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Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

2007; Elsevier BV; Volume: 9; Issue: 2 Linguagem: Inglês

10.2353/jmoldx.2007.060135

1943-7811

Molly Yancovitz, Joanne Yoon, Maryann Mikhail, Weiming Gai, Richard L. Shapiro, Russell S. Berman, Anna C. Pavlick, Paul B. Chapman, Iman Osman, David Polsky,

Cutaneous Melanoma Detection and Management

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients. Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients. Mutations in the BRAF oncogene are the most frequently reported molecular alterations in melanoma. Several groups, including ours, have identified these mutations in primary and metastatic melanoma tumors and melanoma cell lines at rates ranging from 31 to 75%.1Pollock PM Harper UL Hansen KS Yudt LM Stark M Robbins CM Moses TY Hostetter G Wagner U Kakareka J Salem G Pohida T Heenan P Duray P Kallioniemi O Hayward NK Trent JM Meltzer PS High frequency of BRAF mutations in nevi.Nat Genet. 2003; 33: 19-20Crossref PubMed Scopus (1350) Google Scholar2Davies H Bignell GR Cox C Stephens P Edkins S Clegg S Teague J Woffendin H Garnett MJ Bottomley W Davis N Dicks E Ewing R Floyd Y Gray K Hall S Hawes R Hughes J Kosmidou V Menzies A Mould C Parker A Stevens C Watt S Hooper S Wilson R Jayatilake H Gusterson BA Cooper C Shipley J Hargrave D Pritchard-Jones K Maitland N Chenevix-Trench G Riggins GJ Bigner DD Palmieri G Cossu A Flanagan A Nicholson A Ho JW Leung SY Yuen ST Weber BL Seigler HF Darrow TL Paterson H Marais R Marshall CJ Wooster R Stratton MR Futreal PA Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8204) Google Scholar3Gorden A Osman I Gai W He D Huang W Davidson A Houghton AN Busam K Polsky D Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar4Kumar R Angelini S Czene K Sauroja I Hahka-Kemppinen M Pyrhonen S Hemminki K BRAF mutations in metastatic melanoma: a possible association with clinical outcome.Clin Cancer Res. 2003; 9: 3362-3368PubMed Google Scholar5Shinozaki M Fujimoto A Morton DL Hoon DS Incidence of BRAF oncogene mutation and clinical relevance for primary cutaneous melanomas.Clin Cancer Res. 2004; 10: 1753-1757Crossref PubMed Scopus (195) Google Scholar6Dong J Phelps RG Qiao R Yao S Benard O Ronai Z Aaronson SA BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma.Cancer Res. 2003; 63: 3883-3885PubMed Google Scholar7Uribe P Wistuba II Gonzalez S BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin.Am J Dermatopathol. 2003; 25: 365-370Crossref PubMed Scopus (129) Google Scholar8Maldonado JL Fridlyand J Patel H Jain AN Busam K Kageshita T Ono T Albertson DG Pinkel D Bastian BC Determinants of BRAF mutations in primary melanomas.J Natl Cancer Inst. 2003; 95: 1878-1890Crossref PubMed Scopus (530) Google Scholar BRAF, a serine-threonine kinase, activates the mitogen-activated protein kinase cascade, a pathway critical in tumor cell proliferation.9Govindarajan B Bai X Cohen C Zhong H Kilroy S Louis G Moses M Arbiser JL Malignant transformation of melanocytes to melanoma by constitutive activation of mitogen-activated protein kinase kinase (MAPKK) signaling.J Biol Chem. 2003; 278: 9790-9795Crossref PubMed Scopus (108) Google Scholar10Cohen C Zavala-Pompa A Sequeira JH Shoji M Sexton DG Cotsonis G Cerimele F Govindarajan B Macaron N Arbiser JL Mitogen-activated protein kinase activation is an early event in melanoma progression.Clin Cancer Res. 2002; 8: 3728-3733PubMed Google Scholar11Satyamoorthy K Li G Gerrero MR Brose MS Volpe P Weber BL Van Belle P Elder DE 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 The codon 600 mutation (formerly 5994Kumar R Angelini S Czene K Sauroja I Hahka-Kemppinen M Pyrhonen S Hemminki K BRAF mutations in metastatic melanoma: a possible association with clinical outcome.Clin Cancer Res. 2003; 9: 3362-3368PubMed Google Scholar), which accounts for >90% of BRAF mutations identified in melanoma, possesses constitutive kinase activity and can transform primary mouse embryonic fibroblasts.2Davies H Bignell GR Cox C Stephens P Edkins S Clegg S Teague J Woffendin H Garnett MJ Bottomley W Davis N Dicks E Ewing R Floyd Y Gray K Hall S Hawes R Hughes J Kosmidou V Menzies A Mould C Parker A Stevens C Watt S Hooper S Wilson R Jayatilake H Gusterson BA Cooper C Shipley J Hargrave D Pritchard-Jones K Maitland N Chenevix-Trench G Riggins GJ Bigner DD Palmieri G Cossu A Flanagan A Nicholson A Ho JW Leung SY Yuen ST Weber BL Seigler HF Darrow TL Paterson H Marais R Marshall CJ Wooster R Stratton MR Futreal PA Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8204) Google Scholar,12Rajagopalan H Bardelli A Lengauer C Kinzler KW Vogelstein B Velculescu VE Tumorigenesis: rAF/RAS oncogenes and mismatch-repair status.Nature. 2002; 418: 934Crossref PubMed Scopus (1058) Google Scholar13Brose MS Volpe P Feldman M Kumar M Rishi I Gerrero R Einhorn E Herlyn M Minna J Nicholson A Roth JA Albelda SM Davies H Cox C Brignell G Stephens P Futreal PA Wooster R Stratton MR Weber BL BRAF and RAS mutations in human lung cancer and melanoma.Cancer Res. 2002; 62: 6997-7000PubMed Google Scholar14Mercer K Giblett S Green S Lloyd D Darocha Dias S Plumb M Marais R Pritchard C Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts.Cancer Res. 2005; 65: 11493-11500Crossref PubMed Scopus (123) Google Scholar In addition, two studies have demonstrated that knockdown of mutant BRAF V600E expression in cultured human melanoma cell lines inhibits cell growth and invasion and promotes apoptosis.15Hingorani SR Jacobetz MA Robertson GP Herlyn M Tuveson DA Suppression of BRAF(V599E) in human melanoma abrogates transformation.Cancer Res. 2003; 63: 5198-5202PubMed Google Scholar,16Sumimoto H Miyagishi M Miyoshi H Yamagata S Shimizu A Taira K Kawakami Y Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference.Oncogene. 2004; 23: 6031-6039Crossref PubMed Scopus (161) Google Scholar Thus, the BRAF oncogene is an attractive target of new treatments for metastatic melanoma. For clinical trials of treatments with well-defined molecular targets, patient selection based on the mutational status of their metastatic tumor allows for substantially fewer patients to be enrolled to achieve appropriate statistical power.17Simon R Maitournam A Evaluating the efficiency of targeted designs for randomized clinical trials.Clin Cancer Res. 2004; 10: 6759-6763Crossref PubMed Scopus (262) Google Scholar This is based on the fact that agents that target specific molecular alterations in tumor cells have shown the highest response rates in tumors that carry these alterations.18Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Brannigan BW Harris PL Haserlat SM Supko JG Haluska FG Louis DN Christiani DC Settleman J Haber DA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9953) Google Scholar19Tsao MS Sakurada A Cutz JC Zhu CQ Kamel-Reid S Squire J Lorimer I Zhang T Liu N Daneshmand M Marrano P da Cunha Santos G Lagarde A Richardson F Seymour L Whitehead M Ding K Pater J Shepherd FA Erlotinib in lung cancer: molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1731) Google Scholar20Piccart-Gebhart MJ Procter M Leyland-Jones B Goldhirsch A Untch M Smith I Gianni L Baselga J Bell R Jackisch C Cameron D Dowsett M Barrios CH Steger G Huang CS Andersson M Inbar M Lichinitser M Lang I Nitz U Iwata H Thomssen C Lohrisch C Suter TM Ruschoff J Suto T Greatorex V Ward C Straehle C McFadden E Dolci MS Gelber RD Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.N Engl J Med. 2005; 353: 1659-1672Crossref PubMed Scopus (4177) Google Scholar21Kantarjian H Sawyers C Hochhaus A Guilhot F Schiffer C Gambacorti-Passerini C Niederwieser D Resta D Capdeville R Zoellner U Talpaz M Druker B Goldman J O'Brien SG Russell N Fischer T Ottmann O Cony-Makhoul P Facon T Stone R Miller C Tallman M Brown R Schuster M Loughran T Gratwohl A Mandelli F Saglio G Lazzarino M Russo D Baccarani M Morra E Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.N Engl J Med. 2002; 346: 645-652Crossref PubMed Scopus (1819) Google Scholar Some ongoing clinical trials of BRAF inhibitors plan to include tumor mutational typing in their analyses. The final results of these trials are pending.22Hardin EPA Liebes L Osman I Hamilton A Soon J Polsky D Friedman K Wright J Muggia F A phase II trial of BAY 43-9006 in metastatic melanoma with molecularly targeted BRAF status.J Clin Oncol. 2006; 24: 8046Google Scholar23Ahmad T Marais R Pyle L James M Schwartz B Gore M Eisen T BAY 43–9006 in patients with advanced melanoma: The Royal Marsden experience (Meeting Abstract).J Clin Oncol. 2004; 22: 7506Google Scholar24Flaherty KT Redlinger M Schuchter LM Lathia CD Weber BL O'Dwyer PJ Phase I/II, pharmacokinetic and pharmacodynamic trial of BAY 43-9006 alone in patients with metastatic melanoma (Meeting Abstract).J Clin Oncol. 2005; 23: 3037Google Scholar As more potent and specific BRAF inhibitors become available, mutational typing is expected to acquire greater clinical relevance to determine the most suitable patients for these therapies. Although the benefits of molecularly targeted therapies are clear,18Lynch TJ Bell DW Sordella R Gurubhagavatula S Okimoto RA Brannigan BW Harris PL Haserlat SM Supko JG Haluska FG Louis DN Christiani DC Settleman J Haber DA Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N Engl J Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9953) Google Scholar19Tsao MS Sakurada A Cutz JC Zhu CQ Kamel-Reid S Squire J Lorimer I Zhang T Liu N Daneshmand M Marrano P da Cunha Santos G Lagarde A Richardson F Seymour L Whitehead M Ding K Pater J Shepherd FA Erlotinib in lung cancer: molecular and clinical predictors of outcome.N Engl J Med. 2005; 353: 133-144Crossref PubMed Scopus (1731) Google Scholar20Piccart-Gebhart MJ Procter M Leyland-Jones B Goldhirsch A Untch M Smith I Gianni L Baselga J Bell R Jackisch C Cameron D Dowsett M Barrios CH Steger G Huang CS Andersson M Inbar M Lichinitser M Lang I Nitz U Iwata H Thomssen C Lohrisch C Suter TM Ruschoff J Suto T Greatorex V Ward C Straehle C McFadden E Dolci MS Gelber RD Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.N Engl J Med. 2005; 353: 1659-1672Crossref PubMed Scopus (4177) Google Scholar21Kantarjian H Sawyers C Hochhaus A Guilhot F Schiffer C Gambacorti-Passerini C Niederwieser D Resta D Capdeville R Zoellner U Talpaz M Druker B Goldman J O'Brien SG Russell N Fischer T Ottmann O Cony-Makhoul P Facon T Stone R Miller C Tallman M Brown R Schuster M Loughran T Gratwohl A Mandelli F Saglio G Lazzarino M Russo D Baccarani M Morra E Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.N Engl J Med. 2002; 346: 645-652Crossref PubMed Scopus (1819) Google Scholar patient selection based on the mutational status of their tumor can be quite restrictive. A substantial number of patients who might benefit from the treatment will be ineligible because of the lack of an accessible tissue sample. In addition, determining the presence of mutations is limited by the ability of the assay to detect the mutation. In melanoma, samples often have large amounts of contaminating normal tissue. Thus, a highly sensitive and less invasive method of determining patients' mutational status is desirable. In this study, we developed a technique to test patient plasma samples for BRAF mutations. Using a mutant-specific amplification strategy with fluorescent detection, we show that plasma samples from patients with metastatic melanoma contain ample DNA for analysis, and we demonstrate that this assay may be a useful tool for determining the BRAF mutational status of a patient's tumor, especially when an appropriate tissue sample is not available for analysis. The study cohort consisted of 26 metastatic melanoma patients with stage IV disease (age range, 35 to 92; mean, 63; 20 males, six females; tumor biopsy sites included 21 skin or subcutaneous metastases, three lymph node, one brain, and one local recurrence) accrued from either New York University School of Medicine (16 patients) or Memorial Sloan-Kettering Cancer Center (10 patients). The study was approved by the institutional review boards of both institutions, and all patients signed informed consent at time of enrollment. All patients had recurrent stage IV disease at time of blood draw. Peripheral blood samples were collected in two ethylenediamine tetraacetic acid tubes, placed immediately on ice, and transported to the laboratory where samples were centrifuged to separate cells and plasma. The plasma supernatant was transferred to a clean tube and stored at −80°C before use. For DNA extraction, plasma samples were thawed at room temperature, and 1-ml aliquots were placed into 1.5-ml microcentrifuge tubes. Samples were centrifuged at 16,000 rpm in an Eppendorf model 5415R microcentrifuge (Eppendorf-5 Prime, Inc., Boulder, CO) for 30 minutes. The supernatant was discarded, and the pellet was resuspended in 200 μl of Tris-ethylenediamine tetraacetic acid buffer. This material was used to isolate DNA using the QIAmp DNA Blood Mini kit (QIAgen, Valencia, CA). For analysis of tumor samples, hematoxylin and eosin-stained slides were reviewed to ensure enough viable tumor. Unstained cut sections mounted on slides were macrodissected to remove contaminating normal cells when possible, and tissue sections were scraped into a 1.5-ml microcentrifuge tube. Subsequent steps followed the protocol for DNA isolation from paraffin slides in the QIAmp Mini Blood DNA kit. Peaks with an amplitude of 50 or more [default analysis parameters of the Genescan software (Applied Biosystems, Foster City, CA)] were considered positive. All blood and tissue samples were run in triplicate; a positive result required at least two positive replicates. The human melanoma cell line SK-MEL 29, mutant for BRAF, and human placental DNA, wild type for BRAF, were included in every run as positive and negative controls, respectively. Mutant-specific polymerase chain reaction (MS-PCR) was accomplished using two primers: forward, 5′-GGTGATTTTGGTCTAGCTACATA-3′; and reverse, 5′-HEX-GGCCAAAAATTTAATCAGTGGA-3′. The forward, mutant-specific primer was based on a design by Xu et al25Xu X Quiros RM Gattuso P Ain KB Prinz RA High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines.Cancer Res. 2003; 63: 4561-4567PubMed Google Scholar with minor modifications. This design used two bases at the 3′ end of the primer that do not anneal to the wild-type sequence. In the presence of the mutant sequence, the terminal 3′ base will anneal, allowing amplification to proceed. The reverse primer described by Davies et al2Davies H Bignell GR Cox C Stephens P Edkins S Clegg S Teague J Woffendin H Garnett MJ Bottomley W Davis N Dicks E Ewing R Floyd Y Gray K Hall S Hawes R Hughes J Kosmidou V Menzies A Mould C Parker A Stevens C Watt S Hooper S Wilson R Jayatilake H Gusterson BA Cooper C Shipley J Hargrave D Pritchard-Jones K Maitland N Chenevix-Trench G Riggins GJ Bigner DD Palmieri G Cossu A Flanagan A Nicholson A Ho JW Leung SY Yuen ST Weber BL Seigler HF Darrow TL Paterson H Marais R Marshall CJ Wooster R Stratton MR Futreal PA Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8204) Google Scholar was labeled with the HEX fluorochrome to permit detection using an ABI 310 Genetic Analyzer (Applied Biosystems). For amplification of the entire BRAF exon 15, we used the primers of Davies et al.2Davies H Bignell GR Cox C Stephens P Edkins S Clegg S Teague J Woffendin H Garnett MJ Bottomley W Davis N Dicks E Ewing R Floyd Y Gray K Hall S Hawes R Hughes J Kosmidou V Menzies A Mould C Parker A Stevens C Watt S Hooper S Wilson R Jayatilake H Gusterson BA Cooper C Shipley J Hargrave D Pritchard-Jones K Maitland N Chenevix-Trench G Riggins GJ Bigner DD Palmieri G Cossu A Flanagan A Nicholson A Ho JW Leung SY Yuen ST Weber BL Seigler HF Darrow TL Paterson H Marais R Marshall CJ Wooster R Stratton MR Futreal PA Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8204) Google Scholar DNA templates were extracted and purified from the human melanoma cell line SK-MEL 29 and human plasma samples using the QIAamp Mini DNA kit (QIAgen). Human placental DNA (Sigma-Aldrich, St. Louis, MO) was used as a negative control DNA template. Amplification was accomplished using 49 cycles of PCR at an annealing temperature of 62°C. For initial optimization experiments, an annealing temperature gradient was tested using an Eppendorf Mastercycler PCR machine. Subsequent amplifications were performed using a Perkin Elmer 9700 machine (Applied Biosystems). The amplified products were analyzed using ABI 310 Genetic Analyzer and Genescan 3.1 software (Applied Biosystems). Conventional DNA sequencing was accomplished as described by Gorden et al.3Gorden A Osman I Gai W He D Huang W Davidson A Houghton AN Busam K Polsky D Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar Sequences were analyzed using SeqScape software (Applied Biosystems). A gradient of annealing temperatures from 35 to 64°C was tested using DNA from the melanoma cell line SK-MEL 29, which carries the BRAF V600E mutation.3Gorden A Osman I Gai W He D Huang W Davidson A Houghton AN Busam K Polsky D Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar Amplified products could be detected at annealing temperatures ranging from 35 to 62°C, with markedly reduced or absent amplification using annealing temperatures greater than 62°C. The negative control human placental DNA (wild-type BRAF) failed to amplify at any annealing temperature, indicating the highly specific nature of this PCR reaction (Figure 1a). Two approaches were used to characterize the sensitivity of the assay and further define its specificity. In the first, decreasing amounts of DNA from the BRAF mutant cell line SK-MEL 29 were mixed with human placental DNA. Each mixture contained a total of 100 ng of DNA, and these mixtures were used as templates for the MS-PCR assay. The assay was able to detect mutant BRAF alleles for samples containing as little as 0.1 ng of mutant DNA per 100 ng of DNA (Figure 1b). Below this concentration, there was no amplification of the mutant allele. In the second approach, whole blood from a normal healthy volunteer was spiked with decreasing numbers of cells of the BRAF mutant tumor cell line SK-MEL 29. The limit of detection of the assay was 50 melanoma cells mixed in 5 ml of whole blood (10 cells/ml) (Figure 1c). In both of these experiments, there was no allelic amplification at very low concentrations of mutant BRAF, despite the presence of abundant wild-type DNA, confirming the specificity of this assay. In addition, testing of peripheral blood cell samples from 10 normal controls via MS-PCR demonstrated no detectable PCR product (Figure 1d). To compare the sensitivities of the MS-PCR assay and direct sequencing techniques in detecting the BRAF hotspot codon 600 mutation, we applied both techniques to samples composed of a mixture of mutant and wild-type DNA (Figure 2). Direct sequencing with automated base calling could not definitively detect mutations when the sample was comprised of less than 50% of melanoma DNA. Manual inspection of the electropherogram could only detect mutant peaks when at least 30% mutant DNA was present. The MS-PCR could detect mutant alleles in the 10% mixture, and given the results of the sensitivity analysis shown in Figure 1, it can detect mutations at much lower proportions. To test the clinical utility of the MS-PCR assay in melanoma patients, plasma samples from 26 patients with metastatic melanoma were tested for BRAF mutations. To control for DNA quality, all samples also underwent PCR amplification of the entire BRAF exon 15. The electropherograms for 10 patients (Figure 3) and MS-PCR results for all patients screened (Table 1) are provided. Fourteen of 26 (54%) patients had detectable mutant BRAF alleles in their plasma samples. All patients had detectable amplification of the entire BRAF exon 15, indicating adequate DNA quality in each sample. Thus, the assay is able to detect mutant BRAF alleles in plasma samples of patients in the targeted population.Table 1MS-PCR Results of Metastatic Melanoma PatientsPatientPlasma MS-PCRTissue MS-PCR1MutantMutant2MutantMutant3MutantMutant4MutantMutant5MutantMutant6MutantMutant7MutantMutant8MutantWild type9MutantWild type10MutantN/A11MutantN/A12MutantN/A13MutantN/A14MutantN/A15Wild typeWild type16Wild typeWild type17Wild typeWild type18Wild typeMutant19Wild typeMutant20Wild typeMutant21Wild typeMutant22Wild typeMutant23Wild typeN/A24Wild typeN/A25Wild typeN/A26Wild typeN/AN/A, not available. Open table in a new tab N/A, not available. Tumor tissue was available from metastatic lesions of 17 of 26 patients. Of the 17 samples, 12 (71%) had detectable mutant BRAF alleles. Comparison of the plasma and tissue results for these 17 patients revealed concordance of results in 10 of 17 (59%) patients: seven patients had detectable BRAF mutations in both plasma and tumor tissue, and three had wild-type BRAF alleles in both their plasma and tumor tissues. Seven of 17 patients had discordance between the plasma and tissue results: two had mutations detected only in their plasma, and five had mutations detected only in their tumor tissue (Table 1). Given that patients with metastatic cancers have higher concentrations of DNA in plasma than healthy individuals (see review26Ziegler A Zangemeister-Wittke U Stahel RA Circulating DNA: a new diagnostic gold mine?.Cancer Treat Rev. 2002; 28: 255-271Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), it would be clinically valuable to develop assays that use the accessibility of tumor DNA from plasma samples. In this study, we developed a fluorescence-based mutant-specific PCR assay that can detect BRAF mutations in plasma samples from patients with metastatic melanoma. The method has several technical features that make it an attractive assay for future study. It requires only a small amount of DNA, is more sensitive than direct sequencing techniques, and is highly specific for the V600E mutation. A similar technique reported by Miller et al27Miller CJ Cheung M Sharma A Clarke L Helm K Mauger D Robertson GP Method of mutation analysis may contribute to discrepancies in reports of (V599E)BRAF mutation frequencies in melanocytic neoplasms.J Invest Dermatol. 2004; 123: 990-992Crossref PubMed Scopus (38) Google Scholar compared the sensitivity of allele-specific PCR using agarose gel electrophoresis with DNA sequencing. Allele-specific PCR could detect the mutation in as low as a 1% concentration of mutant DNA. Sequencing required at least 5% mutant DNA for visual detection of the mutant allele on the electropherogram. Comparison of BRAF mutation frequencies in melanocytic lesions using these two methods revealed higher detected mutation rates with the allele-specific PCR, as would be expected with a more sensitive assay. We have confirmed this finding by comparing sequencing with our MS-PCR and fluorescent detection system. In addition, the technique we describe can detect BRAF mutations in blood samples, greatly expanding on previous work and broadening the potential uses for BRAF mutation testing. Our detected rate of BRAF mutations [14 of 26 (54%)] in the plasma of patients with metastatic melanoma corresponds well with previously described rates in tumor tissue from this patient population.2Davies H Bignell GR Cox C Stephens P Edkins S Clegg S Teague J Woffendin H Garnett MJ Bottomley W Davis N Dicks E Ewing R Floyd Y Gray K Hall S Hawes R Hughes J Kosmidou V Menzies A Mould C Parker A Stevens C Watt S Hooper S Wilson R Jayatilake H Gusterson BA Cooper C Shipley J Hargrave D Pritchard-Jones K Maitland N Chenevix-Trench G Riggins GJ Bigner DD Palmieri G Cossu A Flanagan A Nicholson A Ho JW Leung SY Yuen ST Weber BL Seigler HF Darrow TL Paterson H Marais R Marshall CJ Wooster R Stratton MR Futreal PA Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8204) Google Scholar3Gorden A Osman I Gai W He D Huang W Davidson A Houghton AN Busam K Polsky D Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar4Kumar R Angelini S Czene K Sauroja I Hahka-Kemppinen M Pyrhonen S Hemminki K BRAF mutations in metastatic melanoma: a possible association with clinical outcome.Clin Cancer Res. 2003; 9: 3362-3368PubMed Google Scholar,28Omholt K Platz A Kanter L Ringborg U Hansson J NRAS and BRAF mutations arise early during melanoma pathogenesis and are preserved throughout tumor progression.Clin Cancer Res. 2003; 9: 6483-6488PubMed Google Scholar In addition, MS-PCR of 17 tissue samples from these patients revealed concordant results in 10 of 17 (59%) patients (seven mutant in blood and tissue, and three wild type in blood and tissue). Among the discordant results, two patients had tumors negative for mutant BRAF but plasma that was positive. Interestingly, one patient had a positron emission tomography/computed tomography scan performed at the time of the subcutaneous tumor biopsy analyzed in this study, revealing multifocal disease with metastases to the lung, peritoneum, and muscle. Thus, it is possible that these other metastatic sites contained the mutation, and it was from one of these sources that the blood-borne mutant BRAF allele originated. In support of this hypothesis, we have shown in a previous study that there is heterogeneity in BRAF mutation status between metastases in the same patient.29Chang DZ Panageas KS Osman I Polsky D Busam K Chapman PB Clinical significance of BRAF mutations in metastatic melanoma.J Transl Med. 2004; 2: 46Crossref PubMed Scopus (59) Google Scholar On the other hand, the mutant BRAF alleles in the bloodstream may have been derived from a portion of the biopsied tumor that was not used for the tissue-based PCR. A recent study examining short-term human melanoma cultures revealed a single culture in which both BRAF and NRAS mutations could be detected (a rare finding); however, these mutations were mutually exclusive at the single-cell level.30Sensi M Nicolini G Petti C Bersani I Lozupone F Molla A Vegetti C Nonaka D Mortarini R Parmiani G Fais S Anichini A Mutually exclusive NRAS(Q61R) and BRAF(V600E) mutations at the single-cell level in the same human melanoma.Oncogene. 2006; 25: 3357-3364Crossref PubMed Scopus (143) Google Scholar These results provide additional direct evidence that some melanomas may be heterogeneous with respect to BRAF mutations. In the two cases presented here, it is possible that the MS-PCR results of the plasma are a more accurate representation of the patients' tumor(s) than the single biopsy specimen available for analysis. The other five patients with discordant results had tumors that were positive for mutant BRAF, but plasma samples were negative. One explanation for this discordance is that shedding of mutant BRAF in the bloodstream may be intermittent. Curry et al31Curry BJ Myers K Hersey P Utility of tests for circulating melanoma cells in identifying patients who develop recurrent melanoma.Recent Results Cancer Res. 2001; 158: 211-230Crossref PubMed Scopus (15) Google Scholar showed in their study of circulating melanoma cells that the sensitivity of detecting circulating tumor markers increased with serial blood draws. In this regard, we are embarking on a study with a larger sample size that will incorporate serial blood draws. In general, we may find that repeated sampling of the blood compartment has the potential to be the easiest and most useful method to detect tumor-associated BRAF mutations in melanoma and other tumor types in which substantial frequencies of BRAF mutations have been detected (eg, thyroid, colon, and ovarian).12Rajagopalan H Bardelli A Lengauer C Kinzler KW Vogelstein B Velculescu VE Tumorigenesis: rAF/RAS oncogenes and mismatch-repair status.Nature. 2002; 418: 934Crossref PubMed Scopus (1058) Google Scholar,25Xu X Quiros RM Gattuso P Ain KB Prinz RA High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines.Cancer Res. 2003; 63: 4561-4567PubMed Google Scholar,32Tanaka H Deng G Matsuzaki K Kakar S Kim GE Miura S Sleisenger MH Kim YS BRAF mutation, CpG island methylator phenotype and microsatellite instability occur more frequently and concordantly in mucinous than non-mucinous colorectal cancer.Int J Cancer. 2006; 118: 2765-2771Crossref PubMed Scopus (114) Google Scholar,33Singer G Oldt III, R Cohen Y Wang BG Sidransky D Kurman RJ Shih IeM Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma.J Natl Cancer Inst. 2003; 95: 484-486Crossref PubMed Scopus (698) Google Scholar Although there is not enough clinical data available today to determine whether therapeutic response to currently available BRAF-inhibiting agents correlates with BRAF mutational status, preclinical testing of these drugs reveals that BRAF V600E is more sensitive to these agents than wild-type BRAF.34da Rocha Dias S Friedlos F Light Y Springer C Workman P Marais R Activated B-RAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin.Cancer Res. 2005; 65: 10686-10691Crossref PubMed Scopus (203) Google Scholar,35Karasarides M Chiloeches A Hayward R Niculescu-Duvaz D Scanlon I Friedlos F Ogilvie L Hedley D Martin J Marshall CJ Springer CJ Marais R B-RAF is a therapeutic target in melanoma.Oncogene. 2004; 23: 6292-6298Crossref PubMed Scopus (372) Google Scholar In addition, given preclinical data showing that BRAF mutant cells are extremely sensitive to mitogen-activated protein kinase kinase inhibition compared with wild-type and RAS mutant cells, it is possible that patient stratification based on BRAF mutation status may ultimately be useful for molecularly targeted therapies aimed at multiple kinases in the mitogen-activated protein kinase pathway.36Solit DB Garraway LA Pratilas CA Sawai A Getz G Basso A Ye Q Lobo JM She Y Osman I Golub TR Sebolt-Leopold J Sellers WR Rosen N BRAF mutation predicts sensitivity to MEK inhibition.Nature. 2006; 439: 358-362Crossref PubMed Scopus (1137) Google Scholar There are important limitations to this assay. Because not all melanomas harbor BRAF mutations, and this assay was specifically designed to distinguish BRAF mutant from BRAF wild-type tumors, it cannot be used as a tool for staging and monitoring all melanoma patients. In addition, not all patients with BRAF mutant tumors will necessarily have BRAF mutant DNA in their circulation. Thus, the potential clinical utility of this assay at this point is as an adjunctive study to tissue-based testing. On the other hand, it could serve as a stand-alone assay in patients with tumors that are unable to be biopsied. Finally, we recognize that although the assay detects the V600E mutation and is predicted to detect the much less common V600K mutation, it is not expected to detect the V600R mutation. This mutation is quite rare; it is estimated to occur in only 3 to 6% of melanomas with BRAF mutations. Thus, the current assay remains a potentially powerful clinical tool3Gorden A Osman I Gai W He D Huang W Davidson A Houghton AN Busam K Polsky D Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar,37Thomas NE Alexander A Edmiston SN Parrish E Millikan RC Berwick M Groben P Ollila DW Mattingly D Conway K Tandem BRAF mutations in primary invasive melanomas.J Invest Dermatol. 2004; 122: 1245-1250Crossref PubMed Scopus (55) Google Scholar and can be modified to detect the more rare mutation if needed. Our laboratory is currently pursuing several avenues of study to expand on this work. We are developing a quantitative PCR assay based on this qualitative technique, which may expand the potential utility of the assay and may be useful for staging and monitoring of patients with BRAF mutations. We are also working to determine whether the presence of circulating mutant BRAF alleles correlates with patients' responses to BRAF inhibitors and clinical outcome. Last, we are also investigating whether testing DNA isolated from peripheral blood cells may be advantageous when compared with plasma-based assays for the detection of BRAF mutations. We are hopeful that with further refinements, the discordance between blood- and tissue-based results will decrease and that in the future, we will be able to add blood-based testing to the techniques used to type patients for targeted therapies. As such, our lab is working to explore these routes to improve blood-based testing of patients with melanoma. We thank the patients for participating in this study. We are grateful to Daniel Freedberg for laboratory assistance and to Caroline Chang and Joanna Spira for help with the clinical data.