC cases, including 5 HGDTC and 2 PDTC cases, had a FN-OFN (n = 6) or a malignant (n = 1) cytologic diagnosis. All 5 patients with a diagnosis of FA or OA on thyroidectomy were alive with no evidence of recurrent or metastatic disease at a median follow-up of 35 months (range, 20-48 months).

Most of the detected genetic alterations in the TERT promoter were the p.C228T (n = 29), with the remaining cases being p.C250T (n = 2) and p.C228A (n = 1) TERT promoter mutation (Supplemental Table 1; all supplementary material is available at American Journal of Clinical Pathology online). TERT promotor mutation by itself was not always associated with malignancy. Among the 4 cases with an isolated TERT promoter mutation, 3 (75%) were malignant (2 OCAs, 1 PTC), and 1 (25%) turned out to be a FA on histopathologic follow-up TABLE 3. No high-grade malignancy was found in cases with isolated TERT promoter mutation. Among 17 cases with a TERT promoter mutation plus 1 additional molecular alteration, the overall risk of malignancy (ROM) was 77%. The diagnoses on histopathologic follow-up included FA or OA in 4 (23%) cases, well-differentiated malignant neoplasms (FCA, OCA, or PTC) in 11 (65%) cases, and HGDTCs in 2 (12%) cases. In 6 cases with TERT mutation plus 2 additional molecular alterations, 5 (83%) were well-differentiated malignant neoplasms (FCA, OCA, or PTC), and 1 (17%) was a HGDTC. In the 5 cases with TERT mutation plus 3 additional molecular alterations, 4 (80%) cases were HGCs (2 HGDTCs and 2 PDTCs), and 1 (20%) case was PTC. There were no benign neoplasms in cases with TERT mutation plus 2 or more molecular alterations, and the ROM was 100%. The HGCs correlated positively with the number of molecular alterations, with 0% in TERT-alone cases, 12% in TERT plus 1 molecular alteration cases, 17% in TERT plus 2 molecular alterations cases, and 80% in TERT plus 3 molecular alterations cases TABLE 3.

The molecular alterations identified aside from TERT promoter mutations included gene expression profile (GE) in 9 (28%), CNV in 8 (25%), BRAF V600E in 5 (16%), BRAF 1485-T488del in 1 (3%), HRAS in 6 (19%), NRAS in 6 (19%), and EIF1AX in 4 (13%) cases FIGURE 1. PIK3CA, EZH1, TP53, PTEN, and TRA2A/THADA were observed in 1 case each. The additional molecular alterations encountered in benign neoplasms were CNV (n = 2), HRAS (n = 1), and EIF1AX (n = 1). Molecular alterations or genes with mutations present in well-differentiated malignant neoplasms were BRAF V600E (n = 4), BRAF 1485-T488del (n = 1), GE (n = 4), NRAS (n = 4), HRAS (n = 3), EIF1AX (n = 3), CNV (n = 1), PTEN (n = 1), EZH1 (n = 1), TRA2A/THADA (n = 1), and PIK3CA (n = 1). Additional molecular alterations associated with HGCs included CNV (n = 5), GE (n = 5), NRAS (n = 2), HRAS (n = 2), BRAF V600E (n = 1), and TP53 (n = 1). The HGCs harbored at least 1 of the 3 following molecular alterations: GE, CNV, or TP53 mutation. Out of 7 cases, 4 (57%) HGDTC or PDTC cases had 3 additional molecular alterations.

DISCUSSION

Molecular testing has emerged as a cornerstone analysis that extends beyond diagnostic information to identify thyroid tumors with aggressive behavior. A substantial number of molecular alterations, such as TERT promoter mutations, have a defined cancer risk and increase the ROM when detected in thyroid FNAs, particularly when seen in combination with other molecular alterations. While TERT promoter mutation represents an early event in glioblastoma, melanoma, and hepatocellular carcinoma, it typically develops late in thyroid carcinogenesis.^25,26 It is infrequently encountered in early-stage well-differentiated PTCs, but the frequency increases significantly as tumors dedifferentiate and become invasive, as well as in advanced forms of disease.^{8,14,17,18,27-29} High-grade thyroid tumors such as HGDTC, PDTC, anaplastic thyroid carcinoma (ATC), and advanced-stage well-differentiated thyroid cancer are almost twice as likely to harbor TERT promoter mutations compared to well-differentiated thyroid carcinomas.^15,18,27 When occurring in the latter, TERT promoter mutations are associated with widely invasive tumors, unlike their minimally invasive counterparts, and are an independent predictor of recurrence, distant metastases, persistent disease, poor prognosis, and cancer‐related mortality.^27,30-35

FNA of the thyroid is the standard diagnostic tool used for evaluating thyroid nodules, and its interpretation is standardized by a uniform tiered classification scheme, the TBSRTC. This reporting system has not only established a uniform category-based reporting system for thyroid FNAs but also expanded on providing an implicit ROM for each of those diagnostic categories. The ROM increases from TBSRTC category II to category VI. The presence of molecular alterations further refines the probability of malignancy in thyroid FNAs with indeterminate cytology. In fact, the combination of indeterminate cytology and molecular alterations results from ThyroSeq v3, GC correlates with the ROMs provided in the TBSRTC but appear to be a better estimation of the true ROMs.³⁶ In our study, the rates of malignancy in TBSRTC categories III, IV, and V/VI were 75%, 88%, and 100%, respectively. However, all category III cases were well-differentiated carcinomas or benign neoplasms on surgical follow-up, while high-grade carcinomas were classified as TBSRTC categories IV (n = 6) and VI (n = 1), reemphasizing the importance of cytomorphologic diagnostic characterization and the TBSRTC system for reporting thyroid cytopathology. Those findings may prove useful in clinical practice since the presence of TERT promoter mutations in TBSRTC category III cases was not associated with a high-grade malignancy in our case series. In contrast, TBSRTC category IV and V/VI cases with TERT promoter mutations consistently showed malignant neoplasms, including high-grade malignancies on surgical follow-up.

There are uncertainties as to how early telomerase activation occurs in thyroid carcinogenesis, and studies have reported an association between TERT promoter mutation and worse survival in thyroid carcinomas without aggressive histology, suggesting that there may be patients with TERT promoter mutations that have not yet manifested an aggressive histopathologic appearance at the time of surgery.³⁷ Furthermore, most studies have failed to identify TERT promoter mutations in benign thyroid lesions,^9,19,30,38 with only rare reports on its presence in adenomas, nodular goiter, or thyroiditis.³⁶ Samples may be biased, as benign thyroid nodules that are sampled on FNA cytology typically do not undergo molecular testing. A case of follicular adenoma with TERT and NRAS mutations was reported in a patient who ultimately developed a recurrence from a follicular thyroid carcinoma and died of disease. On careful retrospective review, the authors confirmed the original diagnosis of follicular adenoma as an encapsulated tumor without high cellularity, mitosis, or aberrant relation to the capsule or vessels, and the absence of carcinoma was confirmed. It is, however, unclear whether the capsule was entirely submitted. While the question of the original pathology diagnosis remains, the authors conclude that the case was a follicular adenoma that progressed to follicular carcinoma, reflected in the alteration of histopathologic and clinical phenotypes.³⁹ Another case of follicular adenoma with TERT, HRAS, and EIF1AX mutations was reported by Topf et al.⁴⁰ The nodule was entirely submitted for microscopic examination, and multiple levels were performed on the sections containing the capsule without capsular or vascular invasion identified.⁴⁰ We have observed similar results and report 5 cases of benign neoplasms with TERT promoter mutations. It is noteworthy that the capsule was entirely submitted in all our cases, and all patients were alive with no evidence of thyroid disease at the last follow-up. The findings suggest that TERT promoter mutation may occur as an early genetic event before the tumor develops histopathologic malignant features. In fact, the presence of TERT promoter mutation in preneoplastic stages has been documented in other organs such as liver and bladder.^41,42 Given that most studies have failed to identify TERT promoter mutations in benign thyroid neoplasms, it is possible that its frequency is quite low. This is further supported by the rare benign thyroid neoplasm cases reported in the literature. Additionally, the presence of TERT mutation in thyroid carcinomas has been established by the multitude of studies seeking to establish the molecular profile of thyroid carcinomas, which would benefit from prognostication and potential targeted therapies, including well-differentiated, advanced, and HGCs.^18,43,44 Alternatively, apart from studies that included benign thyroid lesions for various reasons, testing for molecular alterations in benign thyroid neoplasms is not widely implemented in routine clinical practice. It is therefore possible that the incidence of TERT promoter mutations in benign thyroid neoplasms is underestimated.

In our cohort, there were no high-grade thyroid carcinomas with an isolated TERT promoter mutation. Furthermore, we observed that cases with 1 or 2 molecular alterations were predominantly low-risk malignant neoplasms, and the frequency of high-grade malignancy increased with the number of molecular alterations, with 80% of cases with TERT promoter mutations and 3 co-occurring additional molecular alterations displaying high-grade carcinomas on histopathology. Benign neoplasms were encountered in cases with an isolated TERT promoter mutation or TERT promoter mutations with 1 additional molecular alteration. There were no benign neoplasms with 2 or more additional molecular alterations.

There are reports indicating that molecular alterations increase in frequency from benign to malignant thyroid neoplasms with suggestions that adenomas may represent precancerous lesions that progress to malignancy as molecular alterations accumulate.^45-47 It is possible that the presence of TERT promoter mutations in benign thyroid neoplasms confers a different developmental potential and predisposes to a malignant transformation and an aggressive course as additional genetic alterations occur. Indeed, multiple studies have shown that the presence of simultaneous molecular activations and alterations is oncogenic.⁴⁷ In a study by Nikiforova et al,⁴⁸ well-differentiated PTCs with multiple mutations showed aggressive behavior and typically had metastasis.

Our study provides a focused analysis of thyroid FNAs with TERT promoter mutations, but it has several limitations, including its retrospective nature. First, we only included patients with surgical follow-up. It is unclear whether patients who were lost to follow-up at our institutions had surgery performed elsewhere or whether the pretest probability of neoplasia was lower in those patients and surgery was avoided all together. Second, TBSTRC category VI, malignant, FNAs are not reflexed for molecular analysis. As a result, the incidence of PTCs with TERT promoter mutations may be underrepresented in our study. A third drawback is the inclusion of multiple institutions where diagnostic thresholds are likely different when classifying thyroid FNA specimens. The indeterminate categories, where molecular testing is indicated most, are very heterogeneous, which, despite the TBSRTC definitions, remain subject to interobserver variability. Last, to increase the sensitivity by using a more comprehensive molecular panel, cases tested by molecular platforms other than ThyroSeq v3, GC were excluded from the analysis. This exclusion is reflected in the small number of patients in the study and within each subgroup.

In summary, the presence of TERT promoter mutations in benign thyroid lesions has been occasionally reported, but its significance in the thyroid has not been well established. Our study suggests that TERT promoter mutations occur frequently, but not universally, in association with malignancy, particularly high-grade carcinomas. When TERT promoter mutations are seen in concert with additional genetic/epigenetic alterations and in conjunction with TBSRTC category IV, V, or VI, the risk of high-grade carcinoma is significant. Therefore, the presence of TERT promoter mutations alone or in combination with other molecular alterations in an FNA cytology specimen may help triage patients for proper clinical management.

Conflict of interest disclosure: The authors have nothing to disclose.

References

Author notes