1. AZD6244 Detection of BRAF Mutations in Tumour and Serum of Patients with Advanced Melanoma Dr Ruth Board CMGS Spring Scientific Conference March 26 th – 27 th 2009

2. AZD6244 Introduction  BRAF mutations occur in up to 60% of cutaneous melanomas  AZD6244 (ARRY-142886) is an orally available, potent, selective, ATP- uncompetitive inhibitor of MEK1/2, a downstream activator of BRAF  AZD6244 has shown preclinical activity in BRAF+ tumour models BRAF+, BRAF mutation positive

3. AZD6244 Phase II study of AZD6244 vs temozolomide in patients with advanced melanoma (study D1532C00003)  6 patients randomised to AZD6244 had a clinical response, 5 of whom had BRAF+ tumour 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 (primary endpoint) PFS AZD6244 100 mg BID continuously Temozolomide 200 mg/m 2 for 5 days, q28d First-line advanced melanoma patients randomised (n = 200) Investigator choice of therapy May receive AZD6244

4. 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 3–4 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

5. AZD6244 cfDNA as an alternative to tumour biopsies Fleischhacker M & Schmidt B. Nature Medicine 2008; 14:914–915  Increased levels of cfDNA are detected in the blood of patients with cancer  Source and mechanism of DNA release remains unclear – Direct shedding from tumours or from CTCs  Previous studies have shown that it is possible to detect tumour- specific 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

6. 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

7. AZD6244 A T g. 1799T>A mutation present Perfect match and primer extension Product detected by Taqman probe Allele specific PCR T T No mutation present Mismatch - no primer extension or product

8. AZD6244 A T g. 1799T>A mutation present Perfect match and primer extension Product detected by Taqman probe Allele specific PCR T T No mutation present Mismatch - no primer extension or product Control: Diagnostic: + – +

9. AZD6244 BRAF mutation detection in cfDNA Tumour DNA Total (%) BRAF+ n (%) No BRAF mutation n (%) BRAF unknown n (%) cfDNA BRAF+25 (56)3 (6)5 (16)33 (26) No BRAF mutation 20 (44)46 (94)27 (84)93 (74) Total454932126

10. 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?

11. AZD6244 The presence of circulating BRAF mutations does not affect PFS* *In those patients with BRAF+ tumours where serum was available for analysis † HR calculated using an unadjusted Cox proportional hazards model BRAF+ on tumour only BRAF+ on tumour and cfDNA HR 1.08 (80% CI 0.69, 1.68; two-sided p=0.826 † )

12. AZD6244 Does having BRAF positive cfDNA correlate with known prognostic factors? LDH 126 cfDNA patients: serum mutation result Total population in Phase II advanced melanoma study n = 200 (%) BRAF+ n = 33 (%) BRAF unknown n = 93 (%) LDH < 2xULN24 (73)82 (88)158 (79) LDH ≥ 2xULN8 (24)8 (9)32 (16)

13. 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

14. 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)