1. MOLECULAR DIAGNOSTICS IN ONCOLOGY Dr. Sergey Kovalenko

2. What is the benefit of molecular diagnostics in cancer? Early detection of the disease for hereditary cancer Optimal therapy selection

3. EGFR-initiated signal transduction pathways

4. Anti-EGFR Monoclonal antibodies-based drugs Cetuximab (Erbitux) – Merck Serono Vectibix (Panitumumab) - Amgen

5. 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

6. EGFR-initiated signal transduction pathways Thyrosine kinase domain is the target

7. 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

8. PF Survival of lung cancer patients under Iressa treatment

9. 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

10. Iressa and Tarceva

11. 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

12. Zelboraf (Vemurafenib) The need to analyze BRAF mutations in melanoma patients Melanoma treatment highly effective new drug Vemurafenib (Roche). The drug is effective only for patients with V600 mutations in BRAF gene

13. 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

14. Percentage of malignant cells in 313 lung cancer samples (our lab results) <10%10-25%25-50%>50%

15. Principle of allele-specific RealTime PCR analysis Mutant DNA TaqMan-probe Mutation Wild-type DNA Mutation-specific primer B. Allele-specific PCR Reverse primer Wild-type Mutant DNA Mutation Wild-type DNA Forward primer Reverse primer A. Control PCR TaqMan- probe Wild-type

16. Results of mutations detection by allele-specific Real-time PCR

17. RealLine BRAF V600 PCR test DNA StandardsSkin melanoma Control PCR Mutation-specific PCR WT 1% V600E V600E2 V600E

18. Wild-type blocking PCR Mutant DNA Mutation Wild-type DNA Forward primer Reverse primer Wild-type Oligonucleotide Blocker

19. Enrichment of PCR fragments with mutations

20. Mutations to be analyzed

21. Mutations of BRAF-V600 in skin melanoma Aminoacid Codon Number of tumors, (%)* WTGTG146 (33.7%) V600EGAG236 (54.5%) V600KAAG36 (8.3%) V600RAGG7 (1.6%) V600E2GAA7 (1.6%) V600D1GAT0 (0.0%) V600D2GAC1 (0.2%) Total433 (100.0%) *Sequencing by Sanger and 454 Roche Anderson et al (2012) Arch Pathol Lab Med. Feb 14.

22. Bioron Dia vs competitor (R)- BRAF ParameterBioron DiagnosticsCompetitor Mutation spectrum V600E/K/DV600E/K SensitivityV600E/D -1% V600K – 5% 5% Price<15 Euro/testUp to 300 Euro/test

23. Sensitivity of commercial tests for mutations of BRAF V600 Mutation Analytical sensitivity, % mutant allele Cobas 4800 BRAF V600 (Roche) THxID BRAF (bioMerieux) RealLine BRAF V600 (BIORON) V600E551 V600E26551 V600D1051 V600K35510 V600Rnd*nd *nd – not detected

24. K-Ras Mutations K-Ras Codon Mutation Sequence 12 13 wt---GGT GGC--- G12S--- A GT GGC--- G12R--- C GT GGC--- G12C--- T GT GGC--- G12D---G A T GGC--- G12A---G C T GGC--- G12V---G T T GGC--- G13D---GGT G A C--- codon 13 codon 12

25. Bioron Dia vs competitor (R)- KRAS ParameterBioron Diagnostics Competitor Mutation spectrum 7 mutations (12- 13 codons) Sensitivity1-5%5% - 30% Price<15 Euro/testUp to 300 Euro/test

26. EGFR Mutations

27. Bioron Dia vs competitor EGFR ParameterBioron DiagnosticsCompetitor Mutation spectrum 77 mutations mutations del19 (70 variants), L858R, L861Q, G719A/C/S, S768I, T790M 29 mutations Sensitivity1-5%5% - 30% Price<25 Euro/testUp to 300 Euro/test

28. Sensitivity of EGFR-7R and Therascreen EGFR kits (Qiagen) PCR MixExonMutationSensitivity, % (a) EGFR-7R Thera- screen Del1919Various deletions (b)11-17 L858R21p.L858R16 L861Q21p.L861Q19 G719X18p.G719A 533 p.G719C 5NA (c) p.G719S 5NA S768I20p.S768I18 T790M20p.T790M518 Ins 2020p.H773_V774insHND (d)4 p.D770_N771insGND20 (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;

29. Products for sequencing analysis KRAS 12-13 codons Mut-sequencing enrichment BRAF V600 Mut sequencing enrichment EGFR del746-750 Mut sequencing enrichment

30. KRAS G13D sequencing 5%1% +blocker -blocker 20% 50%

31. 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

32. Thank you for your attention ! BIORON Diagnostics GmbH Rheinhorststrasse 18 - 67071 Ludwigshafen - Germany www.bioron.de – info@bioron.de