High RAS Allele Frequency Signals Increased Risk of TERT Promoter Mutations in Thyroid Tumors
by
, , ,
Coralie Lefebvre
1,*, 
Hannah Greenspoon
2,
Kayla E. Payne
3,
Emily Steinberg
4,
Felicia Tewfik
5,
Gianluca Savoia
1,
Sabrina Daniela da Silva
6, 
Marc Pusztaszeri
7
,
Véronique-Isabelle Forest
6,8 and
Richard J. Payne
6,8 1
Faculty of Medicine, McGill University, Montreal, QC H3A 2M7, Canada
2
Faculty of Arts and Science, Queen’s University, Kingston, ON K7L 3N6, Canada
3
Faculty of Arts, McGill University, Montreal, QC H3A 2M7, Canada
4
Department of Family Medicine, University of Ottawa, Ottawa, ON K1N 6N5, Canada
5
Faculty of Medicine, Université de Montréal, Montreal, QC H3T 1J4, Canada
6
Department of Otolaryngology—Head and Neck Surgery, Sir Mortimer B. Davis—Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada
7
Department of Pathology, Sir Mortimer B. Davis—Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada
8
Department of Otolaryngology—Head and Neck Surgery, Royal Victoria Hospital, McGill University, Montreal, QC H4A 3J1, Canada
*
Author to whom correspondence should be addressed.
Cancers 2025, 17(17), 2851; https://doi.org/10.3390/cancers17172851
Submission received: 13 August 2025
/
Revised: 28 August 2025
/
Accepted: 29 August 2025
/
Published: 30 August 2025
(This article belongs to the Section Molecular Cancer Biology)
Simple Summary
Thyroid cancer is the most common endocrine malignancy. When a thyroid nodule is found to be indeterminate on biopsy, despite the fact that the majority are benign, surgery is often required for definitive diagnosis. To reduce unnecessary surgical interventions, accurate pre-operative risk assessment is critical. Molecular testing plays an increasingly important role by identifying oncogenic mutations and providing insights into their potential malignancy and aggressiveness. RAS mutations are among the most commonly found genetic changes in thyroid nodules and are generally associated with predictable clinical behavior. In contrast, TERT (telomerase reverse transcriptase) promoter mutations in conjunction with a RAS mutation are strongly associated with tumor aggressiveness, recurrence, and poor prognosis. The allele frequency (AF), representing the proportion of mutated DNA within a sample, has been correlated with tumor behavior in certain mutations, such as BRAF V600E. However, its prognostic significance in RAS-mutated tumors remains unclear. This study investigates whether a high RAS AF can serve as a predictive marker for coexisting TERT promoter mutations. Identifying such a correlation could improve pre-operative risk stratification and support more personalized approaches to thyroid cancer management.
Abstract
Background/Objectives: RAS mutations are among the most common genetic alterations in thyroid cancer and are generally associated with less aggressive behavior. However, when co-occurring with TERT (telomerase reverse transcriptase) promoter mutations, known markers of poor prognosis, tumors exhibit markedly more aggressive features. The allele frequency (AF) of RAS may serve as a potential indicator of clonal dominance and the likelihood of additional high-risk mutations, such as TERT mutation. This study aims to assess whether a high RAS AF correlates with the presence of coexisting TERT promoter mutations and other molecular alterations. Methods: A retrospective chart review was performed on 111 patients with thyroid nodules harboring RAS mutations, either alone or in combination with TERT promoter mutations. All patients underwent molecular testing with ThyroSeq v3 and subsequent thyroidectomy at McGill University teaching hospitals. RAS AF was analyzed in relation to TERT mutation status, nodule size, and other molecular alterations including copy number alterations (CNA) and gene expression profiles (GEP). Results: The mean RAS AF was significantly higher in nodules with both RAS and TERT mutations (38.1%) compared to those with RAS mutations alone (22.1%) (p = 0.002). Nodules with coexisting TERT mutations were also significantly larger (mean size: 3.7 cm vs. 2.4 cm; p = 0.005). Malignant nodules, regardless of TERT status, showed a trend toward higher RAS AF than benign nodules (23.0% vs. 16.3%; p = 0.052). Higher RAS AF was also associated with the presence of CNA and/or GEP positivity. Notably, GEP was positive in 100% of nodules with both RAS and TERT mutations, compared to 37.5% in RAS-only nodules (p = 0.002). Conclusions: A high RAS AF increases the likelihood of a TERT promoter mutation and other genetic alterations, highlighting the importance of RAS AF in optimizing patient care and management.
1. Introduction
Thyroid cancer is the most common endocrine malignancy and ranks ninth in overall cancer incidence globally, with a rising prevalence, particularly among women [1]. It is diagnosed at an annual rate of approximately 13.5 cases per 100,000 individuals, while the mortality rate remains low, at 0.5 deaths per 100,000 per year [2]. The biological behavior of thyroid cancer varies widely, ranging from indolent to highly aggressive forms, which can complicate treatment planning [3]. Given the relatively low mortality associated with thyroid cancer, accurate preoperative assessment of thyroid nodules is essential to guide clinical decision-making and optimize patient outcomes. It helps avoid unnecessary surgical interventions that may result in postoperative complications and lifelong thyroid hormone replacement [4].
Molecular testing is an advanced diagnostic tool used to evaluate thyroid nodule samples for genetic alterations, such as point mutations, copy number alterations (CNA) and gene expression profile (GEP) which can serve as markers of benignity or malignancy [5,6]. Among the most clinically relevant mutations, BRAF V600E and TERT promoter mutations are strongly associated with malignancy and aggressive tumor behavior [7,8]. The TERT gene plays a key role in maintaining chromosomal integrity by preventing telomere shortening. While normally silent in most adult tissues, mutations in the TERT promoter can lead to aberrant gene activation, promoting unchecked cellular proliferation and contributing to tumor progression and aggressiveness [9]. In contrast, RAS genes encode signaling molecules involved in regulating cell growth, differentiation, and survival. Activating point mutations in RAS can result in persistent downstream pathway activation, driving tumorigenesis [10]. However, RAS-mutant thyroid tumors typically exhibit a more indolent clinical course compared to those harboring BRAF V600E or TERT mutations [11]. Despite this, RAS mutations are associated with a wide range of malignancy risk, estimated between 37% and 85%, which adds complexity to clinical decision-making and underscores the need for additional risk stratification tools [12].
In the context of molecular testing, allele frequency (AF) refers to the percentage of mutant DNA detected within a collection of cells examined [13]. This parameter helps estimate the degree of intratumoral genetic heterogeneity and how extensively a mutation is distributed across the tumor cell population. Elevated AF values typically indicate that the mutation is present in a large fraction of tumor cells, whereas lower values are more consistent with subclonal alterations confined to a smaller subset of cells [14].
Recent studies have also proposed AF as a potential substitute marker of tumor burden, with increasing AF values possibly associated with more aggressive clinical behavior and poor prognosis [15]. Indeed, AF has recently helped in the identification of aggressive thyroid cancer in the case of certain mutations [16]. For example, studies have shown that an AF higher than 25.8% for a BRAF V600E mutation is indicative of a higher level of invasiveness and aggressiveness [17]. However, in the case of RAS mutations, AF studies have not demonstrated a direct correlation between higher AF and increased tumor aggressiveness. A recent study has postulated that a higher RAS AF may increase the likelihood of coexisting genetic alterations, such CNA and GEP [12]. We therefore hypothesized that a high RAS AF could similarly be associated with a second high-risk mutation, such as a TERT promoter mutation, potentially contributing to more aggressive tumor behavior. Identifying a potential RAS AF threshold predictive of such co-mutations could improve preoperative risk stratification and guide surgical decision-making. This study aims to assess the extent to which a high RAS mutation AF correlates with the presence of a TERT promoter mutation.
2. Materials and Methods
2.1. Patient Samples
A retrospective chart review of 362 surgical patients was conducted at two McGill University teaching hospitals in Montreal, Canada, between January 2021 and March 2025. All selected patients were over 18 years of age, had undergone thyroid surgery, and had molecular testing using ThyroSeq v3. ThyroSeq v3 is a next-generation sequencing-based multigene panel for thyroid cancer used for identifying different alterations, including point mutations, insertions, deletions, gene fusions, CNA and GEP. This molecular test analyzes more than 112 genes, including BRAF, RAS, TERT, RET, and many others. Testing was performed on samples from fine-needle aspiration pre-surgery following the manufacturer’s protocol [18].
Patients were included in the study if their thyroid nodules demonstrated a RAS-like mutation. Molecular testing results for eligible patients included RAS mutation AF and other genetic alterations, such as TERT, CNA, and GEP. Patients with a RAS mutation in combination with a co-mutation other than TERT were excluded from the study.
Ethics approval was obtained by the Research Ethics Committee of the integrated Health and Social Services Network for West-Central Montreal 29 August 2023 (#37-2024-9661).
2.2. Data Collection
Out of 362 patient records reviewed, 111 met the inclusion criteria outlined above and were included in the study. Patients were categorized into two groups: those with a RAS mutation only, and those with both RAS and TERT promoter mutations. Baseline clinical and cytological data were collected, including patient age, sex, nodule size, and Bethesda category. Molecular data were retrieved from the ThyroSeq v3 results, including RAS AF, presence of TERT promoter mutations, GEP, CNA and the specific isoform pattern of RAS. Final surgical pathology reports were reviewed to collect histopathological findings and classify malignancy subtypes.
2.3. Statistical Analysis
Descriptive analyses were conducted to summarize baseline characteristics. Associations between RAS AF and other genetic alterations, including TERT, CNA, and GEP, were assessed. Additional correlations were examined in relation to malignancy status, Bethesda category, nodule size, and cancer types and subtypes. Statistical significance between groups was established as p-value ≤ 0.05 using a logistic regression model with a reported confidence interval of 95%. Associations between categorical variables were assessed using the chi-square test or Fisher’s exact test, as appropriate. Analysis of variance (ANOVA) was used for comparisons involving continuous variables. The analyses were performed using the statistical software package STATA-13 (STATA Corporation, College Station, TX, USA) and IBM Statistical Package for Social Sciences (SPSS), Version 29.0.1.0 (IBM Corp., Armonk, NY, USA).
3. Results
Of the 362 patient files who underwent both a ThyroSeq v3 and a thyroidectomy at one of the McGill University centers, a total of 111 patients fit the above-mentioned criteria. In addition, 7 patients both had TERT and RAS mutations and 104 patients only had RAS mutations. The average age for patients with both TERT and RAS mutations was 56.4 years old and 49.8 years old for patients with only a RAS mutation. Of the patients with TERT and RAS mutations, 5 (71.4%) were female while 2 (28.6%) were male. Regarding the patients with only RAS mutations, 86 (82.7%) were female while 18 (17.3%) were male. Therefore, there were no major differences in age and sex between the two categories of patients.
Regarding the nodule size, the patients with TERT and RAS mutations had a significantly higher size at 3.7 cm compared to 2.4 cm for patients with only a RAS mutation (p = 0.005) (Table 1).
Regarding Bethesda categories, although not statistically significant, there was a trend where patients with TERT and RAS mutations were most often associated with Bethesda IV, while patients with only a RAS mutation were most often linked to Bethesda III. The average RAS mutation AF in the cases where TERT mutation was present was significantly higher (at 38.1%) than the patients without TERT mutation (at 22.1%) (p = 0.002). In addition, all the patients with TERT and RAS mutations had malignant nodules, while 89 (85.6%) patients with only RAS mutation had malignant nodules (Table 2).
Figure 1 highlights that RAS AF is significantly higher in nodules with TERT mutations, clustering from 37% to 45%, compared to a much wider range of AF with a median around 25% in the RAS-only nodules. One outlier in the TERT and RAS category can be seen with an AF of 19%.
The presence of GEP was significantly higher in patients with both TERT and RAS mutations with all (100%) patients compared to 39 patients (37.5%) for those with only RAS mutations (p = 0.002). On the contrary, CNA did not seem to be associated with one category over the other, with 2 (28.6%) and 22 (21.2%) positive CNA in patients with and without a TERT mutation, respectively (Table 3).
Analysis of RAS mutation AF based on the presence of GEP and/or CNA revealed distinct trends. No comparison was possible for GEP status in TERT and RAS co-mutated nodules, since all cases were GEP-positive. However, among RAS-only nodules, higher RAS AF appeared associated with GEP positivity (ANOVA with post hoc p = 0.202).
CNA presence was associated with higher RAS AF in both groups. In TERT and RAS co-mutated nodules, the mean AF was 43.5% with CNA compared to 36.0% without, although this trend did not reach significance (ANOVA with post hoc p = 0.792). In RAS-only nodules, the difference was more pronounced: 30.1% with CNA compared to 19.9% without (ANOVA with post hoc p < 0.001).
Regarding malignancy, because all TERT and RAS co-mutated nodules were malignant, no further comparison could be made. However, among RAS-only nodules, RAS AF was higher in malignant (23.0%) compared to benign (16.3%) cases. ANOVA showed a significant overall difference (p < 0.001), with a borderline post hoc p-value of 0.052 between these two groups.
Finally, we observed a statistically significant increase in RAS AF at 25% for NRAS compared to 21.4% for HRAS and 16.6% for KRAS (Table 4).
Regarding thyroid cancer types, we observed a trend, although not statistically significant, where follicular thyroid carcinoma (FTC) had a higher AF at 30.8% compared to papillary thyroid carcinoma (PTC) with an AF of 22.5%, regardless of the presence of TERT (p = 0.104) (Table 5).
Although no statistically significant results were found regarding AF across malignancy subtypes, the FTC subtypes tended to have a higher RAS AF compared to PTC subtypes.
When evaluating the final pathology results (Table 6), RAS-only nodules demonstrated a higher RAS AF in classical and the solid/trabecular subtypes. Conversely, papillary microcarcinoma, oncocytic subtype and benign follicular nodular disease categories had the lowest RAS AF. With respect to the final pathology of the RAS and TERT-positive nodules, the highest RAS AF, at 45.0%, corresponded to a widely invasive oncocytic carcinoma with a poorly differentiated component. Two cases had a RAS AF of 42.0% and were, respectively, a high-grade widely invasive FTC and a differentiated high-grade follicular variant of papillary thyroid carcinoma (FVPTC). The two cases with both TERT and RAS mutations that had the lowest RAS AF, averaging 28.0%, were classified as minimally invasive FTC.
4. Discussion
In recent years, molecular testing has allowed thyroid specialists to gather more information on the type of mutations present in PTC and FTC, and RAS-like mutations are one of the most common [3]. In addition, RAS mutations can be present in combination with another mutation, such as a TERT promoter mutation. It has been demonstrated that the effect of the TERT co-mutation induces further PTC/FTC progression and distant metastasis [19]. Indeed, studies have shown that RAS with TERT promoter co-mutation may play a synergistic role in thyroid tumorigenesis and increase aggressiveness behavior and poor prognosis [9]. Therefore, understanding the malignancy and aggressiveness of RAS mutations is essential to enhance clinical decisions regarding RAS mutations.
The present study examined the relationship between RAS mutation AF and the presence of a TERT promoter mutation and additional genetic alterations such as GEP and CNA. One of the most important findings of our study was the significant difference in RAS mutation AF between patients with and without a TERT promoter mutation (p = 0.002). The average RAS AF was notably higher in the RAS and TERT group (38.1%) compared to the RAS-only group (22.1%).
Additionally, we observed that in our sample, all cases with a RAS AF of 42% or higher had both RAS and TERT mutations and exhibited aggressive histopathological subtypes. The highest RAS AF observed in the RAS-only group was 41%. While this suggests that a RAS AF above 41% may be associated with an increased likelihood of a coexisting TERT promoter mutation, we did not formally define a cutoff point due to limited sample size. These observations are exploratory in nature and a larger cohort would be required to establish a statistically robust threshold.
Nonetheless, this finding shows that RAS AF may play an important role in determining the likelihood of having a second genetic alteration, such as a TERT promoter mutation, which leads to more aggressive disease [12]. Identification of RAS mutation AF is therefore essential for creating an optimal treatment plan.
In addition, the size of the nodules was found to be significantly higher in the case of patients with both RAS and TERT mutations compared to patients with only RAS mutations (p = 0.005). In accordance with our findings, the TERT promoter mutation has been previously shown to correlate with larger nodule size in other carcinomas such as anaplastic thyroid carcinoma (ATC) [20]. This finding could also potentially explain the high RAS mutation AF in patients with TERT promoter mutations. Previous studies have suggested a positive correlation between increased AF or mutational load and increased tumor size [21,22]. The relationship could also be explained by the length of time the thyroid nodule had to develop, which could lead to a bigger size, a higher AF and a higher likelihood of a second genetic alteration, such as a TERT promoter mutation.
In keeping with the literature, we have found that an increase in the RAS AF led to a higher likelihood of having other genetic alterations, such as CNA and GEP [12]. Regardless of the presence of TERT, the RAS AF was higher when CNA and GEP were positive compared to when they were not. In fact, all patients with both RAS and TERT mutation were also GEP-positive compared to only 37.5% of RAS-only nodules. This could indicate the potential use of GEP in defining the likelihood of aggressive features and malignancy. Furthermore, this finding demonstrates that the higher the RAS mutation AF, the higher the likelihood of having additional genetic alterations. Moreover, we compared the differences in RAS AF between HRAS, NRAS and KRAS. We observed that NRAS had a significantly higher AF compared to HRAS and KRAS (p = 0.018).
As previously discussed, an outlier was observed in the TERT and RAS category, with a RAS mutation AF of 19%. This could be partially explained by differences in histopathological subtypes. Indeed, a subset of TERT and RAS-positive nodules were diagnosed as minimally invasive FTCs, which by definition lack aggressive features [23]. These nodules also exhibited lower RAS AF compared to TERT and RAS cases with aggressive histopathological subtypes, such as widely invasive FTC and differentiated high-grade FVPTC [24,25]. This observation underscores furthermore the potential relevance of RAS AF in clinical decision-making. Although TERT promoter mutations are generally associated with adverse outcomes, cases with low RAS AF did not display aggressive histology on final surgical pathology.
On the other hand, although not statistically significant, a relationship between malignancies and RAS AF was established. Indeed, the average RAS AF was higher when the final surgical pathology was malignant compared to when it was benign. This finding could furthermore help thyroid specialists to guide their treatment plan according to the RAS AF.
In addition to the physical manifestations of malignancy, there are specific quality of life (QOL) issues associated with having a TERT mutation. According to Richard Lazarus’ stress appraisal theory [26], stress is determined by the way individuals perceive and evaluate the event. The primary appraisal is the initial stage, which is the individual’s perception of the nature and significance of the event, while the secondary appraisal refers to an individual’s evaluation of their ability to cope with the perceived threat. Accordingly, this model suggests that individuals experience heightened anxiety when they appraise a situation as threatening, such as having a TERT mutation, and that they lack the coping skills to deal with it. Moreover, the information processing view [27] asserts that knowledge about a situation reduces stress. As a result, knowing the TERT status prior to surgery may lead to less overall stress with an improved QOL.
The main limitations of this study include its retrospective nature and the relatively small sample size. TERT promoter mutations are rare mutations in thyroid cancer, which inherently limits the number of available cases for analysis. Not only are TERT mutations uncommon, but their co-occurrence with RAS mutations is even rarer, as demonstrated in prior studies reporting very few such cases (1 case out of 145, 4 cases out of 243) [28,29]. In our cohort, only seven cases had both TERT and RAS mutations, compared with 104 cases with RAS mutations alone. This imbalance with the exclusion of some cases due to conflicting postoperative findings, reduced the statistical power and precision of subgroup analyses. Therefore, our results should be interpreted with caution and considered hypothesis-generating rather than definitive. However, our findings are consistent with prior studies demonstrating the clinical relevance of TERT mutations, and they underscore the need for larger, multi-institutional cohorts to validate these associations. In addition, the data was collected in one region, leading to geographical bias. Some patients needed to pay out of pocket for the molecular testing, which can lead to demographic bias. In addition, not every molecular testing company indicates additional information such as the AF, CNA and the GEP. Since that data was essential for conducting this research, only the patients who received a ThyroSeq V3 molecular test were admissible. This could introduce biases since not all patients with RAS mutations could be included in this study. In addition, tumor purity and CNA are known to influence the sensitivity of molecular testing and can significantly alter the reported AF [30,31]. However, in ThyroSeq v3, specimen cellularity is provided only as a binary designation (adequate vs. inadequate), and in our cohort all specimens were reported as adequate. Similarly, CNA is reported only as positive or negative, without quantitative information on copy number gains or losses. Therefore, our study is limited by the inability to more precisely account for tumor content or CNA complexity, which may impact the interpretation of molecular results. Finally, the study only includes surgical patients and therefore not all RAS patients could be included.
5. Conclusions
This study indicates that a higher RAS AF is significantly associated with the presence of coexisting TERT promoter mutations. These findings suggest that RAS AF may serve as a surrogate marker for identifying thyroid nodules with more complex and potentially aggressive molecular profiles, particularly valuable when molecular testing platforms do not assess for TERT mutation.
Thyroid nodules with both RAS and TERT mutations were significantly larger, had higher RAS AF, and showed a strong correlation with positive GEP results compared to nodules with RAS mutations alone. Incorporating RAS AF into the molecular evaluation of thyroid nodules may improve the prediction of high-risk co-mutations, thereby improving individualized management and optimizing patient care and QOL.
Author Contributions
Conceptualization, C.L. and E.S.; Methodology, C.L., E.S. and H.G.; Validation, S.D.d.S. and C.L.; Investigation, C.L. and S.D.d.S.; Data curation, C.L. and F.T.; Writing—original draft, C.L. and H.G.; Writing—review and editing, C.L., H.G., K.E.P., F.T., G.S., M.P., R.J.P. and V.-I.F. All authors have read and agreed to the published version of the manuscript.
Funding
This research was supported by the Judith Anne Wright Litvack Bursary, awarded as part of the McGill Faculty of Medicine and Health Sciences Research Bursary Program. This bursary provides funding to students conducting research in the area of cancer. The awarded amount was $2000.
Institutional Review Board Statement
The study complied with the principles of the Declaration of Helsinki and received approval from the Medical-Bioethics Research Ethics Committee (REC) of the Integrated Health and Social Services Network for West-Central Montreal (29 August 2023 #37-2024-9661).
Informed Consent Statement
As noted in the REC’s final project approval, the requirement for patient consent was waived because of the study’s retrospective design.
Data Availability Statement
Data supporting the findings of this study can be obtained from the corresponding author upon reasonable request. Public sharing of the data is restricted in accordance with the ethics approval agreement.
Acknowledgments
The authors would like to acknowledge Maxime Houde, an actuarial student, and Michelle Houde, a retired statistician, for their valuable support in statistical interpretation and advice.
Conflicts of Interest
The authors declare no conflicts of interest.
Abbreviations
| AF | Allele Frequency |
| CNA | Copy Number Alteration |
| CI | Confidence Interval |
| FTC | Follicular Thyroid Carcinoma |
| GEP | Gene Expression Profiling |
| NIFTP | Non-Invasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features |
| PTC | Papillary Thyroid Carcinoma |
| RAS | Rat Sarcoma Virus Oncogene Family |
| SD | Standard Deviation |
| TERT | Telomerase Reverse Transcriptase |
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Figure 1.
Boxplot comparing RAS mutation AF in thyroid nodules with and without TERT promoter mutations. The plot illustrates the distribution of RAS AF values, highlighting potential differences between TERT-mutant and TERT-wildtype groups. Boxes represent the interquartile range (IQR), with the median shown as a horizontal line; whiskers indicate 1.5 × IQR. Outliers are shown as *.
Figure 1.
Boxplot comparing RAS mutation AF in thyroid nodules with and without TERT promoter mutations. The plot illustrates the distribution of RAS AF values, highlighting potential differences between TERT-mutant and TERT-wildtype groups. Boxes represent the interquartile range (IQR), with the median shown as a horizontal line; whiskers indicate 1.5 × IQR. Outliers are shown as *.
Table 1.
Baseline characteristics of 111 thyroid nodules with RAS-only mutation or RAS and TERT co-mutation.
Table 1.
Baseline characteristics of 111 thyroid nodules with RAS-only mutation or RAS and TERT co-mutation.
| Variant | RAS + TERT Co-Mutation (%) (n = 7) | RAS Mutation Only (%) (n = 104) | p-Value |
|---|---|---|---|
| Age (years) | |||
| mean ± SD * (CI) | 56.4 ± 9.4 (95% CI: 47.8–65.1) | 49.8 ± 13.2 (95% CI: 47.2–52.4) | 0.118 |
| Sex | |||
| Female | 5 (71.4) | 86 (82.7) | 0.607 |
| Male | 2 (28.6) | 18 (17.3) | |
| Nodule size (cm) | |||
| mean ± SD * (CI) | 3.7 ± 1.1 (95% CI: 2.6–4.7) | 2.4 ± 1.1 (95% CI: 2.2–2.6) | 0.005 |
* SD: standard deviation, CI: confidence interval, cm: centimeter.
Table 2.
Comparison of clinical and molecular features between RAS-only mutation or RAS and TERT co-mutation.
Table 2.
Comparison of clinical and molecular features between RAS-only mutation or RAS and TERT co-mutation.
| Variant | RAS + TERT Co-Mutation (%) (n = 7) | RAS Mutation Only (%) (n = 104) | p-Value |
|---|---|---|---|
| Bethesda Category | 0.065 | ||
| Bethesda III | 2 (28.6) | 55 (52.9) | |
| Bethesda IV | 5 (71.4) | 31 (29.8) | |
| Bethesda V | 0 (0) | 18 (17.3) | |
| AF (%) of RAS Mean ± SD | 38.1 ± 8.8 | 22.1 ± 10.6 | 0.002 |
| Pathology | 0.591 | ||
| Benign | 0 (0) | 15 (14.4) | |
| Malignant/NIFTP * | 7 (100.0) | 89 (85.6) |
* NIFTP: Non-invasive follicular thyroid neoplasm with papillary-like nuclear features, AF: allele frequency, SD: standard deviation.
Table 3.
Association between RAS mutation and second molecular alterations including CNA and GEP.
| Molecular Alterations | RAS + TERT Co-Mutation (%) (n = 7) | RAS Mutation Only (%) (n = 104) | p-Value |
|---|---|---|---|
| GEP(%) | 0.002 | ||
| Yes | 7 (100.0) | 39 (37.5) | |
| No | 0 (0) | 65 (62.5) | |
| CNA(%) | 0.643 | ||
| Yes | 2 (28.6) | 22 (21.2) | |
| No | 5 (71.4) | 82 (78.8) |
Table 4.
Association between molecular alterations, malignancy, isoform patterns and RAS AF for patients with a RAS mutation.
Table 4.
Association between molecular alterations, malignancy, isoform patterns and RAS AF for patients with a RAS mutation.
| Groups | Average RAS AF (%) | Standard Deviation (SD) | n | p-Value (ANOVA) |
|---|---|---|---|---|
| TERT and GEP comparison | <0.001 | |||
| TERT+/GEP+ | 38.1 | 8.8 | 7 | |
| TERT+/GEP− | - | - | 0 | |
| TERT−/GEP+ | 24.3 | 9.2 | 39 | |
| TERT−/GEP− | 20.7 | 11.1 | 65 | |
| TERT and CNA comparison | <0.001 | |||
| TERT+/CNA+ | 43.5 | 2.1 | 2 | |
| TERT+/CNA− | 36.0 | 9.8 | 5 | |
| TERT−/CNA+ | 30.1 | 6.9 | 22 | |
| TERT−/CNA− | 19.9 | 10.4 | 82 | |
| Pathology and TERT | <0.001 | |||
| TERT+/Benign | - | - | 0 | |
| TERT+/Malignant | 38.1 | 8.8 | 7 | |
| TERT−/Benign | 16.3 | 10.5 | 15 | |
| TERT−/Malignant | 23.0 | 10.3 | 89 | |
| Isoform pattern of RAS | 0.018 | |||
| HRAS | 21.4 | 10.9 | 25 | |
| NRAS | 25.0 | 10.4 | 71 | |
| KRAS | 16.6 | 12.6 | 15 |
Table 5.
Association between RAS Mutation AF and types of thyroid carcinomas.
| Type of Thyroid Cancer | Average RAS AF (%) of Patients | Standard Deviation (SD) | n | p-Values |
|---|---|---|---|---|
| Follicular thyroid carcinoma (FTC) | 30.8 | 12.3 | 5 | 0.104 |
| Papillary Thyroid Carcinoma (PTC) | 22.5 | 10.9 | 105 |
Please note that the final surgical pathology of 1 patient with TERT and RAS mutations is unknown.
Table 6.
RAS Mutation AF in TERT and RAS-positive vs. RAS-only thyroid nodules across PTC and FTC subtypes.
Table 6.
RAS Mutation AF in TERT and RAS-positive vs. RAS-only thyroid nodules across PTC and FTC subtypes.
| Groups | Average RAS AF (%) of Patients with RAS + TERT Co-Mutation | Average RAS AF (%) of Patients with RAS Mutation Only | Standard Deviation (SD) | n | p-Values |
|---|---|---|---|---|---|
| Subtype of FTC | |||||
| Widely invasive FTC | 42.0 | - | N/A | 1 | N/A * |
| Minimally invasive FTC | 28.0 | 28.0 | 12.3 | 4 | |
| Subtypes of PTC/NIFTP | |||||
| NIFTP | - | 23.0 | 9.2 | 19 | |
| FVPTC | - | 24.1 | 10.0 | 44 | |
| Invasive FVPTC | - | 22.3 | 11.5 | 12 | |
| Differentiated high-grade FVPTC | 42.0 | - | N/A | 1 | |
| Oncocytic | 45.0 | 11.3 | 18.2 | 4 | |
| Classical | - | 35.0 | N/A | 1 | |
| Solid/Trabecular | - | 25.5 | 21.9 | 2 | |
| Macrofollicular | - | 19.0 | 14.1 | 3 | |
| Well-differentiated thyroid carcinoma (not otherwise specified) | - | 16.0 | N/A | 1 | N/A * |
| Papillary microcarcinoma | - | 10.0 | N/A | 1 | |
| PTC in adenomatoid nodule | - | 25.0 | N/A | 1 | |
| Benign pathology | |||||
| Follicular nodular disease | - | 12.3 | 11.2 | 6 | N/A * |
| Follicular adenoma | - | 17.8 | 9.9 | 6 | |
| Oncocytic adenoma | - | 21.0 | 11.4 | 3 |
* Statistical analysis was not performed due to low sample size (<5) in a vast majority of the cell counts, which violates the assumptions of the Chi-square test. Two patients in the TERT and RAS group were excluded from Table 6 classification due to unavailable or inaccessible final surgical pathology for the RAS-positive nodule.
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Lefebvre, C.; Greenspoon, H.; Payne, K.E.; Steinberg, E.; Tewfik, F.; Savoia, G.; da Silva, S.D.; Pusztaszeri, M.; Forest, V.-I.; Payne, R.J. High RAS Allele Frequency Signals Increased Risk of TERT Promoter Mutations in Thyroid Tumors. Cancers 2025, 17, 2851. https://doi.org/10.3390/cancers17172851
AMA Style
Lefebvre C, Greenspoon H, Payne KE, Steinberg E, Tewfik F, Savoia G, da Silva SD, Pusztaszeri M, Forest V-I, Payne RJ. High RAS Allele Frequency Signals Increased Risk of TERT Promoter Mutations in Thyroid Tumors. Cancers. 2025; 17(17):2851. https://doi.org/10.3390/cancers17172851
Chicago/Turabian StyleLefebvre, Coralie, Hannah Greenspoon, Kayla E. Payne, Emily Steinberg, Felicia Tewfik, Gianluca Savoia, Sabrina Daniela da Silva, Marc Pusztaszeri, Véronique-Isabelle Forest, and Richard J. Payne. 2025. "High RAS Allele Frequency Signals Increased Risk of TERT Promoter Mutations in Thyroid Tumors" Cancers 17, no. 17: 2851. https://doi.org/10.3390/cancers17172851
APA StyleLefebvre, C., Greenspoon, H., Payne, K. E., Steinberg, E., Tewfik, F., Savoia, G., da Silva, S. D., Pusztaszeri, M., Forest, V.-I., & Payne, R. J. (2025). High RAS Allele Frequency Signals Increased Risk of TERT Promoter Mutations in Thyroid Tumors. Cancers, 17(17), 2851. https://doi.org/10.3390/cancers17172851
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