KRAS and BRAF Mutation Analysis in Routine Molecular Diagnostics
2012; Elsevier BV; Volume: 14; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2012.01.011
ISSN1943-7811
AutoresDaniëlle A.M. Heideman, Irene Lurkin, Marije Doeleman, Egbert F. Smit, Henk M.W. Verheul, Gerrit A. Meijer, Peter J.F. Snijders, Erik Thunnissen, Ellen C. Zwarthoff,
Tópico(s)CAR-T cell therapy research
ResumoAccurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor–derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor–derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach. Accurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor–derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor–derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach. The introduction of novel classes of therapeutic agents for treating cancer, including monoclonal antibodies and small-molecule inhibitors of tyrosine kinases, such as the epidermal growth factor receptor (EGFR), has rapidly changed routine clinical pathology. Because these agents are only effective in subgroups of patients, companion diagnostics with molecular methods are necessary to select those patients who will likely derive the greatest clinical benefit from these targeted therapies. For example, KRAS mutation analysis is a prerequisite for selection of patients with metastasized colorectal cancer (CRC) for EGFR-targeted therapy, as positioned by the European Medicine Agency (EMEA/H/C/000558, approved June 29, 2004; and EMEA/H/C/000741, approved December 3, 2007) and the US Food and Drug Administration (FDA/BLA/125084, approved February 12, 2004; and FDA/BLA/125147, approved September 27, 2006). KRAS mutations are associated with resistance to treatment by monoclonal antibodies against EGFR, such as cetuximab and panitumumab, and are correlated with a shorter progression-free survival.1Bardelli A. Siena S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer.J Clin Oncol. 2010; 28: 1254-1261Crossref PubMed Scopus (561) Google Scholar BRAF mutations also may play a role in treatment decisions, indicating either a hampered response to EGFR-targeted treatment2Sartore-Bianchi A. Bencardino K. Cassingena A. Venturini F. Funaioli C. Cipani T. Amatu A. Pietrogiovanna L. Schiavo R. Di Nicolantonio F. Artale S. Bardelli A. Siena S. Therapeutic implications of resistance to molecular therapies in metastatic colorectal cancer.Cancer Treat Rev. 2010; 36: S1-S5Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 3Di Nicolantonio F. Martini M. Molinari F. Sartore-Bianchi A. Arena S. Saletti P. De Dosso S. Mazzucchelli L. Frattini M. Siena S. Bardelli A. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer.J Clin Oncol. 2008; 26: 5705-5712Crossref PubMed Scopus (1438) Google Scholar or sensitivity in case of treatment with BRAF-selective inhibitors, such as PLX4032 and GSK2118436.4Pratilas C.A. Xing F. Solit D.B. Targeting oncogenic BRAF in human cancer.Curr Top Microbiol Immunol. 2011; ([Epub ahead of press])Google Scholar, 5Flaherty 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. Chapman P.B. Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med. 2010; 363: 809-819Crossref PubMed Scopus (2953) Google Scholar, 6Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maoi 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. Notop 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 (6115) Google Scholar The widespread use of these targeted therapies has generated the need to develop and implement accurate, cost-effective methods for mutation analysis of KRAS and BRAF genes in molecular pathology diagnostic laboratories. Valuable methods include multiplex PCR, followed by SNaPshot primer extension analysis (ie, multiplex mutation assay)7Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar; and melting curve analysis [high-resolution melting (HRM)], followed by cycle sequencing (HRM-sequencing).8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar The latter features a prescreening approach to decide which specimens should be subjected to sequence analysis for confirmation of the nucleotide change,9Reed G.H. Kent J.O. Wittwer C.T. High-resolution DNA melting analysis for simple and efficient molecular diagnostics.Pharmacogenomics. 2007; 8: 597-608Crossref PubMed Scopus (524) Google Scholar allowing a fast turnaround time. The multiplex mutation assay features a straightforward, sensitive genotype system using SNaPshot primer extension analysis with primers that target a sequence immediately upstream of a specific mutation site. This approach is particularly useful when hotspot mutation sites are known, as is the case for KRAS and BRAF. Both the multiplex mutation assay and HRM-sequencing offer inexpensive, sensitive, and rapid screening for mutations; need little DNA input; have a good performance rate on DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue; and have superior mutation analysis by (nested-) PCR and cycle sequencing.7Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar, 8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar, 10Heideman D.A. Thunnissen F.B. Doeleman M. Kramer D. Verheul H.M. Smit E.F. Postmus P.E. Meijer C.J. Meijer G.A. Snijders P.J. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes.Cell Oncol. 2009; 31: 329-333PubMed Google Scholar Based on these findings, both methods can be reliably used as companion diagnostics to select patients for targeted therapy. However, these assays have not been mutually analyzed thus far. In the present study, we compared the SNaPshot-based multiplex mutation assay with HRM-sequencing analysis for identifying KRAS codons 12/13/61 and BRAF codon 600/601 mutations on 195 FFPE tumor–derived DNA samples. Subsequently, we evaluated the performance of a successor assay (HRM-SNaPshot) in which both HRM and SNaPshot analyses are combined. A consecutive series of FFPE tissue–derived DNAs isolated from 152 non–small-cell lung carcinomas and 43 metastasized CRCs, which were evaluated during routine diagnostics in 2009, were selected from the files of the Department of Pathology (VU University Medical Center, Amsterdam, The Netherlands). All samples were used in compliance with the respective institutional ethical regulations for surplus material.11Federa. Code 2011: proper secondary use of human tissue in scientific research Dutch Federation of Biomedical Scientific Societies. Stichting FMWV, Rotterdam, the Netherlands2011Google Scholar Specimens were processed according to a routinely used protocol in which the tumor tissue was manually macrodissected from serial sections guided by an H&E-stained tissue section on which the tumor was marked by a pathologist. DNA was subsequently isolated by proteinase K digestion, followed by a magnetic bead isolation procedure.8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar All DNA concentrations were measured on a Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). DNA specimens isolated from human cancer cell lines HCT116 (KRAS G13D), Calu6 (KRAS Q61K), and RKO (BRAF V600E) were used as positive controls throughout the study. Dilution series of these cell lines [ie, 50%, 25%, 10%, 5%, 2.5%, 1.25%, and 0.625% of mutant DNA in a background of wild-type (WT) DNA extracted from the SiHa cell line] were used to compare the analytical sensitivity of assays. HRM, followed by cycle sequencing of PCR products displaying abnormal melting, was performed at the Department of Pathology, VU University Medical Center, essentially as previously described,8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar using primers and probes as indicated in Table 1.7Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar, 8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar, 12Pichler M. Balic M. Stadelmeyer E. Ausch C. Wild M. Guelly C. Bauernhofer T. Samonigg H. Hoefler G. Dandachi N. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.J Mol Diagn. 2009; 11: 140-147Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Primers for BRAF exon 15 (147 bp) were derived from Pichler et al.12Pichler M. Balic M. Stadelmeyer E. Ausch C. Wild M. Guelly C. Bauernhofer T. Samonigg H. Hoefler G. Dandachi N. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.J Mol Diagn. 2009; 11: 140-147Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Specimens that failed to generate a BRAF exon 15 PCR product were retested using another HRM, generating a shorter (ie, 75-bp) BRAF amplicon (Table 17Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar, 8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar, 12Pichler M. Balic M. Stadelmeyer E. Ausch C. Wild M. Guelly C. Bauernhofer T. Samonigg H. Hoefler G. Dandachi N. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.J Mol Diagn. 2009; 11: 140-147Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To allow direct sequencing of HRM products, M13-tagged forward or reverse primers were used. Unidirectional sequencing of HRM PCR products was performed using the BigDye Terminator version 3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA), M13 forward (−20) primer, Big Dye XTerminator purification kit (Applied Biosystems), and the ABI PRISM 3500 Genetic Analyzer (Applied Biosystems), as previously described.8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar Given that HRM prescreening identifies samples likely to contain a nucleotide substitution, unidirectional sequencing suffices to determine the type of mutation.Table 1Oligonucleotides Used in PCROligonucleotideSequenceProduct size (bp)Concentration (μmol/L)HRM BRAF exon 1512Pichler M. Balic M. Stadelmeyer E. Ausch C. Wild M. Guelly C. Bauernhofer T. Samonigg H. Hoefler G. Dandachi N. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.J Mol Diagn. 2009; 11: 140-147Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Forward 1475′-GGTGATTTTGGTCTAGCTACAG-3′1470.2 Reverse 1475′-GTAAAACGACGGCCAGAGTAACTCAGCAGCATCTCAGG-3′0.2 KRAS exon 28Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar Forward5′-GTAAAACGACGGCCAGTCACATTTTCATTATTTTTATTATAAGGC-3′950.5 Reverse5′-GATTCTGAATTAGCTGTATCGTCAAG-3′0.1 KRAS exon 2 WT probe8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar5′-CTTGCCTACGCCACCAGCTCCAACT-3′-C3 spacer0.5KRAS exon 3 Forward5′-GTAAAACGACGGCCAGCCTTCTCAGGATTCCTACAGGAAGCAAG-3′1000.5 Reverse5′-AGTCCTCATGTACTGGTCCCTCA-3′0.1 WT probe5′-CCTCTTGACCTGCTGTGTCGAGAATAT-3′-C3 spacer0.5BRAF exon 15 Forward 755′-CACAGTAAAAATAGGTGATTTTGG-3′750.2 Reverse 755′-GTAAAACGACGGCCAGAACTGTTCAAACTGATGGGAC-3′0.2Multiplex Mutation Assay BRAF exon 157Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar Forward5′-TCTTCATGAAGACCTCACAGT-3′960.3 Reverse5′-CCAGACAACTGTTCAAACTGA-3′0.3KRAS exon 27Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar Forward5′-GGCCTGCTGAAAATGACTG-3′1630.7 Reverse5′-GGTCCTGCACCAGTAATATG-3′0.7KRAS exon 37Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar Forward5′-CCAGACTGTGTTTCTCCCTT-3′1550.3 Reverse5′-CACAAAGAAAGCCCTCCCCA-3′0.3The M13 moiety is underlined. Open table in a new tab The M13 moiety is underlined. The multiplex PCR for KRAS exon 2 (163 bp) and exon 3 (155 bp), and BRAF exon 15 (96 bp), followed by mutation detection by SNaPshot analysis using a SNaPshot Multiplex kit (Applied Biosystems), was performed at the Department of Pathology, Josephine Nefkens Institute (Erasmus MC, Rotterdam, the Netherlands), according to a routinely used protocol7Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar using primers and probes as indicated in Tables 17Lurkin I. Stoehr R. Hurst C.D. van Tilborg A.A. Knowles M.A. Hartmann A. Zwarthoff E.C. Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes.PLoS One. 2010; 5: e8802Crossref PubMed Scopus (84) Google Scholar, 8Kramer D. Thunnissen F.B. Gallegos-Ruiz M.I. Smit E.F. Postmus P.E. Meijer C.J. Snijders P.J. Heideman D.A. A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.Cell Oncol. 2009; 31: 161-167PubMed Google Scholar, 12Pichler M. Balic M. Stadelmeyer E. Ausch C. Wild M. Guelly C. Bauernhofer T. Samonigg H. Hoefler G. Dandachi N. Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.J Mol Diagn. 2009; 11: 140-147Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar and 2, respectively. Specimens that failed to generate a result for one or more exons were additionally tested by a modified SNaPshot assay with adjusted primer and/or probe sequences and concentrations, as detailed in Table 2.Table 2SNaPshot ProbesProbeSequenceAdjustmentSize (bp)StrandWTMutantConcentration (μmol/L)Original AssayBRAF Position 1799T14: 5′-GGTGATTTTGGTCTAGCTACAG-3′None36SenseTA0.2 Position 1801T21: 5′-GGACCCACTCCATCGAGATT-3′None43AntisenseTG/C0.2KRAS Position 34T30: 5′-GGCACTCTTGCCTACGCCAC-3′None50AntisenseCG/A/T0.4 Position 35T36: 5′-AACTTGTGGTAGTTGGAGCTG-3′None57SenseGC/A/T0.1 Position 37T44: 5′-CAAGGCACTCTTGCCTACGC-3′None64AntisenseCG/A/T1.4 Position 38T49: 5′-CTTGTGGTAGTTGGAGCTGGTG-3′None71SenseGC/A/T0.1 Position 57T54: 5′-ATGATTCTGAATTAGCTGTATCGT-3′None78AntisenseCA1.4 Position 181T63: 5′-CTCATTGCACTGTACTCCTCTT-3′None85AntisenseGC/T0.08 Position 182T73: 5′-ATTCTCGACACAGCAGGTC-3′None92SenseAC/G/T0.2 Position 183T76: 5′-CCTCATTGCACTGTACTCCTC-3′None97AntisenseTG/A0.08Adjusted Assay⁎The PCR products of the three HRM assays were analyzed in separate SNaPshot reactions. Data in parentheses for each probe indicate the target for which it was used.BRAF Position 1799 (BRAFx15)T14: 5′-GGTGATTTTGGTCTAGCTACAG-3′Concentration36SenseTA0.4 Position 1801 (BRAFx15)T21: 5′-GGACCCACTCCATCGAGATT-3′Concentration43AntisenseTG/C0.6KRAS Position 34 (KRASx2)T29: 5′-AAACTTGTGGTAGTTGGAGCT-3′Sequence†To increase peak heights. and concentration50SenseGC/A/T0.4 Position 35 (KRASx2)T36: 5′-AACTTGTGGTAGTTGGAGCTG-3′Concentration57SenseGC/A/T0.4 Position 37 (KRASx2)T44: 5′-CAAGGCACTCTTGCCTACGC-3′Concentration64AntisenseCG/A/T0.8 Position 38 (KRASx2)T49: 5′-CTTGTGGTAGTTGGAGCTGGTG-3′None71SenseGC/A/T0.1 Position 57‡HRM-sequencing and HRM-SNaPshot are unable to detect c.57G>T (p.L19F) because the HRM reverse primer overlaps this codon. Accordingly, no probe specific for this nucleotide change was included in the KRAS exon 2–specific SNaPshot.T54: 5′-ATGATTCTGAATTAGCTGTATCGT-3′Concentration78AntisenseCA2.0 Position 181 (KRASx3)T63: 5′-CTCATTGCACTGTACTCCTCTT-3′Concentration85AntisenseGC/T0.6 Position 182 (KRASx3)T73: 5′-ATTCTCGACACAGCAGGTC-3′Concentration92SenseAC/G/T0.4 Position 183 (KRASx3)T76: 5′-CCTCATTGCACTGTACTCCTC-3′Concentration97AntisenseTG/A1.0 The PCR products of the three HRM assays were analyzed in separate SNaPshot reactions. Data in parentheses for each probe indicate the target for which it was used.† To increase peak heights.‡ HRM-sequencing and HRM-SNaPshot are unable to detect c.57G>T (p.L19F) because the HRM reverse primer overlaps this codon. Accordingly, no probe specific for this nucleotide change was included in the KRAS exon 2–specific SNaPshot. Open table in a new tab HRM PCR products were generated at the Department of Pathology (VU University Medical Center) and subjected to SNaPshot analysis at the Department of Pathology, Josephine Nefkens Institute, in a singleplex format (ie, the PCR amplicons of the different exons were separately analyzed by SNaPshot analysis using only the probes targeting the respective exon). HRM PCR products (3 μL) were treated with 2 μL of ExoSAPit (USB, Cleveland, OH) at 37°C for 30 minutes, followed by 80°C for 15 minutes. This was followed by probe extension using a SNaPshot Multiplex kit and probes, as indicated in Table 2. The mutation detection reactions were performed in a total volume of 10 μL, containing 1 μL of shrimp alkaline phosphatase/ExoI-treated PCR product, 2.5 μL of SNaPshot Multiplex Ready Reaction mix (Applied Biosystems), one times Big Dye sequencing buffer (Applied Biosystems), and 1 μL of probe mix. Thermal cycler conditions were as follows: 35 cycles of 10 seconds at 96°C and 40 seconds at 58.5°C. The labeled extension products were treated with 1 U of SAP at 37°C for 60 minutes and 72°C for 15 minutes. Next, 2 μL was mixed with 9.8 μL of HiDi formamide (Applied Biosystems) and 0.2 μL of Genescan-120LIZ (Applied Biosystems) size standard. Products were denatured at 95°C for 5 minutes and then separated using an ABI PRISM 3130 XL Genetic Analyzer (Applied Biosystems). The fluorescent label on the incorporated ddNTPs indicated the presence or absence of a mutation. For analysis of the data, GeneMarker version 1.70 (2004, SoftGenetics LLC, State College, PA) was used. The mutation assays were performed independently and in a blinded manner, and the results were compared afterward. For feasibility analysis of HRM-SNaPshot, all HRM PCR products were subjected to SNaPshot, irrespective of and blinded for the results of the melting curve analysis by HRM. For diagnostic interest, HRM prescreening was considered in data analysis (ie, only among samples with a suspect profile in HRM, the genotyping data of SNaPshot were considered). Statistical analyses were performed using SPSS statistical package, version 15.0 (2003, SPSS, Inc., Chicago, IL). The level of agreement between genotype findings of different methods was determined using κ statistics. Genotyping results were considered concordant in case of sequence agreement between assays and discordant in case no genotype similarity was observed. In this analysis, only nucleotide substitutions that could be detected by all assays (ie, BRAF c.1799 and c.1801 and KRAS c.34, c.35, c.37, c.38, c.181, c.182, and c.183) were considered (Table 3). Cases for which one or more assays failed to generate a result (referred to as not determined) were excluded from κ statistics. The level of statistical significance was set at 0.05.Table 3Overview of Nucleotide Substitution Detectable by Each Assay and the Respective Frequency Found in this StudyVariableNucleotide substitutionHRM- sequencingValues, no. (%)Multiplex mutation assayValues, no. (%)HRM- SNaPshotValues, no. (%)NotesBRAF codon 600/601KRAS codon 12/13 BRAF position 1799T>AYes2 (1)Yes2 (1)Yes2 (1) BRAF position 1801A>G/CYes—Yes—Yes— KRAS position 34G>AYes2 (1)Yes2 (1)Yes2 (1) KRAS position 34G>TYes17 (9)⁎One specimen had a combination of c.34G>T and c.35G>T.Yes16 (8)Yes18 (9) KRAS position 34G>CYes—Yes—Yes— KRAS position 35G>AYes8 (4)Yes8 (4)Yes8 (4) KRAS position 35G>CYes3 (1.5)Yes3 (1.5)Yes3 (1.5) KRAS position 35G>TYes18 (9)⁎One specimen had a combination of c.34G>T and c.35G>T.Yes17 (9)Yes17 (9) KRAS position 37G>TYes4 (2)Yes4 (2)Yes4 (2) KRAS position 37G>A/CYes—Yes—Yes— KRAS position 38G>AYes9 (4.5)Yes8 (4)Yes9 (4.5) KRAS position 38G>C/TYes—Yes—Yes— KRAS position 181C>G/TYes—Yes—Yes— KRAS position 182A>GYes1 (0.5)Yes1 (0.5)Yes1 (0.5) KRAS position 182A>C/TYes—Yes—Yes— KRAS position 183A>CYes1 (0.5)Yes1 (0.5)Yes2 (1) KRAS position 183A>TYes—Yes—Yes—Others KRAS position 57G>TNo—Yes1 (0.5)No—The HRM reverse primer covers this codon KRAS position 30A>TYes1 (0.5)No—No—No probe included in SNaPshot analysis KRAS position 36T> AYes1 (0.5)No—No—No probe included in SNaPshot analysis KRAS position 39C>AYes1 (0.5)No—No—No probe included in SNaPshot analysis KRAS position 176C>AYes1 (0.5)No—No—No probe included in SNaPshot analysis Region flanked by primer pairAny substitutionYes—No—No—No probe included in SNaPshot analysisNo, a nucleotide alteration cannot be detected by the respective assay; yes, a nucleotide alteration can be detected by the respective assay. One specimen had a combination of c.34G>T and c.35G>T. Open table in a new tab No, a nucleotide alteration cannot be detected by the respective assay; yes, a nucleotide alteration can be detected by the respective assay. Reconstruction experiments using a dilution series of DNA isolated from cell lines demonstrated an analytical sensitivity of HRM-sequencing of 2.5% of mutant DNA in a background of WT DNA. The multiplex mutation assay had an analytical sensitivity of 5%. The type of mutation was 100% correct for both HRM-sequencing and multiplex mutation assay. Next, a series of 195 FFPE tumor–derived DNAs from patients with non–small-cell lung carcinoma or CRC was evaluated for KRAS and BRAF mutations using HRM-sequencing and multiplex mutation assay. The results are summarized in Table 3, Table 4, and detailed assay findings per specimen are depicted in Supplemental Table S1 (available at http://jmd.amjpathol.org). An example of results of both assays on a sample with a KRAS c.35G>A (p.G12D) mutation is shown in Figure 1, A and B. The assays were highly compatible for genotype findings (κ, 0.98; 95% CI, 0.94 to 1) (Table 4).Table 4Comparison of Assay Performance: HRM-Sequencing versus Multiplex Mutation AssayHRM-sequencingMultiplex mutation assayWild typeMutant⁎Only nucleotide substitutions that could be detected by all assays (ie, BRAF c.1799 and c.1801 and KRAS c.34, c.35, c.37, c.38, c.181, c.182, and c.183) were considered.Wild type130†Five specimens were, in part, not determined by the multiplex mutation assay.1Mutant⁎Only nucleotide substitutions that could be detected by all assays (ie, BRAF c.1799 and c.1801 and KRAS c.34, c.35, c.37, c.38, c.181, c.182, and c.183) were considered.3‡Two specimen were not determined by the multiplex mutation assay for a respective exon containing the mutation.61Genotyping agreement: κ, 0.98 (95% CI, 0.94 to 1). Only nucleotide substitutions that could be detected by all assays (ie, BRAF c.1799 and c.1801 and KRAS c.34, c.35, c.37, c.38, c.181, c.182, and c.183) were considered.† Five specimens were, in part, not determined by the multiplex mutation assay.‡ Two specimen were not determined by the multiplex mutation assay for a respective exon containing the mutation. Open table in a new tab Genotyping agreement: κ, 0.98 (95% CI, 0.94 to 1). Given the high agreement in assay findings between HRM-sequencing and multiplex mutation assay, we next assessed the feasibility of a combined approach of HRM with SNaPshot for mutation analysis of KRAS and BRAF. Application of HRM-SNaPshot to the dilution series of mutant DNA revealed that the assay identified the known mutations with 100% accuracy and an analytical sensitivity down to 1.25%. Next, HRM-SNaPshot was performed on the series of 195 FFPE tumor–derived DNAs. For feasibility analysis, SNaPshot analysis was applied to all HRM PCR products, which demonstrated a success rate of 100%. For diagnostic interest, HRM prescreening was considered for interpretation of the data (see Supplemental Tab
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