Detection Of BRAF Mutations Research Articles

Detection of EGFR and BRAF mutations by competitive allele-specific TaqMan polymerase chain reaction in lung adenocarcinoma.

Epithelial growth factor receptor (EGFR)-tyrosine kinase inhibitors are the standard first-line treatment for patients with metastatic non-small cell lung cancer (NSCLC) expressing sensitive EGFR-mutants. Other drugs target different driver mutants, including the serine/threonine-protein kinase B-raf (BRAF) inhibitor dabrafenib, which has exhibited promising efficacy for treating patients with metastatic BRAF-mutated NSCLC. Therefore, identifying patients carrying mutations that may be treated using targeted therapies is important. However, the methods of molecular detection presently applied in clinical practice, particularly detection of BRAF in NSCLC patients, require further investigation. Therefore, more sensitive and economic methods are required. The present study applied the competitive allele-specific TaqMan polymerase chain reaction (CastPCR) technology to the molecular detection of EGFR (del2235-2249, del2236-2250, T790M, L858R) and BRAF (V600E, G469A, D594G) mutations in 144 treatment-naive patients with lung adenocarcinoma, and analyzed the association between the mutation rates and patients' clinicopathological features. 51.4% (74/144) cases were identified harboring EGFR mutations. A total of 40.3% (58/144) patients carried sensitizing mutations (exon 19 deletion or L858R) and 14.6% (21/144) carried T790M mutations. 6.9% (10/144) mutation-positive patients were double-mutated. Total EGFR mutation rate was significantly increased in female compared with that of males (60.9 vs. 43.8%, P<0.05), in non-smokers compared with that of smokers (62.8 vs. 34.5%, P<0.05). In total, 8.3% (12/144) patients were identified with BRAF mutations. 16.7% were V600E (2/12) and 83.3% (10/12) were non-V600E mutants. Among the 10 non-V600E mutations, D594G accounted for 90.0% (9/10) and G469A accounted for 10.0% (1/10). Statistical analysis demonstrated that the BRAF mutation rate was not associated with any of the following clinicopathological features: Sex, age, smoking history, clinical stages, distant metastasis, differentiation degree, tumor size and regional lymph node metastasis (P≥0.05). CastPCR technology is a robust method with high sensitivity for the molecular detection of EGFR and BRAF mutations in clinical formalin-fixed paraffin-embedded samples.

Neoplasias de células histiocitarias y dendríticas: revisión de la literatura

Histiocytic and dendritic cell neoplasms (HDN) are rare and their biology, prognosis, treatment and terminology are still under discussion. They are composed of macrophage and dendritic-derived cells and show a wide range of clinical, morphological and prognostic features. Clinicopathological correlation and a broad immunohistochemical panel are required to establish a correct diagnosis. After the detection of BRAF mutations in Langerhans cell histiocytosis, the potential role of other molecular alterations is being studied. We have reviewed the literature published in the last 10 years to provide an overview of NHD, with particular emphasis their molecular features.

BRAF Mutation Status Concordance Between Primary Cutaneous Melanomas and Corresponding Metastases: A Review of the Latest Evidence

The identification of B-Raf proto-oncongene (BRAF) mutation and the emergence of targeted therapy marked a turning point in the treatment of melanoma. The study of mutation status concordance between primary tumors and metastases in this cancer has major treatment implications as it facilitates the selection of candidates for targeted therapy. This review analyzes the evidence on the level of mutation status concordance between primary tumors and different types of metastases in cutaneous melanoma and provides an overview of the advantages and disadvantages of the various methods used to detect BRAF mutations.

Tumor-suppressive function of UNC5D in papillary thyroid cancer.

BackgroundStudies have shown an association of the UNC5D gene with kidney and bladder cancer and neuroblastoma. We investigated whether UNC5D acts as a tumor suppressor in papillary thyroid carcinoma (PTC).MethodsPrimary PTC tumors and matched normal thyroid tissues were obtained from 112 patients to detect UNC5D mRNA by real-time PCR. Genomic DNA sequencing was performed to detect BRAF mutation in PTC tumors. The association between UNC5D expression and clinicopathological data from PTC patients was reviewed retrospectively. PTC-derived cancer cell lines TPC-1 and K1 with stable transfection of UNC5D were used to investigate the functions of UNC5D. Flow cytometry, CCK-8, Transwell assay and scratch tests were used to examine cell cycle distribution, proliferation and migration.ResultsThe expression of UNC5D was significantly decreased in PTC compared with adjacent normal thyroid tissues. Lower UNC5D expression was significantly associated with aggressive tumor behaviors, such as lymph node metastasis and BRAF mutation. Overexpression of UNC5D significantly suppressed malignant cell behaviors, including cell proliferation and migration, as well as tumor growth in vivo.ConclusionsThese findings suggest a potential tumor suppressor role of UNC5D in PTC progression; and provide insight into potential clinical relevance for the prognosis of PTC.

Concordancia mutacional de BRAF entre melanoma primario cutáneo y sus correspondientes metástasis. Revisión de la evidencia actual

The identification of B-Raf proto-oncongene (BRAF) mutation and the emergence of targeted therapy marked a turning point in the treatment of melanoma. The study of mutation status concordance between primary tumors and metastases in this cancer has major treatment implications as it facilitates the selection of candidates for targeted therapy. This review analyzes the evidence on the level of mutation status concordance between primary tumors and different types of metastases in cutaneous melanoma and provides an overview of the advantages and disadvantages of the various methods used to detect BRAF mutations.

Abstract 5696: Emerging KRAS mutation can play a considerable role to get acquired resistance to EGFR blockade

Abstract Background: Epidermal growth factor receptor (EGFR) blockade can achieve considerable tumor shrinkage in patients with metastatic colorectal cancer (CRC). However, most patients who benefit from EGFR blockade acquire resistance within a year. It was previously reported that nine of 24 (38%) patients whose tumors initially exhibited wild type KRAS developed detectable mutations in KRAS in their sera after acquiring resistance to EGFR blockade. However, the mechanisms of this acquired resistance remain unclear. In this study, we aimed to identify the mechanisms underlying acquired resistance to EGFR blockade by using circulating cell-free (ccf)DNA to track emerging KRAS, BRAF and S492R mutations during chemotherapy. Methods: We enrolled 29 patients with metastatic CRC and no RAS mutations in their primary tumors. Patients were treated with first-line systemic chemotherapy that included EGFR blockade. We purified ccfDNA from 1 mL of serum from each patient before they started chemotherapy, and every 2-3 months during chemotherapy until disease progression. We detected KRAS (codons 12, 13, 61, and 146), BRAF (V600E) and S492R mutations using digital polymerase chain reaction. Results: KRAS mutations were detected in the ccfDNA of three of the 29 patients (10%) before chemotherapy. The response rate was 86% (25/29); the four non-responders were the three patients with KRAS mutations, and another who exhibited ccfDNA BRAF mutations before starting chemotherapy. All patients with no KRAS or BRAF mutations in their ccfDNA before chemotherapy responded to the chemotherapy (25/25). Of these 25 initially responsive patients, 13 (52%) acquired resistance. We detected emerging KRAS mutations in the ccfDNA of 11 of these 13 patients (84%); eight of these patients had multiple mutations. We also detected BRAF mutations in six patients (46%); none of the patients had solo BRAF mutations. Five patients (38%) had S492R mutations; one had mutations in S492R only. Only one patient had no KRAS, BRAF or S492R mutations. Discussion: EGFR blockade has no beneficial effect in patients with KRAS or BRAF mutations in their ccfDNA prior to starting chemotherapy. Emergence of KRAS, BRAF or S492R mutations that were undetectable before the start of chemotherapy may be a mechanism underlying acquisition of resistance to EGFR blockade. Notably, emerging KRAS mutations were detected in most of the patients (84%) who acquired resistance. This indicates that the emergence of KRAS mutations may play a considerable role in the acquisition of resistance to EGFR blockade. Citation Format: Takeshi Yamada, Goro Takahashi, Takuma Iwai, Kouki Takeda, Michihiro Koizumi, Akihisa Matsuda, Seiichi Shinji, Yasuyuki Yokoyama, Masahiro Hotta, Keisuke Hara, Satoshi Matsumoto, Keiichiro Ohta, Eiji Uchida. Emerging KRAS mutation can play a considerable role to get acquired resistance to EGFR blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5696. doi:10.1158/1538-7445.AM2017-5696

BRAF V600 mutation detection in melanoma: a comparison of two laboratory testing methods

AimsThe assessment of B-raf proto-oncogene, serine/threonine kinase (BRAF) gene status is now standard practice in patients diagnosed with metastatic melanoma with its presence predicting a clinical response to treatment with...

KRAS and BRAF mutations in serum exosomes from patients with colorectal cancer in a Chinese population.

The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf serine/threonine kinase proto-oncogene (BRAF) mutation in patients with colorectal cancer (CRC), for which the standard gene testing is currently performed using tumor tissue DNA. The aim of the present study was to compare the presence of KRAS and BRAF mutations in the serum exosome and primary tumor tissue from patients with CRC. Genomic DNA were extracted from the tumor tissues of 35 patients with histologically-confirmed CRC and exosomal mRNA were obtained from peripheral blood, which were collected from the corresponding patients prior to surgery. Three mutations in the KRAS gene (codons 12, 13 and 61) and a mutation in the BRAF gene (codon 600) were detected using a polymerase chain reaction-based sequencing method and their presence were compared between tumor tissues and the matched serum exosomes. The KRAS mutation rates in tumor tissues and the matched serum exosomes were 57.6 and 42.4%, respectively, which was not significantly different (P=0.063). The detection rate of the BRAF mutation was 24.2 and 18.2% in tumor tissues and the matched serum exosomes, respectively, and there was no significant difference (P=0.500). The patients with CRC that had a KRAS mutation of codon 12 in exon 2 in their tumor tissues and serum exosomes were significantly older compared with those without this mutation (tumor tissue, P=0.002; serum exosome, P=0.022). The sensitivity of KRAS and BRAF mutation detection using exosomal mRNA was 73.7 and 75%, respectively. The specificity of the detected mutations exhibited an efficiency of 100%, and the total consistency rate was 94.9 and 93.9% for KRAS and BRAF mutations, respectively. These results suggested that serum exosomal mRNA may be used as a novel source for the rapid and non-invasive genotyping of patients with CRC.

Detection of BRAF mutation in the cytocentrifugation supernatant fluid from fine-needle aspiration of thyroid lesions may enhance the diagnostic yield.

Objective:BRAF mutations using cellular DNA from fine-needle aspiration (FNA) specimens are commonly used to support the diagnosis of papillary thyroid carcinoma (PTC). The goal of this study was to preliminarily evaluate the diagnostic utility of detecting BRAF mutations in the routinely discarded FNA specimen supernatant fluid.Materials and Methods:Seventy-eight FNAs of thyroid lesions were evaluated for BRAF mutations using both cellular and supernatant DNA. BRAF mutation data were correlated with cytology and surgical pathology.Results:Of the 78 samples evaluated, 68 (87%) had amplifiable DNA in the supernatant with 2 (3%) positive for BRAF mutations. These two samples showed no mutations in the cellular counterpart. Among the 11 samples showing morphologic findings (FNA/surgical pathology) suspicious/diagnostic of PTC, 6 (55%) samples (one supernatant and five cellulars) were positive for BRAF mutations. This suggests that testing supernatant DNA in FNA specimens may increase the diagnostic yield by 1/11 (9%) in this setting.Conclusions:The vast majority of routinely discarded FNA supernatants contain amplifiable DNA. In addition, profiling the mutations of BRAF and other genes using supernatant DNA may provide valuable diagnostic information to assist the diagnosis of PTC in patients with clinical/morphologic findings suspicious for malignancies and cellular DNA showing no mutations.

Validation of an NGS mutation detection panel for melanoma

BackgroundKnowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.MethodsWhile polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time. We describe here an Ampliseq hotspot panel for melanoma for use with the IonTorrent Personal Genome Machine (PGM) which covers the mutations currently of most clinical interest.ResultsWe have validated this in 151 cases of skin and uveal melanoma from our files, and correlated the data with PCR based assessment of BRAF status. There was excellent agreement, with few discrepancies, though NGS does have greater coverage and picks up some mutations that would be missed by PCR. However, these are often rare and of unknown significance for treatment.ConclusionsPCR methods are rapid, less time-consuming and less expensive than NGS, and could be used as triage for patients requiring more extensive diagnostic workup. The NGS panel described here is suitable for clinical use with formalin-fixed paraffin-embedded (FFPE) samples.

Tracking emerging KRAS, BRAF and EGFR mutations through ccfDNA in colorectal cancers treated with EGFR blockade.

547 Background: Epidermal growth factor receptor (EGFR) blockade can achieve considerable tumor shrinkage in patients with metastatic colorectal cancer (CRC). However, most patients who benefit from EGFR blockade acquire resistance within a year through emerging KRAS, BRAF or EGFR ( S492R) mutations. We have shown that patients with KRAS or BRAF mutations in circulating cell-free DNA (ccfDNA) do not respond favorably to EGFR blockade. In this study, we aimed to detect acquired resistance to EGFR blockade early by using ccfDNA to track emerging KRAS, BRAF and S492R mutations during chemotherapy. Methods: We enrolled 24 patients with metastatic CRC and no KRAS mutations in their primary tumors who were to be treated with systemic chemotherapy that included EGFR blockade. We purified ccfDNA from 1 mL of serum from each patient before they started chemotherapy. We also extracted ccfDNA from these patients every 2–3 months until disease progression. We detected nine KRAS, BRAF (V600E) and S492R mutations using digital polymerase chain reaction. Results: We detected KRAS mutations in ccfDNA of three patients before chemotherapy (12.5%; 3/24). The response rate was 83% (20/24); the four non-responders comprised the three with KRAS mutations and the other with BRAF mutations in their ccfDNA before chemotherapy. All patients with no KRAS or BRAF mutations in their ccfDNA before chemotherapy responded to the chemotherapy (20/20). Of these 20 initially responsive patients, 12 (60%) acquired resistance. We detected emerging KRAS mutations in the ccfDNA of eight of these 12 patients (67%) prior to relapse, six of them having multiple mutations. Five of these patients had mutations in codon 61 and one had solo codon 61 mutations. We also detected BRAF mutations in five patients; none had solo BRAF mutations. Four patients had S492R mutations; none had solo S492R mutations. Conclusions: If KRAS or BRAF mutation do not detected in primary tumor, EGFR blockade has no beneficial effect in patients with KRAS or BRAF mutations in their ccfDNA prior to commencing chemotherapy. Emerging KRAS, BRAF or S492R mutations that were undetectable before starting chemotherapy are associated with acquired resistance to EGFR blockade.

Comparison of Five Different Assays for the Detection of BRAF Mutations in Formalin-Fixed Paraffin Embedded Tissues of Patients with Metastatic Melanoma.

Metastatic or unresectable melanoma is a serious and deadly disease. Anti-BRAF and immunotherapy improved overall survival in patients with metastatic disease. Thus, BRAF genotyping is important to choose the right therapy. In our study, we assessed and compared BRAF mutations in 59 formalin-fixed and paraffin-embedded tumor samples of patients with metastatic melanoma with next-generation sequencing (NGS), Cobas® 4800 BRAF V600 mutation test CE-IVD commercial kit, high-resolution melting PCR (HRM), multiplex real-time allele specific amplification (multiplexed RT-ASA) and immunohistochemistry (IHC). Thirty-one samples were found bearing a BRAF mutation with NGS (52.5%), 28 with Cobas® test (47.5%), 28 with HRM (47.5%), 29 with multiplexed RT-ASA (49.2%) and 27 with IHC (45.8%). Based on NGS data, 26 (81.2%) were c.1799 T>A (p.Val600Glu), 3 (9.4%) were c. 1798-1799 GT>AA (p.Val600Lys), 1 was c.1789_1790 CT>TC (p.Leu597Ser) and 2 were complex mutations. Sensitivity was 90.3% for Cobas® test, 93.1% for multiplexed RT-ASA and 87.1% for IHC and HRM. Specificity was 100% for Cobas® test, IHC and multiplexed RT-ASA and 96.4% for HRM. The reference assay was NGS. Rare mutations were detected with NGS and HRM: c.1789_1790 CT>TC (p.Leu597Ser) mutation and the complex mutation c.1796 A>T; c.1797_1798 insACT (p.Thr599Thr; p.Thr599_Val600insThr). Our data suggest that multiplexed RT-ASA is the most sensitive assay but specific primers for each mutation are needed. HRM can detect all exon 15 mutations but has a lower sensitivity. Because of its specificity for Val600Glu mutation, IHC may be considered only as a screening tool and testing should be completed by a method able to detect other V600 mutations. BRAF Cobas® assay is Val600Glu-specific and has poor sensitivity for the other V600 mutations; thus, it looks important to use multiplex assays able to detect all V600 mutations because a false-negative result will deprive the patient of an important treatment option.

Rapid BRAF mutation detection in melanoma patients by immunohistochemistry

The V600E mutation is the most common (~90%) activating mutation of the BRAF gene. BRAF mutations have been frequently investigated in melanoma, colorectal cancer and papillary thyroid carcinoma. The importance of the detection of BRAF mutations has been rising by the routine use of Braf inhibitor therapy. We evaluated the usefulness of the BRAF V600E mutation-specific monoclonal antibody (VE1) in metastatic melanoma patients. To confirm the results of immunohistochemistry (IHC), we used COBAS 4800 BRAF V600 mutation test and PCR amplification followed by Sanger sequencing.36 of 105 patients have wild-type BRAF gene, 64 have V600E mutation and 5 of 105 have V600K mutation. Predicting the mutation only by IHC using VE1 antibody, all 58 positively scored specimen were V600E mutant. The V600K, the wild-type patients and 7 patients from the V600E mutant group scored as negative. Thus the specificity is 100% and the positive predictive value is 1 of the IHC method. After processing our data we could establish a cheaper diagnostic algorithm for rapid detection ofBRAF mutation.

Coexistence of KRAS and BRAF Mutations in Colorectal Cancer: A Case Report Supporting The Concept of Tumoral Heterogeneity.

The detection of KRAS and BRAF mutations is a crucial step for the correct therapeutic approach and predicting the epidermal growth factor receptor (EGFR)-targeted therapy resistance of colorectal carcinomas. The concomitant KRAS and BRAF mutations occur rarely in the colorectal cancers (CRCs) with the prevalence of less than 0.001% of the cases. In patients with KRAS-mutant tumors, BRAF mutations should not regularly be tested unless the patient is participating in a clinical trial enriching for the presence of KRAS or BRAF-mutated tumor. The current report demonstrates a case with advanced adenocarcinoma of the colon showing the coexistence of KRAS and BRAF mutations and may have profound clinical implications for disease progression and therapeutic responses.

PUB079 EGFR and BRAF Mutation Detection in FFPE Samples of Lung Adenocarcinoma

EGFR-TKIs have been the standard first-line treatment for metastatic NSCLC patients with sensitive EGFR-mutants, meanwhile, other drugs target different driver mutants like BRAF-inhibitor dabrafenib has shown promising efficacy for metastatic BRAF-mutated NSCLC patients. Therefore, spotting patients carrying the targeted mutations is of great significance. However, molecular detection methods applied in clinical practice, especially detection of BRAF in NSCLC patients needs further exploration. Therefore, more sensitive and economic methods are required. We applied the CastPCR technology to the molecular detection of EGFR (del2235-2249, del2236-2250, T790M, L858R) and BRAF (V600E, G469A, D594G) mutations in 144 treatment-naive adenocarcinomas of the lung, and analyzed the association between the mutation rates and patients' clinicopathological features. EGFR mutation: 74 (51.4%, 74/144) cases were identified harboring EGFR mutations. 58 (40.3%, 58/144) carried sensitizing mutations (19Del or L858R) and 21 (14.6%, 21/144) carried resistant mutations (T790M). 10 (6.9%, 10/144) mutation-positive patients were double-mutated. EGFR mutation rate was significantly higher in female compared with that of males (60.9% v.s. 43.8%, P＜0.05), in non-smokers compared with that of smokers (62.8% v.s. 34.5%, P＜0.05). BRAF mutation:12 (8.3%, 12/144) patients were identified with BRAF mutations. 2 were V600E (16.7%, 2/12) and 10 (83.3%, 10/12) were non-V600E. Among the 10 non-V600E mutations, D594G accounted for 9 (90.0%，9/10) and G469A accounted for only 1 (10.0%, 1/10). Statistical analysis demonstrated that BRAF mutation rate was not associated with any of the clinicopathological features: age, gender, smoking status, differentiation degree, tumor size, regional lymph nodes metastasis and distant metastasis of (P≥0.05). The CastPCR technology is a robust method with high sensitivity for molecular detecting of EGFR and BRAF mutations in clinical FFPE samples.

DNA extraction, polymerase chain reaction, and sequencing: workshop in clinical genetics

Abstract DNA extraction, Polymerase Chain Reaction (PCR), and Sequencing are basic methods in molecular biology and genetics. Those there are routinely performed as basic methods in genetic research and currently also for diagnostic lab especially for pathology and human genetics. With the advance in the genetics and clinical service for cancer management, mutation analysis is very important not only for diagnosis but also for prediction of therapeutic response. Detection of KRAS, BRAF, EGFR , and c-KIT mutations is presently performed in almost every molecular pathology lab as part of daily clinical service in cancer management. In this workshop we will discuss tips and tricks for those three basic lab methods. How to improve amount and purity of DNA extraction from blood and tissues, how to avoid DNA degradation during the procedure and storage, how to perform PCR, factors and substance that inhibit polymerases during PCR, how to design effective primer pairs, and how basic theory for sequencing, and interpretation of sequencing will be discussed. Although it has been widely discussed, this workshop is especially important for clinicians who previous do not have hands-on laboratory experience. In addition, number of labs with ability to perform and serve basic genetic and molecular analysis are still limited in Indonesia. With this workshop, we expect to improve knowledge and skill in DNA extraction, PCR, and Sequencing. Keywords : DNA, PCR, sequencing

Detection of mutations in the BRAF gene in patients with KIT and PDGFRA wild-type gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are characterized by mutations in exons 9, 11, 13, and 17 of KIT or exons 12, 14, and 18 of PDGFRA gene. However, approximately 10 to 15% of GISTs lack the mutations in KIT and PDGFRA, and these are referred to as wild-type GISTs which are less sensitive to tyrosine-kinase inhibitors. The aim of this study was to detect BRAF mutations in patients with wild-type GISTs. We applied a sensitive allele-specific PCR, which was optimized using the V600E mutation-harboring cell line RKO, followed by verification of the results by dideoxy sequencing. We selected 149 GIST patients without detectable mutations in KIT and PDGFRA genes from the Slovak national GIST register and analyzed biopsy specimens for the presence of BRAF mutations in exon 15. We identified nine patients with the V600E mutation. The BRAF-driven GISTs were primary gastric (n=3), small intestinal (n=3), colon (n=1), and of uncertain origin (n=1). We also included a liver metastasis of a patient with a simultaneous KIT exon 11-mutated intra-abdominal metastasis. We conclude that genome analysis of wild-type GISTs for mutations should include the BRAF gene, as its mutation status contributes to understanding of pathogenesis and might be important for decisions on therapy.

Mitogen-Activated Protein Kinase Signaling Pathway in Cutaneous Melanoma: An Updated Review.

The mitogen-activated protein kinase (MAPK) signaling pathway is a cascade of protein kinases that act in a sequential and predominantly linear fashion, albeit displaying some cross talk with other signaling cascades. Mutations in proteins integral to the MAPK signaling pathway are present in more than 50% of cutaneous melanomas. The most frequently mutated protein is v-raf murine sarcoma viral oncogene homolog B (BRAF), followed by neuroblastoma Ras viral oncogene homolog (NRAS). Recently, the development of targeted drugs for the treatment of BRAF-mutant melanoma has led to the widespread implementation of molecular assays for the detection of specific BRAF mutations. There have been some attempts to standardize testing of BRAF mutations, but this has not been achieved so far. Here we provide an updated review on the role of the MAPK signaling pathway in the pathogenesis of cutaneous melanoma, focusing on several different BRAF mutations and their diagnostic and therapeutic implications.

1174P - Detection of NRAS, KRAS and BRAF mutations in FFPE derived DNA with a novel targeted resequencing-based diagnostics assay

Identification of cancer-associated mutations has become standard care for cancer treatment. Somatic mutations in NRAS, KRAS and BRAF genes are important to decide the treatment options for patients with cancer. Mutations in NRAS, KRAS or BRAF help to determine the treatment options for patients when EGFR inhibitor or BRAF inhibitor treatment is considered. The novel SOMATIC 1 MASTRTM Plus Dx® assay has been developed to allow analysis of the hotspot mutations or of the full coding region of KRAS, NRAS and BRAF genes in combination with Next Generation Sequencing (NGS). To assess the performance characteristics of the SOMATIC 1 MASTRTM Plus Dx® assay, a multicenter study was performed. The performance of SOMATIC 1 MASTR Plus Dx to detect single nucleotide variants (SNV) and small insertions and/or deletions (indels) in the NRAS, KRAS and BRAF genes at a variant allele frequency (VAF) as 5% in the entire coding region and for specific hotspot mutations was evaluated. The sample population comprised 252 formalin-fixed, paraffin-embedded (FFPE) clinical samples derived from a variety of different cancer types and 4 proficiency samples (Tru-Q Reference Standards, Horizon Discovery). Amplicon libraries were analysed using Illumina MiSeq platform. Data analysis was performed with the SeqNext module (JSI Medical Systems) and the Sophia DDM platform (ILL1MR1S5_somatic1_v2). SOMATIC 1 MASTRTM Plus Dx® showed amplification uniformity of 97.8% (% of amplicons covered at 0.2x of the mean coverage) and a target specificity of 99.1%. The overall limit of detection (LOD) of the assay was determined as 2% VAF, and showed to be as low as 1% for hotspot mutations. Diagnostically, the assay showed 99.96% (95 % CI ≥ 99.90 %) accuracy, 99.96% (95 % CI ≥ 99.90 %) specificity and 100% (95 % CI ≥ 98.54 %) sensitivity. Furthermore, Somatic 1 MASTRTM Plus Dx® assay showed to be 99.99% (95 % CI ≥ 99.97 %) repeatable and 99.96% (95 % CI ≥ 99.91%) reproducible. We have established a targeted and cost-effective NGS assay for the detection of KRAS, NRAS, and BRAF somatic mutations in clinical FFPE samples that can be routinely incorporated into clinical practice.

Clinical significance of BRAF mutation status in circulating tumor DNA of metastatic melanoma patients at baseline.

Circulating tumor DNA is a promising non-invasive tool for cancer monitoring. The main objective of our work was to investigate the relationship between mutant BRAF DNA in plasma and clinical response. Thirty-eight stage IV patients with a V600 mutated BRAF melanoma were included prior to any treatment. DNA was extracted from plasma and mutant DNA was detected using the amplification-refractory mutation system method. Before the beginning of any treatment, the corresponding BRAF mutation was detected in 29 of the 38 tested plasma samples (76.3% positive per cent agreement). We observed a strong correlation between the presence of circulating mutated DNA and overall survival (OS; P=.02), and with the number of metastatic sites (P=.01). The presence of circulating mutated DNA was also strongly correlated with serum LDH activity (P<.01) and S100 protein concentration (P<.01). Finally, seven patients presented discordant BRAF status in different tumor sites. In all these patients, the test performed on ctDNA was positive, suggesting that ctDNA analysis might be less sensitive to tumor heterogeneity. Altogether, these results suggest that plasmatic mutant BRAF DNA is a prognostic factor of OS, correlated with tumor burden. In addition, it represents an interesting alternative source of DNA to detect BRAF mutations before treatment.