BRAF inhibitor activity in V600R metastatic melanoma

Activating mutations in the BRAF gene occur in approximately 50% of melanomas. More than 70% of BRAF mutations are V600E and 10–30% are V600K. Potent and selective BRAF inhibitors have demonstrated significant clinical benefits in patients with V600E and V600K BRAF-mutated melanoma. V600R mutations constitute approximately 3–7% of all BRAF mutations and the activity of BRAF inhibitors in patients with this mutation is unknown. We have treated 45 patients with V600 mutated melanoma including patients with V600R mutation between July 2011 and October 2012 with the selective BRAF inhibitor dabrafenib (n = 43) or vemurafenib (n = 2) via a compassionate access programme. The overall response rate was 50% for the whole population with a progression-free survival of 5.5 months. Five objective responses were seen in six assessable patients with V600R BRAF mutation (n = 9). Our experience suggests that patients with V600R BRAF mutations can be treated successfully with oral BRAF inhibitors, and molecular diagnostic assays should include detection of this type of mutation.     

Distribution of some activating <Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> mutations in Slovene patients with colorectal cancer

Numerous clinical studies have shown that anti-EGFR therapies are effective only in a subset of patients with colorectal cancer. Even though mutations in the KRAS gene have been confirmed as negative predictors of the response to EGFR-targeted therapies, not all KRAS wild-type (wt-KRAS) patients will respond to treatment. Recent studies have demonstrated that additionally wild-type BRAF (wt-BRAF) genotype is required for response to panitumumab or cetuximab, suggesting that BRAF genotype criteria should be used together with KRAS genotype for selecting the patients who are about to benefit from the anti-EGFR therapy. In this study, 239 samples obtained from 215 patients with metastatic colorectal cancer were tested for the presence of the seven most common mutations in the KRAS gene and the V600E mutation in the BRAF gene. Among the tested patients, 53.8% of patients had wt-KRAS genotype and 46.2% were KRAS mutants. Around five percent (5.1%) of the tested patients bore the V600E mutation in BRAF gene. All the patients showing to have the V600E mutation in BRAF were wt-KRAS. The concordance of KRAS and BRAF mutational status between primary and metastatic tumor tissue samples was 100%. We have shown that the proportions of mutated and non-mutated KRAS in Slovene patients, as well as the proportion of V600E mutations in BRAF is similar to genotyping results reported by other authors. The tested seven KRAS mutations on codons 12 and 13 were mutually exclusive with the V600E mutation in the BRAF gene. Summing up the results about the KRAS and the BRAF mutation carriers from our study, the portion of potentially non-responsive patients for the anti-EGFR treatment is 51.3%.     

Optimizing targeted therapeutic development: Analysis of a colorectal cancer patient population with the BRAFV600E mutation

BRAF^V600E mutations are found in 10% of colorectal cancers (CRCs). The low frequency of this mutation therefore makes it a challenging target for drug development, unless subsets of patients with higher rates of BRAF^V600E can be defined. Knowledge of the concordance between primary–metastasis pairs and the impact of BRAF^V600E on outcome would also assist in optimal drug development. We selected primary CRCs from 525 patients (stages I–IV) evenly matched for age (<70 and ≥70), gender and tumor location (right, left and rectum), and 81 primary–metastasis pairs. BRAF^V600E, KRAS mutation and microsatellite instability (MSI) were determined and correlated with clinical features and patient outcomes. In multivariate analyses, increasing patient age (p = 0.04), female gender (p = 0.0005) and right‐sided tumor location (p < 0.0001) were independently associated with BRAF^V600E. The prevalence of BRAF^V600E was considerably higher in older (age > 70) females with KRAS wild‐type right‐sided colon cancers (50%) compared to the unselected cohort (10%). BRAF^V600E was associated with inferior overall survival in metastatic CRC (HR = 2.02; 95% CI 1.26–3.26), particularly evident in patients treated with chemotherapy, and is independent of MSI status. BRAF status was concordant in all primary tumors and matched metastases (79 wild‐type pairs and two mutant pairs). Clinicopathological and molecular features can identify CRC patients with a higher prevalence of BRAF^V600E. Patients with BRAF^V600E wild‐type primary tumor do not appear to acquire the mutation in their metastases, and BRAF^V600E is associated with poorer outcomes in metastatic patients. Our findings are timely and will help inform the rational development of BRAF^V600E inhibitors in CRC.     

The use of BRAF V600E mutation‐specific immunohistochemistry in pediatric Langerhans cell histiocytosis

BRAF p.V600E mutations are detected in greater than 50% of pediatric Langerhans cell histiocytosis (LCH) lesions. However, the use of mutation‐specific BRAF V600E immunohistochemistry (IHC) as a surrogate for molecular testing in pediatric LCH is unknown. We tested the mutation‐specific BRAF V600E monoclonal antibody (clone VE1) in formalin‐fixed, paraffin‐embedded LCH samples from 26 pediatric patients (14 males and 12 females, ages 7 mo–17 y) using allele‐specific real‐time polymerase chain reaction (PCR) with a limit of detection of 0.5% as the comparative gold standard. BRAF VE1 staining was scored for both intensity (0‐3+) and percentage of immunoreactive tumor cells (0%‐100%). BRAF VE1 immunoreactivity was determined using both lenient (≥1+, ≥1%) and stringent (≥2+, ≥10%) scoring criteria. Using lenient‐scoring criteria, we found that the sensitivity and specificity of IHC compared with allele‐specific real‐time PCR were 100.0% and 18.2%, respectively. The poor specificity of lenient IHC analysis was attributable to weak, 1+ staining in both BRAF‐mutated and wild‐type LCH. Using stringent‐scoring criteria, we found that specificity improved to 100.0% at the expense of sensitivity that decreased to 80.0%. Stringent scoring generated 3 false‐negative results, but in all cases, neoplastic tissue comprised less than 5% of the stained section and/or the specimen was decalcified. In conclusion, highly sensitive molecular assays remain the gold standard for BRAF mutation analysis in LCH paraffin‐embedded lesions. To avoid false‐positive results, unequivocal VE1 staining of 2+ intensity in greater than or equal to 10% neoplastic histiocytes is required. However, negative VE1 results require additional studies to exclude false‐negatives, and stringent‐scoring criteria may not be optimal for scant or decalcified specimens.     

Combined analysis of specific KRAS mutation,BRAF and microsatellite instability identifies prognostic subgroups of sporadic and hereditary colorectal cancer

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAF^V600E have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAF^V600E and specific KRAS mutation (Gly → Asp; G12D, Gly → Asp, G13D; Gly → Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAF^V600E was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild‐type cancers (p = 0.038). With MSI, specific KRAS and BRAF^V600E mutations, 3 distinct prognostic subgroups were observed in univariate (p = 0.006) and multivariable (p = 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAF^V600E mutation, (ii) KRAS/BRAF^V600E wild‐type or KRAS G13D mutations in MSS/MSI‐L and (iii) MSI‐H and KRAS G13D mutations. Moreover, none of the sporadic MSI‐H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAF^V600E and MSI may identify sporadic CRC patients with poor clinical outcome.     

Prognostic value of BRAF and KRAS mutation status in stage II and III microsatellite instable colon cancers

Microsatellite instability (MSI) has been associated with favourable survival in early stage colorectal cancer (CRC) compared to microsatellite stable (MSS) CRC. The BRAF V600E mutation has been associated with worse survival in MSS CRC. This mutation occurs in 40% of MSI CRC and it is unclear whether it confers worse survival in this setting. The prognostic value of KRAS mutations in both MSS and MSI CRC remains unclear. We examined the effect of BRAF and KRAS mutations on survival in stage II and III MSI colon cancer patients. BRAF exon 15 and KRAS exon 2–3 mutation status was assessed in 143 stage II (n = 85) and III (n = 58) MSI colon cancers by high resolution melting analysis and sequencing. The relation between mutation status and cancer‐specific (CSS) and overall survival (OS) was analyzed using Kaplan–Meier and Cox regression analysis. BRAF V600E mutations were observed in 51% (n = 73) and KRAS mutations in 16% of cases (n = 23). Patients with double wild‐type cancers (dWT; i.e., BRAF and KRAS wild‐type) had a highly favourable survival with 5‐year CSS of 93% (95% CI 84–100%), while patients with cancers harbouring mutations in either BRAF or KRAS, had 5‐year CSS of 76% (95% CI 67–85%). In the subgroup of stage II patients with dWT cancers no cancer‐specific deaths were observed. On multivariate analysis, mutation in either BRAF or KRAS vs. dWT remained significantly prognostic. Mutations in BRAF as well as KRAS should be analyzed when considering these genes as prognostic markers in MSI colon cancers.     

Incorporation of somatic <Emphasis Type="Italic">BRAF</Emphasis> mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.     

Allele frequencies of BRAFV600 mutations in primary melanomas and matched metastases and their relevance for BRAF inhibitor therapy in metastatic melanoma

Background

The detection of BRAFV600 mutations in patients with metastatic melanoma is important because of the availability of BRAF inhibitor therapy. However, the clinical relevance of the frequency of BRAFV600 mutant alleles is unclear.

Patients and Methods

Allele frequencies of BRAFV600 mutations were analyzed by ultra-deep next-generation sequencing in formalin-fixed, paraffin-embedded melanoma tissue (75 primary melanomas and 88 matched metastases). In a second study, pretreatment specimens from 76 patients who received BRAF inhibitors were retrospectively analyzed, and BRAFV600 allele frequencies were correlated with therapeutic results.

Results

Thirty-five patients had concordantly BRAF-positive and 36 (48%) patients had concordantly BRAF-negative primary melanomas and matched metastases, and four patients had discordant samples with low allele frequencies (3.4–5.2%). Twenty-six of 35 patients with concordant samples had BRAFV600E mutations, three of whom had additional mutations (V600K in two patients and V600R in one) and nine patients had exclusively non-V600E mutations (V600K in eight patients and V600E -c.1799_1800TG > AA- in one patient). The frequency of mutated BRAFV600 alleles was similar in the primary melanoma and matched metastasis in 27/35 patients, but differed by >3-fold in 8/35 of samples. BRAFV600E allele frequencies in pretreatment tumor specimens were not significantly correlated with treatment outcomes in 76 patients with metastatic melanoma who were treated with BRAF inhibitors.

Conclusions

BRAFV600 mutation status and allele frequency is consistent in the majority of primary melanomas and matched metastases. A small subgroup of patients has double mutations. BRAFV600 allele frequencies are not correlated with the response to BRAF inhibitors.     

BRAFV600E mutation: A promising target in colorectal neuroendocrine carcinoma

To determine the role of BRAF^V600E mutation and MAPK signaling as well as the effects of BRAF and MEK directed therapy in gastroenteropancreatic neuroendocrine neoplasia (GEP-NEN), with a focus on highly aggressive gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC). Using Sanger sequencing of BRAF exon 15 we determined the frequency of BRAF^V600E mutations in 71 primary GEP-NENs. MEK phosphorylation was examined by immunohistochemistry in corresponding tissue samples. To evaluate the biological relevance of BRAF^V600E mutation and MAPK signaling in GEP-NECs, effects of a pharmacological BRAF and MEK inhibition were analyzed in NEC cell lines both in vitro and in vivo. BRAF^V600E mutation was detected in 9.9% of all GEP-NENs. Interestingly, only NECs of the colon harbored BRAF^V600E mutations, leading to a mutation frequency of 46.7% in this subgroup of patients. In addition, a BRAF^V600E mutation was significantly associated with high levels of MEK phosphorylation (pMEK) and advanced tumor stages. Pharmacological inhibition of BRAF and MEK abrogated NEC cell growth, inducing G1 cell cycle arrest and apoptosis only in BRAF^V600E mutated cells. BRAF inhibitor dabrafenib and MEK inhibitor trametinib prevented growth of BRAF^V600E positive NEC xenografts. High frequencies of BRAF^V600E mutation and elevated expression levels of pMEK were detected in biologically aggressive and highly proliferative colorectal NECs. We provide evidence that targeting BRAF oncogene may represent a therapeutic strategy for patients with BRAF mutant colorectal NECs.     

MGL ligand expression is correlated to BRAF mutation and associated with poor survival of stage III colon cancer patients

Colorectal cancer (CRC) is the third most prevalent cancer type worldwide with a mortality rate of approximately 50%. Elevated cell-surface expression of truncated carbohydrate structures such as Tn antigen (GalNAcα-Ser/Thr) is frequently observed during tumor progression. We have previously demonstrated that the C-type lectin macrophage galactose-type lectin (MGL), expressed by human antigen presenting cells, can distinguish healthy tissue from CRC through its specific recognition of Tn antigen. Both MGL binding and oncogenic BRAF mutations have been implicated in establishing an immunosuppressive microenvironment. Here we aimed to evaluate whether MGL ligand expression has prognostic value and whether this was correlated to BRAF^V600E mutation status. Using a cohort of 386 colon cancer patients we demonstrate that high MGL binding to stage III tumors is associated with poor disease-free survival, independent of microsatellite instability or adjuvant chemotherapy. In vitro studies using CRC cell lines showed an association between MGL ligand expression and the presence of BRAF^V600E. Administration of specific BRAF^V600E inhibitors resulted in decreased expression of MGL-binding glycans. Moreover, a positive correlation between induction of BRAF^V600E and MGL binding to epithelial cells of the gastrointestinal tract was found in vivo using an inducible BRAF^V600E mouse model. We conclude that the BRAF^V600E mutation induces MGL ligand expression, thereby providing a direct link between oncogenic transformation and aberrant expression of immunosuppressive glycans. The strong prognostic value of MGL ligands in stage III colon cancer patients, i.e. when tumor cells disseminate to lymph nodes, further supports the putative immune evasive role of MGL ligands in metastatic disease.     

BRAF V600E-mutated lung adenocarcinoma with metastases to the brain responding to treatment with vemurafenib

Somatic BRAF mutations have been reported in 1–4% of non-small cell lung cancer (NSCLC), primarily in adenocarcinomas with the BRAF (V600E) mutation in about 50% of the cases. The role of BRAF mutation in NSCLC and the treatment for tumors with such mutations is still evolving. Our patient had metastatic NSCLC with metastases to her brain. Due to the BRAF (V600E) mutation in her tumor and her poor functional status, we offered her off-label treatment with vemurafenib, a BRAF inhibitor approved for use in metastatic melanoma. Our patient's visceral disease improved supporting vemurafenib's efficacy in the treatment of metastatic BRAF-mutated NSCLC. The regression of intracranial disease indicated vemurafenib was able to cross the blood–brain barrier and was efficacious in treating brain metastases in this patient with lung cancer.     

BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status

Background  

BRAF is a member of RAF family of serine/threonine kinases and mediates cellular responses to growth signals through the RAS-RAF-MAP kinase pathway. Activating mutations in BRAF have recently been found in about 10% of colorectal cancers, with the vast majority being a V600E hotspot mutation. The aim of the present study was to evaluate the clinical, pathological and molecular phenotype of colorectal tumors with BRAF mutations.     

Demographic trends and KRAS/BRAFV600E mutations in colorectal cancer patients of South China: A single-site report

The incidence of colorectal cancer (CRC) is increasing in China. Here, we aimed to evaluate the latest demographic trends and KRAS/BRAF mutations status of Chinese CRC. Five thousand five hundred and forty-six CRC patients diagnosed from 2010 to 2017 were involved. KRAS exon 2 and BRAF^V600E mutations were detected by Sanger sequencing and high-resolution melting analysis or allelic-specific probe method. Gene mutation profiles and clinicopathologic characteristics of 5495 patients were analyzed. The joinpoint regression model was used to predict the demographic data in 2018. We found KRAS exon 2 and BRAF^V600E mutation rates were 37.7 and 2.8% in CRC patients. Tumors with KRAS exon 2 mutations were more likely to be present in female and patients aged older than 75 years, right colon and have well-differentiated histology. Tumors with BRAF^V600E mutations were more likely to be present in the female, right colon and have poorly differentiated histology. From 2010 to 2017, the percentage of colon cancer and tubular adenocarcinoma in CRC increased substantially (from 39.3 to 51.8%, from 78.6 to 93.4%, respectively). The percentage of right colon cancer increased from 18.3 to 20.5%, which predictively may keep at 22.6% in 2018. The rise trends for patients with moderate differentiation tumor or KRAS exon 2 mutated tumor were apparent (from 50.3 to 78.6%, from 32.8 to 39.7%, respectively). In conclusion, in recent 8 years, there is a shift to the colon, especially right colon in the incidence of Chinese CRC. Moreover tubular adenocarcinoma is becoming the primary histology type.     

Multicenter retrospective analysis of metastatic colorectal cancer (CRC) with high-level microsatellite instability (MSI-H)

BackgroundThe microsatellite instability-high (MSI-H) phenotype, present in 15% of early colorectal cancer (CRC), confers good prognosis. MSI-H metastatic CRC is rare and its impact on outcomes is unknown. We describe survival outcomes and the impact of chemotherapy, metastatectomy, and BRAF V600E mutation status in the largest reported cohort of MSI-H metastatic colorectal cancer (CRC).Patients and methodsA retrospective review of 55 MSI-H metastatic CRC patients from two institutions, Royal Melbourne Hospital (Australia) and The University of Texas MD Anderson Cancer Center (United States), was conducted. Statistical analyses utilized Kaplan–Meier method, Log-rank test, and Cox proportional hazards models.ResultsMedian age was 67 years (20–90), 58% had poor differentiation, and 45% had stage IV disease at presentation. Median overall survival (OS) from metastatic disease was 15.4 months. Thirteen patients underwent R0/R1 metastatectomies, with median OS from metastatectomy 33.8 months. Thirty-one patients received first-line systemic chemotherapy for metastatic disease with median OS from the start of chemotherapy 11.5 months. No statistically significant difference in progression-free survival or OS was seen between fluoropyrimidine, oxaliplatin, or irinotecan based chemotherapy. BRAF V600E mutation was present in 14 of 47 patients (30%). BRAF V600E patients demonstrated significantly worse median OS; 10.1 versus 17.3 months, P = 0.03. In multivariate analyses, BRAF V600E mutants had worse OS (HR 4.04; P = 0.005), while patients undergoing metastatectomy (HR 0.11; P = <0.001) and patients who initially presented as stage IV disease had improved OS (HR 0.27; P = 0.003).ConclusionsPatients with MSI-H metastatic CRC do not appear to have improved outcomes. BRAF V600E mutation is a poor prognostic factor in MSI-H metastatic CRC.     

Functional characterization of the novel BRAF complex mutation,BRAFV600delinsYM,identified in papillary thyroid carcinoma

An activating mutation in the BRAF gene is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The mutation in PTCs is almost a c.1799T>A transversion, resulting in a p.V600E amino acid substitution (BRAF^V600E). Here, we report a novel complex BRAF mutation identified in 4/492 Japanese PTC cases (0.81%). The mutation was comprised of one nucleotide substitution at position 1798, followed by an in‐frame insertion of three nucleotides, c.1798delinsTACA in Exon 15, resulting in p.V600delinsYM. In silico three‐dimensional protein structure prediction implied altered kinase activity of this mutant. In vitro kinase assay and western blotting revealed that this mutation conferred high kinase activity on the BRAF protein, leading to constitutive activation of the MAPK signaling pathway. The mutation also showed high transforming ability in focus formation assay using NIH3T3 cells. The degree of all the functional characteristics was comparable to that of BRAF^V600E, and treatment with a BRAF inhibitor Sorafenib was also equally effective in this mutant. These findings suggest that the novel BRAF mutation, BRAF^V600delinsYM, is a gain‐of‐function mutation and plays an important role in PTC development.     

BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease

Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis.     

BRAF‐V600 mutational status affects recurrence patterns of melanoma brain metastasis

Brain metastasis is common and carries a poor prognosis in melanoma. A single institution, retrospective cohort of 225 melanoma patients was analyzed to determine if BRAF‐V600 mutational status was associated with brain metastasis. Eighty‐three of the 225 patients (37%) had BRAF‐V600 mutations. At initial diagnosis, BRAF‐V600 mutations were associated with younger age (p ≤ 0.001), higher proportion of females (p = 0.0037), higher AJCC stage (p = 0.030), regional lymph node involvement (p = 0.047), and family history of cancer (p = 0.044). Compared to BRAF‐WT, BRAF‐V600 patients had an increased risk of brain metastasis in multivariate analysis (OR = 2.24; 95% CL = 1.10–4.58; p = 0.027). However, BRAF‐V600 patients treated with a selective BRAF inhibitor (BRAFi) had a similar risk of brain metastasis compared to BRAF‐WT patients (OR = 1.00; 95% CL = 0.37–2.65; p = 0.98). Moreover, treatment with BRAFi significantly prolonged the time from initial diagnosis to brain metastasis diagnosis (HR = 0.30; 95% CL = 0.11–0.79; p = 0.015). Compared to other tissues, the brain was the most frequent site of metastasis in BRAF‐V600 patients without BRAFi (42% 7%). The frequency of brain metastasis was lower in BRAF‐WT and BRAF‐V600 patients with BRAFi (25% 4% and 25% 8%, respectively). The proportion of patients with brain metastasis as the only site was 40%, 60%, and 0% in the BRAF‐WT, BRAF‐V600 without BRAFi, and BRAF‐V600 with BRAFi groups, respectively. This study provides evidence on the clinical importance of BRAF‐V600 mutations and BRAF inhibition in the progression to melanoma brain metastasis.     

High prevalence of BRAF mutation in a Brazilian cohort of patients with sporadic papillary thyroid carcinomas

BACKGROUND:

Although several studies undoubtedly demonstrated that BRAF mutation is an important genetic event in the pathogenesis of papillary thyroid carcinoma (PTC), its prognostic significance and correlation with less differentiated states remains unclear. It has been suggested that the discrepancy may be at least partially due to the insufficient number of cases analyzed, epidemiologic factors, and a combination of different variants of PTC included in these studies.

METHODS:

In this context, the prevalence of the BRAF mutation in a Brazilian cohort of PTCs (n = 120) was first assessed and correlated with clinicopathologic features. The BRAF exon 15 mutation was evaluated by direct sequencing. Furthermore, using quantitative polymerase chain reaction, the issue of whether the expression level of the iodide‐metabolizing genes (NIS and TSHR) was correlated with BRAF mutational status was investigated.

RESULTS:

A high prevalence of the BRAF mutation was found in PTC cases (48%). The BRAF mutation was found to be significantly associated with the classic variant of PTC (66%; P < .0001), although it was found in the follicular variant as well (21%). Subtype stratification demonstrated that the BRAF V600E mutation was associated with tumor size, extrathyroid invasion, the presence of lymph node metastasis and risk of disease recurrence, and mortality in patients with the classic variant of PTC. Moreover, the expression levels of NIS and TSHR were remarkably lower in PTCs harboring the BRAF V600E mutation.

CONCLUSIONS:

These findings provide further evidence that BRAF might be associated with a more aggressive phenotype and less differentiated state due to decreased expression of iodide‐metabolizing genes. The search for a BRAF mutation in the current study population appears to be valuable for predicting prognosis and guiding management in patients with PTC. Cancer 2009. © 2009 American Cancer Society.     

Detecting BRAF Mutations in Formalin-Fixed Melanoma: Experiences with Two State-of-the-Art Techniques

Background

Melanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition.

Patients and Methods

We used 2 methods to analyze the BRAF mutation status in 52 of 62 melanoma patients. Method 1 (mutation-specific real-time PCR) specifically detects the most frequent BRAF mutations, V600E and V600K. Method 2 (denaturing gel gradient electrophoresis and direct sequencing) identifies any mutations affecting exons 11 and 15.

Results

Eighteen BRAF mutations and 15 wild-type mutations were identified with both methods. One tumor had a double mutation (GAA) in codon 600. Results of 3 samples were discrepant. Additional mutations (V600M, K601E) were detected using method 2. Sixteen DNA samples were analyzable with either method 1 or method 2. There was a significant association between BRAF V600E mutation and survival.

Conclusion

Standardized tissue fixation protocols are needed to optimize BRAF mutation analysis in melanoma. For melanoma treatment decisions, the availability of a fast and reliable BRAF V600E screening method may be sufficient. If other BRAF mutations in exons 11 and 15 are found to be of predictive value, a combination of the 2 methods would be useful.Key Words: BRAF mutations, V600E, Mutation detection methods, Melanoma