Genetic alterations and allele frequency of BRAF V600E and TERT mutation in papillary thyroid carcinoma with intermediate-to-high recurrence risk: a retrospective study

In recent decades, there has been a global increase in the occurrence of thyroid cancer [1]. Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer, and the BRAF V600E mutation is the most frequent genetic mutation in PTC [2]. The BRAF gene encodes B-Raf protein kinase, which is a crucial member of the MAPK signaling pathway that regulates cell proliferation and differentiation [3]. Research suggests an association between PTC with BRAF V600E mutation and tumor volume, invasiveness and metastasis [4, 5]. However, it remains controversial whether BRAF V600E accurately reflects the malignancy and prognosis of the tumor [6‐8]. The TERT gene encodes telomerase, which is a crucial enzyme for maintaining chromosomal telomere length. Activation of TERT gene mutations can sustain telomere length, leading to unlimited cell proliferation. Research has shown that mutations in the TERT gene are related to increased tumor size, invasiveness, lymph node metastasis and resistance to radiotherapy [9, 10]. However, the severity of PTC with BRAF V600E mutation can be variable, and patients with BRAF mutations may also present with different conditions, possibly related to the individual frequency of BRAF V600E in each patient [9]. Allele frequency (AF) is calculated by dividing the number of mutated molecules by the total number of wild-type molecules at a specific position in the genome [11, 12]. In this retrospective review of gene test results from Chinese patients with intermediate-to-high recurrence risk PTC [13], we analyzed and explored the impact of BRAF V600E and TERT mutation AF, aiming to identify reliable indicators that reflect the invasiveness and prognosis of PTC.

This study included 94 patients with intermediate-to-high recurrence risk in PTC, with a male-to-female ratio of 1:1.35. The mean age was 43.99 ± 15.35 years, and the average follow-up time was 1.90 ± 1.75 years. No distant metastases were detected in patients during diagnosis or follow-up. Among these patients, 88 (93.62%) patients exhibited at least one genetic alteration. 78 (82.98%) patients had gene mutations, 11 (11.70%) patients had gene fusions, 1 (1.06%) patient had both gene mutation and fusion, and 6 (6.38%) patients showed no genetic abnormalities. The gene mutation events observed included BRAF V600E missense mutation (n = 75), TERT promoter mutation (n = 17), AKT1 missense mutation (n = 1), KRAS missense mutation (n = 1), TP53 missense mutation (n = 1), PIK3CA missense mutation (n = 2), and HRAS missense mutation (n = 1). The gene fusion events observed in this study included CCDC6-RET gene fusion (n = 7), NCOA4-RET gene fusion (n = 2), and ETV6-NTRK3 gene fusion (n = 2).

The incidence of PTC is higher in females than in males, with a ratio of 2–3 to 1. However, in patients with intermediate-to-high recurrence risk PTC, females are only 1.36 times than males, suggesting that more males develop intermediate-to-high risk PTC. Some studies suggested that male is considered one of the risk factors for lymph metastasis in thyroid cancer [14]. We observed no significant differences in the allele frequencies of BRAF V600E and TERT mutations between males and females, which indicates that the severity of thyroid cancer in males may be associated with other factors. The occurrence rates of TERT mutations in thyroid cancer do not differ significantly among Asians, Europeans, and Americans [9, 10]. However, the occurrence rate of BRAF V600E mutations varies significantly across different races. The incidence of BRAF V600E mutation varies among different populations. In American patients with PTC, the incidence is approximately 50.8% [3]. In Korean patients, the incidence is around 72% [4], while in Japanese patients, it is about 38% [5]. In Italian patients, the incidence is around 38.1% [7], and in Saudi Arabian patients, it is about 59.5% [6]. In Chinese PTC patients, the incidence is approximately 72.4% [11], which is similar to the rate observed in our retrospective analysis (76.5%). Although BRAF V600E is considered a major driver in Chinese PTC populations, BRAF V600E AF in Chinese PTC patients is not significantly higher than in other regions and is even lower in our study. The reported BRAF V600E AF in PTC in other regions is approximately 26% in Canada [12] and around 27% in Italy [15]. While our study found that the AF of BRAF V600E in Chinese patients was approximately 19.36%, demonstrating inconsistency between its AF and incidence characteristics.

Gene mutations and fusions often do not occur simultaneously, exhibiting mutual exclusivity. Only 1 patient was found to have both TERT mutation and ETV6-NTRK3 fusion, but this patient's condition was not severe. Among the patients analyzed, the most common gene fusion was CCDC6-RET fusion (63.6%), followed by NCOA4-RET fusion (18.2%) and ETV6-NTRK3 fusion (18.2%), which may be related to a history of neck exposure [16, 17].

The AF of BRAF V600E mutation is suspected to be associated with metastasis in PTC, leading to higher tumor staging and poor outcome [5, 11, 12]. However, some studies have contradicted this claim, stating that the AF of BRAF V600E mutations does not significantly impact the prognosis and invasiveness of PTC [15, 18]. These discrepancies may be related to the diversity of populations in different countries or variations in the risk stratification of patients. Tumors form due to genomic instability in somatic cells, which can lead to the emergence of aggressive clones that can survive and outcompete other cells in the microenvironment. With the competition among cells, various genomic compositions (Allele Frequency) have emerged. The overall density of somatic mutations is relatively low, and this is considered the biological basis for the indolent clinical behavior observed PTC [18‐21]. The efficacy of AF varies in different literature, which may be due to its unclear efficacy in low-risk thyroid cancer patients, while its effect becomes more distinct in intermediate-to-high-risk patients, clearly demonstrating its association with the disease. Therefore, we focused on intermediate-to-high-risk population to avoid interference from the large low-risk PTC population. In addition, genetic testing is a costly procedure, and although we found that BRAF V600E AF can predict patient prognosis, it is apparently unnecessary for low-risk PTC patients, since next generation sequencing techniques are mainly applicable to populations with intermediate-to-high risk recurrence patients. However, as this is a single-center, single-region, single-ethnicity study, it has certain limitations. Future research could include multiple populations, regions, and larger sample sizes to further investigate the overall impact of BRAF and TERT gene frequencies on disease. In addition, 94.1% of TERT mutation carriers had other types of genetic alterations, which may be due to the small sample size of TERT mutations. The lack of significant association between TERT promoter mutation AF and disease prognosis may be influenced by other mutation types, which requires further investigation.

We analyzed the impact of BRAF V600E mutation AF on tumor invasion, co-occurring mutations, and recurrence in patients with PTC in intermediate and high-risk recurrence categories. For each 1% increase in BRAF V600E mutation frequency, the risk of tumor invasion, recurrence, and co-occurring gene mutations increases by 10.0%, 8.0%, and 8.8%, respectively. The combination of BRAF mutation with other gene mutations significantly increases the risk of thyroid cancer invading the thyroid membrane and recurrence, with TERT mutations being the most common co-occurring mutation. When the frequency of the BRAF V600E mutation is greater than 28.2%, the risk of thyroid cancer recurrence increases sixfold compared to when the frequency is 28.2% or lower. Using AF of BRAF V600E mutation > 28.2% to predict the recurrence of intermediate and high risk PTC has a sensitivity of 60% and a specificity of 80%, making it a potential new indicator for predicting the risk of thyroid cancer recurrence.

The study indicates that BRAF mutations are the primary genetic mutation events in PTC, followed by TERT mutations, while other types occur less frequently. TERT mutations are associated with older age (average 61.41 years) and a higher AF (average 46.71%). Research indicates that TERT mutation is infrequent in children and teenagers [22], while combined with BRAF mutation, they contribute to increased malignancy in thyroid cancer [23, 24]. Our study suggested that TERT mutations are often accompanied by other mutations. The higher malignancy of TERT mutations in PTC may be due to the co-occurrence of other mutations [25]. The co-occurrence of BRAF mutations with TERT mutations is the most common scenario, and there is a positive correlation between the AF of BRAF V600E and the risk of co-occurring TERT mutations. According to extensive data reviewed by scholars, there appears to be no significant correlation between BRAF mutation and distant metastasis in PTC patients, while TERT mutation has been associated with distant metastasis. But our study identified a potential association between a high allele frequency of BRAF V600E and TERT mutation, which is consistent with the findings of previous research, BRAF mutations may lead to abnormal overexpression of the TERT promoter [26]. However, among the 94 patients we reviewed, none showed evidence of distant metastasis. Further research is needed to explore the relationship between BRAF, TERT mutations and distant metastasis [27].

Although the incidence of BRAF V600E mutation varies across different regions, the AF of BRAF V600E mutation is similar between Asian and Western patients. AF of BRAF V600E is positively correlated with invasiveness and the risk of recurrence. It may induce other gene mutations, such as TERT mutations, thereby enhancing the invasive capabilities of the tumor and leading to a poorer prognosis. The AF of BRAF V600E shows potential as a novel indicator for predicting tumor invasiveness and prognosis.