Metaanalysis of BRAF mutations and clinicopathologic characteristics in primary melanoma

8540 Background: Prognosis of AJCC stage III melanoma is heterogeneous. BRAFV600 mutations are frequent in melanomas. BRAFV600-targeted therapy has dramatic, but often transitory, efficacy in stage IV patients (pts). We aimed to determine for the first time the prognostic value of BRAFV600 mutations and other known prognostic criteria in stage III pts with sufficient nodal invasion. Methods: We searched all pts with cutaneous melanoma who had radical lymphadenectomy in our institution between 1/1/00 and 15/6/10 and included those with a nodal deposit >2 mm. BRAFV600 mutations were detected by DNA sequencing and pyrosequencing in formalin-fixed nodal samples containing >60% melanoma cells. Samples were considered mutated when >15% of DNA was positive. Endpoints were overall survival (OS) and distant metastasis free survival (DMFS). 92 patients had to be included to demonstrate a doubling of OS in patients without (40 months (m)) and with BRAFV600 mutation (20 m). Log-rank test and multivariate Cox proportional hazards regression model were used. Results: 105 consecutive pts were included, with 72% prospectively followed-up pts. BRAFV600 mutations (E: 83%; K: 14% of pts) were detected in 40% of pts. Median follow-up was 19 m (range: 3-139). Death occurred in 83% and 60% of pts with and without BRAF mutations, respectively, with median OS of 1.4 and 2.8 years. Pts’ age, primary melanoma ulceration, number of invaded nodes, AJCC staging, and BRAF status influenced OS and DMFS in the univariate analysis. The multivariate analysis showed the major prognostic role of BRAF status and of the number of invaded nodes (table). Conclusions: Provided our findings are independently replicated, BRAFV600 status should be used to stage melanoma pts with nodal metastasis. Our results also help to plan adjuvant trials with BRAFV600-targeted therapy in such patients, for whom the low tumor load may induce longer efficacy of BRAF-targeted therapies. [Table: see text]

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Analysis of BRAF mutations in circulating tumor cells selected by size from patients with melanoma and comparision to the primary tumor.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e21014 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e21014-e21014

Author(s):

Janine Wechsler ◽

Naoual Benali-Furet ◽

Fei Ye ◽

Marie-Françoise Avril ◽

Francoise Boitier ◽

...

Keyword(s):

Primary Tumour ◽

Primary Melanoma ◽

Melanoma Cells ◽

Molecular Target ◽

Tumour Cells ◽

Benign Lesions ◽

Braf Mutations ◽

Tumour Removal ◽

Healthy Donors ◽

The Mean

e21014 Background: The efficacy of anti-BRAF therapies in patients with melanoma has emphasized the need to identify BRAF mutations in the tumour. As circulating tumour cells (CTC) derive from the primary tumour (PT), their analysis may represent a non-invasive tool to follow the molecular-target in blood after tumour removal. Methods: This prospective study included 26 patients: 24 patients had confirmed melanoma, 2 patients had benign lesions (1 naevus and 1 pyogenic granuloma). Blood samples were taken at time of the PT surgical excision; 12 healthy donors were included as controls. The 24 patients, 13 females and 11 males, had invasive melanoma (13 superficial spreading melanoma, 7 nodular melanoma, 1 lentigo malignant melanoma, 1 desmoplastic melanoma, 2 unclassified melanoma.). The mean thickness was 7.8 mm. Cytology, immunophenotyping and analysis of BRAF mutations on exon 15 were performed comparatively in primary tumour and CTC from the same patient. CTC were selected by the ScreenCell method, a single-use innovative system to isolate rare tumour cells through a filter-membrane on the basis of their size. Nucleic acids DNA/RNA were extracted from live CTC after filtration. Results: Isolated CTC and microemboli were found in 21/24 patients and were found comparable to the primary melanoma cells. No CTC was found in 10 healthy donors and 2 patients with benign lesions. The mean number of CTC was 2 CTC per ml, the maximum was 4 CTC and 7 microemboli per ml. Malignancy of CTC was assessed on classic criteria: cell size larger than 15 microns, nucleo-cytoplasmic ratio > 0.5, presence of clusters composed of pleomorphic cells. CTC were positively immunostained either by S-100 or MART1/melanA or HMB45 antibodies; 4 patients were studied for BRAF mutations in both melanoma and CTC. The results (2 negative and 2 positive) were concordant in primary melanoma cells and CTC. The V600E-BRAF mutation was found in the 2 positive cases. Conclusions: The ScreenCell method of CTC selection by size allows to detect BRAF mutations in CTC of patients with melanoma. This simple method could be proposed to follow the molecular-target during the course of evolution and treatment after tumour removal.

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Analysis of BRAF, NRAS, cKIT, and EGFR alterations in melanoma lung metastases.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e19033 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e19033-e19033

Author(s):

Alessandra Ulivieri ◽

Giuseppe Cardillo ◽

Liborio Manente ◽

Andrea Petricca Mancuso ◽

Leonardo Vigna ◽

...

Keyword(s):

Lung Metastases ◽

Direct Sequencing ◽

Primary Melanoma ◽

High Response Rate ◽

Braf V600e Mutation ◽

Braf V600e ◽

Fish Analysis ◽

Braf Mutations ◽

Curative Intent ◽

Free Survival

e19033 Background: About 25% of pts will eventually develop lung metastases from primary melanoma. Activating mutations in BRAF, NRAS, CKIT and EGFR genes are involved in melanoma progression. Trials with drugs targeting oncogenic BRAF in melanoma have shown that a V600E mutation may identify a subgroup of pts with a high response rate. Methods: We analysedtheprevalence of BRAF, NRAS and CKIT mutations by direct sequencing and of EGFR copy number alterations by FISH, in pts affected by melanoma lung metastases, and explored their correlation with lung metastasis-free survival (LMFS) and post surgical progression-free survival (psPFS). Results: Over 10 years, 33 cases of pts with melanoma lung metastases were diagnosed at our institution, of which 22 underwent surgery with curative intent. The mean age was 64 years, and 19 (57.5%) were male. 51% carried a BRAF V600E mutation while 18% had a NRAS codon 61 mutation. The two events were mutually exclusive. No alterations were observed in exon 11 of the CKIT gene. FISH analysis indicated an EGFR low amplification in 3 cases and chromosome 7 polysomy in 13 cases. The presence of a BRAF V600E mutation was associated with a longer LMFS (median 6.1 vs 4.4 years, p<0.05), but it had no impact on psPFS (26.9 vs 29.6 months, p=0.6). NRAS mutations were associated with a reduction in both LMFS (3.7 years vs 4.7 years, p = 0.008) and, more significantly, in psPFS (12,5 months vs 41,1 months, p = 0.005; hazard ratio, 2.7). Conclusions: The frequency of mutations in BRAF (51%) and NRAS (18%) observed in lung metastatic melanomas was similar to the rates reported in other metastatic sites. BRAF mutations don't appear to be related with a more aggressive clinical course, while NRAS might be an independent negative prognostic factor after resection of melanoma lung metastases. The presence of BRAF mutations should be tested in melanoma lung metastases to identify the patients eligible for anti BRAF targeted therapy.

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Clinical characteristics of melanoma patients with non-V600E/K BRAF mutations.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e20036 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e20036-e20036 ◽

Cited By ~ 2

Author(s):

Sasan Nowroozi ◽

Zhuang Zuo ◽

Keyur Patel ◽

Sapna Pradyuman Patel ◽

Rajyalakshmi Luthra ◽

...

Keyword(s):

Clinical Characteristics ◽

Primary Melanoma ◽

Stage Iv ◽

Distant Metastases ◽

Unknown Primary ◽

Sequencing Analysis ◽

Superficial Spreading ◽

Braf Mutations ◽

Number Of Patients ◽

Melanoma Patients

e20036 Background: BRAF mutations are found in ~50% of melanoma. V600E/K substitutions are the most common and well-characterized. We analyzed the clinical characteristics of patients with non-V600E/K BRAF mutations. Methods: We identified 38 melanoma patients whose tumor contained a non-V600E/K BRAF mutation and reviewed the clinical characteristics. The sequencing analysis was performed with either pyrosequencing or next generation sequencing assay. Results: 38 patients were identified with non-V600E/K BRAF mutations. Mutations detected in more than 1 patient included V600R (n=14, 37%), K601E (5, 13%), G469E (3, 8%); L597S (3, 7%), D594G (2, 5%); 11 other mutations were identified in single patients. Median age was 57 yrs; 82% were men; 95% were white. The common primary subtypes were nodular (26%), superficial spreading (24%), unknown primary (21%); no one were acral, mucosal or uveal melanomas. Ten (26%) of 27 with known ulceration status had ulceration, and 3 (7%) of 22 with known mitosis status had < 1 mitosis /mm2. The sites of primary melanoma were located mostly in the head/neck and the trunk (63%), extremities (16%) and unknown primary (21%). The stage at diagnosis was I /II (29%), III (40%), IV (18%) and unknown (13%). Among 33 (87%) patients who ultimately developed distant metastases, 23(67%) had metastasis in the soft tissue/nodes, 21 (63%) in the lung, 9 (24%) in the brain, 7 (21%) in the liver, 6 (16%) in the bone, and 5 (15%) in the adrenal gland. Among patients (n=25) with initial stage I-III melanoma who later developed distant metastasis, the median duration between the time of initial diagnosis and distant metastases was 36 months. Among the 32 (84%) of the patients who developed stage IV melanoma, the median survival from the time of stage IV diagnosis was 18 months. Five patients received vemurafenib treatment, and 2 patients (K601E; T599_V600ins2) had stable disease and 2 patients (L597Q; G466E) had disease progression and 1 lost to follow up. Conclusions: Unexpectedly higher proportion of the patients with non-V600E/K mutant-melanoma had unknown primary melanoma and higher mitotic rate. In a small number of patients who received vemurafenib, the clinical response appears to be low.

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Clinical, pathological and prognostic features of rare BRAF mutations (MTs) in metastatic colorectal cancer (mCRC): A bi-institutional retrospective analysis (REBUS study).

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.3554 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 3554-3554

Author(s):

Brunella Di Stefano ◽

Maria Alessandra Calegari ◽

Michele Basso ◽

Armando Orlandi ◽

Alessandra Boccaccino ◽

...

Keyword(s):

Retrospective Analysis ◽

Survival Data ◽

Kinase Activity ◽

Radical Resection ◽

Braf V600e ◽

Lower Grade ◽

Braf Mutations ◽

Clinicopathologic Characteristics ◽

Prognostic Features ◽

Resection Of Metastases

3554 Background: Recently, 3 classes of BRAF MTs have been described. BRAF V600 MTs, which identify mCRC with poor prognosis and not benefitting from anti-EGFR drugs, belong to class 1. Class 2 and 3 include BRAF non-V600 MTs, which occur in about 1-2% mCRC and are associated to favourable prognosis and specific clinicopathologic features. Class 2 and 3 differ in kinase activity and sensitivity to anti-EGFR: class 2 are activated and RAS-independent MTs; class 3 are kinase-dead and sensitive to inhibition of This study aims to retrospectively evaluate features and prognostic role of rare BRAF non-V600 compared to BRAF V600E MTs in mCRC pts treated at 2 Italian Institutions. Methods: mCRC pts harboring BRAF MTs, assessed by means of NGS, pyrosequencing or RT-PCR, treated between Jan-13 and Dec-18 at 2 Italian Institutions, were retrospectively analyzed. Clinico-pathological and treatment characteristics and survival data were collected. Results: 55 pts bearing BRAF MTs were identified. Of those, 46 (84%) harbored a V600E and 9 (16%) a non-V600 MT. Within the non-V600 group, 3 MTs (K601E, G469A, G469R) belonged to class 2, while 5 MTs (G466E, G466A, 2 D594G, D594N), belonged to class 3. One pt harboured a T599I MT, whose kinase activity is unknown. Compared to BRAF V600E mCRC, BRAF non-V600 mCRC were more frequently left-sided ( p .017) and displayed a lower grade ( p .045). In addition, non-V600 mCRC pts had a lower tumor burden (involving one metastatic site) ( p .026) and underwent more frequently to resection of metastases with radical intent (77.7 vs 18%; p .000175). mOS was significantly longer in the non-V600 compared to the V600E group (61.3 vs 20.4 m; HR 0.41, 95%CI 0.18-0.93; p .05). No difference in activity and efficacy of anti-EGFR agents was observed between class 2 and 3. Conclusions: Despite the small size of our retrospective analysis, the results were consistent with previous evidences. BRAF non-V600 MTs identified a subgroup of mCRC, differing both in terms of clinicopathologic characteristics and prognosis from BRAF V600 mCRC. Interestingly, the better prognostic features allowed more frequently radical resection of metastases, positively impacting on survival.

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Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry

Oncotarget ◽

10.18632/oncotarget.14094 ◽

2016 ◽

Vol 8 (5) ◽

pp. 8069-8082 ◽

Cited By ~ 18

Author(s):

William Bruno ◽

Claudia Martinuzzi ◽

Virginia Andreotti ◽

Lorenza Pastorino ◽

Francesco Spagnolo ◽

...

Keyword(s):

Primary Melanoma ◽

Molecular Methods ◽

Braf Mutations

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BRAF Mutation (V600E) Prevalence in Mexican Patients Diagnosed with Melanoma

Case Reports in Oncology ◽

10.1159/000445939 ◽

2016 ◽

Vol 9 (1) ◽

pp. 241-245 ◽

Cited By ~ 3

Author(s):

Priscilla Denise Zepeda-Lopez ◽

Julio Cesar Salas-Alanis ◽

Sonia Toussaint-Caire ◽

Daniela Gutierrez-Mendoza ◽

Elisa Vega-Memije ◽

...

Keyword(s):

Braf Mutation ◽

Statistical Significance ◽

Primary Melanoma ◽

Braf V600e ◽

Molecular Diagnostic ◽

Braf Mutations ◽

Diagnostic Laboratory ◽

Dermatology Department ◽

Skin Biopsies ◽

Hospital General

Background: B-Raf is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway. It has been shown that 50% of melanomas harbor activating BRAF mutations, with over 90% being the V600E mutation. Objective: The goal of this research was to determine the prevalence of the BRAF V600E mutation in patients from Central Mexico diagnosed with primary melanoma. Methods: Skin biopsies from 47 patients with melanoma were obtained from the dermatology department of the Hospital General ‘Dr. Manuel Gea González' in Mexico City. For BRAF mutation determination, after DNA isolation, the gene region where the mutation occurs was amplified by PCR. Subsequently, the presence or absence of the V600E mutation was detected by Sanger sequencing performed at the private molecular diagnostic laboratory Vitagénesis in Monterrey, Mexico. Results: Of the 47 patients sampled, 6.4% harbored the V600E mutation. No statistical significance was found between mutations and the type of tumor.

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Different prevalence of BRAF and NRAS somatic mutations in melanomas according to the patients’ origin.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e20013 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e20013-e20013

Author(s):

Giuseppe Palmieri ◽

Amelia Lissia ◽

Antonio Cossu ◽

Paolo Antonio Ascierto ◽

Gerardo Botti ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Somatic Mutations ◽

Primary Melanoma ◽

Cancer Genes ◽

Nras Mutation ◽

Braf Mutations ◽

Paired Samples ◽

Melanoma Metastases ◽

Melanoma Cell Lines

e20013 Background: Genetic factors predisposing to melanoma at germline level have been demonstrated to be geographically heterogeneous. We here evaluated the spectrum of NRAS and BRAF mutations at somatic level, in a large subset of melanoma tissues from patients originating from different Italian geographical areas: Sardinia, whose population is genetically homogeneous, and Middle-South Italy, with a genetically heterogeneous population. Methods: Patients were enrolled consecutively between June 2008 and December 2012. Genomic DNA was isolated from tumor tissues [primary melanomas (N=439) and melanoma metastases (N=269)] or melanoma cell lines (N=32). Paired samples of primary melanomas (n=140) and synchronous or asynchronous metastases from the same patients (n=203) were included. The full coding sequences and splice junctions of NRAS (exons 2-3) and BRAF (exon 15) genes were screened for mutations through automated sequencing. Results: BRAF/NRAS mutations were identified in 63% of primary melanomas (48% BRAF; 15% NRAS), 67% melanoma metastases (51% BRAF; 16% NRAS), and 68% melanoma cell lines (56% BRAF; 12% NRAS). A non-significant increase in mutation frequency after progression from primary melanoma was observed. However, distribution of BRAF/NRAS mutations varied between in vivo tumors and melanoma cell lines, suggesting a preponderant role for BRAF activation in highly proliferating cultured melanoma cells. Among paired samples, consistency of BRAF/NRAS mutation patterns between metastatic and primary melanomas ranged from 71% (skin metastases) to about 90% (lymph node and visceral metastases). A significant inverse distribution of BRAF/NRAS mutation rates was observed in our series on the basis of the geographical origin of patients: for BRAF, 58% Sardinian vs. 42% non-Sardinian cases (p=0.045); for NRAS, 2% Sardinian vs. 21% non-Sardinian cases (p<0.001). Conclusions: Our findings provide additional insights into the spectrum and distribution of BRAF/NRAS mutations in melanoma; moreover, they support the hypothesis that differences in patients’ origins and related genetic backgrounds may contribute to even determine the incidence rate of somatic mutations in such cancer genes.

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