Metaanalysis of BRAF mutations and clinicopathologic characteristics in primary melanoma

ORIGINAL

ARTICLE

Metaanalysis of BRAF mutations and clinicopathologic characteristics in primary melanoma Soo Young Kim, MD,a Soo Nyung Kim, MD, PhD,b Hyung Jin Hahn, MD,a Yang Won Lee, MD, PhD,a,c Yong Beom Choe, MD, PhD,a,c and Kyu Joong Ahn, MD, PhDa,c Seoul, Korea Background: BRAF mutations occur in some melanomas. We hypothesized that BRAF mutation rates may differ in melanomas found in Asian compared to white populations. Objective: We performed a metaanalysis of BRAF mutations and their associations with the clinicopathologic characteristics of primary melanoma (PM), with a subgroup analysis to compare Asian and white patients with PM. Methods: The PubMed, EMBASE, and Cochrane databases were searched up to November 2013. The incidence rates and odds ratios (ORs) of BRAF mutations were calculated using a fixed or random effects model. Results: BRAF mutation was associated with younger age (OR = 1.734; P \ .001), trunk location (OR = 2.272; P \ .001), nonechronically sun damaged skin (OR = 2.833; P \ .001), superficial spreading melanoma (OR = 2.081; P \.001), and advanced melanoma stage (OR = 1.551; P = .003). The incidence of BRAF mutations in Asian patients with PM was half that of white patients with PM, but it was linked to the same clinicopathologic characteristics. Limitations: Only a small number of studies have been conducted on Asian patients with PMs. Conclusions: The BRAF mutation in PM was associated with age, anatomic site based on ultraviolet radiation exposure, histologic subtype, and advanced stage of melanoma. The clinicopathologic associations with BRAF mutations were similar in Asian and white patients with PM. ( J Am Acad Dermatol http://dx.doi.org/10.1016/j.jaad.2015.02.1113.) Key words: Asians; BRAF mutation; melanoma; metaanalysis; race; systematic review.

elanoma accounts for \5% of all skin cancers but is responsible for the majority of deaths from skin-related malignancies.1 The enhanced understanding of the molecular pathogenesis of primary melanoma (PM) over the past decade has led to the introduction of serine/ threonine-protein kinase B-Raf (B-raf) inhibitors, such as vemurafenib and dabrafenib, which have recently been approved by the US Food and Drug Administration.2-4 BRAF inhibitors have shown

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From the Departments of Dermatologya and Obstetrics and Gynecology,b Konkuk University School of Medicine, and the Research Institute of Medical Science,c Konkuk University, Seoul. This work was supported by Konkuk University. Conflicts of interest: None declared. Accepted for publication February 13, 2015. Correspondence to: Yang Won Lee, MD, PhD, Department of Dermatology, Konkuk University School of Medicine, 120-1

Abbreviations used: ALM: CI: CSD: I2: LMM: NM: OR: PM: SSM: UV:

acral lentiginous melanoma confidence interval chronic sun damage degree of inconsistency lentigo malignant melanoma nodular melanoma odds ratio primary melanoma superficial spreading melanoma ultraviolet

Neungdong-ro, Gwangjin-gu, Seoul 143-729, Korea. E-mail: [email protected]. Published online March 24, 2015. 0190-9622/$36.00 Ó 2015 by the American Academy of Dermatology, Inc. http://dx.doi.org/10.1016/j.jaad.2015.02.1113

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significant improvement in both overall and were included based on following criteria: (1) BRAF progression-free survival compared to dacarbazine mutation in patients with PM, (2) examination of chemotherapy.2-4 In a phase III trial of vemurafenib tissue samples, and (3) clear data available for the versus dacarbazine chemotherapy, vemurafenib incidence of BRAF mutation according to clinicopresented a relative reduction of 63% in the risk pathologic parameters. Studies in which metastatic of death and 74% in the risk of either death or melanoma or rare types of melanoma (eg, Spitzoid, disease progression compared to dacarbazine.2 desmoplastic, and spindle cell melanoma) and those BRAF mutation is one of the examining cell lines rather best-defined molecular abthan tissue samples were CAPSULE SUMMARY normalities contributing to excluded. We also excluded the pathogenesis of melastudies that only included BRAF mutations are found in noma. Mutation of B-Raf inmucosal melanoma, which approximately 40% of cutaneous duces hyperactivity of the is rarely experienced in dermelanomas. mitogen-activated protein kimatology, such as uveal melBRAF mutation in primary melanoma is nase (MAPK) extracellular anoma, choroidal melanoma, associated with younger age, trunk signaling pathway, which and intestinal melanoma. Our location, non-chronically sun damaged promotes the proliferation of study followed the skin, superficial spreading melanoma, tumor cells. The most preMetaanalysis of and advanced stage of tumor. Clinical valent mutation is a T1799A Observational Studies in correlates of BRAF mutations were the transversion in exon 15, which Epidemiology8 guidelines same in Asian and white populations. causes a V600E (Val600Glu) for the reporting of metaanalamino acid substitution.1,5 yses of observational studies. Primary melanoma with BRAF mutation Numerous studies have presents unique clinical associations examined the association Data extraction independent of race. between BRAF mutation in Two of the authors indemelanoma and its clinicopendently performed the sepathologic characteristics. lection and review of the studies, with disagreements However, there remain inconsistencies between subsequently resolved by discussion. Study selection studies, most of which have been conducted on was initially performed by reviewing titles. The white populations. There are a few studies of Asian abstracts of the candidate studies were then repatients with PM that have described differences in viewed and selected for data retrieval. The following the incidence and clinical and histologic types of data were extracted from all of the studies: the melanoma compared to white patients.6,7 We hyauthor(s), year of publication, and methods, the pothesized that clinicopathologic differences beage, sex, and race of the patients, and the clinicotween races could reflect different molecular and pathologic details of the melanoma (ie, tumor genetic backgrounds. In the present study, we location, chronic sun damage [CSD], histologic subinvestigated the association between the frequency type, ulceration, thickness, and stage). of BRAF mutation and the clinicopathologic characteristics of patients with PM by conducting a systemStatistical analysis atic review and metaanalysis. In addition, a subgroup Data regarding the incidence and number of analysis was performed to elucidate the differences BRAF mutations were extracted for each study. The in this regard between the Asian and white patients studies were assessed for homogeneity and comwith melanoma. bined in a metaanalysis; odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. We assessed the interstudy heterogeneity using the I2 METHODS Data collection and eligibility criteria statistic,9-11 such that if I2 $50%, the assumption of The PubMed, EMBASE, and Cochrane databases homogeneity was deemed invalid and the random were searched up to November 20, 2013 using the effects model was applied; otherwise the fixed keywords ‘‘melanoma’’ and ‘‘BRAF’’; the search was model was used. We also performed sensitivity limited to articles published in English and those analysis to examine the influence of each study on conducted on humans. The reference lists of the the pooled OR by serially omitting individual studies retrieved articles were reviewed manually to obtain and pooling the remaining studies. Possible publiany additional relevant references. Studies examcation bias was scrutinized by visual assessment of a ining the association between clinicopathologic funnel plot and the Egger test for the degree of characteristics and the prevalence of BRAF mutation asymmetry (P \.05). If the results diverged between d

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association was found among the Asian populations (OR = 1.669 [95% CI, 1.230-2.263]; P = .001 and OR = 3.700 [95% CI, 0.956-14.328]; P = .058, respectively; Fig 2). The incidence of BRAF mutation according to sex was compared in 33 studies. BRAF mutation was seen in 839 of 2325 (36.1%) men and in 770 of 2025 (38.0%) women. No relationship was found between sex and BRAF mutation (OR = 0.879 [95% CI, 0.7701.003]; P = .055). There was no statistical heterogeneity among the studies (I2 = 11.560). The subgroup analysis of 27 studies of whites showed statistical significance (OR = 0.854 [95% CI, 0.740-0.986]; P = .032), whereas 6 studies of Asians revealed that there was no significant association between BRAF mutation and sex (OR = 1.022 [95% CI, 0.733-1.424]; P = .898, respectively). Fig 1. Flowchart of the study selection process.

methods, the Egger test overruled visual inspection.10 We also conducted subgroup analysis by race (ie, Asian vs white). Statistical analyses were performed using Comprehensive Meta-Analysis Software (v 2.0; Biostat, Englewood, NJ). P \ .05 was considered statistically significant.

RESULTS Fig 1 is the flowchart of studies accessed through the review process. Of the 1929 articles retrieved, 45 met our eligibility criteria, including 8 articles on Asian populations. The eligible studies are summarized in Table I (available at http://www.jaad.org). Overall, BRAF mutation was detected in 2090 (36.4%) of all 5744 PM patients from all of the included studies. Subgroup analysis revealed that BRAF mutation occurred in 1881 of 4672 (40.3%) white patients with PM and 209 of 1072 (19.5%) Asian patients with PM. Age and sex The frequency of BRAF mutation was dichotomized according to patient age (ie, \50 and $50 years of age). Because many studies noted the mean age of patients, we could include only 11 studies. BRAF mutation was observed in 130 of 352 (36.9%) patients \50 years of age and in 182 of 720 (25.3%) patients $50 years of age. The incidence of BRAF mutation was significantly higher in the younger age group (OR = 1.734 [95% CI, 1.288-2.335]; P \ .001). No statistical heterogeneity was detected among the studies (I2 = 14.050). The subgroup analysis of 9 white and 2 Asian studies revealed that BRAF mutation was significantly associated with younger age among whites, but only a tendency toward this

Location The frequency of BRAF mutation according to anatomic tumor location was described in 19 of the included studies. The incidence of BRAF mutation was highest for lesions in the trunk (580/1128; 51.4%), followed by the extremities (365/928; 39.3%) and the head and neck (156/636; 24.5%). The incidence of BRAF mutation was significantly related to the tumor site. The OR for BRAF mutation in the trunk was 2.272 (95% CI, 1.918-2.691; P\.001; Fig 3), whereas those for the head and neck and extremities were significantly lower (OR = 0.410 [95% CI, 0.332-0.507]; P \ .001 and OR = 0.784 [95% CI, 0.656-0.938]; P = .008, respectively). There was no evidence of interstudy heterogeneity between any of the groups classified according to tumor site (I2 = 31.539, 19.094, and 0.000, respectively). A subgroup analysis by race was not performed because of the 19 studies, only 1 was performed in an Asian population. Ultraviolet exposure The occurrence of BRAF mutation relative to the degree of ultraviolet (UV) radiation exposure, as confirmed by histologic examination of tissue containing solar elastosis, was investigated in 14 studies. BRAF mutation was found in 177 of 620 (28.5%) cases with CSD and in 750 of 1444 (51.9%) non-CSD cases. Significant statistical heterogeneity was found among the studies (I2 = 54.203). Random effects modeling revealed that BRAF mutation was significantly associated with non-CSD (OR = 2.833 [95% CI, 1.947-4.122]; P \ .001). BRAF mutation was more frequently detected in non-CSD white patients (655/ 1195; 54.8%) than in non-CSD Asian patients (95/ 249; 38.2%), and BRAF mutation was significantly associated with non-CSD in both groups (OR = 2.968

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Fig 2. Forest plot for BRAF mutation and age (A) overall and (B) for the subgroup analysis: Asian (race 1) versus whites (race 2).

[95% CI, 2.384-3.695]; P \.001 and OR = 3.253 [95% CI, 1.415-7.478]; P = .005, respectively; Fig 4). Histologic subtype Twenty-nine studies produced data on BRAF mutation relative to histologic subtype. The frequency of BRAF mutation was highest in superficial spreading melanoma (SSM; 945/1875 [50.4%]), followed by nodular melanoma (NM; 279/686 [40.7%]), lentigo malignant melanoma (LMM; 75/384 [19.5%]), and acral lentiginous melanoma (ALM; 67/460 [14.6%]). BRAF mutation was closely related to SSM (OR = 2.081 [95% CI, 1.772-2.445]; P \.001; Fig 5, A). The ORs for BRAF mutation in LMM and in ALM were significantly low (OR = 0.331 [95% CI, 0.252-0.436]; P

\ .001 and OR = 0.348 [95% CI, 0.243-0.499]; P \ .001, respectively). NM was not associated with BRAF mutation (OR = 1.039 [95% CI, 0.858-1.257], P = .696). There was no evidence of statistical heterogeneity among the studies for SSM, NM, LMM, and ALM (I2 = 44.843, 10.480, 45.515, and 0.000, respectively). In a subgroup analysis of 4 Asian and 25 white studies, the incidence of BRAF mutation differed markedly with histologic type among Asians and whites: 33.3% (16/48) versus 50.8% (929/1827) in SSM, 10.0% (2/20) versus 20.1% (73/364) in LMM, 12.2% (35/288) versus 18.6% (32/172) in ALM, and 16.7% (7/42) versus 42.2% (272/644) in NM. BRAF mutation was strongly associated with SSM in both

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Fig 3. Forest plot for BRAF mutation and trunk location overall.

Asians and whites (OR = 3.989 [95% CI, 1.9138.317]; P \ .001 and OR = 2.014 [95% CI, 1.7072.376]; P \ .001, respectively; Fig 5, B). The OR for BRAF mutation in LMM and ALM was not statistically significant in Asians, but was significant in whites (OR = 0.417; P = .195 vs OR = 0.328; P \ .001 and OR = 0.587; P = .103 vs OR = 0.274; P \ .001, respectively). BRAF mutation was not associated with NM in either group (OR = 1.239; P = .632 and OR = 1.030; P = .769, respectively). Because SSM and NM constitute the majority of melanoma cases in whites, we also performed a subgroup analysis between SSM and NM in whites. Interestingly, SSM was still highly associated with BRAF mutation with OR 1.265 (P = .023) over NM (Fig 5, C ). Ulceration and thickness An analysis of the 14 selected studies on ulceration revealed that BRAF mutation was seen in 266 of 736 (36.1%) patients with ulceration and in 474 of 1285 (36.9%) patients without ulceration. BRAF mutation was not associated with ulceration (OR = 1.174 [95% CI, 0.950-1.451]; P = .138). There was no statistical heterogeneity among the studies (I2 = 47.543). Subgroup analysis of whites and Asians revealed no relationship between BRAF mutation and ulceration (OR = 1.060; P = .657 and OR = 1.452; P = .050, respectively).

Eight studies produced data on the incidence of BRAF mutation relative to tumor thickness. The incidence rates of BRAF mutation in cases with tumor thicknesses of #1 mm and [1 mm were 50.5% (239/473) and 41.3% (300/727), respectively. There was statistical heterogeneity among the studies (I2 = 54.833). Random effects modeling revealed that BRAF mutation was not associated with tumor thickness (OR = 1.508 [95% CI, 0.9232.465]; P = .101). The subgroup analysis also revealed that BRAF mutation and tumor thickness were not related in either the white or Asian populations (OR = 1.250; P = .078 and OR = 2.158; P = .326, respectively). Stage Comparison of 12 studies revealed that BRAF mutation was present in 323 of 925 (34.9%) cases with in situ stage I and II tumors, and in 155 of 419 (37.0%) cases with stage III and IV tumors. BRAF mutation was significantly associated with advanced stage melanoma (OR = 1.551 [95% CI, 1.166-2.064]; P = .003). There was no statistical heterogeneity among the studies (I2 = 0.000). A subgroup analysis found that BRAF mutation was significantly associated with advanced stage tumors in Asians, and showed a tendency toward an association in whites (OR = 1.640 [95% CI, 1.106-2.433]; P = .014 and OR = 1.459 [95% CI, 0.964-2.208]; P = .074, respectively; Fig 6).

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Fig 4. Forest plot for BRAF mutation and nonechronic sun damage (CSD) (A) overall and (B) for the subgroup analysis: Asian (race 1) versus whites (race 2).

DISCUSSION Our study has provided potentially important information. First, the incidence of BRAF mutation appears to differ, at least between white and Asian populations. Second, BRAF mutation is associated with distinct clinical and histopathologic characteristics, such as younger age, localization to the trunk, SSM subtype, non-CSD, and advanced stages of melanoma, but not with presence of ulceration or tumor thickness. Third, we found that the pattern of BRAF mutation associations in PM is similar in Asians and whites. The incidence of BRAF mutation in white patients with PM was 40.3%, in line with a previous

metaanalysis by Lee et al,55 while that for Asian patients with PM was much lower (19.5%). This marked difference could be explained by the different proportions of common histologic subtypes of PM between the 2 races.56 It was suggested in previous studies that melanoma develops via one of several pathways that have different patterns of association with host and environmental risk factors.22,50 As one of these causal molecular pathways, BRAF mutations appear to be linked with younger patients who have had intermittent exposure to the sun.22,55 In the present study, we confirmed this pattern of linkage of BRAF mutation. The OR for age \50 years was 1.7, and that

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Fig 5. Forest plot for BRAF mutation and superficial spreading melanoma (SSM) (A) overall, (B) for the subgroup analysis: Asian (race 1) versus whites (race 2), and (C) SSM versus nodular melanoma in whites (race 2).

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Fig 5. (continued).

for non-CSD was almost 3.0. We only included studies with CSD confirmed histologically, because the criteria used to classify CSD/non-CSD according to anatomic site differed between studies and there was evidence of inconsistency between anatomic and histologic classifications. In addition, UV radiation intensity differs according to geographic location.24,29,39,43 According to Curtin et al,22 this result could be explained by skin exposed intermittently to solar radiation being more susceptible to UV radiation, leading to a higher probability of acquiring a BRAF mutation. In the same context, we observed that BRAF mutation was predominantly associated with the trunk as a tumor location, which is commonly considered to be non-CSD skin. It is well known that SSM is closely related to BRAF mutation and is the most common subtype of PM in whites. In our study, although BRAF mutation in SSM was found more frequently in whites than in Asians (50.8% vs 33.3%), it was closely linked to SSM in both groups. On the other hand, in ALM, which is the major PM subtype among Asians, the frequency of BRAF mutation was approximately 15%, revealing a similarly low risk of BRAF mutation in the 2 groups. The association between tumor stage and BRAF mutation was not clear-cut. We found that BRAF mutation was associated with relatively advanced melanoma stages, regardless of race. Although there are suggestions that BRAF mutations are an early event in the pathogenesis of melanoma,6,57 our

findings support the argument that BRAF mutation may be acquired during the development of metastasis with evidence that a higher frequency of BRAF mutation was seen in metastasis than in PM.16 Although Breslow thickness is related to the poor prognosis, the SSM subtype—which tends to be thin—might have an effect on the discrepancy between BRAF mutation, thickness, and stage. Our study was subject to several limitations. Only 8 studies on Asian patients were included, and conclusions must therefore be drawn with caution for that group. In addition, the metaanalysis was based on observational studies, and therefore we cannot prove causality. There may be some minor phenotypic differences between V600E, V600K, and V600R. However, only a few studies presented the clinical characteristics according to each mutation separately, and we could not perform a metaanalysis of the clinicopathologic characteristics between these BRAF mutations. In summary, the incidence rates of BRAF mutation were 19.5% and 40.3% among Asians and whites, respectively. We can confirm that BRAF mutation was associated with distinct clinical characteristics: younger age, non-CSD skin and site (specifically the trunk), SSM histologic type, and advanced stage of melanoma. Moreover, the clinicopathologic associations with BRAF mutation were similar in Asian and white patients with PM. This finding implies that PM with BRAF mutation is a genetically distinct subtype

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Fig 6. Forest plot for BRAF mutation and stage (A) overall and (B) for the subgroup analysis: Asian (race 1) versus whites (race 2).

of the disease that presents with a unique pattern of clinical associations that is independent of race. Additional studies across diverse populations are required to clarify the molecular pathways in melanoma, to enable the provision of better options for future targeted therapies.

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53. Massi D, Pinzani P, Simi L, et al. BRAF and KIT somatic mutations are present in amelanotic melanoma. Melanoma Res. 2013;23:414-419. 54. Zebary A, Omholt K, Vassilaki I, et al. KIT, NRAS, BRAF and PTEN mutations in a sample of Swedish patients with acral lentiginous melanoma. J Dermatol Sci. 2013;72:284-289. 55. 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. 56. Kukita A, Ishihara K. Clinical features and distribution of malignant melanoma and pigmented nevi on the soles of the feet in Japan. J Invest Dermatol. 1989;92:210S-213S. 57. Rajagopalan H, Bardelli A, Lengaurer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature. 2002;418:934.

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Table I. Characteristics of the individual studies included in the metaanalysis BRAF mutation

Study (first author and year)

Country

Method

Uribe et al, 200312 Maldonado et al, 200313 Omholt et al, 200314 Reifenberger et al, 200415 Shinozaki et al, 200416 Thomas et al, 200417 Sasaki et al, 20046 Deichmann et al, 200418 Libra et al, 200519 Akslen et al, 200520 Takata et al, 200521 Lang and MacKie, 20057 Curtin et al, 200522 Goel et al, 200623 Deichmann et al, 200624 Poynter et al, 200625 Saldanha et al, 200626 Edlundh-Rose et al, 200627 Liu et al, 200728 Thomas et al, 200729 Akslen et al, 200830 Fargnoli et al, 200831 Viros et al, 200832 Beadling et al, 200833 Kannengiesser et al, 200834 Dadzie et al, 200935 Daniotti et al, 200936 Uribe et al, 200937 Lazar et al, 200938

Chile United States, Japan Sweden Germany

PCR-RFLP Sequencing SSCP, sequencing Sequencing

United States United States Japan* Germany Italy Europe Japan* UK United States United States Germany United States United Kingdom Sweden

Sequencing Sequencing Sequencing SSCP, sequencing Sequencing SSCP, sequencing Sequencing AS-PCR, sequencing Sequencing Sequencing SSCP, sequencing Sequencing SSCP, RFLP Pyrosequncing

Hacker et al, 201039

Australia

Broekaert et al, 201040 Ellerhorst et al, 201141 Bauer et al, 201142 Qi et al, 201143 Devitt et al, 201144 Si et al, 201245 Ashida et al, 201246 Lee et al, 201247 Jin et al, 201348

United States, Europe United States United States, Europe, Australia China* Australia China* Japan* Korea* Korea*

Feller et al, 201349

United States

Hacker et al, 201350

Australia

Greaves et al, 201351 Niu et al, 201352

United States China*

Massi et al, 201353

Italy

Australia AS-PCR United States SSCP, sequencing Europe SSCP, sequencing Italy Sequencing United States, Germany Sequencing United States Sequencing France Sequencing United States Italy Chile Hungary

Sequencing Sequencing Sequencing Real-time PCR, sequencing AS-PCR, iPLEX genotyping Sequencing Sequencing Sequencing Sequencing Duplex AS-PCR Sanger sequencing Sequencing Real-time PCR Pyrosequencing, sequencing AS-PCR, Sanger sequencing AS-PCR, iPLEX genotyping Pyrosequencing Mass spectrometric genotyping Real-time PCR

Positive/total

V600E

V600K

V600R

Others

14/25 32/115 41/71 8/15

13 24 37 8

1 2 1 0

0 6 1 0

0 0 2 0

18/59 17/37 9/35 19/50 15/23 15/51 3/28 13/52 34/126 33/58 32/101 19/81 22/59 116/213

17 13 9 12 15 13 3 13

0 2 0 5 0 2 0 0

0 1 0 0 0 0 0 0

1 1 0 2 0 0 0 0

33 21 16 22 103

0 6 3 0 11

0 0 0 0 1

0 5 0 0 1

112 64 17

0 15 5

0 2 0

0 11 5

2 30

0 0

0 0

0 2

9 5

0 0

0 0

0 0

18

5

2

0

96

9

0

4

27 112 98

0 0 3

0 0 0

0 0 9

10 20

0 2

0 0

0 0

6/35

6

0

0

0

81/375

56

19

3

3

81/217 8/58

6

1

0

1

19/33

19

0

0

0

112/251 92/214 27/124 92/165 142/302 2/57 32/69 9/18 5/10 14/24 25/68 40/123 157/350 109/220 261/544 27/180 112/249 110/432 20/79 10/58 22/202

Continued

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VOLUME jj, NUMBER j

Table I. Cont’d BRAF mutation

Study (first author and year)

Country

Method

Zebary et al, 201354 Total (%)

Sweden

Sequencing 2090/5744 (36.4)

Subgroup (%)

Asian White

209/1072 (19.5) 1881/4672 (40.3)

Positive/total

15/88

V600E

V600K

V600R

Others

13 1 0 1 1092 93 16 48 87.4% 7.4% 1.3% 3.8%

iPLEX, Sequenom Inc, San Diego, CA. AS-PCR, Allele-specific polymerase chain reaction; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SSCP, single-strand conformational polymorphism. *Study was conducted on an Asian population.