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Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas

2012; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês

10.1016/j.jmoldx.2012.08.003

ISSN

1943-7811

Autores

Johanne Lade‐Keller, Kirsten M. Rømer, Per Guldberg, Rikke Riber‐Hansen, Lise Lotte Hansen, Torben Steiniche, Henrik Hager, Lasse S. Kristensen,

Tópico(s)

Computational Drug Discovery Methods

Resumo

Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings. Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings. Cutaneous melanoma is a malignancy with a favorable prognosis if it presents as a localized disease, but with a dramatically worse prognosis if it metastasizes.1Balch C.M. Gershenwald J.E. Soong S.J. Thompson J.F. Atkins M.B. Byrd D.R. Buzaid A.C. Cochran A.J. Coit D.G. Ding S. Eggermont A.M. Flaherty K.T. Gimotty P.A. Kirkwood J.M. McMasters K.M. Mihm Jr., M.C. Morton D.L. Ross M.I. Sober A.J. Sondak V.K. Final version of 2009 AJCC melanoma staging and classification.J Clin Oncol. 2009; 27: 6199-6206Crossref PubMed Scopus (3664) Google Scholar So far, no effective treatment exists for the group of melanoma patients with metastasizing disease, although several non-specific chemotherapy regimens and immunotherapies are able to prolong survival at least for a short period of time.2Davies M.A. Gershenwald J.E. Targeted therapy for melanoma: a primer.Surg Oncol Clin N Am. 2011; 20: 165-180Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar In normal melanocytes, the stimulation of receptor tyrosine kinases by several growth factors released from the microenvironment induces the mitogen-activated protein kinase (MAPK) pathway to give weak growth-promoting signals to the nucleus.3Bogenrieder T. Herlyn M. The molecular pathology of cutaneous melanoma.Cancer Biomark. 2011; 9: 267-286Crossref Scopus (25) Google Scholar However, melanoma cells frequently become independent of growth factors because of activating mutations within key players of this pathway, such as BRAF and NRAS, which induce an ongoing stimulation of the MAPK pathway with subsequently increased proliferation and cellular growth.3Bogenrieder T. Herlyn M. The molecular pathology of cutaneous melanoma.Cancer Biomark. 2011; 9: 267-286Crossref Scopus (25) Google Scholar In recent years, these findings have paved the way for the development of selective inhibitors of the MAPK pathway such as BRAF and MEK inhibitors. In particular, the selective inhibitors of the BRAF kinase, Vemurafenib (PLX4032/RG7204) and Dabrafenib (GSK2118436) have attracted attention.4Eggermont A.M. Robert C. New drugs in melanoma: It's a whole new world.Eur J Cancer. 2011; 47: 2150-2157Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Chapman 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. BRIM-3 Study GroupImproved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6142) Google Scholar, 6Kefford R. Arkenau H. Brown M.P. Millward M. Infante J.R. Long G.V. Ouellet D. Curtis M. Lebowitz P.F. Falchook G.S. Phase I/II study of GSK2118436, a selective inhibitor of oncogenic mutant BRAF kinase, in patients with metastatic melanoma and other solid tumors (abstract).J Clin Oncol. 2010; 28: 8503Crossref Google Scholar, 7Sosman 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 (1780) Google Scholar In a recent phase III clinical trial, Vemurafenib proved to significantly increase overall survival and progression-free survival of cutaneous melanoma patients compared with standard chemotherapy using the alkylating agent dacarbazine.5Chapman 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. BRIM-3 Study GroupImproved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6142) Google Scholar, 7Sosman 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 (1780) Google Scholar One of the major criteria for inclusion in the clinical trials of these BRAF inhibitors has been the presence of a BRAF c.1799T>A (V600E) mutation,4Eggermont A.M. Robert C. New drugs in melanoma: It's a whole new world.Eur J Cancer. 2011; 47: 2150-2157Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Chapman 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. BRIM-3 Study GroupImproved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6142) Google Scholar which accounts for approximately 90% of all BRAF mutations so far detected in cutaneous melanoma.5Chapman 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. BRIM-3 Study GroupImproved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6142) Google Scholar This has resulted in a rapid increase in the demand for mutation testing in both the clinical setting and in research. Vemurafenib was recently approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for treatment of advanced metastatic melanoma (non-resectable stage III/IV). The FDA approved Vemurafenib, along with the Cobas 4800 BRAF V600 Mutation Test (Roche Diagnostics) for patient selection. Surprisingly, Vemurafenib and other BRAF inhibitors may function as "double-edged swords" by activating the MAPK pathway in cells with wild-type BRAF.8Poulikakos P.I. Zhang C. Bollag G. Shokat K.M. Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.Nature. 2010; 464: 427-430Crossref PubMed Scopus (1404) Google Scholar, 9Hatzivassiliou G. Song K. Yen I. Brandhuber B.J. Anderson D.J. Alvarado R. Ludlam M.J. Stokoe D. Gloor S.L. Vigers G. Morales T. Aliagas I. Liu B. Sideris S. Hoeflich K.P. Jaiswal B.S. Seshagiri S. Koeppen H. Belvin M. Friedman L.S. Malek S. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.Nature. 2010; 464: 431-435Crossref PubMed Scopus (1288) Google Scholar, 10Heidorn S.J. Milagre C. Whittaker S. Nourry A. Niculescu-Duvas I. Dhomen N. Hussain J. Reis-Filho J.S. Springer C.J. Pritchard C. Marais R. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.Cell. 2010; 140: 209-221Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar This important finding may have two implications. First, squamous-cell carcinoma of the skin and keratoacanthoma, which have been observed in a significant proportion of patients treated with Vemurafenib,5Chapman 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. BRIM-3 Study GroupImproved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6142) Google Scholar, 7Sosman 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 (1780) Google Scholar, 11Flaherty K.T. Puzanov I. Kim K.B. Ribas A. McArthur G.A. Sosman J.A. O'Dwyer P.J. Lee R.J. Grippo J.F. Nolop K. Chapman P.B. Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010; 363: 809-819Crossref PubMed Scopus (2962) Google Scholar, 12Ribas A. Kim K.B. Schuchter L.M. Gonzalez R. Pavlick A.C. Weber J.S. McArthur G.A. Hutson T.E. Flaherty K.T. Moschos S.J. Lawrence D.P. Hersey P. Kefford R.F. Chmielowski B. Puzanov I. Li J. Nolop K.B. Lee R.J. Joe A.K. Sosman J.A. BRIM-2: An open-label, multicenter phase II study of vemurafenib in previously treated patients with BRAF V600E mutation-positive metastatic melanoma (abstract).J Clin Oncol. 2011; 29: 8509Crossref Google Scholar may be a result of an activation of the MAPK pathway in BRAF wild-type preneoplastic cells in these patients.5Chapman 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. BRIM-3 Study GroupImproved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6142) Google Scholar Second, intratumor heterogeneity with respect to the BRAF mutation may result in non-response or relapse. However, it has not been investigated as to whether a correlation between drug response and the ratio of mutant to wild-type alleles exists, as mutational status in the clinical setting has been evaluated only using the purely qualitative Cobas test. The BRAF c.1799T>A mutation frequency in primary cutaneous melanoma varies in different studies, from 22% to 72%.13Lee J.H. Choi J.W. Kim Y.S. Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis.Br J Dermatol. 2011; 164: 776-784Crossref PubMed Scopus (331) Google Scholar This variance may be caused by the use of different histological tumor subtypes in these studies, but may also be a result of differences among the mutation detection methods applied.13Lee J.H. Choi J.W. Kim Y.S. Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis.Br J Dermatol. 2011; 164: 776-784Crossref PubMed Scopus (331) Google Scholar The proportion of tumor cells relative to normal cells is subject to much variation among samples, and the analytical sensitivities of the different methods applied for mutation detection varies tremendously. In addition, it is also possible that various degrees of intratumor heterogeneity with respect to the BRAF mutation exist in cutaneous melanoma.14Yancovitz M. Litterman A. Yoon J. Ng E. Shapiro R.L. Berman R.S. Pavlick A.C. Darvishian F. Christos P. Mazumdar M. Osman I. Polsky D. Intra- and inter-tumor heterogeneity of BRAF(V600E)mutations in primary and metastatic melanoma.PLoS One. 2012; 7: e29336Crossref PubMed Scopus (240) Google Scholar The frequency of detected BRAF mutations in primary cutaneous melanoma is therefore likely to be influenced by the analytical sensitivity of the method used. We have recently shown that this is indeed the case when studying KRAS mutations in colorectal cancer.15Kristensen L.S. Daugaard I.L. Christensen M. Hamilton-Dutoit S. Hager H. Hansen L.L. Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products.Hum Mutat. 2010; 31: 1366-1373Crossref PubMed Scopus (33) Google Scholar A number of different detection methods are being used in melanoma research for detection of the BRAF c.1799T>A mutation, including Sanger sequencing, pyrosequencing, high-resolution melting (HRM) analysis, and various allele-specific PCR-based methods. Sanger sequencing, pyrosequencing, and HRM analysis generally are characterized by a relatively low sensitivity, and allele-specific PCR assays may be susceptible to false-positive results if false amplification of wild-type DNA occurs in early PCR cycles. Recently, a modified PCR protocol, known as co-amplification at lower denaturation temparature-PCR (COLD-PCR), has been developed, which is capable of selectively amplifying mutant alleles based on melting temperature differences between mutant and wild-type amplicons. This greatly improves the sensitivity of downstream detection methods.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 (320) Google Scholar However, when the mutation in question is melting temperature retaining (such as the BRAF c.1799T>A mutation) or melting temperature increasing, only a modest increase in sensitivity may be observed.17Stadelmeyer E. Heitzer E. Wolf P. Dandachi N. COLD-HRM PCR versus conventional HRM PCR to detect the BRAF V600E mutation A real improvement?.J Mol Diagn. 2011; 13 (author reply 244): 243Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar We have recently described a novel allele-specific PCR methodology, called Competitive Amplification of Differentially Melting Amplicons (CADMA), which works well independent of the melting properties of the mutation to be detected. The principle of the method is to add another competitive primer partially targeting the same sequence as the mutation-specific primer, to limit false-priming events. In addition, the mutation-specific primer is designed to introduce melting temperature–decreasing mutations, thereby enabling mutation calling based on high-resolution melting (HRM) analysis, and the use of COLD-PCR to further increase analytical sensitivity.18Kristensen L.S. Andersen G.B. Hager H. Hansen L.L. Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.Hum Mutat. 2012; 33: 264-271Crossref PubMed Scopus (19) Google Scholar In the clinical setting, the only BRAF c.1799T>A mutation detection method used so far is the Cobas test. To our knowledge, only two studies have evaluated the performance of the Cobas test relative to Sanger sequencing. This was, in part, done by testing discordant results using next-generation sequencing.19Halait H. Demartin K. Shah S. Soviero S. Langland R. Cheng S. Hillman G. Wu L. Lawrence H.J. Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma.Diagn Mol Pathol. 2012; 21: 1-8Crossref PubMed Scopus (130) Google Scholar, 20Anderson 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 (119) Google Scholar In the present study, we have evaluated whether the frequency of detected BRAF V600E mutations in cutaneous melanoma is influenced directly by the analytical sensitivity of the applied method. For this purpose, we used five different methods, the Cobas 4800 BRAF V600 Mutation Test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and CADMA. The analytical sensitivity of each method was determined using a serial dilution of mutated cell line DNA in a wild-type background. We also investigated how the percentages of tumor cells in primary cutaneous melanoma DNA samples derived from formalin-fixed paraffin-embedded (FFPE) tissues influenced the detection capabilities of the methods applied. Finally, we have evaluated whether tumors may be BRAF mutation positive as a result of only a small fraction of the tumor cells carrying the BRAF mutation, as such tumors may not respond to Vemurafenib. A total of 28 FFPE cutaneous melanoma tissue samples (collected from 2009 to 2010) were obtained from the pathology archives at Aarhus University Hospital. The clinicopathological features are shown in Table 1.Table 1Clinocopathological Characteristics of the 28 Study PatientsCharacteristicNo. (%)Sex Male16 (57) Female12 (43)Breslow (mm) Mean (95% CI)1.14 (1.03–1.27) Range (min–max)0.60–1.89 Median1.12Clark level 3 9 (32) 419 (68)Ulceration Yes 2 (7) No26 (93)Regression Yes 2 (7) No26 (93)Histological subtype Superficial spreading23 (82) Nodular 5 (18)Total28 (100) Open table in a new tab To assess the analytical sensitivity of each of the methods, a standard dilution of mutant alleles in a wild-type background was prepared using DNA extracted from the cell line FM82, which is heterozygous for the BRAF c.1799T>A mutation. DNA extracted from peripheral blood, obtained from Danish medical students in their first year of Medical school at Aarhus University, was used as wild-type DNA. Using pyrosequencing, it was estimated that the cell line does indeed contain 50% mutant alleles. However, if the fraction of BRAF copies relative to the overall amount of genomic material in the cell line is not the same as in the blood sample, this will result in a bias between the methods that amplify both mutated and wild-type DNA and the methods that amplify only or preferably mutated DNA. To correct for this potential bias, we made three dilutions of the cell line DNA into wild-type DNA (40%, 30%, and 20% mutant alleles) and measured the allele frequencies using pyrosequencing. These measurements were then used to calculate the concentration for the wild-type DNA sample to avoid a bias, before serially diluting the cell line DNA into wild-type DNA to the following fractions (50%, 20%, 10%, 5%, 2.5%, 1.25%, 0.62%, 0.31%, 0.15%, and 0.078%). The DNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). To assess whether the CADMA and TaqMan assays show cross-reactivity with the c.1798_1799GT>AA (V600K) mutation, a standard dilution of mutated alleles in a wild-type background was prepared using DNA extracted from the cell line FM88, which is homozygous for the c.1798_1799GT>AA (V600K) mutation.21Christensen C. Guldberg P. Growth factors rescue cutaneous melanoma cells from apoptosis induced by knockdown of mutated (V 600 E) B-RAF.Oncogene. 2005; 24: 6292-6302Crossref PubMed Scopus (45) Google Scholar Dilutions of 100%, 50%, 25%, and 12.5% cell line DNA were prepared, and five replicates of each dilution were evaluated. The percentage of tumor tissue was estimated semiquantitatively on H&E-stained slides with two different methods and subdivided into the following intervals: 50%. In the first method, the tumor tissue area was estimated as a fraction of all visible tissues at the slide. In the second method, the tumor tissue area was estimated as a fraction of cell-dense tissue only (fatty tissue and cell-deprived tissue were excluded). Each FFPE sample was sectioned using a microtome (five slides 10 μm thick), and DNA was extracted using a commercial kit (Qiamp DNA FFPE Tissue Kit; Qiagen AB, Sollentuna, Sweden) following the manufacturer's protocol. The DNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Primers for the pyrosequencing assay were designed using the PyroMark Assay Design Software v2.0.6 (Qiagen). The amplification primers (forward: 5′-TTCATGAAGACCTCACAGTAAAAA-3′, reverse: 5′-GGCCAAAAATTTAATCAGTGGAA-3′) amplify a 152-bp region of BRAF containing the c.1799T>A variant. The forward primer was 5′ biotin-labeled to enable preparation of a single-stranded template. Amplification was performed in 25-μL reactions containing 200 nmol/L each primer, 0.1 mmol/L each dNTP, 1 unit of HotStarTaq DNA Polymerase (Qiagen), and 25 ng of DNA. Reactions were started by denaturation at 95°C for 15 minutes, followed by 45 cycles at 95°C for 10 seconds, 56°C for 20 seconds, and 72°C for 20 seconds. Pyrosequencing was performed on a PyroMark Q24 platform (Qiagen), using PyroMark Gold Q24 Reagents and the sequencing primer 5′-GCCAGGTCTTGATGTACT-3′, with the following dispensation order: 5′-GCATCTGT-3′. Data analysis was performed with PyroMark Q24 Software. Sequencing was performed in the reverse direction. Samples that were borderline positive (6% to 7%) were repeated. If the second run gave 6% or more, the sample was scored as positive. The TaqMan-based allele-specific PCR assay used herein has been published.22Lang A.H. Drexel H. Geller-Rhomberg S. Stark N. Winder T. Geiger K. Muendlein A. Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF.JMol Diagn. 2011; 13: 23-28Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar BRAF c.1799T>A–mutated samples are discriminated from wild-type samples based on a mutation-specific primer, which have a 3′ mismatch against wild-type alleles. The mutation specific primer is equally well matched against the rare c.1799_1800TG>AA (V600E) and c.1799_1800TG>AT (V600D) mutations. On the other hand, the primer has a mismatch against c.1798_1799GT>AA (V600K) and c.1798_1799GT>AG (V600R) at the position adjacent to the 3′ end. We used the same PCR conditions and real-time PCR instrument, as described by Lang et al,22Lang A.H. Drexel H. Geller-Rhomberg S. Stark N. Winder T. Geiger K. Muendlein A. Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF.JMol Diagn. 2011; 13: 23-28Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar using 50 ng of each sample for analysis. This assay determines mutation status using a predetermined cutoff ΔCt value (Ct [allele-specific assay]−Ct [reference assay]) of 9 as described. The analytical sensitivity of the assay was reported to be 1% mutant alleles in a wild-type background.22Lang A.H. Drexel H. Geller-Rhomberg S. Stark N. Winder T. Geiger K. Muendlein A. Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF.JMol Diagn. 2011; 13: 23-28Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Samples were analyzed in duplicate for all TaqMan experiments. The Competitive Amplification of Differentially Melting Amplicons (CADMA) assay discriminates BRAF ∼1799T>A mutated samples from wild-type samples based on a mutation-specific primer, which has a 3′ mismatch against wild-type alleles. The mutation-specific primer is equally well matched against the rare c.1799_1800TG>AA (V600E) and c.1799_1800TG>AT (V600D) mutations. On the other hand, the primer has a mismatch against c.1798_1799GT>AA (V600K) and c.1798_1799GT>AG (V600R) at the position adjacent to the 3′ end. Two melting temperature–decreasing mutations are introduced by the mutation-specific primer to facilitate further mutation enrichment by COLD-PCR and to allow mutation calling based on HRM. The primer competes with another overlapping primer for target binding. As the overlapping primer does not cover the bases of codon 600, the CADMA assay may potentially detect other codon 600 mutations, albeit at a lower analytical sensitivity. PCR cycling and HRM analysis were performed on the LightCycler 480 (Roche Applied Science, Mannheim, Germany). The reaction mixtures consisted of 25 ng DNA using a 1x final concentration of the LC480 HRM Scanning Master (Roche), and a final MgCl2 concentration of 2.5 mmol/L. Primer concentrations were as follows: 200 nmol/L reverse overlapping primer (5′-TGATGGGACCCACTCCATCG-3′), 400 nmol/L mutation specific reverse primer (5′-TGAGACCCACTCTATCGAGATTTCT-3′), and 400 nmol/L common forward primer (5′-AGGTGATTTTGGTCTAGCTACAG-3′). The cycling protocol was initiated by one cycle at 95°C for 10 minutes, followed by 15 standard PCR cycles at 95°C for 10 seconds, 69°C for 20 seconds, and 72°C for 20 seconds, followed by 35 fast COLD-PCR cycles at 78°C for 10 seconds, 69°C for 20 seconds, 72°C for 20 seconds, and a final denaturation step at 95°C for 1 minute. The HRM step was performed from 65°C to 95°C using 30 acquisitions per degree Celsius. Samples were analyzed in duplicate for all CADMA experiments. The analytical sensitivity of the CADMA assay was determined as the dilution point at which both replicates could be distinguished from 10 wild-type replicates (Figure 1A ). Cutaneous melanoma samples were scored as mutation positive if the melting profiles deviated more from the wild-type melting curves than the standard containing approximately 0.15% mutant alleles in a wild-type background. PCR amplicons were generated using the following primers: forward: 5′-AGGTGATTTTGGTCTAGCTACAG-3′ and reverse: 5′-GTTGAGACCTTCAATGACTTTCTAG-3′, and were analyzed on the ABI Genetic Analyzer 3130 XL (Applied Biosystems, Foster City, CA), using the BigDye terminator kit v1.1 (Applied Biosystems) according to the manufacturer's instructions with slight modifications; the single-stranded PCR was performed using 1 μL of the BigDye terminator in a final volume of 10 μL. Sequencing was performed in the reverse direction only. The Cobas 4800 BRAF V600 Mutation Test (Molecular Diagnostics, Roche Diagnostics A/S, Hvidovre, Denmark) is a real-time PCR analysis using TaqMan probes for specificity, and it is designed for detecting the presence of the c.1799T>A mutation. Results are binary (mutation detected or mutation not detected). Except for the preparation of the tissue (cutting the paraffin blocks and DNA extraction), all analyses were done according to the manufacturer protocol including dilution and standardization of the samples so that at least 125 ng DNA was used from each sample. Statistical analyses were performed using Stata/IC 11 (Stata Corp, College Station, TX). Inter- and intraobserver variations were calculated using non-weighted κ-statistics, and were interpreted as poor, slight, fair, moderate, substantial, or almost perfect, according to previously defined groups.23Landis J.R. Koch G.G. The measurement of observer agreement for categorical data.Biometrics. 1977; 33: 159-174Crossref PubMed Scopus (51397) Google Scholar The zero hypothesis of no difference in tumor size (Breslow thickness) was estimated using a two-sample Student's t-test on a logarithmically transf

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