Detection of the BRAF V600E mutation is required for use of the BRAF inhibitor, vemurafenib, in patients with metastatic melanoma. Although the Roche Cobas 4800 BRAF V600 Mutation Test is approved, it detects primarily the single-nucleotide V600E mutation and could miss other potentially relevant V600 mutations. To assess the detection rate of the cobas assay for V600 mutations in clinical specimens, we compared the results of this assay with Sanger sequencing in 295 melanoma FFPE samples. Twenty samples were excluded because of invalid results on the cobas (n = 3), sequencing (n = 15), or both (n = 2). V600 mutations were detected by the cobas test in 96 (34.9%) of 275 samples and by Sanger sequencing in 118 (42.9%) of 275 samples. Thus, relative to Sanger sequencing, the cobas test exhibited 80.5% sensitivity (95% CI, 72.4% to 86.6%) and 99.4% specificity (95% CI, 96.5% to 99.9%). Of 23 samples with positive sequencing results but negative cobas results, 21 harbored dinucleotide mutations (V600E in 6, V600K in 10, and V600R in 5); the other two involved single-nucleotide mutations (V600E and V600G). These findings indicate that the cobas assay may miss many V600 mutations in clinical specimens. In our study, the addition of Sanger sequencing for samples with negative cobas results increased the detection rate to 42.9%. This approach could help maximize the number of patients who benefit from BRAF inhibitor treatment. Detection of the BRAF V600E mutation is required for use of the BRAF inhibitor, vemurafenib, in patients with metastatic melanoma. Although the Roche Cobas 4800 BRAF V600 Mutation Test is approved, it detects primarily the single-nucleotide V600E mutation and could miss other potentially relevant V600 mutations. To assess the detection rate of the cobas assay for V600 mutations in clinical specimens, we compared the results of this assay with Sanger sequencing in 295 melanoma FFPE samples. Twenty samples were excluded because of invalid results on the cobas (n = 3), sequencing (n = 15), or both (n = 2). V600 mutations were detected by the cobas test in 96 (34.9%) of 275 samples and by Sanger sequencing in 118 (42.9%) of 275 samples. Thus, relative to Sanger sequencing, the cobas test exhibited 80.5% sensitivity (95% CI, 72.4% to 86.6%) and 99.4% specificity (95% CI, 96.5% to 99.9%). Of 23 samples with positive sequencing results but negative cobas results, 21 harbored dinucleotide mutations (V600E in 6, V600K in 10, and V600R in 5); the other two involved single-nucleotide mutations (V600E and V600G). These findings indicate that the cobas assay may miss many V600 mutations in clinical specimens. In our study, the addition of Sanger sequencing for samples with negative cobas results increased the detection rate to 42.9%. This approach could help maximize the number of patients who benefit from BRAF inhibitor treatment. Melanoma is one of the most common cancers in the United States and appears to be increasing in incidence.1Simard E.P. Ward E.M. Siegel R. Jemal A. Cancers with increasing incidence trends in the United States: 1999 through 2008.CA Cancer J Clin. 2012; 62: 118-128Crossref Scopus (593) Google Scholar, 2Cokkinides V.E. Geller A.C. Jemal A. Trends in melanoma mortality among non-Hispanic whites by educational attainment, 1993–2007.Arch Dermatol. 2012; 148: 587-591Crossref PubMed Scopus (12) Google Scholar, 3Jemal A. Saraiya M. Patel P. Cherala S.S. Barnholtz-Sloan J. Kim J. Wiggins C.L. Wingo P.A. Recent trends in cutaneous melanoma incidence and death rates in the United States, 1992–2006.J Am Acad Dermatol. 2011; 65: S17-S25Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar Although melanoma is the most aggressive form of skin cancer, it remains highly curable if it is detected early: 5-year survival is approximately 90% overall and exceeds 98% when disease is detected at the localized stage (84% of cases).4Howlader N, Noone AM, Krapcho M, Garshell J, Neyman N, Altekruse SF, et al (eds). SEER Cancer Statistics Review 1975-2009. Table 16.8 Melanoma of the Skin (Invasive): 5-Year Relative and Period Survival (Percent) by Race, Sex, Diagnosis Year, Stage and Age. Bethesda, MD: National Cancer Institute. Available at: http://seer.cancer.gov/csr/1975_2009_pops09/browse_csr.php?section=16&page=sect_16_table.08.html. Accessed July 17, 2012.Google Scholar However, approximately 4% of melanoma cases are diagnosed after distant metastasis, when 5-year survival is only 15%4Howlader N, Noone AM, Krapcho M, Garshell J, Neyman N, Altekruse SF, et al (eds). SEER Cancer Statistics Review 1975-2009. Table 16.8 Melanoma of the Skin (Invasive): 5-Year Relative and Period Survival (Percent) by Race, Sex, Diagnosis Year, Stage and Age. Bethesda, MD: National Cancer Institute. Available at: http://seer.cancer.gov/csr/1975_2009_pops09/browse_csr.php?section=16&page=sect_16_table.08.html. Accessed July 17, 2012.Google Scholar; median survival in patients with metastatic disease is <1 year.5Altekruse S.F. Kosary C.L. Krapcho M. Neyman N. Aminou R. Waldron W. 2010 SEER Cancer Statistics Review, 1975–2007. National Cancer Institute, Bethesda2010Google Scholar Increased knowledge of the molecular changes that drive cancer development can allow development of targeted therapeutics that can improve survival in patients with advanced melanoma.6Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O’Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6111) Google Scholar, 7Hauschild A. Grob J.J. Demidov L.V. Jouary T. Gutzmer R. Millward M. Rutkowski P. Blank C.U. Miller Jr., W.H. Kaempgen E. Martin-Algarra S. Karaszewska B. Mauch C. Chiarion-Sileni V. Martin A.M. Swann S. Haney P. Mirakhur B. Guckert M.E. Goodman V. Chapman P.B. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial.Lancet. 2012; 380: 358-365Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar Mutations in the BRAF oncogene are the most frequently reported molecular alterations in melanoma. More than 50% of malignant melanomas harbor an activating V600 mutation in the BRAF gene.8Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8270) Google Scholar BRAF plays important roles in the proliferation and survival of melanoma cells through activation of the RAF/MEK/ERK mitogen-activated protein kinase pathway.9Dhomen N. Marais R. BRAF signaling and targeted therapies in melanoma.Hematol Oncol Clin North Am. 2009; 23 (ix): 529-545Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10Cohen C. Zavala-Pompa A. Sequeira J.H. Shoji M. Sexton D.G. Cotsonis G. Cerimele F. Govindarajan B. Macaron N. Arbiser J.L. Mitogen-activated protein kinase activation is an early event in melanoma progression.Clin Cancer Res. 2002; 8: 3728-3733PubMed Google Scholar, 11Satyamoorthy K. Li G. Gerrero M.R. Brose M.S. Volpe P. Weber B.L. Van B.P. Elder D.E. Herlyn M. Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation.Cancer Res. 2003; 63: 756-759PubMed Google Scholar These factors make BRAF an attractive candidate for specific targeted therapies. Treatment with the BRAF inhibitor, vemurafenib, has been reported to improve survival relative to chemotherapy in patients with metastatic melanoma harboring the most common activating BRAF mutation, V600E.6Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O’Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6111) Google Scholar, 12Sosman J.A. Kim K.B. Schuchter L. Gonzalez R. Pavlick A.C. Weber J.S. McArthur G.A. Hutson T.E. Moschos S.J. Flaherty K.T. Hersey P. Kefford R. Lawrence D. Puzanov I. Lewis K.D. Amaravadi R.K. Chmielowski B. Lawrence H.J. Shyr Y. Ye F. Li J. Nolop K.B. Lee R.J. Joe A.K. Ribas A. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1768) Google Scholar According to vemurafenib labeling, the V600E mutation must be identified using a Food and Drug Administration (FDA)–approved test to establish eligibility for treatment in patients with inoperable or metastatic melanoma. At present, the Cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics, Pleasanton, CA) is the only FDA-approved assay for this purpose. Although this oligonucleotide probe-based test primarily detects V600E, variant V600 mutations (eg, V600K, V600R, V600E2, and V600D) are inconsistently detected as well. The detection rate for the most common of these variants, V600K, has been reported to be 65.8%.6Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O’Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6111) Google Scholar Overall, V600K might account for 6% to 30% of V600 mutations.13Rubinstein J.C. Sznol M. Pavlick A.C. Ariyan S. Cheng E. Bacchiocchi A. Kluger H.M. Narayan D. Halaban R. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032.J Transl Med. 2010; 8: 67Crossref PubMed Scopus (211) Google Scholar, 14Amanuel B. Grieu F. Kular J. Millward M. Iacopetta B. Incidence of BRAF p.Val600Glu and p.Val600Lys mutations in a consecutive series of 183 metastatic melanoma patients from a high incidence region.Pathology. 2012; 44: 357-359Crossref PubMed Scopus (49) Google Scholar Because patients harboring V600K have been documented to respond to vemurafenib,6Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O’Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6111) Google Scholar, 12Sosman J.A. Kim K.B. Schuchter L. Gonzalez R. Pavlick A.C. Weber J.S. McArthur G.A. Hutson T.E. Moschos S.J. Flaherty K.T. Hersey P. Kefford R. Lawrence D. Puzanov I. Lewis K.D. Amaravadi R.K. Chmielowski B. Lawrence H.J. Shyr Y. Ye F. Li J. Nolop K.B. Lee R.J. Joe A.K. Ribas A. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1768) Google Scholar alternative approaches to detect V600 mutations in cases with negative results on the cobas test could help identify additional patients who might benefit from BRAF inhibitor therapy. Several other methods can be used to detect BRAF mutations. Among them, Sanger DNA sequencing is one of the most well-established methods and is widely used to identify mutations in DNA derived from formalin-fixed, paraffin-embedded (FFPE) tumor specimens. Until recently, Sanger sequencing was the primary method used by clinical laboratories to detect BRAF V600 mutations. Unlike the cobas test, Sanger sequencing can differentiate V600E from alternative V600 mutations that may be of potential clinical relevance. Our initial validation studies of the cobas assay demonstrated that it did not detect V600 mutations in a subset of specimens that were positive by Sanger sequencing. In this study, we compared the V600 mutation detection rate of the cobas assay with that of Sanger sequencing in a large series of deidentified FFPE samples from patients with metastatic melanoma. Our intent was to better understand the performance of the Cobas 4800 BRAF V600 Mutation Test in a clinical laboratory setting, and to determine whether Sanger sequencing could provide additional information in cases negative for mutation with the cobas test. Consecutive deidentified FFPE tissues (n = 295) submitted to Quest Diagnostics Nichols Institute (San Juan Capistrano, CA; and Chantilly, VA) for BRAF mutation analysis were included in this study. All FFPE specimens contained histologically confirmed melanoma. The median age of patients was 66 (range, 32 to 96) years, and 184 patients (62.4%) were men. H&E-stained slides from each submitted sample were reviewed by a pathologist, and tumor tissue was identified for analysis. FFPE samples containing <50% tumor cells were subjected to macrodissection. Each tissue was tested using the cobas method and Sanger sequencing. For all 295 tissue specimens, the cobas assay was performed on total DNA extracted from sections (5 μm thick) using the cobas extraction kit (Roche Molecular Diagnostics). For the first 102 samples, Sanger sequencing was also performed on DNA extracted with the cobas extraction kit. For the other 193 samples, Sanger sequencing was performed on DNA extracted with the alternative extraction protocol (ie, the standard extraction protocol used for Sanger sequencing in our laboratory). This approach was meant to address the concern that differences in V600 mutation detection rates between Sanger and cobas sequencing might be due to the extraction process used. For the alternative extraction method, total DNA was extracted from a section (10 μm thick) using Agencourt extraction kits (Beckman Coulter, Brea, CA) or FFPE DNA extraction kits (Qiagen, Valencia, CA). DNA was quantified on a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). V600 mutations were detected using the Cobas 4800 BRAF V600 Mutation Test, according to the manufacturer’s protocol. The cobas results were reported as follows: i) V600E mutation detected, ii) V600E mutation not detected, or iii) invalid (ie, no result was obtained on the cobas test). Bidirectional Sanger sequencing of BRAF mutations was performed using a laboratory-developed method. Briefly, PCR was performed to detect mutations at exon 15 of the BRAF gene with the following forward and reverse primers: BRAF 15-forward, 5′-CCTAAACTCTTCATAATGCTTGCT-3′; and BRAF 15-reverse, 5′-AGTAACTCAGCAGCATCTCAGG-3′. PCR was performed in a 50-μL volume containing 50 to 100 ng genomic DNA; 20 pmol/L forward and 20 pmol/L reverse primers; 50 μmol/L (each) deoxy-ATP, deoxy-CTP, and deoxy-GTP; 400 μmol/L deoxy-UTP; 1.5 mmol/L MgCl2; 1× ABI PCR buffer containing 1.5 mmol/L MgCl2; and 2.5 U HotStarTaq DNA polymerase (Qiagen). The PCR amplification was performed under the following conditions: 1 cycle at 95°C for 15 minutes; 40 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds; and a final extension step at 72°C for 7 minutes. Amplified products were purified using the ABI cleanup kit and sequenced in both directions using the BigDye Terminator, version 3.1, Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on ABI3730 running ABI Prism DNA Sequence Analysis Software version 2.6 (Applied Biosystems). Results for the V600 mutation are reported as mutation not detected, mutation detected (V600 E, K, or R), or invalid (ie, no sequencing data obtained). Differences in mutation detection frequencies between Sanger sequencing and the cobas assay were assessed using McNemar’s test. The R statistical package (http://cran.r-project.org, last accessed February 15, 2012) was used for all statistical analyses. BRAF V600 mutation status was determined using the cobas test and Sanger sequencing in a total of 295 melanoma FFPEs. For all specimens, the cobas test was performed on DNA extracted by the cobas protocol. Because we normally use a different DNA extraction procedure for Sanger sequencing, we first determined whether the extraction technique would affect agreement between Sanger sequencing and the cobas test. Sanger sequencing was, thus, performed on DNA extracted using the cobas extraction protocol in 102 of the 295 samples and an alternative protocol in the other 193 samples. Between-method agreement for V600 mutation status was similar regardless of which extraction procedure was used for Sanger sequencing: 91.4% for samples extracted using the cobas method and 91.2% for those extracted using the alternative procedure. Thus, results were pooled for subsequent analyses. Among the 295 samples, invalid results were obtained by the cobas assay alone in 3, by Sanger sequencing alone in 15, and by both methods in another 2. These 20 samples were excluded from further analysis. Among the remaining 275 specimens, 118 (42.9%) were positive and 157 (57.1%) were negative for a V600 mutation by Sanger sequencing. The cobas method detected a V600 mutation in 95 of the 118 specimens that were positive by Sanger sequencing, as well as one sample that was negative by Sanger sequencing. Thus, relative to Sanger sequencing, the cobas test demonstrated 80.5% sensitivity (95% CI, 72.4% to 86.6%) and 99.4% specificity (95% CI, 96.5% to 99.9%). Based on the V600 mutation prevalence of 42.9% (by Sanger sequencing) in this sample set, the positive predictive value of the cobas test for the BRAF V600 mutation was 98.9% (95% CI, 94.3% to 99.9%) and the negative predictive value was 87.2% (95% CI, 81.5% to 91.3%). Overall agreement between the cobas and Sanger sequencing was 91.3% (Table 1).Table 1Agreement of Cobas V600 Mutation Test and Sanger Sequencing for Detection of BRAF V600 Mutations in FFPE Melanoma SamplesCobas testSanger sequencingNot detectedDetectedInvalidNot detected1562311Detected1954Invalid212 Open table in a new tab Sanger sequencing yielded a significantly greater detection rate (42.9%, 118/275) for V600 mutations than did the cobas method (34.9%, 96/275) (P < 0.001), detecting V600 mutations in 23 samples that were negative by the cobas assay (Table 2). Conversely, only one specimen was positive for the V600 mutation by the cobas method but negative by Sanger sequencing. The cobas assay detected only 62.9% of V600K mutations by Sanger sequencing and no V600R/G variants (Table 2). By Sanger sequencing, V600E was the most common mutation (72.0%), followed by V600K (22.9%) and V600R/G (5.1%).Table 2Detailed Summary of Cobas and Sanger Sequencing Mutation ResultsCobas testSanger sequencingNot detectedV600EV600KV600R/GNot detected1567∗Of seven mutations, six had dinucleotide mutations (GTG>GAA) and one had a GAG mutation.10†All had dinucleotide mutations (GTG>AAG).6‡Five V600R had dinucleotide mutations (GTG>AGG) and V600G had GTG>GGG.V600 detected178170∗ Of seven mutations, six had dinucleotide mutations (GTG>GAA) and one had a GAG mutation.† All had dinucleotide mutations (GTG>AAG).‡ Five V600R had dinucleotide mutations (GTG>AGG) and V600G had GTG>GGG. Open table in a new tab Six of seven samples with a V600E mutation detected by Sanger sequencing, but not by the cobas assay, had dinucleotide mutations (GTG>GAA), whereas all 78 samples with a V600E mutation detected by both methods had single-nucleotide mutations (GTG>GAG) (Figure 1). Dinucleotide mutations were also noted in all samples with V600K (GTG>AAG) and V600R (GTG>AGG) mutations. The V600G had a single-nucleotide mutation (GTG>GGG). Two samples had invalid results with both methods. Of the 15 samples with invalid Sanger sequencing results, four were V600 mutation positive by the cobas assay. Conversely, the cobas assay produced three invalid results, one of which was positive for V600E by Sanger sequencing. The results of this study demonstrate that Sanger sequencing can detect V600 mutations in many melanoma samples with negative results on the Cobas 4800 BRAF V600 Mutation Test. The overall frequency of V600 mutations in our study was 42.9% by Sanger sequencing and 34.9% by the cobas assay. These values are consistent with previously reported V600 mutation frequencies in melanoma, which have ranged from approximately 20% to 80%.15Platz A. Egyhazi S. Ringborg U. Hansson J. Human cutaneous melanoma: a review of NRAS and BRAF mutation frequencies in relation to histogenetic subclass and body site.Mol Oncol. 2008; 1: 395-405Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar Variations in reported BRAF mutation rates may be attributable to differences in the detection methods used. Sanger sequencing is one of the most widely used methods to identify mutations in DNA derived from FFPE. It can detect different mutations in the sequence analyzed. However, sequencing has relatively low analytical sensitivity, meaning that a mutation must be present in >15% to 20% of tumor content to be detected.16Li J. Wang L. Mamon H. Kulke M.H. Berbeco R. Makrigiorgos G.M. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.Nat Med. 2008; 14: 579-584Crossref PubMed Scopus (319) Google Scholar Low frequencies of BRAF mutation were reported in studies that used sequencing as the sole method of mutation analysis.13Rubinstein J.C. Sznol M. Pavlick A.C. Ariyan S. Cheng E. Bacchiocchi A. Kluger H.M. Narayan D. Halaban R. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032.J Transl Med. 2010; 8: 67Crossref PubMed Scopus (211) Google Scholar Several real-time PCR methods have been developed to detect BRAF mutations, including allele-specific PCR, pyrosequencing,17Spittle C. Ward M.R. Nathanson K.L. Gimotty P.A. Rappaport E. Brose M.S. Medina A. Letrero R. Herlyn M. Edwards R.H. Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma.J Mol Diagn. 2007; 9: 464-471Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar competitive allele-specific TaqMan,18Didelot A. Le C.D. Luscan A. Cazes A. Pallier K. Emile J.F. Laurent-Puig P. Blons H. Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples.Exp Mol Pathol. 2012; 92: 275-280Crossref PubMed Scopus (95) Google Scholar and amplification-refractory mutation system-PCR.19Ellison G. Donald E. McWalter G. Knight L. Fletcher L. Sherwood J. Cantarini M. Orr M. Speake G. A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples.J Exp Clin Cancer Res. 2010; 29: 132Crossref PubMed Scopus (135) Google Scholar Increased BRAF mutation rates have been reported in studies using these methods, which can detect mutations present at levels as low as 1% of total cells.20Dong J. Phelps R.G. Qiao R. Yao S. Benard O. Ronai Z. Aaronson S.A. BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma.Cancer Res. 2003; 63: 3883-3885PubMed Google Scholar However, unlike Sanger sequencing, these methods detect only the mutations they were designed to interrogate. Carbonell and colleagues21Carbonell P. Turpin M.C. Torres-Moreno D. Molina-Martinez I. Garcia-Solano J. Perez-Guillermo M. Conesa-Zamora P. Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.J Mol Diagn. 2011; 13: 467-473Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar also reported detection of V600 mutations using denaturing high-performance liquid chromatography, high-resolution melting, TaqMan, and sequencing analysis of archival colorectal cancer tissue.21Carbonell P. Turpin M.C. Torres-Moreno D. Molina-Martinez I. Garcia-Solano J. Perez-Guillermo M. Conesa-Zamora P. Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.J Mol Diagn. 2011; 13: 467-473Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The TaqMan assay was the most effective of the four methods for determining V600 mutation status, followed by denaturing high-performance liquid chromatography, high-resolution melting, and sequencing. When considering all V600 variants, standard Sanger sequencing detected mutations in 23 specimens identified as mutation not detected using the cobas assay in the current study. Of these 23 specimens, seven harbored V600E, the mutation the cobas assay was designed to detect. Differences in detection rates between methods may be related to the type of mutation present: six of the seven V600E mutations detected by Sanger sequencing, but not by the cobas assay, were dinucleotide substitutions (GTG>GAA), whereas all 78 samples with a V600E mutation detected by both methods had a single-nucleotide mutation (GTG>GAG). The cobas test is a single-oligonucleotide probe-based test that has greater analytical sensitivity than Sanger sequencing. However, as our findings suggest, its specificity for a single-nucleotide mutation means that it may miss relevant dinucleotide mutations in some cases. Sanger sequencing can detect single-nucleotide and dinucleotide mutations in the same region, which accounts for its higher detection rate in this study. This difference is also consistent with the relatively low detection rate for other dinucleotide substitutions at codon V600 by the cobas method. In fact, much of the difference in V600 detection rates between Sanger sequencing and the cobas assay was attributable to V600 mutations other than V600E. Only 62.9% of the V600K mutations identified by Sanger sequencing were detected by the cobas assay; this is marginally lower than in previous reports, in which the cobas method detected approximately 66% to 70% of samples with V600K.22Anderson S. Bloom K.J. Vallera D.U. Rueschoff J. Meldrum C. Schilling R. Kovach B. Lee J.R. Ochoa P. Langland R. Halait H. Lawrence H.J. Dugan M.C. Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma.Arch Pathol Lab Med. 2012; 136: 1385-1391Crossref PubMed Scopus (118) Google Scholar Again, the low detection rate of V600K may stem from the fact that this mutation involves dinucleotide substitutions (GTG>AAG). The relevance of the low detection rate for V600 mutations other than V600E depends on their prevalence in the patient population. Reported frequencies of non-V600E mutations vary among studies. In our study, V600K accounted for approximately one quarter of mutations in our series. This rate is higher than reported in several previous studies, including clinical trials of BRAF (7.5%)12Sosman J.A. Kim K.B. Schuchter L. Gonzalez R. Pavlick A.C. Weber J.S. McArthur G.A. Hutson T.E. Moschos S.J. Flaherty K.T. Hersey P. Kefford R. Lawrence D. Puzanov I. Lewis K.D. Amaravadi R.K. Chmielowski B. Lawrence H.J. Shyr Y. Ye F. Li J. Nolop K.B. Lee R.J. Joe A.K. Ribas A. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib.N Engl J Med. 2012; 366: 707-714Crossref PubMed Scopus (1768) Google Scholar and MEK (12%) inhibitors.23Flaherty K.T. Robert C. Hersey P. Nathan P. Garbe C. Milhem M. Demidov L.V. Hassel J.C. Rutkowski P. Mohr P. Dummer R. Trefzer U. Larkin J.M. Utikal J. Dreno B. Nyakas M. Middleton M.R. Becker J.C. Casey M. Sherman L.J. Wu F.S. Ouellet D. Martin A.M. Patel K. Schadendorf D. METRIC Study GroupImproved survival with MEK inhibition in BRAF-mutated melanoma.N Engl J Med. 2012; 367: 107-114Crossref PubMed Scopus (1712) Google Scholar However, reported rates of V600K have varied from approximately 6% to 30%.13Rubinstein J.C. Sznol M. Pavlick A.C. Ariyan S. Cheng E. Bacchiocchi A. Kluger H.M. Narayan D. Halaban R. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032.J Transl Med. 2010; 8: 67Crossref PubMed Scopus (211) Google Scholar, 14Amanuel B. Grieu F. Kular J. Millward M. Iacopetta B. Incidence of BRAF p.Val600Glu and p.Val600Lys mutations in a consecutive series of 183 metastatic melanoma patients from a high incidence region.Pathology. 2012; 44: 357-359Crossref PubMed Scopus (49) Google Scholar, 24Long G.V. Menzies A.M. Nagrial A.M. Haydu L.E. Hamilton A.L. Mann G.J. Hughes T.M. Thompson J.F. Scolyer R.A. Kefford R.F. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.J Clin Oncol. 2011; 29: 1239-1246Crossref PubMed Scopus (825) Google Scholar, 25Menzies A.M. Haydu L.E. Visintin L. Carlino M.S. Howle J.R. Thompson J.F. Kefford R.F. Scolyer R.A. Long G.V. Distinguishing clinicopathologic features of patients with V600E and V600K BRAF-mutant metastatic melanoma.Clin Cancer Res. 2012; 18: 3242-3249Crossref PubMed Scopus (355) Google Scholar Reasons for this variation are unclear, because most studies used Sanger sequencing or other methods that should efficiently detect such variants. Differences in the patient age distributions could play a role: one study found that the frequency of non-V600E mutation genotypes increases with age, comprising <20% of V600 mutations in patients <50 years but >40% in patients ≥70 years.25Menzies A.M. Haydu L.E. Visintin L. Carlino M.S. Howle J.R. Thompson J.F. Kefford R.F. Scolyer R.A. Long G.V. Distinguishing clinicopathologic features of patients with V600E and V600K BRAF-mutant metastatic melanoma.Clin Cancer Res. 2012; 18: 3242-3249Crossref PubMed Scopus (355) Google Scholar The median age of patients in the current study was 66 years. High rates of V600K mutations could have important implications for determining eligibility for treatment with BRAF or MEK inhibitors. In preclinical studies, vemurafenib was reported to strongly inhibit melanoma cell lines expressing V600K (or other V600 variants), in addition to those expressing V600E.26Yang H. Higgins B. Kolinsky K. Packman K. Go Z. Iyer R. Kolis S. Zhao S. Lee R. Grippo J.F. Schostack K. Simcox M.E. Heimbrook D. Bollag G. Su F. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models.Cancer Res. 2010; 70: 5518-5527Crossref PubMed Scopus (357) Google Scholar This response is consistent with limited evidence from two clinical trials of vemurafenib, in which 4 of 10 patients carrying V600K showed a response.6Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O’Day S.J. Sosman J.A. 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Therefore, inhibition of these proteins also blocks the effects of BRAF activation. In the trametinib trial, patients with V600K showed improvements in progression and overall survival rates similar to those of patients with V600E.23Flaherty K.T. Robert C. Hersey P. Nathan P. Garbe C. Milhem M. Demidov L.V. Hassel J.C. Rutkowski P. Mohr P. Dummer R. Trefzer U. Larkin J.M. Utikal J. Dreno B. Nyakas M. Middleton M.R. Becker J.C. Casey M. Sherman L.J. Wu F.S. Ouellet D. Martin A.M. Patel K. Schadendorf D. METRIC Study GroupImproved survival with MEK inhibition in BRAF-mutated melanoma.N Engl J Med. 2012; 367: 107-114Crossref PubMed Scopus (1712) Google Scholar In contrast to our findings, clinical studies by Anderson et al22Anderson S. Bloom K.J. Vallera D.U. Rueschoff J. Meldrum C. Schilling R. Kovach B. Lee J.R. Ochoa P. Langland R. Halait H. Lawrence H.J. Dugan M.C. Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma.Arch Pathol Lab Med. 2012; 136: 1385-1391Crossref PubMed Scopus (118) Google Scholar found that the cobas assay detected more V600 mutations than Sanger sequencing. Several considerations may contribute to the difference between their results and ours. First, the selection of melanoma samples differed. The specimens used in our study were consecutive clinical FFPE samples that were sent to Quest Diagnostics Nichols Institute for BRAF mutation analysis. Second, the protocol for FFPE specimen preparation and Sanger sequencing may have differed between the two sites. Despite its long history of successful use for detection of mutations in DNA derived from FFPE samples, Sanger sequencing has variable sensitivity that is influenced by assay design and the specific protocol used.8Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8270) Google Scholar Although we did not select samples with greater than average tumor content, we did enhance tumor content through macrodissection when necessary in samples with small amounts of tumor tissue. The DNA extraction procedure might also affect sensitivity. To account for this possibility, we performed both cobas testing and Sanger sequencing on 102 DNA samples extracted with the cobas procedure. The agreement between the cobas assay and Sanger sequencing was similar to that obtained when we used an alternative method for DNA extraction for Sanger sequencing, suggesting that the extraction procedure did not significantly affect assay concordance. Although the findings of this study suggest that Sanger sequencing can detect V600 mutations in more melanoma cases than can cobas, several factors prevent us from recommending its use as a first-line test. First, the primary reason to determine V600 mutation status is to document eligibility for treatment with vemurafenib. At present, the labeling for vemurafenib requires that this mutation be documented with an FDA-approved test because the trial used to approve the drug used this test. Second, as previously noted, Sanger sequencing requires a relatively large tumor content, and variations in sample preparation among different laboratories could lead to differences in assay success rate. In our study, many samples (15/275, 5.5%) could not be successfully Sanger sequenced, although most of these yielded valid results with the cobas assay. Nevertheless, our results suggest that performing Sanger sequencing on samples negative by the cobas assay could identify many patients with V600 mutations who would otherwise be ineligible for treatment with vemurafenib. If this approach had been used to determine vemurafenib eligibility in the samples in this study, an additional 23 patients would have been eligible; this represents a 23% increase relative to determination with the cobas test alone. However, use of vemurafenib in these cases would require the clinical judgment of the clinician. In conclusion, the sensitivity of the cobas test relative to Sanger sequencing was 80.5%, and specificity was 99.4%. The Sanger sequencing method detected 23 additional V600 mutations not detected by cobas. These findings suggest that sole reliance on the cobas assay could miss identification of many patients who might benefit from therapy with BRAF inhibitors. Use of Sanger sequencing as a second-line test for samples that are negative on the cobas assay could be a rational approach to maximizing the potential benefit of vemurafenib. We thank Jeff Radcliff (Quest Diagnostics) for critical review and suggestions on the manuscript.