In this study, we evaluated a prospective screening program that analyzed miFTC/miOTC tumors for TERT promoter mutations. We included 84 patients with an overall TERT promoter mutational frequency of 11.9%. This frequency is lower than in previous study cohorts containing all FTC subtypes, ranging between 15 and 20% [4, 6, 8, 9, 17, 24, 25]. The frequency is, however, similar compared to other study cohorts that have described the frequency of TERT mutations in the miFTC subtype alone, ranging between 9.7 and 10.3% [8, 17].

In contrast to most other studies on this topic, we opted for ddPCR as the method of choice in our screening program. This highly sensitive technique enables the detection of mutations with VAFs < 10%. This stands in clear contrast to conventional Sanger sequencing, which typically cannot detect mutations occurring in less than 10% of the interrogated DNA [21]. Interestingly, three out of ten (30%) tumors with TERT promoter mutations exhibited VAFs < 10%, thus arguing that a substantial subset of these tumors potentially may exhibit subclonal mutations not easily detected by conventional Sanger. Similar findings in FT-UMPs have been reported by us earlier [21]. As no metastatic events were recorded for any miFTC/miOTC with mutations in our series, the value of detecting this aberrancy in a fraction of tumor cells remains unclear. However, it is evident that ddPCR increases the likelihood of detecting this high-risk molecular aberration compared to traditional Sanger sequencing. As the objective of this study was to specifically target a single, high-risk genetic event, we did not use next-generation sequencing (NGS) to screen for a broader range of genetic alterations. While NGS analyses may reveal additional genetic events of interest, they are not yet of universal use in clinical routine at our department, which is guided by overall cost–benefit considerations and the long turnaround time.

In our cohort, one of the eleven miFTC/miOTC tumors measuring less than 20 mm harbored a potential subclonal TERT promoter mutation with a VAF of 1.3%. This patient did not exhibit metastases, and therefore, it is not known if the presence of this very low-abundant TERT promoter variant might affect the risk of metastases in the future. The finding of a potentially subclonal TERT promoter variant in a pT1 tumor may not necessitate the need to diverge from existing guidelines, as the tumor is still considered low-risk and not in need of further treatment. However, it is still intriguing to find a fraction of tumor cells with a high-risk genetic variant in such a clinically indolent case. In fact, our national guidelines would leave room for a more personalized follow-up approach for this individual patient, which would involve carefully discussing the pros and cons of total thyroidectomy and RAI ablation with the patient. If the patient is treated with lobectomy alone, we will monitor them through clinical examinations and imaging (ultrasound, CT scans, etc.) as appropriate. Although thyroglobulin measurements are unreliable in this context, a significant increase over time could suggest recurrent disease and may warrant consideration. As mentioned above, the importance of these subclonal mutations is elusive, and since no mutated tumors were diagnosed with metastases, it is uncertain if TERT promoter mutations have the same significance in miFTCs/miOTCs as in other FTC subtypes. Therefore, caution is necessary to avoid overestimating the risk until the role of fractional TERT promoter mutations in thyroid neoplasia has been characterized.

The overall metastatic rate in our series was 3.6%, which is lower than the 17.9% published previously in a mixed cohort of all FTC subtypes [2]. In our small series, TERT promoter mutations were not associated with metastatic disease in this study since none of the metastatic tumors had a TERT promoter mutation. This is not entirely expected, since the presence of mutation has repeatedly been shown to be a poor prognostic factor in previous studies of other subtypes of thyroid tumors—but this observation could also merely mirror our limited sample size and limited follow-up time. One of the TERT promoter wild-type tumors measuring less than 20 mm exhibited metastasis already at the time of diagnosis. This tumor harbored an NRAS codon 61 mutation, which has been associated with a higher rate of metastases compared to other RAS mutations in FTCs [26]. In the small cohort of pT1 tumors, the metastatic rate was 9.1%. This is a similar rate compared to a pT1 group with a rate of 12.1% that includes all FTC subtypes [2]. Although small miFTCs are typically regarded as low-risk, a fraction of cases can still present with metastasis at the time of diagnosis. Interestingly, the patient with a pT1 tumor presenting with metastatic disease did not display any known risk factors for aggressive clinical behavior according to current clinical risk stratification. Unfortunately, TERT promoter mutational analysis did not provide insights in this regard. As no miFTCs with distant metastases were found to exhibit vascular invasion upon histological re-evaluation, one must acknowledge a small but biologically relevant risk of dissemination even in these tumors with only limited capsular invasion and no TERT promoter mutation.

We must emphasize the limitations of our study, particularly the small sample size and short follow-up period, which partially restrict our ability to draw definitive conclusions regarding TERT promoter genotypes and their impact on patient outcomes. However, given that previous retrospective analyses of large thyroid cancer cohorts have established TERT promoter mutations as a high-risk event linked to poor prognosis, we instead aimed to conduct a prospective study to assess the real-world outcomes of a positive mutational finding in indolent tumor types like miFTC and miOTC. Our results are further complicated by the presence of low VAFs in some tumors, possibly indicating subclonal mutations of uncertain clinical significance.

In our series, TERT promoter mutations were associated with older age, which is consistent with previous studies [25]. Surprisingly, other known risk factors for a TERT promoter mutation did not differ between the groups with mutated or wild-type tumors. Usually, TERT promoter mutations are restricted to larger thyroid tumors in older patients. In this study, the tumor size difference between the mutated and wild-type groups did not reach statistical significance. This may be attributed, at least in part, to the discovery of a 17 mm tumor with a subclonal TERT promoter mutation, which, to our knowledge, represents one of the smallest miFTC/miOTC with a TERT promoter mutation to date. Further investigations using the more sensitive ddPCR methodology could potentially uncover whether subclonal mutations are more prevalent in smaller tumors and younger patients than previously believed and whether this phenomenon carries any clinical significance.

Since TERT promoter mutations were more frequent in larger tumors and rare in the smallest ones, the screening program was most valuable for larger tumors. Although the mutated tumors did not exhibit any metastases, the relatively short follow-up and small sample size may affect the results. However, it is clear from this study that the presence of a TERT promoter mutation does not have the same impact in this FTC subtype compared to a mixed study cohort including all subtypes. In a previous study [17], the authors analyzed all three subtypes of FTC separately for TERT promoter mutations. They found that the presence of TERT promoter mutations did not affect the prognosis in miFTC, while it was of clinical importance in eaiFTC and wiFTC. As TERT promoter mutations did not impact the prognosis of our miFTC/miOTC patients, other tumor groups might be more suitable for mutational screening, such as larger tumors with vascular invasion. Future studies are required to draw more definitive conclusions.

We conclude that a proportion of miFTCs/miOTCs carry TERT promoter mutations that may be subclonal, and the mutation is associated with a higher patient age. Although mutations are rare in small tumors, we discovered a mutated tumor among those measuring less than 20 mm. Although our molecular risk stratification cannot be stated to contradict the current approach of releasing small, low-risk lesions as outpatients without additional treatment, the finding of single pT1 tumors with potential, low-abundance mutations suggests that TERT promoter mutational testing might be important regardless of tumor size. However, the true value of detecting these genetic alterations in FTCs and OTCs with limited capsular invasion only remains to be determined, and studies with longer follow-up are needed to investigate the prognostic implications of our findings.