TCF12 is mutated in anaplastic oligodendroglioma

Abstract

Anaplastic oligodendroglioma (AO) are rare primary brain tumours that are generally incurable, with heterogeneous prognosis and few treatment targets identified. Most oligodendrogliomas have chromosomes 1p/19q co-deletion and an IDH mutation. Here we analysed 51 AO by whole-exome sequencing, identifying previously reported frequent somatic mutations in CIC and FUBP1. We also identified recurrent mutations in TCF12 and in an additional series of 83 AO. Overall, 7.5% of AO are mutated for TCF12, which encodes an oligodendrocyte-related transcription factor. Eighty percent of TCF12 mutations identified were in either the bHLH domain, which is important for TCF12 function as a transcription factor, or were frameshift mutations leading to TCF12 truncated for this domain. We show that these mutations compromise TCF12 transcriptional activity and are associated with a more aggressive tumour type. Our analysis provides further insights into the unique and shared pathways driving AO.

ARTICLE

Received 22 Feb 2015 | Accepted 17 Apr 2015 | Published 12 Jun 2015

DOI: 10.1038/ncomms8207 OPEN

TCF12 is mutated in anaplastic oligodendroglioma

Karim Labreche1,2,3,4,5,*, Iva Simeonova2,3,4,5,*, Aurlie Kamoun6,*, Vincent Gleize2,3,4,5,*, Daniel Chubb1,

Eric Letouz6, Yasser Riazalhosseini7,8, Sara E. Dobbins1, Nabila Elarouci6, Francois Ducray9,Aurlien de Reynis6, Diana Zelenika10, Christopher P. Wardell11, Mathew Frampton1, Olivier Saulnier2,3,4,5, Tomi Pastinen7,8, Sabrina Hallout2,3,4, Dominique Figarella-Branger12,13, Caroline Dehais14,Ahmed Idbaih2,3,4,5,14, Karima Mokhtari2,3,4,15, Jean-Yves Delattre2,3,4,5,14,**, Emmanuelle Huillard2,3,4,5,**,

G. Mark Lathrop7,8,**, Marc Sanson2,3,4,5,14,**, Richard S. Houlston1,** & POLA Networkw

Anaplastic oligodendroglioma (AO) are rare primary brain tumours that are generally incurable, with heterogeneous prognosis and few treatment targets identied. Most oligodendrogliomas have chromosomes 1p/19q co-deletion and an IDH mutation. Here we analysed 51 AO by whole-exome sequencing, identifying previously reported frequent somatic mutations in CIC and FUBP1. We also identied recurrent mutations in TCF12 and in an additional series of 83 AO. Overall, 7.5% of AO are mutated for TCF12, which encodes an oligodendrocyte-related transcription factor. Eighty percent of TCF12 mutations identied were in either the bHLH domain, which is important for TCF12 function as a transcription factor, or were frameshift mutations leading to TCF12 truncated for this domain. We show that these mutations compromise TCF12 transcriptional activity and are associated with a more aggressive tumour type. Our analysis provides further insights into the unique and shared pathways driving AO.

1 Division of Genetics and Epidemiology, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. 2 Inserm, U 1127, ICM, F-75013 Paris, France. 3 CNRS, UMR 7225, ICM, F-75013 Paris, France. 4 Institut du Cerveau et de la Moelle pinire ICM, Paris 75013, France. 5 Sorbonne Universits, UPMC Universit Paris 06, UMR S 1127, F-75013 Paris, France. 6 Programme Cartes dIdentit des Tumeurs (CIT), Ligue Nationale Contre Le Cancer, 75013 Paris, France.

7 Department of Human Genetics, McGill University, Montreal, Quebec, Canada H3A 0G1. 8 McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada H3A 0G1. 9 INSERM U1028, CNRS UMR5292, Service de Neuro-oncologie, Hopital neurologique, Hospices civils de Lyon, Lyon Neuroscience Research Center, Neuro-Oncology and Neuro-Inammation Team, 69677 Lyon, France. 10 Centre National de Gnotypage, IG/CEA, 2 rue Gaston Crmieux, CP 5721, Evry 91057, France. 11 Division of Molecular Pathology, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. 12 AP-HM, Hpital de la Timone, Service danatomie pathologique et de neuropathologie, 13385 Marseille, France. 13 Universit de la Mditerrane, Aix-Marseille, Facult de Mdecine La Timone, CRO2, UMR 911 Marseille, France. 14 AP-HP, Groupe Hospitalier Piti-Salptrire, Service de neurologie 2-Mazarin, 75013 Paris, France. 15 AP-HP, Groupe Hospitalier Piti-Salptrire, Laboratoire de Neuropathologie R. Escourolle, 75013 Paris, France. * These authors contributed equally to this work. ** These authors jointly supervised this work. w A full list of consortium members appears at the end of the paper. Correspondence and requests for materials should be addressed to R.S.H. (email: mailto:[email protected]

Web End [email protected] ).

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Anaplastic oligodendrogliomas (AO; World Health Organization grade III oligodendrogliomas) are rare primary malignant brain tumours with a highly variable

overall prognosis. The emblematic molecular alteration in oligodendrogliomas is 1p/19q co-deletion, which is associated with a better prognosis and response to early chemotherapy with procarbazine, lomustine and vincristine13. Recent high-throughput sequencing approaches have identied IDH (IDH1 and IDH2), CIC, FUBP1 and TERT promoter mutations in oligodendroglioma (75, 50, 10 and 75%, respectively)2,4,5, IDH mutation status typically being associated with a better clinical outcome6. Identifying additional driver genes and altered pathways in oligodendroglioma offers the prospect of developing more effective therapies and biomarkers to predict individual patient outcome.

Here we perform whole-exome and transcriptome sequencing of AO to search for additional tumour driver mutations and pathways disrupted. In addition to previously reported recurrently mutated genes, we report the identication of somatic mutations in TCF12 in AO. These mutations compromise TCF12 transcriptional activity and confer a more aggressive AO phenotype.

ResultsIn accordance with conventional clinical practice, we considered three molecular subtypes for our analyses: (i) IDH-mutated 1p/19q co-deleted (IDHmut-codel); (ii) IDH-mutated 1p/19q non-co-deleted (IDHmut-non-codel) and (iii) IDH-wild type (IDHwt)7. Assignment of IDH-mutated (dened by IDH1 R132 or IDH2 R172 mutations), 1p/19q and TERT promoter mutation (dened by C228T or C250T) status in tumours was determined using conventional sequencing and single-nucleotide polymorphism (SNP) array methods.

Mutational landscape. We performed whole-exome sequencing of 51 AO tumours (Supplementary Data 1) and matched germ-line DNA, targeting 318,362 exons from 18,901 genes. The mean sequencing coverage across targeted bases was 57 , with 80% of

target bases above 20 coverage (Supplementary Fig. 1). We

identied a total of 4,733 mutations (with a mean of 37 non-silent mutations per sample) equating to a mean somatic mutation rate of 1.62 mutations per megabase (Mb) (Fig. 1). Although the tumours of two patients (3,063 and 3,149) had high rates of mutation (9.1 and 12.4, respectively), this was not reective of tumour site (both frontal lesions as were 68% of the whole series) or treatment. Excluding these two cases the mean rate of non-silent mutations per tumour was 3314, which is similar to the number found in most common adult brain tumours. The mutation spectrum in AO tumours was characterized by a predominance of C4T transitions, as observed in most solid cancers (Fig. 1)8,9. While few of the tumours were IDHwt, these did not harbour a signicantly higher number of mutations compared with IDHmut-1p/19q co-deleted and IDHmut-non-1p/19q co-deleted tumours (Fig. 1). Intriguingly, one tumour (2,688) was co-mutated for IDH1 (R132H) and IDH2 (P162S), but exhibited no distinguishing phenotype in terms of clinicopathology or mutation rate.

We used MutSigCV version 1.4 (ref. 8) to identify genes harbouring more non-synonymous mutations than expected by chance given gene size, sequence context and mutation rate of each tumour for the three molecular subtypes, respectively. As expected, we observed frequent mutations of the tumour suppressors FUBP1 (22%) located on 1p, and CIC (32%) located on 19q, which have been reported in the context of 1p/19q co-deletion (Fig. 1; Supplementary Fig. 2); these were not

mutually exclusive events (Fig. 1). Also within the IDHmut-codel group, 37 of tumours tested carried TERT C228T or C250T promoter mutations (72%), none of which also carried an ATRX mutation, concordant with the previously reported nding that these are mutually exclusive events2.

In addition to the mutation of IDH1 (78%), IDH2 (17%), CIC (32%) and FUBP1 (22%), TCF12 was also signicantly mutated (Q-valueo0.1; Fig. 1; Supplementary Table 2). Heterozygous somatic mutations in TCF12, which encodes the basic helix loophelix (bHLH) transcription factor 12 (aliases HEB, HTF4 and ALF1) were identied in ve (1 missense, R602M; 2 splice-site, c.825 5G4T, c.1979-3_1979-delTA and 2 frameshift,

E548fs*13, S682fs*14) of the 46 IDH-mutated 1p/19q co-deleted. Intriguingly, germline mutations of residues E548 and R602 have been previously shown to cause coronal craniosynostosis10.

The availability of high-quality tumour material allowed us to generate SNP array and expression data on 31 of the cases exome sequenced. In addition to co-deletion of chromosome arms 1p/19q, we identied several other recurrent genomic alterationsmainly loses of chromosomes 4 (29%), 9p (28%) and 14q (19%); Supplementary Fig. 3; Supplementary Table 1). Notably, tumours featuring mutation of Notch-pathway genes showed signicant chromosome 4 loss (P 0.02, w2-test).

To identify fusion transcripts, we analysed RNA-sequencing (RNA-seq) data, which was available for 36 of the 51 tumours. After ltering, the only chimeric transcript identied was the predicted driver FGFR3TACC3 fusion, previously described in IDH wild-type gliomas1113, which was seen in two of the IDHwtnon-1p/19q co-deleted tumourspatients 2463 and 2441; Of note was that patient 2463 carried an IDH2 intron-5 mutation(c.679-28C4T).

Incorporation of TCGA mutation data. To explore the mutational spectra of AO in an independent series, we made use of data generated by The Cancer Genome Atlas (TCGA) study of low-grade glioma, which provides exome sequencing data on a further 43 AO tumours. Two of these 43 tumours harboured frameshift mutations in TCF12 (E548R and D171fs) (Supplementary Table 2). As with our series, these TCF12 mutations were exclusive to IDH-1p/19q co-deleted tumours. In a combined analysis, mutations in PI3KCA, NOTCH1 and TP53 were signicantly overrepresented when analysed using MutSigCV (Q-valueo0.1; Supplementary Table 2). In addition, mutation of ATRX and RBPJ were of borderline signicance.

A bias towards variants with functional impact (FM) is a feature of cancer drivers14. To increase our ability to identify cancer drivers and delineate associated oncogenic pathways for AO, we incorporated mutation data from multiple tumour types using Oncodrive-fm14 implemented within the IntOGen-mutations platform15 (Fig. 2). The most recurrently mutated genes according to MutSig were also detected by Oncodrive-fm as signicantly mutated (Q-valueo0.05). Oncodrive-fm also identied a number of other important mutated genes (that is, displaying high FM bias) including SETD2, NOTCH2, RBPJ, ARID1A, ARID1B, HDAC2 and SMARCA4 (Fig. 2).

Using all mutation results, we performed an analysis to identify pathways or gene ontologies that were signicantly enriched in mutated genes. As expected, the most signicantly altered pathways were linked to the tricarboxylic acid cycle and isocitrate metabolic process as a consequence of IDH mutation. Consistent with the other genes that were found signicantly mutated by MutSigCV and Oncodrive-fm analysis, the Notch signalling pathway (P 1.0 10 5, binomial test), genes involved in

neuron differentiation (P 2.0 10 5, binomial test) and genes

involved in chromatin organization (P 0.02, binomial test) were

also signicantly enriched for mutations (Supplementary Data 3).

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8207 ARTICLE

Mutation per MB

12

10

8

6

4

2

0

T-2402

T-2688

Age

50

57

42

59

33

40

56

45

28

33

58

46

45

60

61

60

46

35

64

39

78

52

46

40

27

31

61

47

52

55

49

38

33

51

33

35

42

44

58

35

45

43

53

78

45

44

43

42

38

53

49

Male

Female

Sex

TERT

1p19q codel

T-2532

T-2472

T-2669

T-2716

T-2821

T-2866

T-2965

T-3122

T-2626

T-2671

T-2691

T-2702

T-2485

T-2755

T-2775

T-2795

T-2830

T-2832

T-2877

T-2878

T-2911

T-2915

T-2921

T-2971

T-3149

T-3463

T-2708

T-2842

T-2747

T-2819

T-2694

T-3000

T-2898

T-2807

T-3443

T-3126

T-3016

T-2728

T-3063

T-2497

T-2551

T-2896

T-3237

T-3401

T-2463

T-3338

T-2470

T-3130

T-2441

IDH1 (78%)

IDH2 (17%)

CIC (32%)

FUBP1 (22%)

TCF12 (10%)

100 75 50 25 0

TransverIndel+nullA>G*CpG>T *Cp(A/C/T)>T 0.0

0.2

Mutation frequency

Promoter mutation 1p19q codeleted Non 1p19q codeleted Missense Frame shift Inframe indel Intron

Splice

1.0

0.6

%

0.8

0.4

Figure 1 | Signicantly mutated genes in anaplastic oligodendroglioma by molecular subtype. Signicantly mutated genes (Q-valueo0.1) identied by exome sequencing are listed by Q-value. The percentage of AO samples with mutation detected by automated calling is detailed on the left. Samples are displayed as columns, with the mutation rate plotted at the top. Samples are arranged to emphasize mutual exclusivity. Mutation types are indicated in different colours (see legend). White colour indicates no information available. Also shown is the relative proportion of base-pair substitutions within mutation categories for each tumour.

Validation of TCF12 in an additional series of AO. To identify additional TCF12-mutated AO tumours, we conducted targeted sequencing of a further 83 AO. Five tumours harboured TCF12 mutationsG48fs*38, M260fs*5, R326S, D455fs*59 and delN606 (Supplementary Data 1). On the basis of our combined sample of 134 tumours, the mutation frequency of TCF12 in AO is 7.5% (95% condence interval 3.613.2%). No signicant difference in patient survival in 1p/19q co-deleted AO was associated with TCF12 mutation in 69 patients (Supplementary Fig. 4). While our power to demonstrate a statistically signicant relationship was limited (that is, B40% for a hazard ratio of 2.0, stipulating

P 0.05), we noted that patients having either TCF12 mutated or

TCF12 loss of heterozygosity (LOH) tended to be associated with shorter survival (Supplementary Fig. 4). To gain further insight into the role of TCF12 mutation in oligodendroglioma, we sequenced 75 grade II tumours identifying one mutation carrier (P212fs*31; Supplementary Data 1). The observation that the frequency of TCF12 mutations is higher in AO as compared with grade II tumours (P 0.049, w2-test) is compatible with TCF12

participating in the generation of a more aggressive phenotype.

TCF12 bHLH mutants compromised transactivation. To explore the functional consequences of TCF12 mutation, we tested the transcriptional activity of several mutants (Fig. 3). We tested the frameshift mutations M260fs*5 and E548fs*13, which in the germline cause coronal craniosynostosis10 and S682fs*14, since introduction of a C-terminal premature stop codon may result in escape from non-sense-mediated decay. We also tested the missense mutation R602M, which is predicted to destabilize

the bHLH domain required for DNA binding and dimerization (Fig. 3) and whose adjacent residue (R603) has been found recurrently mutated in colon cancer16. Finally, we tested the missense mutation R326S, since mutations of adjacent G327 have been reported in lung adenocarcinoma17. The frameshift mutants M260fs*5 and E548fs*13 completely abolished TCF12 transactivation, consistent with the lack of bHLH DNA-binding domain (Fig. 3). R602M retained only 34% of WT transcriptional activity (P 0.0018, Students t-test; Fig. 3). We did not observe

signicant modulation of transactivation for the R326S and S682fs*14 mutants, although the latter consistently showed decreased activity (Fig. 3).

Downregulation of pathways in TCF12 bHLH mutants. We proled gene expression in 8 TCF12-mutated and 45 wild-type tumours within 1p/19q co-deleted samples (Supplementary Table 1). TCF12 mutation was associated with signicant enrichment of immune response pathways (Supplementary Data4). Restricting the analysis to tumours with the TCF12-altered bHLH domain (n 6), we found downregulation of pathways

featuring known partners of TCF12, such as TCF21, EZH2 and BMI1 (ref. 18) (Supplementary Table 2). Interestingly, we found decreased activity of genes sets related to E-cadherin (CDH1), which is a TCF12 target gene associated with tumour pheno-type18. Since the promotor sequences of CDH1 and BMI1 feature E-box motifs and are modulated by the bHLH binding19,20, this provides a mechanistic basis for change in gene expression associated with mutant TCF12.

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TCGA-CS-5396

TCGA-DU-6393

TCGA-DU-6394

TCGA-DU-6397

TCGA-DU-6410

TCGA-DU-7018

TCGA-DU-7300

TCGA-DU-7302

TCGA-DU-8168

TCGA-E1-5311

TCGA-FG-5962

TCGA-FG-7638

TCGA-HT-7468

TCGA-HT-7471

TCGA-HT-7616

TCGA-HT-7620

TCGA-HT-7677

TCGA-HT-7687

TCGA-HT-7694

TCGA-HT-7874

TCGA-HT-8105

TCGA-HT-A4DV

TCGA-HT-8109

TCGA-HW-A5KJ

TCGA-P5-A5EX

T-2402

T-2472

T-2485

T-2497

T-2532

T-2551

T-2626

T-2669

T-2671

T-2688

T-2691

T-2702

T-2708

T-2716

T-2728

T-2747

T-2755

T-2775

T-2795

T-2819

T-2821

T-2832

T-2830

T-2842

T-2866

T-2877

T-2878

T-2896

T-2911

T-2915

T-2921

T-2965

T-2971

T-3063

T-3122

T-3126

T-3149

T-3237

T-3401

T-3463

TCGA-DB-A4XG

TCGA-DH-5141

TCGA-HT-7856

TCGA-HT-A5R9

TCGA-DU-6408

TCGA-FG-8191

TCGA-HT-7470

TCGA-HT-7688

TCGA-DU-6404

TCGA-DU-7309

TCGA-DU-8165

TCGA-DU-A5TT

TCGA-FG-6692

TCGA-HT-7469

TCGA-HT-7882

TCGA-HT-8019

T-2694

T-2807

T-2898

T-3000

T-3016

T-3443

T-2441

T-2463

T-2470

T-3130

T-3338

Codel FM Pvalue < 1E16

< 1E16 < 1E16

1.02E10

6.087E125.894E105.071E73.357E62.482E53.278E51.809E38.288E39.72E30.0160.0280.0470.0470.0470.0470.0470.0470.0470.0470.047

0.051

IDH1 IDH2

CIC FUBP1

TP53 ARID1A

TCF12

ATRX

RBPJ NOTCH1

SETD2 PIK3R1 SMARCA4

ARID1B NEO1

NF1 NIPBL DOPEY1

HDAC2

BCOR

BRE NOTCH2

GLUD1 CCDC30

TCGA-DB-A64P

TCGA-DH-5144

1p19 codeleted

Non-1p19 codeleted

2

5

0

MA score

FM P-value

Figure 2 | FM-biased genes and gene modules in AO identied by Oncodrive-fm using data from this study and tumours proled by TCGA. Heatmap shows tumours in columns and genes in rows, the colour reecting the MutationAssessor (MA) scores of somatic mutations. FM ext. qv, correctedP values of the FM bias analysis using the external null distribution.

Mutant TCF12 proteins show subcellular localization changes. We evaluated TCF12 expression and subcellular localization for all of our 11 TCF12-mutated tumours (10 AO and 1 oligodendroglioma grade II) and 11 TCF12 wild-type tumours by immunohistochemistry. All TCF12 wild-type tumours showed nuclear expression in a heterogeneous cell population (Fig. 4; Supplementary Fig. 5), whereas several TCF12-mutated tumours showed nuclear and cytoplasmic staining (Fig. 4; Supplementary Fig. 5). Interestingly, mutations abolishing transcriptional activity were associated with increased staining, suggesting inactive mutant protein accumulation.

TCF12 mutations associate with aggressive tumour phenotype. We proled the extent of necrosis, microvascular proliferation and the mitotic index available for TCF12 wild-type or mutated tumours. A signicant increase in palisading necrosis (Fig. 5) as well as a trend towards a higher mitotic index was associated with TCF12 mutation, consistent with a more aggressive phenotype (Fig. 5). Intriguingly, tumours harbouring disruptive bHLH domain mutations exhibited the highest proportion of palisading necrosis and mitotic gures.

DiscussionOur genome sequencing of AO has conrmed the mutually exclusive mutational prole in IDHmut-1p/19q co-deleted and IDHmut non-1p/19q co-deleted tumour subtypes, which reect distinct molecular mechanisms of oncogenesisconsistent with the requirement for either 1p/19q co-deletion or TP53 mutation post IDH mutation. Moreover, as previously proposed, the genomic abnormalities in IDHmut-1p/19p co-deleted tumours are consistent with one common mechanism of tumour initiation being through 1p/19q loss, mutation of IDH1 or IDH2 and TERT activation through promoter mutation2, which in turn

predisposes to deactivation of CIC, FUBP1, NOTCH and activating mutations/amplications in the PI3K pathway.

We identied and replicated mutations in TCF12, a bHLH transcription factor that mediates transcription by forming homo- or heterodimers with other bHLH transcription factors. Tcf12 is highly expressed in neural progenitor cells during neural development21 and in cells of the oligodendrocyte lineage22.

We found that mutations generating truncated TCF12 lacking the bHLH DNA-binding domain abrogate the transcriptional activity of TCF12. In addition, single residue substitutions such as R602M within the bHLH domain also dramatically reduce TCF12 transcriptional ability. Finally, we found that the loss of TCF12 transcriptional activity was associated with a more aggressive tumour phenotype. Although speculative, our expression data provides evidence that the effects of TCF12 mutation on AO development may be mediated in part through E-cadherin related pathway. Indeed, this was one of the pathways down-regulated in mutated tumours and intriguingly CDH1 has been implicated in metastatic behaviour in a number of cancers18,23. It is likely that some TCF12 mutations may have subtle effects on bHLH function or act through independent pathways. Irrespective of the downstream effects of TCF12 mutation on glioma, our data are compatible with TCF12 having haploinsufcient tumour suppressor function. TCF12 haploinsufciency has previously been reported in patients with coronal craniosynostosis and in their unaffected relatives10. Strikingly, 3 of the 11 mutations we identied in AO, which concern residues M260, E548 and R602, cause coronal craniosynostosis10,24. Although speculative, collectively these data raise the possibility that carriers of germline TCF12 mutations may be at an increased risk of developing AO.

To our knowledge, this study represents the largest sequencing study of AO conducted to date. However, given the number of

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8207 ARTICLE

1

1

1

1

1

1

109

160

329

523

540

570

599

655

706

706

706

AD1

AD2

AD2

TCF12 WT

TCF12 M260fs

TCF12 R326S

TCF12 E548fs

TCF12 R602M

TCF12 S682fs AD1

AD1

Rep

Rep

bHLH

S682fs*14

265

AD1

M260fs*5

AD1

bHLH

R326S

561

AD2

AD2

AD2

AD1

E548fs*13

Rep

bHLH

R602M

696

Rep

bHLH

Relative luminescence

300

200

100

0 Eb

TCF12 WT + Eb

TCF12 M260fs + Eb

TCF12 R326s + Eb

TCF12 E548fs + Eb

TCF12 R602M + Eb

TCF12 S682fs + Eb

R602M

WT

**

***

***

Figure 3 | TCF12 mutations altering the bHLH domain result in impaired transactivation. (a) Schematic view of the wild-type and mutant TCF12 proteins for which the transactivation capacity has been assessed. Upper panel: wild-type human TCF12, functional domains in greyactivation domain 1 (AD1), activation domain 2 (AD2), repressor domain (Rep) and bHLH domain (bHLH). Lower panel: resulting truncated proteins. Black boxes indicate non-related amino-acid sequences resulting from frameshift mutations (fs), and truncated proteins size is in italic. (b) Schematic structure of the bHLH domain of TCF12 (blue) bound to DNA (grey). WT R602 (yellow) and mutant M602 (purple) residues are indicated. (c) E-box-luciferase reporter plasmid (Eb) was transfected alone or in combination with TCF12 wild-type or mutant expression plasmids. Both frameshift mutants that lack the bHLH DNA binding domain completely abolish TCF12 transcriptional activity. All samples were run in triplicate in four independent experiments. Data were normalized to control renilla luciferase. Values are means.d. ***P 0.0002, **P 0.0018 (Students t-test).

TCF12 WT TCF12 E548fs TCF12 R602M

TCF12 M260fs;LOH

TCF12 S682fs

TCF12 R326S

Figure 4 | TCF12 is highly expressed in a subset of anaplastic oligodendroglioma. Representative TCF12 immunostainings are shown: (a) wild-type TCF12 tumours show nuclear staining in a heterogeneous cell population. (be) Mutant TCF12 tumours show strong nuclear and cytoplasmic staining. (f) Mutant M260fs (resulting in a truncated protein) is associated with 15q21.3 LOH and shows no staining. Scale bar, 50 mm.

tumour-normal pairs we have analysed and the mutational frequency in AO, we were only well powered to identify genes that have a high-frequency mutations (that is, 410%). Hence

further insights into the biology of AO should be forthcoming through additional sequencing initiatives and meta-analyses of these data.

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Palisading necrosis Mitotic index

80

** *

25

*

% Of tumors

TCF12 WT (65)

Nb of mitotic figures/HPF

60

20

15

40

10

20

5

0

0

TCF12m (8)

altbHLH TCF12m (6)

TCF12 WT (62)

TCF12m (7)

altbHLH TCF12m (6)

Figure 5 | TCF12 mutation correlates with a higher necrotic and mitotic index. (a) Percentage of palisading necrosis in tumours with wild-type TCF12, all tumours mutated for TCF12 or only altered bHLH TCF12 mutants; *P 0.02, **P 0.004. (b) Mitotic index in TCF12 wild-type, TCF12-

mutated and altered bHLH TCF12 mutants; *P 0.039, means.e.m. CN,

copy number; LOH, loss of heterozygosity; HPF, high-power eld. The number of samples is indicated in parenthesis.

Methods

Patient samples and consent. Samples were obtained with informed and written consent and the study was approved by Comit de Protection des Personnes Ile de France-VI (October 2008) of respective hospitals participating in the Prise en charge des oligodendrogliomes anaplasiques (POLA) network. All patients were aged 18 years or older at diagnosis, and tumour histology was centrally reviewed and validated according to World Health Organization (WHO) guidelines25. Exome sequencing was conducted on samples from 51 AO patients (33 male; median age 49 years at diagnosis, range 2781). For targeted follow-up analyses, we studied the tumours from an additional 83 AO patients and 75 patients with grade II tumours. A summary of each of the tumour cohorts and respective pathological information on the patients is provided in Supplementary Table 1.

DNA and RNA extraction. Germline DNA was extracted from EDTA-venous blood samples using QIAquick PCR Purication Kits (Qiagen Ltd). Tumour DNA was extracted from snap-frozen tumour samples using the iPrep ChargeSwitchH Forensic Kit, according to manufacturers recommendations. DNAs were quantied and qualied using a NanoVue Plus spectrophotometer (GE Healthcare Life Sciences) and gel electrophoresis. RNA was extracted from tumours lysed by Lysing Matrix D tube and FastPrep instrument (MP Biomedicals) using the iPrep Trizol Plus RNA Kit (Life Technologies). Stringent criteria for RNA quality were applied to rule out degradation, specically a 28S/18S ratio 41.8.

SNP array analysis. In total, 115 samples from tumours were genotyped using Illumina SNP microarrays: 32 samples with Illumina 370-Duo 1.0 BeadChips,31 with Human610-Quad, 46 with HumanOmniexpress-12V1 and 6 with HumanCore-12v1. Raw uorescent signals were imported into BeadStudio software (Illumina) and normalized to obtain log R ratio and B-allele frequency (BAF) values. The tQN normalization procedure was then applied to correct for asymmetry in BAF signals due to bias between the two dyes used in Illumina assays. Genomic proles were divided into homogeneous segments by applying the circular binary segmentation algorithm to both log R ratio and BAF values. We then used the Genome Alteration Print method to determine the ploidy of each sample, the level of contamination with normal cells and the allele-specic copy number of each segment. Chromosome aberrations were dened using empirically determined thresholds as follows: gain, copy number Zploidy 1; loss, copy

number rploidy 1; high-level amplication, copy number 4ploidy 2;

homozygous deletion, copy number 0. Finally, we considered a segment to have

undergone LOH when the copy number of the minor allele was equal to 0. Lists of homozygous deletions and focal amplications, dened by at least ve consecutive probes, were generated and veried manually to remove doubtful events. Signicantly recurrent copy number changes were identied using the GISTIC2.0 algorithm26.

TERT promoter mutation sequencing. Characterized mutations in the TERT promoter, C228T and C250T variants with G4A nucleotide substitutions at genomic positions 1,295,228 bp and 1,295,250 bp (hg19), respectively, were obtained by Sanger sequencing. Primer sequences were: TERT-F50-GGCCGA

TTCGACCTCTCT-30 and TERT-R 50-AGCACCTCGCGGTAGTGG-30.

Whole-exome sequencing. DNA was quantied using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). Libraries were generated robotically using the SureSelectXT Automated Human All Exon Target Enrichment for Illumina

Paired-End Multiplexed Sequencing (Agilent) as per the manufacturers recommendations. Libraries were quantied using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (D-Mark). Average size of the fragment was determined using a LaChip GX (PerkinElmer) instrument. Sequencing was performed by pooling four libraries per lane at a 9-pM dilution on an Illumina HiSeq 2,000 instrument for 2 100 cycles using the recommended manufacturers conditions. PhiX control

was added at 1% on each lane. BCL2FASTQ (Illumina) was used to convert bcl les to fastqs (v 1.8.4). Coverage statistics are summarized in Supplementary Fig. 1. Paired-end fastq les were extracted using Illumina CASAVA software (v.1.8.1, Illumina) and aligned to build 37 (hg19) of the human reference genome using Stampy and BurrowsWheeler Aligner27, and PCR duplicates were removed with PicardTools 1.5. We assessed coverage of consensus coding sequence bases using Genome Analysis Toolkit28 v2.4-9. Somatic single-nucleotide variants were called using MuTect29 and the Genome Analysis Toolkit v2.4-9, and indels using IndelGenotyper. We excluded potential Covaris-induced mutations as per Costello et al.30 using in-house scripts. Conrmation of selected single-nucleotide variants including TCF12, CIC, FUBP1, SYNE1, FAT1, SETD2, RBPJ, NOTCH1, IDH1 and IDH2 was performed by Sanger sequencing implemented on ABI 3,300 l

platforms (Applied Biosystems, Foster City, USA). Primer sequences are detailed in Supplementary Data 5. In all cases, Sanger sequencing was 100% concordant with next-generation sequencing.

We used MutSigCV8 version 1.4 to identify genes harbouring more non-synonymous mutations than expected by chance, given gene size, sequence context and the mutation rate. We used as genomic covariates the mean expression level of each gene in our AO expression data set, the DNA replication time and the HiC statistic of chromatin state available in MutSig reference les. To increase our ability to identify cancer drivers and delineate associated oncogenic pathwaysfor AO, we incorporated mutation data from multiple tumour types using Oncodrive-fm14 implemented within the IntOGen-mutations platform15.

Transcriptome sequencing. Extracted RNA was cleaned using the RNeasy MinElute Cleanup Kit (Qiagen) and the RNA integrity assessed using an Agilent 2,100 Bioanalyzer and quantied using a Nanodrop 1,000. Libraries for stranded total RNA-seq were prepared using the Illumina Stranded Total RNA protocol (RS-122-2301). Libraries were assessed by the Agilent 2,100 Bioanalyzer. Sequencing was performed by pooling four libraries per lane at a 9-pM dilution on an Illumina HiSeq 2,000 instrument for 2 100 cycles using the recommended

manufacturers conditions. PhiX control was added at 1% on each lane. BCL2FASTQ was used to convert bcl les to fastqs (v 1.8.4). Paired-end reads from RNA-seq were aligned to the following database les using BurrowsWheeler Aligner 0.5.5: (i) the human GRCh37-lite reference sequence, (ii) RefSeq, (iii) a sequence le representing all possible combinations of non-sequential pairs in RefSeq exons and (iv) the AceView database at le downloaded from UCSC, representing transcripts constructed from human expressed sequence tag (ESTs). The mapping results from databases (ii)-(iv) were aligned to human reference genome coordinates. The nal BAM le was constructed by selecting the best alignment. To identify fusion transcripts, we analysed RNA-seq data using Chimerascan software31 (version 0.4.5). As advocated, algorithmic output was analysed for high-condence fusion transcripts imposing lters: (i) spanning reads 42 (ii)

total supported reads Z10 (ref. 32). In absence of corresponding paired normal tissue samples, we made use of data from the human body map project data to identify fusions seen in normal tissue.

TCF12 sequencing in the validation series. PCR amplication of 21 amplicons covering each exon of TCF12 on DNA extracted from fresh-frozen tumours were performed using Fluidigm technology according to the manufacturers recommendations. The 21 PCR products from one tumour sample were then equimolarly pooled and submitted to the MiSeq (Illumina) sequencing as per the manufacturers protocol. All mutations were validated by Sanger sequencing. Somatic mutations were conrmed using paired constitutional DNA.

mRNA expression proling. Gene expression proles of 71 samples were analysed using Affymetrix Human Genome U133 Plus 2.0 arrays. All samples were normalized in batches using the RMA algorithm (Bioconductor affy package), and probe set intensities were then averaged per gene symbol.

Identication of signicantly mutated pathways. Gene set member lists were retrieved online from MSigDB33, GO34 and SMD35 databases. We searched for gene sets harbouring more damaging mutations than expected by chance. Given the set G of all the genes sequenced with sufcient coverage, the set S of tumour samples (of size n) and any gene set P, we calculated the probability of observing a number of mutations equal or greater to that observed in P across the n samples according to a binomial law B(k, p), with k n L(P) and the mutation rate

p A(G, S)/(n L(G)), where L(X) is the sum of the lengths (in bp) of all genes/

exons from a gene set X, and A(G, S) is the total number of mutations observed in all the targeted sequences across all the samples from S.

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Deregulated gene sets in TCF12 mutant samples. We performed a moderate t-test using LIMMA R package to identify signicantly differentially expressed genes between TCF12 mutant samples and TCF12 wild-type samples (Po0.05 and absolute log fold change 40.6). Biological pathways and gene set member lists were retrieved online from MSigDB33, GO34 and SMD35 databases. Enrichment P values were computed from a hypergeometric test between those gene sets and the initial list of differentially expressed genes. To visualize gene set activity, for each gene set dened as target genes of either CDH1, TCF21, BMI1, EZH2 and found to be signicantly deregulated in TCF12 bHLH-altered samples compared with TCF12 wild-type samples in O3 samples with co-deletion, we retrieved the complete member list from MSigDB33 and computed a global mean gene expression value in each sample. We then ranked the samples according to the later global mean expression value for each of these gene sets.

Structure modelling. The Swiss Model36 server was used to model mutated TCF12 and VMD software37 used to align the structures of wild-type and mutated TCF12 proteins with STAMP (STructural Alignment of Multiple Proteins)38. Prediction of the functional effect of the R602M mutation on TCF12 was made using Project HOPE39.

Statistical analysis. Statistical analysis was carried out using R3.0.1 software.

A P value r0.05 was considered to be signicant. Continuous variables were analysed using the Students t-test or MannWhitney test. Categorical data were compared using Fishers exact test or the w2-test. Overall survival of patients was the end point of the analysis. Survival time was calculated from the date of tumour diagnosis to the date of death. Patients who were not deceased were censored at the date of last contact. Mean follow-up time was computed among censored observations only. KaplanMeier survival curves according to genotype were generated and the homogeneity of the survival curves between genotypes was evaluated using the log-rank test. Power to demonstrate a relationship between mutation status and overall survival was estimated using sample size formulae for comparative binomial trials40.

Cell culture. Human embryonic kidney HEK293T cell line (American Type Culture Collection) was maintained in a 5% CO2-regulated incubator in DMEM

Glutamax (Life Technologies), completed with 10% fetal bovine serum and penicillin/streptomycin (Life Technologies).

Plasmid construction. To construct the TCF12 wild-type plasmid, we cloned, by Gateway recombination (Life Technologies), a pENTR221 TCF12 Ultimate ORF Clone (Life Technologies) into a pDEST12 lentiviral vector (kind gift fromP. Ravassard), under the control of hCMV promoter. The M260fs*5 and R326S mutations were generated by PCR mutagenesis using the Q5 Site-directed Mutagenesis kit (New England Biolabs) on pENTR221 TCF12 plasmid (primer sequences are detailed in Supplementary Data 5) and then cloned into the pDEST12 vector by LR Gateway cloning. Synthetic NdeI/MfeI fragments (encompassing sequences from exon 16 to the TAG stop codon of the ENST00000438423 isoform), containing the mutations E548fs*13, R602M and S683fs*14, were obtained from GeneCust, then substituted into pENTR221and nally cloned by Gateway recombination into the pDEST12 plasmid. All expression plasmids were sequenced before use.

Luciferase expression assays. For each experiment, 105 exponentially growing HEK293T cells were seeded in 12-well plates and transfected 24 h later using Fugene6 (Promega), according to manufacturers instructions, with 0.3 mg of a reporter plasmid encoding rey luciferase under the control of an E-box-responsive element (Eb, kind gift from A. Lasorella), or 0.3 mg of Eb plasmid and0.7 mg of a TCF12 wild-type expression plasmid, or 0.3 mg of Eb plasmid and 0.7 mg of either TCF12 mutant (M260fs*5, R326S, E548fs*13, R602M or S628fs*14) expression plasmid. For all points, data were normalized by adding 30 ng of renilla luciferase expression plasmid (pGL4.73, Promega, gift from F. Toledo). Cells were harvested 24 h after transfection, and luminescence was monitored using the Dual-Glo Luciferase assay system (Promega), according to the manufacturers instructions, on a Spectramax M4 instrument and SoftMax Pro 6.2.2 software. All samples were run in triplicate, in four independent experiments.

Immunohistochemistry. Parafn-embedded tumour sections were deparafnized using standard protocols. Heat-mediated antigen retrieval was achieved by boiling sections in a pressure cooker with Citrate buffer at pH 6. Sections were blocked in 10% goat serum in PBS 0.5% Triton X-100 for 30 min prior to incubation with

an anti-TCF12 antibody (Proteintech Cat no.: 14419-1-AP) and then revealed using the Polink-2 HRP Plus Rabbit DAB Detection System (GBI Labs:D39-6). Photographs were taken at 400 magnication and processed using AxioVision

software (Zeiss). The mitotic index in tumours was recorded as the number of mitotic gures in 10 high-power elds.

TCGA data. To complement our analysis, we made use of exome sequencing data on AO tumours generated by the TCGA (Supplementary Data 2).

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Acknowledgements

This work is part of the national program Cartes dIdentit des Tumeurs (CIT) (http://cit.ligue-cancer.net

Web End =http://cit.ligue-cancer.net), Prise en charge des oligodendrogiomes anaplasiques (POLA) Network, POLA Tumor Bank, OncoNeuroTek tumorothque du systme nerveux central ICM APHP and the Institut National du Cancer (INCa) (http://www.e-cancer.fr

Web End =http://www.e-cancer.fr). Research in Huillard and Sanson labs has received funding from the program Investissements davenir ANR-10-IAIHU-06. Grant support from Gnome Qubec,le Ministre de lEnseignement suprieur, de la Recherche, de la Science et de la Technologie (MESRST) Qubec and McGill University is also acknowledged. At The Institute of Cancer Research, work was primarily supported by Cancer Research UK (C1298/A8362 Bobby Moore Fund for Cancer Research UK). D.C. is supported by Leukaemia Lymphoma Research. C.P.W. is funded by Myeloma UK. We are indebted toA. Lasorella and A. Iavarone for helpful discussion, technical advices and for providing the E-box-responsive reporter plasmid. We thank P. Ravassard, S Rozenberg andV. Lejour for discussion and technical advice, and A. Nadaradjane for the TCF12 structure modelling. I.S. is supported by a fellowship from the Ligue Nationale Contre

le cancer. V.G. is supported by a fellowship from the Fondation ARC pour la Recherche sur le Cancer. Research in Huillard lab is supported by the Ligue Nationale Contre le Cancer, Fondation ARC pour la Recherche sur le Cancer, Institut National de la Sant et de la Recherche Mdicale (INSERM) and European Union (FP7-PEOPLE-CIG-2012). Research in Sanson lab has been supported by grants from the Ligue Nationale Contre le Cancer, Fondation ARC pour la Recherche sur le Cancer and the Institut National du Cancer. The results published here are in whole or part based upon data generated by The Cancer Genome Atlas (TCGA) pilot project established by the NCI and NHGRI. Information about TCGA and the investigators and institutions that constitute the TCGA research network can be found at http://cancergenome.nih.gov/

Web End =http://cancergenome.nih.gov/ .

Author contributions

M.S., A.I., J.-Y.D., G.M.L., E.H. and R.S.H conceived the study. R.S.H., K.L., I.S., A.K., E.H. and M.S. wrote the manuscript. K.L., A.K., D.C., E.L. and A.d.R. designed and reviewed statistical and bioinformatic analyses. I.S., V.G., D.Z., T.P., Y.R., O.S. and S.H. performed experiments. K.L., D.C., S.E.D., C.W., M.F., A.K. and E.L. performed bioinformatic analyses. D.F.-B., F.D. and C.D. performed sample preparation. N.E. reviewed samples annotations and performed data management. All authors reviewed and contributed to the manuscript.

Additional information

Accession codes: All whole-exome sequencing and transcriptome data have been deposited at the European Genome-phenome Archive (EGA), which is hosted by the European Bioinformatics Institute (EBI), under the accession code EGAS00001001209. mRNA expression and SNP data can be accessed through ArrayExpress under accession numbers E-MTAB-2768 for mRNA expression data, and E-MTAB-3457, E-MTAB-3458, E-MTAB-2772 and E-MTAB-2771 for SNP data.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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POLA NetworkClovis Adam16, Marie Andraud17, Marie-Hlne Aubriot-Lorton18, Luc Bauchet19, Patrick Beauchesne20, Claire Blechet21, Mario Campone22, Antoine Carpentier23, Catherine Carpentier24, Ioana Carpiuc25, Marie-Pierre Chenard26, Danchristian Chiforeanu27, Olivier Chinot28, Elisabeth Cohen-Moyal29, Philippe Colin30, Phong Dam-Hieu31, Christine Desenclos32, Nicolas Desse33, Frederic Dhermain34, Marie-Danile Diebold35, Sandrine Eimer36, Thierry Faillot37, Mlanie Fesneau38, Denys Fontaine39, Stphane Gaillard40, Guillaume Gauchotte41, Claude Gaultier42, Francois Ghiringhelli43, Joel Godard44, Edouard Marcel Gueye45, Jean Sebastien Guillamo46, Selma Hamdi-Elouadhani47, Jerome Honnorat48, Jean Louis Kemeny49, Touk Khallil50, Anne Jouvet51, Francois Labrousse52, Olivier Langlois53, Annie Laquerriere54, Emmanuelle Lechapt-Zalcman55, Caroline Le Gurinel56, Pierre-Marie Levillain57, Hugues Loiseau58, Delphine Loussouarn59, Claude-Alain Maurage60, Philippe Menei61, Marie Janette Motsuo Fotso62, Georges Noel63, Fabrice Parker64, Michel Peoch65, Marc Polivka66, Isabelle Quintin-Rou67, Carole Ramirez68, Damien Ricard69, Pomone Richard70, Valrie Rigau71, Audrey Rousseau72, Gwenaelle Runavot73, Henri Sevestre74, Marie Christine Tortel75, Emmanuelle Uro-Coste76, Fanny Burel-Vandenbos77, Elodie Vauleon78, Gabriel Viennet79, Chiara Villa80, Michel Wager57

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16Hpital Bictre, Pathology Department, 94275 Le Kremlin-Bictre, France. 17CHU Saint-Pierre de la Runion, Pathology Department, Saint-Pierre de la Runion, 97410 France. 18CHU Dijon, Pathology Department, 21000 Dijon, France. 19CHU de Montpellier, Neurosurgery Department, 34295 Montpellier, France. 20CHU Nancy, Neuro-oncology Department, 54035 Nancy, France. 21CHR Orlans, Pathology Department, 45000 Orlans, France. 22Centre Ren Gauducheau, Medical Oncology Department, 44805 Saint-Herblain, France. 23Hpital Avicenne, Neurology Department, 93009 Bobigny, France.

24Universite Pierre et Marie Curie, Centre de Recherche de linstitut du Cerveau et de la Moelle Epiniere and INSERM UMRS 975/CNR, 75013 Paris, France.

25Clinique des Cdres, Medical Oncology Department, 31700 Cornebarrieu, France. 26CHU Strasbourg, Pathology Department, 67098 Strasbourg, France.

27CHU Rennes, Pathology Department, 35033 Rennes, France. 28Hpital de la Timone, Assistance PubliqueHpitaux de Marseille, Neuro-oncology Department, 13385 Marseille, France. 29Institut Claudius Regaud, Radiotherapy Department, 31059 Toulouse, France. 30Clinique de Courlancy, Radiotherapy Department, 51100 Reims, France. 31Hpital de la cavale blanche, CHU Brest, Neurosurgery Department, 29609 Brest, France. 32Hpital Nord, CHU Amiens, Neurosurgery Department, 80054 Amiens, France. 33HIA Sainte-Anne, Neurosurgery Department, 83800 Toulon, France. 34Institut Gustave Roussy, Radiotherapy Department, 94805 Villejuif, France. 35CHU Reims, Pathology Department, 51092 Reims, France. 36CHU de Bordeaux-GH Pellegrin, Pathology Department, 33000 Bordeaux, France. 37Hpital Beaujon, Neurosurgery Department, 92110 Clichy, France. 38CHR Orlans, Radiotherapy Department, 45000 Orlans, France. 39CHU Nice, Neurosurgery Department, 06002 Nice, France. 40Hpital Foch, Neurosurgery Department, 92151 Suresnes, France.

41CHU Nancy, Pathology Department, 54035 Nancy, France. 42CH Colmar, Neurology Department, 68024 Colmar, France. 43Centre Georges-Franois Leclerc, Medical Oncology, 21079 Dijon, France. 44Hpital Jean Minjoz, CHU Besanon, Neurosurgery Department, 25030 Besanon, France. 45Hpital Dupuytren, CHU de Limoges, Neurosurgery Department, 87042 Limoges, France. 46CHU de Caen, Neurology Department, 14033 Caen, France. 47Hpital Lariboisire, Neurosurgery Department, 75475 Paris, France. 48Hospices Civils de Lyon, Hpital Neurologique, Neuro-oncology Department, 69677 Bron, France. 49CHU Clermont-Ferrand, Pathology Department, 63003 Clermont-Ferrand, France. 50CHU Clermont-Ferrand, Neurosurgery Department, 63003 Clermont-Ferrand, France. 51Hospices Civils de Lyon, Hpital Neurologique, Pathology and Neuropathology Department, 69677 Bron, France. 52Hpital Dupuytren, CHU de Limoges, Pathology Department, 87042 Limoges, France. 53CHU Charles Nicolle, Neurosurgery Department, 76000 Rouen, France.

54CHU Charles Nicolle, Pathology Department, 76031 Rouen, France. 55CHU de Caen, Pathology Department, Caen, 14033 France. 56Hpital Henri Mondor, Neurosurgery Department, 94010 Henri Mondor, France. 57CHU Poitiers, Neurosurgery Department, 86000 Poitiers, France. 58CHU de Bordeaux-GH Pellegrin, Neurosurgery Department, 33000 Bordeaux, France. 59CHU Nantes, Pathology Department, 44093 Nantes, France. 60CHU de Lille, Pathology Department, 59037 Lille, France. 61CHU Angers, Neurosurgery Department, 49933 Angers, France. 62Hpital Nord, CHU Saint-tienne, Neurosurgery Department, 42270 Saint-Priest en Jarez, France. 63Centre Paul Strauss, Radiotherapy Department, 67065 Strasbourg, France. 64Hpital Bictre, Neurosurgery Department, 94275 Le Kremlin-Bictre, France. 65Hpital Nord, CHU Saint-tienne, Pathology Department, 42270 Saint-Priest en Jarez, France. 66Hpital Lariboisire, Pathology Department, 75475 Paris, France. 67Hpital de la cavale blanche, CHU Brest, Pathology Department, 29609 Brest, France. 68CHU de Lille, Neurosurgery Department, Lille, 59037 France. 69HIA du Val de Grce, Neurology Department, 75230 Paris, France. 70Laboratoire les Feuillants, Pathology Department, 31023 Toulouse, France. 71CHU de Montpellier, Pathology Department, 34295 Montpellier, France. 72CHU Angers, Pathology Department, 49933 Angers, France. 73CHU Saint-Pierre de la Runion, Neurology Department, 97410 Saint-Pierre de la Runion, France. 74Hpital Nord, CHU Amiens, Pathology Department, 80054 Amiens, France. 75CH Colmar, Pathology Department, 68024 Colmar, France. 76Hpital Rangueil, CHU Toulouse, Pathology Department, 31059 Toulouse, France. 77CHU Nice, Pathology Department, 06002 Nice, France. 78Centre Eugne Marquis, Medical Oncology, 35042 Rennes, France. 79Hpital Jean Minjoz, CHU Besanon, Pathology Department, 25030 Besanon, France. 80Hpital Foch, Pathology Department, 92151 Suresnes, France.

NATURE COMMUNICATIONS | 6:7207 | DOI: 10.1038/ncomms8207 | http://www.nature.com/naturecommunications

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