Detection of BRAF Mutations in Tumour and Serum of Patients with Advanced Melanoma Dr Ruth Board CMGS Spring Scientific Conference March 26th 27th 2009 AZD6244 Introduction BRAF+, BRAF mutation positive BRAF mutations occur in up to 60% of cutaneous melanomas AZD6244 (ARRY-142886) is an orally available, potent, selective, ATPuncompetitive inhibitor of MEK1/2, a downstream activator of BRAF AZD6244 has shown
preclinical activity in BRAF+ tumour models AZD6244 Phase II study of AZD6244 vs temozolomide in patients with advanced melanoma (study D1532C00003) First-line advanced melanoma patients randomised (n = 200) AZD6244 100 mg BID continuously Temozolomide 200 mg/m2 for 5 days, q28d Investigator choice of therapy Investigator choice of therapy May receive AZD6244
PFS (primary endpoint) Follow up for overall survival Primary Objective: To compare the efficacy of AZD6244 versus temozolomide by evaluation of: (i) primary outcome variable PFS using site measurements and (ii) secondary outcome variables, including overall survival and response rate 6 patients randomised to AZD6244 had a clinical response, 5 of whom had BRAF+ tumour AZD6244 Mutation detection in tumour samples Invasive procedure Access to tumour samples
Often archival, FFPE Often at multiple hospitals, difficult and slow to access Requires further processing and 34 days extraction DNA from FFPE samples often poor quality and low yield Mutation status of archival tumour may not reflect current mutational status Emergence of EGFR resistance mutations Heterogeneous nature of tumour samples Differences in mutations between sites of metastases and within metastases EGFR, epidermal growth factor receptor; FFPE, formalin fixed paraffin embedded AZD6244 cfDNA as an alternative to tumour biopsies Increased levels of cfDNA are detected in the blood of patients with cancer Source and mechanism of DNA release remains unclear
Fleischhacker M & Schmidt B. Nature Medicine 2008; 14:914915 Direct shedding from tumours or from CTCs Previous studies have shown that it is possible to detect tumourspecific mutations in cfDNA Provides the opportunity to develop mutation detection tests which are: less invasive quicker to process real time assessment cfDNA, circulating free DNA; CTCs, circulating tumour cells AZD6244 Study design
Access to 126 serum samples from patients enrolled in the AZD6244 Phase II advanced melanoma study Included 94 cases with known BRAF tumour data 47.9% BRAF+ The aim of this study was to assess the feasibility of using cfDNA for BRAF mutation detection in patients with advanced melanoma AZD6244 Allele specific PCR T A g. 1799T>A mutation present Perfect match and primer extension
T T No mutation present Mismatch - no primer extension or product Product detected by Taqman probe AZD6244 Allele specific PCR T T A T g. 1799T>A mutation present No mutation present
Perfect match and primer extension Mismatch - no primer extension or product Product detected by Taqman probe Control: Diagnostic: + + + AZD6244 BRAF mutation detection in cfDNA Tumour DNA cfDNA Total
BRAF+ n (%) No BRAF mutation n (%) BRAF unknown n (%) Total (%) BRAF+ 25 (56) 3 (6) 5 (16) 33 (26) No BRAF mutation 20 (44) 46 (94)
27 (84) 93 (74) 45 49 32 126 AZD6244 Questions Does the presence of a serum BRAF mutation reflect prognosis? If patients are pre-selected on the basis of serum BRAF status, will this enrich for a population of patients with a worse outcome and/or poorer prognostic factors? AZD6244 The presence of circulating BRAF
mutations does not affect PFS* BRAF+ on tumour only BRAF+ on tumour and cfDNA HR 1.08 (80% CI 0.69, 1.68; two-sided p=0.826) *In those patients with BRAF+ tumours where serum was available for analysis HR calculated using an unadjusted Cox proportional hazards model AZD6244 Does having BRAF positive cfDNA correlate with known prognostic factors? 126 cfDNA patients: serum mutation result BRAF+ n = 33 (%) BRAF unknown n = 93 (%) Total population in Phase II advanced melanoma study n = 200 (%) LDH < 2xULN
24 (73) 82 (88) 158 (79) LDH 2xULN 8 (24) 8 (9) 32 (16) LDH AZD6244 Conclusions We have demonstrated the potential to use cfDNA for BRAF mutation detection in patients with advanced melanoma Future work will include:
Further validation of cfDNA as an alternative to tissue biopsies Use of plasma derived cfDNA Alternative technologies for mutation detection Other cancer types and mutations AZD6244 Acknowledgements AstraZeneca R&D Genetics Laura Blockley Gillian Ellison Simon Dearden Emma Donald Gael McWalter
Vicky Williams PhD supervisors Caroline Dive Malcolm Ranson Andrew Hughes AZD6244 study team Maria Orr Mireille Cantarini Karin Kemsley Clive Morris All of the patients and investigators involved in the AZD6244 Phase II trial in advanced melanoma (study 3) AZD6244
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