transcriptomic definition of molecular subgroups of small

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Transcriptomic definition of molecular subgroups ofsmall round cell sarcomas

Sarah Watson, Virginie Perrin, Delphine Guillemot, Stéphanie Reynaud,Jean-Michel Coindre, Marie Karanian, Jean-Marc Guinebretière, Paul

Fréneaux, Francois Le Loarer, Megane Bouvet, et al.

To cite this version:Sarah Watson, Virginie Perrin, Delphine Guillemot, Stéphanie Reynaud, Jean-Michel Coindre, etal.. Transcriptomic definition of molecular subgroups of small round cell sarcomas. The Journalof pathology and bacteriology, John Wiley & Sons, 2018, 245 (1), pp.29-40. �10.1002/path.5053�.�inserm-02440510�

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Watson et al. 1

Title: Transcriptomic definition of molecular subgroups of small round cell sarcomas Running Title: Molecular classification of sarcoma subtypes Authors Sarah Watson1,2, Virginie Perrin1,2, Delphine Guillemot3, Stephanie Reynaud3, Jean-Michel Coindre4,5, Marie Karanian6, Jean-Marc Guinebretière7, Paul Freneaux8, François Le Loarer4,5, Megane Bouvet3, Louise Galmiche-Rolland9,10, Frédérique Larousserie11, Elisabeth Longchampt12, Dominique Ranchere-Vince6, Gaelle Pierron3*, Olivier Delattre1,2,3,13*, Franck Tirode1,2,14* *Co-senior authors Affiliations 1 INSERM U830, Laboratory of Genetics and Biology of Cancer, F-75005, Paris, France 2 Institut Curie, Paris Sciences et Lettres, F-75005, Paris, France 3 Institut Curie, Unité de génétique somatique, F-75005, Paris, France 4 Institut Bergonié, Department of Pathology, F-33000, Bordeaux, France. 5 Université Bordeaux 2, F-33000, Bordeaux, France. 6 Centre Leon Bérard, Department of Pathology, F-69008, Lyon, France. 7 Service de Pathologie, Hôpital René-Huguenin, Institut Curie, F-92210, Saint-Cloud, France. 8 Département de Biologie des Tumeurs, Institut Curie, Service d'anatomie pathologique, F-75005, Paris, France 9 Service d’Anatomie Pathologique, Hôpital Necker Enfants malades, F-75015, Paris, France 10 Université Paris Descartes, F-75006, Paris, France 11 Service d'Anatomie Pathologique, Hôpital Cochin, F-75014, Paris, France 12 Service d'Anatomie et de Cytologie Pathologiques, Hôpital Foch, F-92151, Suresnes, France 13 Ligue Contre le Cancer, Equipe labellisée 14 Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Cancer Research Center of Lyon, Centre Léon Bérard, F-69008, Lyon, France Corresponding authors: Franck Tirode, Ph.D. Biology of Rare Sarcomas Group Cancer Research Center of Lyon, INSERM U1052 Centre Léon Bérard, 28 Rue Laënnec, 69373 Lyon Cedex 08 Tel.: +33 4 69 85 61 45 Email: [email protected] Olivier Delattre, M.D., Ph.D. INSERM U830 “Genetics and Biology of Cancers” Institut Curie Research Centre 26 rue d’Ulm, 75248 Paris cedex 05, France Tel.: +33 1 56 24 66 79 Fax.: +33 1 56 24 66 30 Email: [email protected] Conflict of Interest statement:

Authors have nothing to disclose

Watson et al. 2

Word count: 3929

Abstract

Sarcoma represents a highly heterogeneous group of tumours. We report here the first

unbiased and systematic search for gene fusions combined with unsupervised expression

analysis of a series of 184 small round cell sarcomas. Fusion genes were detected in 59%

percent of samples, with half of them being observed recurrently. We identified biologically

homogeneous groups of tumours such as the CIC-fused (to DUX4, FOXO4 or NUTM1) and

BCOR-rearranged (BCOR-CCNB3, BCOR-MAML3, ZC3H7B-BCOR and BCOR internal

duplication) tumour groups. VGLL2-fused tumours represented a more biologically and

pathologically heterogeneous group. This study also refined the characteristics of some

entities such as EWSR1-PATZ1 spindle cell sarcoma or FUS-NFATC2 bone tumours that

are different from EWSR1-NFATC2 tumours and transcriptionally resemble CIC-fused

tumour entities. We also describe a completely novel group of epithelioid and spindle-cell

rhabdomyosarcomas characterized by EWSR1- or FUS-TFCP2 fusions. Finally, expression

data identified some potentially new therapeutic targets or pathways.

Keywords

Sarcoma, Fusion genes, FET-TFCP2, FUS-NFATC2, EWSR1-PATZ1, VGLL2-NCOA2,

BCOR-Rearranged, CIC-Fused, RNAseq.

Introduction

Small round cell sarcoma is a heterogeneous group of tumours mostly affecting children and

young adults and characterized by an overall poor prognosis [1]. They remain challenging for

Watson et al. 3

pathologists since those tumours share overlapping morphological and immunophenotypical

features. The identification of specific fusion transcripts has considerably improved their

diagnosis as many subtypes are characterized by a specific fusion gene, such as

EWSR1/FUS-ETS, SS18-SSX and PAX-FOXO1 fusions in Ewing sarcoma [2], synovial

sarcoma [3] and aRMS [4], respectively, or the more recently described CIC-DUX4 [5],

EWSR1-NFATc2 [6] and BCOR-CCNB3 [7] in “Ewing-like” tumours. As a result, diagnostic

evaluation of specific chromosome translocations by FISH or of the resulting fusion

transcripts by RT-PCR has become an asset for the molecular assessment of small round

cell sarcoma. However, a number of sarcomas remains uncharacterised, raising both

diagnostic and therapeutic issues.

In the present study, we report RNA-sequencing of 184 small round cell sarcoma samples

from three different cohorts. By applying several fusion algorithms and performing

unsupervised clustering analysis, we were able to show that recurrent fusions define

homogeneous groups of tumours based on pathological features and expression profile

analyses, including FUS-NFATC2-positive, EWSR1-PATZ1-positive, CIC-fused or BCOR-

rearranged tumours. We also identified a new epithelioid rhabdomyosarcoma group

characterized by a EWSR1- or FUS-TFCP2 fusion gene. Finally, we propose genes and

pathways specifically expressed in a variety of different entities that may serve as biomarkers

or potential therapeutic targets.

Materials and Methods

Tumour samples. Tumour samples were chosen based on their pathological diagnosis either

as unclassified sarcoma or as known sarcoma that did not carry pathognomonic genetic

aberrations or suspicious for sarcoma with simple genetic or monomorphic features

Watson et al. 4

(supplementary material, Figure S1). Samples were used in accordance with the French

Biobank legislation.

Paired-end RNA sequencing. Total RNAs were isolated from crushed frozen tumours using a

Trizol reagent kit (Life technologies). Library constructions were performed following the

TruSeq Stranded mRNA LS protocol (Illumina). Sequencing were performed on either HiSeq

2500 (100nt paired-end) or NextSeq 500 (150nt paired-end) Illumina sequencing machines.

All fastq files were deposited on the European Genome-phenome Archive

(https://www.ebi.ac.uk/ega/studies/EGAS00001002189). Raw sequencing files of

SMARCA4-DTS and MRT control samples were previously deposited in the Sequence Read

Archive (#SRP052896).

Bioinformatics analyses. For fusion gene discovery, sequencing reads were injected into

both the deFuse tools [8] and the FusionMap tool [9] with hg19 genome as reference. Only

fusion transcripts supported by both tools with at least 2 split reads (and 3 spanning pairs for

defuse) were considered. Gene expression values were extracted by Kallisto v0.42.5 [10]

with GRCh38 release 79 genome annotation. Clustering, BGA, PCA and t-SNE were

computed using R packages Cluster v2.0.3, made4 version 1.44.0, FactoMiner v1.31.4 and

Rtsne version 0.10, respectively. Gene ontology analyses were performed online

(https://david.ncifcrf.gov/) with DAVID v6.7 tool [11].

Fusion validation: PCR amplification of the fusion point using specifically designed primers

and 50ng of cDNA were then performed with AmpliTaq Gold® DNA Polymerase with Buffer II

(Thermo Fisher Scientific, Waltham, MA USA 02451) prior to Sanger sequencing on an

Applied 3500XL Genetic Analyzer.

https://www.ebi.ac.uk/ega/studies/EGAS00001002189

Watson et al. 5

Immunohistochemistry. Immunohistochemistry was performed using the following antibodies

and methods: NUTM1 (C52B1, Cell Signaling, dilution 1:50) and ALK (5A4, Cell Signaling,

dilution 1:50) IHC were performed on a Ventana BenchMark platform with Cell Conditioning

Solution 1 pre-treatment. NUTM1 was incubated for 56min and ALK for 80min prior to be

revealed with the ultraView Universal DAB Detection Kit. ETV4 (clone 16, Santa Cruz

Biotechnology, dilution 1:15) IHC was performed overnight at 4°C, following a pre-heating at

98°C for 30 min in Trisbuffer pH9, and revealed with REAL EnVision kit (Dako, Glostrup,

Denmark).

Results

In molecular diagnostic routine, primitive small round or spindle cell sarcomas are

systematically tested for the most common fusion genes using multiplexed Q-PCR

(supplementary material, Table S1). In the present study, we performed whole transcriptome

sequencing to investigate its efficacy for proper tumour classification. Our investigation

cohort was composed of 94 samples randomly selected from about 700 retrospective

samples in which no common fusion gene could be identified by standard diagnostic

investigation (see the general scheme of the analysis in supplementary material, Figure S1).

We first investigated the presence of gene fusions and the retained fusion candidates were

subsequently validated and searched for within the remaining available samples (around 600

cases) using specific RT-PCR assay. The new cases thus identified were also RNA-

sequenced and generated our follow-up cohort (12 samples). Basic clinical data, initial

diagnosis and results of molecular analyses for all the 184 cases sequenced in this study are

summarized in the supplementary material, Table S2. We next performed expression profile

analyses that we compared using t-Distributed Stochastic Neighbour Embedding [12] and

unsupervised clustering analyses (Figure 1A and B, supplementary material, Figure S2) to a

Watson et al. 6

subset of 78 well defined cases composing our control cohort. For each group, differential

expression analyses against each of all other tumour types were performed. Top variant

genes are reported in the supplementary material, Table S3 together with ontology

enrichment and gene set enrichment analyses (GSEA).

A fusion gene could be identified in almost 3/5 of the investigation cohort samples

We identified fusion genes in 55 out of the 94 tumours of the investigation cohort. Except for

one case (SARC036) for which numerous fusions were found on chromosome 1, suggestive

of chromothripsis, the mean number of fusion events detected per sample was 1.8. Forty

samples carried a single fusion gene, while multiple (2 to 9) gene fusions were detected in 14

samples (supplementary material, Table S2). All fusion genes were subsequently confirmed

by RT-PCR and Sanger sequencing.

Most of the RNAseq fusion-positive samples (26/55) expressed previously described fusion

genes that were not included in our RT-PCR routine procedure, namely single cases of each

of the following fusions: EWSR1-PBX1, ACTB-GLI1, PAX3-MAML3, COL1A1-PDGFB,

VGLL2-CITED2, FUS-NFATc2, BCOR-MAML3, ZC3H7B-BCOR, TPR-NTRK1, BRD3-

NUTM1, KIAA1549-BRAF; two cases with EWSR1-PATZ1, TPM3-NTRK1, EML4-ALK or

NAB2-STAT6 fusions; and three cases with VGLL2-NCOA2 or CIC-NUTM1 fusions. We also

identified a variant of a SS18-SSX2 fusion with an atypical SS18 gene breakpoint. RT-PCR

screening of these fusions in the initial cohort led to the identification of eleven additional

cases: 3 EWSR1-PATZ1, 1 EML4-ALK, 2 FUS-NFATC2, 3 VGLL2-NCOA2 and 2 CIC-

NUTM1 (supplementary material, Table S2).

Novel fusions were detected in 29 samples (supplementary material, Table S2). Two cases

presented variants of known fusions: UXT-TFE3, a variant of the ASPSCR1- or SFPQ-TFE3

fusions found in alveolar soft part sarcomas and renal cell carcinoma; and IKBKG-ALK, a

Watson et al. 7

new variant among the numerous ALK fusions found in inflammatory myofibroblastic

tumours. In two cases, we identified a completely new fusion between EWSR1 or FUS and

TFCP2. Eleven samples harboured previously undescribed fusions, involving genes relevant

for tumorigenesis, but for which no additional sample could be identified during the RT-PCR

screen. These fusions were then considered as private to their respective tumour. Finally, in

the remaining 14 fusion-positive cases, we were unable to propose a definitive driver event

due to our lack of knowledge on the implication in cancer of the fused genes.

Emerging VGLL2-NCOA2/CITED, CIC-fused and BCOR-rearranged sarcoma subtypes

A total of 7 samples harbouring a VGLL2 fusion with either NCOA2 (n=6) or CITED (n=1)

were identified (supplementary material, Figure S3A) with two samples forming a

primary/relapse couple (SARC070_Primary and SARC070_Relapse). These fusion genes

characterized tumours occurring in very young children (below 5yo), as previously described

[13]. Centralized review of cases highlighted two subtypes with specific pathological aspects.

In three cases (SARC061, SARC065 and SARC070_primary), the tumours were composed

of a large amount of fibrous stroma with scarce tumour cells (Figure 2A). Tumour cells

presented neither atypia nor abnormal mitotic figures. They were negative for desmin,

whereas few cells (below 1%) showed a nuclear positivity for myogenin. Fewer than 25% of

cells were Ki67 positive. In contrast, in the four other cases (SARC070 at relapse, SARC085,

SARC088 and SARC102), the cellular mass was far denser with cells presenting numerous

atypia and mitoses. Myogenin staining was strongly positive in around 10% of cells and

desmin and Ki67 immuno-reactivity was displayed by over 30% of tumour cells (Figure 2B).

Consistently with this histological heterogeneity, t-SNE analysis and unsupervised clustering

revealed a relatively disparate group of tumours (Figure 1A and supplementary material,

Figure S2A) which samples could further be separated according to the histology, when

Watson et al. 8

different clustering conditions were applied (supplementary material, Figure S2C-D). When

considering these two histological subtypes separately it appeared that the “fibrous” subtype

was enriched in immune/inflammatory response genes, while the “dense” subtype was

enriched in cell cycle/proliferation genes (supplementary material, Table S3). Supervised

group comparisons and between group analyses (BGA) indicated that VGLL2-fused tumours

expressed numerous muscle-related genes as well as genes involved in extracellular matrix

and in the epithelial-mesenchymal transition process. Despite clear positivity for muscle

differentiation markers, none of the two subtypes clustered with rhabdomyosarcoma

samples.

A total of five CIC-NUTM1 (supplementary material, Figure S3B) fusions were retrieved. All

but one case were observed in young children, at various locations (supplementary material,

Table S2). Tumours harbouring CIC-NUTM1 fusion clustered tightly with the other CIC-

DUX4- or -FOXO4-positive samples (Figure 1). All these tumours overexpressed ETS family

members of the PEA3 type [5,14,15] as well as a variety of secreted and matrix protein

genes such as VGF, BMP2, glypican 3/4, NRG1, PTX3, pleiotrophin and spondins. GSEA

revealed enrichment in genes of extracellular matrix but also in genes overlapping

proliferation/response to drug/immune response categories (supplementary material, Table

S3).

The seven samples presenting a rearrangement of BCOR, including BCOR-MAML3 or

ZC3H7B-BCOR fusions [16] and internal tandem duplication (ITD) [17–19] (supplementary

material, Figure S3C), grouped together with the BCOR-CCNB3-positive samples from the

control cohort, describing a very-well defined cluster. These samples also shared some

pathological features, as previously described [17]. Among the most significantly enriched

gene ontologies for this BCOR-rearranged group were “developmental protein” and

Watson et al. 9

“homeobox” (supplementary material, Table S3) with a strong overexpression of HOX-A, -B,

-C and -D family genes as well as HMX1, PITX1, ALX4 or DLX1. More specifically, we

observed very strong gene sets and ontology enrichments for the morphogenesis,

development and differentiation of neurons and for the skeletal system development. Finally,

different genes encoding membrane receptors including RET, FGFR2/3, EGFR, PDGFRA,

NTRK3, KIT or NGFR were also highly and constantly overexpressed in these tumours and

may hence constitute actionable target genes.

Identification of a FUS-NFATC2 fusion gene in a new bone tumour entity

transcriptionally distinct from EWSR1-NFATC2-positive tumours

We identified FUS-NFATC2 fusion genes in three tumours of the femur of adult patients

(median age 38.3yo), like the only case described in the literature (17). Tumours displayed

areas composed of round tumour cells arranged in sheets or short fascicles. Tumour cells

were embedded in a variably myxoid stroma (Figure 3A). Focal hemangiopericytic vascular

network was present in all cases (Figure 3B). More distinctively, these tumours harboured

focal myxohyaline foci raising suspicion of cartilaginous differentiation (Figure 3C). All

tumours displayed brisk mitotic activity and necrosis. Altogether, these tumours were

histologically reminiscent of CIC-fused sarcomas. Clustering analyses indicated that FUS-

NFATC2-positive tumours grouped together and were clearly distinct from all other FET-

fused samples, including EWSR1-NFATC2-positive tumours (Figure 1). While the EWSR1-

NFATC2 tumours were strongly enriched in genes associated with inflammatory and immune

responses (supplementary material, Tables S3 and S4), the FUS-NFATC2 tumours were

enriched in proliferation and drug resistance signatures. In accordance with the potential

areas of cartilaginous differentiation, genes involved in the extracellular matrix of

cartilaginous tissues (like ACAN, COL9A2, MATN3, COMP or CILP2) or encoding secreted

Watson et al. 10

proteins (pleiotrophin, DKK3, ANGPTL2, SBSPON or WNT5B) were preferentially expressed

in FUS-NFATC2-positive tumours.

EWSR1-PATZ1 fusion gene is found in a new tumour group unrelated to any other

EWSR1-fused tumour.

While only single cases of tumour carrying an EWSR1-PATZ1 fusion have been previously

reported [21–23], we identified in our cohorts a total of 5 cases (Figure 4A and

supplementary material, Figure S3D). All tumours were from soft tissues and occurred

across a very broad age range (from 0.9 to 68.5yo). Centralized pathological review of three

cases indicated three different morphological aspects (Figure 4B): i) A first tumour was

composed of bundles of relatively monomorphic spindle cells with an eosinophilic cytoplasm

and enlarged hyperchromatic nuclei; ii) A second case consisted of a diffuse proliferation

with several vessels forming a “haemangioma-like” vasculature. Tumour cells were mostly

round, and focally spindle, with scant cytoplasm and an enlarged nucleus containing one

nucleolus; iii) The last case resided in a diffuse proliferation of spindle cells displaying an

eosinophilic cytoplasm, oval nuclei with an irregular chromatin distribution with one or several

nucleoli. Some nests of epithelioid cells with abundant, eosinophilic or clear, cytoplasm and

atypical nuclei could be seen. Nevertheless, two features were observed consistently: a

fibrous stroma and the presence of at least one component of spindle-shaped cells. All cases

were negative for EMA and AE1/AE3 and inconsistent for PS100, cytoplasmic CD99 and

vimentin staining. Ki67 was high (between 20% and 70%). In agreement with this

heterogeneous histological presentation, the proposed diagnoses for EWSR1-PATZ1-

positive tumours were quite variable including unclassified spindle cell sarcoma, myxoid

liposarcoma, Ewing-like sarcoma, or unclassified malignant neuroectodermal tumours.

Nevertheless, all five tumours tightly clustered together and away from other EWSR1-fused

Watson et al. 11

tumours, indicative of a transcriptionally different entity. GSEA identified enrichment of genes

correlated with SMARCA2 expression in prostate cancer (supplementary material, Table S3).

A new “epithelioid rhabdomyosarcoma” entity characterized by EWSR1/FUS-TFCP2

fusion

New fusions involving either EWSR1 (exon 5) or FUS (exon 6) and TFCP2 (exon2) were

identified in three cases (Figure 5A and supplementary material, Figure S4A), linking part of

the low complexity domains of EWSR1/FUS to the CP2 DNA binding and the SAM/pointed

domains of TFCP2. The three tumour samples formed a discrete cluster away from any other

EWSR1/FUS-fused tumour samples (Figure 1 and supplementary material, Figure S2).

EWSR1/FUS-TFCP2-positive tumours arose in young adult females (age range 16-38yo)

and developed in either the pelvic region, chest wall or the sphenoid bone. All three tumours

were extremely aggressive, since patient survival did not exceed 5 months. Upon review, all

three tumours were composed of an epithelioid proliferation arranged in small sheets or short

fascicles. Tumour cells presented monotonous round nuclei with high grade features and

prominent nucleoli (Figure 5B). They were associated with variable amounts of fibrous

stroma with focal sclerosing areas that were present in all cases. The three tumours stained

positive for desmin, MYOD1 and myogenin. Gene expression profile comparison confirmed a

strong expression of MYOD1 and DES as well as an impressive overexpression of TERT

and ALK (supplementary material, Figure S4B), the latter being heterogeneously expressed

with both a cytoplasmic and membranous staining (Figure 5B). In silico functional analyses

highlighted T cell immune response as well as keratin intermediate filament enrichment

(supplementary material, Table S3). Altogether the observation of positivity for both muscle

and epithelial markers suggests that this EWSR1/FUS-TFCP2 fusion defines a new

aggressive “epithelioid rhabdomyosarcoma” entity.

Watson et al. 12

Expression profiling reveals distinct transcriptomic patterns and potential biomarkers

Supervised group comparisons and between group analyses (BGA) highlighted genes

specifically expressed in the molecularly defined entities (Figure 6; supplementary material,

Table S3). More specifically, crossing pairwise differential analysis and BGA analysis, we

identified genes that were specifically expressed in each tumour type including VGLL2-fused

tumours (LANCL2), CIC-fused tumours (ETV4), BCOR-rearranged (HES7), FUS-NFATC2-

positive sarcomas (CD8B), EWSR1-PATZ1-positive tumours (GPR12) and FET-TFCP2-

positive tumours (REQL), but also for almost all of the other tumour entities (supplementary

material, Figure S5).

Discussion

We have used RNA sequencing to investigate unclassified small round or spindle cell

sarcomas. We first confirm that sarcomas, like haematological malignancies [24],

demonstrate a high incidence of gene fusions as compared to carcinomas. In this respect, it

is noteworthy that the mesenchyme, from which sarcomas are derived, and haematopoiesis,

from which arise leukaemias and lymphomas, both originate from the mesoderm. One

hypothesis may rely on the activity of specific recombinases in mesodermal derived tissues.

In this respect we can mention the role of the RAG1 recombinase in the generation of

lymphoma-specific translocations [25] and the recently suspected role of the PGBD5

recombinase in malignant rhabdoid tumours [26].

Combining fusion gene discovery and expression profiling, our analysis resulted in the

delineation of biologically homogeneous groups of tumours. While the CIC-NUTM1 fusion

was discovered as a new brain tumor entities [14], we show here that they are encompassed

in an homogeneous CIC-fused group of tumors together with CIC-DUX4- and CIC-FOXO4-

positive samples [27]. The overexpression of genes of the PEA3 type of the ETS family

Watson et al. 13

observed in all CIC-fused samples is known to be a consequence of a loss of function of CIC

[28], which was also involved in resistance to MAPK inhibitors [28,29] or in the promotion of

metastasis [30]. Further analyses should confirm whether a dominant negative effect on wild

type CIC is a consequence of CIC-fusions and if it may be correlated with the

aggressiveness of CIC-fused tumors. The BCOR-rearranged group of tumors includes

various BCOR-fusion genes and BCOR-ITD. BCOR-ITD were described in CCSK [19], in a

new brain tumor entity CNS HGNET-BCOR [14] and in soft tissue undifferentiated round cell

sarcoma of infancy sharing strong similarities with CCSK [17]. This homogeneous biological

entity may also present clinical specificities depending on the type of genetic lesions. Indeed,

while BCOR-CCNB3 was exclusively observed in bone, BCOR-ITD was only observed in soft

tissues. BCOR rearrangements may lead to an abnormal activity of the non-conventional

polycomb repressive BCOR (or PRC1.1) complex [31]. In this regard, the observation that

BCOR-rearrangements are associated with increased expression of genes correlated with

SMARCA2 expression (Figure 6A) may suggest that impairment of PRC1.1 activity lead to

an increased activity of the antagonist SWI/SNF complex. Thanks to a unique collection, we

found several samples carrying FUS-NFATC2 or EWSR1-PATZ1 fusions, which formed tight

groups. FUS-NFATC2-positive tumours present different morphologies but with features

rather reminiscent of CIC-fused tumours and are definitively different from EWSR1-NFATC2-

positive tumours. EWSR1-PATZ1 tumours present relatively divergent cell morphologies,

rendering their pathological identification challenging, though areas of spindle cells found in

all three samples may guide pathologists during diagnosis. The identification of more cases

is nevertheless mandatory to improve the characterization of these ultra-rare FET-fused

sarcomas.

Watson et al. 14

We also describe here a new entity carrying an EWSR1- or FUS-TFCP2 fusion gene. TFCP2

(also known as LSF or LBP1) does not resemble any of the usual FET-fusion partners. It was

first described as an activator of the late SV40 promoter and later found to bind globins and

HIV-1 promoters [32,33]. TFCP2 is ubiquitously expressed and plays determinant roles in

lineage-specific gene expression or cell cycle regulation [34]. Potential involvement of

TFCP2 in oncogenesis has been suggested and may depend on tumor type: In

hepatocellular carcinomas, TFCP2 is overexpressed [35] and its inhibition by small

molecules leads to cell cycle arrest [36], whereas in skin melanoma TFCP2 is under-

expressed, and its re-expression induces growth arrest [37]. Interestingly, in addition to the

expression of MYOD1 (supplementary material, Table S3), a number of up-regulated genes

in these FUS/EWSR1-TFCP2-positive tumours are involved in muscle biology (such as

Cholinergic Receptor Nicotinic subunit alpha1, delta and gamma or sarcoglycan alpha). This

may suggest either a muscle cellular origin of these tumours or a muscle differentiation

program triggered by the fusion protein. Consistently with the genes expressed, pathological

review confirmed that TFCP2-rearranged tumours were an epithelioid variant of

rhabdomyosarcoma, although the morphological spectrum of these tumours needs to be

assessed on a larger scale. Despite a common muscle phenotype, these samples do not

cluster together with other rhabdomyosarcomas. Considering potential therapeutic targets, it

is noteworthy that ALK and TERT are highly expressed in these tumours. Moreover, recently

developed small molecules impeding the DNA-binding activity of TFCP2 [36,38], which

remains in the fusion protein, might also be effective in hindering EWSR1-TFCP2 binding

and possibly in inhibiting the development of these very aggressive tumours.

Samples carrying VGLL2-NCOA2 or VGLL2-CITED2 fusion genes demonstrate some

transcriptome heterogeneity related to two different histological subtypes: one presenting

Watson et al. 15

only few tumour cells surrounded by fibrous stroma, rather suggestive of relatively benign

tumours, the other with a spindle cell rhabdomyosarcoma-like morphology similar to that

reported [13] and exhibiting expression profiles enriched in cell cycle genes. Interestingly,

one fibrous tumour and one rhabdomyosarcoma-like tumour represented the primary and the

relapse tumours from the same patient, respectively. It is therefore likely that the

rhabdomyosarcoma-like tumour represents a more aggressive evolution of the fibrous

tumour. Further analyses including exome-sequencing of the two tumours may enable the

identification of additional genetic mutations accounting for this evolution.

In addition to the fundamental role of expert surgical pathology, molecular analysis is now

becoming an integral part of the diagnosis of small round cell tumours, in particular for

resolving frequent diagnostic dilemmas. With the increase of sarcoma subclasses, it

becomes difficult for the pathologist to ascertain a precise diagnosis in a reliable and cost

effective way. In this respect, and depending on the expertise and technical availabilities at

each pathology department, two molecular approaches may be proposed. One may rely on

the design of a specific panel of genes, based on our selection of genes that are specifically

expressed in each tumour group, tested in a simple assay (p.e., using Nanostring

technology. Alternatively, thanks to the constant decrease of whole transcriptome costs and

the availability of robust bioinformatics tools, we strongly believe that RNA-sequencing is a

key approach. Indeed, our work indicates that the convergent information from gene fusion

detection and expression profiles-based clustering is extremely powerful to identify

biologically homogeneous groups of tumours. Hence, in the short term, RNA-sequencing is

expected to constitute a mandatory methodology for an efficient molecular diagnosis of

sarcoma, a requirement recently proposed in the GENSARC study [39]. Such a precise

subgrouping of sarcomas is essential i) to investigate the clinical characteristics of each

Watson et al. 16

subgroup, particularly regarding the risk of evolution and response to current treatment

options, ii) to identify new therapeutic opportunities, as potentially ALK overexpression in

EWSR1/FUS-TFCP2 sarcomas, iii) to construct relevant animal models to design robust pre-

clinical studies, and iv) to design highly specific assays to monitor circulating cell and tumour

DNA during treatment. Further increasing the number of samples, within large international

consortia, is essential to identify groups of tumours of sufficient size to enable robust

conclusions and to allow the collection of otherwise “orphan” samples.

Acknowledgments

The authors wish to thank pathologists who provided tumour material: E. Angot, H. Antoine-

Poirel, S. Aubert, A. Babik, J.P. Barbet, C. Bastien, A.M. Bergemer-Fouquet, D. Berrebi, L.

Boccon-Gibod, C. Bossard, S. Boudjemaa, E. Cassagnau, J. Champigneulle, M.A.

Chrestian, A. Clemenson, S. Collardeau-Frachon, S. Corby, J.F. Cote, A. Coulomb-

Lhermine, A. Croue, C. Daniliuc, A. De Muret , A. Dhouibi, C. Douchet, F. Dujardin, J.M.

Dumollard, H. Duval, M. Fabre, C. Fernandez, S. Fraitag, F. Galateau-Salle, L. Gibault, A.

Gomez-Brouchet, C. Guettier, M.F. Heymann, J.F. Ikoli, J.F. Jazeron, C. Jeanne-Pasquier,

A. Jouvet, R. Kaci, M. Karanian-Philippe, O. Kerdraon, J. Klijanienko, C. Labit-Bouvier, M.

Lae, T. Lazure, F. Le Pessot , F. Lemoine, A. Liprandi, F. Llamas - Gutierrez, M.C. Machet,

A. Maran-Gonzalez, L. Marcellin, P. Marcorelles, C. Marin, A. Maues De Paula, C.A.

Maurage, A. Moreau, A. Neuville, H. Perrochia, J.M. Picquenot, C. Renard, V. Rigau, J.

Riviere, C. Rouleau, A. Rouquette, H.Sartelet, I. Serre, N. Stock, H. Szabo, P. Terrier, M.

Terrier-Lacombe, V. Thomas De Montpreville, J. Tran Van Nhieu, E. Uro-Coste, P. Validire, I.

Valo, V. Verkarre, J.M. Vignaud, M.O. Vilain, M.L. Wassef, D. Zachar, L. Zemoura and the

tumorothèque Necker-Enfants Malades. We also thank Brigitte Manship for the careful

Watson et al. 17

reading of the manuscript. This work was supported by the Institut National de la Santé et de

la Recherche Médicale, the Institut Curie, the Ligue National Contre Le Cancer, the Institut

National du Cancer and la Direction générale de l'offre de soins (INCa-DGOS_5716), the

European PROVABES (ERA- 649 NET TRANSCAN JTC-2011), ASSET (FP7-HEALTH-

2010-259348), and EEC (HEALTH-F2-2013-602856) projects. U830 is also indebted to the

Société Française des Cancers de l’Enfant, Enfants et Santé, Courir pour Mathieu, Dans les

pas du Géant, La course de l’espoir du mont Valérien, Au nom d'Andréa, Association

Abigaël, Association Marabout de Ficelle, Les Bagouz à Manon, Les amis de Claire, the

association Adam. SW was supported by a grant from the Fondation Nuovo-Soldati. High-

throughput sequencing has been performed by the NGS platform of Institut Curie, supported

by the grants ANR-10-EQPX-03 and ANR10-INBS-09-08 from the Agence Nationale de la

Recherche (investissements d’avenir) and by the Canceropôle Ile-de-France.

Author contributions

SW, GP, OD and FT designed the study and wrote the manuscript. SW, VP, DG, SR and MB

performed all the experiments. SW, GP and FT, acquired and analysed the data. JMC, MK,

JMG, PF, DRV and FLL provided samples and were the reference senior pathologists. LGR,

FL and EL recruited patients and provided numerous samples and clinical information.

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Figure Legend

Figure 1: Fusion genes define tumour groups.

Kallisto-extracted expression values were used for all genes having a CDS annotation in

Ensembl GRCh38p5. A. t-Distributed Stochastic Neighbour Embedding (t-SNE) analysis.

Tumour samples (coloured spheres) carrying identical or similar fusion genes are linked to

the centroid of the group (small grey sphere). Fusion genes defining major groups are

indicated. Unclassified sarcomas are not represented here for the sake of clarity but were

taken into account in the analysis. An enlarged view of the centre of the figure is presented in

Watson et al. 21

the supplementary material, Figure S2A. B. Hierarchical clustering. 1-pearson correlation and

Ward’s method were used as distance and clustering method, respectively; identified

recurrent fusion genes are indicated. An enlarged view of TFE3-fused, CIC-fused and

BCOR-rearranged samples are presented below (see supplementary material, Figure S2B

for the full annotated cluster).

Figure 2: Subtypes of VGLL2-NCOA2-positive samples

A. Representative images of fibrous tumour samples. SARC070_primary and SARC065

tumour samples contained abundant fibrous stroma, displayed low Ki67 and were mostly

negative for myogenin and desmin. B. Representative images of dense tumour samples.

SARC070_relapse and SARC102 cells were packed and strongly positive for Ki67, with

numerous cells being positive for myogenin and desmin. Scale bar: 100 µm

Figure 3: FUS-NFATC2-positive tumours

Morphology (hematoxylin/eosin staining) of FUS-NFATC2 tumours. A. Proliferation of round

tumour cells embedded in a variable myxoid stroma. B. Round to spindle tumour cells

arranged in sheets associated with haemangioperycitic vessels. C. Tumour cells were focally

embedded within myxohyaline stroma reminiscent of cartilaginous differentiation. Scale bars:

100 µm

Figure 4: EWSR1-PATZ1-positive tumours

A. Schematic diagram of native EWSR1 and PATZ1 proteins indicating fusion point of

EWSR1-PATZ1 fusion gene. LC: Low complexity domain; RRM: RNA recognition motif;

RGG: Arg-Gly-Gly rich region; Zn: Zinc Finger, RanBP2-type; BTB/POZ: Broad-Complex,

Tramtrack and Bric a brac / POxvirus and Zinc finger; H: AT hook; C2H2: Cys2-His2 Zinc-

finger. Exon number based on the indicated RefSeq accession number is indicated below

Watson et al. 22

each protein scheme together with amino acid length scale. B. H & E staining of three

EWSR1-PATZ1-positive samples demonstrating a variety of morphologies (scale bar: 100

µm).

Figure 5: FET-TFCP2 fusion

A. Schematic diagram of native proteins and fusion proteins. EWSR1-TFCP2 fusion was

identified in SARC049 while FUS-TFCP2 was identified in RNA009_16_062 and

RNA020_16_136 samples. LC: Low complexity domain; RRM: RNA recognition motif; RGG:

Arg-Gly-Gly rich region; Zn: Zinc Finger, RanBP2-type; SAM: Sterile alpha motif / pointed

domain. Exon number based on the indicated RefSeq accession number is indicated below

each protein scheme. Amino acid length scale is presented at the bottom. B. Morphological

spectrum of FET-TFCP2 tumours. Hematoxylin/eosin staining (left panels) illustrating

epithelioid (top), spindling (middle) and sclerosing (bottom) areas and immunohistochemistry

(right panels) for MYOD1 (top), desmin (middle), and ALK (bottom) seen in all cases are

illustrated in EWSR1-TFCP2-positive sample SARC049. ALK staining was cytoplasmic-

positive with nuclear and membrane exclusion. Morphological aspects were evocative of an

epithelioid and spindled variant of rhabdomyosarcoma, with numerous mitotic figures. Scale

bar: 40 µm

Figure 6: Expression profiles functional analyses

Heatmap representing the specificity of the top 100 genes (detailed in the supplementary

material, Table S3) identified in the between group analysis for each 24 tumour groups

containing at least two samples. Data were centred and scaled (in row direction) prior

analysis. Colour key of the scaled expression values is shown.

Watson et al. 23

Supporting information:

Supplementary Figure S1: Constitution of the cohorts

Supplementary Figure S2: Details of the unsupervised analyses

Supplementary Figure S3: Details of VGLL2-NCOA2/CITED2, CIC-NUTM1, BCOR-ITD and

EWSR1-PATZ1 fusion points.

Supplementary Figure S4: Sequence of the fusion points and genes expressed in FET-

TFCP2-positive tumors

Supplementary Figure S5: Expression of the most specific gene for each tumor entity (with

more than 2 samples)

Supplementary Table S1: List of the fusion genes routinely tested by RT-PCR at the Institut

Curie Unité de Génétique Somatique.

Supplementary Table S2: Description of the samples from the three cohorts

Supplementary Table S3: Differentially expressed genes in the BGA and supervised

analyses and Gene ontology / GSEA enrichment for all sarcomas subtypes

Supplementary Table S4: Differentially expressed genes and gene ontology enrichment for

FUS-NFATC2- vs EWSR1-NFATC2-positive tumors

A

B

BCOR-CCNB3

CIC-DUX4

EWSR1-WT1

BAF-deficient

FET-ETS

EWSR1-PATZ1

NAB2-STAT6

FET-NR4A3

HEY1-NCOA2

FET-TFCP2

BRD3/4-

NUTM1

FUS-NFATC2

EWSR1-NFATC2

VGLL2-NCOA2/CITED2

BCOR-ITD

BCOR-MAML3 /

ZC3H7B-BCOR

EML4-ALK

EWSR1-

CREB1/ATF1

FET-CREB3L1/2

FET-DDIT3

SS18-SSX

CIC-NUTM1

Tsne

2

Tsne 1-60 60

40

-60

-60

0

0

0

UT

X-T

FE

3

AS

PS

CR

1-T

FE

3

SF

PQ

-TF

E3

CIC

-DU

X4

CIC

-NU

TM

1

CIC

-FO

XO

4

BC

OR

-CC

NB

3

BC

OR

-MA

ML3

ZC

3H

7B

-BC

OR

BC

OR

-IT

D

FE

T-E

TS

CIC

-Fu

sed

EW

SR

1-W

T1

BC

OR

-Rearr

anged

FU

S-N

FA

TC

2

HE

Y1-N

CO

A2

SS

18-S

SX

EW

SR

1-P

TA

TZ

1

PA

X-F

OX

O

TF

E3-F

used

NA

B2-S

TA

T6

FE

T-N

R4A

3

BR

D3/4

-NU

T

EM

L4-A

LK

VG

LL2-F

used

FE

T-T

FC

P2

BA

F-d

eficie

nt

ER

MS

EW

SR

1-C

RE

B1/

EW

SR

1-N

FA

TC

2

AT

/RT

Ward

'sdis

tance

0.0

0.2

0.4

0.6

0.8

AT

F1

Watson et al. Figure 1

A

B

HES Ki67 Desmin

HES Myogenin

SA

RC

070 (

rela

pse)

SA

RC

102

Ki67

SARC070 (primary) SARC065

Ki67

HES Desmin

Myogenin

HES

Ki67


A

B

C


54321 6 7 8 910 13 1411 12 15 16 17 NM_005243

EWSR1 RRM ZnLC RGG RGG RGG

200 300 4001000 500 687600

54321NM_014323

BTB/POZ C2H2 PATZ1

1610 1620 1630 1640 1650 1660 1670 1680 1690 1700

* * * * * * * * * * * * * * * * * * *

accagcctccagctgggctacatcgaccttcctcctccgaggctgggtgagaatgggctacccatctctgaagaccccgacggcccccgaaag

T S L Q L G Y I D L P P P R L G E N G L P I S E D P D G P R K

320 330 340 NM_014323

H

A

B

SA

RC

041

SA

RC

017

G155_R

NA

045_17_001


SARC049: EWSR1-TFCP2

54321 191817161514131211109876

CP2 transcription factor SAMLC

RNA009_16_062 & RNA020_16_136 : FUS-TFCP2CP2 transcription factor SAMLC

201918171615141312111098754321 6

200 300 4001000 500 700600

A

B

54321 6 7 8 910 13 1411 12 15 16 17 NM_005243

EWSR1 RRM ZnLC RGG RGG RGG

54321 6 7 8 9 10 13 1411 12 15 NM_004960

FUSRRM ZnLC RGGRGG

MYOD1

desmin

ALK

HES

HES

HES

151413121110987654321 NM_005653

CP2 transcription factor TFCP2SAMCP2 transcription factor


0-1-2-3 1 2 3

TF

E3-F

used

Em

bry

onalrh

abdom

yosarc

om

a

BC

OR

-rearr

anged

CIC

-Fu

sed

EW

SR

1-W

T1

EM

L4-A

LK

PA

X-F

OX

O

FE

T-E

TS

EW

SR

1-P

AT

Z1

EW

SR

1-N

FA

TC

2

FE

T-N

R4A

3

FE

T-T

FC

P2

NT

RK

3-F

used

FU

S-N

FA

TC

2

FE

T-C

RE

B3L1/2

NT

RK

1-F

used

HE

Y1

-NC

OA

2

FE

T-C

RE

B3L2/3

BR

D3/4

-NU

TM

1

NA

B2-S

TA

T6

SS

18-S

SX

VG

LL2-F

used s

arc

om

aclu

ste

r1

VG

LL2-F

used s

arc

om

aclu

ste

r2

EW

SR

1-C

RE

B1/A

TF

1

Scaled expression value (Log2)


Selection of 78 samples:

Ewing Sarcoma (n=7),

CIC-DUX4 sarcoma (n=6)

Rhabdomyosarcoma (n=6)

BCOR-CCNB3 sarcoma (n=5)

Clear cell sarcoma (n=5)

Solitary fibrous tumor (n=5)

EWSR1-NFATC2 Ewing-like sarcoma (n=4)

Extraskeletal myxoid chondrosarcoma (n=4)

NUT midline carcinoma (n=4)

Alveolar soft part sarcoma (n=3)

Renal cell carcinoma (n=3)

Desmoplastic small round cell tumor (n=3)

Myxoid liposarcoma (n=3)

Congenital fibrosarcoma (n=2)

Low-grade fibromyxoid sarcoma (n=2)

Mesenchymal chondrosarcoma (n=2)

Synovial sarcoma (n=1)

SMARCA4-DTS (n=4)

Malignant rhabdoid tumor (n=7)

SCCOHT (n=1)

+ CIC-FOXO sarcoma (n=1)**

Identification of a pathognomonic

alteration

No pathognomonic alteration identified

Investigation cohort N=94

Robust fusion gene

detected in 55 cases

No robust fusion gene


Single fusion gene


Multiple fusion genes


(1 sample with

chromothripsis)

Sarcoma samples received for molecular

diagnosis

700+ retrospective samples without

detectable fusion with good quality RNA

Follow-up cohort N=12

Watson et al., Supplementary Figure S1

Supplementary Figure S1: Constitution of the cohorts

* See Supplementary table S1

** : RNAseq raw fastq files kindly provided by Yasuhito Arai and Tadashi Hasegawa [27]

*** Screened fusions were:

BCOR-MAML3, CDKN2A-CCDC12, CIC-NUTM1, EML4-ALK, EWSR1-PATZ1, EWSR1-

TFCP2, FUS-TFCP2, FUS-NFATC2, KMT2A-YAP1 / YAP1-KMT2A, MN1-TAF3, NF1-

RHOT1, PARG-BMS1, VGLL2-NCOA2 and VGLL2-CITED2.

Control cohort N=78

Random selection

of 94 cases

RNA-sequencing

RNA-sequencing

PCR screening***

RNA-sequencing

yes no

Molecular investigations including

QPCR on known fusion transcripts *

Pool of samples without

detectable fusion


A

-40

20

0

-20

0

0

Tsne 1

Tsne

3

VGLL2-Fused

NTRK3-Fused

NTRK1-Fused

BRD3/4-NUTM1

PAX-FOXO

FET-CREB3L1/2

FET-DDIT3

EWSR1-CREB1/ATF1

ERMS

Ward's distance

0.0

0.2

0.4

0.6

0.8

RNA010_16_052

RNA011_16_065

RNA011_16_066

SARC066

RNA010_16_053

RNA011_16_067

RNA010_16_054

RNA001_16_002

RNA004_16_011

SARC055

SARC069

SARC058

RNA007_16_037

RNA002_16_002

RNA003_16_007

SARC046

RNA004_16_010

SARC053

SARC079

SARC067

SARC087

SARC011

SARC059

SARC043

RNA002_16_003

RNA006_16_025

RNA006_16_026

RNA004_16_009

SARC056

SARC060

RNA006_16_027

RNA005_16_019

RNA003_16_003

SARC042

SARC022

SARC002

SARC019

SARC024

SARC031

SARC073

SARC062

SARC003

SARC090

SARC033

SARC047

RNA004_16_012

SARC085

SARC102

RNA004_16_013

SARC061

SARC065

SARC070_(Primary)

SARC070_(Relapse)

RNA001_16_004

SARC005

SARC063

RNA009_16_062

SARC049

RNA020_16_136

SARC006

SARC016

SARC012

SARC021

SARC071

RNA002_16_004

RNA034_16_245

RNA041_16_300

RNA041_16_303

RNA003_16_005

SARC098

SARC010

SARC091

SARC020

SARC089

SARC072

SARC095

RNA007_16_034

SARC039

SARC035

ASPSCR1-TFE3

ASPSCR1-TFE3

ASPSCR1-TFE3

UXT-TFE3

ASPSCR1-TFE3

ASPSCR1-TFE3

SFPQ-TFE3

EWSR1-ATF1

EWSR1-ATF1

EWSR1-CREB1

EWSR1-CREB1

EWSR1-ATF1

TAF15-NR4A3

TAF15-NR4A3

EWSR1-NR4A3

EWSR1-NR4A3

PAX3-MAML3

BRD4-NUTM1

BRD3-NUTM1

TANC1-DAPL1;FAM172A-THBS4;GABRA1-PSMB3 ;ARHGEF28-EFCAB3;ICK-BTBD9

BRD4-NUTM1

BRD4-NUTM1

BRD4-NUTM1

EML4-ALK

EML4-ALK

EML4-ALKETV6-NTRK3

TPM3-NTRK1

IKBKG-ALK

KMT2A-YAP1

PDLIM5-USP8

FUS-DDIT3

EWSR1-PBX1

EWSR1-CREB3L1

FUS-CREB3L2

VGLL2-CITED2

VGLL2-NCOA2

VGLL2-NCOA2

VGLL2-NCOA2

VGLL2-NCOA2

FUS-DDIT3

EWSR1-DDIT3

ACTB-GLI1

FUS-TFCP2

EWSR1-TFCP2

FUS-TFCP2

GNB1-PTGER3;RERE-CAMTA1;FAR1-ACSM5

SEC14L1-CYP39A1

MAPKAPK5-ACAD10

CREB3L2-AUTS2;TAX1BP1-RARB;PACS1-SEPT9

EWSR1-NFATC2

EWSR1-NFATC2

EWSR1-NFATC2

EWSR1-NFATC2

CDKN2A-CCDC12

PVT1-KIAA0125

FET-ETS

CIC-Fused

EWSR1-WT1

BCOR-Rearranged

FUS-NFATC2

HEY1-NCOA2SS18-SSX

EWSR1-PTATZ1

PAX-FOXO

TFE3-Fused

NAB2-STAT6

FET-NR4A3

BRD3/4-NUT

EML4-ALK

VGLL2-Fused

FET-TFCP2

BAF-deficient

ERMS

EWSR1-CREB1/ATF1

EWSR1-NFATC2

AT/RT


B Fusion Gene SampleID

Ward's distance

0.0

0.2

0.4

0.6

0.8

RNA001_16_005RNA006_16_032RNA006_16_030RNA006_16_031

SARC036RNA005_16_020RNA006_16_029

SARC086RNA007_16_040

SARC025RNA007_16_036

SARC097SARC075

SMARCA4_MRT02SMARCB1_MRT05SMARCB1_MRT04

RNA009_16_064SARC007SARC027SARC082SARC015SARC096

RNA003_16_001RNA004_16_015RNA004_16_014RNA004_16_016RNA003_16_002

SARC074SARC030SARC078SARC068SARC048SARC050

RNA009_16_058SARC028SARC032


RNA006_16_028SARC040SARC076

RNA005_16_018SARC083

RNA009_16_063SARC099SARC100SARC004SARC081


RNA045_17_001SARC041

RNA034_16_244RNA002_16_005RNA005_16_023RNA007_16_039

SARC044SARC037

RNA007_16_038SARC052

RNA001_16_001RNA001_16_006RNA005_16_017RNA005_16_021RNA001_16_008

SARC064SARC080

SMARCA4_MRT01RNA005_16_024

SARC018SARC093SARC008SARC014

SMARCB1_MRT01SMARCB1_MRT02SMARCB1_MRT03

RNA020_16_134RNA022_16_147


RNA001_16_003RNA002_16_008RNA002_16_007

EWN1069TSARC051SARC013SARC038SARC084

EWN1072T4SXT1

INI42RNA012_16_073RNA003_16_008

SARC101RNA012_16_074

PAX3-FOXO1PAX7-FOXO1

PAX3-FOXO1

PPFIBP1-TUBA1BPDLIM5-LOC285419

RASSF3-XPOT;IGF2AS-LSP1STAG1-ATF7

TPM3-NTRK1

Chr1-chromothripsisCCNT2-KYNU;RGL1-CCDC149;SMARCD1-KNTC1

KPNA6-EPHB2;XPR1-IGSF21;TNFRSF14-ACOT7;CAMSAP1L1-CSRP1;EPB41-RBM34

BCOR-CCNB3BCOR-CCNB3BCOR-CCNB3BCOR-CCNB3BCOR-CCNB3

BCOR_ITDBCOR_ITDBCOR_ITDBCOR_ITD

BCOR-MAML3BCOR_ITD

ZC3H7B-BCOREPC2-PHC2;ETNK2-IKBKE

TRPS1-RIMBP2;RIMBP2-TRPS1MN1-TAF3

FAM133B-DRG1;LATS1-SNX9;HPS4;LOC96610;HADH-TBC1D24;TRIM4;CALN1;LARGE;TPST1

NF1-RHOT1AKAP13-ABHD2

ETV6-NTRK3KIAA1549-BRAF

MEMO1-BCL11A1H19-COL1A

COL1A-PDGFB

TPR-NTRK1

PVT1-ATP10BSS18-SSX2SS18-SSX2

HEY1-NCOA2HEY1-NCOA2

EWSR1-PATZ1EWSR1-PATZ1EWSR1-PATZ1EWSR1-PATZ1EWSR1-PATZ1

NAB2-STAT6NAB2-STAT6NAB2-STAT6NAB2-STAT6NAB2-STAT6NAB2-STAT6NAB2-STAT6EWSR1-FLI1EWSR1-FLI1EWSR1-FLI1EWSR1-FLI1EWSR1-FLI1

FUS-ERGFUS-ERG

FUS-NFATC2FUS-NFATC2FUS-NFATC2

EWSR1-WT1EWSR1-WT1EWSR1-WT1

CIC-DUX4CIC-DUX4CIC-DUX4CIC-DUX4

CIC-NUTM1CIC-DUX4

CIC-FOXO4CIC-DUX4

CIC-NUTM1CIC-NUTM1CIC-NUTM1CIC-NUTM1

FET-ETS

CIC-Fused

EWSR1-WT1

BCOR-Rearranged

FUS-NFATC2

HEY1-NCOA2SS18-SSX

EWSR1-PTATZ1

PAX-FOXO

TFE3-Fused

NAB2-STAT6

FET-NR4A3

BRD3/4-NUT

EML4-ALK

VGLL2-Fused

FET-TFCP2

BAF-deficient

ERMS

EWSR1-CREB1/ATF1

EWSR1-NFATC2

AT/RT


B (continued) Fusion Gene SampleID

Cohort

Sequencin

g_R

un

Te

chnolo

gy

Tu

mor_

tissue

Tu

mor

site

Gender

Age

Tu

mor

Type

0 1 2 3 4 5

ward's distance

SARC017_EWSR1.PATZ1SARC026_EWSR1.PATZ1SARC041_EWSR1.PATZ1

RNA009_16_058_ZC3H7B.BCOR

RNA009_16_063_COL1A.PDGFBRNA006_16_028_ETV6.NTRK3

SARC040_KIAA1549.BRAFSARC076_MEMO1.BCL11A1

SARC083RNA005_16_018_H19.COL1ARNA003_16_004_MN1.TAF3


SARC007SARC082_CCNT2.KYNU;RGL1.CCDC149;SMARCD1.KNTC1

SARC027_Chr1.chromothripsisSARC096

RNA009_16_064_TPM3.NTRK1RNA007_16_037

SARC064_FUS.ERGSARC080_FUS.ERG

RNA001_16_001_EWSR1.FLI1.1RNA001_16_006_EWSR1.FLI1.2

RNA005_16_021_EWSR1.FLI1RNA005_16_017_EWSR1.FLI1

RNA001_16_008_EWSR1.FLI1.1SARC013_CIC.DUX4EWN1069_CIC.DUX4SARC051_CIC.DUX4

INI42_CIC.DUX4RNA012_16_073_CIC.NUTM1

EWN1072_CIC.DUX4SARC038_CIC.DUX4

4SXT1_CIC.FOXO4SARC101_CIC.NUTM1

RNA012_16_074_CIC.NUTM1RNA003_16_008_CIC.NUTM1

SARC084_CIC.NUTM1SARC093

SMARCA4_MRT01RNA005_16_024

SARC014SARC018SARC008

SMARCB1_MRT01SMARCB1_MRT02SMARCB1_MRT03

SARC039_PVT1.KIAA0125SARC067_BRD4.NUTM1

RNA006_16_026_BRD4.NUTM1RNA006_16_025_BRD4.NUTM1RNA002_16_003_BRD4.NUTM1

SARC043_TANC1.DAPL1;FAM172A.THBS4;GABRA1.PSMB3 ;ARHGEF28.EFCAB3;ICK.BTBD9 RNA003_16_001_BCOR.CCNB3RNA004_16_015_BCOR.CCNB3RNA004_16_014_BCOR.CCNB3RNA003_16_002_BCOR.CCNB3

SARC074_BCOR_ITDRNA004_16_016_BCOR.CCNB3

SARC050_BCOR_ITDSARC068_BCOR_ITDSARC078_BCOR_ITDSARC030_BCOR_ITD

SARC048_BCOR.MAML3

SARC066_UXT.TFE3RNA011_16_065_ASPSCR1.TFE3RNA011_16_066_ASPSCR1.TFE3RNA010_16_053_ASPSCR1.TFE3RNA011_16_067_ASPSCR1.TFE3

RNA010_16_054_SFPQ.TFE3SARC069_EWSR1.CREB1SARC055_EWSR1.CREB1

RNA001_16_002_EWSR1.ATF1SARC046_EWSR1.NR4A3

RNA003_16_007_TAF15.NR4A3RNA002_16_002_TAF15.NR4A3

RNA004_16_010_EWSR1.NR4A3SARC052B2.STAT6

RNA007_16_038B2.STAT6SARC037B2.STAT6SARC044B2.STAT6

RNA007_16_039B2.STAT6RNA002_16_005B2.STAT6RNA005_16_023B2.STAT6

RNA010_16_052_ASPSCR1.TFE3SARC079

SARC058_EWSR1.ATF1RNA004_16_011_EWSR1.ATF1

SARC020SARC102_VGLL2.NCOA2

RNA002_16_007_EWSR1.WT1SARC087_BRD3.NUTM1

RNA006_16_027_ETV6.NTRK3RNA005_16_019_TPM3.NTRK1

RNA003_16_003SARC063_ACTB.GLI1

SARC089SARC035

RNA007_16_034RNA034_16_245_EWSR1.NFATC2RNA041_16_300_EWSR1.NFATC2RNA002_16_004_EWSR1.NFATC2RNA041_16_303_EWSR1.NFATC2

SARC053_PAX3.MAML3SARC056_EML4.ALKSARC060_EML4.ALK

RNA004_16_009_EML4.ALKSARC042_IKBKG.ALKSARC062_FUS.DDIT3


SARC019_PDLIM5.USP8SARC024SARC031

SARC002_KMT2A.YAP1RNA004_16_012RNA004_16_013

SARC065_VGLL2.NCOA2SARC061_VGLL2.NCOA2

SARC070 (Primary)SARC033_EWSR1.CREB3L1

SARC047_FUS.CREB3L2SARC003_EWSR1.PBX1

SARC090SARC005_EWSR1.DDIT3

RNA001_16_004_FUS.DDIT3SARC059SARC072SARC095SARC099

SARC100_TPR.NTRK1SARC049_EWSR1.TFCP2

RNA009_16_062_FUS.TFCP2RNA020_16_136_FUS.TFCP2SARC012_SEC14L1.CYP39A1

SARC016SARC006_GNB1.PTGER3;RERE.CAMTA1;FAR1.ACSM5

SARC070 (Relapse)_VGLL2.NCOA2SARC021_MAPKAPK5.ACAD10

SARC071_CREB3L2.AUTS2;TAX1BP1.RARB;PACS1.SEPT9RNA001_16_005_PAX3.FOXO1RNA006_16_032_PAX7.FOXO1

RNA006_16_030RNA006_16_031_PAX3.FOXO1

SARC036RNA005_16_020_PPFIBP1.TUBA1B

RNA006_16_029_PDLIM5.LOC285419SARC086

SARC025_STAG1.ATF7RNA007_16_040_RASSF3.XPOT;IGF2AS.LSP1

SARC097RNA007_16_036

SARC075RNA001_16_003_EWSR1.WT1RNA002_16_008_EWSR1.WT1

SARC094_FUS.NFATC2RNA020_16_134_FUS.NFATC2RNA022_16_147_FUS.NFATC2

SARC054SARC085_VGLL2.CITED2


RNA003_16_005_CDKN2A.CCDC12SARC077_FAM133B.DRG1;LATS1.SNX9;HPS4;LOC96610;HADH.TBC1D24;TRIM4;CALN1;LARGE;TPST1

SARC028_EPC2.PHC2;ETNK2.IKBKESARC009_NF1.RHOT1

SARC001SARC023_AKAP13.ABHD2

SARC032_TRPS1.RIMBP2;RIMBP2.TRPS1SARC057SARC011

SARC015_KPNA6.EPHB2;XPR1.IGSF21;TNFRSF14.ACOT7;CAMSAP1L1.CSRP1;EPB41.RBM34SARC034_SS18.SSX2

RNA001_16_007_SS18.SSX2SARC004

SARC081_PVT1.ATP10BSARC029_HEY1.NCOA2SARC045_HEY1.NCOA2

RNA045_17_001_EWSR1-PATZ1

RNA034_16_244_EWSR1-PATZ1

To

pH

at/C

ufflin

ks e

xp

ressio

n

C


SARC017_EWSR1-PATZ1RNA045_17_001_EWSR1-PATZ1

SARC026_EWSR1-PATZ1RNA034_16_244_EWSR1-PATZ1

SARC041_EWSR1-PATZ1

SARC066_UXT.TFE3RNA010_16_052_ASPSCR1.TFE3RNA011_16_065_ASPSCR1.TFE3RNA011_16_066_ASPSCR1.TFE3RNA010_16_053_ASPSCR1.TFE3RNA011_16_067_ASPSCR1.TFE3

RNA010_16_054_SFPQ.TFE3SARC053_PAX3.MAML3

SARC079SARC058_EWSR1.ATF1

RNA001_16_002_EWSR1.ATF1RNA004_16_011_EWSR1.ATF1

SARC033_EWSR1.CREB3L1SARC047_FUS.CREB3L2

SARC002_KMT2A.YAP1SARC003_EWSR1.PBX1

SARC069_EWSR1.CREB1SARC055_EWSR1.CREB1

SARC090RNA007_16_037

RNA002_16_007_EWSR1.WT1SARC087_BRD3.NUTM1

SARC020SARC102_VGLL2.NCOA2


SARC070 (Relapse)_VGLL2.NCOA2SARC085_VGLL2.CITED2

SARC088SARC067_BRD4.NUTM1

RNA006_16_026_BRD4.NUTM1RNA006_16_025_BRD4.NUTM1RNA002_16_003_BRD4.NUTM1

SARC011SARC015_KPNA6.EPHB2;XPR1.IGSF21;TNFRSF14.ACOT7;CAMSAP1L1.CSRP1;EPB41.RBM34

SARC057SARC059

SARC043_TANC1.DAPL1;FAM172A.THBS4;GABRA1.PSMB3 ;ARHGEF28.EFCAB3;ICK.BTBD9SARC056_EML4.ALKSARC060_EML4.ALK

RNA004_16_009_EML4.ALKSARC042_IKBKG.ALKSARC062_FUS.DDIT3


SARC019_PDLIM5.USP8SARC024SARC031

RNA004_16_012RNA004_16_013

SARC065_VGLL2.NCOA2SARC061_VGLL2.NCOA2

SARC070 (Primary)SARC049_EWSR1.TFCP2

RNA009_16_062_FUS.TFCP2RNA020_16_136_FUS.TFCP2SARC012_SEC14L1.CYP39A1

SARC016SARC006_GNB1.PTGER3;RERE.CAMTA1;FAR1.ACSM5

SARC092SARC021_MAPKAPK5.ACAD10

SARC071_CREB3L2.AUTS2;TAX1BP1.RARB;PACS1.SEPT9RNA034_16_245_EWSR1.NFATC2RNA041_16_300_EWSR1.NFATC2RNA002_16_004_EWSR1.NFATC2RNA041_16_303_EWSR1.NFATC2

RNA006_16_027_ETV6.NTRK3RNA005_16_019_TPM3.NTRK1

SARC063_ACTB.GLI1RNA003_16_003

SARC035RNA007_16_034

SARC039_PVT1.KIAA0125RNA009_16_063_COL1A.PDGFB

RNA006_16_028_ETV6.NTRK3SARC040_KIAA1549.BRAF

SARC076_MEMO1.BCL11A1SARC083

RNA005_16_018_H19.COL1ARNA003_16_004_MN1.TAF3

SARC099SARC100_TPR.NTRK1

SARC029_HEY1.NCOA2SARC045_HEY1.NCOA2

SARC032_TRPS1.RIMBP2;RIMBP2.TRPS1SARC034_SS18.SSX2

RNA001_16_007_SS18.SSX2SARC004

SARC081_PVT1.ATP10BSARC009_NF1.RHOT1

SARC001SARC023_AKAP13.ABHD2

SARC077_FAM133B.DRG1;LATS1.SNX9;HPS4;LOC96610;HADH.TBC1D24;TRIM4;CALN1;LARGE;TPST1SARC028_EPC2.PHC2;ETNK2.IKBKE

SARC046_EWSR1.NR4A3RNA003_16_007_TAF15.NR4A3RNA002_16_002_TAF15.NR4A3

RNA004_16_010_EWSR1.NR4A3SARC005_EWSR1.DDIT3

RNA001_16_004_FUS.DDIT3SARC098

RNA003_16_005_CDKN2A.CCDC12SARC052B2.STAT6

RNA007_16_038B2.STAT6SARC037B2.STAT6SARC044B2.STAT6

RNA007_16_039B2.STAT6RNA002_16_005B2.STAT6RNA005_16_023B2.STAT6

RNA001_16_005_PAX3.FOXO1RNA006_16_032_PAX7.FOXO1

RNA006_16_030RNA006_16_031_PAX3.FOXO1

SARC036RNA005_16_020_PPFIBP1.TUBA1B

RNA006_16_029_PDLIM5.LOC285419SARC086

SARC025_STAG1.ATF7RNA007_16_040_RASSF3.XPOT;IGF2AS.LSP1

SARC097RNA007_16_036


SARC096RNA009_16_064_TPM3.NTRK1RNA001_16_003_EWSR1.WT1RNA002_16_008_EWSR1.WT1

SARC094_FUS.NFATC2RNA020_16_134_FUS.NFATC2RNA022_16_147_FUS.NFATC2

SARC007SARC027_Chr1.chromothripsis

SARC082_CCNT2.KYNU;RGL1.CCDC149;SMARCD1.KNTC1SARC054SARC075

SMARCA4_MRT01SARC093SARC014SARC018SARC008

RNA005_16_024SMARCB1_MRT02SMARCB1_MRT03SMARCB1_MRT01

SARC064_FUS.ERGSARC080_FUS.ERG

RNA001_16_001_EWSR1.FLI1.1RNA001_16_006_EWSR1.FLI1.2

RNA005_16_021_EWSR1.FLI1RNA001_16_008_EWSR1.FLI1.1

RNA005_16_017_EWSR1.FLI1SARC013_CIC.DUX4EWN1069_CIC.DUX4SARC051_CIC.DUX4

SARC084_CIC.NUTM1EWN1072_CIC.DUX4SARC038_CIC.DUX4

4SXT1_CIC.FOXO4INI42_CIC.DUX4

RNA012_16_073_CIC.NUTM1SARC101_CIC.NUTM1

RNA012_16_074_CIC.NUTM1RNA003_16_008_CIC.NUTM1

RNA003_16_001_BCOR.CCNB3RNA004_16_015_BCOR.CCNB3RNA004_16_014_BCOR.CCNB3RNA003_16_002_BCOR.CCNB3RNA004_16_016_BCOR.CCNB3

SARC074_BCOR_ITDSARC068_BCOR_ITDSARC078_BCOR_ITDSARC030_BCOR_ITDSARC050_BCOR_ITD

SARC048_BCOR.MAML3RNA009_16_058_ZC3H7B.BCOR

0 1 2 3 4 5

ward's distance

Cohort

Sequencin

g_R

un

Te

chnolo

gy

Tu

mor_

tissue

Tu

mor

site

Gender

Age

Tu

mor

Type

Kalllis

toexp

ressio

n

D


Supplementary Figure S2: Detailed unsupervised analyses.

A) Zoom on the center region of the TSNE analysis (Figure 1A) with another angle, demonstrating

dispersion of EWSR1-CREB3L1/2, NTRK1-, NTRK3- and VGLL2-fused tumors, and at a lesser extend

EWSR1-CREB1/ATF1 and FET-DDIT3-fused tumors, as compared to the more defined EWSR1-NR4A3,

EWSR1-WT1 or EWSR1-PATZ1 groups. B). Details of the clustering analysis (identical to Figure 1B)

showing all the fusion genes identified. C&D) Expression profiles were generated using two different

methods: in C) sequencing reads were aligned with TopHat2 and expression profiles were extracted using

Cufflinks2 tool. In D) expression profiles were extracted with Kallisto tools that does not require prior

alignment. In both cases, hierarchical clustering using the ten percent most variant genes based on

interquartile range, proved to be quite similar. The main difference with Figure 1 concerned the VGLL2-fused

samples that split in two or more groups. No clustering bias due to either patient ‘s clinical data (age, tumor

site, gender or tissue type) or experimental condition (technology used or sequencing run) could be

observed.

Age

<3<10<25>25

Gender

MF

Tumor site

Head and neckTrunkDistal limbsProximal limbs

Tumor tissue

soft tissuebonebrain tissue

Technology

HiSeq PE100NextSeq PE150

Cohort

INVESTIGATION COHORTCONTROL COHORTSCREENED SAMPLES

Legend

Tumor Type Sequencing Run

Inflammatory myofibroblastic tumorFIC

Myxoid Liposarcoma

Low-grade fibromyxoid sarcoma

Myoepithelioma

FET-TFCP2Extraskeletal myxoid chondrosarcoma

MRT Extracerebral

Lipofibromatosis-like Neural Tumor

EWSR1-PATZ1

Mesenchymal chondrosarcoma

EWSR1-NFATc2 sarcoma

Biphenotypic sinonasal sarcoma

Alveolar soft part sarcoma

Clear cell sarcoma

DSCRTEML4-ALK sarcoma

Dermatofibrosarcoma protuberans

Ewing sarcoma

CIC-Fused sarcoma

AT/RTARMS

ERMS

BCOR-Rearranged sarcoma

Unclassified SarcomaVGLL2-Fused

NUT midline carcinoma

Solitary fibrous tumorSMARCA4-DTSSCCOHT

Synovial Sarcoma

Pericytoma

Renal cell carcinoma

HiSeq.RunB127

NextSeq.Run46

NextSeq.Run49

NextSeq.Run1

NextSeq.Run41

NextSeq.Run34

NextSeq.Run35

HiSeq.RunB102

HiSeq.RunB137

HiSeq.RunB68

HiSeq.RunB72

NextSeq.Run48

HiSeq.RunB135

HiSeq.RunB70

HiSeq.RunA119

HiSeq.RunA53

EXT.HASEGAWA

HiSeq.RunB143

NextSeq.Run54

NextSeq.Run29

NextSeq.Run38

HiSeq.RunA125

NextSeq.Run118

NextSeq.Run142

HiSeq.RunA304

NextSeq.Run73

NextSeq.Run81

NextSeq.Run43

NextSeq.Run45



C

SARC084 & RNA012_16_074

GACATCTTCACCTTTGACCGTACAGCATCTGCATTGCCGGGACCGGATATG

D I F T F D R T A S A L P G P D M

CIC exon 18 NUTM1 exon 3

SARC101

GACATCTTCACCTTTGACCGTACAGTGTACATTCCGAAGAAGGCAGCCTCC

D I F T F D R T V Y I P K K A A S

CIC exon 18 NUTM1 exon 6

RNA003_16_008

CGCAAGAAGAGGAAGAACTCCACGGTGTACATTCCGAAGAAGGCAGCCTCC

R K K R K N S T V Y I P K K A A S

NUTM1 exon 6CIC exon 17

RNA012_16_073

CCCAGCCCCGCAGGGGGCCCAGACCACGGACCTCCTGCTCCTGAGGCACCC

P S P A G G P D H G P P A P E A P

NUTM1 exon 6CIC exon 20

B

WT CSKDLEAFNPESKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYW*

SARC068 CSKDLEAFNPESKELLDLVEFTNEIQTLLGSSVSLEAFNPESKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYW*

SARC050 CSKDLEAFNPESKELLDLVEFTNEIQTLLGSSVFMEAFNPESKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYW*

SARC078 CSKDLEAFNPESKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYW*

SARC074 CSKDLEAFNPESKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDELLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYW*

SARC030 CSKDLEAFNPESKELLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYWLDLVEFTNEIQTLLGSSVEWLHPSDLASDNYW*

A

SARC061

SARC070

SARC088

SARC102VGLL2 exon 2 NCOA2 exon 14

CCAAGGAGCTCTGGGCCCTGGCGAGGAATGATTGGTAACAGTGCTTCTCGG

P R S S G P W R G M I G N S A S R

VGLL2 exon 2 NCOA2 exon 13

CCAAGGAGCTCTGGGCCCTGGCGAGCTTTTAATAACCCACGACCAGGGCAA

P R S S G P W R A F N N P R P G Q

GGTCTGGGCCTCAGCGTGGACTCAGGAATGATTGGTAACAGTGCTTCTCGG

G L G L S V D S G M I G N S A S R

VGLL2 exon 3 NCOA2 exon 14

SARC065


D

Supplementary Figure S3: Details of VGLL2-NCOA2/CITED2 (A), CIC-NUTM1 (B), BCOR-ITD (C) and

EWSR1-PATZ1 (D) fusion points.

Sanger sequences of the fusion points are given in A, B and D together with amino acid sequences of the

translated fusion genes. Amino acid sequences of the internal tandem duplications in exon 15 of BCOR are

shown for BCOR-ITD-positive tumors (C).

EWSR1 exon 8 PATZ exon 1

SARC041

CGGGGAGGAGGACGCGGTGGAATGGGAATGAACTTCAAAATTAAAGACGGCCCCCGAAAGAGG

R G G G R G G M G M N F K I K D G P R K R

EWSR1 intron 8

(936nt from exon8)


SARC026

CGGGGAGGAGGACGCGGTGGAATGGGAAGTGACCCCGACGGCCCCCGAAAG

R G G G R G G M G S D P D G P R K


RNA045_17_001

CGGGGAGGAGGACGCGGTGGAATGGGCCTCCAGCTGGGCTACATCGACCTT

R G G G R G G M G L Q L G Y I D L

CGGGGAGGAGGACGCGGTGGAATGGGTCCCACTTTTCATTATGCTGCCGGGGGCCCCCGAAAGAGGAGCCGG

R G G G R G G M G P T F H Y A A G G P R K R S R


RNA034_16_244

EWSR1 intron 8 (1689nt from exon8)

EWSR1 exon 9 (NM_001163287)

GCTGGGTGAGAATGGGCTACCCATC

L G E N G L P I


SARC017

CCTTCCTCCTCCGAGGCTGGGTGAG

L P P P R L G E

CGGGGAGGAGGACGCGGTGGAATGGG

R G G G R G G M G


CGTGGAGGCAGAGGTGGCATGGGTGATGTCCTTGCATTGCCCATTTTTAAG

R G G R G G M G D V L A L P I F K

FUS exon 6 TFCP2 exon 2

RNA020_16_136 & RNA009_16_062

CCAGCAGCCACTGCACCTACAAGTGATGTCCTTGCATTGCCCATTTTTAAG

P A A T A P T S D V L A L P I F K

EWSR1 exon 5 TFCP2 exon 2

SARC049

B

AV

GLL2-F

used c

luste

r 2 (

4)

BC

OR

-Rearr

anged

sarc

om

a(1

2)

NU

T m

idlin

ecarc

inom

a(5

)

FIC

(2)

VG

LL2-F

used c

luste

r 1 (

3)

DS

RC

T (

3)

EW

SR

1-N

FA

TC

2 s

arc

om

a(4

)

extr

askele

tal m

yxoid

chondro

sarc

om

a(4

)

Uncla

ssifie

dsarc

om

a(6

0)

Mesenchym

alchondro

sarc

om

a(2

)

LP

F-N

T (

3)

Low

gra

de f

ibro

myxoid

sarc

om

a(2

)F

US

-NF

AT

C2 s

arc

om

a(3

)

BA

F-D

eficie

nt(1

2)

Solit

ary

fib

rous

tum

or

(7)

EW

SR

1-P

AT

Z1 s

arc

om

a(5

)R

enalcell

carc

inom

a(3

)C

IC-F

used

(12)

Myxoid

Lip

osarc

om

a (

3)

Synovia

l S

arc

om

a(2

)A

lveola

rsoft

part

sarc

om

a(4

)C

lear

cell

sarc

om

a(5

)E

RM

S (

2)

FE

T-E

TS

Ew

ing s

arc

om

a(7

)E

ML4

-ALK

sarc

om

a(3

)A

RM

S (

4)

FE

T-T

FC

P2 (

3)

2

4

6

8 ENSG00000171094 (ALK)

Inte

nsitie

sin

Log2(T

PM

+2) ENSG00000164362 (TERT)

FU

S-N

FA

TC

2 s

arc

om

a(3

)

NU

T m

idlin

ecarc

inom

a(5

)

DS

RC

T (

3)

FE

T-E

TS

Ew

ing s

arc

om

a(7

)

Synovia

l S

arc

om

a(2

)

EW

SR

1-P

AT

Z1 s

arc

om

a(5

)

Renalcell

carc

inom

a(3

)

AR

MS

(4)

Alv

eola

rsoft

part

sarc

om

a(4

)

Myxoid

Lip

osarc

om

a (

3)

CIC

-Fu

sed

(12)

VG

LL2-F

used c

luste

r 2 (

4)

BA

F-D

eficie

nt(1

2)

Cle

ar

cell

sarc

om

a(5

)E

WS

R1-N

FA

TC

2 s

arc

om

a(4

)

extr

askele

tal m

yxoid

chondro

sarc

om

a(4

)

Uncla

ssifie

dsarc

om

a(6

0)

BC

OR

-Rearr

anged

sarc

om

a(1

2)

EM

L4

-ALK

sarc

om

a(3

)

Solit

ary

fib

rous

tum

or

(7)

FIC

(2)

LP

F-N

T (

3)

Mesenchym

alchondro

sarc

om

a(2

)

VG

LL2-F

used c

luste

r 1 (

3)

Low

gra

de f

ibro

myxoid

sarc

om

a(2

)

ER

MS

(2)

FE

T-T

FC

P2 (

3)

2

4

6

8

Inte

nsitie

sin

Log2(T

PM

+2)

**

**

Supplementary Figure S4: Sequence of the fusion points and genes expressed in FET-TFCP2-positive

tumors

A. Sanger sequencing of EWSR1-TFCP2-positive sample (SARC049) and FUS-TFCP2-positive

samples (RNA020_16_136, similar to RNA009_16_062)

B. Boxplots for ALK and TERT gene expression level as log2(transcript per million + 2) across all

tumor samples demonstrate strong signals in FET-TFCP2 tumor samples. Number of samples for

each boxplot is indicated under brackets. ** : Welsh t-test p-value < 0.01.

B

C D

E F

G H

ENSG00000175832 (ETV4)

2

4

6

8

10

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

**

ENSG00000179111 (HES7)

2

4

6

8

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

ENSG00000183337 (BCOR)

2

4

6

8

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

n.s.

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

*****

ENSG00000132434 (LANCL2)

3

4

5

6

7

2

4

6

8

**ENSG00000162624 (LHX8)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

ENSG00000172116 (CD8B)

2

4

6

8

Inte

nsitie

sin

Lo

g2

(FP

KM

+2

)

2

4

6

8

10

ENSG00000004700 (RECQL)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

12 ***

2

4

6

8 ENSG00000132975 (GPR12)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***


A

I

J

K L

M N

OP


ENSG00000165553 (NGB)

2

4

6

8

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

ENSG00000158022 (TRIM63)

2

4

6

8

10

12

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

2

4

6

8

10 ENSG00000179761 (PIPOX)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

ENSG00000151715 (TMEM45B)

2

4

6

8

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

2

4

6

8

10 ENSG00000188992 (LIPI)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

ENSG00000197696 (NMB)

2

4

6

8

10

12

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

***

2

4

6

8

10ENSG00000119866 (BCL11A)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

**

2

4

6

8

10ENSG00000181092 (ADIPOQ)

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

*

Supplementary Figure S5: Expression of the most specific gene for each tumor entity (with more than 2 samples)

A. Expression level of LANCL2 gene throughout all samples demonstrating its specificity for VGLL2-Fused tumors

B. Expression level of ETV4 gene throughout all samples demonstrating its specificity for CIC-Fused tumors

C. Expression level of BCOR gene throughout all samples demonstrating its lack of specificity for BCOR-rearranged tumors

D. Expression level of HES7 gene throughout all samples demonstrating its specificity for BCOR-rearranged tumors

E. Expression level of LHX8 gene throughout all samples demonstrating its specificity for EML4-ALK-positive tumors

F. Expression level of CD8B gene throughout all samples demonstrating its specificity for FUS-NFATC2-positive tumors

G. Expression level of GPR12 gene throughout all samples demonstrating its specificity for EWSR1-PATZ1-positive tumors

H. Expression level of RECQL gene throughout all samples demonstrating its specificity for FET-TFCP2-positive tumors

I. Expression level of TRIM63 gene throughout all samples demonstrating its specificity for TFE3-fused sarcoma

J. Expression level of NGB gene throughout all samples demonstrating its specificity for EWSR1-WT1 desmoplastic small

round cell tumors

K. Expression level of PIPOX gene throughout all samples demonstrating its specificity for PAX-FOXO alveolar

rhabdomyosarcoma

L. Expression level of TMEM45 gene throughout all samples demonstrating its specificity for EWSR1-NFATC2-positive

tumors

M. Expression level of NMB gene throughout all samples demonstrating its specificity for FET-NR4A3 extraskeletal myxoid

chondrosarcoma

N. Expression level of LIPI gene throughout all samples demonstrating its specificity for FET-ETS Ewing sarcoma

O. Expression level of ADIPOQ gene throughout all samples demonstrating its specificity FET-DDIT3 myxoid liposarcoma

P. Expression level of BCL11A gene throughout all samples demonstrating its specificity for NUTM1-BRD3/4 NUT-midline

carcinoma

Q. Expression level of NEMP2 gene throughout all samples demonstrating its specificity for NAB2-STAT6 Solitary Fibrous

Tumors

R. Expression level of GLP1R gene throughout all samples demonstrating its specificity for FET-CREB1/ATF1 clear cell

sarcoma

Number of tumors by groups are indicated under brackets. *, ** and ***: Welsh t-test p-value < 0.05, 0.01 and 10-4,

respectively, n.s.: not significant

Q

***

R

ENSG00000189362 (NEMP2)

2

4

6

8

10

Inte

nsitie

sin

Lo

g2

(TP

M+

2)

ENSG00000112164 (GLP1R)

2

4

6

8

Inte

nsitie

sin

Lo

g2

(TP

M+

2) *


transcriptomic definition of molecular subgroups of small

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