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

1
Detection of BRAF Mutations in Tumour and Serum
of Patients with Advanced Melanoma

• Dr Ruth Board CMGS 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
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 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 receptorFFPE,
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 14914915
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. 1799TgtA 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. 1799TgtA mutation present
No mutation present
Perfect match and primer extension
Mismatch - no primer extension or product
Product detected by Taqman probe
Control Diagnostic


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BRAF mutation detection in cfDNA
Tumour DNA Tumour DNA Tumour DNA Total ()
BRAF n () No BRAF mutationn () BRAF unknown n () Total ()
cfDNA BRAF 25 (56) 3 (6) 5 (16) 33 (26)
cfDNA No BRAF mutation 20 (44) 46 (94) 27 (84) 93 (74)
Total 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
12
Does having BRAF positive cfDNA correlate with
known prognostic factors?
LDH 126 cfDNA patients serum mutation result 126 cfDNA patients serum mutation result Total population in Phase II advanced melanoma studyn 200 ()
LDH BRAF n 33 () BRAF unknown n 93 () Total population in Phase II advanced melanoma studyn 200 ()
LDH lt 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

14
Acknowledgements

• AZD6244 study team
• Maria Orr
• Mireille Cantarini
• Karin Kemsley
• Clive Morris

• AstraZeneca
• RD Genetics
• Laura Blockley
• Gillian Ellison
• Simon Dearden
• Emma Donald
• Gael McWalter
• Vicky Williams

• PhD supervisors
• Caroline Dive
• Malcolm Ranson
• Andrew Hughes

• All of the patients and investigators involved in
  the AZD6244 Phase II trial in advanced melanoma
  (study 3)