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Allele frequency in thyroid cancer: mechanisms, challenges, and applications in cancer therapy

Thyroid Research volume 18, Article number: 19 (2025) Cite this article

Abstract

Allele Frequency (AF) is the percentage of sequence reads with a specific mutation relative to the read depth at that locus, reflecting the proportion of gene mutation. This review explores the AF characteristics of different mutations in thyroid cancer, investigating their connection with tumor features and clinical characteristics. BRAF mutation AF is associated with tumour malignancy and prognosis, exhibiting a relatively low peak value. TERT mutations in AF are associated with invasive characteristics, and the combination between BRAF and TERT mutations AF improved the diagnostic value in identifying patients' risk of recurrence and tumour malignancy. RET mutation is frequently observed in medullary carcinoma, and RET mutation AF is associated with partial tumour characteristics. RAS mutation is prevalent in follicular tumors, but the association between RAS mutation AF and tumour characteristics is relatively weak. TP53 mutation is more frequently occurred in poorly differentiated and anaplastic carcinoma, and its AF might be associated with the dedifferentiation process. We also concentrated on the mutually exclusive and synergistic effect between different mutations. The mutation rate of TERT increases with the elevation of BRAF mutation AF. Finally, the detection and assessment of AF by NGS in clinical practice helps to provide a reference for individualised targeted therapy plans.

Facts

  1. 1.

    The allele frequency of genetic mutations may provide a more precise reflection of tumour malignancy and prediction of prognosis, due to its quantitative nature and detailed biological insights.

  2. 2.

    The allele frequency of BRAF mutations in thyroid carcinoma is associated with tumor characteristics, and exhibit a low peak value, which is different from melanoma. Increased chromosome 7 copy number is more prevalent in melanoma with high BRAF mutations allele frequency, which may be a significant factor contributing to the disparities in AF characteristics.

  3. 3.

    The allele frequency of TERT mutations in thyroid carcinoma is associated with tumor characteristics. Allele frequency exceeding 50% indicate the potential of biallelic mutations, copy number variation and loss of heterozygosity.

  4. 4.

    The association between RAS mutation AF and tumour characteristics is relatively weak. RET mutations AF in MTC is associated with partial tumor characteristics.

  5. 5.

    Interactions between different gene mutations are also reflected in their allele frequency. The combined testing for TERT and BRAF mutations improved the diagnostic value in identifying patients' risk of recurrence and tumour malignancy.

Open questions

  1. 1.

    Does high mutation rate imply high allele frequency? What is the relationship between these two metrics?

  2. 2.

    What factors contribute to the differences in BRAF mutations allele frequencies between different tumors? Is it caused by increased copy number or extrinsic environmental factors?

  3. 3.

    Does high allele frequency significantly impact the malignancy of tumors? Could this lead to changes in treatment strategies?

  4. 4.

    How do interactions between different gene mutations affect their allele frequency? What are the specific mechanisms underlying these interactions?

  5. 5.

    Is the percentage of tumor cells a reliable indicator for assessing the accuracy of allele frequency? Additionally, is an 80% threshold appropriate for this purpose?

Introduction

The pathological types of thyroid carcinoma include papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), poorly differentiated thyroid carcinoma (PDTC), anaplastic thyroid carcinoma (ATC) originating from follicular epithelium, and medullary thyroid carcinoma (MTC) originating from parafollicular C cells. PTC accounts for approximately 84% of all thyroid carcinomas, with the most common BRAF V600E mutation, resulting from a T1799A missense mutation in the BRAF gene [1]. The BRAF V600E mutation has been extensively studied and applied in clinical diagnosis and treatment of thyroid carcinoma. It has been proved to be associated with tumor multifocality, vascular invasion, lymph metastasis, extrathyroidal extension and prognosis [2,3,4]. The other prevalent mutations, including RAS, TERT, RET, TP53, AKT, and PIK3CA mutations, among others, are associated with different types of thyroid tumors. The novel genetic testing methodologies, including Amplification Refractory Mutation System quantitative Polymerase Chain Reaction (ARMS-qPCR), digital droplet Polymerase Chain Reaction (ddPCR), and Next-Generation Sequencing (NGS), permit the precise detection of gene mutations and the estimation of mutation frequency, which is also known as allele frequency (AF) or variant allele frequency (VAF). AF is defined as the percentage of sequence reads with a specific mutation over the read depth at that locus, reflecting the proportion of gene mutations [5]. As a quantitative indicator, AF reflects the quantity and proportion of mutations in individuals. Even under the same positive conditions for BRAF mutations, there is a significant difference in the impact of high VAF compared to low VAF. Higher VAF may imply more abnormal transcription and expression of variant proteins, which could be associated with tumor malignancy and poor prognosis [6]. Currently, AF has received widespread focus in gastrointestinal and breast cancers. Research on the AF of BRAF, RET, and RAS mutations in thyroid cancers is in progress, while relatively less research has been conducted on mutations such as TERT, TP53, AKT1, and PIK3CA mutations.

BRAF mutation and AF

BRAF mutation and AF characteristics in different regions

The mutation rates of BRAF V600E in different countries exhibit considerable variation. For instance, the mutation rate of BRAF V600E in Chinese PTC patients is approximately 60.1–79.8% [6,7,8]; in Canada it's approximately 42.9% [9]; in America it's approximately 48.5%; in Korea it's approximately 84.5%; in Italy it's approximately 46.7%; in Australia it's approximately 62.3% [10]. According to the researches, the average AF of BRAF V600E mutations in Italian PTC patients is approximately 21.9% [11]; in Canadian patients it's approximately 21.4–26% [9, 12]; in Chinese patients it's approximately 19.36–31.20% [6,7,8], shown as Table 1. As demonstrated in Fig. 1, which presents a comparison of the BRAF mutation rate and AF, while the BRAF mutation rate is higher in PTC patients in some regions, their average BRAF mutations AF is lower. The collective influence of factors such as race, genetics, living environment and dietary habits across diverse regions gives rise to variations in the propensity of cells to develop BRAF V600E mutations. However, subsequent to the occurrence of the mutation, the micro-environment conducive to cell growth and the inter-cellular interactions may impede the quantity accumulation of the BRAF V600E mutation through specific mechanisms, thereby achieving a new dynamic equilibrium.

Table 1 Characteristics of the BRAF V600E mutation AF in different studies
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Fig. 1
figure 1

The characteristics of BRAF V600E mutation rate and AF in studies from different regions

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Association between AF of BRAF mutation and tumor feature

BRAF mutations are closely associated with tumor malignancy. In addition to activating the RAF/MEK/ERK signaling pathway, BRAF may also be involved in upregulating the expression of extracellular matrix-related genes and inhibiting immune pathways to increase the invasiveness of thyroid carcinoma [13, 14]. The average BRAF mutation AF in invasive thyroid carcinoma in Canada is reported to be approximately 26(± 14.6)%, which is significantly higher than the average AF level of 10(± 13.5)% in non-invasive tumors [12]. Even in the case of benign nodules with BFAR mutations, the BRAF mutation AF is less than 5%, while average AF in thyroid malignant nodules is 31.2% [8]. These results suggest a strong correlation between BRAF mutation AF and tumor benignity or malignancy as well as aggressiveness.

In clinical characteristics, BRAF mutation AF is significantly associated with clinical TN stages, thyroid capsule penetration and recurrence, while showing no significant association with gender, age and multifocality [6,7,8,9, 11, 12, 15], as shown in Table 2. Besides, AF of BRAF mutation is significantly higher in aggressive subtypes, such as hobnail and tall cell subtypes, and associated with multi-loci mutations [6, 9, 12]. The association between AF and lymphatic metastasis remains controversial. In PTC patients with intermediate to high risk of recurrence, those with a BRAF V600E mutation AF of > 28.2% have a sixfold higher risk of recurrence compared to those with a mutation AF of ≤ 28.2%, exhibiting 60% sensitivity, 80% specificity and 77.3% accuracy in predicting tumor recurrence [6]. Therapeutically, patients exhibiting a high AF of BRAF mutations demonstrate a greater response to BRAF inhibitor therapy in comparison to those presenting with a low AF of BRAF mutations [16]. Quantitative analysis of the BRAF mutation AF has the potential to serve as a valuable tool in selecting patients who are most likely to benefit from MAPK inhibitor therapy, and functions as a biomarker for long-term response in clinical practice [16, 17].

Table 2 Association between BRAF mutation and tumor feature from different studies
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The Peak Value of BRAF mutation AF in thyroid carcinoma

A total of ten studies from China, Italy, and Canada [6,7,8,9, 11, 12, 15,16,17,18] reported on 892 thyroid cancer patients with BRAF mutations. An unique phenomenon is that the AF of BRAF mutations was less than 50% in almost every patient, which is different from melanoma. According to the report in 368 patients [16], BRAF mutation AF was greater than 60% in 19% of patients with melanoma, and chromosome 7 polysomy was observed in 54% of BRAF mutant cases with AF > 60%. Increased chromosome 7 copy number is more prevalent in BRAF mutant melanoma, particularly in patients with high BRAF mutation AF, which may be a significant factor contributing to the disparities in AF characteristics between thyroid carcinoma and melanoma [16, 17, 19]. In addition, as melanoma is strongly associated with sun exposure, the environmental factors may also play an important role in the BRAF mutation process [20]. An AF of less than 50% supports that BRAF mutations in thyroid cancer typically occur in the heterozygous state [9, 16]. However, current analyses of chromosomal alterations associated with BRAF mutations in thyroid cancer are currently inadequate and require further study.

RAS mutation and AF

RAS mutation and AF characteristics

RAS mutations occurred in different types of thyroid carcinoma, with mutation rates of about 49% in FTC, 13% in PTC, and about 20% in MTC [21]. The major types include HRAS, KRAS, and NRAS mutations, which were mutually exclusive. The average AF of RAS mutations in thyroid carcinoma was 23.2% [22]. The range of AF for NRAS mutations was 0.6–51.5%, for HRAS mutations was 0.4–43.6%, and for KRAS mutations was 0.2–46.6%. The differences were not statistically significant, and the maximum AF are close to each other [23]. In a separate study focusing on MTC [24], the mutation rate of RAS was found to be 27%, with an average AF of 35.3%, ranging from 0 to 60%. Of these RAS mutations, HRAS accounted for 70.5%, KRAS accounted for 27.3%, and NRAS accounted for 2.2%.

Association between AF of RAS mutation and tumor feature

As an oncogene, the RAS family encoding RAS proteins induces thyroid cancer transformation by activating the MAPK signaling pathway [14]. However, isolated RAS mutations are rarely associated with aggressive tumors, with a lower carcinogenic potential compared to BRAF V600E mutations. The average AF of RAS mutations reported in thyroid carcinoma is approximately 23.2%, while in benign nodules it is approximately 20.1%. Even when divided by RAS sub-types (NRAS, HRAS, KRAS), the AF differences between benign and malignant tumors are not statistically significant [23]. Regardless of whether the AF is low (AF < 1%) or high, the risk of malignant thyroid cancer remains similarly high, suggesting that these factors may not be used alone to differentiate between malignant and benign tumors [23]. Furthermore, there is a deficiency of evidence demonstrating a clear association between RAS mutation AF and the occurrence of extrathyroidal extension, lymph node metastasis, capsular invasion, or neural invasion in thyroid cancer. The association between AF and thyroid cancer recurrence requires further research [22,23,24].

Research indicates that the AF of RAS mutations increases with tumor diameter in MTC [23]. Specifically, tumors smaller than 1 cm had an average RAS mutation AF of 29.8%; tumors sized 1–2 cm had an average AF of 33.87%; tumors sized 2–3 cm had an average AF of 36.13%; and tumors larger than 3 cm had an average AF of 46.62%. Besides, there was a trend towards an increased occurrence of secondary genetic abnormalities with increasing RAS mutation AF, although these trends did not reach statistical significance [24]. This suggests that RAS may induce dedifferentiation in thyroid malignancies, contributing to the transition of thyroid cancer towards PDTC/ATC.

In conclusion, RAS mutations or AF may not fully reflect the malignant potential of tumors. Consequently, they should not be considered in isolation when making clinical decisions. When RAS mutations are combined with BRAF and TERT mutation analysis, the sensitivity for diagnosing malignant tumors reaches 70.5%, with a specificity of 88.8%, exhibiting an improvement in sensitivity of 30.2% while reducing specificity by 11.2% [23].

Association between RAS and other mutations

RAS mutations exhibit mutual exclusivity with RET mutations in MTC and BRAF mutations in ATC [24, 25]. In ATC, EIF1AX is most commonly found to co-mutate with RAS family mutations. Additionally, RAS mutations can synergistically activate TERT mutations via the PI3K pathway, thereby enhancing tumor malignancy. These mutations occur in combinations such as TERT + HRAS/KRAS/HRAS, albeit with much lower prevalence compared to TERT + BRAF mutations [21, 25]. According to the report, 14.6% of PTC cases exhibit coexistence of RAS + BRAF mutations; 9.8% exhibit RAS + TERT mutations; and 4.9% exhibit RAS + BRAF V600E + TERT mutations [21]. Notably, the concurrent presence of RAS mutations with BRAF and/or TERT promoter mutations tends to be enriched in high-risk malignancies such as the tall-cell subtype of PTC, FTC, MTC, oncocytic carcinoma and ATC [6, 21]. In the event that RAS mutations occur independently or in conjunction with TERT mutations, the AF may be slightly elevated (approximately 32.3–41.5%, observed in FTC, PTC, and ATC.). When co-occurring with BRAF mutations, AF may be relatively low (approximately 13.8–16.7%, observed in PTC and PDTC) [6, 24], which may be attributed to the mutual exclusivity between BRAF and RAS mutations.

TERT mutation and AF

TERT mutation and AF characteristics

TERT mutations can be categorized into C228T and C250T mutations, which are mutually exclusive. In PTC, the mutation rates of TERT C228T and C250T mutations are 9.7% and 2.1%, respectively [26]. Telomerase activity and TERT expression are typically undetectable in normal thyroid tissue and benign thyroid nodules, but are more prevalent in PDTC and ATC [26,27,28]. The mutation rate of TERT in PTC exhibits variability across different regions, with reported ranges of approximately 2–11.3% in China, 6.1–12% in the United States, 12.1–17.4% in Italy, 4.2–9.8% in Korea, 10.1–12.6% in Japan, and 7.7–12.1% in Portugal [27]. The discrepancies between different regions are not clearly discernible. AF of TERT mutation appears to be relatively high, with a range of 28.65% to 73.04% and a mean of 46.71% in PTC [6]. However, the lack of research on TERT mutation AF makes it difficult to identify definitive characteristics.

Association between AF of TERT mutation and tumor feature

TERT promoter mutations contribute to TERT over-expression, initiating a cascade of effects that result in telomerase reactivation [29, 30]. These alterations increase the risk of tumour vascular invasion, distant metastasis, and a poorer prognosis [31,32,33]. Besides, TERT mutations were also posited as a pivotal oncogenic driver of dedifferentiation in thyroid carcinoma, associated with a lower degree of differentiation, increased chromosomal instability, and a propensity for mesenchymal transition [25, 34]. TERT mutations were reported in 13.0% of thyroid tumors, and the AF ranged from 0.13% to 54.7%, with 30.6% exhibiting low AF levels (≤ 1.0%) and 69.4% exhibiting high AF levels (> 1.0%.) [9]. AF levels of TERT mutations have been shown to be significantly associated with aggressive pathological features of PTC, including lymphovascular invasion, perineural invasion, lymph node metastasis, extra-thyroidal invasion, and advanced TNM staging [9]. In addition, thyroid nodules presenting TERT and/or BRAF V600E mutations at a VAF of 0.03% or higher could provide a definitive cancer diagnosis [9].

Association between TERT and other mutations

TERT mutations have been frequently observed in conjunction with BRAF and RAS mutations [35]. Approximately 76.47–81.63% of TERT mutations coexist with BRAF mutations in thyroid tumors[6, 9]. BRAF mutations have been identified as a catalyst for TERT over-expression through two mechanisms: first, by increasing the expression of ETS transcription factors via the MAPK pathway, which bind to the C228T/C250T region in the TERT promoter, enhancing transcription and expression; second, by inducing the transcription factor c-FOS through the MAPK pathway, leading to an up-regulation of GABPB. The elevated level of the GABPA-GABPB complex facilitates the transcription of mutated TERT [26, 36]. This synergistic effect is also exhibiting in AF, where the average AF of BRAF mutations in the BRAF + TERT group (29.23%) significantly surpasses that of the BRAF single mutation group (17.54%) [6]. For each 1% increase in the AF of BRAF mutations, there is a corresponding 1.16% increase in the risk of combining TERT mutations [6].

PTC patients with concurrent BRAF and TERT mutations have exhibited increased invasive characteristics, an elevated Ki-67 index, advanced TNM staging, and an increased propensity for extrathyroidal extension, distant and lymphatic metastasis and a poorer prognosis compared to patients with either mutation alone [37,38,39]. Additionally, PTC cases with BRAF + TERT mutations have shown a higher postoperative thyroglobulin level and reduced sensitivity to iodine uptake [40]. Combined testing for BRAF and TERT mutations has significantly improved the diagnostic value in identifying patients' risk of recurrence, exceeding testing for BRAF or TERT mutations alone [9]. In addition, a significant correlation has been identified between high AF of either mutation individually or different AF for both mutations in coexistence and aggressive tumor features [9].

AF Greater than 50% and biallelic mutations

In intermediate-to-high-risk PTC patients, 23.5% had a TERT mutation with an AF greater than 50%, with a maximum of 73.04% [6]. When AF was calculated by NGS, all the specific genetic loci present on homologous chromosomes were included in the denominator of the calculation. Consequently, an AF greater than 50% may indicate biallelic mutations, copy number variation (CNV) and loss of heterozygosity (LOH). CNV, in which sequences of the genome are repeated, can be caused by genomic instability, the selective advantages conferred by aneuploidy, gene amplification, and chromosomal structural rearrangements. The occurrence of thyroid cancer is accompanied by CNV [41,42,43]. LOH involves the loss of the wild-type allele, which subsequently leads to an increase in AF. This phenomenon can be triggered by mitotic errors, chromosomal fragmentation, gene conversion, and improper repair of DNA breaks [44, 45]. It has been reported that the incidence of LOH significantly increases in thyroid cancers associated with lymph node metastasis [46]. Besides, another important scenario to consider is biallelic mutation, where TERT mutations are present on both homologous chromosomes.

It has been proposed that TERT mutations occur predominantly as heterozygotes and are expressed monoallelically, whereas expression of both alleles of TERT is uncommon [47]. AF > 50% may be caused by events such as CNV and LOH [48,49,50]. A comparative study of various tumour cell lines with either single-allele or biallelic TERT mutations has not identified significant disparities in TERT mRNA expression levels [51], corroborating this opinion. Another hypothesis is that a biallelic mutation with AF > 50% may initiate TERT reactivation and the recruitment of GABPA/B1 transcription factors, thereby facilitating biallelic expression [36]. The biallelic TERT mutations exhibit reduced levels of DNA methylation and an activated chromatin modification H3K4me2/3 compared to monoallelic mutations [52]. In addition, research in mice has suggested that biallelic HRAS mutations can achieve biallelic expression [53]. Further research is required to elucidate the chromosomal alterations of TERT mutations and to determine whether malignancy will be further exacerbated after the AF exceeds 50%.

Other mutations and AF

RET mutation and AF

The mutation and fusion of the proto-oncogene RET play a pivotal role in the development of various types of tumors, occurring in a mutually exclusive manner [54]. In MTC, RET mutations are particularly prevalent, with a mutation rate as high as 95% to 98% in hereditary MTC (hMTC), and 25% to 40% in sporadic MTC (sMTC). RET M918T mutation is the most common type, accounting for 94.7% of all RET mutations and is considered the primary driver of carcinogenesis in sMTC [24, 54]. RET mutation significantly promotes cell proliferation. Specifically, cells with RET mutations exhibit a faster rate of proliferation, a markedly increased Ki-67 index, larger tumor size, and greater invasiveness [24, 54]. In MTC, the average AF of RET mutations is 35.9%, with a wide range of fluctuation from 4.4% to 90% [24, 55]. A positive correlation exists between tumor diameter and the increase in AF. For instance, the average AF for tumors less than 1 cm in diameter is 23.42%, while for tumors larger than 3 cm, the average AF is 42.48% [24]. An increasing AF is significantly associated with distant metastasis of tumor and a poorer prognosis. Among patients with sMTC, patients with RET mutations have a higher disease-specific mortality rate compared to patients without RET mutations, and the prognosis deteriorates with an increase in AF [24, 55].

TP53 mutation and AF

TP53 is a tumor suppressor, encodes the p53 protein, which plays a crucial role in regulating the cell cycle, promoting DNA repair, and inducing apoptosis. TP53 mutations are more frequently observed in PDTC and ATC, closely associated with radio-iodine refractory and poor prognosis [56,57,58]. Some studies suggest considering TP53 mutations as an event that distinguishes differentiated thyroid carcinoma from ATC [59,60,61,62]. While study on TP53 mutation AF in thyroid carcinomas is limited, in breast carcinoma, the AF of TP53 mutations varies significantly, ranging from 0 to 80%, and is correlated with circulating free DNA (cfDNA) levels [63], can be used to reflect tumor burden and assess treatment efficacy. Similar findings in ATC indicate that TP53 mutations detected via cfDNA are highly consistent with tissue NGS results, potentially serving as an important molecular tool [64].

PIK3CA/AKT mutation and AF

Mutations in the PI3K-AKT-mTOR pathway are commonly observed in ATC, with a mutation rate of approximately 18% for PIK3CA, which facilitates the development of ATC and are associated with a poor prognosis [59, 64,65,66]. PIK3CA mutations and KRAS mutations are mutually exclusive, but tend to synergize with BRAF V600E mutations. In ATC, BRAF mutations are most commonly observed in conjunction with PIK3CA mutations [24]. Following BRAF inhibitor therapy, there is also an increased mutation rate in PI3K-AKT-mTOR, which may contribute to resistance [66]. In PTC, lower rates of PIK3CA and AKT1 mutations (2% and 1%, respectively) are observed with AF ranging from 6.7% to 12.2%, in conjunction with BRAF and TERT mutations [6].

The magnitude of AF, driver mutations and passenger mutations

An objective standard for defining high and low AF is currently lacking. An AF of 1% was frequently used to classify variants as high or low because variants with an AF of 1% or less were hardly identified by immunohistochemistry staining or Sanger sequencing [9, 21]. However, we believe that an optimal threshold for distinguishing should be based on the indicators actually being evaluated. For instance, a threshold of 0.03% AF for BRAF and TERT mutations can effectively differentiate between benign and malignant tumors, while a threshold of 1% AF for BRAF mutations can help to differentiate aggressive histopathologic features and intermediate to high risks of recurrence [9]. A study have calculated a 28.2% BRAF mutation AF by ROC curve, which is the optimal threshold for assessing PTC recurrence [6]. Therefore, the specific threshold value to be taken in practical application depends on the purpose of the assessment.

In addition, AF has a natural advantage in distinguishing between driver and passenger mutations. Genetic mutations can be divided into driver and passenger mutations. Driver mutations directly contribute to the development and progression of cancer and usually give cancer cells a growth advantage, whereas passenger mutations occur concurrently in cancer cells due to genetic instability and do not directly promote cancer development or progression [66, 67]. Cells carrying driver mutations have been favoured by natural selection and have increased in proportion over time and therefore typically exhibit a higher AF. In contrast, passenger mutations have not been subject to positive selection and, although present in cancer cells, tend to exhibit a lower AF [68,69,70]. A Japanese study suggested that the VAF above 5% can be used as a criterion to distinguish driver from passenger mutations [25], and this criterion may help to identify the most important driver mutations in both PDTC and ATC among a variety of mutations. According to this criterion, 72.3% of ATC cases are strongly associated with multiple driver mutations, including BRAF, RAS family, NF1, SDHA, FGFR1, PIK3CA, AKT1, TERT and TP53 mutations [25].

Discussion

While we focus on the occurrence of genetic mutations, the development of genetic detection technologies has highlighted AF as a quantitative indicator, which also holds the potential to become a crucial factor in diagnostic and therapeutic processes [5]. In clinical practice, PDTC patients with targetable signaling pathway mutations in NGS and received targeted therapy—such as tyrosine kinase receptor inhibitors (pazopanib, sorafenib, vandetanib), anti-BRAF and MEK inhibitors (vemurafenib, trametinib), or mTOR inhibitors (everolimus) showed improved duration of overall survival than similarly treated PDTC patients with no detectable mutations within targetable signaling pathways in NGS [71]. Besides, patients with a high AF of BRAF mutations show a greater response to BRAF inhibitor therapy compared to those with a low AF of BRAF mutations [16], suggesting that the detection and assessment of AF by NGS in clinical practice helps to provide a reference for individualised targeted therapy plans [5, 72, 73]. Furthermore, the investigation of VAF can facilitate the distinction between passenger and driver mutations, thereby offering a more profound comprehension of the occurrence, development, and evolution of thyroid cancer [25]. Research currently concentrated on the precision of AF as a predictive tool and establishing a effective threshold values in thyroid cancers [6,7,8,9, 11, 12, 15,16,17,18]. A growing body of research is emerging on the AF of BRAF, RAS, and RET mutations, as demonstrated by the average AF shown in Fig. 2. The BRAF V600E mutation, despite being the most prevalent mutation in PTC, exhibits the lowest average AF [6,7,8,9, 11, 12, 15,16,17,18]. The polysomy and increased copy number of chromosome 7 in melanoma results in a BRAF mutation AF greater than 50% [9, 16]. However, this alteration may be absent in thyroid cancer, where almost all BRAF mutation AFs are below 50% [6,7,8,9, 11, 12, 15,16,17,18]. Other mutations with high AF may be associated with biallelic mutations, CNV and LOH [48,49,50]. In addition, the chromosomal alterations associated with an TERT VAF greater than 50% and their impact on malignancy require further investigation [47, 51, 52].

Fig. 2
figure 2

Average AF of different mutations in thyroid cancer. The average AF of BRAF mutation is calculated as the sum of the sample size of each study multiplied by its respective average AF, divided by the total sample size of all studies. Other average AF data are directly sourced from the references. BRAF mutations have the highest occurrence rate but the lowest average AF among these mutations (18.2%), while TERT mutations have the highest average AF (46.7%)

Full size image

The development of a tumour is attributed to the instability of the somatic genome, which can result in the emergence of aggressive clones with the capacity to survive and outcompete other cells in the micro-environment. The relatively low density of somatic mutations is thought to underlie the biology of the observed inert clinical behaviour of PTC [74,75,76], and the accumulation of somatic mutations in PTC can be manifested by the AF. The BRAF V600E mutation AF has been demonstrated to be associated with thyroid capsule penetration, vascular and neural invasion, incorporation of other mutations, recurrence and metastasis [6,7,8,9, 11, 12, 15,16,17,18]. Of these, BRAF V600E mutations are most commonly combined with TERT mutations, and the risk of co-occurrence of BRAF and TERT mutations is positively correlated with BRAF V600E mutations [6, 21]. Conducting a combined evaluation of BRAF and TERT for VAF greater than 0.03% enhances the accuracy of FNA in diagnosing malignant tumours, and offers a more substantial diagnostic value in evaluating the prognosis of PTC when compared to the use of BRAF or TERT alone [9]. In BRAF and TERT mutations, a significant correlation has been identified between high AF of either mutation individually or different AF for both mutations in coexistence and aggressive tumor features [9]. The utilisation of an AF of 28.2% for the BRAF V600E mutation as a cutoff value to predict the risk of recurrence in patients with PTC in clinical practice helps to adjust therapy plans and follow-up intervals [6]. In FTC, RAS mutations are frequently observed, while in MTC, RET mutations predominate, followed by RAS. The elevated AF levels in both RAS and RET mutations indicates the identity of the driver mutations [24, 73]. However, RAS mutation association with malignancy degree is weak; neither RAS mutations nor AF can be used as a sole indicator of prognosis, requiring a comprehensive assessment in conjunction with BRAF mutation and other indicators [77,78,79,80].

Despite the decline in the expense of NGS compared to first-generation sequencing, it remains a costly diagnostic procedure, thus necessitating accurate identification of indications. The histopathological types and risk stratification of thyroid cancer are essential factors in determining subjects. PTC patients with intermediate to high recurrence risks may benefit from NGS, whereas decisions for low-risk patients should be made with caution [6, 9]. Furthermore, the variant loci differs between poorly differentiated and well-differentiated carcinoma, necessitating a corresponding adjustment in the focus of testing loci. For PTC, high-frequency mutation loci including BRAF, TERT, and RAS, should be prioritized for testing [6, 21]. In contrast, for other low-frequency mutation loci, the scope of testing may be appropriately narrowed or omitted based on clinical necessity. For PDTC and ATC, which involve a larger number of genetic loci, extensive gene sequencing is recommended [25]. The reduction in the scope of testing loci based on the human genomic characteristics helps to conserve limited medical resources and facilitates the integration of NGS into routine diagnostic and therapeutic procedures [81,82,83].

Several limitations are noteworthy. Firstly, the AF of certain mutations in thyroid cancer, such as those in the AKT, RAS, TERT, and TP53 genes, has not been extensively reported. Further research with larger sample sizes is necessary to elucidate the underlying patterns. Secondly, the purity of tumor cells in the sample is crucial when calculating AF, as contamination with normal cells can affect the AF results. It is essential to introduce indicators such as tumor cell purity for quality control. However, most relevant research papers have yet to provide clear descriptions of quality control standards, and a standardized protocol for genetic testing is currently lacking [5]. Therefore, in order to avoid the drawing of imprecise conclusions due to inconsistent quality control standards, no statistical analysis algorithms were employed in the interpretation and comparison of results. We suggest that relevant indicators be introduced and quality control standards be unified in future studies to enhance the reliability of research findings. A study have proposed that the reproducibility of assessing tumor cell content using an 80% cutoff is relatively high, while a 70% cutoff is only moderate [16]. Furthermore, tumors exhibit heterogeneity, which may affect AF values. However, the literature generally lacks descriptions of quality control indicators for tumor heterogeneity, a problem that needs attention. For larger tumors, multiple sampling points across regions are recommended to obtain an average value. For smaller tumors, sampling should sufficiently cover the tumor.

Conclusion

The AF of BRAF mutation is associated with tumour malignancy and prognosis, exhibiting a relatively low peak value. TERT mutations in AF are associated with invasive characteristics, and the combination between BRAF and TERT mutations AF improved the diagnostic value in identifying patients' risk of recurrence and tumour malignancy. RET mutation is frequently observed in medullary carcinoma, and RET mutation AF is associated with partial tumour characteristics. RAS mutation is prevalent in follicular tumors, but the association between RAS mutation AF and tumour characteristics is relatively weak. TP53 mutation is more frequently occurred in poorly differentiated and anaplastic carcinoma, and its AF might be associated with the dedifferentiation process. We also concentrated on the mutually exclusive and synergistic effect between different mutations. The mutation rate of TERT increases with the elevation of BRAF mutation AF. Finally, the detection and assessment of AF by NGS in clinical practice helps to provide a reference for individualised targeted therapy plans.

Prospects

Yet preoperative NGS analysis through fine-needle aspiration also has the potential to assess the malignant potential of tumors. Despite the significant reduction in cost associated with second-generation sequencing compared to first-generation sequencing, comprehensive screening remains a costly endeavor. It is essential to consider the cost and actual utility of genetic testing in practical applications. The indications for genetic testing must be strictly defined, focusing on high-risk thyroid cancer and pathological types with poor prognosis, analyzing significant gene loci, to ensure the rational use of resources and achieve personalized precision treatment [84, 85]. Furthermore, the dynamic changes in AF can reveal the occurrence, development, and evolution of tumors. Future research is expected to gain new insights from these progresses and feed back into clinical practice, thereby optimizing diagnostic and treatment strategies.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

AF:

Allele Frequency

VAF:

Variant allele frequency

PTC:

Papillary Thyroid Carcinoma

FTC:

Follicular Thyroid Carcinoma

PDTC:

Poorly Differentiated Thyroid Carcinoma

ATC:

Anaplastic Thyroid Carcinoma

MTC:

Medullary Thyroid Carcinoma

sMTC:

Sporadic Medullary Thyroid Carcinoma

ARMS-qPCR:

Amplification Refractory Mutation System quantitative Polymerase Chain Reaction

ddPCR:

digital droplet Polymerase Chain Reaction

NGS:

Next-Generation Sequencing

CNV:

Copy number variation

LOH:

Loss of heterozygosity

sMTC:

sporadic Medullary Thyroid Carcinoma

hMTC:

hereditary Medullary Thyroid Carcinoma

cfDNA:

circulating free DNA

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Acknowledgements

This research was funded by the “1+X” project (2022LCJSGC05 and 2022MDTQL02) provided by the Second Hospital of Dalian Medical University.

Funding

This research was funded by the “1 + X” project (2022LCJSGC05 and 2022MDTQL02) provided by the Second Hospital of Dalian Medical University.

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  1. Jiayu Huang and Jiazhi Wang contributed equally to this work and should be considered co-first authors.

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  1. Department of Thyroid Surgery, The Second Hospital of Dalian Medical University, Dalian, Liaoning Province, China

    Jiayu Huang, Jiazhi Wang, Guangzhi Wang & Yongfu Zhao

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Jiayu Huang, Jiazhi Wang designed and wrote the manuscript, Guangzhi Wang, Yongfu Zhao revise and edited the manuscript. All authors contributed to the article and approved the submitted version. Jiayu Huang and Jiazhi Wang made equal contributions to this study.

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Huang, J., Wang, J., Wang, G. et al. Allele frequency in thyroid cancer: mechanisms, challenges, and applications in cancer therapy. Thyroid Res 18, 19 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13044-025-00237-8

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Keywords

Thyroid Research

ISSN: 1756-6614