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MOLECULAR DIAGNOSTICS IN ONCOLOGY Dr. Sergey Kovalenko What is the benefit of molecular diagnostics in cancer? • Early detection of the disease for hereditary cancer • Optimal therapy selection EGFR-initiated signal transduction pathways Anti-EGFR Monoclonal antibodies-based drugs • Cetuximab (Erbitux) – Merck Serono • Vectibix (Panitumumab) - Amgen Activation mutations in KRAS gene • Mutations can be found in 40% cases • Anti-EGFR therapy is not effective if KRAS mutations appear • Package inserts to Cetuximab and Vectibix clearly specify the need to analyze KRAS mutation before drugs prescriptions EGFR-initiated signal transduction pathways Thyrosine kinase domain is the target Activation mutations in EGFR gene • Mutations in TK domain of EGFR can be found in 10-40% cases • TK inhibitors (Iressa, Tarceva) are effective only for patients with mutations • Package inserts to Iressa and Tarceva clearly specify the need to analyze EGFR mutations before drugs prescriptions PF Survival of lung cancer patients under Iressa treatment Cetuximab and Vectibix • The medications are effective only for CRC patients without KRAS mutations in tumor cells • PF and Overall Survival under Cetuximab and Vectibix is much better in comparison with Best Available Treatment The need to analyze KRAS mutations in CRC tumors Iressa and Tarceva Iressa and Tarceva • The medications are effective only for Lung Cancer patients with EGFR mutations in tumor cells • PF and OS under Iressa and Tarceva is much better in comparison with Best Available Treatment The need to analyze EGFR mutations in lung cancer tumors Zelboraf (Vemurafenib) • Melanoma treatment • highly effective new drug Vemurafenib (Roche). • The drug is effective only for patients with V600 mutations in BRAF gene The need to analyze BRAF mutations in melanoma patients Problems in mutations analysis in tumor samples • The sample contains a mixture of malignant and normal cells – sequencing is problematic • Just a few cells are suitable for analysis • The sample is fixed in paraffin Percentage of malignant cells in 313 lung cancer samples (our lab results) 160 151 140 120 100 71 80 60 48 43 40 20 0 <10% 10-25% 25-50% >50% Principle of allele-specific RealTime PCR analysis A. Control PCR TaqMan- probe Forward primer Mutant DNA Wild-type DNA Mutation Reverse primer Wild-type B. Allele-specific PCR Mutation-specific primer Mutant DNA Mutation Wild-type DNA Wild-type TaqMan-probe Reverse primer Results of mutations detection by allele-specific Real-time PCR RealLine BRAF V600 PCR test DNA Standards Skin melanoma WT V600E2 1% V600E V600E Control PCR Mutation-specific PCR Wild-type blocking PCR Forward primer Oligonucleotide Blocker Wild-type DNA Wild-type Mutant DNA Mutation Reverse primer Enrichment of PCR fragments with mutations Mutations to be analyzed Mutations of BRAF-V600 in skin melanoma Codon Number of tumors, (%)* WT V600E V600K GTG GAG AAG 146 (33.7%) 236 (54.5%) 36 (8.3%) V600R V600E2 V600D1 AGG GAA GAT 7 (1.6%) 7 (1.6%) 0 (0.0%) V600D2 GAC 1 (0.2%) Aminoacid Total *Sequencing by Sanger and 454 Roche 433 (100.0%) Anderson et al (2012) Arch Pathol Lab Med. Feb 14. Bioron Dia vs competitor (R)- BRAF Parameter Bioron Diagnostics Competitor Mutation spectrum V600E/K/D V600E/K Sensitivity V600E/D -1% V600K – 5% 5% Price <15 Euro/test Up to 300 Euro/test Sensitivity of commercial tests for mutations of BRAF V600 Analytical sensitivity, % mutant allele Cobas 4800 BRAF V600 (Roche) THxID BRAF (bioMerieux) RealLine BRAF V600 (BIORON) V600E 5 5 1 V600E2 65 5 1 V600D 10 5 1 V600K 35 5 10 V600R nd* nd nd Mutation *nd – not detected K-Ras Mutations K-Ras Mutation Codon Sequence 12 13 wt ---GGT GGC--- G12S ---AGT GGC--G12R ---CGT GGC--G12C ---TGT GGC--- codon 12 G12D ---GAT GGC--G12A ---GCT GGC--G12V ---GTT GGC--G13D ---GGT GAC--- codon 13 Bioron Dia vs competitor (R)- KRAS Parameter Bioron Diagnostics Competitor Mutation spectrum 7 mutations (1213 codons) 7 mutations (1213 codons) Sensitivity 1-5% 5% - 30% Price <15 Euro/test Up to 300 Euro/test EGFR Mutations Bioron Dia vs competitor EGFR Parameter Bioron Diagnostics Competitor Mutation spectrum 77 mutations mutations del19 (70 variants), L858R, L861Q, G719A/C/S, S768I, T790M 29 mutations Sensitivity 1-5% 5% - 30% Price <25 Euro/test Up to 300 Euro/test Sensitivity of EGFR-7R and Therascreen EGFR kits (Qiagen) PCR Mix Exon Mutation Del19 19 Various deletions (b) 1 1-17 L858R L861Q 21 21 p.L858R p.L861Q G719X 18 p.G719A 1 1 5 6 9 33 p.G719C 5 NA (c) p.G719S 5 NA p.S768I p.T790M 1 5 8 18 S768I T790M 20 20 Ins 20 20 Sensitivity, % (a) EGFR-7R Therascreen p.H773_V774insH ND (d) p.D770_N771insG ND (a) % of mutant allele; (b) 70 variants of del19 in EGFR-7R and 19 variants of del19 in Therascreen; (c) NA – not available; (d) ND – not detected; 4 20 Products for sequencing analysis • KRAS 12-13 codons Mut-sequencing enrichment • BRAF V600 Mut sequencing enrichment • EGFR del746-750 Mut sequencing enrichment KRAS G13D sequencing 1% -blocker +blocker 5% 20% 50% CONCLUSIONS (1) Bioron tests for mutations analysis cover comprehensive spectrum of mutations (2) The sensitivity of tests is superior due to the use of “enrichment technique” (3) The prices of the tests are strongly competitive (4) Enrichment technique can be used in sequencing labs on the basis of BIORON reagents Thank you for your attention ! BIORON Diagnostics GmbH Rheinhorststrasse 18 - 67071 Ludwigshafen - Germany www.bioron.de – [email protected]