Advertisement

Diagnostic Pathology

, 14:74 | Cite as

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

  • Meiling Huang
  • Changjiao Yan
  • Jingjing Xiao
  • Ting WangEmail author
  • Rui LingEmail author
Open Access
Research
  • 121 Downloads

Abstract

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.

Keywords

BRAF V600E mutation TERT mutation NRAS mutation Co-mutations Papillary thyroid carcinoma 

Abbreviations

FFPE

Formalin-fixed and paraffin-embedded

PCR

Polymerase chain reaction

PTC

Papillary thyroid cancer

TERT

Telomerase reverse transcriptase

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.

Materials and methods

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.
Table 1

Baseline characteristics

Index

Data (N = 483)

Sex

 Male

127 (26.3%)

 Female

356 (73.7%)

Age

 Average age

43.15 ± 11.25

 Median age

43 (14–79)

Pathology

 PTC

187 (38.7%)

 PTMC

296 (61.3%)

Lesion number

 Single lesion

342 (70.8%)

 Multiple lesions

141 (29.2%)

Lesion location

 Unilateral

404 (83.6%)

 Bilateral

79 (16.4%)

Gene mutation

 BRAF V600E mutation alone

419 (86.7%)

 BRAF V600E/TERT co-mutation

10 (2.1%)

 BRAF V600E/NRAS co-mutation

6 (1.2%)

 No mutations in BRAF V600E/TERT/NRAS

48 (9.9%)

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 by xylene-ethanol extraction, and lysed overnight with 20 μL proteinase K in a 56 °C rotating incubator. DNA purification was performed using the QIAamp DNA Mini Kit (Qiagen GmBH, Hilden, Germany), according to the manufacturer’s instructions. The yielded DNA with sufficient quantity and quality was stored at − 40 °C.

Detection of the BRAF V600E mutation

BRAF V600E mutation was determined by polymerase chain reaction (PCR) assay. The gene was performed in a final volume of 50 μl using as template 100–300 ng of genomic DNA, with 1× buffer including 1.5 mM MgCl2, 0.2 mM dNTPs, 25 pmoles of each (Forward, Reverse) primer and 1 unit of Taq polymerase (Kapa Biosystems). PCR was run with a step-down protocol: 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. DNA sequence was read on ABI PRISM 3700 DNA Analyzer (Applied Biosystems). PCR efficiency was assessed according to the Ct value of FAM signal. BRAF V600E was regarded as positive when Ct value lowered than 28.

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 PCR-amplified using primers 5′-AGTGGATTCGCGGGCACAGA-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.

Detection of NRAS mutation by real-time PCR

When genomic DNA isolation was finished, the detection of NRAS mutation in exon 2~4 was performed by AmoyDx® NRAS Mutation Detection Kit (Amoy Diagnostics, Xiamen, China). DNA (5 μL) was added to 35 μL PCR master mix, which contained PCR primers, fluorescent probes, PCR buffer, and DNA polymerase. The PCR cycling conditions were: 5 min denaturation at 95 °C, followed by 15 cycles of 95 °C for 25 s, 64 °C for 20 s, 72 °C for 20 s, 31 cycles of 93 °C for 25 s, 60 °C for 35 s, and 72 °C for 20 s. The PCR experiment was performed on ABI 7500 real-time instrument (Life Technologies, Carlsbad, CA, USA). Fluorescent signal was collected from FAM and HEX channels. NRAS mutation assay was determined according to the FAM Ct value.

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 χ2 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.

Results

BRAF V600E gene mutation alone

As shown in Fig. 1, 435 (90.1%) patients with PTC harbored BRAF V600E mutation, including 419 patients with BRAF V600E mutation alone and 16 patients with double mutations. Interestingly, TERT and NRAS mutations were likely to coexist with BRAF V600E mutation in PTC. BRAF V600E and NRAS promoter double mutations were identified in 6 patients, with a prevalence of 1.2%. The mutant site of NRAS gene referred Exon2 G12D/G12S, Exon2 G12X/G13X and Exon3 Q61X. The average Ct value of NRAS gene was 24.23 ± 1.379. Meanwhile, we identified 10 (2.1%) cases of patients with TERT and BRAF V600E co-mutations, the most common mutant site of which was TERT C228T.
Fig. 1

Distribution of BRAF V600E, TERT, NRAS mutations

Combined mutation of BRAF V600E with TERT or NRAS

Here we screened 16 (3.3%) patients with double mutations. Among them, 14/16 (87.5%) were female. The average age and BMI were 56.0 ± 11.0 and 25.38 ± 2.06, respectively. Other clinical characteristics were listed in Table 2. It seems like that PTCs with concurrent promoter mutations were associated with increased tumor aggressiveness. A majority of patients with double mutations possessed multiple lesions, metastatic lymph nodes, and achieved total thyroidectomy surgery.
Table 2

Clinical features and treatment patterns of 11 patients double mutations

No

Sex

Age

Blood type

BMI

Surgery

Operation time (min)

Invasion

Lesion location

Lesion number

Pathology

Stage

LNM

LNM location

Gene mutation

131I

1

F

29

O

27.5

a

100

Yes

Bilateral

Multiple

PTC + HT

mpT1bN1

1/1

lateral cervical LN

BRAF p.V600E(c.1800 T > A) (Ct = 18.31),

NRAS Exon2 G12D/G12S (Ct = 21.88)

Yes

2

F

62

AB

24.4

a

60

Yes

Bilateral

Multiple

PTMC

T1a(3)N1a

1/4

Central LN

BRAF p.V600E (Ct = 19.46),

NRAS Exon2 G12D/G12S (Ct = 24.5)

Yes

3

F

69

O

24.6

a

60

Yes

Bilateral

Multiple

PTC

T1b(m)N1b

11/15

Prelaryngeal and lateral cervical LN

BRAF p.V600E(c.1799 T > A) (Ct = 19.07),

NRAS Exon3 Q61X (Ct = 26.57)

Yes

4

F

54

B

24.8

a

120

No

Bilateral

Multiple

PTC

T1bNx

1/2

Central LN

BRAF p.V600E) (Ct = 18.34),

NRAS Exon3 Q61X (Ct = 24.03)

Yes

5

M

56

B

27.5

a

80

Yes

Unilateral

Single

PTC + HT

T1aN0

No

No

BRAF p.V600E(c.1800 T > A) (Ct = 18.03),

TERT C228T

Yes

6

F

42

B

22.9

a

70

Yes

Unilateral

Single

PTC

T1bN1a

1/4

Central LN

BRAF p.V600E(Ct = 17.12),

TERT C228T

Yes

7

F

66

A

27.1

a

180

Yes

Unilateral

Multiple

PTC

T3b(2) N1

1/12

lateral cervical LN

BRAF p.V600E(Ct = 19.11),

TERT C228T,C250T

Yes

8

F

56

AB

23.0

b

85

No

Unilateral

Single

PTMC

T1aN1a

1/2

Central LN

BRAF p.V600E(Ct = 17.84),

TERT C228T

No

9

F

62

B

23.4

a

160

Yes

Bilateral

Multiple

PTMC

T1a(2)N1

6/24

Central and lateral cervical LN

BRAF p.V600E(Ct = 18.7),

TERT C228T

Yes

10

F

46

B

23.9

a

150

Yes

Unilateral

Multiple

PTC

T2 N1

7/16

Central and lateral cervical LN

BRAF p.V600E(Ct = 18.45),

TERT C228T

Yes

11

F

58

A

28.8

a

60

No

Bilateral

Multiple

PTC

T1b(2)Nx

No

No

BRAF p.V600E(Ct = 18.72),

TERT C228T

Yes

12

F

60

A

24.17

a

105

No

Unilateral

Single

PTMC

T1aN0

No

No

BRAF p.V600E (Ct = 17.7),

NRAS Exon2 G12X/G13X (Ct = 23.8)

No

13

F

56

B

25.08

a

160

Yes

Unilateral

Multiple

PTC

T1bN0

No

No

BRAF p.V600E(Ct = 19.78),

TERT C228T

No

14

F

43

A

27.05

b

75

No

Unilateral

Single

PTMC

T1aN0

No

No

BRAF p.V600E (Ct = 19.73),

NRAS Exon3 Q61X (Ct = 24.57)

No

15

M

72

B

22.84

a

155

Yes

Unilateral

Single

PTC

T2 N1b

4/4

lateral cervical LN

BRAF p.V600E(Ct = 17.51),

TERT C228T

Yes

16

F

67

O

29.07

b

80

No

Unilateral

Single

PTMC

T1aN0

No

No

BRAF p.V600E(Ct = 20.47),

TERT C228T

No

a total thyroidectomy, b near-total thyroidectomy, PTC Papillary thyroid cancer, PTMC Papillary thyroid microcarcinoma, HT Hashimoto thyroiditis, LNM Lymph node metastasis

Compared the thyroid function before and after surgery (2.45 ± 1.2 months) of these 16 patients, the TSH [3.03 ± 1.65 (uIU/mL) vs 19.77 ± 39.7 (uIU/mL), F = 17.328, P < 0.01], T4 [109.86 ± 12.45(nmol/L) vs 108.98 ± 53.94 (nmol/L), F = 9.410, P = 0.005], FT4 [16.77 ± 2.05 (pmol/L) vs 20.01 ± 9.26 (pmol/L), F = 11.389, P = 0.003], FT3 [4.63 ± 0.51 (pmol/L) vs 4.28 ± 1.68 (pmol/L), F = 8.108, P = 0.009], Tg value [68.37 ± 137.06 (ng/mL) vs 1.25 ± 2.50 (ng/mL), F = 7.921, P = 0.01] were significantly different. The PTH [61.88 ± 31.5 (pg/mL) vs 48.8 ± 38.7 (pg/mL), F = 0.099, P = 0.76], T3[1.97 ± 0.23 (nmol/L) vs 1.51 ± 0.61 (nmol/L), F = 3.432, P = 0.076], TPO [54.01 ± 78.59 (U/mL) vs 54.08 ± 61.16 (U/mL), F = 0.129, P = 0.722], Atg [719.96 ± 1175.4(U/mL) vs 521.70 ± 778.51 (U/mL), F = 0.692, P = 0.414] remained relatively stable. So far, no recurrence, metastasis and mortality were observed.

Relationship of gene mutations with clinicopathological outcomes of PTC

The risk factors for different gene mutations were explored. As Table 3 indicated, age (F = 16.704, P < 0.001), pathology (χ2 = 6.207, P = 0.045), number of lesions (χ2 = 7.169, P = 0.028), location of lesion (χ2 = 8.988, P = 0.011), lymph node metastasis (χ2 = 9.983, P = 0.007), and radiotherapy achievement (χ2 = 7.463, P = 0.024) were significantly different between 3 groups.
Table 3

Relationship between gene mutations and clinicopathologic features of PTC

 

No gene mutation

(N = 48)

BRAF V600E mutation alone(N = 419)

Double mutations

(N = 16)

χ 2 /F

P

Sex

 Male

13 (27.1%)

112 (26.7%)

2 (12.5%)

1.627

0.443

 Female

35 (72.9%)

307 (73.3%)

14 (87.5%)

Average age

37.9 ± 12.6

43.2 ± 10.7

56.0 ± 11.0

16.704

< 0.001

Average BMI

22.8 ± 3.57

24.9 ± 5.08

25.38 ± 2.06

3.521

0.316

Pathology

 PTC

23 (47.9%)

154 (36.8%)

10 (62.5%)

6.207

0.045

 PTMC

25 (52.1%)

265 (63.2%)

6(37.5%)

Lesion number

 Single lesion

38 (79.2%)

293(69.9%)

7 (43.8%)

7.169

0.028

 Multiple lesions

10 (20.8%)

126(30.1%)

9 (56.2%)

Lesion location

 Unilateral

38 (79.2%)

353 (84.2%)

9 (56.2%)

8.988

0.011

 Bilateral

10 (20.8%)

66 (15.8%)

7(43.8%)

Surgery

 Total thyroidectomy

39 (81.3%)

332 (79.2%)

13 (81.3%)

0.138

0.933

 Near-total thyroidectomy

9 (18.7%)

87(20.8%)

3 (18.7%)

LNM

 Yes

31 (64.6%)

180 (43.0%)

10 (62.5%)

9.983

0.007

 No

17 (35.4%)

239 (57.0%)

6(37.5%)

131I radiotherapy

 Yes

24 (50.0%)

163(38.9%)

11 (68.8%)

7.463

0.024

 No

24 (50.0%)

256 (61.1%)

5 (31.2%)

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], and 131I radiotherapy [OR = 7.628, 95% CI = 2.721–21.378; P < 0.001] were significantly different between patients with or without BRAF V600E mutation (Table 4). For double mutant group, age [OR = 1.135, 95% CI = 1.069–1.205; P < 0.001], number of lesion (multiple/single) [OR = 4.128, 95% CI = 1.026–16.614; P = 0.046], and pathology (PTC/PTMC) [OR = 3.954, 95% CI = 1.235–12.654; P = 0.021] were independent factors (Table 5).
Table 4

Logistic regression analyses between BRAF V600E mutation group and BRAF V600E wild group

Index

β

SE

Wals

Sig.

HR

95%CI

upper

lower

Lymph node metastasis

−2.363

0.526

20.154

< 0.001

0.094

0.034

0.264

131I radiotherapy

2.032

0.526

14.930

< 0.001

7.628

2.721

21.378

Pathology

(PTC/PTMC)

−0.418

0.334

1.564

0.211

0.659

0.342

1.267

Table 5

Logistic regression analyses between BRAF V600E mutation alone and double mutant group

Index

β

SE

Wals

Sig.

HR

95%CI

upper

lower

Age

0.126

0.031

17.008

< 0.001

1.135

1.069

1.205

Location of leision (bilateral/unilateral)

0.616

0.710

0.754

0.385

1.852

0.461

7.443

Number of leision (multiple/single)

1.418

0.710

3.983

0.046

4.128

1.026

16.614

Lymph node metastasis

−0.711

1.427

0.248

0.619

0.491

0.030

8.058

131I radiotherapy

0.840

1.487

0.319

0.572

2.315

0.126

42.70

Pathology (PTC/PTMC)

1.375

0.594

5.365

0.021

3.954

1.235

12.654

Literature review of co-existence of BRAF V600E and TERT promoter mutations

Systematic review was conducted to explore the impact of double gene mutations on clinicopathological features. Fifteen studies with 5057 participants, from inception to October 2018 were included [9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. Statistically meaningful association was found between BRAF V600E /TERT promoter co-mutations and lymph node metastasis (OR = 2.24, 95%CI = 1.53–3.29, P < 0.01, I2 = 8%, Fig. 2a), multifocality (OR = 1.52, 95%CI = 1.07–2.16, P = 0.02, I2 = 57%, Fig. 2b), dead of disease (OR = 12.63, 95%CI = 6.85–23.27, P < 0.01, I2 = 22%, Fig. 2c), distant metastasis (OR = 10.17, 95%CI = 5.39–19.22, P < 0.01, I2 = 39%, Fig. 3a), tumor recurrence (OR = 8.20, 95%CI = 4.97–13.54, P < 0.01, I2 = 66%, Fig. 3b), and extrathyroidal extension (OR = 5.02, 95%CI = 3.32–7.59, P < 0.01, I2 = 0%, Fig. 3c). Vascular invasion (OR = 1.18, 95%CI = 0.61–2.28, P = 0.61, I2 = 47%, Fig. 3d) was found without relationship with mutation coexistence.
Fig. 2

Systematic analysis of the association of BRAF promoter mutation alone or BRAF/TERT coexistence with clinicopathological features in thyroid cancer. a Lymph node metastasis, b Multifocality, c Dead of disease

Fig. 3

a Meta-analysis results of the relationship between BRAF V600E/TERT promoter mutations and distant metastasis. b Meta-analysis results of the relationship between BRAF V600E/TERT promoter mutations and tumor recurrence. c Meta-analysis results of the relationship between BRAF V600E/TERT promoter mutations and extrathyroidal extension. d Meta-analysis results of the relationship between BRAF V600E/TERT promoter mutations and vascular invasion

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 et al. reported the BRAF V600E mutation rate in Korean PTC patients was 80.8% [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 recurrence, extrathyroidal extension, and dead of disease. The meta-analysis by Vuong HG et al. achieved the same results. The combination of BRAF V600E and TERT promoter mutations could classify PTCs into four distinct risk groups with decreasing aggressiveness as follows: coexisting BRAF V600E and TERT > BRAF V600E alone > no mutations [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 131I 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.

Notes

Acknowledgments

We thanks the clinical pathologist from pathology department in Xijing Hospital.

Authors’ contributions

TW and RL designed the protocol and supervised the progress. JX collected the clinical information of participants. MH and CY analyzed the patient data and write the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Science Foundation of China (No. 81572917).

Ethics approval and consent to participate

Ethical approval for the study was provided by the Ethical Committees of Xijing Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

  1. 1.
    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69:7-34.Google Scholar
  2. 2.
    Roman BR, Morris LG, Davies L. The thyroid cancer epidemic, 2017 perspective. Curr Opin Endocrinol Diabetes Obes. 2017;24:332-36.Google Scholar
  3. 3.
    Li DD, Zhang YF, Xu HX, et al. The role of BRAF in the pathogenesis of thyroid carcinoma. Front Biosci (Landmark Ed). 2015;20:1068-78.Google Scholar
  4. 4.
    Liu R, Xing M. TERT promoter mutations in thyroid cancer. Endocr Relat Cancer. 2016;23:R143-55.Google Scholar
  5. 5.
    Xing M. Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer. 2013;13:184-99.Google Scholar
  6. 6.
    Jang EK, Song DE, Sim SY, et al. NRAS codon 61 mutation is associated with distant metastasis in patients with follicular thyroid carcinoma. Thyroid. 2014;24:1275-81.Google Scholar
  7. 7.
    Mond M, Alexiadis M, Fuller PJ, et al. Mutation profile of differentiated thyroid tumours in an Australian urban population. Intern Med J. 2014;44:727-34.Google Scholar
  8. 8.
    Qasem E, Murugan AK, Al-Hindi H, et al. TERT promoter mutations in thyroid cancer: a report from a Middle Eastern population. Endocr Relat Cancer. 2015;22:901-8.Google Scholar
  9. 9.
    Argyropoulou M, Veskoukis AS, Karanatsiou PM, et al. Low Prevalence of TERT Promoter, BRAF and RAS Mutations in Papillary Thyroid Cancer in the Greek Population. 2018.Google Scholar
  10. 10.
    Liang J, Cai W, Feng D, et al. Genetic landscape of papillary thyroid carcinoma in the Chinese population. 2018;244:215-26.Google Scholar
  11. 11.
    Jin A, Xu J, Wang Y. The role of TERT promoter mutations in postoperative and preoperative diagnosis and prognosis in thyroid cancer. Medicine (Baltimore). 2018;97:e11548.Google Scholar
  12. 12.
    Sun J, Zhang J, Lu J, et al. BRAF V600E and TERT Promoter Mutations in Papillary Thyroid Carcinoma in Chinese Patients. PLoS One. 2016;11:e0153319.Google Scholar
  13. 13.
    Vuong HG, Altibi AMA Prognostic implication of BRAF and TERT promoter mutation combination in papillary thyroid carcinoma-A meta-analysis. 2017;87:411-17.Google Scholar
  14. 14.
    Gandolfi G, Ragazzi M, Frasoldati A, et al. TERT promoter mutations are associated with distant metastases in papillary thyroid carcinoma. Eur J Endocrinol. 2015;172:403-13.Google Scholar
  15. 15.
    Hahn SY, Kim TH, Ki CS, et al. Ultrasound and clinicopathological features of papillary thyroid carcinomas with BRAF and TERT promoter mutations. Oncotarget. 2017;8:108946-57.Google Scholar
  16. 16.
    Jin L, Chen E, Dong S, et al. BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients. Oncotarget. 2016;7:18346-55.Google Scholar
  17. 17.
    Liu R, Bishop J, Zhu G, et al. Mortality Risk Stratification by Combining BRAF V600E and TERT Promoter Mutations in Papillary Thyroid Cancer: Genetic Duet of BRAF and TERT Promoter Mutations in Thyroid Cancer Mortality. JAMA Oncol. 2016;2:202-8.Google Scholar
  18. 18.
    Liu X, Qu S, Liu R, et al. TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer. J Clin Endocrinol Metab. 2014;99:E1130-6.Google Scholar
  19. 19.
    Marques IJ, Moura MM, Cabrera R, et al. Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas. 2017;87:394-99.Google Scholar
  20. 20.
    Melo M, Gaspar da Rocha A, Batista R, et al. TERT, BRAF, and NRAS in Primary Thyroid Cancer and Metastatic Disease. J Clin Endocrinol Metab. 2017;102:1898-907.Google Scholar
  21. 21.
    Nasirden A, Saito T, Fukumura Y, et al. In Japanese patients with papillary thyroid carcinoma, TERT promoter mutation is associated with poor prognosis, in contrast to BRAF (V600E) mutation. Virchows Arch. 2016;469:687-96.Google Scholar
  22. 22.
    Ren H, Shen Y, Hu D, et al. Co-existence of BRAF(V600E) and TERT promoter mutations in papillary thyroid carcinoma is associated with tumor aggressiveness, but not with lymph node metastasis. Cancer Manag Res. 2018;10:1005-13.Google Scholar
  23. 23.
    Rusinek D, Pfeifer A, Krajewska J, et al. Coexistence of TERT Promoter Mutations and the BRAF V600E Alteration and Its Impact on Histopathological Features of Papillary Thyroid Carcinoma in a Selected Series of Polish Patients. Int J Mol Sci. 2018;19.Google Scholar
  24. 24.
    Shen X, Liu R, Xing M. A six-genotype genetic prognostic model for papillary thyroid cancer. Endocr Relat Cancer. 2017;24:41-52.Google Scholar
  25. 25.
    Song YS, Lim JA, Choi H, et al. Prognostic effects of TERT promoter mutations are enhanced by coexistence with BRAF or RAS mutations and strengthen the risk prediction by the ATA or TNM staging system in differentiated thyroid cancer patients. Cancer. 2016;122:1370-9.Google Scholar
  26. 26.
    Xing M, Liu R, Liu X, et al. BRAF V600E and TERT promoter mutations cooperatively identify the most aggressive papillary thyroid cancer with highest recurrence. J Clin Oncol. 2014;32:2718-26.Google Scholar
  27. 27.
    Fagin JA, Wells SA. Biologic and Clinical Perspectives on Thyroid Cancer. N Engl J Med. 2016;375:1054-67.Google Scholar
  28. 28.
    Poller DN, Glaysher S. Molecular pathology and thyroid FNA. Cytopathology. 2017;28:475-81.Google Scholar
  29. 29.
    Yip L, Nikiforova MN, Yoo JY, et al. Tumor genotype determines phenotype and disease-related outcomes in thyroid cancer: a study of 1510 patients. Ann Surg. 2015;262:519-25; discussion 24-5.Google Scholar
  30. 30.
    Fraser S, Go C, Aniss A, et al. BRAF(V600E) Mutation is Associated with Decreased Disease-Free Survival in Papillary Thyroid Cancer. World J Surg. 2016;40:1618-24.Google Scholar
  31. 31.
    Ilera V, Dourisboure R, Colobraro A, et al. BRAF V600E mutation in thyroid nodules in Argentina. Medicina (B Aires). 2016;76:223-9.Google Scholar
  32. 32.
    Lee SE, Hwang TS, Choi YL, et al. Molecular Profiling of Papillary Thyroid Carcinoma in Korea with a High Prevalence of BRAF(V600E) Mutation. Thyroid. 2017;27:802-10.Google Scholar
  33. 33.
    Colombo C, Muzza M, Proverbio MC, et al. Impact of Mutation Density and Heterogeneity on Papillary Thyroid Cancer Clinical Features and Remission Probability. Thyroid. 2019;2:237-51.Google Scholar
  34. 34.
    Tobias B, Halaszlaki C, Balla B, et al. Genetic Alterations in Hungarian Patients with Papillary Thyroid Cancer. Pathol Oncol Res. 2016;22:27-33.Google Scholar

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Department of Thyroid, Breast, and Vascular Surgery, Xijing HospitalThe Fourth Military Medical UniversityXi’anChina

Personalised recommendations