Relevance and clinicopathologic relationship of BRAF V600E, TERT and NRAS mutations for papillary thyroid carcinoma patients in Northwest China

Background To determine the relevance of the single or combination mutations of BRAF V600E, TERT, and NRAS genes and the clinicopathologic relationship in papillary thyroid cancer (PTC). Methods Patients with PTC were enrolled into the study between February 2018 and April 2019. Based on the number of mutant genes, we classified the participants into single BRAF V600E mutation group, double mutations group and no mutation group. Single factor and multiple logistic regression analyses were applied to explore the independent factors. Review Manager 5.3 was used for meta-analysis to review the clinical efficacy of gene co-mutations. Results Finally, 483 patients were enrolled into the study and 419 (86.7%) of them harbored BRAF V600E mutation. TERT or NRAS mutation was likely to coexist with BRAF V600E mutation in PTC. BRAF V600E and NRAS promoter co-mutations was identified in 6 patients, with a prevalence of 1.2%. Prevalence of BRAF V600E and TERT coexistence in PTC was 2.1%. Significant differences were found among age, pathology, multifocality, bilateral lesions, lymph node metastasis, and 131I radiotherapy, P < 0.01. Multiple logistic regression analyses demonstrated that age [odds ratio (OR) = 1.044, 95% confidence interval (CI) = 1.013–1.076; P = 0.006], lymph node metastasis [OR = 0.094, 95% CI = 0.034–0.264; P < 0.001], 131I radiotherapy [OR = 7.628, 95% CI = 2.721–21.378; P < 0.001] were risk factors for BRAF V600E mutation. Besides, age [OR = 1.135, 95% CI = 1.069–1.205; P < 0.001], multiple leisions [OR = 4.128, 95% CI = 1.026–16.614; P = 0.046], pathology [OR = 3.954, 95% CI = 1.235–12.654; P = 0.021] were independent factors for combination mutations. Meta-analysis showed significant association of BRAF V600E+/TERT+ co-mutations with lymph node metastasis, multifocality, distant metastasis, tumor recurrence, extrathyroidal extension, and dead of disease. Conclusions Prevalence of BRAF V600E mutation in Northwest China was higher than other areas. Age, multiple lesions, and pathology were independent factors for double mutation of BRAF V600E/TERT or BRAF V600E/NRAS. Coexistence of BRAF V600E and TERT promoter mutations was significantly correlated with poor outcome.


Introduction
Thyroid cancer is the most common endocrine malignancy, and its global incidence has rapidly increased in recent decades [1]. Papillary thyroid carcinoma (PTC), which is derived from the follicular epithelium, represents 80 to 85% of thyroid malignancies. Although PTC is highly curable in general, approximately 10% of patients are destined as progressive disease [2]. Thus, the molecular-based risk stratification has been emphasized to compare treatment-associated benefits. Recently, improved understanding of the molecular pathogenesis and the identification of molecular markers are of high clinical significance, indicating the diagnosis and prognosis of PTC.
Molecular markers have been focused so far, such as BRAF V600E, telomerase reverse transcriptase (TERT) and NRAS, which might be potential prognostic factors for FTC. BRAF V600E mutation was correlated with more aggressive and iodine-resistant phenotypes, providing valuable prognostic information for thyroid cancer [3]. Similarly, TERT promoter mutation was associated with aggressive thyroid tumor characteristics, tumor recurrence, and patient mortality [4]. NRAS gene, the most frequent mutant gene of the RAS gene family, was related to increased risk of distant metastasis [5,6]. However, features of gene mutation from different regions are different. In Australian urban population, 68% of PTC patients were identified with BRAF V600E mutation [7]. In Middle Eastern, TERT promoter mutation was harbored in 6.5% PTC patients [8]. For PTC patients from Greek, low prevalence of TERT promoter (3.4%), BRAF V600E (17%), and RAS mutations (3.4%) was detected [9]. In China, data of gene mutation for PTC was relatively limited. In 2018, Liang J et al. reported 72.4% of BRAF V600E mutation and 2.8% of RAS mutation among 355 Chinese PTC patients [10]. In China, it is essential to achieve more evidence of genetic events as trustworthy prognostic markers for risk stratification and patient management.
Considering the synergistic effects of mutant genes, coexistence of gene mutation should be emphasized. BRAF V600E promoter mutation, in combination with TERT or RAS mutation, was recognized as clinically important diagnostic and prognostic genetic markers for thyroid cancer. TERT, a predominant determinant for controlling the activity of telomerase, was likely to coexist with BRAF V600E mutation in thyroid cancer [11]. In 2016, Sun J et al. found that 94.7% PTC patients with TERT promoter mutation were detected with BRAF V600E mutation [12]. In 2017, Vuong HG et al. claimed that the combination of BRAF V600E and TERT promoter mutations indicated increasing risk of aggressiveness of PTC than TERT or BRAF V600E mutation alone [13]. In this study, we focused on the prevalence of BRAF V600E, TERT and NRAS mutations and their association with clinicopathological features in PTC patients from Northwest China.

Participants
This retrospective study included 483 patients (127 men and 356 women) admitted to Xijing Hospital, between February 2018 and April 2019. The fundamental features were shown in Table 1. All patients underwent preoperative ultrasound and fine-needle aspiration biopsy tests. Total or near-total thyroidectomy, cervical lymph node dissection, and radioiodine therapy were pursued as clinically determined. Pathological diagnosis was established following the World Health Organization criteria and confirmed by expert thyroid cancer pathologists. All patients provided written informed consent. Ethical approval for the study was provided by the Ethical Committees of Xijing Hospital.

Genomic DNA isolation
Formalin-fixed and paraffin-embedded (FFPE) tumor tissue was achieved for human genomic DNA isolation, using the AmoyDx® FFPE DNA Kit (Amoy Diagnostics Co., Ltd., Xiamen, China). Selection of the most representative areas was made by an experienced thyroid pathologist. Before DNA isolation, paraffin was removed

Detection of the TERT mutations
TERT promoter C228T and C250T mutations were identified on genomic tumor DNA using standard PCR. Briefly, a 235-bp region of TERT promoter, containing the hotspots of C228T and C250T mutations, was PCRamplified using primers 5′-AGTGGATTCGCGGGCA-CAGA-3′ (sense) and 5′-CAGCGCTGCCTGAAACTC-3′ (antisense). The thermal cycling conditions were as follows: 95°C for 5 min × 1 cycle, 95°C for 25 s, 64°C for 20 s, and 72°C for 20 s × 15 cycles; 93°C for 25 s, 60°C for 35 s, and 72°C for 20 s × 31 cycles. After quality confirmation by agarose gel electrophoresis, PCR products were subjected to Sanger sequencing using ABI3500xl Dx Genetic Analyzer (Thermo Fisher, USA). When mutation was identified, an independent PCR amplification/sequencing, both in forward and reverse directions, was performed to confirm the result.

Statistical analysis
Quantitative data were expressed as means (±SD) for normally distributed variables or as medians and percentiles for non-normally distributed variables. The t-test was applied for variables that were normally distributed. Categorical variables were compared using χ 2 tests. All P values were 2-sided and P less than 0.05 was considered significant. Analyses were performed using SPSS version 22.0 (SPSS Inc., Chicago, IL) and GraphPad Prism version 5 (GraphPad Software Inc., San Diego, USA). Review Manager 5.3 (Cochrane Collaborative, Oxford, UK) was used for meta-analysis.

BRAF V600E gene mutation alone
As shown in Fig. 1

Relationship of gene mutations with clinicopathological outcomes of PTC
The risk factors for different gene mutations were explored. As Table 3 (Table 5).

Discussion
In recent decades, the incidence of thyroid cancer has increased significantly, raising an imperative need to explore its pathogenesis, diagnosis, and treatment [27]. Genetic abnormalities maybe crucial in the tumorigenesis of thyroid cancer. Many molecule therapeutics, such as BRAF, has already undergone clinical trials, indicating the need to discover other markers for diagnosis and treatment prediction [28]. So far, the coexistence of gene mutation was focused. In 2014, Xing MZ et al. claimed firstly that the coexisting of BRAF V600E and TERT C228T mutations present the worst clinicopathologic outcomes [26]. Therefore, exploring the function of genetic events as prognostic markers for risk stratification and patient management is essential.
BRAF V600E mutation is the most frequent molecular alteration detected in PTC. But the mutation rate varies around the world. In 2015, Yip L et al. found the most common mutations were BRAF V600E (644/1039, 62%) in thyroid cancer patients from USA [29]. Identically, 62% BRAF V600E mutation was detected in Australia [30]. For Argentinean, 77% of patients operated for PTC harbored BRAF V600E mutation [31]. In 2017, Lee SE   [32]. Presently, the prevalence of BRAF V600E mutation of PTC patients was up to 88.2%, even higher than that in Korea. Hence, it is of great significance to obtain more evidence-based support of gene mutation in PTC patients. Several studies have reported the coexistence of BRAF V600E and TERT gene mutations. However, it is still unclear why TERT promoter mutations most likely occur in cooperation with BRAF V600E mutation. In 2018, Ren H et al. found 3.5% PTC patients with co-existence of BRAF V600E and TERT promoter mutations [22]. In 2019, Colombo C et al. demonstrated that the double mutation rate of BRAF V600E and TERT promoter in aggressive PTC was 12% [33]. In this study, we observed 2.1% patients with BRAF V600E and TERT double mutations, lower than reported data around the world. Importantly, conflicting results were reported involving the clinical effects of BRAF V600E/TERT coexistence. In 2018, Jin A thought that patients with combined mutations were more likely to have a poor prognosis and outcome [11]. On the contrary, Nasirden A et al. found TERT/BRAF V600E double mutant tumors showed lower disease-free survival rate than BRAF V600E mutant tumors [21]. Presently, our meta-analysis provided strong evidence that BRAF V600E/TERT promoter mutations were significantly correlated with lymph node metastasis, multifocality, distant metastasis, tumor    [13].
There are limited studies about NRAS gene mutation in PTC, still less about BRAF V600E and NRAS gene co-mutation. In 2017, Melo M et al. reported 1.2% mutation frequency of NRAS in primary PTCs [20]. Tobiás B et al. found 3.1% NRAS mutation in Hungarian Patients with PTC [34]. In 2018, NRAS promoter mutations were identified in 2 PTC cases, with a prevalence of 3.4% in the Greek Population [9]. In this study, the prevalence of NRAS mutation was 1.2%. NRAS promoter mutation was also likely to coexist with BRAF V600E mutation in PTC. However, the limited number of NRAS mutation interfered the research of its clinicopathological relationship. Because of the small number of NRAS mutation, we could not perform the clinicopathological relationship analysis. With the enlargement of mutant participants, we could obtain more promising evidence in the near future.
In conclusion, prevalence of BRAF V600E mutation in Northwest China was higher than other areas. Age, lymph node metastasis, and 131 I radiotherapy were risk factors for BRAF V600E mutation. Age, multiple lesions, and pathology were independent factors for combination mutations. Coexistence of BRAF V600E and TERT promoter mutations were significantly correlated with lymph node metastasis, multifocality, distant metastasis, tumor recurrence, extrathyroidal extension, and dead of disease. The predictive value of NRAS combinational mutation with BRAF V600E needs more evidence.