Intra-tumoral heterogeneity of KRAS and BRAF mutation status in patients with advanced colorectal cancer (aCRC) and cost-effectiveness of multiple sample testing

KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE) tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor). We also investigated the utility and efficiency of genotyping a 'DNA cocktail' prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1%) showed intratumoral heterogeneity; 5 (7.2%) at KRAS codons 12, 13 and 2 (2.9%) at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in 'DNA cocktail' samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a 'DNA cocktail' from two or more tumor blocks, improves mutation detection at minimal extra cost.     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

Trametinib with or without Vemurafenib in BRAF Mutated Non-Small Cell Lung Cancer

V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) mutated lung cancer is relatively aggressive and is resistant to currently available therapies. In a recent phase II study for patients with BRAF-V600E non-small cell lung cancer (NSCLC), BRAF V600E inhibitor demonstrated evidence of activity, but 30% of this selected group progressed while on treatment, suggesting a need for developing alternative strategies. We tested two different options to enhance the efficacy of vemurafenib (BRAF V600E inhibitor) in BRAF mutated NSCLC. The first option was the addition of erlotinib to vemurafenib to see whether the combination provided synergy. The second was to induce MEK inhibition (downstream of RAF) with trametinib (MEK inhibitor). We found that the combination of vemurafenib and erlotinib was not synergistic to the inhibition of p-ERK signaling in BRAF-V600E cells. Vemurafenib caused significant apoptosis, G1 arrest and upregulation of BIM in BRAF-V600 cells. Trametinib was effective as a single agent in BRAF mutated cells, either V600E or non-V600E. Finally, the combination of vemurafenib and trametinib caused a small but significant increase in apoptosis as well as a significant upregulation of BIM when compared to either single agent. Thus, hinting at the possibility of utilizing a combinational approach for the management of this group of patients. Importantly, trametinib alone caused upregulation of p-AKT in BRAF non-V600 mutated cells, while this effect was nullified with the combination. This finding suggests that, the combination of a MEK inhibitor with a BRAF inhibitor will be more efficacious in the clinical setting for patients with BRAF mutated NSCLC.     

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.     

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.     

High-frequency microsatellite instability and BRAF mutation (V600E) in unselected Serbian patients with colorectal cancer

Microsatellite instability (MSI) is a genetic consequence of a MisMatch Repair defect in colorectal cancer (CRC). We compared clinicopathohistological features with MSI status of CRC and evaluated prognostic significance of MSI status and BRAF mutation in the group of MSI-H tumors. 155 primary CRCs were excised surgically, 2006–2008. MSI analysis was carried out using a fluorescence-based pentaplex polymerase chain reaction technique. BRAF mutation (V600E) was analyzed by direct sequencing in MSI-H tumors. For all patients were evaluated: age, gender, localization, tumor cell type, tumor differentiation, mucin production, lymphocytic infiltration (TILs) and TNM stage. Patients’ disease-free survival (DFS) was compared according to MSI and BRAF status using Kaplan–Meier test. Of the 155 CRCs, 19 (12.3%) were MSI-H, and 136 (87.7%) were MSS/L. BRAF mutations were found in 4 of the MSI-H tumors. Patients with MSI-H CRC had lower recurrence rate (log rank test; P = 0.04) than MSS/L group. Patients with MSI-H tumor and BRAF mutation had worse DFS than MSI-H tumors without this mutation (log rank test; P = 0.01). Most of the clinicopathologic characteristics of MSI-H CRC in Serbian patients are similar to those reported in previous studies. Patients with MSI tumor phenotype had favourable prognosis, but in those with BRAF mutation higher recurrence rate was observed.     

Combined ipilimumab and nivolumab first‐line and after BRAF‐targeted therapy in advanced melanoma

The combination of ipilimumab and nivolumab is a highly active systemic therapy for metastatic melanoma but can cause significant toxicity. We explore the safety and efficacy of this treatment in routine clinical practice, particularly in the setting of serine/threonine‐protein kinase B‐Raf (BRAF)‐targeted therapy. Consecutive patients with unresectable stage IIIC/IV melanoma commenced on ipilimumab and nivolumab across 10 tertiary melanoma institutions in Australia were identified retrospectively. Data collected included demographics, response and survival outcomes. A total of 152 patients were included for analysis, 39% were treatment‐naïve and 22% failed first‐line BRAF/MEK inhibitors. Treatment‐related adverse events occurred in 67% of patients, grade 3–5 in 38%. The overall objective response rate was 41%, 57% in treatment‐naïve and 21% in BRAF/MEK failure patients. Median progression‐free survival was 4.0 months (95% CI, 3.0–6.0) in the whole cohort, 11.0 months (95% CI, 6.0‐NR) in treatment‐naïve and 2.0 months (95% CI, 1.4–4.6) in BRAF/MEK failure patients. The combination of ipilimumab and nivolumab can be used safely and effectively in a real‐world population. While first‐line efficacy appears comparable to trial populations, BRAF‐mutant patients failing prior BRAF/MEK inhibitors show less response.     

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21

Aims

The aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors.

Main methods

The IC₅₀ values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1.

Key findings

Gossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G₂/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21^Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21^Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G₂/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis.

Significance

Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21^Cip1 is a prerequisite for a positive response to its clinical application.     

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.     

An immune-active tumor microenvironment favors clinical response to ipilimumab

Purpose

Ipilimumab, a fully human monoclonal antibody specific to CTLA-4, has been shown to improve overall survival in metastatic melanoma patients. As a consequence of CTLA-4 blockade, ipilimumab treatment is associated with proliferation and activation of peripheral T cells. To better understand various tumor-associated components that may influence the clinical outcome of ipilimumab treatment, gene expression profiles of tumors from patients treated with ipilimumab were characterized.

Experimental design

Gene expression profiling was performed on tumor biopsies collected from 45 melanoma patients before and 3?weeks after the start of treatment in a phase II clinical trial.

Results

Analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab. Furthermore, ipilimumab appeared to induce two major changes in tumors from patients who exhibited clinical activity: genes involved in immune response showed increased expression, whereas expression of genes for melanoma-specific antigens and genes involved in cell proliferation decreased. These changes were associated with the total lymphocyte infiltrate in tumors, and there was a suggestion of association with prolonged overall survival in these patients. Many IFN-γ-inducible genes and Th1-associated markers showed increased expression after ipilimumab treatment, suggesting an accumulation of this particular type of T cell at the tumor sites, which might play an important role in mediating the antitumor activity of ipilimumab.

Conclusions

These results support the proposed mechanism of action of ipilimumab, suggesting that cell-mediated immune responses play an important role in the antitumor activity of ipilimumab.     

A Novel Approach for the Detection and Genetic Analysis of Live Melanoma Circulating Tumor Cells

Background

Circulating tumor cell (CTC) detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring. We therefore sought to establish the feasibility of a telomerase-based assay for detecting and isolating live melanoma CTCs.

Methods

The telomerase-based CTC assay utilizes an adenoviral vector that, in the presence of elevated human telomerase activity, drives the amplification of green fluorescent protein. Tumor cells are then identified via an image processing system. The protocol was tested on melanoma cells in culture or spiked into control blood, and on samples from patients with metastatic melanoma. Genetic analysis of the isolated melanoma CTCs was then performed for BRAF mutation status.

Results

The adenoviral vector was effective for all melanoma cell lines tested with sensitivity of 88.7% (95%CI 85.6-90.4%) and specificity of 99.9% (95%CI 99.8-99.9%). In a pilot trial of patients with metastatic disease, CTCs were identified in 9 of 10 patients, with a mean of 6.0 CTCs/mL. At a cutoff of 1.1 CTCs/mL, the telomerase-based assay exhibits test performance of 90.0% sensitivity and 91.7% specificity. BRAF mutation analysis of melanoma cells isolated from culture or spiked control blood, or from pilot patient samples was found to match the known BRAF mutation status of the cell lines and primary tumors.

Conclusions

To our knowledge, this is the first report of a telomerase-based assay effective for detecting and isolating live melanoma CTCs. These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials

Purpose

Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.

Methods

The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.

Results

Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.

Conclusion

We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.     

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.     

Survival According to BRAF-V600 Tumor Mutations – An Analysis of 437 Patients with Primary Melanoma

The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies.     

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.     

NRAS and BRAF Mutations in Melanoma-Associated Nevi and Uninvolved Nevi

According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF^V600E-mutation specific antibody VE1. The BRAF^V600E mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF^V600E-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.