Mutation of BRAF is a predominant event in cancers with poor prognosis such as melanoma and colorectal cancer. BRAF mutation leads to a constitutive activation of mitogen activated protein kinase pathway which is essential for cell proliferation and tumor progression. Despite tremendous efforts made to target BRAF for cancer treatment, the correlation between BRAF mutation and patient survival is still a matter of controversy.
Clinical studies on the correlation between BRAF mutation and patient survival were retrieved from MEDLINE and EMBASE databases between June 2002 and December 2011. One hundred twenty relevant full text studies were categorized based on study design and cancer type. Publication bias was evaluated for each category and pooled hazard ratio (HR) with 95% confidence interval (CI) was calculated using random or fixed effect meta-analysis based on the percentage of heterogeneity. Twenty six studies on colorectal cancer (11,773 patients) and four studies on melanoma (674 patients) were included in our final meta-analysis. The average prevalence of BRAF mutation was 9.6% in colorectal cancer, and 47.8% in melanoma reports. We found that BRAF mutation increases the risk of mortality in colorectal cancer patients for more than two times; HR = 2.25 (95% CI, 1.82–2.83). In addition, we revealed that BRAF mutation also increases the risk of mortality in melanoma patients by 1.7 times (95% CI, 1.37–2.12).
We revealed that BRAF mutation is an absolute risk factor for patient survival in colorectal cancer and melanoma.
Citation: Safaee Ardekani G, Jafarnejad SM, Tan L, Saeedi A, Li G (2012) The Prognostic Value of BRAF Mutation in Colorectal Cancer and Melanoma: A Systematic Review and Meta-Analysis. PLoS ONE 7(10): e47054. doi:10.1371/journal.pone.0047054
Editor: Amanda Ewart Toland, Ohio State University Medical Center, United States of America
Received: July 24, 2012; Accepted: September 7, 2012; Published: October 9, 2012
Copyright: © Safaee Ardekani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Canadian Institutes of Health Research (MOP-93810, MOP-110974 and CCI-117958) and Canadian Dermatology Foundation to GL; Canadian Institute of Health Research - Skin Research Training Centre Trainee Award (GSA, SMJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The mitogen activated protein kinase (MAPK) pathway is one of the most crucial pathways in regulation of cancer cell proliferation and survival . Constitutive activation of the MAPK pathway in cancers has been frequently observed in various malignancies which is usually due to activating mutations in upstream factors such as RAS and RAF . Accordingly, mutations in BRAF are reported in up to 70% of cancer cell lines  and they are highly prevalent in most common cancers with poor prognosis such as malignant melanoma , . Mutations in BRAF have been reported in up to 60% of melanoma cases, between 40 to 70% of thyroid carcinomas, and up to 18% of colorectal cancers , .
So far, over 50 distinct mutations have been identified in the BRAF gene, which are present either in the glycine-rich P-loop of the N lobe or the activating segment in the exon 15 region . Most of these mutations increase BRAF activity by 1.5 to 700 folds depending on the type of the mutation . Of all BRAF activating mutations, a transitional mutation in nucleotide 1799 (T-A), also known as BRAF-V600E, is the most common change. In fact, this single mutation dramatically increases BRAF activity and accounts for more than 80% of all reported BRAF mutations in tumors , . This point mutation results in a valine to glutamic acid substitution that exposes the active site (normally sealed in a hydrophobic pouch) and implicates the constitutive activation of BRAF. As a result, malignant cells with V600E mutation proliferate in a growth factor-independent manner in culture as well as in tumors in animal models . In addition, it has been demonstrated that BRAF mutation is highly involved in main steps of cancer development and progression . Together, these reports nominate the BRAF-V600E mutation as a very promising therapeutic target in BRAF mutated cancers. So far, BRAF inhibitor PLX4032 is one of the only few promising treatments for malignant melanoma approved by the US Food and Drug Administration.
Although there are multiple reports on the correlation of BRAF mutation with a variety of cancer progression steps, the correlation between BRAF mutation and cancer patient survival is still a matter of controversy in different reports –. In this study, we used systematic review and meta-analysis as the most reliable approach to investigate whether BRAF-V600E mutation is associated with patient outcome. A pool of studies published between 2002 and 2011 on the association between BRAF-V600E mutation and patient survival in colorectal cancer, malignant melanoma and papillary thyroid carcinoma were reviewed and analyzed for this study. We found that BRAF mutation increases the risk of mortality in colorectal cancer patients by more than two-fold. In addition, we revealed that BRAF-V600E mutation also increases the risk of mortality in melanoma patients by 1.7 times, while its effect on papillary thyroid carcinoma still requires further investigation.
Search Strategy and Selection Criteria
We conducted a comprehensive search of medical literature on studies evaluating the effect of BRAF-V600E mutation on cancer patient survival. We searched MEDLINE and EMBASE using the terms “BRAF”, “BRAF mutation”, “BRAF V600E”, “cancer”, “patient survival”, “colorectal cancer”, “melanoma”, and “papillary thyroid carcinoma” in different combinations from June 2002 to December 2011. We initially narrowed our search based on research title followed by abstract and finally full texts were reviewed if they were categorized as relevant reports. We did not restrict the language in our research. All of the references from review papers and original reports were checked for further relevant studies in the systematic review.
Studies were excluded if contained no clinicopathologic data, survival analysis, or no comparison between wild type and mutant BRAF. In addition, studies which only reported a progression free survival as well as in vitro and animal reports were also excluded. For more information in detail please refer to PRISMA checklist (Table S1).
Data Extraction and Study Assessment
Two independent reviewers (GSA and LT) reviewed each full text report for eligibility and extracted required data. For each study the data on the number of patients in each group, mean survival time, hazard ratio and mean progression free survival time for randomized controlled trials (RCT), cancer type and study design were obtained and a consensus was achieved on all items. In the cases of incomplete required information, authors were contacted for additional information which was added as best as possible. Duplication of data was avoided by matching the author’s name and the name of the research centers.
We started summarizing the effect of BRAF-V600E mutation on patient survival separately based on study design RCT versus cohort and cancer type. We evaluated the publication bias using funnel plot analysis. We also assessed the heterogeneity of the studies using chi-square test of heterogeneity and I2 measure of inconsistency. Significant heterogeneity was defined as a Chi-square test P value of <0.10 or as an I2 measure >50%. Estimated hazard ratio (HR) was calculated using odds ratio and confidence interval in studies where HR was not available. In the absence of heterogeneity HRs and CIs were calculated according to a fixed model  which assumes that results across studies differ only by sampling error. In those studies where only the survival curve was available with no other detailed information, survival rates were extracted over multiple time periods in order to reconstruct HR and its variance with the assumption that patient censor rate was constant during study follow-up. This method has been described previously by Parmar et al.  to extract summary statistics for meta-analysis. A HR>1 was considered as a risk factor for worse survival in patient with positive BRAF mutation. In the end we used a log hazard ratio in the pooled data for the final analysis using R software (2011, The R Foundation for Statistical Computing). The impact of BRAF mutation on patient survival was considered statistically significant if 95% confidence interval for individual or overall log HR did not overlap zero.
Number of Studies
A total of 565 studies were retrieved from our electronic search. Of these, 120 abstracts were considered relevant and full texts were reviewed in detail. By the end of the review 26 studies on colorectal cancer (5 RCTs and 21 cohorts; 11,773 patients) met our inclusion criteria for meta-analysis. In addition, four studies on melanoma (1 RCT and 3 cohorts; 674 patients) including one study published at the time of statistical analysis  were incorporated in our final meta-analysis (Figure 1). Please also refer to complete PRISMA flow diagram (Figure S1) for more information. We were able to extract the overall survival information from two studies on papillary thyroid carcinoma , . However, we did not perform meta-analysis on papillary thyroid carcinoma subject due to the small number of studies (Table 1). The funnel plot for colorectal cancer but not for melanoma studies showed a publication bias in our collected data.
Figure 1. Flow diagram demonstrates the study selection process.doi:10.1371/journal.pone.0047054.g001
Table 1. Summary of studies that evaluated the impact of BRAF mutation on overall patient survival in colorectal cancer and melanoma.doi:10.1371/journal.pone.0047054.t001
Impact of BRAF-V600e Mutation on Colorectal Cancer Patient Survival
In our pooled data for colorectal cancer only one paper reported a protective HR (less than one) for BRAF mutation. Accordingly, Zlobec et al  observed a protective HR of 0.53 (0.3–1.3) for left side colon cancer. However, they reported a higher HR of 2.82 (1.5–5.5) for BRAF mutation as a risk factor for right side colon cancer in the same report. We considered these two analyses as separate reports in our final analysis. The pooled log HR of BRAF mutation effect on patient survival in colorectal cancer for cohort and RCT studies were 0.88 (0.60–1.16) and 0.61 (0.28–0.94), respectively. The final log HR for all studies on colorectal cancer was 0.81 (0.60–1.03) which corresponds to a HR of 2.24 (1.82–2.83, 95% CI). The heterogeneity of data on colorectal cancer was significant (P<0.0001) and I2 estimate of variation between analyzed studies was 74.3% (Figure 2).
Figure 2. Random effect model of Log hazard ratio (LogHR) with 95% confidence interval for studies comparing the effect of BRAF-V600E mutation on overall survival of colorectal cancer patients.
A LogHR <0 implies a survival benefit for patients with BRAF mutation. The square size indicates the power of each study in meta-analysis based on the number of patients in that study. The center of diamond shape at the lowest part indicates the combined LogHR for meta-analysis and its extremities the 95% confidence interval.doi:10.1371/journal.pone.0047054.g002
Impact of BRAF-V600e Mutation on Melanoma Patient Survival
One RCT study  compared BRAF mutation in patients’ serum level with tumor samples but had no data on wild type BRAF status. Two other RCTs evaluated progression-free survival (PFS) with either no overall survival information  and non-significant PFS or no overall survival data on wild type BRAF group . One cohort study used age <55 years as a surrogate marker for BRAF mutation while others either reported PFS or non-significant difference with no detailed information or survival curve graphs (Table 1). Pooled log HR for BRAF mutation effect on patient survival in melanoma for cohort studies was 0.57 (0.35–0.80) and the final pooled log HR including one RCT was 0.53 (0.32–0.75) corresponding to a HR of 1.70 (1.37–2.12, 95% CI). The heterogeneity of the data was not significant (P = 0.467) and I2 estimate of variation between analyzed studies was 0.0% (Figure 3).
Figure 3. Random effect model of Log hazard ratio (LogHR) with 95% confidence interval for studies comparing the effect of BRAF-V600E mutation on overall survival in melanoma patients.
A LogHR <0 implies a survival benefit for patients with BRAF mutation. The square size indicates the power of each study in meta-analysis based on the number of patients in that study. The center of diamond shape at the lowest part indicates the combined LogHR for meta-analysis and its extremities the 95% confidence interval.doi:10.1371/journal.pone.0047054.g003
Impact of BRAF-V600e Mutation on Papillary Thyroid Carcinoma Patient Survival
One study  reported no death in wild type BRAF group after almost 221 months of follow up. Another study  reported just one death in wild-type BRAF group after 20 years of follow up with odds ratio of 14.63 (1.28–167.29) for mutant BRAF. The study by Musholt et al  reported no difference in overall survival (HR = 1.04), while two other reports ,  showed no difference in disease-free survival between mutant and wild-type BRAF patients. However, another study by Abubaker et al  found BRAF mutation as a risk factor for disease free survival and Costa et al  reported that BRAF mutation would affect patient survival only if it is considered in combination with other mutations but not alone. In addition, Wang et al  reported that patients with synchronous bilateral papillary thyroid carcinoma, which harbor more BRAF mutation, have worse survival compared with those who have unilateral papillary thyroid carcinoma (Table 2).
BRAF mutation has become an important research topic in cancer biology since the original observation by Davies et al  in 2002. They revealed that high frequency of BRAF mutation is a common phenomenon in multiple types of cancers. Since then, numerous studies investigated the role of BRAF mutation in cancer development and progression. In mechanistic point of view, BRAF-V600E mutation, as the most prevalent BRAF mutation, changes the inactive conformation of BRAF kinase to a very active state . This simple point mutation leads to a constitutive activation of whole MAPK pathway, which mediates the cell surface growth signals to transcriptional activity of cell cycle regulatory genes. The key regulatory role of BRAF mutation in MAPK activation especially in melanoma generated a tremendous research effort to block this signaling pathway for cancer treatment. The usage of most available multi-kinase inhibitor at that time, sorafenib, was the first step toward targeted BRAF inhibition. Despite the first promising results in cell culture and animal studies, sorafenib was found to be unsuccessful in melanoma patients treatment even among those harboring mutant BRAF , . A number of other small molecule inhibitors have been tested for targeted BRAF inhibition; however, so far only PLX4032 and GSK2118436 have successfully been used in clinical stages , . Taking everything into account, the main goal in cancer treatment is to increase patient survival, while the idea of whether BRAF mutation per se actually affects patient survival has been a matter of debate. In this study, by conducting meta-analysis on data reported in 30 independent studies, we evaluated the effect of BRAF-V600E mutation on patient survival in colorectal cancer and melanoma. We also reviewed another 10 independent studies on papillary thyroid carcinoma in which BRAF mutation is prevalent.
In a population of 11,773 patients from 26 independent studies, we found that the risk of mortality in colorectal cancer patients harboring BRAF-V600E mutation is more than two times higher than those with wild-type BRAF. We also demonstrated that melanoma patients with BRAF mutation have a 1.7 times higher risk of mortality when compared with their counterparts without BRAF mutation in a population of 674 patients from the pooled result of 4 studies. In fact, this significant hazard ratio for BRAF mutation in our study can indirectly explain the previously reported promising improvement of melanoma patient survival harboring BRAF mutation after selective BRAF inhibitor treatments –. However, short period of symptom free survival and resistance to drug therapy are new emerging problems in BRAF specific inhibitor treatments in melanoma patients. Although the preliminary results for BRAF inhibitor treatments were promising, resistance to drug treatment usually appears in almost all cases , . Typically a reactivation of MAPK pathway happens in resistant cases through other mechanisms including RAS or MEK1 mutations, COT overexpression or BRAF truncation –. Nevertheless, the response rate of colon cancer patients harboring BRAF-V600E mutation to BRAF inhibitor treatment is much lower than melanoma patients , . In fact, over activation and crosstalk of parallel pathways like phosphatidylinositol 3-kinase (PI3 kinase) – AKT with MAPK in colorectal cancer is playing a main role in the observed different response to BRAF inhibitor treatments in colorectal cancer. Likewise, a very recent study by Prahallad et al  revealed the important role of epidermal growth factor receptor (EGFR) activation in colon cancer patients as well. They showed that a feedback activation of EGFR occurs in colon cancer cells after BRAF-V600E inhibition very quickly. In fact, this feedback activation of EGFR in colon cancer cells leads to a continuous malignant cell proliferation even in the presence of BRAF-V600E inhibition. However, this mechanism would not be applicable to melanoma cells as they express a very low level of EGFR .
BRAF mutation in papillary thyroid cancer was reported to be a risk factor for worse survival in two studies , . Notwithstanding a notably long term follow-up of patients for 18 to 20 years in these studies from Australia and Italy, the authors either did not observe any death  or only one death  in BRAF wild-type group of patients. Authors reported only one death in a population of 64 or no death among 41 wild-type BRAF patients while Standardized Death Rate for general population in Australia was found to be 6.9 and 4.7 per 1000 standard populations for male and female respectively (http://www.abs.gov.au/ausstats/abs@.nsf/Lookup/by+Subject/4125.0~Jul+2011~Main+Features~Death+rate~3210). Also, based on the report from the Centers for Disease Control and Prevention, age specific mortality rate for normal population within the same age group as patients in these two studies (45 to 54 years) is 420.9 per 100,000 of population (http://www.cdc.gov/nchs/nvss/mortality_tables.htm). Altogether, it seems that more studies with larger sample size are required to determine the significance of BRAF-V600E mutation effect on papillary thyroid carcinoma patient survival.
The number of studies comparing molecular and clinicopathological difference between right and left side colon cancers have been increased during the past two decades. For instance, a higher frequency of microsatellite instability, which is a poor prognostic factor for colon cancer, has been reported to be more prevalent in right side compared with left side colon cancer , . A number of studies also reported more prevalent BRAF mutations in right side colon cancer , . Although different biological and clinicopathological characteristics have been described for right and left side colon cancer, this issue is still a matter of controversy. Accordingly, investigating a large number of patients (29,568) in a recent study, Benedix et al  revealed a remarkable clinicopathological variation among colonic subsites irrespective of the side of tumor (right versus left). They showed that these differences are more related to the anatomical site of the cancer origin rather than a simple right and left categorization , . Despite a number of descriptive reports on the prevalence of BRAF mutation and its correlation with clinicopathological characteristics, there has been no comprehensive comparison on the effect of BRAF mutation on patient survival in separate groups of right and left side colon cancers. Accordingly, a controversial favorable effect for BRAF mutation on patient survival on left side colon cancer (P = 0.084) has been reported by Zlobec et al , while in the same study they observed a significant negative effect of BRAF mutation on patient survival for right side colon cancer (P = 0.01). They did observe a significant protective effect for BRAF mutation on left side colon cancer considering other risk factors in a multifactorial analysis (HR, 0.53; P = 0.109). However, the negative effect of BRAF mutation on right side colon cancer patient survival was persistently significant in multifactorial analysis (HR, 2.82; P = 0.002) . A number of other researchers from our pooled studies also observed a considerable decrease in patient survival with BRAF mutation compared with wild-type BRAF; however, the specific HR for wild-type or mutant BRAF was not determined , , , . Based on the significantly poor patient survival in mutant BRAF group in those studies according to survival curves and reported survival time difference, we estimated the HR of mutant BRAF in our meta-analysis.
With respect to reports on melanoma, Ellerhorst et al  reported no difference in patient survival between a group of patients with either BRAF mutation (109 cases) or NRAS mutation (31 cases) and wild-type BRAF/NRAS group (80 cases). There was no data available for the effect of BRAF mutation alone on patient survival in this report. In a very similar study, Houben et al  evaluated the effect of combined mutation of BRAF and NRAS mutation in 200 patients and reported a poor overall survival prognosis for metastatic samples which harbor either BRAF or NRAS mutation. However, they did not observe the same pattern in primary melanoma patients. As these two reports did not provide any information on the effect of BRAF mutation per se on patient survival we did not include them in our final meta-analysis. The inconsistency of results in these reports could be due to the fact that they combined BRAF and NRAS mutation and classified this group of patients together. In addition, Akslen et al  and Chang et al  reported no difference in patient survival in 69 and 68 cases respectively according to their BRAF mutation status. However, no details on patient survival have been provided in these reports. Akslen et al  mostly focused on different BRAF and NRAS mutations and their combinations and possible correlation with clinicopathologic characteristics. They reported that BRAF and NRAS mutations are mutually exclusive except for one case but they did not find any correlation with tumor cell proliferation, thickness or vascular invasion. Although they reported a median follow-up time of 76 months for the patients, no detailed information on mean survival time in each arm of the study was provided. There was no survival curve available in this report either. In a separate study, Chang et al  observed a significant trend for liver metastasis and tendency for multiple organ metastasis in BRAF mutant group but they did not detect a significant difference in either clinicopathological characteristics or in patient survival. Basically in this study authors chose a descriptive method to explain their observation and just mentioned that they did not find any correlation between patient survival and BRAF mutation. Unfortunately, no more detailed information including mean survival time in each group of study or a survival graph has been provided by the authors. A need for a conclusive meta-analysis on the effect of BRAF mutation on melanoma patient survival has been emerged due to the controversial reports on this issue. In our meta-analysis, we combined the results of four independent studies and measured the pooled risk of BRAF mutation on melanoma patient survival. So far our report is the first study on this issue which demonstrates the correlation between BRAF mutation and poor melanoma patient survival in a reliable statistical point of view. The number of reports on BRAF mutation and colorectal cancer were enough to pool the results together and perform a meta-analysis. Therefore, our findings in the pooled data suggest that with successful BRAF inhibition we would be able to increase the survival of colorectal cancer and melanoma patients harboring BRAF mutation.
BRAF plays a very important role in cancer initiation and progression. Mutation of BRAF is detected in all stages of melanocytic lesions including nevi, primary and metastatic melanoma. It is known to be involved in the multiple stages of tumor progression such as cell proliferation  and invasion . Interestingly, BRAF has also been shown to be involved in the progression of melanoma toward metastasis by enhancing its migration . However, cancer is a complex disease with multiple markers being involved in its formation and progression. Therefore, simultaneous study of other factors involved in BRAF network is crucial for a better understanding of its role in cancer. For instance, the cooperation between BRAF mutation and PTEN loss in melanoma progression has been identified . Since improving patient survival is the main goal in cancer treatment, further meta-analysis evaluation on the combination of markers involved in this critical network including RAS and PTEN with BRAF seems necessary for future planning in cancer treatment and drug development.
In summary, we used systematic review and meta-analysis approach to investigate possible association between BRAF-V600E mutation and cancer patient survival. We found that BRAF-V600E mutation increases the risk of mortality in colorectal cancer patients for more than two-fold. In addition, we revealed that BRAF-V600E mutation also significantly increases the risk of mortality in melanoma patients. This data highlights the important role of mutant BRAF in patient survival and suggest that with successful BRAF inhibition we may be able to increase the survival of colorectal cancer and melanoma patients harboring BRAF mutation.
Complete PRISMA search for Pubmed and EMBASE 2002–2011.
Conceived and designed the experiments: GSA SMJ GL. Performed the experiments: GSA LT. Analyzed the data: GSA LT AS. Contributed reagents/materials/analysis tools: AS. Wrote the paper: GSA SMJ GL.
- 1. Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, et al. (1992) Raf-1 activates MAP kinase-kinase. Nature 358: 417–421.
- 2. Malumbres M, Barbacid M (2003) RAS oncogenes: the first 30 years. Nat Rev Cancer 3: 459–465.
- 3. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, et al. (2002) Mutations of the BRAF gene in human cancer. Nature 417: 949–954.
- 4. Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, et al. (2002) BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 62: 6997–7000.
- 5. Cohen Y, Xing M, Mambo E, Guo Z, Wu G, et al. (2003) BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 95: 625–627.
- 6. Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, et al. (2004) Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116: 855–867.
- 7. Wellbrock C, Ogilvie L, Hedley D, Karasarides M, Martin J, et al. (2004) V599EB-RAF is an oncogene in melanocytes. Cancer Res 64: 2338–2342.
- 8. Hoeflich KP, Gray DC, Eby MT, Tien JY, Wong L, et al. (2006) Oncogenic BRAF is required for tumor growth and maintenance in melanoma models. Cancer Res 66: 999–1006.
- 9. Van Cutsem E, Kohne CH, Lang I, Folprecht G, Nowacki MP, et al. (2011) Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol 29: 2011–2019.
- 10. Stanojevic B, Dzodic R, Saenko V, Milovanovic Z, Pupic G, et al. (2011) Mutational and clinico-pathological analysis of papillary thyroid carcinoma in Serbia. Endocr J 58: 381–393.
- 11. Barault L, Charon-Barra C, Jooste V, de la Vega MF, Martin L, et al. (2008) Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases. Cancer Res 68: 8541–8546.
- 12. French AJ, Sargent DJ, Burgart LJ, Foster NR, Kabat BF, et al. (2008) Prognostic significance of defective mismatch repair and BRAF V600E in patients with colon cancer. Clin Cancer Res 14: 3408–3415.
- 13. Zlobec I, Bihl MP, Schwarb H, Terracciano L, Lugli A (2010) Clinicopathological and protein characterization of BRAF- and K-RAS-mutated colorectal cancer and implications for prognosis. Int J Cancer 127: 367–380.
- 14. Akslen LA, Angelini S, Straume O, Bachmann IM, Molven A, et al. (2005) BRAF and NRAS mutations are frequent in nodular melanoma but are not associated with tumor cell proliferation or patient survival. J Invest Dermatol 125: 312–317.
- 15. Chang DZ, Panageas KS, Osman I, Polsky D, Busam K, et al. (2004) Clinical significance of BRAF mutations in metastatic melanoma. J Transl Med 2: 46.
- 16. Greenland S (1998) Meta-analysis. In: Rothman KJ, Greenland, S., editor. Modern epidemiology. 2nd ed. Philadelphia: Lippincott-Raven. 643–673.
- 17. Parmar MK, Torri V, Stewart L (1998) Extracting summary statistics to perform meta-analyses of the published literature for survival endpoints. Stat Med 17: 2815–2834.
- 18. Si L, Kong Y, Xu X, Flaherty KT, Sheng X, et al. (2012) 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 48: 94–100.
- 19. Musholt TJ, Schonefeld S, Schwarz CH, Watzka FM, Musholt PB, et al. (2010) Impact of pathognomonic genetic alterations on the prognosis of papillary thyroid carcinoma. ESES vienna presentation. Langenbecks Arch Surg 395: 877–883.
- 20. Elisei R, Ugolini C, Viola D, Lupi C, Biagini A, et al. (2008) BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: a 15-year median follow-up study. J Clin Endocrinol Metab 93: 3943–3949.
- 21. Board RE, Ellison G, Orr MC, Kemsley KR, McWalter G, et al. (2009) Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study. Br J Cancer 101: 1724–1730.
- 22. Amaravadi RK, Schuchter LM, McDermott DF, Kramer A, Giles L, et al. (2009) Phase II Trial of Temozolomide and Sorafenib in Advanced Melanoma Patients with or without Brain Metastases. Clin Cancer Res 15: 7711–7718.
- 23. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, et al. (2010) Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 363: 809–819.
- 24. O'Neill CJ, Bullock M, Chou A, Sidhu SB, Delbridge LW, et al.. (2010) BRAF(V600E) mutation is associated with an increased risk of nodal recurrence requiring reoperative surgery in patients with papillary thyroid cancer. Surgery 148: 1139–1145; discussion 1145–1136.
- 25. Ito Y, Yoshida H, Maruo R, Morita S, Takano T, et al. (2009) BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients. Endocr J 56: 89–97.
- 26. Abubaker J, Jehan Z, Bavi P, Sultana M, Al-Harbi S, et al. (2008) Clinicopathological analysis of papillary thyroid cancer with PIK3CA alterations in a Middle Eastern population. J Clin Endocrinol Metab 93: 611–618.
- 27. Costa AM, Herrero A, Fresno MF, Heymann J, Alvarez JA, et al. (2008) BRAF mutation associated with other genetic events identifies a subset of aggressive papillary thyroid carcinoma. Clin Endocrinol (Oxf) 68: 618–634.
- 28. Wang W, Zhao W, Wang H, Teng X, Chen X, et al. (2012) Poorer Prognosis and Higher Prevalence of BRAF (V600E) Mutation in Synchronous Bilateral Papillary Thyroid Carcinoma. Ann Surg Oncol 19: 31–36.
- 29. Sharma A, Trivedi NR, Zimmerman MA, Tuveson DA, Smith CD, et al. (2005) Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors. Cancer Res 65: 2412–2421.
- 30. Hauschild A, Agarwala SS, Trefzer U, Hogg D, Robert C, et al. (2009) Results of a phase III, randomized, placebo-controlled study of sorafenib in combination with carboplatin and paclitaxel as second-line treatment in patients with unresectable stage III or stage IV melanoma. J Clin Oncol 27: 2823–2830.
- 31. Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, et al. (2010) Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467: 596–599.
- 32. Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, et al. (2012) Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 380: 358–365.
- 33. Young K, Minchom A, Larkin J (2012) BRIM-1, -2 and -3 trials: improved survival with vemurafenib in metastatic melanoma patients with a BRAF(V600E) mutation. Future Oncol 8: 499–507.
- 34. Richman SD, Seymour MT, Chambers P, Elliott F, Daly CL, et al. (2009) KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol 27: 5931–5937.
- 35. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, et al. (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 364: 2507–2516.
- 36. Paraiso KH, Fedorenko IV, Cantini LP, Munko AC, Hall M, et al. (2010) Recovery of phospho-ERK activity allows melanoma cells to escape from BRAF inhibitor therapy. Br J Cancer 102: 1724–1730.
- 37. Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, et al. (2010) COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468: 968–972.
- 38. Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, et al. (2011) RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480: 387–390.
- 39. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, et al. (2011) Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol 29: 3085–3096.
- 40. Kopetz S, Desai J, Chan E, Hecht JR, O'Dwyer PJ, et al.. (2010) PLX4032 in metastatic colon cancer patients with mutant BRAF tumors. J Clin Oncol 28: abstract 3534.
- 41. Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, et al. (2010) Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 28: 466–474.
- 42. Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, et al. (2012) Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 483: 100–103.
- 43. Nash GM, Gimbel M, Cohen AM, Zeng ZS, Ndubuisi MI, et al. (2010) KRAS mutation and microsatellite instability: two genetic markers of early tumor development that influence the prognosis of colorectal cancer. Ann Surg Oncol 17: 416–424.
- 44. Rampazzo E, Bertorelle R, Serra L, Terrin L, Candiotto C, et al. (2010) Relationship between telomere shortening, genetic instability, and site of tumour origin in colorectal cancers. Br J Cancer 102: 1300–1305.
- 45. Farina-Sarasqueta A, van Lijnschoten G, Moerland E, Creemers GJ, Lemmens VE, et al. (2010) The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol 21: 2396–2402.
- 46. Benedix F, Schmidt U, Mroczkowski P, Gastinger I, Lippert H, et al. (2011) Colon carcinoma-classification into right and left sided cancer or according to colonic subsite?-Analysis of 29,568 patients. Eur J Surg Oncol 37: 134–139.
- 47. Benedix F, Meyer F, Kube R, Kropf S, Kuester D, et al.. (2012) Influence of anatomical subsite on the incidence of microsatellite instability, and KRAS and BRAF mutation rates in patients with colon carcinoma. Pathol Res Pract (in press).
- 48. Loupakis F, Ruzzo A, Cremolini C, Vincenzi B, Salvatore L, et al. (2009) KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer 101: 715–721.
- 49. Price TJ, Hardingham JE, Lee CK, Weickhardt A, Townsend AR, et al. (2011) Impact of KRAS and BRAF Gene Mutation Status on Outcomes From the Phase III AGITG MAX Trial of Capecitabine Alone or in Combination With Bevacizumab and Mitomycin in Advanced Colorectal Cancer. J Clin Oncol 29: 2675–2682.
- 50. Ellerhorst JA, Greene VR, Ekmekcioglu S, Warneke CL, Johnson MM, et al. (2011) Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res 17: 229–235.
- 51. Houben R, Becker JC, Kappel A, Terheyden P, Brocker EB, et al. (2004) Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J Carcinog 3: 6.
- 52. Kumar R, Angelini S, Snellman E, Hemminki K (2004) BRAF mutations are common somatic events in melanocytic nevi. J Invest Dermatol 122: 342–348.
- 53. Arozarena I, Sanchez-Laorden B, Packer L, Hidalgo-Carcedo C, Hayward R, et al. (2011) Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A. Cancer Cell 19: 45–57.
- 54. Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, et al. (2009) Braf(V600E) cooperates with Pten loss to induce metastatic melanoma. Nat Genet 41: 544–552.
- 55. De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, et al. (2010) Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 11: 753–762.
- 56. Ferracin M, Gafa R, Miotto E, Veronese A, Pultrone C, et al. (2008) The methylator phenotype in microsatellite stable colorectal cancers is characterized by a distinct gene expression profile. J Pathol 214: 594–602.
- 57. Laurent-Puig P, Cayre A, Manceau G, Buc E, Bachet JB, et al. (2009) Analysis of PTEN, BRAF, and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer. J Clin Oncol 27: 5924–5930.
- 58. Liao W, Liao Y, Zhou JX, Xie J, Chen J, et al. (2010) Gene mutations in epidermal growth factor receptor signaling network and their association with survival in Chinese patients with metastatic colorectal cancers. Anat Rec (Hoboken) 293: 1506–1511.
- 59. Liou JM, Wu MS, Shun CT, Chiu HM, Chen MJ, et al. (2011) Mutations in BRAF correlate with poor survival of colorectal cancers in Chinese population. Int J Colorectal Dis 26: 1387–1395.
- 60. Maestro ML, Vidaurreta M, Sanz-Casla MT, Rafael S, Veganzones S, et al. (2007) Role of the BRAF mutations in the microsatellite instability genetic pathway in sporadic colorectal cancer. Ann Surg Oncol 14: 1229–1236.
- 61. Maughan TS, Adams RA, Smith CG, Meade AM, Seymour MT, et al. (2011) Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet 377: 2103–2114.
- 62. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, et al. (2009) CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 58: 90–96.
- 63. Park JH, Han SW, Oh DY, Im SA, Jeong SY, et al. (2011) Analysis of KRAS, BRAF, PTEN, IGF1R, EGFR intron 1 CA status in both primary tumors and paired metastases in determining benefit from cetuximab therapy in colon cancer. Cancer Chemother Pharmacol 68: 1045–1055.
- 64. Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, et al. (2005) Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res 65: 6063–6069.
- 65. Saridaki Z, Tzardi M, Papadaki C, Sfakianaki M, Pega F, et al. (2011) Impact of KRAS, BRAF, PIK3CA mutations, PTEN, AREG, EREG expression and skin rash in >/ = 2 line cetuximab-based therapy of colorectal cancer patients. PLoS One 6: e15980.
- 66. Shaukat A, Arain M, Thaygarajan B, Bond JH, Sawhney M (2010) Is BRAF mutation associated with interval colorectal cancers? Dig Dis Sci 55: 2352–2356.
- 67. Souglakos J, Philips J, Wang R, Marwah S, Silver M, et al. (2009) Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. Br J Cancer 101: 465–472.
- 68. Tie J, Gibbs P, Lipton L, Christie M, Jorissen RN, et al. (2011) Optimizing targeted therapeutic development: analysis of a colorectal cancer patient population with the BRAF(V600E) mutation. Int J Cancer 128: 2075–2084.
- 69. Tol J, Dijkstra JR, Klomp M, Teerenstra S, Dommerholt M, et al. (2010) Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab. Eur J Cancer 46: 1997–2009.
- 70. Tran B, Kopetz S, Tie J, Gibbs P, Jiang ZQ, et al.. (2012) Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer. Cancer (in press).
- 71. Yokota T, Ura T, Shibata N, Takahari D, Shitara K, et al. (2011) BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer. Br J Cancer 104: 856–862.
- 72. Kumar R, Angelini S, Czene K, Sauroja I, Hahka-Kemppinen M, et al. (2003) BRAF mutations in metastatic melanoma: a possible association with clinical outcome. Clin Cancer Res 9: 3362–3368.
- 73. Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, et al. (2011) Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol 29: 1239–1246.
- 74. von Moos R, Seifert B, Simcock M, Goldinger SM, Gillessen S, et al. (2012) First-line temozolomide combined with bevacizumab in metastatic melanoma: a multicentre phase II trial (SAKK 50/07). Ann Oncol 23: 531–536.
- 75. Oler G, Cerutti JM (2009) High prevalence of BRAF mutation in a Brazilian cohort of patients with sporadic papillary thyroid carcinomas: correlation with more aggressive phenotype and decreased expression of iodide-metabolizing genes. Cancer 115: 972–980.
- 76. Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, et al. (2005) BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 90: 6373–6379.