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

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

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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

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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

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The Royal Marsden

The complexity of human cancer genomes

S tratified Medicine 2

The complexity of human cancer genomes

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The Royal Marsden S tratified Medicine 3

Molecular diagnostics in cancer Molecular diagnostics in cancer

Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

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The Royal Marsden S tratified Medicine 4

Molecular diagnostics in cancer Molecular diagnostics in cancer

Vemurafenib in BRAFV600E Melanoma Crizotinib in ALK+ NSCLC Vemurafenib in BRAF Melanoma Crizotinib in ALK+ NSCLC

Chapm an P et al N Engl J Med 2011;364:2507-16 C id DR t l L t O l

Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

Chapm an P et al N Engl J Med 2011;364:2507-16 Cam idge DR et al, Lancet Oncology 2011

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The Royal Marsden S tratified Medicine 5

Molecular diagnostics in cancer Molecular diagnostics in cancer

TP53 mutations in CLL

0.8 0.9 1.0 p=0.002

CLLU1 expression in CLL

0.4 0.5 0.6 0.7 mulative Proportion Surviving Low (n=247) High (n=268) 12 24 36 48 60 72 84 96 108 120 Months from Randomization 0.0 0.1 0.2 0.3 Cum High (n 268)

Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013 D Gonzalez et al, J Clin Oncol 2011 D Gonzalez et al, Haemat ologica 2013

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The Royal Marsden S tratified Medicine 6

Molecular diagnostics in cancer Molecular diagnostics in cancer

BCR-ABL monitoring in CML

Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

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The Royal Marsden

Development and validation of new tests

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Development and validation of new tests

Mat t ocks C et al. European Journal of Human Genet ics (2010) 18, 1276– 1288

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The Royal Marsden

The importance of “Target” validation

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The importance of Target validation

L Friboulet et al. N Engl J Med 2013;368:1101-10.

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The Royal Marsden

The importance of “Target” validation

S tratified Medicine 9

The importance of Target validation

L Friboulet et al. N Engl J Med 2013;368:1101-10. Olaussen KA et al. N Engl J Med 2006;355:983-91.

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The Royal Marsden S tratified Medicine 10

The CR-UK S tratified Medicine programme The CR UK S tratified Medicine programme

Research infrastructure S ervice delivery

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The Royal Marsden S tratified Medicine 11

The CR-UK S tratified Medicine programme The CR UK S tratified Medicine programme

Colorectal Lung Breast Prostate Ovarian Melanoma BRAF KRAS EML4-ALK BRAF BRAF TP53 BRAF TMPRS S 2- ERG BRAF TP53 BRAF KIT NRAS PIK3CA EGFR KRAS PIK3CA PTEN ERG PTEN PTEN

PIK3CA

NRAS PIK3CA PIK3CA TP53 KRAS

DDR2 + iBRAF

PTEN

PIK3CA

PIK3CA

Each panel for £300

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The Royal Marsden S tratified Medicine 12

The CR-UK S tratified Medicine programme: The CR UK S 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 i 3 4% NSC C C i i ib

  • ALK fusions

3-4% NSCLC Crizotinib

  • DDR2 + iBRAF mutations

2-4% SCC Dasatinib trial DDR2 + iBRAF mutations 2-4% SCC Dasatinib trial

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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 10000 12000

Mar‐13 Apr‐13 May‐13

8000 10000

Monthly recruitment 648 678 651 Monthly samples sent 585 565 531

6000

Monthly results returned 524 757 561

4000

Paired samples 115 (1.7%) 119 (1.6%) 128 (1.7%)

2000 Stratified Medicine 13

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The Royal Marsden S tratified Medicine 15

The CR-UK S tratified Medicine programme The CR UK S tratified Medicine programme COLORECTAL CANCER

3% 1%

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The Royal Marsden S tratified Medicine 16

The CR-UK S tratified Medicine programme The CR UK S tratified Medicine programme COLORECTAL CANCER

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The Royal Marsden S tratified Medicine 17

CRUK-S MP results in malignant melanoma CRUK S MP results in malignant melanoma

Distribu on

  • f

muta ons in melanoma (CRUK‐SMP) N=362 Distrubu ons

  • f

muta ons in melanoma across TH

BRAF 46% WT 29%

40% 50% 60%

NRAS 23% PIK3CA 2%

20% 30% 40%

KIT 0%

0% 10% BRAF KIT NRAS PIK3CA

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The Royal Marsden

Molecular suitability of FFPE samples

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Molecular suitability of FFPE samples

Lung carcinomas Tertiary referrals (n=250) Lung carcinomas Specialised center (n=70) ( ) ( )

EGFR KRAS BRAF ALK EGFR KRAS BRAF ALK

8-12% 20-25% 1-2% 2-4%

21% 0% Failure rate Failure rate

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The Royal Marsden S tratified Medicine 20

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

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The Royal Marsden S tratified Medicine 21

New tools: Next Generation S equencing New tools: Next Generation S equencing

C i O Current sequencing One gene/ patient at a time £150 per gene/ patient £150 per gene/ patient N t G ti Next Generation seq All genes/ patients at the same time same time £20-50 per gene/ patient

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The Royal Marsden S tratified Medicine 22

The CR-UK S tratified Medicine programme: The CR UK S tratified Medicine programme:

Next generation sequencing

Colorectal Lung Breast Prostate Ovarian Melanoma BRAF KRAS EML4-ALK BRAF BRAF TP53 BRAF TMPRS S 2- ERG BRAF TP53 BRAF KIT

BRAF KRAS EGFR NRAS

NRAS PIK3CA EGFR KRAS PIK3CA PTEN ERG PTEN PTEN NRAS PIK3CA

BRAF KRAS EGFR NRAS PIK3CA TP53 PTEN KIT DDR2

PIK3CA TP53 KRAS PTEN PIK3CA

Based on TS CA technology by Illumina

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The Royal Marsden

Challenges: sources of material

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Challenges: sources of material

Slide kindly provided by Dr Taniere

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The Royal Marsden S tratified Medicine 24

Next Generation S equencing in the clinic: Next Generation S 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

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The Royal Marsden S tratified Medicine 25

Next Generation S equencing in the clinic: Next Generation S equencing in the clinic:

S equencing artefacts

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The Royal Marsden S tratified Medicine 26

Next Generation S equencing in the clinic Next Generation S 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 Variants Variants Raw 228 191 70 All target regions 213 179 70 g g Tumour specific genes 84 71 17 Predicted functional 38 42 3 consequence 38 42 3

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The Royal Marsden S tratified Medicine 27

Next Generation S equencing in the clinic Next Generation S 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% 7 55259515 T G EGFR L858R 17K 17K 10% 13% 17K 13%

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The Royal Marsden S tratified Medicine 28

Next Generation S equencing in the clinic: Next Generation S equencing in the clinic:

Advantages over conventional sequencing

PTEN

Capillary sequencing Illumina sequencing

p.Asn276Met Negative control

Capillary sequencing Illumina sequencing

p.Asn276Ile and p.Asn276Lys Prostate tumour Prostate tumour

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The Royal Marsden S tratified Medicine 29

Translocations and amplifications (FIS H) Translocations and amplifications (FIS H)

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The Royal Marsden

Detection of structural variants by NGS

S tratified Medicine 30

Detection of structural variants by NGS

Chromosomal translocations in multiple myeloma

Walker BA et al. Blood 2013;121:3413-19.

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The Royal Marsden

Detection of structural variants by NGS

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Detection of structural variants by NGS

Chromosomal translocations in lymphoid malignancies

Disease entity Translocation F lli l l h t(14 18) Follicular lymphoma t(14;18) Mantle cell lymphoma t(11;14) Burkitt’s lymphoma t(8;14) Burkitt s lymphoma t(8;14) MALT lymphoma t(11;18) Anaplastic large cell t(2;5) lymphoma Multiple myeloma, CLL, ALL t(14q;?) Others MLL TP53 ATM BCL3 Others MLL, TP53, ATM, BCL3, BCL6, BRAF, ABL1, RARA…

D Gonzalez et al. EuroClonalit y Consort ium.

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The Royal Marsden

Detection of structural variants by NGS

S tratified Medicine 32

Detection of structural variants by NGS

Chromosomal translocations in lymphoid malignancies

FFPE FF BCL2 IGHV3-21

D Gonzalez et al. EuroClonalit y Consort ium.

IGHV3 21

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The Royal Marsden

Detection of mutations and CNV by NGS

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Detection of mutations and CNV by NGS

TP53 mutation and 17p deletion

FFPE FF

D Gonzalez et al. EuroClonalit y Consort ium.

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Resistance mechanisms to TKi Resistance mechanisms to TKi

Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

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The Royal Marsden S tratified Medicine 35

Resistance mechanisms to TKi Resistance mechanisms to TKi

Popat S et al. Lung Cancer, 2013

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The Royal Marsden S tratified Medicine 36

Morpho-Molecular profiling and Adaptive therapies Morpho Molecular profiling and Adaptive therapies

Gonzalez, Clarke, Al-Lazikani, Workman; Clin Pharm Ther 2013

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The Royal Marsden S tratified Medicine 37

Immunotherapeutics: the holy grail? Immunotherapeutics: the holy grail?

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The Royal Marsden S tratified Medicine 38

Immunotherapeutics: the holy grail? Immunotherapeutics: the holy grail?

Freeman and S harpe, Nat ure Immunology 2012

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The Royal Marsden S tratified Medicine 39

Acknowledgments Acknowledgments

Molecular Diagnostics Molecular Diagnostics

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

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The Royal Marsden S tratified Medicine 40

Next Generation S equencing in the clinic: Next Generation S equencing in the clinic:

The need for good quality FFPE samples

40000 25000 30000 35000 40000 10000 15000 20000 Mean 5000x 5000

90000 100000 40000 50000 60000 70000 80000 Mean 5000x 10000 20000 30000