Molecular Stratification of Cancer in the Clinical Setting Clinical - PowerPoint PPT Presentation

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

2 The Royal Marsden S tratified Medicine The complexity of human cancer genomes The complexity of human cancer genomes

3 The Royal Marsden S tratified Medicine Molecular diagnostics in cancer Molecular diagnostics in cancer Gonzalez, Clarke, Al-Lazikani, Workman ; Clin Pharm Ther 2013

4 The Royal Marsden S tratified Medicine Molecular diagnostics in cancer Molecular diagnostics in cancer Vemurafenib in BRAF V600E Melanoma Vemurafenib in BRAF Melanoma Crizotinib in ALK+ NSCLC Crizotinib in ALK+ NSCLC Chapm an P et al N Engl J Med 2011;364:2507-16 Chapm an P et al N Engl J Med 2011;364:2507-16 C Cam idge DR et al, Lancet Oncology 2011 id DR t l L t O l Gonzalez, Clarke, Al-Lazikani, Workman ; Clin Pharm Ther 2013

5 The Royal Marsden S tratified Medicine Molecular diagnostics in cancer Molecular diagnostics in cancer TP53 mutations in CLL CLLU1 expression in CLL 1.0 0.9 p=0.002 0.8 mulative Proportion Surviving 0.7 0.6 0.5 Low (n=247) 0.4 High (n=268) High (n 268) Cum 0.3 0.2 0.1 0.0 0 12 24 36 48 60 72 84 96 108 120 Months from Randomization D Gonzalez et al, J Clin Oncol 2011 D Gonzalez et al, Haemat ologica 2013 Gonzalez, Clarke, Al-Lazikani, Workman ; Clin Pharm Ther 2013

6 The Royal Marsden S tratified Medicine Molecular diagnostics in cancer Molecular diagnostics in cancer BCR-ABL monitoring in CML Gonzalez, Clarke, Al-Lazikani, Workman ; Clin Pharm Ther 2013

7 The Royal Marsden S tratified Medicine Development and validation of new tests Development and validation of new tests Mat t ocks C et al. European Journal of Human Genet ics (2010) 18, 1276– 1288

8 The Royal Marsden S tratified Medicine The importance of “Target” validation The importance of Target validation L Friboulet et al. N Engl J Med 2013;368:1101-10.

9 The Royal Marsden S tratified Medicine The importance of “Target” validation The importance of Target validation Olaussen KA et al. N Engl J Med 2006;355:983-91. L Friboulet et al. N Engl J Med 2013;368:1101-10.

10 The Royal Marsden S tratified Medicine The CR-UK S The CR UK S tratified Medicine programme tratified Medicine programme Research infrastructure S ervice delivery

11 The Royal Marsden S tratified Medicine The CR-UK S The CR UK S tratified Medicine programme tratified Medicine programme Colorectal Lung Breast Ovarian Melanoma Prostate BRAF EML4-ALK BRAF BRAF BRAF BRAF TMPRS S 2- KRAS BRAF TP53 TP53 KIT ERG ERG NRAS EGFR PIK3CA PTEN NRAS PTEN PIK3CA PIK3CA PIK3CA PIK3CA KRAS KRAS PTEN PTEN PIK3CA PIK3CA DDR2 + TP53 iBRAF Each panel for £300

12 The Royal Marsden S tratified Medicine The CR-UK S The CR UK S tratified Medicine programme: tratified Medicine 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 f ALK fusions i 3-4% NSCLC 3 4% NSC C C i Crizotinib i ib - DDR2 + iBRAF mutations DDR2 + iBRAF mutations 2-4% SCC 2-4% SCC Dasatinib trial Dasatinib trial

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 12000 Mar ‐ 13 Apr ‐ 13 May ‐ 13 10000 10000 Monthly 648 678 651 recruitment Monthly 8000 585 565 531 samples sent Monthly results 524 757 561 6000 returned 115 119 128 Paired samples (1.7%) (1.6%) (1.7%) 4000 2000 0 13 Stratified Medicine

15 The Royal Marsden S tratified Medicine The CR-UK S The CR UK S tratified Medicine programme tratified Medicine programme COLORECTAL CANCER 1% 3%

16 The Royal Marsden S tratified Medicine The CR-UK S The CR UK S tratified Medicine programme tratified Medicine programme COLORECTAL CANCER

17 The Royal Marsden S tratified Medicine CRUK-S CRUK S MP results in malignant melanoma MP results in malignant melanoma Distribu on� of� muta ons� in� melanoma� (CRUK ‐ SMP)� Distrubu ons� of� muta ons� in� melanoma� across� TH� N=362� 60%� WT� 50%� 29%� BRAF� 46%� 40%� 40%� 30%� NRAS� PIK3CA� 23%� 20%� 2%� 10%� KIT� 0%� 0%� BRAF� KIT� NRAS� PIK3CA�

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

20 The Royal Marsden S tratified Medicine Panel testing in the clinical setting: Panel testing in the clinical setting: What can you expect to find? Alrifai D et al Lung Cancer, 2013

21 The Royal Marsden S tratified Medicine New tools: Next Generation S New tools: Next Generation S equencing equencing Current sequencing One C i O gene/ patient at a time £150 per gene/ patient £150 per gene/ patient Next Generation seq N t G ti All genes/ patients at the same time same time £20-50 per gene/ patient

22 The Royal Marsden S tratified Medicine The CR-UK S The CR UK S tratified Medicine programme: tratified Medicine programme: Next generation sequencing Colorectal Lung Breast Ovarian Melanoma Prostate BRAF EML4-ALK BRAF BRAF BRAF BRAF TMPRS S 2- KRAS BRAF TP53 TP53 KIT BRAF BRAF KRAS EGFR NRAS KRAS EGFR NRAS ERG ERG NRAS EGFR PIK3CA PTEN NRAS PTEN PIK3CA TP53 PTEN KIT DDR2 PIK3CA PIK3CA KRAS KRAS PTEN PTEN PIK3CA PIK3CA TP53 Based on TS CA technology by Illumina

23 The Royal Marsden S tratified Medicine Challenges: sources of material Challenges: sources of material Slide kindly provided by Dr Taniere

24 The Royal Marsden S tratified Medicine Next Generation S Next Generation S equencing in the clinic: equencing 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

25 The Royal Marsden S tratified Medicine Next Generation S Next Generation S equencing in the clinic: equencing in the clinic: S equencing artefacts

26 The Royal Marsden S tratified Medicine Next Generation S Next Generation S equencing in the clinic equencing in the clinic Non-degraded DNA BUT slight deaminat ion (subopt imal f ixat ion) Original Repeat Common Mean Coverage 9,733x 9,496x Tiles <500x 0 0 Variants Variants Raw 228 191 70 All target regions g g 213 179 70 Tumour specific genes 84 71 17 Predicted functional 38 38 42 42 3 3 consequence

27 The Royal Marsden S tratified Medicine Next Generation S Next Generation S equencing in the clinic equencing in the clinic Non-degraded DNA BUT slight deaminat ion (subopt imal f ixat ion) Chr Pos Ref Alt Gene Change Depth Frequency 17 7572985 T TG TP53 G375fs*7 5K 2% 10 89692830 G A PTEN C105Y 2K 2% 17K 10% 7 55259515 T G EGFR L858R 17K 17K 13% 13%

28 The Royal Marsden S tratified Medicine Next Generation S Next Generation S equencing in the clinic: equencing in the clinic: Advantages over conventional sequencing PTEN Capillary sequencing Capillary sequencing Illumina sequencing Illumina sequencing p.Asn276Met p.Asn276Ile and p.Asn276Lys Prostate tumour Negative control Prostate tumour

29 The Royal Marsden S tratified Medicine Translocations and amplifications (FIS Translocations and amplifications (FIS H) H)

30 The Royal Marsden S tratified Medicine Detection of structural variants by NGS Detection of structural variants by NGS Chromosomal translocations in multiple myeloma Walker BA et al. Blood 2013;121:3413-19.

31 The Royal Marsden S tratified Medicine Detection of structural variants by NGS Detection of structural variants by NGS Chromosomal translocations in lymphoid malignancies Disease entity Translocation Follicular lymphoma F lli l l h t(14 18) t(14;18) Mantle cell lymphoma t(11;14) Burkitt s lymphoma Burkitt’s lymphoma t(8;14) t(8;14) MALT lymphoma t(11;18) Anaplastic large cell t(2;5) lymphoma Multiple myeloma, CLL, ALL t(14q;?) Others Others MLL TP53 ATM BCL3 MLL, TP53, ATM, BCL3, BCL6, BRAF, ABL1, RARA… D Gonzalez et al. EuroClonalit y Consort ium.

32 The Royal Marsden S tratified Medicine Detection of structural variants by NGS Detection of structural variants by NGS Chromosomal translocations in lymphoid malignancies FFPE FF BCL2 IGHV3-21 IGHV3 21 D Gonzalez et al. EuroClonalit y Consort ium.

33 The Royal Marsden S tratified Medicine Detection of mutations and CNV by NGS Detection of mutations and CNV by NGS TP53 mutation and 17p deletion FFPE FF D Gonzalez et al. EuroClonalit y Consort ium.

34 The Royal Marsden S tratified Medicine Resistance mechanisms to TKi Resistance mechanisms to TKi Gonzalez, Clarke, Al-Lazikani, Workman ; Clin Pharm Ther 2013