A more sensitive platform for the detection of low-abundance BRAFV600E mutations

Identifying low-abundance mutations is important for the therapy and diagnose of cancer. Since the potential for tumor heterogeneity, the efficient detection of cancer-relevant mutations largely depends on the sensitivity of the methods employed. To confirm whether the mutation detection platforms affect the perceived prevalence of the BRAF(V600E) and its correlation with clinicopathologic features in papillary thyroid carcinomas (PTC), we compared Sanger Sequencing (SS), Pyrosequencing (PS), and a newly built allele-specific real-time PCR (AS-qPCR) apparatus for the detection of BRAF(V600E) in a Chinese cohort of conventional variant PTC. Accurate plasmid standards were built to assess the limit of detection of the three platforms. In this research, AS-qPCR has been found both the most sensitive and reliable at detecting mutation. The mutations detected by AS-qPCR which were not detected by SS or PS due to low abundance were confirmed by mutation enrichment platform COLD-PCR followed by SS. When analyzed by AS-qPCR, BRAF(V600E) was associated with a more aggressive phenotype. Our results indicate that the reported prevalence of the BRAF(V600E) mutations in PTC has been underestimated and more sensitive methods such as AS-qPCR should be applied in clinical settings.     

Tumor Homogeneity between Primary and Metastatic Sites for BRAF Status in Metastatic Melanoma Determined by Immunohistochemical and Molecular Testing

BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF^V600 mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF^V600E antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF^V600E (45.2%), c.1799_1800TG>AA, BRAF^V600E2 (3.0%), c.1798_1799GT>AA, BRAF^V600K (3.0%), c.1801 A>G, BRAF^K601E (1.3%), c.1789_1790CT>TC, BRAF^L597S (0.4%), c.1780G>A, BRAF^D594N (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF^V600E/E2 mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF^V600E mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF^V600E/E2 mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF^V600E detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.     

A modified screening strategy for Lynch syndrome among MLH1-deficient CRCs: Analysis from consecutive Chinese patients in a single center

BackgroundThe low prevalence of the BRAF V600E mutation in colorectal cancers (CRCs) in Chinese populations has stimulated concern about the efficacy of BRAF mutation analysis for Lynch syndrome (LS) screening.MethodsIn total, 169 of 4104 consecutive CRC patients with absent MLH1 staining were analyzed to compare the utility of the BRAF V600E mutation testing with MLH1 promoter methylation analysis in the Chinese population. Germline genetic testing was performed in patients with wild-type BRAF/methylated MLH1.ResultsCompared with BRAF genotyping, the use of MLH1 methylation testing alone to evaluate patients with MLH1 deficiency reduced referral rates for germline testing by 1.8-fold (82.8% vs. 47.1%). However, 6 patients harboring MLH1 promoter methylation were verified to have LS through germline genetic testing. It is notable that all 6 patients had a family history of CRC in at least 1 first-degree relative (FDR) or second-degree relative (SDR). The combination of MLH1 promoter methylation analysis and a family history of CRC could preclude significantly more patients from germline genetic testing than from BRAF mutation testing alone (45.5% vs. 17.2%, p<0.001) and decrease the number of misdiagnosed LS patients with MLH1 promoter methylation.ConclusionThe combination of a family history of CRC with MLH1 promoter methylation analysis showed better performance than BRAF mutation testing in the selection of patients in the Chinese population for germline genetic testing.     

BRAFV600E Mutation Analysis in Patients with Metastatic Colorectal Cancer (mCRC) in Daily Clinical Practice: Correlations with Clinical Characteristics,and Its Impact on Patients’ Outcome

Background

To prospectively evaluate the usefulness of the BRAFV600E mutation detection in daily clinical practice in patients with metastatic Colorectal Cancer (mCRC).

Patients and Methods

504 mCRC patients treated with systemic chemotherapy ± biologics were analyzed.

Results

A statistically significant higher incidence of the BRAF mutation was observed in patients with ECOG-PS 2 (p=0.001), multiple metastatic sites (p=0.002),> 65 years old (p=0.004), primary tumors located in the colon (p<0.001), high-grade tumors (p=0.001) and in those with mucinous features (p=0.037). Patients with BRAFV600E mutated tumors had a statistically significantly reduced progression-free survival (PFS) compared to wild-type (wt) ones (4.1 and 11.6 months, respectively; p<0.001) and overall survival (OS) (14.0 vs. 34.6 months, respectively; p<0.001). In the multivariate analysis the BRAFV600E mutation emerged as an independent factor associated with reduced PFS (HR: 4.1, 95% CI 2.7–6.2; p<0.001) and OS (HR: 5.9, 95% CI 3.7–9.5; p<0.001). Among the 273 patients treated with salvage cetuximab or panitumumab, the BRAFV600E mutation was correlated with reduced PFS (2.2 vs. 6.0 months; p<0.0001) and OS (4.3 vs. 17.4 months; p<0.0001).

Conclusions

The presence of BRAFV600E-mutation in mCRC characterizes a subgroup of patients with distinct biologic, clinical and pathological features and is associated with very poor patients’ prognosis.     

BRAF mutation and RASSF1A expression in thyroid carcinoma of southern Italy

Aim of this work is to provide a detailed comparison of clinical‐pathologic features between well‐differentiated and poorly differentiated tumors according to their BRAF and RASSF1A status. We analyzed RASSF1A methylation by MSP and BRAF mutation by LCRT‐PCR with LightMix® kit BRAF V600E in neoplastic thyroid tissues. Immunohistochemical evaluation of RASSF1A expression was also performed by standard automated LSAB‐HRP technique. An overall higher degree of RASSF1A over‐expression than normal thyroid parenchyma surrounding tumors (P < 0.05) has been found in all malignant well‐differentiated lesions. Moreover, statistically significant higher levels of RASSF1A expression were observed in differentiated cancers associated to an inflammatory autoimmune background (P = 0.01). Amplifiable DNA for LC PCR with LightMix® kit BRAF V600E was obtained in nine PTCs, four FVPTCs, five ATCs, and one control. The V600E mutation was found in 13 of 18 (72%) tumors. BRAF was mutated in 6 of 9 (66%) classical PTC, in 2 of 4 (50%) follicular variant PTC and in all ACs (100%). The overall frequency of RASSF1A promoter methylation observed was 20.5% (9 cases out 44). Hypermethylation of RASSF1A in primary tumors was variable according to histotypes ranging from100% (5/5) in ACs to only 12.5% (4/32) in PTCs. We show a correlation between RASSF1A methylation status and RASSF1A protein expression. Finally, we conclude that BRAF V600E mutation and RASSF1A methylation were pathogenetic event restricted to a subgroup of PTC/FVPTCs in early stage and to clinically aggressive ATCs. J. Cell. Biochem. 114: 1174–1182, 2013. © 2012 Wiley Periodicals, Inc.     

BRAF Mutation Analysis of Fine-Needle Aspiration Biopsies of Papillary Thyroid Carcinoma: Impact on Diagnosis and Prognosis

Objective: The BRAF V600E mutation has been associated with aggressive disease in papillary thyroid carcinoma (PTC). Molecular testing has been proposed as a useful adjunct to cytology in the diagnosis of malignancy and for tailoring clinical management. The aims of our study were to evaluate the BRAF mutational status using archived fine-needle aspiration biopsy (FNAB) material from patients with long-term follow-up and to correlate it with the original cytology diagnosis, clinicopathological stage at surgery, and prognosis. Study Design: FNAB material from 52 cases of PTC, with a mean follow-up of 8.4 years, was used in this study. DNA was extracted from archival cytology slides. Mutation analysis was performed by standard sequencing and locked nucleic acid-PCR/sequencing. Results: The BRAF V600E mutation was present in 46% of cases, but it was absent in all FNABs diagnosed originally as atypical and in 14 of 17 suspicious cases. Recurrence was significantly more frequent (p = 0.006) in cases with BRAF mutations and 54% of these cases presented with stage 2 or higher. Conclusion: The BRAF V600E mutation is associated with a higher pathological stage at surgery and a higher rate of recurrence. BRAF mutation analysis did not provide a significant increase in the accuracy of thyroid FNABs diagnosed as suspicious or atypical in our institution.     

Prevalence of class I–III BRAF mutations among 114,662 cancer patients in a large genomic database

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer.Impact statementThese data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.     

Prevalence and Clinicopathological Characteristics of HER2 and BRAF Mutation in Chinese Patients with Lung Adenocarcinoma

Aims

To determine the prevalence and clinicopathological characteristics of BRAF V600E mutation and HER2 exon 20 insertions in Chinese lung adenocarcinoma (ADC) patients.

Methods

Given the fact that the driver mutations are mutually exclusive in lung ADCs, 204 EGFR/KRAS wild-type cases were enrolled in this study. Direct Sanger sequencing was performed to examine BRAF V600E and HER2 exon 20 mutations. The association of BRAF and HER2 mutations with clinicopathological characteristics was statistically analyzed.

Results

Among the 204 lung ADCs tested, 11 cases (5.4%) carried HER2 exon 20 insertions and 4 cases (2.0%) had BRAF V600E mutation. HER2 mutation status was identified to be associated with a non-smoking history (p<0.05). HER2 mutation occurs in 9.4% of never smokers (10/106), 8.7% of female (8/92) and 2.7% of male (3/112) in this selected cohort. All four BRAF mutated patients were women and three of them were never-smokers. No HER2 mutant patients harbor BRAF mutation.

Conclusions

HER2 and BRAF mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer and the availability of targeted therapy, Chinese lung ADC patients without EGFR and KRAS mutations are recommended for HER2 and BRAF mutations detection, especially for those never smokers.     

Associations of the BRAFV600E Mutation with Sonographic Features and Clinicopathologic Characteristics in a Large Population with Conventional Papillary Thyroid Carcinoma

Objective

To evaluate the association of the BRAF^V600E mutation with sonographic features and clinicopathologic characteristics in a large population with conventional papillary thyroid carcinoma (PTC).

Methods

We retrospectively reviewed the sonographic features, clinicopathologic characteristics, and presence of the BRAF^V600E mutation in 688 patients who underwent thyroidectomy for conventional PTC between January and July 2010 at a single institution. The incidence of the BRAF^V600E mutation was calculated. The sonographic features and clinicopathologic characteristics were compared between BRAF-positive and BRAF-negative patients. BRAF-positive patients were subdivided into those with papillary thyroid microcarcinoma (the PTMC group) and those with PTC larger than 10 mm (the PTC>10 mm group), and their sonographic features were compared.

Results

The BRAF^V600E mutation was detected in 69.2% of patients (476 of 688). Sonographic features were not significantly different between BRAF-positive and BRAF-negative PTC, nor between PTMC and PTC>10 mm groups. The BRAF^V600E mutation was associated with male sex (P = 0.028), large tumor size, extrathyroidal extension, central and lateral lymph node metastasis, and advanced tumor stage (P<0.0001).

Conclusion

The BRAF^V600E mutation was significantly associated with several poor clinicopathologic characteristics, but was not associated with sonographic features, regardless of tumor size. We recommend that patients with a thyroid nodule with any suspicious sonographic feature undergo preoperative BRAF^V600E testing for risk stratification and to guide the initial surgical approach in PTC.     

Focus: Personalized Medicine: Simultaneously Detection of 50 Mutations at 20 Sites in the BRAF and RAS Genes by Multiplexed Single-Nucleotide Primer Extension Assay Using Fine-Needle Aspirates of Thyroid Nodules

Fine-needle aspiration (FNA) is commonly used for primary evaluation of thyroid nodules. Twenty to 30 percent of thyroid nodules remain indeterminate after FNA evaluation. Studies show the BRAF p.V600E to be highly specific for papillary thyroid carcinoma (PTC), while RAS mutations carry up to 88 percent positive predictive value for malignancy. We developed a two-tube multiplexed PCR assay followed by single-nucleotide primer extension assay for simultaneous detection of 50 mutations in the BRAF (p.V600E, p.K601E/Q) and RAS genes (KRAS and NRAS codons 12, 13, 19, 61 and HRAS 61) using FNA smears of thyroid nodules. Forty-two FNAs and 27 paired formalin-fixed, paraffin-embedded (FFPE) tissues were tested. All BRAF p.V600E-positive FNA smears (five) carried a final diagnosis of PTC on resection. RAS mutations were found in benign as well as malignant lesions. Ninety-two percent concordance was observed between FNA and FFPE tissues. In conclusion, our assay is sensitive and reliable for simultaneous detection of multiple BRAF/RAS mutations in FNA smears of thyroid nodules.     

GNAS and KRAS Mutations are Common in Intraductal Papillary Neoplasms of the Bile Duct

Intraductal papillary neoplasms of the bile duct (IPNB) shows favorable prognosis and is regarded as a biliary counterpart of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Although activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of IPMNs of the pancreas, there have been few studies on GNAS mutations in IPNBs. This study investigates the status of GNAS and KRAS mutations and their association with clinicopathological factors in IPNBs. We examined the status of GNAS mutation at codon 201 and KRAS mutation at codon 12&13, degree of mucin production and immunohistochemical expressions of MUC mucin core proteins in 29 patients (M/F = 15/14) with IPNB in intrahepatic and perihilar bile ducts (perihilar IPNB) and 6 patients (M/F = 5/1) with IPNB in distal bile ducts (distal IPNB). GNAS mutations and KRAS mutations were detected in 50% and 46.2% of IPNBs, respectively. There was no significant correlation between the status of GNAS mutation and clinicopathological factors in IPNBs, whereas, the status of KRAS mutation was significantly inversely correlated with the degree of MUC2 expression in IPNBs (p<0.05). All IPNBs with GNAS mutation only showed high-mucin production. Degree of mucin production was significantly higher in perihilar IPNBs than distal IPNBs (p<0.05). MUC2 and MUC5AC expression was significantly higher in IPNBs with high-mucin production than those with low-mucin production (p<0.01 and p<0.05, respectively). In conclusions, this study firstly disclosed frequent GNAS mutations in IPNBs, similarly to IPMNs. This may suggest a common histopathogenesis of IPNBs and IPMNs. The status of KRAS mutations was inversely correlated to MUC2 expression and this may suggest heterogeneous properties of IPNBs. IPNBs with high-mucin production are characterized by perihilar location and high expression of MUC2 and MUC5AC, irrespective of the status of GNAS and KRAS mutations.     

BRAF V600E protect from cell death via inhibition of the mitochondrial permeability transition in papillary and anaplastic thyroid cancers

BRAF T1799A mutation is the most common genetic variation in thyroid cancer, resulting in the production of BRAF V600E mutant protein reported to make cells resistant to apoptosis. However, the mechanism by which BRAF V600E regulates cell death remains unknown. We constructed BRAF V600E overexpression and knockdown 8505C and BCPAP papillary and anaplastic thyroid cancer cell to investigate regulatory mechanism of BRAF V600E in cell death induced by staurosporine (STS). Induced BRAF V600E expression attenuated STS‐induced papillary and anaplastic thyroid cancer death, while BRAF V600E knockdown aggravated it. TMRM and calcein‐AM staining showed that opening of the mitochondrial permeability transition pore (mPTP) during STS‐induced cell death could be significantly inhibited by BRAF V600E. Moreover, our study demonstrated that BRAF V600E constitutively activates mitochondrial ERK (mERK) to inhibit GSK‐3‐dependent CypD phosphorylation, thereby making BRAF V600E mutant tumour cells more resistant to mPTP opening. In the mitochondria of BRAF V600E mutant cells, there was an interaction between ERK1/2 and GSKa/ß, while upon BRAF V600E knockdown, interaction of GSKa/ß to ERK was decreased significantly. These results show that in thyroid cancer, BRAF V600E regulates the mitochondrial permeability transition through the pERK‐pGSK‐CypD pathway to resist death, providing new intervention targets for BRAF V600E mutant tumours.     

BRAF initiating mutations in the papillary thyroid carcinoma

Among genetic alterations most important for the initiation of papillary thyroid carcinoma (PTC) is mutation T1799A in the BRAF gene which is the most frequent event (54.5%) in this type of thyroid cancer. It is seen in all stages, from microcarcinoma through clinically overt disease to anaplastic cancer. It has been shown that BRAF mutation is correlated with PTC histotype. It is identified most frequently in classical PTC and in tall cell variant. Moreover, BRAF mutation is described more often in older patients, whereas in young patients RET/PTC rearrangements dominate. In PTC cases with BRAF mutation V600E the prognosis is poorer, with more cancer invasiveness, metastasis and recurrence. The presence of BRAF mutation is related to the specific gene expression signature, different than in cancer cases showing RET/PTC rearrangement or no known initiating mutation.     

Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma

The effect of NRAS mutations on the pathological features and clinical outcomes in patients with cutaneous melanoma was compared with that of tumors containing BRAF(V600E) mutations and tumors wild type for both (WT). Clinical outcome data were obtained from a prospective cohort of 249 patients. Mutations involving NRAS and BRAF(V600E) were detected by PCR and were sequence verified. Cox proportional hazards regression was performed to relate NRAS and BRAF mutations to clinical outcome. Seventy-five percentage of NRAS mutations occurred in tumors >1 mm thick (BRAF(V600E) 40%, WT 34%); 75% of NRAS mutations had >1 mitosis/mm(2) (BRAF(V600E) 40%, WT 55%). When compared to WT, multivariate analysis of melanoma-specific survival (MSS) identified NRAS mutations as an adverse prognostic factor [hazard ratio (HR) 2.96; P = 0.04] but not BRAF(V600E) mutations (HR 1.73; P = 0.23). NRAS mutations were associated with thicker tumors and higher rates of mitosis when compared to BRAF(V600E) and WT melanoma and independently of this, with shorter MSS.     

RASSF1A Promoter Hypermethylation Is a Strong Biomarker of Poor Survival in Patients with Salivary Adenoid Cystic Carcinoma in a Chinese Population

In addition to the clinicopathological parameters, molecular biomarkers are becoming increasingly important in the prognostic evaluation of cancer patients. This study aimed to determine the molecular alterations in the RAS association domain family protein1A gene (RASSF1A) in salivary adenoid cystic carcinoma (ACC) and to evaluate the potential of such alterations as prognostic markers. One hundred and sixty-seven ACC tumor tissues and 50 samples of matched normal salivary gland tissues from the same patients were analyzed for RASSF1A promoter methylation status by bisulfite sequencing PCR (BSP) and/or methylation-specific PCR (MSP). Fifty ACC tumor tissues and matched normal salivary gland tissues were analyzed for loss of heterozygosity (LOH) by examining two microsatellite markers (D3S1478, D3S1621) at 3p21. RASSF1A gene mutations were detected by direct sequencing of all six exons in 50 tumor and normal tissue specimens. Over-all, RASSF1A promoter hypermethylation was detected in 35.3% (59/167) of ACC tissues and was associated with histologically solid tumor pattern (P = 0.002) and advanced TNM stage (P = 0.014). RASSF1A LOH was observed in 18.0% (9/50) of cases, and no somatic mutation of RASSF1A was detected in any cases. RASSF1A promoter methylation was associated with the poor over-all survival (Log-rank test, P <0.001) and disease-free survival (Log-rank test, P <0.001) and identified as an independent predicator of over-all patient survival (P = 0.009) and disease-free survival (P <0.001). It was concluded that RASSF1A methylation is involved in the development, differentiation and progression of ACC and is a strong independent biomarker of poor survival in ACC patients in a Chinese population.     

Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAFV600

Malignant melanoma is a highly-aggressive type of malignancy with considerable metastatic potential and frequent resistance to cytotoxic agents. BRAF mutant protein was recently recognized as therapeutic target in metastatic melanoma. We present a newly-developed U-BRAF^V600 approach – a universal pyrosequencing-based assay for mutation detection within activation segment in exon 15 of human braf. We identified 5 different BRAF mutations in a single assay analyzing 75 different formalin-fixed paraffin-embedded (FFPE) samples of cutaneous melanoma metastases from 29 patients. We found BRAF mutations in 21 of 29 metastases. All mutant variants were quantitatively detectable by the newly-developed U-BRAF^V600 assay. These results were confirmed by ultra-deep-sequencing validation (^∼60,000-fold coverage). In contrast to all other BRAF state detection methods, the U-BRAF^V600 assay is capable of automated quantitative identification of at least 36 previously-published BRAF mutations. Under the precaution of a minimum of 3% mutated cells in front of a background of wild type cells, U-BRAFV600 assay design completely excludes false wild-type results. The corresponding algorithm for classification of BRAF-mutated variants is provided. The single-reaction assay and data analysis automation makes our approach suitable for the assessment of large clinical sample sizes. Therefore, we suggest U-BRAF^V600 assay as a most powerful sequencing-based diagnostic tool to automatically identify BRAF state as a prerequisite to targeted therapy.     

Clinicopathological characteristics associated with BRAFK601E and BRAFL597 mutations in melanoma

BRAF mutations at codons L597 and K601 occur uncommonly in melanoma. Clinical and pathological associations of these mutations were investigated in a cohort of 1119 patients with known BRAF mutation status. A BRAF mutation was identified in 435 patients; Mutations at L597 and the K601E mutation were seen in 3.4 and 3.2% of these, respectively. K601E melanomas tended to occur in male patients, a median age of 58 yr, were generally found on the trunk (64%) and uncommonly associated with chronically sun‐damaged (CSD) skin. BRAF L597 melanomas occurred in older patients (median 66 yr), but were associated with CSD skin (extremities or head and neck location – 73.3%, P = 0.001). Twenty‐three percent of patients with V600E‐ and 43% of patients with K601E‐mutant melanomas presented with nodal disease at diagnosis compared to just 14% of patients with BRAF wild‐type tumors (P = 0.001 and 0.006, respectively). Overall, these mutations represent a significant minority of BRAF mutations, but have distinct clinicopathological phenotypes and clinical behaviors.