Somatic mutations in the telomerase reverse transcriptase (TERT) promoter increase telomerase activity, which maintains telomere length. Maintenance of telomere length is very important for the immortalization of cancer cells.^1-3) TERT promoter mutations have been identified in more than 50 human cancers including thyroid cancer.⁴⁾ In meta-analyses, they were found in 10-20% of differentiated thyroid carcinoma (DTC) and 40% of poorly differentiated thyroid carcinoma (PDTC) or anaplastic thyroid carcinoma (ATC).^5-7) They are highly prevalent in large tumors, old age, aggressive histology, advanced stages, and distant metastasis.^5,6) They are also strongly associated with tumor recurrence and mortality in thyroid cancer.^8-14)

TERT promoter mutations were independently associated with anaplastic transformation of papillary thyroid carcinoma (PTC).¹⁵⁾ PTC-derived ATCs are characterized by BRAF mutation and TERT promoter mutations, which occur prior to anaplastic transformation.

ATC has the worst prognosis, with a median survival of 3 to 6 months.^16,17) All patients with ATC are classified as stage IV (A, B, or C) by the American Joint Committee on Cancer TNM system. The introduction of multimodal treatment including tyrosine kinase inhibitors and immune checkpoint inhibitors improves survival.^18-25) However, molecular markers that can predict ATC prognosis have not been elucidated.

Therefore, we conducted this study to evaluate whether TERT promoter mutations, known as a poor prognostic factor in DTC, also act as a poor prognostic factor in ATC. We also re-evaluated the prognostic factors affecting survival in ATC, including TERT promoter mutations.

Patient Selection

We reviewed the medical records of 102 patients diagnosed with ATC at Samsung Medical Center between November 1995 and December 2022. Among the 102 ATC patients, those who were transferred to another hospital during treatment or lost to follow-up, who previously underwent treatment for DTC, with other fatal diseases, or who could not be tested for TERT promoter mutations due to difficulty in DNA extraction from pathological tissue were excluded. Finally, we included 41 patients with ATC in this study. The clinicopathological characteristics of the 41 patients who underwent the TERT promoter mutations test were not different from those of the 61 patients who did not (Supplementary Table 1).

The patients’ clinical information was obtained from electronic medical records. Cancer staging was conducted using the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. The Institutional Review Board at Samsung Medical Center approved this study (SMC-IRB 2020-10-033).

Diagnosis of ATC

The diagnosis of ATC adhered to the 2021 American Thyroid Association guidelines.²⁶⁾ Coexistent DTC was checked. When a sarcomatoid component or squamous carcinoma was identified without concurrent DTC, investigations were conducted to ascertain the potentiality of metastasis to the thyroid from a distinct origin. If necessary, immunohistochemistry (IHC) markers such as low molecular-mass cytokeratin, PAX-8, thyroglobulin, thyroid-transcription factor 1 (TTF-1), Ki-67, or p53 were tested to exclude alternative cancers.^27,28) Also, assessments such as tumor necrosis, atypical mitoses, and mitotic rates were conducted as needed. Such additional analyses were conducted at the discretion of the pathologists.

Detection of TERT Promoter Mutation

Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue using a Qiagen DNA FFPE Tissue Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) sequencing was carried out to identify TERT promoter mutations using an iTERT mutation detection kit (Geninus Inc., Seoul, Korea). The PCR reactions were assembled on ice and preincubated at 94°C for 15 minutes, followed by 40 cycles at 94°C for 20 seconds, 58°C for 40 seconds, 72°C for 1 minute, and a final extension at 72°C for 5 minutes using a C1000 Touch Thermal Cycler Kit (Bio-Rad, Hercules, CA, USA). Bidirectional sequencing was performed using a BigDye Terminator v.3.1 Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130xl Genetic Analyzer. The sample was considered mutation-positive if mutations were detected in both the forward and reverse DNA strands.⁷⁾

Treatments

Patients diagnosed with ATC received multimodal treatment including surgery, radiotherapy (RT), and systemic therapy. Among the 41 patients, 29 (70.8%) received total thyroidectomy. The extent of lymph node resection was determined according to surgeon’s decision. A total of 23 patients (56.1%) received external radiotherapy, and the median cumulative dose was 59.40 Gy (range, 24.20-66.00 Gy). Patients prior to 2002 received mainly 2D RT, and patients after 2002 received either 3D conformal RT or intensity-modulated RT.

A total of 25 patients (61.0%) was treated with varying types of systemic therapy. Among them, five patients received conventional chemotherapy, eight patients received chemotherapy and tyrosine kinase inhibitor (TKI), and 12 patients received TKI. Conventional chemotherapy included doxorubicin, cisplatin, paclitaxel, and carboplatin. Doxorubicin was administered at 60 mg/m² with 60 mg/m² of cisplatin every three weeks. Paclitaxel of 175 mg/m² was administered with carboplatin every three weeks. TKIs included dabrafenib plus trametinib, lenvatinib, and vemurafenib. Dabrafenib plus trametinib was taken 150 mg twice daily plus 2 mg once daily, lenvatinib was taken 10 to 24 mg once daily, and vemurafenib was taken 480 to 960 mg twice daily by oral administration.²⁴⁾ We divided the 41 patients into two groups according to the multimodal therapy: an aggressive treatment group and a non-aggressive treatment group. The definition of aggressive treatment was based on a previous study.²⁴⁾ The aggressive treatment group consisted of patients who underwent surgery, external radiotherapy, and systemic therapy. The non-aggressive treatment group consisted of patients who either did not receive treatment or who received one or two of the following therapy types: surgery, radiotherapy, and systemic therapy.

Outcomes and Statistical Analysis

The response evaluation was based on guidelines outlined in the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1.²⁹⁾ Overall survival (OS) was defined as the time from diagnosis (pathologically confirmed) to the date of death for deceased study members or the date of last observation for surviving patients. The cause of death was identified from the Korea National Statistical Office and hospital medical records.

To compare the distribution between the TERT-mutant and wild-type groups, a Chi-square test was performed for nominal variables, and a Mann-Whitney U test was performed for continuous variables. The OS was analyzed by Kaplan–Meier method and log-rank test. The evaluation of factors affecting survival was verified by the Cox regression analysis method.

p-values less than 0.05 were considered statistically significant. An additional analysis was conducted to compare the differences in patient and disease characteristics between those who underwent the TERT promoter mutations test and those who did not. Statistical analysis was conducted using R version 4.3.0 (R Foundation for Statistical Computing, Vienna, Austria).

Clinicopathological Characteristics of 41 Patients with ATC Who Underwent TERT Promoter Mutation Testing

The clinicopathological characteristics of the 41 patients are shown in Table 1. The median age was 69 years (range, 42-91 years), and 25 patients (61.0%) were female. Twenty-six patients (63.4%) had concomitant DTC histologically. The median tumor size was 4.9 cm (range, 0.8-11.0 cm). Lymph node metastasis and distant metastasis at the time of diagnosis were found in 33 (80.5%) and 23 patients (56.1%), respectively. There was no one with stage IVA disease, but 18 patients (43.9%) had stage IVB, and the remaining 23 patients (56.1%) had stage IVC. Of the 41 patients, TERT promoter mutations were detected in 15 patients (36.6%, TERT-mutant group) but not in 26 patients (63.4%, wild-type group). The variables that showed significant differences between the TERT-mutant and wild-type groups were tumor size (p=0.010) and coexistent DTC (p=0.044). The median tumor size in the TERT-mutant group was 5.1 cm (range, 3.0-11.0 cm), which was significantly larger than that of the wild-type group (4.2 cm, range 0.8-8.0 cm). In terms of treatment multimodality, 13 patients (31.7%) received aggressive treatment and 28 patients (68.3%) received non-aggressive treatment. Aggressive treatment was performed more frequently in the TERT-mutant group; however, there was no statistically significant difference (53.3% vs. 19.2%, p=0.056).

Although TERT promoter mutations have been strongly associated with tumor recurrence and mortality in DTC, they did not affect survival in patients with ATC. When we analyzed various factors including TERT promoter mutations affecting survival by Cox regression analysis, distant metastasis was a significant factor for predicting poor outcomes. However, TERT promoter mutations did not affect survival in ATC patients.

Several studies have previously investigated the effects of TERT promoter mutations in ATC patients. Liu et al.³⁰⁾ reported that the TERT-mutant group in ATC patients had a shorter survival than the TERT wild-type group, but the difference was not significant (p=0.129). However, the number of patients was small (n=20), and distant metastasis and treatment multimodality were not considered. Shi et al.³¹⁾ demonstrated an association of TERT promoter mutations with several high-risk factors of thyroid cancer, including the BRAFV600E mutation, old age, and distant metastasis in ATC patients. However, the independent prognostic roles of TERT promoter mutations and OS according to TERT promoter mutations were not described. In addition, treatment multimodality was not considered. In 2022, Xu et al.³²⁾ compared ATC patients with long-term survival (LTS) greater than 2 years to control patients who survived for less than 2 years. The proportion of patients with TERT promoter mutations was not different between the two groups (LTS 60% vs. control 80%, p=0.080), but co-existence of the BRAF or RAS mutation with TERT promoter mutation was significantly less frequent in the LTS group (LTS 35% vs. control 67%, p=0.019).

In our study, the TERT-mutant group received relatively more aggressive treatment (53.3% vs. 19.2%, p=0.056), but this treatment trend was not intentional and was discovered incidentally in the retrospective analysis. Although the TERT-mutant group received more aggressive treatment and tended to have higher median OS compared to the wild-type group, there was no significant difference in OS between the two groups (p=0.458).

In the present study, the TERT-mutant group exhibited a larger tumor size compared to the TERT-wild type group. Although the exact reason for the larger tumor size in the TERT-mutant group remains unclear, this finding aligns with previous studies indicating a higher frequency of TERT promoter mutations in larger tumors in total thyroid carcinoma or PTC.^6,14,33)

Additionally, we observed a higher rate of co-existence with DTC in the TERT-mutant group, supporting the hypothesis that TERT promoter mutations contribute to the dedifferentiation process from DTC to ATC.^15,34)

In this study, cases where patients had previously been treated for DTC and later recurred with ATC were not included. This exclusion was due to the difficulty in clearly assessing the therapeutic outcomes for ATC following prior treatment for DTC and the ambiguity in staging criteria.

The strength of this study was that it included treatment multimodality as a prognostic factor that could affect the outcome. It also focused on how the presence of TERT promoter mutations affects prognosis in ATC patients. However, there were a few limitations in this study. First, it was a retrospective study. Second, it included a small number of patients and is limited in demonstrating statistical significance. Third, the effects of genetic mutations other than the TERT promoter mutations (BRAF mutation or RAS mutation) could not be considered. Because a large number of patients did not undergo a BRAF mutation test, it was difficult to derive significant results.

Despite these limitations, we found that TERT promoter mutations alone did not have any effect on survival in ATC, unlike in DTC. 

This study was conducted in accordance with the 1964 Declaration of Helsinki. This study was approved by the Institutional Review Board of Samsung Medical Center (SMC-IRB 2020-10-033), and written informed consent was waived due to the retrospective nature of this study.

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

No potential conflict of interest relevant to this article was reported.