Multicenter Evaluation of the Idylla NRAS-BRAF Mutation Test in Metastatic Colorectal Cancer
2018; Elsevier BV; Volume: 20; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2018.05.008
ISSN1943-7811
AutoresIván Prieto‐Potín, Clara Montagut, Beatríz Bellosillo, Matthew Evans, Matthew R. Smith, Linea Cecilie Melchior, Réiltín Werner, Michael Bennett, Veronica Pennati, Franca Castiglione, Karl-Friedrich Bürrig, Ulrike Cooper, Barbara Dockhorn‐Dworniczak, Christiana Rossenbach, Claudia Maribel Luna-Aguirre, Hugo A. Barrera‐Saldaña, José Carlos Machado, José Luís Costa, Rinat Yacobi, Hilla Tabibian‐Keissar, Simonetta Buglioni, Livia Ronchetti, Lotte Douglas-Berger, Hendrikus J. Dubbink, Mohammed Alorini, J.C. Sabourin, Federico Rojo,
Tópico(s)Cancer Genomics and Diagnostics
ResumoTreatment of colorectal cancer (CRC) with monoclonal antibodies against epidermal growth factor receptor requires the assessment of the mutational status of exons 2, 3, and 4 of the NRAS and KRAS oncogenes. Moreover, the mutational status of exon 15 of the BRAF oncogene is a marker of poor prognosis in CRC. The Idylla NRAS-BRAF Mutation Test is a reliable, simple (<2 minutes hands-on time), and quick (<2 hours turnaround time) sample-to-result solution, enabling the detection of clinically relevant mutations in NRAS (18 mutations) and BRAF (5 mutations). A multicenter study was conducted in 14 centers using the Idylla NRAS-BRAF Mutation Test to assess the NRAS and BRAF mutational status of 418 formalin-fixed, paraffin-embedded tissue samples from CRC patients. Results were compared with those obtained earlier by routine reference methods, including next-generation sequencing, pyrosequencing, mass spectrometry–based assays, PCR-based assays, and Sanger sequencing. In case of discordance, additional tests were performed by digital droplet PCR. Overall, after testing confirmation and excluding invalids/errors by design, concordances between the Idylla NRAS-BRAF Mutation Test and the reference test results were found in almost perfect agreement. In conclusion, the Idylla NRAS-BRAF Mutation Test enables the routine detection of all NRAS and BRAF mutations deemed clinically relevant according to the latest clinical guidelines, without necessitating molecular expertise or infrastructure. Treatment of colorectal cancer (CRC) with monoclonal antibodies against epidermal growth factor receptor requires the assessment of the mutational status of exons 2, 3, and 4 of the NRAS and KRAS oncogenes. Moreover, the mutational status of exon 15 of the BRAF oncogene is a marker of poor prognosis in CRC. The Idylla NRAS-BRAF Mutation Test is a reliable, simple (<2 minutes hands-on time), and quick ( 2 years.4Gustavsson B. Carlsson G. Machover D. Petrelli N. 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Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti–epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Summary.J Oncol Pract. 2016; 12: 180-181Crossref PubMed Scopus (20) Google Scholar genotyping of tumor tissue (primary or metastatic) for the presence of the above mentioned KRAS and NRAS mutations is mandatory before initiating anti-EGFR monoclonal antibody treatment of mCRC patients.10Van Cutsem E. Cervantes A. Adam R. Sobrero A. Van Krieken J.H. Aderka D. et al.ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.Ann Oncol. 2016; 27: 1386-1422Abstract Full Text Full Text PDF PubMed Scopus (2095) Google Scholar, 11Benson A.B. Venook A.P. Cederquist L. Chan E. Chen Y.-J.J. Cooper H.S. Deming D. Engstrom P.F. Enzinger P.C. Fichera A. Grem J.L. Grothey A. Hochster H.S. Hoffe S. Hunt S. Kamel A. Kirilcuk N. Krishnamurthi S. Messersmith W.A. Mulcahy M.F. Murphy J.D. Nurkin S. Saltz L. Sharma S. Shibata D. Skibber J.M. Sofocleous C.T. Stoffel E.M. Stotsky-Himelfarb E. Willett C.G. Wu C.S. Gregory K.M. Freedman-Cass D. Colon cancer, version 1.2017, NCCN clinical practice guidelines in oncology.J Natl Compr Canc Netw. 2017; 15: 370-398Crossref PubMed Scopus (520) Google Scholar, 12Allegra C.J. Rumble R.B. Schilsky R.L. Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti–epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Summary.J Oncol Pract. 2016; 12: 180-181Crossref PubMed Scopus (20) Google Scholar The v-Raf murine sarcoma viral oncogene homolog B (BRAF) V600E mutation is a significant biomarker of poor prognosis in CRC.13Di Nicolantonio F. Martini M. Molinari F. Sartore-Bianchi A. Arena S. Saletti P. De Dosso S. Mazzucchelli L. Frattini M. Siena S. 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Aderka D. et al.ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.Ann Oncol. 2016; 27: 1386-1422Abstract Full Text Full Text PDF PubMed Scopus (2095) Google Scholar Substantial literature evidence indicates that BRAF mutations are mutually exclusive with RAS mutations,15Morkel M. Riemer P. Bläker H. Sers C. Similar but different: distinct roles for KRAS and BRAF oncogenes in colorectal cancer development and therapy resistance.Oncotarget. 2015; 6: 20785-20800Crossref PubMed Scopus (88) Google Scholar, 16Cisowski J. Sayin V.I. Liu M. Karlsson C. Bergo M.O. Oncogene-induced senescence underlies the mutual exclusive nature of oncogenic KRAS and BRAF.Oncogene. 2016; 35: 1328-1333Crossref PubMed Scopus (58) Google Scholar although exceptions have been reported.17Grellety T. Gros A. Pedeutour F. Merlio J.P. Duranton-Tanneur V. Italiano A. Soubeyran I. 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Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8310) Google Scholar Both the NRAS and BRAF proteins are components of the mitogen-activated protein kinase cascade that is implicated in a wide variety of cellular functions, such as cell proliferation and cell survival.21Peyssonnaux C. Eychène A. The Raf/MEK/ERK pathway: new concepts of activation.Biol Cell. 2001; 93: 53-62Crossref PubMed Scopus (615) Google Scholar The membrane-bound NRAS protein (189 amino acids) has GTPase activity and functions as a mediator in cell signal transduction. RAS proteins are activated by ligand binding to an upstream transmembrane tyrosine kinase receptor (eg, EGFR), which, in turn, leads to activation of downstream effectors, including BRAF, a serine/threonine-specific protein kinase (765 amino acids) belonging to the Raf family.22Chong H. Vikis H.G. Guan K.L. Mechanisms of regulating the Raf kinase family.Cell Signal. 2003; 15: 463-469Crossref PubMed Scopus (345) Google Scholar Activated Raf proteins activate mitogen-activated protein kinase/extracellular signal-regulated kinase in a further step in the signal cascade.21Peyssonnaux C. Eychène A. The Raf/MEK/ERK pathway: new concepts of activation.Biol Cell. 2001; 93: 53-62Crossref PubMed Scopus (615) Google Scholar Mutations of NRAS or BRAF may lead to a constitutive activation, as well as to an increased affinity toward downstream effectors of the mitogen-activated protein kinase signaling pathway, which is a crucial event in many cancers.23Fernandez-Medarde A. Santos E. Fernández-Medarde A. Santos E. RAS in cancer and developmental diseases.Genes Cancer. 2011; 2: 344-358Crossref PubMed Scopus (612) Google Scholar Routine genotyping of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples is performed by laboratory-based assays or commercially available CE-IVD kits, and there is a rapid uptake of extended RAS mutation testing in addition to KRAS exon 2 mutations.24Boleij A. Tack V. Taylor A. Kafatos G. Jenkins-Anderson S. Tembuyser L. Dequeker E. van Krieken J.H. RAS testing practices and RAS mutation prevalence among patients with metastatic colorectal cancer: results from a Europe-wide survey of pathology centres.BMC Cancer. 2016; 16: 825Crossref PubMed Scopus (26) Google Scholar Routine methods include Sanger sequencing, pyrosequencing, next-generation sequencing (NGS), immunohistochemistry, and tests based on real-time PCR, with each method having its sensitivity, specificity, cost, turnaround time, level of automation and multiplexing, and need for specialized equipment and skilled staff. In particular, turnaround time is an important parameter in this population of rapidly progressing metastatic patients. In a recent study in 96 laboratories across 24 European countries, the turnaround times from sample reception to report for RAS testing were ≤10 working days for 90% of the institutions and ≤5 working days for 47% of the institutions,24Boleij A. Tack V. Taylor A. Kafatos G. Jenkins-Anderson S. Tembuyser L. Dequeker E. van Krieken J.H. RAS testing practices and RAS mutation prevalence among patients with metastatic colorectal cancer: results from a Europe-wide survey of pathology centres.BMC Cancer. 2016; 16: 825Crossref PubMed Scopus (26) Google Scholar whereas guidelines require a turnaround time for expanded RAS analysis of ≤7 working days for >90% of the specimens.10Van Cutsem E. Cervantes A. Adam R. Sobrero A. Van Krieken J.H. Aderka D. et al.ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.Ann Oncol. 2016; 27: 1386-1422Abstract Full Text Full Text PDF PubMed Scopus (2095) Google Scholar The Idylla Mutation Tests performed on the Idylla platform (Biocartis, Mechelen, Belgium) do not require time-consuming FFPE processing steps, because they integrate analytical processes in a single cartridge and offer a fully automated sample-to-result solution. Earlier, the CE-IVD Idylla BRAF Mutation Test for the detection of BRAF V600 mutations in melanoma patients25Melchior L. Grauslund M. Bellosillo B. Montagut C. Torres E. Moragón E. Micalessi I. Frans J. Noten V. Bourgain C. Vriesema R. van der Geize R. Cokelaere K. Vercooren N. Crul K. Rüdiger T. Buchmüller D. Reijans M. Jans C. Multi-center evaluation of the novel fully-automated PCR-based IdyllaTM BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.Exp Mol Pathol. 2015; 99: 485-491Crossref PubMed Scopus (38) Google Scholar, 26Schiefer A.I. Parlow L. Gabler L. Mesteri I. Koperek O. von Deimling A. Streubel B. Preusser M. Lehmann A. Kellner U. Pauwels P. Lambin S. Dietel M. Hummel M. Klauschen F. Birner P. Möbs M. Multicenter evaluation of a novel automated rapid detection system of BRAF status in formalin-fixed, paraffin-embedded tissues.J Mol Diagn. 2016; 18: 370-377Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and the CE-IVD Idylla KRAS Mutation Test enabling detection of 21 clinically relevant KRAS mutations in mCRC27Solassol J. Vendrell J. Märkl B. Haas C. Bellosillo B. Montagut C. Smith M. O'Sullivan B. D'Haene N. Le Mercier M. Grauslund M. Melchior L.C. Burt E. Cotter F. Stieber D. Schmitt F.L. Motta V. Lauricella C. Colling R. Soilleux E. Fassan M. Mescoli C. Collin C. Pagès J.C. Sillekens P. Multi-center evaluation of the fully automated PCR-based IdyllaTM kras mutation assay for rapid KRAS mutation status determination on formalin-fixed paraffin-embedded tissue of human colorectal cancer.PLoS One. 2016; 11: e0163444Crossref PubMed Scopus (32) Google Scholar were successfully launched. The Idylla NRAS-BRAF Mutation Test has been developed to support therapeutic decisions made by physicians in mCRC: the NRAS mutational result is meant to aid identifying patients who may or may not benefit from anti-EGFR therapy, whereas the BRAF mutational result supports recognizing patients with poor prognosis. The Idylla NRAS-BRAF Mutation Test has been launched as CE-marked IVD. In the current study, the performance of the Idylla NRAS-BRAF Mutation Test, for Investigational Use Only, was evaluated in a multicenter study testing archived FFPE tumor samples from CRC patients. Results were compared with data previously obtained from the same samples screened with local routine reference methods. The study was conducted in 14 clinical centers and included archived clinical FFPE materials from 418 mCRC patients: Hospital del Mar (Barcelona, Spain; n = 30); Queen Elizabeth Hospital (Birmingham, UK; n = 30); Rigshospitalet (Copenhagen, Denmark; n = 30); Cork University Hospital (Cork, Ireland; n = 30); Careggi's Hospital (Firenze, Italy; n = 30); Institute of Pathology (Hildesheim, Germany; n = 30); Center for Pathology (Kempten, Germany; n = 28); Fundación Jiménez Díaz University Hospital (Madrid, Spain; n = 30); Vitagénesis (Monterrey, Mexico; n = 30); Institute of Molecular Pathology and Immunology of the University of Porto (Porto, Portugal; n = 31); Sheba Medical Center (Ramat Gan, Israel; n = 30); Regina Elena National Cancer Institute (Rome, Italy; n = 29); Erasmus MC Cancer Institute (Rotterdam, the Netherlands; n = 30); and Rouen University Hospital Centre (Rouen, France; n = 30). Each center was due to select at least three mutated samples for NRAS and a minimum of five mutated samples for BRAF. Nevertheless, two centers did not adhere to the condition. The use of these patient samples was approved by the respective local Ethics Committee and was in accordance with the Declaration of Helsinki. FFPE tissue sections (mostly one or two, up to seven), of 5 to 30 μm of thickness, were sampled mostly consecutive (within the same FFPE block) to the sections used earlier to generate the routine method reference result, and they were placed directly into the Idylla cartridge (Biocartis) following the assay instructions of the manufacturer. Tumor content and area of the specimen were determined on hematoxylin-eosin–stained slides by a pathologist and, if needed, macrodissection was performed (138 cases) to achieve a neoplastic cell content of at least 10%. The tissue area of the specimen should be between 50 and 600 mm2 when 5-μm FFPE tissue sections are used and between 25 and 300 mm2 when 10-μm FFPE tissue sections are used; multiple FFPE tissue sections can be used to meet this requirement. The required number of samples for a diagnostic agreement study with a precision value of 5% and an α error of 0.05 at a 95% CI is 384. Because of plausible sample exclusion or invalid runs, 5% of sample loss was assumed, so the target number of samples to be tested was set at 404. Each center might include approximately 30 samples with a known mutation on either NRAS or BRAF to comply with the established requirements. The Idylla NRAS-BRAF Mutation Test performed on the Biocartis Idylla System (Biocartis) is an in vitro sample-to-result diagnostic test for the qualitative detection of the 23 mutations in the NRAS and BRAF oncogenes considered clinically relevant according to the latest clinical guidelines.10Van Cutsem E. Cervantes A. Adam R. Sobrero A. Van Krieken J.H. Aderka D. et al.ESMO consensus guidelines for the management of patients with metastatic colorectal cancer.Ann Oncol. 2016; 27: 1386-1422Abstract Full Text Full Text PDF PubMed Scopus (2095) Google Scholar, 11Benson A.B. Venook A.P. Cederquist L. Chan E. Chen Y.-J.J. Cooper H.S. Deming D. Engstrom P.F. Enzinger P.C. Fichera A. Grem J.L. Grothey A. Hochster H.S. Hoffe S. Hunt S. Kamel A. Kirilcuk N. Krishnamurthi S. Messersmith W.A. Mulcahy M.F. Murphy J.D. Nurkin S. Saltz L. Sharma S. Shibata D. Skibber J.M. Sofocleous C.T. Stoffel E.M. Stotsky-Himelfarb E. Willett C.G. Wu C.S. Gregory K.M. Freedman-Cass D. Colon cancer, version 1.2017, NCCN clinical practice guidelines in oncology.J Natl Compr Canc Netw. 2017; 15: 370-398Crossref PubMed Scopus (520) Google Scholar, 12Allegra C.J. Rumble R.B. Schilsky R.L. Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti–epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Summary.J Oncol Pract. 2016; 12: 180-181Crossref PubMed Scopus (20) Google Scholar, 28Sepulveda A.R. Hamilton S.R. Allegra C.J. Grody W. Cushman-Vokoun A.M. Funkhouser W.K. Kopetz S.E. Lieu C. Lindor N.M. Minsky B.D. Monzon F.A. Sargent D.J. Singh V.M. Willis J. Clark J. Colasacco C. Rumble R.B. Temple-Smolkin R. Ventura C.B. Nowak J.A. Molecular biomarkers for the evaluation of colorectal cancer: guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology.J Clin Oncol. 2017; 35: 1453-1486Crossref PubMed Scopus (215) Google Scholar NRAS mutations detected by the Idylla NRAS-BRAF Mutation Test include G12D (c.35G>A), G12C (c.34G>T), G12S (c.34G>A), G12A/V (c.35G>C or c.35G>T), G13D (c.38G>A), G13R/V (c.37G>C or c.38G>T), A59T (c.175G>A), Q61K (c.181C>A), Q61R (c.182A>G), Q61L (c.182A>T), Q61H (c.183A>C or c.183A>T), K117N (c.351G>C or c.351>G>T), and A146T/V (c.436G>A or c.437C>T). The assay also identifies BRAF mutations V600E/D (c.1799T>A, c.1799_1800delinsAA, or c1799_1800delinsAC) and V600K/R (c.1798_1799delinsAA or c.1789_1799delinsAG). Disposable Idylla NRAS-BRAF Mutation Test cartridges were loaded with FFPE human CRC tissue sections, without needing prior manual deparaffinization or FFPE preprocessing, and the cartridge was inserted in the Idylla instrument, according to the instructions of the manufacturer. Inside the Idylla cartridge, DNA is liberated from FFPE material by a combination of reagents, enzymes, heat, and high-intensity focused ultrasound. Next, within the cartridge, five allele-specific multiplex PCRs are performed, which are designed for the specific amplification of NRAS and BRAF gene sequences with a mutation. Simultaneously, conserved regions of the NRAS and BRAF gene are amplified to serve as sample processing controls, as a measure for amplifiable DNA in the sample, and in the analysis of the mutation status of the sample. All reagents needed for sample preparation, and for real-time PCR amplification and detection, are provided in the cartridges. The NRAS-BRAF–specific software automatically analyzes the collected signals from the fluorescently labeled probes. The obtained fluorescent signals are evaluated for PCR curve validity. A cycle of quantification value (Cq) is calculated for every valid curve. Sample processing control signals are used to verify adequate completion of the sample-to-result process. The presence of an NRAS or BRAF mutant genotype is determined by calculating the ΔCq [ie, the difference between the sample processing control Cq and the Cq value obtained for the mutant signal(s)]. The mutant signal is considered detected if the ΔCq value is within a predefined range, and the sample will then be reported as mutation positive; the first mutant signal (ie, the lowest ΔCq value) for each gene is called. Samples with a valid control signal but a ΔCq value outside the predefined range for all mutant signals are reported as mutation negative. The NRAS and BRAF mutational status levels are reported separately. Apart from the mutation and wild-type calls, the Idylla software may also generate an invalid result call. This call might be attributable to the presence of inhibitors in the sample, an insufficient amount of amplifiable DNA in the sample (eg, because of severe DNA fragmentation, potentially caused by too long fixation time), the incorrect placement of the sample in the cartridge, or a sample volume that is out of range. As well, incorrect storage of cartridges, use of cartridges that exceeded their in-use period after removal from the pouch, or cartridge malfunctioning may cause invalid result calls. The results obtained in the current study with the Idylla NRAS-BRAF Mutation Test, for Investigational Use Only, on archival material were not used for diagnostic purposes of any kind. The Idylla Explore application (registration required; last accessed April 10, 2018) offers visualization of PCR curves and corresponding Cq and ΔCq values from Idylla test results, as depicted in Figure 1. This tool is not meant for data interpretation. The limit of detection (LOD) is defined as the lowest allelic frequency at which the mutant alleles can consistently be detected in ≥95% of the test cases. An input of 16,000 copies of wild-type FFPE background in each of the five multiplex PCRs of one Idylla NRAS-BRAF Mutation Test cartridge is representative for one FFPE reference standard specimen. To estimate the LOD for each of the 23 mutations detected by the Idylla NRAS-BRAF Mutation Test, serial dilutions containing a range of allelic frequencies for each mutant were tested at 10% to 1% in 16,000 copies (three dilutions). If mutations were commercially available, the serial dilutions were prepared from liquefied mutant FFPE reference standard; and in the other cases, they were prepared from synthetic target oligonucleotides harboring the mutation and spiked-in liquefied wild-type FFPE reference standard. A dose range study with 6 repeats for each dilution was performed, followed by a refined study with 12 repeats for a narrower range of dilutions near the estimated LOD. Cartridges originating from three different Idylla NRAS-BRAF Mutation Test lots were used. Mutation positivity rate of the serial dilutions was used to calculate the LOD by logistic regression. The most prevalent mutations (ie, ≥3% among NRAS- or BRAF-mutated CRC patients) were included to confirm the estimated LOD level. At least one mutation for each codon was evaluated. This was performed by testing 25 replicates near the corresponding upper limit of the 95% CI with an input level representative for an FFPE reference standard spec
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