Identification of BRAF mutations in melanoma lesions with the Roche z480 instrument Márta Széll IAP, Siófok May 14, 2012
Multifactorial skin diseses: much more frequent then genodermatoses exhibit familial aggregation BUT do not follow the mendelian rules frequent gene variants (polymorphisms) in the background environmental and life style factors skin symptomes
Malignant melanoma 90 % of melanoma cases - sporadic melanoma, several low penetrance polymorphisms of different genes as susceptibility genetic factors 10% of cases familial melanoma, high penetrance mutations of the CDKN2A gene skin symptomes
Mutations in the pathogenesis of melanoma CDKN2A MC1R TYR MTAP germline mutations Széll et al, 2007 Balogh et al, 2011 Csoma et al, 2011 Balogh et al, 2012 somatic mutations
Mutations in the pathogenesis of melanoma CDKN2A MC1R TYR MTAP germline mutations Széll et al, 2007 Balogh et al, 2011 Csoma et al, 2011 Balogh et al, 2012 somatic mutations
Melanoma subtypes and mutations in the growth factor signaling pathways ~15-20% ~15% ~60% ~80% GNAQ GNA11 Shepherd, 2010
The V600E mutation of B-RAF gene as a therapeutic target Exon 15 1799 T/A V600E affects the kinase domain of the protein, a gain-of-function mutation 10,7x higher kinase activity
Drug development for the SPECIFIC inhibition of the V600E mutant B-RAF kinase scaffold based drug discovery method for the screening of the putative small (150-350 D) molecules (appr. 20.000) Tsai, 2008
A biochemical, cellular and in vivo tests to measure the inhibitory effect of PLX4720 Name of kinase IC50 nm B-RAF V600E 13 B-RAF 160 BRK 130 FRK 1300 CSK 1500 B-RAF V600E B-RAF wilde type Green calcein = living cells Red EtBr = apoptotic cells B-RAF V600E B-RAF wild type Tsai, 2008
Phase I, II and III clinical trials Vemurafenib is an approved drug in the US since August 2011 and in Europe since February 2012 Indications: unresectable or metestatic melanoma of adult patients mutation positive for V600E Side effects: arthralgias (58%), skin rash (52%), photosensitivity (52%), skin lesions - carcinoma basocellulare (32%) Bollag, 2010
Advanced Melanoma A New Era of Personalized Therapy Prior to 2011 Patients were treated with FDA-approved treatments or enrolled in clinical trials Limited treatment options Poor outcomes High unmet medical need 2011 and beyond Treatment personalized according to tumor molecular characteristics MAPK pathway has emerged as a key driver of melanoma pathology and as an attractive target Implications Molecular testing of melanoma tissue required to personalize treatment for improved outcome MAPK = mitogen-activated protein kinase. ZELBORAF (vemurafenib) PI. 2011. MAPK = mitogen-activated protein kinase. ZELBORAF (vemurafenib) PI. 2011.
BRAF V600E Mutations Clinical Pharmacology, Mechanism of Action RTK Some mutations in the BRAF gene, including V600E, result in constitutively activated BRAF proteins, which can cause cell proliferation in the absence of growth factors that would normally be required for proliferation ZELBORAF (vemurafenib) is a low-molecular-weight, orally available inhibitor of some mutated forms of BRAF serine-threonine kinase, including BRAFV600E ZELBORAF RAS BRAF RAF V600E MEK ERK RTK = receptor tyrosine kinase. ZELBORAF (vemurafenib) PI. 2011 Abnormal Cellular Cellular Proliferation Arrested
Efficacy of Vemurafenib in Treatment-Naive Patients BRAFV600E Mutation-Positive Melanoma Efficacy Endpoint Overall Survival (OS) ZELBORAF (n=337) Dacarbazine (n=338) P value Number of Deaths 78 (23%) 121 (36%) Hazard Ratio (95% CI) 0.44 (0.33, 0.59) <0.0001 Median Survival in Months (95% Cl) Not Reached (9.6 not reached) 7.9 (7.3, 9.6) Median Follow up (Months) (range) 6.2 (0.4, 13.9) 4.5 (<1, 11.7) Progression Free Survival Hazard Ratio (95% CI) 0.26 (0.2, 0.33) <0.0001 Median PFS (Months) 5.3 (4.9, 6.6) 1.6 (1.6, 1.7) Major Efficacy outcomes were overall survival (OS) and progression free survival (PFS) Most patients were male (56%) and Caucasian (99%), the median age was 54 years (24% were 65 years. Majority of patients had metastatic disease
Phase 3 Study of ZELBORAF Kaplan-Meier Curves of Overall Survival OS (%) 100 90 80 70 60 50 40 30 20 10 0 HR 0.44 (0.33-0.59) Log-rank test P<0.0001 Dacarbazine 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 7.9 (95% CI, 7.3-9.6) Months ZELBORAF 6-month OS rate 83% (95% CI, 79%-87%) for ZELBORAF vs 63% (95% CI, 57%-69%) for dacarbazine Median OS for ZELBORAF not reached <10% of patients have follow-up beyond 9.5 months ZELBORAF (vemurafenib) PI. 2011.
ZELBORAF (vemurafenib) Indications and Usage Indication statement ZELBORAF (vemurafenib) is indicated for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test Contraindications None Limitation of use ZELBORAF is not recommended for use in patients with wild-type BRAF melanoma ZELBORAF (vemurafenib) PI. 2011.
cobas V600 BRAF Mutation Test Clinically validated, accurate and reliable results Oncologists Specimen selection Laboratory Test Method Platform & Result Report Clear benefits in accuracy of results Specified tumor cell content, quality, amount & interpretation Standard processes & consistent training of staff Single-source reagents, test methods & standardized quality control Validated platform, results are interpreted manually & subjectively
cobas 4800 BRAF V600 Mutation Test Performance Characteristics & Features Tissue section Tumor content required Total Nucleic Acid (DNA) Mutation coverage Performance Characteristics and Features 5 mm section (minimum 2, 1 for DNA extraction, 1 for H&E) 50% tumor area 125 ng per reaction / 5 ng/ml (25mL) Highly sensitive to V600E May also cross-react with V600K, V600D Time to result Control / Result Interpretation 8 hours from receipt of sample Automatic Sensitivity Cell Line DNA 3.9 ng / 25µL DNA (1:32 dilution of recommended DNA input) Sensitivity FFPET >5% mutant DNA (with tumor content of 15%) Test used to prospectively select patients in phase 2 and 3 clinical studies of ZELBORAF (vemurafenib)
cobas 4800 BRAF V600 Mutation Test Workflow ~1 hour 50% ~4 hours 30 minutes 1.5 hours
Interpretation of Specimen Results cobas 4800 BRAF V600 Mutation Test Result Interpretation Mutation Detected V600E Mutation Detected in the BRAF codon 600 site in exon 15 Mutation Not Detected V600E Mutation Not Detected in the BRAF codon 600 site in exon 15 Invalid Failed Result is invalid. Repeat the testing of specimens with invalid results following the instructions outlined in the Retesting of Specimens with Invalid Results section below. Failed run due to hardware or software failure
Sensitivity of the cobas 4800 BRAF V600 Mutation Test using Cell Line Blend Cell Line Blend Cell Line Blend Mean Percent Mutation 5% Amount of DNA in the Panel Member Mutation Detected Rate (n=60) 125.0 ng/25 µl 97% 31.3 ng/25 µl 100% 15.6ng/25 µl 95% 7.8 ng/25 µl 98% 3.9 ng/25 µl 95% 2.0 ng/25 µl 82% 1.0 ng/25 µl 78% 0.5 ng/25 µl 77% Sensitivity of 3.9ng / 25µL 95% detection (1:32 dilution of recommended input)
BRAF V600E FFPET Specimens cobas BRAF Testing Results with Various Percent Tumor Content V600E Specimen Number Tumor Content Test Result 1 5% / 5% Mutation Not Detected 2 5% / 5% Mutation Not Detected 3 5% / 5% Mutation Not Detected 4 10% / 10% Mutation Not Detected 5 10% / 10% Mutation Detected 6 15% / 10% Mutation Detected 7 15% / 15% Mutation Detected 8 15% / 15% Mutation Detected 9 15% / 15% Mutation Detected 10 15% / 15% Mutation Detected 11 15% / 15% Mutation Detected 12 15% / 20% Mutation Detected 13 20% / 20% Mutation Detected 14 20% / 20% Mutation Detected 15 25% / 20% Mutation Detected 16 25% / 25% Mutation Detected 17 30% / 25% Mutation Detected 18 30% / 30% Mutation Detected 19 30% / 35% Mutation Detected 20 30% / 35% Mutation Detected V600E Specimen Number Tumor Content Test Result 21 35% / 30% Mutation Detected 22 35% / 35% Mutation Detected 23 35% / 35% Mutation Detected 24 35% / 35% Mutation Detected 25 35% / 40% Mutation Detected 26 40% / 35% Mutation Detected 27 40% / 35% Mutation Detected 28 40% / 40% Mutation Detected 29 40% / 40% Mutation Detected 30 40% / 40% Mutation Detected 31 40% / 45% Mutation Detected 32 45% / 45% Mutation Detected 33 50% / 40% Mutation Detected Sensitivity >5% mutant DNA (with tumor content of 15%)
cobas 4800 system for BRAF Real-time PCR Reproducible result with LightCycler Technology cobas z 480 analyzer PN: 05200881001 System Software v2.0 cobas 4800 BRAF AP V1.0.0 CD Five optical channels to detect multiple targets Wild-type BRAF V600 V600E mutation sequence Remote Diagnostics with Connect2 Additional diagnostic options: KRAS, EGFR, PI3K
Mutations in the pathogenesis of melanoma germline mutations somatic mutations CDKN2A MC1R TYR MTAP Széll et al, 2007 Balogh et al, 2011 Csoma et al, 2011 Balogh et al, 2012 BRAF somatic mutation status of CDKN2A germline mutation+ melanoma patients?
CDKN2A mutation+ pedigrees P48T Széll et al, 2007 IVS1+37 G>C Balogh et al, 2012 R24P Balogh et al, 2011 BRAF V600E (-) BRAF V600E (-) BRAF V600E (+) Future plans: detection of superimposing somatic mutations over the existing germline CDKN2A mutations
Many thanks to Prof. Dr. Lajos Kemény Prof. Dr. Attila Dobozy Dr. Judit Oláh Dr. Erika Varga Dr. Klára Balogh Hilda Polyánka Department of Dermatology and Allergology, University of Szeged Dermatological Research Group of the Hungarian Academy of Sciences