FROM KRAS TO RAS Europan experience in RAS-testing and quality assessment Han van Krieken, Nijmegen Inhibition of EGFR dimerisation and downstream signalling that includes RAS proteins EGF EGFR TGF-α EGFR Homodimer Panitumumab EGFR RAS GTP RAS GDP RAF Nck MEK PI3K Rac PAK Proliferation Anti-apoptosis Angiogenesis Survival Metastasis PLCγ PTEN AKT JNKK ERK JNK PKC S6K mTOR Myc Elk Jun Fos Yang XD, et al. Cancer Res 1999; 59:1236-43; Foon KA, et al. Int J Radiat Oncol Biol Phys 2004; 58:984-90; Hecht JR, et al. Proc Am Soc Clin Oncol 2004; 22:A3511; Crawford J, et al. Proc Am Soc Clin Oncol 2004; 22:A7083. ©2007 Amgen Inc. All rights reserved RAS Is Important in Growth and Cell Division—Mutations Can Cause Cancer inactive GDP Normal: – Growth – Proliferation – Differentiation active GTP inactive GDP active * GTP ABNORMAL: – Growth – Proliferation – Differentiation • Ras proteins are GTPases • Ras family members include: KRAS, NRAS, and HRAS • Normal cycle occurs between a GDP-bound (inactive) and a GTPbound (active) form of Ras • Specific mutations in the KRAS gene result in a constitutively active protein Schubbert S, et al. Nature Rev Cancer. 2007;7:295-308. Statements 1 • RAS-testing is now standard • RAS-testing is a easy as KRAS testing • RAS-testing is commonly done using commercial kits/methods KRAS & NRAS mutation hotspots EXON 1 EXON 2 12 12 KRAS 59 59 EXON 2 12 12 1313 4% 59 61 61 EXON 4 117117 146146 5% 6% EXON 3 EXON 4 40% EXON 1 NRAS 13 13 EXON 3 59 61 61 4% 117 117 146146 0% Among WT KRAS exon 2 patients, an additional 17% of tumours with RAS mutations were found There are many different RAS testing methods IVD methods Laboratory based methods Non-commercial “in-house” methods Commercial test kits Conform to Directive 98/97/EC and carry CE marking RUO methods Not validated for use in patient IVD CE-IVD RAS & KRAS only test kits CE-IVD kit CRC RAScan™ ® ® 1 (SURVEYOR /WAVE ) CRC RASseq™ 1 ® Therascreen KRAS & NRAS Pyro ® 2 Therascreen KRAS RGQ PCR ® Cobas KRAS Mutation Test 3 4 Randox Biochip Array ® INFINITI KRAS 5 KRAS StripAssay ®6 ® EnteroGen KRAS 7 ®8 KRAS LightMix PNAClamp™ 9 RealQuality RI-KRASMuST 10 ®2 KRAS NRAS Mutant enrichment Detection method Yes LOD* Yes None Mismatched heteroduplex † cleavage & DHPLC 2-5% Yes Yes None Sanger sequencing 5-10% Yes Yes None Pyrosequencing 1-7% Yes No Allele-specific probes RT-PCR 1-6% Yes No PCR clamp (TaqMelt™) Melting curve analysis-based 5% Yes No None Array hybridization 1% Yes No None Array hybridization NS Yes No PCR clamp (blocker) Strip hybridization 1% Yes No Allele-specific probes RT-PCR Yes No PCR clamp (LNA) Melting curve analysis-based Yes No PCR clamp (PNA) RT-PCR < 1% Yes No Allele-specific probes RT-PCR 1% < 1% 1% CE-IVD RAS & KRAS only test kits Detected codons in KRAS LOD† Exon 2 Exon 3 Detected codons in NRAS Exon 4 Exon 2 Exon 3 Exon 4 p.12 p.13 p.59 p.61 p.117 p.146 p.12 p.13 p.59 p.61 p.117 p.146 ® CRC RAScan™ SURVEYOR /WAVE CRC RASseq™ 1 ® Therascreen NRAS & KRAS Pyro ® 2 Therascreen KRAS RGQ PCR ® Cobas KRAS Mutation Test Randox Biochip Array ® INFINITI KRAS 3 4 5 KRAS StripAssay ®6 ® EnteroGen KRAS 7 ®8 KRAS LightMix PNAClamp™ ®1 9 RealQuality RI-KRASMuST 10 ®2 2-5% all all all all all all all all all all all all 5-10% all* all* all* all* all* all* all* all* all* all* all* all* 1-7% 6 1 6 6 1-6% 6 1 5% 6 6 7 1% 6 3 4 NS 7 6 6 1% 8 2 3 < 1% 6 1 5 1% 7 2 < 1% 6 1 1% 6 1 5 2 6 Laboratory based RAS testing methods Gel electrophoresis Sequencing • Temporal temperature gradient • Denaturing gradient • Constant denaturant capillary electrophoresis • SSCP • Sanger sequencing 15 - 20%3 Limit of detection • Pyrosequencing 5%3 Allele-specific PCR Other • Allele-specific amplification based on primer design (ARMS, REMS, FLAG, RFLP) • Ligation detection reaction (LDR, LDR spFRET) • Coamplification at lower denaturation temperature (COLD) • Hybridization assays 0.1 - 1%2 • HRMA (screening) ~10%3 Conventional ‘Sanger’ sequencing PCR KRAS EGFR BRAF Sequence TP53 Next generation sequencing PCR TP53 EGFR KRAS BRAF Sequence OncoNetwork Consortium: 8 experienced labs in colon and lung cancer diagnosis Prof. Ian Cree Warwick Medical School United Kingdom Dr. Marjolijn Ligtenberg & Dr. Bastiaan Tops Radboud University Nijmegen Medical Centre The Netherlands Prof. Orla Sheils Trinity College Dublin, Ireland Dr. Cristoph Noppen & Dr. Henriette Kurth VIOLLIER AG Basle, Switzerland Dr. Ludovic Lacroix Institut Gustave Roussy Paris, France Prof. Aldo Scarpa ARC-NET University of Verona Italy Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy Prof. Pierre Laurent Puig Université Paris Descartes, France Alain Rico Rosella Petraroli Statements 2 • My laboratory participates in external quality assessment • My laboratoria performs well in external quality assessments ESP Quality Assurance Programme 2008: Multidisciplinary group forms and makes proposal for a European QA Programme with support from Amgen 2009: First European KRAS EQA scheme pilot tests in 11 expert centres Regional EQA roll-out approves 55 centres 2012: The multidisciplinary group establishes guidelines to harmonize EQA schemes in molecular pathology 2010-12: Annual KRAS EQA schemes award 296 approvals in 32 countries 2013: ESP Colon EQA scheme expands to include KRAS, NRAS and BRAF testing RAS testing • A tumour sample is sent to the laboratory for analysis • The test can use fresh, frozen or paraffin-embedded tissue • A pathologist confirms that the tissue is cancerous and selects test material • A sample of DNA is prepared for the RAS test • The polymerase chain reaction (PCR) is used to amplify the DNA sample and test for RAS mutation status The ESP Colon EQA scheme exemplifies best practice ESP / European QA Council European QA Coordinator for KRAS testing Regional QA programmes Centralised QA programme 10 Participating laboratories Results 16 14 12 Numer of labs 10 8 6 4 2 0 Austria Belgium Denmark France Greece The Netherlands UK Spain Sweden 3 mistakes 0 0 0 0 0 0 1 0 0 2 mistakes 0 2 1 0 0 0 0 1 0 1 mistake 0 2 3 0 3 3 0 1 2 all samples correct 7 5 3 5 0 11 0 5 5 Genotyping results Number of laboratories and countries for each year Scheme Number of labs Number of countries % of labs reported all genotypes correctly 2009 61 9 69 2010 76 14 67 2011 124 17 72 2012 105 26 75 * Numerical scoring system - 1 point correct genotype or in case mutation was not screened and identified as wild type - 0 points incorrect genotype - 0 points in case of technical failure in samples of unambiguous quality * Average genotype scores on 10 samples over the years 19 Listing on the website All labs with > 90% genotype score are listed on: http://kras.eqascheme.org/info/public/eqa/previous_participants.xhtml Belgian laboratories 20 Scheme Number of labs % of labs reported all genotypes correctly Average genotyping score On web site (%) 2009 10 50 87 70 2010 13 38.5 95 92.3 2011 12 66.7 94 83.3 2012 12 91.7 96.7 91.7 prof dr E Dequeker Evaluation of the reports Scores of important criteria in written reports sent by the participants. Analysis of the reports was based on: • ISO 15189:2007 • Guideline document CAP regarding reporting of molecular results (Gulley ML, et al Clinical laboratory reports in molecular pathology. Arch Pathol Lab Med. 2007 Jun;131(6):852863). Evaluation of diagnostic reports Requested are (mock) reports as sent to treating physician 2011 prof dr E Dequeker 22 More information on the website http://kras.eqascheme.org Statements 3 • No special preparation of biopsies/resection specimens is needed for molecular testing Why is the preanalytical phase more relevant nowadays? • • • • Quality of DNA • PCR • ISH Quality of RNA • Expression array • EBER Quantification of proteins • IHC • proteomics -omics • Genomics • Proteonomics • Metabolomics 25 Targeted therapy needs different pathology • DNA-based tests • Quality • Quantity • Immunohistochemistry • Quantitative • Localization • FISH • Tissue based • proteolysis 26 Preparing pathology for personalised medicine: possibilities for improvement of the preanalytical phase Groenen et al. Histopathology 2011 Key words: • Control • Standardisation 27 A “Chain of custody” for histological specimens 16 Issues tissue processing • • • • Time Temperature Solvents Microwave enhanced 29 Issues immunohistochemistry • Warm ischaemia affects proteins • Fixation affects proteins • Microwave enhanced tissue processing affects mildly • Quantitative immunohistochemistry • In principle doesn’t exist!! (at most semi-) • Needed (?) for some targeted drugs • cErb2 (Her2) • c-met 30 Issues DNA/RNA testing • • • • • Limited problem for mutations, translocations Major problem for copy number changes, NGS Some problems for FISH Expression array not reliable on FFPE material EBER: internal control 31 Conclusion • We need standardisation and control • Tissue safe is a good start 32 Pathology now • Integretion morphology, immunophenotype and molecular pathology • Care for the preanalytic phase • Importance of external quality assurance
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