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Prevalence of BRAF V600E mutation in Chinese melanoma patients: Large scale analysis of BRAF and NRAS mutations in a 432-case cohort

European Journal of Cancer, 1, 48, pages 94 - 100

Abstract

Background

Mutations of NRAS and BRAF have been described in Caucasian melanomas. However, the status and the clinical significance of BRAF and NRAS mutations in the Asian population have not been investigated on a large scale.

Methods

Melanoma samples (n = 432) were analysed for mutations in exons 11 and 15 of the BRAF gene, and exons 1 and 2 of the NRAS gene in genomic DNA by polymerase chain reaction (PCR) amplification and Sanger sequencing. Mutations of BRAF and NRAS genes were correlated to clinicopathologic features and prognosis of the patients.

Results

The incidence of somatic mutations within the BRAF and NRAS genes was 25.5% (110/432) and 7.2% (31/432), respectively. Among the 110 patients with BRAF mutations, 98 patients (89.1%) had V600E mutations. Melanomas without chronic sun-induced damage (Non-CSD) were more likely (P < 0.01) to show BRAF mutations while NRAS mutation frequency was unbiased between melanoma subtypes. Patients with genetic mutations in BRAF (P < 0.01) or NRAS (P = 0.04) gene are more likely to have ulceration as compared to patients without BRAF or NRAS mutations, respectively. Both BRAF (P = 0.003) and NRAS mutations (P = 0.031) are inversely correlated to overall survival.

Conclusions

BRAF mutation is frequent while mutations in NRAS gene are rare. The most prevalent BRAF mutation type is V600E. Patients with mutations in BRAF or NRAS gene are frequently present with ulceration, and mutation in BRAF or NRAS gene is indicator for poor prognosis. Our study may warrant a clinical trial of kinase inhibitors targeting BRAF V600E in Chinese and Asian melanoma patients.

Keywords: Melanoma, BRAF, NRAS, Mutation, Overall survival.

1. Introduction

RAS mutations typically occur at codons 12, 13 or 61 and maintain RAS in a constitutively active state.1 and 2 Mutations K-RAS of are the most frequent in many human malignancies,1 and 2 mutations of NRAS are rare in melanomas.3, 4, 5, 6, 7, 8, 9, and 10 Meanwhile, BRAF somatic missense mutations are detected in 66% of malignant melanomas, with a single substitution (V600E) accounting for 80% of all BRAF mutations.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22

The genetic mutations in BRAF and NRAS genes have been correlated to clinicopathologic features and prognosis of melanomas.5, 6, 8, 11, 12, 13, 14, 15, 16, 19, 21, and 22 However, these observations are conducted in Caucasian populations, and the mutation status of BRAF and NRAS genes and the clinical significance of these mutations have not been established in other populations (e.g. Asians). It has been demonstrated that acral melanomas account for only 1–7% of all cutaneous melanomas in Caucasians, whereas the percentage is significantly higher in Asian.23, 24, 25, 26, and 27 Curtin et al. reported that mutations of KIT gene were detected in about 29% of melanomas while our study in a Chinese cohort suggested that the incidence was about 11%, indicating that oncogene mutation status may also be different between Caucasians and Asians.28 and 29

Generally, studies regarding examination of NRAS and BRAF mutations have garnered intense attention. Given that kinase inhibitors for BRAF, especially the BRAF V600E-specific inhibitors PLX4032 and GSK2118436, have been demonstrated to be effective in clinical trials in Caucasian population,30, 31, and 32 identification of mutations in BRAF and NRAS mutations may be of great translational relevance for future clinical trails targeting BRAF or NRAS. To further explore the potential targets for melanoma treatments, we evaluated genetic mutations of BRAF and NRAS in a non-Caucasian, Chinese patient population, and correlated the BRAF/NRAS mutations to clinicopathologic features and prognosis of Chinese melanoma patients.

2. Patients and methods

2.1. Patients and tumour tissue samples

This study involved samples from primary lesions of 432 melanoma patients, hospitalised during January 2007 and January 2010 at the Peking University Cancer Hospital & Institute ( Supplementary Table S1 ). These samples were confirmed for the diagnosis of melanoma as described previously. 29 Clinical data, including age, sex, TNM (tumour-node-metastases) stage, thickness (Breslow), ulceration and survival (follow-up persisted until December 2011 or until the missing of follow-up or death of patients) were collected. This study was approved by the medical ethics committee of the Beijing Cancer Hospital & Institute and was conducted according to the Declaration of Helsinki Principles.

2.2. DNA preparation and mutation screening

Genomic DNA was extracted from formalin-fixed, paraffin-embedded sections using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). To detect hotspot mutations, we amplified exons 11 and 15 of the BRAF gene, and exons 1 and 2 of NRAS gene, by PCR in at least two separate preparations of genomic DNA as described previously.9 and 21 The primer sequences are listed in Supplementary Table S2 .

2.3. Statistical analysis

All the statistical analyses were performed using SPSS 13.0 software. Categorical data are described using frequencies and percentages. Continuous data such as age are described using means ± standard deviations or median (range) for normally distributed data. The χ2 test, Fisher’s exact test or Kruskal–Wallis test was used to differentiate the rates of different groups, and differences in measurement data of two groups were evaluated by unpaired t test or mann–whitney test. Survival curves were established using the Kaplan–Meier method and compared by the log-rank test. All statistical analyses were two sided and significance was assigned at P < 0.05.

3. Results

3.1. Genetic mutations of BRAF and NRAS genes in melanoma

Typical sequencing results for mutations are shown in Supplementary Fig. S1–S4 . The overall mutation frequency was 25.5% (110/432) for BRAF gene and 7.2% (31/432) for NRAS gene, respectively ( Table 1 ). The highest mutation frequency was detected in Non-CSD melanoma (57.1%), which was in consistent with the recent reports showing that BRAF-mutated melanomas occur on skin without marked solar elastosis.21 and 33 However, the high frequency of BRAF mutation in Non-CSD melanoma may not necessarily indicate that BRAF mutation was unrelated to chronic sun exposure, given that BRAF mutation could also be detected in CSD melanomas (18.2%) and that BRAF mutations in melanocytic lesions could arise from DNA damage induced by ultraviolet radiation. 34 Of note, the frequencies of BRAF mutations detected in acral (15.5%) and mucosal (12.5%) melanomas were also comparable with the corresponding rates previously reported in Caucasian patients. 17 On the contrary, NRAS mutation was generally rare, with the lowest mutation incidence detected in Non-CSD melanomas (2.0%).

Table 1 Genetic mutations of BRAF and NRAS in melanoma subtype.

Subtype BRAF mutation NRAS mutation
No. Positive % No. Positive %
Acral 148 23 15.5 148 13 8.8
Mucosal 120 15 12.5 120 11 9.2
CSD 22 4 18.2 22 1 4.5
Non-CSD 98 56 57.1 98 2 2.0
UP 44 12 27.3 44 4 9.1
Total 432 110 25.5 432 31 7.2

Abbreviations: CSD, melanomas on skin with chronic sun-induced damage; Non-CSD, melanomas on skin without chronic sun-induced damage; UP, melanoma of unknown primary.

Considering that the primary sites of mucosal melanoma are usually variable, we analysed the BRAF mutation frequencies in 120 cases of mucosal melanomas. We found that the highest frequency (46.7%) of BRAF mutation was detected in mucosal melanomas located in sinonasal mucosa among the 15 mucosal melanomas bearing BRAF mutation ( Supplementary Table S3 ).

3.2. Mutation types of BRAF and NRAS in melanoma

In our study, 11 different types of BRAF mutations and 12 different NRAS mutations were detected ( Table 2 ). We found that, among the detected BRAF mutations, three novel mutations in exon 11 (G442S, W450Stop and I457T) were detected in addition to eight previously reported BRAF mutation types.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 Acral and mucosal melanomas demonstrated more variable mutations while CSD and Non-CSD melanomas demonstrated either V600E or V600K mutation ( Table 2 ). One novel mutation in exon 1 (Y4C) and one novel mutation in exon 2 (A59D) of NRAS were detected in addition to 10 previously reported NRAS mutations.3, 8, 9, 10, 15, 18, and 21 Acral and mucosal melanomas demonstrated more variable NRAS mutations while Non-CSD melanomas mainly demonstrated mutations in codon 61 ( Table 3 ).

Table 2 Mutation types of BRAF gene in melanoma.

DNA mutation Protein mutation No. % a Exon affected Subtype
G1324A G442S 1 0.9 11 Acral
G1349A W450Stop 1 0.9 11 Acral
T1370C I457T 1 0.9 11 Mucosal
G1396A G466R 1 0.9 11 Acral
T1782G D594E 1 0.9 15 Mucosal
A1781G D594G 1 0.9 15 Acral
T1799A V600E 98 89.1 15 Acral; mucosal; CSD; Non-CSD; UP
G1798A;T1799A V600K 3 2.7 15 Mucosal; Non-CSD
A1801G K601E 1 0.9 15 Mucosal
G1811A W604Stop 1 0.9 15 Acral
T1846C S616F 1 0.9 15 Mucosal

a The percentage indicates for the cases showing the indicated mutation type to the total of 110 cases bearing BRAF mutations.

Abbreviations: CSD, melanomas on skin with chronic sun-induced damage; Non-CSD, melanomas on skin without chronic sun-induced damage; UP, melanoma of unknown primary.

Table 3 Mutation types of NRAS gene in melanoma.

DNA mutation Protein mutation No. % a Exon affected Subtype
A11G Y4C 1 3.2 1 Mucosal
G35C G12A 1 3.2 1 Mucosal
G34T G12C 1 3.2 1 Acral
G35A G12D 3 9.7 1 Mucosal; CSD
G34A G12S 1 3.2 1 Mucosal
G37C G13R 3 9.7 1 Acral; mucosal
A50G S17N 1 3.2 1 UP
A176G A59D 2 6.5 2 Acral; mucosal
A183T Q61H 1 3.2 2 Mucosal
C181A Q61K 2 6.5 2 Acral; UP
A182T Q61L 4 12.9 2 Acral; mucosal; Non-CSD
A182G Q61R 11 35.5 2 Acral; Non-CSD; UP

a The percentage indicates for the cases showing the indicated mutation type to the total of 31 cases bearing NRAS mutations.

Abbreviations: CSD, melanomas on skin with chronic sun-induced damage; Non-CSD, melanomas on skin without chronic sun-induced damage; UP, melanoma of unknown primary.

The BRAF V600E mutation was the single most common genetic alteration in this cohort, detected in 98 samples (89.1%) among the 110 samples positive for BRAF somatic mutations ( Table 2 ). Another common mutation of BRAF is V600K, which was detected in three samples (2.7%). Notably, both V600E and V600K are sensitive to specific BRAF inhibitor GSK2118436.30, 31, and 32 For the patients containing NRAS mutations, 58.1% of them demonstrated mutations in codon 61, with Q61R as the most frequent mutation in NRAS (35.5%, Table 3 ). Moreover, we detected two cases of mucosal melanomas that simultaneously harbour mutations in both BRAF and NRAS genes (D594E in BRAF plus G13R in NRAS; V600E in BRAF plus Y4C in NRAS), which was consistent with the previous study reported by Lin et al., 35 but was in contrary to the notion that BRAF and NRAS mutations were mutually exclusive.8, 21, and 36

3.3. Correlation of BRAF and NRAS mutations to the clinicopathologic features of melanoma

In our cohort, the mean age (N = 432) was not significantly different between patients with BRAF mutation and those without BRAF mutation ( Table 4 ). The mean age (48.9 ± 14.4) of patients bearing BRAF mutations was similar to that of a recent report on an American population. 21 On the contrary, another recent report suggests that BRAF-mutated melanomas occur in a younger age group on skin without marked solar elastosis 33 , indicating that more investigations may needed to determine the relationship between BRAF mutation and the age of melanoma patients. Additionally, the mean age (N = 432) in the group showing NRAS mutations was significantly older than the group showing wild type NRAS (P = 0.02).

Table 4 Correlation of BRAF and NRAS genotype to clinicopathologic features of melanoma.

Clinicopathologic factor BRAF genotype NRAS genotype
Mutation Wild type P value a Mutation Wild type P value a
Age (year) 48.9 ± 14.4 51.9 ± 14.3 0.39 56.5 ± 14.3 50.8 ± 14.5 0.02
Gender (F, %) 59 (52.6) 165 (51.2) 0.75 16 (51.6) 207 (51.6) 0.86
Thickness (mm) 5.0 (0.5–10) 4.5 (1.0–10) 0.18 5.0 (1.5–10) 5.0 (0.5–10) 0.69
Ulceration N (%) 58 (61.1) 117 (40.9) <0.01 16 (64.0) 145 (40.7) 0.04
Stages N (%)
 I 6 (5.5) 16 (5.3) 0.90 0 (0) 22 (5.7) 0.37
 II 35 (32.1) 129 (42.4) 0.13 10 (33.3) 154 (40.2) 0.19
 III 36 (33.0) 77 (25.3) 0.18 14 (46.7) 99 (25.8) 0.02
 IV 32 (29.4) 82 (27.0) 0.73 6 (20.0) 108 (28.2) 0.46
Subtypes N (%)
 Acral 23 (20.9) 125 (38.8) <0.01 13 (41.9) 134 (33.4) 0.63
 Mucosal 15 (13.6) 105 (30.6) <0.01 11 (35.5) 105 (26.2) 0.38
 CSD 4 (3.6) 18 (5.6) 0.60 1 (3.2) 21 (5.2) 0.95
 Non-CSD 56 (50.9) 42 (13.0) <0.01 2 (6.5) 92 (22.9) 0.06
 UP 12 (10.9) 32 (9.9) 0.92 4 (12.9) 4 (1.0) <0.01

a For evaluation of age and thickness, the unpaired t or t’ tests were used. For evaluation of gender, ulceration, stages and subtypes, the chi-square tests or Fisher’s exact tests were used.

Abbreviations: F, female; CSD, melanomas on skin with chronic sun-induced damage; Non-CSD, melanomas on skin without chronic sun-induced damage; UP, melanoma of unknown primary.

The average thickness of all 381 available samples was more than 5 mm, which was much thicker than the previous reports but was the actual status for Chinese patients upon hospitalisation.27 and 29 The median thickness of samples without BRAF or NRAS mutations was not significantly different to that of samples with BRAF or NRAS mutations, respectively ( Table 4 ).

In our cohort, the overall ulceration rate was 59.7% (228/383). The ulceration rate in patients with BRAF mutations (61.1%) was significantly higher (P < 0.01) than that in patients without BRAF mutations (40.9%). Moreover, the ulceration rate in patients with NRAS mutations (64.0%) was significantly higher (P = 0.04) than that in patients without NRAS mutations (40.7%). These data suggest that patients harbouring either BRAF or NRAS mutations were prone to have ulceration in melanoma lesions.

Among the 110 patients with BRAF mutations and with the clinical stage data available, the percentages of patients at stage I, II, III and IV were 5.5% (6 cases), 32.1% (35 cases), 33.0% (36 cases) and 29.4% (32 cases), respectively, which were not significantly different from those without a BRAF mutation ( Table 4 ). For the patients with NRAS mutations, the percentages of the four stages were 0% (0 case), 33.3% (10 cases), 46.7% (14 cases) and 20.0% (6 cases), respectively ( Table 4 ). The frequency of NRAS mutations in patients at Stage III (46.7%) was significantly higher (P = 0.02) than that of wild type NRAS in patients at Stage III (25.8%), indicating that patients with NRAS mutations were more likely to be at disease stage III.

We went further to examine whether BRAF or NRAS mutations may be associated with particular melanoma subtypes. The percentages of acral (20.9%) and mucosal (13.6%) melanomas in patients harbouring BRAF mutations were significantly lower (P < 0.01) than those in patients showing wild type BRAF; the percentage of Non-CSD (50.9%) melanomas in patients harbouring BRAF mutations was significantly higher (P < 0.01) than that in patients showing wild type BRAF ( Table 4 ). However, NRAS mutation frequency was unbiased between melanoma subtypes.

3.4. Prognostic significance of BRAF and NRAS mutations for overall survival of melanoma

The survival data were collected for patients (N = 395) who were diagnosed as primary melanoma or melanoma of unknown primary ( Supplementary Table S1 ) from the first time of diagnosis as melanoma to December 2011. The median follow-up period was 24.0 (range: 3.0–229.0) months. We found that the median survival time for patients with BRAF mutations (33.0 months) was significantly shorter than that for patients with wild-type tumours (53.0 months; P = 0.005, Fig. 1 A). In addition, patients with NRAS mutations had a worse survival (33.0 months) than patients with wild-type tumours (48.0 months; P = 0.031, Fig. 1 B). These data suggest that BRAF and NRAS mutations may be of prognostic significance for melanoma patients.

gr1

Fig. 1 Overall survival of melanoma patients in relation to BRAF and NRAS mutations. Solid line: wild type (WT); dotted line: mutation (Mut).

We have reported previously that KIT mutations may be independent prognostic factors for melanoma. 29 Among the 432 cases of melanoma, 43 cases (10.0%) demonstrated KIT mutations, among which four cases simultaneously showed BRAF mutations, indicating that KIT mutations and BRAF mutations may be not mutually exclusive. When analysed with COX regression of survival data for this 432-case population, both KIT mutation (P = 0.003) and BRAF mutation (P = 0.01) were independent prognostic factors with the hazard ratios of 1.989 (95% confidence interval (CI): 1.236, 3.131) and 1.536 (95% CI: 1.110, 2.124), respectively.

4. Discussion

BRAF and NRAS mutations have been documented in all subtypes of melanoma.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 However, most of the studies are conducted in Caucasian population, and indicate for the requirement of deciphering oncogenic differences between Caucasian and Asian populations for proper treatment of melanomas. Given that our study has examined the largest size of samples in Chinese/Asian patients, BRAF may be the major mutated gene in Chinese melanomas. Our study is thus of significance for the understanding of tumourigenesis of melanomas worldwide.

Codon 61 is the most common position of NRAS alterations in melanoma and mutations in this residue lock the RAS protein in the GTP-bound state with a subsequent continuous activation of its downstream effectors.1, 3, 4, 5, 6, 7, 8, 9, and 10 Approximately 90% of reported BRAF mutations occur at residue 600, which is located in the activation domain of this kinase.1 and 9 Consistent with these studies, our data confirm that BRAF mutation in codon 600 (V600E and V600K, comprised 91.8% of all BRAF mutations) and NRAS mutations in codon 61 (Q61H, Q61K, Q61L and Q61R, comprised 58.1% of all NRAS mutations) are the major mutations in melanoma. A most recent report, which examined the BRAF mutational status in a Chinese Han population (195 cases of malignant melanoma and 379 cases of melanocytic nevi), suggested that 15.0% of malignant melanoma harboured the BRAF V600E mutation while BRAF mutation may be unrelated to melanocyte transformation. 37 The BRAF V600E mutation frequency in our cohort (25.5%) was higher than that (15.0%) reported by Qi et al., 37 which may be due to different sample size and difference in regional selection (all regions of China in our cohort versus northeast region of China in Qi’s cohort). Since we have not examined the BRAF mutational status in melanocytic nevi, we could not evaluate the significance of BRAF mutation in initiation of melanoma. It should be noted that mutations detected in our study may not necessarily lead to activation of BRAF and NRAS. For example, two kinds of point mutations in BRAF (W450Stop and W604Stop) can result in termination of BRAF translation, which may lead to loss of BRAF kinase activities. S17N mutation in NRAS may serve as a dominant negative form of NRAS and may inhibit the NRAS activity. Several novel mutations in BRAF and NRAS have also been detected by us. Further investigations of these mutations biochemically and clinically may help to establish therapies targeting BRAF or NRAS.

For therapeutic purposes, BRAF mutations may be of clinical importance in identifying patients who may benefit from small molecule inhibitors (e.g. BRAF V600E inhibitors). Although Sorafenib is a RAF kinase inhibitor, phase II clinical trials suggest that Sorafenib may be ineffective in melanoma treatment, with a clinical response of about 19% stable disease. 38 In contrast, selective RAF inhibitors, including PLX4032 and GSK2118436, demonstrated much better clinical response, with the overall response rate of about 80% and 63%, respectively.30, 31, and 32 The prevalence of BRAF V600E mutation in Chinese melanoma may indicate that clinical trials of PLX4032 or GSK2118436 may be reasonable and ideal for Chinese and Asian melanoma patients.

Previously, correlations of NRAS mutations to Clark level of invasion, Breslow thickness, ulceration rate and mean age at diagnosis have been observed.5, 8, 18, and 21 However, other reports could not demonstrate any association of NRAS mutation with these clinicopathological factors.6 and 39 In our study, NRAS mutation positive patients show higher mean age at diagnosis and higher ulceration rates as compared to patients without NRAS mutations. However, we did not find the association of NRAS mutations with gender and Breslow thickness. For BRAF mutations, most of the studies suggest that BRAF mutation may be not correlated to thickness, age, gender, ulceration and prognosis.8, 12, 15, and 40 However, a most recent study suggests that BRAF mutation may be correlated to ulceration and Brelow thickness, but not overall survival. 21 It has also been reported that the presence of BRAF mutations in melanoma metastases was associated with shortened survival from the time point of removal of the lesions or from the diagnosis of stage IV disease.11 and 13 We found that BRAF mutation may be correlated to ulceration rates and overall survival, with BRAF mutation positive patients showing higher ulceration rates and worse prognosis. However, we could not find the association of BRAF mutation with age, gender and thickness. The consistent findings for BRAF and NRAS mutations are the association of BRAF and NRAS mutations to ulceration and overall survival in Chinese melanoma. These differences and consistencies between our studies and others may represent the unique features of Chinese melanoma. Specifically, the samples in our cohort are mainly derived from the primary lesion sites but not metastatic sites, indicating that our data are of significance for primary melanoma.

In conclusion, our study has confirmed that the most prevalent mutation type of BRAF and NRAS is V600E and G61R, respectively. We found that BRAF and NRAS mutations are unrelated to the gender and thickness of primary melanomas. Patients with mutations in BRAF or NRAS genes are more likely to have ulceration. Most importantly, genetic mutations in BRAF/NRAS may be independent adverse prognostic factors in melanoma. Our study also highlights that there are numerous types of BRAF/NRAS mutations present in this population, with variable mutations of BRAF/NRAS in acral and mucosal melanomas but with limited mutation types in Non-CSD melanomas.

Contributions of authors

Jun Guo, Lu Si, Yan Kong, Xiaowei Xu and Keith T. Flaherty were involved in the conception and design of the study. Jun Guo, Lu Si and Yan Kong wrote the manuscript. Zhihong Chi, Chuanliang Cui, Xinan Sheng, Siming Li and Lili Mao provided study material and quality control. Jun Guo, Yan Kong and Lu Si collected, analysed and interpreted the data. All authors validated the report.

Conflict of interest statement

None declared.

Acknowledgements

This work is supported by the Program for New Century Excellent Talents in University (985-2-085-113), National Natural Science Foundation of China (30973483), Novartis Oncology in China, Program for Beijing Medical Disciplines Leaders (2011-2-25) and Program for Beijing Science Topicis. We thank the staff in the Department of Pathology of our hospital for help in collection and pathological analysis of tissue samples.

Appendix A. Supplementary data

 

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Supplementary data 1 Supplementary Tables and Figures

References

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Footnotes

a Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Renal Cancer and Melanoma, Peking University Cancer Hospital & Institute, Beijing, China

b Department of Pathology and Laboratory Medicine, Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA, USA

c Massachusetts General Hospital Cancer Center, Boston, MA, USA

lowast Corresponding author: Address: Department of Renal Cancer and Melanoma, Peking University Cancer Hospital & Institute, 52 Fucheng Road, Haidian District, Beijing 100142, China. Tel.: +86 1088196317; fax: +86 1088122437.

d These authors contributed equally to this work.