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Detection of BRAF Mutations in Detection of BRAF Mutations in Tumour and Serum of Patients Tumour and Serum of Patients

with Advanced Melanomawith Advanced Melanoma

Dr Ruth Board

CMGS Spring Scientific Conference March 26th – 27th 2009

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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

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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

AZD6244100 mg BID

continuously

Temozolomide200 mg/m2 for5 days, q28d

First-line advanced melanoma patients

randomised

(n = 200)

Investigator choice of therapy

Investigator choice of therapy

May receive AZD6244

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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

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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

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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

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AT

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

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AT

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:

+ +– +

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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)

Total 45 49 32 126

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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?

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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†)

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Does having BRAF positive cfDNA correlate with known prognostic factors?

LDH

126 cfDNA patients:

serum mutation result Total population in Phase II advanced

melanoma studyn = 200 (%)

BRAF+ n = 33 (%)

BRAF unknown n = 93 (%)

LDH < 2xULN 24 (73) 82 (88) 158 (79)

LDH ≥ 2xULN 8 (24) 8 (9) 32 (16)

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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

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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)