Melanoma is the most deadly form of skin cancer, and is a major health problem worldwide. Over the last decade, the incidence and mortality rates of melanoma have increased in individuals of European origin 1. Approximately 40–70% of melanomas contain activating mutations of the BRAF gene 2–5, which might be caused by prolonged periods of exposure to solar ultraviolet radiation 6–8. However, BRAF mutations were detected in melanomas in nonchronic sun-damaged skin, whereas NRAS mutations were present in melanomas in chronic sun-damaged skin 9. BRAF encodes a growth factor (B-Raf protein) that regulates the MAP kinase/ERK-signalling pathway, which is involved in cell division, differentiation and secretion. BRAF mutations encode mutated B-Raf proteins that have elevated kinase activity and can transform cell lines; because of this, BRAF was identified as a common oncogene in human melanoma and other human cancers 2. The most common mutation in BRAF causes the substitution of glutamic acid for valine at amino acid 600 (BRAF V600), resulting in the constitutive activation of BRAF and uncontrollable melanocyte cell growth, eventually leading to tumour formation 10.
A number of clinical trials of treatments for melanoma have been reported, including dabrafenib, a BRAF inhibitor, which improved progression-free survival in metastatic melanoma patients with the BRAF V600 mutation 11; ipilimumab, an anti-CTLA-4 antibody, which improved overall survival, but had a high incidence of adverse events 12; or a MAPK kinase inhibitor, trametinib, for patients with metastatic melanoma with BRAF V600E/V600K mutations, which improved progression-free and overall survival 13. Clinical trials investigating combination therapy showed that dabrafenib plus trametinib promoted longer progression-free survival with reduced incidence and severity of toxicity when compared with respective monotherapies 14, whereas ipilimumab plus dacarbazine, an alkylating agent, improved overall survival but showed similar adverse events to ipilimumab alone with elevated liver enzymes 15.
Three multicentre clinical trials (phase 1, 2 and 3) of a BRAF V600 kinase inhibitor, vemurafenib, in melanoma patients showed that treatment resulted in complete or partial tumour regression and improvements in both overall survival and progression-free survival in the majority of patients carrying the BRAF V600 mutation, thus indicating its potent antitumour activity 16–18. Vemurafenib was approved by the Food and Drug Administration in 2011 and was codeveloped with the Cobas 4800 BRAF V600 Mutation Test (an in-vitro assay based on TaqMan real-time PCR technology) to measure BRAF V600 mutations with a high sensitivity for selecting patients for treatment 19. Melanomas are categorized by the World Health organization into four subtypes as follows: acral lentiginous melanomas (ALMs), superficial spreading melanomas (SSMs), lentigo maligna melanomas (LMMs) and nodular melanomas (NMs) 20. A meta-analysis of 19 studies of different melanoma subtypes and the incidence of BRAF mutations concluded that BRAF mutations were associated with SSM [odds ratio (OR) 2.021, 95% confidence interval (CI) 1.440–2.835, P<0.001]. The ORs for BRAF mutation in LMM, ALM and NM were 0.422 (95% CI 0.291–0.614, P<0.001), 0.385 (95% CI 0.237–0.626, P<0.001) and 0.980 (95% CI 0.730–1.316, P=0.893), respectively 9. BRAF mutations were present in 49% of SSM, 41% of NM, 22% of LMM and 20% of ALM patients 9. By contrast, a study not included in the meta-analysis indicated that both SSM and NM were significantly associated with mutant BRAF (OR 9.8, 95% CI 2.0–47.2, P<0.005) by multivariate analysis of 137 Australian patients with metastatic melanoma 21.
Of note, in a previous study 22, BRAF V599E mutations in primary melanomas surgically removed from 35 Japanese patients were observed at lower frequencies than in a western study 2, and different BRAF V599E mutation frequencies were observed in different melanoma subtypes. BRAF V599E mutations were most common in SSM, but less frequent in LMM, NM and ALM 22. One explanation for this might be a difference in the histological subtypes of melanoma between Japanese and other races.
In Japan, the occurrence of melanoma is rare; the estimated annual incidence is ∼1500–2000 cases compared with the worldwide annual incidence of 132 000. One potential reason for the lower incidence might be skin colour as pigmented skin has increased protection from ultraviolet light. Currently, the frequency and distribution of BRAF V600 mutations among Japanese melanoma patients are unknown. However, a recent study indicated that malignant skin tumours are increasing in frequency in the Japanese population aged 30–39 years and particularly in those between 60 an 69 years of age, although the reason for this increase is unclear 23.
The present study investigated the frequency of BRAF V600 mutations using the Cobas 4800 BRAF V600 Mutation Test in Japanese patients with the four subtypes of melanoma (ALM, SSM, LMM and NM) as the primary endpoint. The associations between the BRAF V600 mutation and clinical or pathologic features were determined as the secondary endpoint.
This observational study used formalin-fixed, paraffin-embedded (FFPE) specimens from 80 Japanese melanoma patients who underwent resection or biopsy between May 2005 and December 2012. The BRAF mutation rate in the USA and European Union is ∼40–70% 2,3,5. Therefore, we assumed that 80 specimens would be required to provide at least one positive BRAF mutation in each subtype. A sample list containing patient demographic data and the incidence of melanoma subtypes in patient specimens (Table 1) from the Department of Dermatological Oncology, National Cancer Center Hospital, Tokyo, Japan, was presented to Roche Diagnostics (Tokyo, Japan) and each sample was assigned an enrolment number.
Six slides per patient containing 5-µm FFPE sections were prepared (one for haematoxylin and eosin staining and five for BRAF testing) at the Department of Pathology and Clinical Laboratories, National Cancer Center Hospital. Sections for BRAF testing were subjected to DNA extraction and mutation detection using the Cobas 4800 BRAF V600 Mutation Test (Roche Molecular Systems, Pleasanton, California, USA), which was performed at Roche Diagnostics.
Patients and tissue samples
Of all Japanese patients newly diagnosed with melanoma between 2005 and 2012 at the National Cancer Center Hospital, 134 patients had samples available in the hospital archives. Therefore, 134 samples from 134 Japanese patients diagnosed with melanoma were screened.
Inclusion criteria included FFPE tissue resected in operation or biopsies and pathologically diagnosed as melanoma, and agreement for use after written informed consent from patients. Of the 134 patients, four patients refused consent, six were unable to comply with the protocol, seven were excluded because the samples from these patients were on loan to other hospitals during the study period and 37 were excluded as their specimens were deemed inappropriate by an investigator because of decalcification of the fingers (n=35) or because of spontaneous regression of the primary lesion (n=2). Thus, 80 FFPE archived samples from 80 Japanese patients were eligible. One sample was considered invalid. Thus, 79 samples were used for the analyses in this study. The study was approved by the ethics committee of each participating institute and was carried out according to the institutional review board guidelines. The diagnosis of melanoma stage III/IV disease was made according to the American Joint Cancer Committee/Union Internationale Contre le Cancer 24,25 and melanoma subtypes were determined according to the definitions proposed by Clark et al.20.
Cobas 4800 BRAF V600 Mutation Test
The Cobas 4800 BRAF V600 Mutation Test was performed as described previously 19 (Fig. 1). Briefly, DNA was extracted using the Cobas DNA Sample Preparation Kit (Roche Molecular Systems, Branchburg, New Jersey, USA). Specimens containing less than 50% tumour area were macrodissected before DNA extraction. BRAF mutations were detected with the Cobas 4800 (z480) System (Roche Diagnostic Systems, Branchburg, New Jersey, USA) using Cobas 4800 BRAF V600 Mutation Test reagents (Roche Diagnostic Systems), and the results were analysed using Cobas 4800 BRAF Analysis Package Software (version 2.0, Roche Diagnostic Systems). In cases of invalid results, specimen testing was repeated. An invalid result from retesting was excluded from the analysis as an invalid sample.
The primary endpoint was the frequency of BRAF V600 mutations in Japanese patients with melanoma. The secondary endpoint was the relationships between BRAF V600 mutations and clinical or pathological features of melanoma.
Patient demographic data are shown in Table 1. Of 79 melanoma patients (53.2% men) grouped by age, 35.4% (28/79) were older than 69 years. The most common melanoma subtypes observed were ALM (40.5%, 32/79) and SSM (43.0%, 34/79), whereas NM (10.1%, 8/79) and LMM (6.3%, 5/79) were less common. All tumours were primary tumours and not metastatic. The Cobas 4800 BRAF V600 Mutation Test required at least 50% tumour content in the FFPE sections used; otherwise, macrodissection was necessary before DNA extraction. The tumour content of FFPE sections was mostly less than 10% (60.8%, 48/79), with 31.6% containing 10–50% tumour content and only 7.6% with greater than 50% tumour content. Eighty FFPE specimens were collected for analysis by the Cobas 4800 BRAF V600 Mutation Test. Seventy-nine specimens were positively evaluated and one invalid specimen was excluded (1.3%, 1/80) because neither wild-type nor mutated DNA was amplified, indicating that the sample was damaged before analysis.
Overall BRAF V600 mutation status
Overall, BRAF V600 mutations were detected in 41.8% (33/79) of the tested FFPE sections. BRAF V600 mutations were not detected in the remaining 58.2% (46/79) of samples (data not shown). The mutation rate in men was 40.5% and that in women was 43.2%, suggesting no sex difference in the mutation rates. The mutation rate was more than 60% in patients aged less than 60 years (Table 2).
BRAF V600 mutation status stratified by melanoma subtype
When mutations were stratified by melanoma subtype, BRAF V600 mutations were detected in 18.8% (6/32) of the ALM samples, 64.7% (22/34) of the SSM samples, 50.0% (4/8) of the NM samples and 20.0% (1/5) of the LMM samples (Table 3).
BRAF V600 mutation status stratified by melanoma stage
Of the 79 patients, 41 were classified as having melanoma stage III/IV disease (Table 4). Although the overall mutation rate was relatively low (18.8%) among ALM patients, when the mutation rate was stratified by subtype for those with stage III/IV disease, 36.4% (4/11) of ALM samples were BRAF V600 mutation positive compared with 65.2% (15/23) of SSM samples, 66.7% (4/6) of NM samples and all (1/1) LMM samples (Table 4). Thus, although ALM was still the melanoma type with the lowest frequency of BRAF V600 mutations, the relative frequency was twice as high and much closer to those in other melanoma types with stage III/IV disease. When stratified by early melanoma stage (I/II), the BRAF mutation frequency was only 9.5% in patients with early-stage ALM (Table 4). By contrast, there was little difference in the BRAF mutation frequency between late-stage (65.2%) and early-stage SSM (63.6%).
Association between the BRAF V600 mutation and melanoma location
Finally, the relationship between BRAF V600 mutations and the location of melanomas in Japanese patients was established. BRAF V600 mutations were commonly observed in the trunk (86.7%), lower limb and hip (64.3%), and the head and neck (61.9%), but were less frequent in the upper limbs (40%) and sole, heel and feet (18.8%) (Table 5).
In the present study, we investigated the frequency of BRAF V600 mutations in Japanese patients with melanoma. We found that BRAF V600 mutations occurred in only 41.8% of melanomas, which is slightly lower than the percentage (49.7%) of melanomas bearing BRAF V600 mutations observed in a study of European and US melanoma patients 26 and higher than in a previous study of Japanese melanoma cases 22. Interestingly, although the frequency of BRAF mutations in the current study was lower (26%, 9/35) than that in the USA or Europe, it was similar to the rate in Chinese melanoma patients (25.5%, 110/432) 27. Other studies of Asian melanoma patients have reported lower BRAF mutation rates in melanomas of Korean patients (11.9%, 24/202) 28 and Chinese Han (14.7%, 16/109) 29, indicating a lower incidence of BRAF mutations in melanomas of Asian individuals.
A number of studies have shown that ALMs have a lower BRAF mutation frequency than do SSMs (reviewed by Platz et al.10). For example, a study in European and US melanoma patients showed that 58.1% of SSM samples had BRAF V600 mutations, whereas only 16.7% of ALM samples did 26. In this study, we found a similar BRAF V600 mutation rate in each subtype (ALM 18.8%, SSM 64.7%). This study investigated the BRAF V600 mutation rate of primary melanomas. A recent study investigating differences in the incidence of the BRAF V600 mutation in primary and metastatic melanoma showed that BRAF status was related to metastatic burden and poor prognosis, and therefore, could be used to identify stage III melanoma patients 30. This indicated that BRAF mutations might be involved in the spread of the primary melanoma to distant sites. However, because small sample numbers were used, these results (n=72) and ours (n=79) should be confirmed by future studies using larger numbers of patients.
A previous study showed that ALM was more prevalent than SSM in Japan (∼45 vs. ∼25%, respectively) and that the ratio of Japanese men to women with melanoma ranged from 1 : 0.97 to 1 : 1.1 23, similar to that observed in the current study (1 : 0.88). In this study, 35 finger specimens with ALM were ineligible, requiring additional acid treatment by the investigator to soften the bones. A diagnosis of melanoma is made on the basis of pathological features; thus, acid treatment was performed on the FFPE samples, especially ALM. Unfortunately, the acid treatment caused DNA fragmentation and the samples were unsuitable for DNA testing. Therefore, the ratio of ALM decreased, and so we observed that the prevalence of ALM was similar to that of SSM (40.5 and 43.0%, respectively). We considered this to be one reason why we observed a similar BRAF V600 mutation rate to that in Europe and the USA. These 35 finger samples were decalcified and therefore were not appropriate for the detection of oncogenes. Thus, the samples for oncogene detection need to be managed carefully.
In the current study, when the mutation rate was stratified by melanoma subtype for those with late-stage melanoma (stage III/IV), 36.4% (4/11) of ALM tumours were found to be BRAF V600 mutation positive. Interestingly, the BRAF mutation frequency was only 9.5% in patients with early-stage ALM (Table 4). By contrast, there was little difference in the BRAF mutation frequency between late-stage (65.2%) and early-stage SSM (63.6%) (Table 4). To avoid missing BRAF V600 mutations in late-stage melanoma, we recommend performing a second biopsy.
This study had some limitations. The study had a relatively small sample size (n=80) and therefore might not be reflective of the Japanese population as a whole, possibly explaining why the frequencies of SSM and ALM were similar, in contrast to previous studies.
Future studies should be carried out to determine whether the majority of V600 mutations are V600E or are other types of V600 mutations such as V600K or V600D, which might distinguish these samples from other ethnic populations. Furthermore, the status of mitogen-activated protein kinase kinase kinase 8 (MAP3K8, COT) expression in patients with BRAF V600 mutations should be assessed to determine whether vemurafenib would be effective in the Japanese population or if resistance would occur rapidly 26. Finally, a comparison of the Cobas 4800 BRAF V600 Mutation Test with other techniques to identify BRAF V600 mutations such as circulating free DNA by amplification refractory mutation testing system 31 or immunohistochemistry 32 should be performed.
In summary, with the emergence of molecular-targeted therapeutics, genetic insights such as those provided by the present study are expected to provide more effective treatment options for the melanoma patients harbouring these mutations. Specifically, analysis using the Cobas 4800 BRAF V600 Mutation Test showed an increased frequency of BRAF V600 mutations in late-stage melanoma patients, especially stage III/IV ALM, who might benefit from vemurafenib treatment.
The authors thank Ludo Croxford, PhD, of Edanz Group Ltd for editorial support in the preparation of this manuscript.
This study was funded by Chugai Pharmaceutical Co. Ltd, Tokyo, Japan, and Roche Diagnostics K.K., Tokyo, Japan.
Conflicts of interest
N.Y. has received speaking fees from Chugai Pharmaceutical Co. Ltd, Bristol-Myers K.K., GlaxoSmithKline K.K. and Takeda Pharmaceutical Company Limited. T.O. and A.H. are salaried employees of Roche Diagnostics K.K. N.N. is a salaried employee of Chugai Pharmaceutical Co. Ltd. For the remaining authors there are no conflicts of interest.
1. Thompson JF, Scolyer RA, Kefford RF. Cutaneous melanoma in the era of molecular profiling. Lancet 2009; 374:362–365.
2. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al.. Mutations of the BRAF gene in human cancer. Nature 2002; 417:949–954.
3. Young J, Barker MA, Simms LA, Walsh MD, Biden KG, Buchanan D, et al.. Evidence for BRAF mutation and variable levels of microsatellite instability in a syndrome of familial colorectal cancer. Clin Gastroenterol Hepatol 2005; 3:254–263.
4. Liu W, Kelly JW, Trivett M, Murray WK, Dowling JP, Wolfe R, et al.. Distinct clinical and pathological features are associated with the BRAF(T1799A(V600E)) mutation in primary melanoma. J Invest Dermatol 2007; 127:900–905.
5. Hoeflich KP, Herter S, Tien J, Wong L, Berry L, Chan J, et al.. Antitumor efficacy of the novel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer Res 2009; 69:3042–3051.
6. Karram S, Novy M, Saroufim M, Loya A, Taraif S, Houreih MA, et al.. Predictors of BRAF mutation in melanocytic nevi: analysis across regions with different UV radiation exposure. Am J Dermatopathol 2013; 35:412–418.
7. Giles N, Pavey S, Pinder A, Gabrielli B. Multiple melanoma susceptibility factors function in an ultraviolet radiation response pathway in skin. Br J Dermatol 2012; 166:362–371.
8. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, et al.. A landscape of driver mutations in melanoma. Cell 2012; 150:251–263.
9. Lee JH, Choi JW, Kim YS. Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis. Br J Dermatol 2011; 164:776–784.
10. Platz A, Egyhazi S, Ringborg U, Hansson J. Human cutaneous melanoma; a review of NRAS and BRAF mutation frequencies in relation to histogenetic subclass and body site. Mol Oncol 2008; 1:395–405.
11. Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al.. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012; 380:358–365.
12. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al.. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010; 363:711–723.
13. Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al.. Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med 2012; 367:107–114.
14. Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, et al.. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med 2012; 367:1694–1703.
15. Robert C, Thomas L, Bondarenko I, O’Day S, Weber JW, Garbe C, et al.. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 2011; 364:2517–2526.
16. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, et al.. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363:809–819.
17. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al.. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib
. N Engl J Med 2012; 366:707–714.
18. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al.. Improved survival with vemurafenib
in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364:2507–2516.
19. Halait H, Demartin K, Shah S, Soviero S, Langland R, Cheng S, et al.. Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib
in metastatic melanoma. Diagn Mol Pathol 2012; 21:1–8.
20. Clark WH Jr, From L, Bernardino EA, Mihm MC. The histogenesis and biologic behavior of primary human malignant melanomas of the skin. Cancer Res 1969; 29:705–727.
21. Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, Mann GJ, et al.. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol 2011; 29:1239–1246.
22. Sasaki Y, Niu C, Makino R, Kudo C, Sun C, Watanabe H, et al.. BRAF point mutations in primary melanoma show different prevalences by subtype. J Invest Dermatol 2004; 123:177–183.
23. Ishihara K, Saida T, Otsuka F, Yamazaki NPrognosis and Statistical Investigation Committee of the Japanese Skin Cancer Society. Statistical profiles of malignant melanoma and other skin cancers in Japan: 2007 update. Int J Clin Oncol 2008; 13:33–41.
24. Balch CM, Buzaid AC, Soong SJ, Atkins MB, Cascinelli N, Coit DG, et al.. Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma. J Clin Oncol 2001; 19:3635–3648.
25. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, et al.. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 2009; 27:6199–6206.
26. Broekaert SM, Roy R, Okamoto I, van den Oord J, Bauer J, Garbe C, et al.. Genetic and morphologic features for melanoma classification. Pigment Cell Melanoma Res 2010; 23:763–770.
27. Si L, Kong Y, Xu X, Flaherty KT, Sheng X, Cui C, et al.. Prevalence of BRAF V600E mutation in Chinese melanoma patients: large scale analysis of BRAF and NRAS mutations in a 432-case cohort. Eur J Cancer 2012; 48:94–100.
28. Jin SA, Chun SM, Choi YD, Kweon SS, Jung ST, Shim HJ, Yun SJ. BRAF mutations and KIT aberrations and their clinicopathological correlation in 202 Korean melanomas. J Invest Dermatol 2013; 133:579–582.
29. Qi RQ, He L, Zheng S, Hong Y, Ma L, Zhang S, et al.. BRAF exon 15 T1799A mutation is common in melanocytic nevi, but less prevalent in cutaneous malignant melanoma, in Chinese Han. J Invest Dermatol 2011; 131:1129–1138.
30. Picard M, Pham Dang N, D’Incan M, Mansard S, Dechelotte P, Pereira B, et al.. Is BRAF a prognostic factor in stage III skin melanoma? A retrospective study of 72 patients after positive sentinel lymph node dissection. Br J Dermatol 2014 doi: 10.1111/bjd.12939. [Epub ahead of print].
31. Aung KL, Donald E, Ellison G, Bujac S, Fletcher L, Cantarini M, et al.. Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system. J Mol Diagn 2014; 16:343–349.
32. Chen Q, Xia C, Deng Y, Wang M, Luo P, Wu C, et al.. Immunohistochemistry as a quick screening method for clinical detection of BRAF(V600E) mutation in melanoma patients. Tumour Biol 2014; 35:5725–5733.