THE CHORDOMA GENOME

PETER CAMPBELL,WELLCOME TRUST SANGER INSTITUTE

Chordoma• 1% of primary malignant bone

tumours. • 0.5-1/1000,000/year.• 0 - 90 yrs: 55, any age, • Male to female ratio 2:1

29%

33%

32%

• 37% metastasize

• 5% dedifferentiation

• Overall median survival ~7 years

Chordoma

BRACHYURY

H&E

Coding point mutations – 23 chordomas

1. Whole exome sequencing

2. Variant discovery

3. Validation and confirmation

Sample ascertainment

Sample QC

Whole exome library

preparation

Massively parallel sequencing (Illumina)

NGS data QC & filtering

Whole exome

mapping

Normal –Tumour sequence

comparison

Raw variant discovery

Variant annotation

Variant selection

Variant resequencing Roche 454

Somatic variant

confirmation

Define driver mutations

Targeted resequencing

studies

Calculate prevalence

Number of coding mutations per patient

Mutations in known cancer genes

PIK3CA mutations

E545K

M1043I

Recurrently mutated genes

sam

ple

gene

gene

freq

(exc

l si

lent

)

gene

cds

(bp)

wt a

llele

mut

alle

le

cons

eque

nce

p. c.

PD3814a AUTS2 2 3780 C A missense p.T1047N c.3140C>APD7186a AUTS2 2 3780 T C essential splice p.? c.660+2T>CPD3820a DCC 2 4344 C T missense p.A455V c.1364C>TPD7186a DCC 2 4344 A G missense p.T1110A c.3328A>GPD3819a DNAH17 2 13458 G A missense p.R1515C c.4543C>TPD7185a DNAH17 2 13458 ag deletion D_ag 2PD4929a FLG 2 12186 T C missense p.H2505R c.7514A>GPD4929a FLG 2 12186 G A missense p.S3149F c.9446C>TPD4929a GPR110 2 2736 G C missense p.N437K c.1311C>GPD7184a GPR110 2 2736 C T missense p.G253R c.757G>APD4188a ITGA10 2 3504 tc deletion D_tc 2PD4188a ITGA10 2 3504 T insertion I_T 1PD4187a LYST 2 11406 ac deletion D_ac 2PD4927a LYST 2 11406 ataac deletion D_ataac 5PD3814a PIK3CA 2 3207 G A missense p.E545K c.1633G>APD7187a PIK3CA 2 3207 G T missense p.M1043I c.3129G>TPD3814a PRICKLE3 2 1885 C A missense p.V130L c.388G>TPD7185a PRICKLE3 2 1885 G T missense p.A301D c.902C>APD3804a PTPRZ1 2 6948 C A missense p.T957N c.2870C>APD6370a PTPRZ1 2 6948 C G missense p.A2267G c.6800C>GPD4187a RELN 2 10383 C G missense p.E2910Q c.8728G>CPD6370a RELN 2 10383 A T missense p.S594T c.1780T>APD3808a SCN2A 2 6018 C T missense p.S1974L c.5921C>TPD4929a SCN2A 2 6018 g deletion D_g 1PD4928a SLC9A10 2 3534 G A missense p.R721C c.2161C>TPD4929a SLC9A10 2 3534 T C missense p.I911V c.2731A>GPD3807a SLCO5A1 2 2547 G C missense p.P341A c.1021C>GPD4926a SLCO5A1 2 2547 C T missense p.R596Q c.1787G>APD4928a VCAN 2 10191 G A missense p.V132I c.394G>APD7185a VCAN 2 10191 T A missense p.D1366E c.4098T>APD3815a XIRP2 2 10650 C A missense p.N2480K c.7440C>APD3821a XIRP2 2 10650 G T missense p.Q1680H c.5040G>T

Summary of exome data• 10-30 coding mutations per patient

• No frequently mutated novel genes

• Recurrent mutations in genes regulating histone modification

• Recurrent mutations leading to activation of PI3K signalling

Finding genomic rearrangements

Chr 1

Chr 4 Chr 1 Chr 4

500bp

Chordoma

Other chordomas

Hallmarks of chromothripsis• Massive genomic rearrangement in localised chromosomal regions

• Whole chromosomes, chromosome arms or chromosome bands

• Alternating copy number states• 2, 3 or occasionally 4 discrete states with many switches

• Retention of heterozygosity in higher copy number state

• Clustering of breakpoints

• Ends essentially randomly joined in random orientation• Approx equal numbers of rearrangements with ‘deletion-type’, ‘tandem-

duplication-type’ and ‘inverted’ orientation

Non-homologous end joining

Catastrophic chromosome breakage

Catastrophe model

Rearrangement screens in sarcoma

Histology Number Number with chromothripsis

Osteosarcoma 10 3

Chordoma 14 2

Myxofibrosarcoma 5 3

Pleomorphic sarcoma 2 2

MPNST 2 2

Liposarcoma 2 0

Leiomyosarcoma 2 2

Chondrosarcoma 2 0

Solitary fibrous tumour 1 0

Total 40 14 (35%)

Triple jeopardy – chordoma

Role of inherited genetic factors

SNP upstream of T (brachyury)

Level of association – rs2305089

CASESA allele freq

CONTROLSA allele freq P value OR 95% CI

Discovery 0.88 0.53 4.4x10-9 6.1 3.1-12.1

Replication 0.83 0.53 2.8x10-4 4.1 1.8-9.5

Combined 4.6x10-12 5.3 3.1-8.9

SNP associated with T expression

Conclusions• Chordomas show 10-30 coding mutations per patient

• Frequent incidence of chromothripsis

• Germline predisposition with brachyury SNP

Acknowledgements

Cancer Genome Project• Jose Tubio & Susie Cooke• Patrick Tarpey• David McBride• John Marshall & Keiran Raine• Adam Butler & Jon Teague• Lucy Stebbings & Catherine Leroy• Sarah O’Meara, Laura Mudie• Mike Stratton & Andy Futreal

University College London• Adrienne Flanagan• Roberto Tibrabosco• Fernanda Amary • Nischalan Pillay

Chordoma Foundation• Josh Sommer