Effect of Having Concurrent Mutations on the Degree of Aggressiveness in Patients with Thyroid Cancer Positive for TERT Promoter Mutations

Simple Summary As molecular testing of thyroid nodules becomes more common, thyroid specialists must be able to interpret and understand the clinical implications of the results. A telomerase reverse transcriptase (TERT) promoter mutation can strongly predict thyroid cancer aggressiveness. However, the reason why some thyroid cancers with TERT promoter mutations are more aggressive than others remains unclear. This study aimed to examine whether TERT promoter mutations coexisting with other mutations are linked to more aggressive disease than TERT promoter mutations alone. The medical records of patients who had thyroid surgery and TERT promotor mutations were examined. Our findings showed that the likelihood of aggressive thyroid cancers was 10 times higher in patients with TERT promoter and other concurrent mutations. Thyroid specialists can use our results to accurately interpret the molecular testing of thyroid nodules, provide appropriate counseling, and discuss possible management options accordingly. Abstract This study aimed to examine whether concurrent mutations with a TERT promoter mutation are associated with a greater likelihood of more aggressive disease than a TERT promoter mutation alone. The medical records of 1477 patients who underwent thyroid surgery at two tertiary hospitals between 2017 and 2022 were reviewed. Twenty-four patients had TERT promoter mutations based on molecular profile testing. Clinicodemographic data, mutational profiles, and histopathological features were assessed. Descriptive analysis, Fisher’s exact test, and binary logistic regression were performed. Seven patients had single-gene TERT promoter mutations, and 17 had concurrent mutations, including BRAF V600E, HRAS, NRAS, PIK3CA, and EIF1AX. The overall prevalence of malignancy was 95.8%, of which 78.3% were aggressive thyroid cancers. There was a statistically significant association between concurrent mutations and disease aggressiveness. The odds of having aggressive disease were 10 times higher in patients with a TERT promoter mutation and a concurrent molecular alteration than in those with a TERT promoter mutation alone. This is an important finding for thyroid specialists to consider when counseling patients concerning risk stratification and management options.


Introduction
The incidence of thyroid cancer has increased rapidly in the last few decades, and it is expected to be the fourth most common cancer by 2030. This significant rise is mainly attributed to advances in imaging modalities that can easily detect thyroid nodules and, therefore, thyroid cancer. Although thyroid cancer has an excellent prognosis in most cases, it is estimated that up to 20% of thyroid cancer cases will recur, up to 30% will metastasize regionally, less than 4% will metastasize to distant organs, and a small percentage will result in mortality (0.4%) [1][2][3][4]. Therefore, it is important to establish prognostic indicators of disease aggressiveness to guide risk stratification and to determine the extent of surgery and adjuvant treatment regimens for thyroid cancer. To date, no single factor has been attributed to thyroid cancer outcomes; instead, a group of factors are known to affect prognosis. Several factors, including patient characteristics, mutational profiles, and histopathological features, have been identified and described in the literature.
As molecular testing of thyroid nodules becomes more common, it is of paramount importance for thyroid specialists to be able to interpret and understand the clinical implications of the results. Several studies have established the diagnostic values and risk stratification abilities of different molecular profile tests for thyroid cytology specimens. To decrease the substantial number of unnecessary thyroid surgeries performed worldwide, molecular profile tests aim to differentiate patients who are more likely to require surgery from those more likely to require conservative management. Validation studies using the ThyroSeq v3 testing platform have confirmed its ability to rule in disease with a positive predictive value of 66% and rule out disease with a negative predictive value of 97% compared with ThyGeNEXT, which has a positive predictive value of 97% and a negative predictive value of 75% [5,6]. It should be emphasized that these percentages are not fixed and are essentially dependent on each institution's disease prevalence.
Abnormal activation of the mitogen-activated protein kinase (MAPK) signaling pathway has been described in carcinogenesis in multiple body sites, including the thyroid. Many fundamental mutations in thyroid cancer target different constituents of the MAPK signaling pathway, including BRAF V600E and RAS gene point mutations, RET::PTC and PAX8::PPARγ chromosomal rearrangements, and telomerase reverse transcriptase (TERT) promoter mutations. Varying levels of growth factors, hormones, and cytokines interact with cell surface receptor tyrosine kinases in the MAPK signaling pathway, which is responsible for regulating cell proliferation, differentiation, and apoptosis [7,8].
TERT is a protein subunit of telomerase that adds telomeres to the ends of chromosomes to maintain their length. It is mainly found in germ lines and stem cells and only rarely in most human somatic cells. The addition of telomeric repeats to the ends of chromosomes in each cell cycle prevents cell death. Enhancing the telomerase function in cancer allows cancer cells to obtain "replicative immortality". TERT promoter mutations exist in human cancer cells responsible for melanoma, bladder cancer, and glioblastoma [9][10][11][12]. X Lie et al. reported, for the first time, the detection of TERT promoter mutations in thyroid cancer. Moreover, the mutation was not present in benign thyroid tumors (0 out of 85 patients) [13]. The prevalence of TERT promoter mutations in papillary thyroid cancer is estimated to be between 5% and 25%, varying among subtypes. It is more common in aggressive subtypes, such as the tall cell variant [14]. The prevalence of TERT promoter mutations in benign thyroid tumors, follicular thyroid cancer, poorly differentiated thyroid cancer, and anaplastic thyroid cancer is estimated to be 0%, 20%, 20-50%, and 30-75%, respectively. In one study, the prevalence was significantly higher in tumors with aggressive histological features (32.7%) than in those with nonaggressive histological features (15.3%) [15].
The modified initial risk stratification system for differentiated thyroid cancer published in the American Thyroid Association (ATA) 2015 guidelines recommends using the TERT promoter mutation status, if available, to improve risk estimates [16]. Many studies have revealed that a TERT promoter mutation is a predictor of thyroid cancer aggressiveness [8,17]. In their systematic review and meta-analysis, Yin et al. reported significant associations with TERT promoter mutations: lymph node metastasis, extrathyroidal extension, distant metastasis, poor outcome (persistent or recurrent disease), and mortality [18]. Additionally, Ebina et al. identified 133 patients with TERT promoter mutations who exhibited significantly worse 10-year cause-specific survival (73.7% vs. 98.1%) and 10-year disease-free survival (53.7% vs. 93.3%) compared to those without mutations. However, only seven patients exhibited a TERT promoter mutation alone [19].
Why some thyroid cancers with TERT promoter mutations tend to be more aggressive remains unclear. It has been shown that anaplastic thyroid carcinoma possesses a higher number of genetic alterations than papillary thyroid carcinoma, which can be rationalized by the widely accepted progression theory of cancer that implies an ongoing buildup of mutations during progression from differentiated to dedifferentiated cancer [20]. Recent meta-analyses have revealed that thyroid cancers with concurrent BRAF V600E or RAS and TERT promoter mutations were associated with increased tumor aggressiveness compared to those harboring BRAF V600E, RAS, or TERT promoter mutations alone [21]. However, most previous studies did not account for the presence of other rare mutations that may also influence the disease outcomes of thyroid cancer with TERT promoter mutations. The present study aimed to examine whether concurrent mutations with TERT promoter mutations are associated with more aggressive thyroid cancers compared to TERT promoter mutations alone. We hypothesized that the presence of any other molecular alterations, hence the accumulation of mutations during carcinogenesis, would influence the aggressiveness of thyroid cancer with TERT promoter mutations. To our knowledge, this is the first study to evaluate the role of coexisting mutations, taking into account all possible mutations available in the commercial molecular tests (ThyGeNEXT or ThyroSeq v3) in patients with thyroid cancer positive for TERT promoter mutations in the development of aggressive features.

Study Participants and Data Collection
This multi-center study was approved by the McGill University Health Centre (MUHC) Research Ethics Committee (File Number: MP-37-2021-7665). A multi-center retrospective chart review of 1477 patients who underwent thyroid surgery at two university teaching hospitals in Montreal, Canada, between January 2017 and May 2022 was performed. The study included adult patients who underwent ultrasound-guided fine needle aspirations of dominant thyroid nodules and tested positive for one of the two TERT promoter mutations located at hotspots chr5, 1,295,228 C > T (C228T) and 1,295,250 C > T (C250T) in molecular profile testing. Clinical and demographic data, such as age, sex, Bethesda category of thyroid nodules, genetic alterations on molecular profile testing, and presence of other molecular mutations, were collected. The types of treatment and extent of thyroid surgeries were also noted. Thyroid nodules were categorized as benign or malignant based on postoperative histopathological findings. The Strengthening the Reporting of Observational Studies in Epidemiology checklist was used to guide the reporting of this study.

Molecular Profile Testing Technique
Informed consent was obtained from all patients before molecular profiling. Fine needle aspiration was performed in the clinic under ultrasound guidance by fellowshiptrained Otolaryngology-Head and Neck surgeons. An alcohol swab was used for skin preparation, followed by injection of the subcutaneous tissue with xylocaine 1% epinephrine 1/100,000 concentration. A 21-gauge needle with a beveled tip measuring 1.5 inches and a 10 cc syringe with a luer lock tip were used. Ultrasound guidance using a linear highfrequency transducer in a parallel technique was used to identify the most suspicious nodule and obtain the biopsy. To improve the quality of the biopsy, the suction effect was usually maintained throughout sampling by pulling back the plunger by 1 to 2 cc. Samples were then analyzed at a commercial laboratory (Interpace Diagnostics) at the University of Pittsburgh Medical Center, Pittsburgh, PA, USA, for ThyroSeq v3 ® testing, or Parsippany, NJ, USA, for ThyGeNEXT ® testing.

Aggressive Thyroid Cancer
The thyroid cancer was classified as aggressive if it exhibited at least one of the following features: evidence of aggressive papillary carcinoma variants (tall cell, hobnail, solid, diffuse sclerosing), poorly differentiated carcinoma, extensive vascular invasion, gross extrathyroidal extension, and presence of regional lymph node or distant metastases. The predefined criteria were based on methods used in recent publications by our group of researchers [22]. Features on histopathology used to define aggressive disease would generally warrant a more aggressive standard treatment approach according to the 2015 ATA guidelines [16], including total thyroidectomy, neck dissection, and adjuvant radioactive iodine, as opposed to hemithyroidectomy alone. Anaplastic carcinoma was excluded due to its rarity and the design of the study.

Statistical Analysis
Descriptive statistics are reported as means ± standard deviations (SDs). An independent-sample t-test was used to compare the mean age and tumor size between patients with single-gene TERT promoter mutations versus those with concurrent mutations. Fisher's exact test was used for categorical variables to assess the associations among thyroid cancer aggressiveness, sex, Bethesda scores, and other mutations. Binary logistic regression was performed to quantify the association between the presence of co-mutations and thyroid cancer aggressiveness. Two-sided p-values < 0.05 were considered to indicate statistical significance.

TERT Promoter Mutation Detection Rate
This was a chart review of 1477 patients who underwent thyroid surgery. Of those, 720 had undergone molecular testing, and 24 tested positive for the TERT promoter mutation. Therefore, the detection rate in our sample was 24/720 (3.33%). Table 1 summarizes the baseline patient characteristics. Most patients were female (75%), and the mean age of the patients was 68 years. The minimum and maximum ages at the time of surgery were 53 and 83 years, respectively.

Patients' Characteristics and Molecular Profile Distribution
The mean size of the dominant thyroid nodule was 3.7 cm. The minimum and maximum nodule sizes on the final pathological examination were 1 cm and 7.8 cm, respectively. The frequencies of each cytological category are summarized in Table 1. Figure 1 shows the molecular profile distribution. Single-gene TERT promoter mutations were found in seven patients on molecular testing. The remaining 17 patients had concurrent mutations, including BRAF V600E (in 12 patients), RAS (in four patients: three NRAS and one HRAS), or PIK3CA and EIF1AX (in one patient) mutations. Eighty-three percent of patients underwent total thyroidectomy. The overall prevalence of malignancy was 95.8%. All patients, except one, had malignant disease on postoperative histopathological examination. The sole patient with benign disease had a Bethesda category of IV and a single-gene TERT promoter mutation on molecular profile testing. Among the remaining 23 patients with malignancy, 18 had at least one of the predefined aggressive features.

Effect of Mutational Profile on Degree of Aggressiveness
The results of Fisher's exact test are presented in Table 2. The Bethesda categories and molecular profiles were significantly associated with disease aggressiveness, with pvalues of 0.001 and 0.038, respectively. Of the patients who had aggressive disease on

Effect of Mutational Profile on Age, Sex, Thyroid Nodule Size, and Bethesda Category
Patients with single-gene TERT promoter mutations were significantly older by 13.6 years than those with co-mutations, with mean ages of 77.6 (SD: 4.7) and 64 (SD: 9) years, respectively (p-value of 0.001). There was no statistically significant association between mutational status and sex. Furthermore, no statistically significant difference was found between the mean dominant thyroid nodule size of patients with single-gene TERT promoter mutations versus those with concurrent mutations (4.16 cm ± 1.77 versus 3.64 cm ± 1.61, p-value > 0.05). Finally, the mutational status association with the Bethesda category reached statistical significance; the presence of concurrent mutations was associated with a higher Bethesda category of 5 or 6, as opposed to a Bethesda category of 3 or 4 (p-value of 0.009).

Effect of Mutational Profile on Degree of Aggressiveness
The results of Fisher's exact test are presented in Table 2. The Bethesda categories and molecular profiles were significantly associated with disease aggressiveness, with p-values of 0.001 and 0.038, respectively. Of the patients who had aggressive disease on postoperative histopathological examination, 83.3% had co-existing mutations, as opposed to 16.7% of those with a TERT promoter mutation alone. On the other hand, of those who had nonaggressive disease on postoperative histopathological examination, 33.3% of patients had co-existing mutations, as opposed to 66.7% of those with a TERT promoter mutation alone. Binary logistic regression was performed to quantify the association between concurrent mutations and aggressiveness, revealing statistical significance with a p-value of 0.0318 and an odds ratio of 10 (1.22, 81.81) (coefficient 2.3, standard error 1). Fisher's exact test was also used to assess the relationship between the mutational profile and lymph node status. The presence of concurrent mutations was significantly associated with lymph node status, with a p-value of 0.0059. All seven patients with single-gene TERT promoter mutations had no positive lymph nodes on final histopathology. Due to the small sample size, subgroup analysis for each pathology type and distant metastasis was impossible. In our study sample, sex was not significantly associated with disease aggressiveness. Nonetheless, all male patients had aggressive disease.

Discussion
This study supports the hypothesis that thyroid nodules positive for both TERT promoter mutations and other mutations are more likely to harbor aggressive features than thyroid nodules positive for TERT promoter mutations alone. Furthermore, the odds of having aggressive disease were 10 times higher in patients with TERT promoter mutations in addition to concurrent mutations than in those with TERT promoter mutations alone.
In our study, more than half of the patients with single-gene TERT promoter mutations had nonaggressive features. Moreover, one patient with a Bethesda category IV thyroid nodule had benign disease on final pathology. This observation led us to attempt to recognize potential factors that may influence the degree of aggressiveness of thyroid cancer with TERT promoter mutations to more accurately identify patients with poor prognoses.
Of the studies examining TERT promoter mutations in patients with follicular adenomas, the reported occurrence is~0% [13,23,24]. In fact, out of the 552 cases of follicular adenomas studied in these different series, only one study reported a positive case for a TERT promoter mutation. Wang et al. found one patient expressing a C228T mutation with a postoperative histopathological diagnosis of follicular adenoma. Of note, the single patient described in the literature subsequently died from metastatic follicular thyroid carcinoma [25]. This supports the theory that a TERT promoter mutation may be the initial genetic event in carcinogenesis that promotes tumor cells to acquire other aggressive mutations. Thyroid carcinogenesis occurs through a cascade of steps that requires multiple genetic alterations. The cancer process is initiated by driver mutations, followed by a cascade of secondary mutations, leading to the progression from differentiated to undifferentiated cancer cells. However, the precise timing of when the TERT promoter mutation occurs is uncertain. Hence, whether it is a driver mutation or a secondary mutation has yet to be determined [26,27].
When a TERT promoter mutation is identified in a malignancy without another identified mutation, it is probable that the TERT promoter mutation is promoting the progression of a mutation that is not yet detectable with the currently available molecular tests. As a result, when a TERT promoter mutation is the only mutation detected, it is likely that other mutations are present but not detected. Numerous commercial molecular tests are available. These include ThyroSeq v3, Afirma GSC/XA, and ThyGenNEXT/ThyraMIR. The two tests used in our study were ThyroSeq v3 and ThyGenNEXT/ThyraMIR. One hundred and twelve thyroid-related genes are evaluated routinely by the next-generation sequencing assay in the third version of the ThyroSeq test. ThyGenNEXT incorporates a mutation panel consisting of the most common thyroid cancer-related mutations, along with the ThyraMIR microRNA classifier test. Although these tests can widely screen for molecular alterations, ranging from gene fusions, copy number alterations, and gene expression alterations, more accurate molecular profile testing methods are required to better predict disease outcomes [28,29].
Many studies have consistently demonstrated a significant association between TERT promoter mutations and older patients. In a recent meta-analysis, the mean patient age was 59.2 ± 15.5 years versus 44.9 ± 15.6 years in patients with TERT promoter mutations versus those without such mutations [8]. In our study population, the mean age at the time of surgery was 68 ± 10 years. Additionally, the seven patients with TERT promoter mutations alone were significantly older than the 14 patients who had concurrent mutations (77.6 ± 4.7 years versus 64 ± 9 years, respectively). Miyauchi et al. showed that the age at diagnosis could be used to assess lifetime disease progression probabilities, which were 8% for patients in their 60s and 4% for patients in their 70s. This is another reason to support the hypothesis that patients with thyroid cancer positive for TERT promoter mutations may not have aggressive disease. [30,31] We detected an association between poorer prognoses and TERT promoter mutations when they coexist with other mutations, highlighting the supportive role of concurrent mutations in developing aggressive thyroid cancer. It has been ascertained that RAS oncogene mutations exhibit a low-risk phenotype, with a reported malignancy prevalence of 47%. This prevalence reaches 100% when nodules are positive for a BRAF V600E mutation [32]. Furthermore, Radkay et al. demonstrated that the risk of cancer is highly dependent on the RAS mutation subtype, with KRAS (41.7%) being associated with the lowest risk, followed by NRAS (86.8%) and HRAS mutations (95.5%) [33]. Four patients in our sample exhibited either NRAS or KRAS mutations. All had confirmed malignancy, with a 75% incidence of aggressive disease. Zhao et al. studied the significance of concurrent mutations in thyroid cancer. Their findings showed that TERT + RAS was highly associated with distant metastases and mortality. This is consistent with our findings [8]. Two of our study patients had distant metastases from their thyroid cancer. Both patients had a mutation in one of the RAS proto-oncogenes; one metastasized to the mandible and the other to the chest wall, mediastinum, and heart. This observation again validated the synergistic effects of the different genetic mutations, demonstrating their greater impact on disease prognosis.
Our study was also in keeping with other studies in the literature that showed poor clinicopathological outcomes for papillary thyroid cancer with concurrent TERT and BRAF V600E mutations. However, inconsistent results have been reported by other researchers who could not find a particular relationship or association between the two mutations [14,34,35].
One patient in our sample had simultaneous PIK3CA/EIF1AX mutations and nonaggressive disease on final pathology. The role of these specific mutations has yet to be elucidated. The literature proposes that the occurrence of PIK3CA mutations may be less useful than TERT promoter mutations for assessing the risk of anaplastic transformation in papillary carcinoma [19]. Nonetheless, García-Rostán et al. suggested that PIK3CA mutations are more common in anaplastic thyroid cancer (5-25%) than in less aggressive thyroid carcinomas (0-5% in papillary thyroid cancer) [36].
Molecular testing can effectively impact decision making regarding the extent of thyroidectomy required. It has been shown in the literature that utilizing molecular testing in thyroid cancer can help guide the optimal surgery in 91.86% of patients, compared to 61.11% of patients who did not undergo molecular testing [37]. Complications of thyroid surgery, such as hyperparathyroidism, recurrent laryngeal nerve palsy, and lifelong thyroid hormone replacement therapy, can be prevented by reducing overtreatment by thyroidectomy when considered unnecessary [19]. Our findings support the need for more extensive thyroidectomy in nodules with TERT promoter mutations and other concurrent mutations, given the high possibility of aggressive disease, but not necessarily in patients with TERT promoter mutations alone, given the significantly lower likelihood of aggressive disease. Based on our results, the finding of aggressive disease on postoperative histopathology (and hence the occurrence of a histopathological diagnosis that would warrant at least a total thyroidectomy as a standard treatment approach) was higher in patients with co-existing mutations, as opposed to those with TERT promoter mutations alone. Moreover, in cases of single-gene TERT promoter mutations, when there are no worrisome clinicodemographic features (e.g., young age or family history of thyroid cancer), one could consider limited treatment with hemithyroidectomy and close observation after shared decision making (i.e., a thorough discussion of treatment options and their benefits and harms with a patient based on best available evidence and a consideration of the patient's preferences).
Our study has several limitations. First, analysis of other co-mutations and subanalysis of specific co-mutations was not possible in our study because of the rarity of TERT promoter mutations, with its prevalence reported to be between 4.2% and 25% in welldifferentiated thyroid cancer. Rent et al. reported the overall prevalence of TERT promoter mutations to be 3.5% in their papillary thyroid carcinoma patients [35]. Another limitation is that a significant proportion (42%) of patients in our cohort had indeterminate cytology (Bethesda III and IV), where the prevalence of high-risk mutations, such as TERT or BRAF V600E, is expected to be lower than in patients with suspicious or malignant cytology (Bethesda IV and VI), where molecular testing is less commonly performed. This is also reflected in our results, which show that 22.2% of Bethesda III/IV cases were aggressive cancers, while 77.8% of Bethesda V/VI cases were aggressive cancers ( Table 2, p = 0.001). Finally, the detection of GC-rich TERT promoter regions by molecular profiling is difficult in general, which may further contribute to the low detection rate.
Other limitations of our study were the small sample size and the high degree of selection bias based on including a nonrandomized sample. Moreover, most patients undergoing thyroid surgery did not undergo molecular testing. Additionally, the retrospective nature of our study may preclude the generalization of our significant results; however, we consider that our results should be perceived as potential findings based on which larger-scale prospective studies can be conducted. Future genetic discoveries will likely result in more accurate molecular panels, and the ongoing understanding of carcinogenesis will allow clinicians to develop more robust management plans and disease predictions.

Conclusions
In conclusion, thyroid nodules positive for TERT promoter mutations concurrent with other mutations had more aggressive malignancies when compared to those positive for TERT promoter mutations alone. Moreover, not all patients with a TERT promoter mutation alone had a malignancy. Our findings challenge the generalized belief that TERT promoter mutations are an independent indicator of aggressive disease. The finding that the aggressiveness of a thyroid tumor with a TERT promoter mutation may be dependent on the presence of concurrent mutations is an important concept for thyroid specialists to consider when counseling patients regarding risk stratification, management, and extent of surgery.

Informed Consent Statement:
A waiver for Informed consent was granted because of the retrospective nature of the study and the analysis used anonymous clinical data.

Data Availability Statement:
The data sets used and/or analyzed during this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.