Molecular Stratification of Cancer in the

Transcription

1 Molecular Stratification of Cancer in the Clinical Setting David Gonzalez de Castro Molecular Diagnostics NHS FT and The Institute of Cancer Research, London, UK 1

2 2 The complexity of human cancer genomes

3 3 Molecular diagnostics in cancer Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

4 4 Molecular diagnostics in cancer Vemurafenib in BRAF V600E Melanoma Crizotinib in ALK+ NSCLC Chapman P et al N Engl J Med 2011;364: Camidge DR et al, Lancet Oncology 2011 Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

5 5 Molecular diagnostics in cancer TP53 mutations in CLL CLLU1 expression in CLL p= Cum mulative Proportion Surviving High (n=268) Low (n=247) Months from Randomization D Gonzalez et al, J Clin Oncol 2011 D Gonzalez et al, Haematologica 2013 Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

6 6 Molecular diagnostics in cancer BCR-ABL monitoring in CML Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

7 7 Development and validation of new tests Mattocks C et al. European Journal of Human Genetics (2010) 18,

8 8 The importance of Target validation L Friboulet et al. N Engl J Med 2013;368:

9 9 The importance of Target validation Olaussen KA et al. N Engl J Med 2006;355: L Friboulet et al. N Engl J Med 2013;368:

10 10 The CR-UK programme Research infrastructure Service delivery

11 11 The CR-UK programme Colorectal Lung Breast Prostate Ovarian Melanoma BRAF EML4-ALK BRAF BRAF BRAF BRAF KRAS BRAF TP53 TMPRSS2- ERG TP53 KIT NRAS EGFR PIK3CA PTEN PTEN NRAS PIK3CA TP53 KRAS DDR2 + ibraf PTEN PIK3CA PIK3CA Each panel for 300

12 12 The CR-UK programme: Lung cancer panel - EGFR mutations (exons 18-21) 13% NSCLC TK-inhibitor - KRAS mutations (codons 12/13/61) 30% NSCLC MEK1-i trials - BRAF mutations (exon 15) 2-3% NSCLC BRAF-i - ALK fusions 3-4% NSCLCC Ci Crizotinibi ib - DDR2 + ibraf mutations 2-4% SCC Dasatinib trial

13 10510 patients consented and 7962 samples sent for testing Target for samples sent Patients consented Consented minus patient drop out Samples sent for testing Results returned Mar 13 Apr 13 May Monthly recruitment Monthly samples sent Monthly results returned Paired samples 115 (1.7%) 119 (1.6%) 128 (1.7%)

14 15 The CR-UK programme COLORECTAL CANCER 1% 3%

15 16 The CR-UK programme The CR UK programme COLORECTAL CANCER

16 17 CRUK-SMP results in malignant melanoma Distribu onofmuta onsinmelanoma(cruk SMP) N=362 Distrubu onsofmuta onsinmelanomaacrossth 60% WT 29% BRAF 46% 50% 40% 30% NRAS PIK3CA 23% 2% 20% KIT 0% 10% 0% BRAF KIT NRAS PIK3CA

17 19 Molecular suitability of FFPE samples Lung carcinomas Lung carcinomas Tertiary referrals Specialised center (n=250) (n=70) EGFR KRAS BRAF ALK 8-12% 20-25% 1-2% 2-4% 21% 0% Failure Failure rate rate

18 20 Panel testing in the clinical setting: What can you expect to find? Alrifai D et al Lung Cancer, 2013

19 21 New tools: Next Generation Sequencing Current sequencing One gene/patient at a time 150 per gene/patient Next Generation seq All genes/patients at the same time per gene/patient

20 22 The CR-UK programme: Next generation sequencing Colorectal Lung Breast Prostate Ovarian Melanoma BRAF EML4-ALK BRAF BRAF BRAF BRAF KRAS NRAS PIK3CA BRAF TP53 TMPRSS2- ERG TP53 BRAF KRAS EGFR NRAS EGFR PIK3CA PTEN PTEN KRAS PTEN PIK3CA KIT NRAS PIK3CA TP53 PTEN KIT DDR2 TP53 Based on TSCA technology by Illumina

21 23 Challenges: sources of material Slide kindly provided by Dr Taniere

22 24 Next Generation Sequencing in the clinic: The need for good quality FFPE samples Tissue taken (POOR) FIXATION WITH FORMALDEHYDE Excessive Crosslinking Deamination DNA Degradation C>T / G>A artefacts

23 25 Next Generation Sequencing in the clinic: Sequencing artefacts

24 26 Next Generation Sequencing in the clinic Non-degraded DNA BUT slight deamination (suboptimal fixation) Original Repeat Common Mean Coverage 9,733x 9,496x Tiles <500x 0 0 Variants Raw All target regions Tumour specific genes Predicted functional consequence

25 27 Next Generation Sequencing in the clinic Non-degraded DNA BUT slight deamination (suboptimal fixation) Chr Pos Ref Alt Gene Change Depth Frequency T TG TP53 G375fs*7 5K 2% G A PTEN C105Y 2K 2% T G EGFR L858R 17K 10% 17K 13%

26 28 Next Generation Sequencing in the clinic: Advantages over conventional sequencing PTEN Capillary sequencing p.asn276met Negative control Illumina sequencing p.asn276ile and p.asn276lys Prostate tumour Prostate tumour

27 29 Translocations and amplifications (FISH)

28 30 Detection of structural variants by NGS Chromosomal translocations in multiple myeloma Walker BA et al. Blood 2013;121:

29 31 Detection of structural variants by NGS Chromosomal translocations in lymphoid malignancies Disease entity Translocation Follicular l lymphoma t(14;18) 18) Mantle cell lymphoma Burkitt s lymphoma MALT lymphoma Anaplastic large cell lymphoma Multiple myeloma, CLL, ALL Others t(11;14) t(8;14) t(11;18) t(2;5) t(14q;?) MLL, TP53, ATM, BCL3, BCL6, BRAF, ABL1, RARA D Gonzalez et al. EuroClonality Consortium.

30 32 Detection of structural variants by NGS Chromosomal translocations in lymphoid malignancies FFPE FF BCL2 IGHV3-21 D Gonzalez et al. EuroClonality Consortium.

31 33 Detection of mutations and CNV by NGS TP53 mutation and 17p deletion FFPE FF D Gonzalez et al. EuroClonality Consortium.

32 34 Resistance mechanisms to TKi Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

33 35 Resistance mechanisms to TKi Popat S et al. Lung Cancer, 2013

34 36 Morpho-Molecular Molecular profiling and Adaptive therapies Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

35 37 Immunotherapeutics: the holy grail?

36 38 Immunotherapeutics: the holy grail? Freeman and Sharpe, Nature Immunology 2012

37 39 Acknowledgments Molecular Diagnostics GI and Lymphoma unit Lisa Thompson Lung unit Debbie Mair Dörte Wren Haemato-Oncology Sue Lillis Rob Hollifield Breakthrough Melanoma unit Belen Gomez Signal Transduction Damian Brookes Chantal Hooper Thomas Jones Jolyon Travis Dee Collins Daphne Webster Manisha Maurya Lisa Grady Stephanie Ogwuru Keeda Dover Immunophenotyping Cytogenetics Histopathology Sarcoma unit Phase I / DDU Paediatric Oncology Molecular Pathology CRUK Cancer Therapeutics

38 40 Next Generation Sequencing in the clinic: The need for good quality FFPE samples Mean 5000x Mean 5000x