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Detection of BRAF Mutations in Tumour and Serum of Patients with Advanced Melanoma

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. Introduction. BRAF mutations occur in up to 60% of cutaneous melanomas

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Detection of BRAF Mutations in Tumour and Serum of Patients with Advanced Melanoma

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

  2. 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. AZD6244100 mg BIDcontinuously Investigator choice of therapy First-line advanced melanoma patients randomised (n = 200) Temozolomide200 mg/m2 for5 days, q28d Investigator choice of therapy May receive AZD6244 Follow up for PFS overall survival (primary endpoint) 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 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

  4. 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. 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 • 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 Fleischhacker M & Schmidt B. Nature Medicine 2008; 14:914–915 cfDNA, circulating free DNA; CTCs, circulating tumour cells

  6. 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. 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

  8. 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: + + – +

  9. BRAF mutation detection in cfDNA

  10. 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. BRAF+ on tumour only BRAF+ on tumour and cfDNA HR 1.08 (80% CI 0.69, 1.68; two-sided p=0.826†) 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

  12. Does having BRAF positive cfDNA correlate with known prognostic factors?

  13. 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. Acknowledgements • AZD6244 study team • Maria Orr • Mireille Cantarini • Karin Kemsley • Clive Morris • AstraZeneca • R&D Genetics • Laura Blockley • Gillian Ellison • Simon Dearden • Emma Donald • Gael McWalter • Vicky Williams • All of the patients and investigators involved in the AZD6244 Phase II trial in advanced melanoma (study 3) • PhD supervisors • Caroline Dive • Malcolm Ranson • Andrew Hughes

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