Detection of KRAS and BRAF Mutations in Colorectal Carcinoma
2011; Elsevier BV; Volume: 13; Issue: 1 Linguagem: Inglês
10.1016/j.jmoldx.2010.11.005
ISSN1943-7811
AutoresMaria E. Arcila, Christopher Lau, Khédoudja Nafa, Marc Ladanyi,
Tópico(s)Cancer Genomics and Diagnostics
ResumoKRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that is suitable for broad mutation screening. Clinical cases (n = 308) previously tested for KRAS and BRAF by standard sequencing were retested by LNA-PCR sequencing incorporating an LNA oligonucleotide to suppress amplification of nonmutant DNA, and by a Sequenom assay panel targeting common mutations in both genes. Standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations; retesting with the LNA-based method and the Sequenom assay detected 19 (140/308, 45%) and 6 (127/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay. The analytical sensitivities were 0.3% for both KRAS and BRAF by LNA-PCR and from 1% to 10% for the Sequenom assays, depending on the specific mutation. Given these results, standard sequencing is suboptimal for mutation detection in metastatic and treated lesions even with predissection for tumor enrichment. High-sensitivity LNA-PCR sequencing detects significantly more mutations, whereas the Sequenom platform shows intermediate sensitivity but offers significant advantages for broader mutation screening. KRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that is suitable for broad mutation screening. Clinical cases (n = 308) previously tested for KRAS and BRAF by standard sequencing were retested by LNA-PCR sequencing incorporating an LNA oligonucleotide to suppress amplification of nonmutant DNA, and by a Sequenom assay panel targeting common mutations in both genes. Standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations; retesting with the LNA-based method and the Sequenom assay detected 19 (140/308, 45%) and 6 (127/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay. The analytical sensitivities were 0.3% for both KRAS and BRAF by LNA-PCR and from 1% to 10% for the Sequenom assays, depending on the specific mutation. Given these results, standard sequencing is suboptimal for mutation detection in metastatic and treated lesions even with predissection for tumor enrichment. High-sensitivity LNA-PCR sequencing detects significantly more mutations, whereas the Sequenom platform shows intermediate sensitivity but offers significant advantages for broader mutation screening. Colorectal carcinoma (CRC) is one of the leading causes of cancer-related death worldwide, with a 5-year survival rate of less than 10% for patients with metastatic disease.1Venook A.P. 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Landi B. Emile J.F. Cote J.F. Tomasic G. Penna C. Ducreux M. Rougier P. Penault-Llorca F. Laurent-Puig P. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer.Cancer Res. 2006; 66: 3992-3995Crossref PubMed Scopus (1915) Google Scholar, 33Punt C.J. Tol J. Rodenburg C.J. Cats A. Creemers G. Schrama J.G. Erdkamp F.L. Vos A. Mol L. Antonini N.F. Randomized phase III study of capecitabine, oxaliplatin, and bevacizumab with or without cetuximab in advanced colorectal cancer (ACC), the CAIRO2 study of the Dutch Colorectal Cancer Group (DCCG).J Clin Oncol. 2008; 26 (abstract LBA4011)Google Scholar, 35Van Cutsem E. Kohne C.H. Hitre E. Zaluski J. Chang Chien C.R. Makhson A. D'Haens G. Pinter T. Lim R. Bodoky G. Roh J.K. Folprecht G. Ruff P. Stroh C. Tejpar S. Schlichting M. Nippgen J. Rougier P. 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Taylor B.S. Pao W. Toyooka S. Ladanyi M. Gazdar A. Rosen N. Solit D.B. Genetic predictors of MEK dependence in non-small cell lung cancer.Cancer Res. 2008; 68: 9375-9383Crossref PubMed Scopus (212) Google Scholar, 34Solit D.B. Garraway L.A. Pratilas C.A. Sawai A. Getz G. Basso A. Ye Q. Lobo J.M. She Y. Osman I. Golub T.R. Sebolt-Leopold J. Sellers W.R. Rosen N. BRAF mutation predicts sensitivity to MEK inhibition.Nature. 2006; 439: 358-362Crossref PubMed Scopus (1147) Google Scholar Mutations in KRAS and BRAF are mutually exclusive,28Di 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 (1443) Google Scholar supporting sequential testing as a rational algorithm. Based on these studies, and further stimulated by the American Society of Clinical Oncology 2008 guidelines, there is currently intense interest in rapid, reliable, and accurate methods of KRAS and BRAF mutation screening. A wide spectrum of technical approaches can be applied to the routine detection of point mutations in KRAS and BRAF,40Do H. Krypuy M. Mitchell P.L. Fox S.B. Dobrovic A. High-resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies.BMC Cancer. 2008; 8: 142Crossref PubMed Scopus (194) Google Scholar, 41Fox J.C. England J. White P. Ellison G. Callaghan K. Charlesworth N.R. Hehir J. McCarthy T.L. Smith-Ravin J. Talbot I.C. Snary D. Northover J.M. Newton C.R. Little S. The detection of K-ras mutations in colorectal cancer using the amplification-refractory mutation system.Br J Cancer. 1998; 77: 1267-1274Crossref PubMed Scopus (34) Google Scholar, 42Marsh S. Pyrosequencing applications.Methods Mol Biol. 2007; 373: 15-24PubMed Google Scholar, 43Ogino S. Kawasaki T. Brahmandam M. Yan L. Cantor M. Namgyal C. Mino-Kenudson M. Lauwers G.Y. Loda M. Fuchs C.S. Sensitive sequencing method for KRAS mutation detection by pyrosequencing.J Mol Diagn. 2005; 7: 413-421Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 44Plesec T.P. Hunt J.L. KRAS mutation testing in colorectal cancer.Adv Anat Pathol. 2009; 16: 196-203Crossref PubMed Scopus (88) Google Scholar each with inherent advantages and disadvantages, depending on individual detection limits, labor requirements, turnaround time, expense of reagents and equipment, and potential for automation and multiplexing. Sanger sequencing of PCR-amplified DNA is the classic and most widely used method of mutation detection.22Bardelli A. Siena S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer.J Clin Oncol. 2010; 28: 1254-1261Crossref PubMed Scopus (562) Google Scholar, 45Garcia J. Riely G.J. Nafa K. Ladanyi M. KRAS mutational testing in the selection of patients for EGFR-targeted therapies.Semin Diagn Pathol. 2008; 25: 288-294Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar However, it has several drawbacks in this particular setting, primarily because of suboptimal sensitivity and its relatively labor-intensive, low-throughput nature. Although the problem of sensitivity can be overcome by microdissection in most cases, it remains a major challenge in the context of recurrent CRC, when testing is commonly based on post-treatment specimens in which scant tumor cells are intimately admixed with abundant non-neoplastic cells. Not infrequently, these tests yield false-negative results despite attempts at predissecting areas richer in tumor. Thus, many institutions and commercial laboratories reject samples in which the tumor cell percentage does not exceed a preset standard. For those institutions that continue routinely to predissect, this process can be time consuming. Furthermore, as the testing volume increases and additional specific markers of response and targetable pathways continue to be discovered, the low throughput inherent in standard sequencing is becoming a major issue. We need optimal testing strategies that are reliable, appropriately sensitive, and can adequately address the testing volume expected from the high prevalence of CRC. To address these issues, we developed and evaluated two mutation genotyping assays: a modified PCR-sequencing assay using a locked nucleic acid (LNA) probe for mutant enrichment and a mass spectrometry-based genotyping assay (Sequenom, San Diego, CA) suitable for large-scale mutation screening. Herein we describe our experience with these two assays and compare them with traditional Sanger sequencing for routine mutation testing of CRC specimens. We demonstrate that both techniques have a higher analytical sensitivity for the detection of mutant DNA and, at different levels, both techniques are less labor intensive. Metastatic CRC samples (n = 334) received for routine clinical KRAS and BRAF testing at Memorial Sloan-Kettering Cancer Center between January and May 2009 were selected for the study. H&E-stained sections of formalin-fixed paraffin-embedded tissue were reviewed for each sample to identify and circle the areas of highest tumor density, ensuring at least 50% tumor content. Macrodissection was performed using the tip of a blade to scrape off the selected tumor areas on corresponding unstained sections. Genomic DNA was extracted using the DNeasy Tissue kit (Qiagen, Valencia, CA), following the manufacturer's standard protocol. The entire coding region of exon 2 of KRAS was amplified using the following forward and reverse intronic primers (custom oligos, Proligo, Boulder, CO): KRAS-F, 5′-GTGTGACATGTTCTAATATAGTCA-3′; and KRAS-R, 5′-CTGTATCAAAGAATGGTCCTGCAC-3′. All samples were tested in duplicate. Each PCR reaction was performed in a 50-μL volume mixture containing 100 ng of genomic DNA; forward and reverse primers (20 pmol each); 50 μmol/L each of dATP, dCTP, dGTP; 400 μmol/L of dUTP; 1.5 mmol/L MgCl2; 1X Qiagen PCR buffer containing 1.5 mmol/L MgCl2 and 2.5 units of HotStarTaq DNA polymerase (Qiagen). AmpErase uracil N-glycosylase (Applied Biosystems, Foster City, CA) (0.5 unit) was added to each reaction mixture for carryover prevention, and they were incubated at room temperature for 10 minutes. The PCR amplification was carried out under the following conditions: 1 cycle at 95°C (15 minutes); 40 cycles at 94°C (30 seconds), at 56°C (30 seconds), and at 72°C (30 seconds); and a final extension step at 72°C (7 minutes). Amplified products were purified using Spin Columns (Qiagen) and sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the manufacturer's protocol, on an ABI3730 running ABI Prism DNA Sequence Analysis Software (Applied Biosystems). BRAF mutation analysis was carried out following the same procedure described for KRAS, using the forward and reverse primers (custom oligos; Proligo, Boulder, CO); BRAF/15F, 5′-TCATAATGCTTGCTCTGATAGG-3′; and BRAF/15R, 5′-GGCCAAAAATTTAATCAGTGG-3′ to amplify the entire coding region of exon 15 of BRAF. For these assays we used the MassARRAY system (Sequenom), which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This system was originally designed to analyze single-nucleotide polymorphisms (SNPs) in amplified DNA fragments,48Nelson M.R. Marnellos G. Kammerer S. Hoyal C.R. Shi M.M. Cantor C.R. Braun A. Large-scale validation of single nucleotide polymorphisms in gene regions.Genome Res. 2004; 14: 1664-1668Crossref PubMed Scopus (70) Google Scholar but it is also wel
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