EWS erg and EWS Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations

EWS-erg and EWS-Fli1 fusion transcripts in

Ewing's sarcoma and primitive

neuroectodermal tumors with variant

translocations.

M Giovannini, … , B S Emanuel, G A Evans

J Clin Invest.

1994;

94(2)

:489-496.

https://doi.org/10.1172/JCI117360

.

We have determined the frequency of EWS fusion transcripts in a series of primary Ewing's

sarcomas and peripheral primitive neuroectodermal tumors and cells lines. Type 1 and 2

EWS-Fli1 fusions were demonstrated in 8 cell lines and 14 patient samples. Five patients

with cytogenetically characterized rearrangements of chromosome 22 that did not involve

chromosome 11 were included in these studies. A novel EWS-Fli1 in-frame isoform fusing

EWS to exon 8 of Fli1 was isolated from a tumor with a variant t(12;22;22)(q14;p1;q12)

translocation. Three in-frame isoforms of a novel hybrid transcript derived from the fusion of

EWS with the ETS domain of the human erg gene were identified in patient samples and a

cell line with cytogenetically unidentified or cryptic translocations involving chromosomes

21 and 22. Interphase analysis by fluorescent in situ suppression hybridization using two

overlapping erg yeast artificial chromosome clones demonstrated disruption of the erg gene

on chromosome 21 in a patient sample with monosomy 22. Our results provide new

information about the involvement of EWS in small round cell tumors involving exchange of

its putative RNA-binding domain with DNA-binding domains derived from different members

of the ETS family of transcription factors. These studies emphasize the utility of reverse

transcriptase PCR analysis and fluorescent in situ hybridization as additional diagnostic

tools for differential diagnosis among small round cell tumors.

[…]

Research Article

Find the latest version:

EWS-erg and EWS-Flil Fusion Transcripts in Ewing's Sarcoma and Primitive

Neuroectodermal Tumors with Variant Translocations

Marco Giovannini,* Jaclyn A. Biegel,* Massimo Serra,§ Jian-Ying Wang,*Yalin H. Wei,* Lynn Nycum,* Beverly S.

Emanuel,*

and Glen A. Evans*

*MolecularGeneticsLaboratory, TheSalkInstitutefor Biological Studies, La Jolla, California 92037;tDivisionofHuman Genetics and MolecularBiology, Children's Hospital, and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104;and§Laboratory of Oncological Research, IstitutiOrtopedici Rizzoli, Bologna, 40136Italy

Abstract

We have determined the frequency of EWS fusion

tran-scriptsinaseries ofprimary Ewing's sarcomas and periph-eralprimitiveneuroectodermal tumors and cell lines. Type 1 and 2EWS-Flil fusions were demonstrated in 8 cell lines and 14 patient samples. Fivepatients with cytogenetically characterized rearrangements of chromosome 22 that did

notinvolve chromosome 11 wereincludedin these studies. Anovel EWS-Flil in-frame isoformfusing EWS to exon 8 of Fliu was isolated from a tumor with a variant

t(12;22;22)(q14;pl;q12) translocation. Three in-frame isoforms ofanovelhybridtranscriptderived fromthe fusion of EWS with the ETS domain of the human erg genewere

identified inpatient samples andacellline with

cytogeneti-cally unidentified orcryptic translocations involving chro-mosomes 21 and 22. Interphase analysis by fluorescent in situ suppression hybridization using two overlapping erg yeast artificial chromosome clones demonstrateddisruption ofthe erg geneonchromosome 21 inapatient samplewith monosomy 22. Our results provide newinformation about theinvolvementof EWS insmall round celltumors

involv-ing exchange of its putative RNA-binding domain with DNA-binding domains derived from different members of the ETSfamily oftranscriptionfactors. These studies

em-phasize the utility of reverse transcriptase PCR analysis

andfluorescentinsituhybridizationasadditional diagnostic tools for differential diagnosis among small round cell

tu-mors.(J. Clin. Invest. 1994.94:489-496.)Key words: small round cell tumors *chromosomaltranslocations * yeast arti-ficial chromosomes * fluorescent in situ hybridization * in-terphasecytogenetics

Introduction

Ewing's sarcoma

(ES)'

is ahighly malignant tumor of bone and soft tissues that mostoften affects young adolescents (1).

Address correspondence to Dr. Marco Giovannini, (present address) Laboratoirede

Gendtique

des Tumeurs, Institut Curie, 26 Rue d'Ulm, 75231 Paris Cedex 05, France.

Receivedfor publication 22 November 1993 and in revised form 2 March 1994.

1.Abbreviations used in this paper: AT, Askin's tumor; ES, Ewing's sarcoma;FISSH, fluorescent in situ suppression hybridization; PN, pe-ripheral neuroepithelioma; PNET, primitive neuroectodermal tumors;

RT-PCR, reverse transcription PCR; SRCT, small round cell tumors; YAC, yeast artificial chromosomes.

Approximately 83% of all cases are associated with a

t(1l;22)(q24;q12) reciprocal translocation (2-4). Cytogenet-ically identical translocationshave been described in othersmall round celltumors (SRCT) such asprimitive neuroectodermal tumor(PNET),peripheral neuroepithelioma (PN),and Askin's tumor(AT) (5).The recentcloningof the EWS andFliu genes

disrupted by the t(11;22)(q24;q12) reciprocal translocation

characteristic of these SRCT has provided new insight into

possible mechanismsoftumorigenesisin solid tumors(6).

Fur-ther, the cloningofhybridfusiontranscriptsbetween EWS and

theATF- 1 transcriptionfactorgeneinmalignantmelanoma of

soft partswith a balancedt(12;22)(q13;q12)translocation(7), the fusion of TLS (translocated in liposarcoma) gene with

CHOP gene (member of the c/EBPfamily oftranscriptional activators) as aresult of thet(12;16)(q13;pll) translocation

inmyxoid liposarcoma (8), and the fusion of PAX3 gene toa

member of the forkhead genefamily in alveolar

rhabdomyosar-comas withat(2;13)(q35;q14) translocation (9, 10) suggest

a commonmechanism ofactivation oftranscriptionfactors that

isresponsible fordevelopmentof different tumortypes. Human Fill (11) is a memberof the ETS familyof

tran-scription factors that exhibit homology within a conserved DNA-binding domain, termed the 3'-ETS domain(12).EWS

(6) is a gene of unknown function with extensive sequence similaritytoTLS(8). Both belongto a newsubclass of

RNA-binding proteins thatcontain a conserved 80-amino acid

do-main in their carboxyl terminus (13). The ES- and PNET-associated t(11;22)(q24;q12) translocation results in ahybrid

mRNA transcribed from the derivative chromosome 22, in which the3' DNA-binding domain of Flil replaces the 3' RNA-binding domain of EWS (6). Hybrid EWS-Flil constructs

in-duce malignant transformation when introduced into murine fibroblasts ( 14). EWS-Flil fusion genes are heterogeneous since three differentbreakpointsinFliu andtwobreakpointsin

EWShave been describedsofar, resulting in four unique fusion sequences (6, 14).Theincidence of the different fusion

tran-scripts inalarge number of patients hasnotyet been determined. Variant and complex translocations have been reported in 9% of all ES and PNET cases (4, 15). In most instances they involve chromosome 22ql2, suggesting that the fusion gene

onthe derivative chromosome 22is related to tumorigenesis. Molecular studies published todate demonstrated that all ES

and PNETwith typicalt(11;22)(q24;ql2) translocations and

oneES cellline withacomplext(11;22; 14) translocation con-tained EWS-Flil fusion transcripts (6, 14, 16). Rearrangements of theEWS andFliu geneswerealsofound intwo tumors with

variant t(7;22) andt(14;22) translocations and in one tumor withanormalkaryotype (17). These findings are similar to the

casesofchronic myeloid leukemia in which cytogenetic analy-sisdoesnotdemonstrate the typicalt(9;22)(q34;ql1) translo-cation yet contains bcr-abl chimeric transcripts (18).

J. Clin. Invest.

©DThe AmericanSocietyforClinical Investigation, Inc.

0021-9738/94/08/0489/08 $2.00

TableI. Pathological and Cytogenetic Characteristics of the Cell Lines and Tumors

Age/sex Diagnosis Partialkaryotype Fusion

Cell line (reference)

TC-71 (5) 22/M ES ofbone t(1 1;22)(q24;qI2) Type 1 6647 (5) 14/F ES ofbone t(l1;22)(q24;ql2),22q+ Type 2

SK-ES-1 18/M ES ofbone t(11;22)(q24;q12) Type 2

RD-ES 19/M ES ofbone 22q- Type 2 TC-32 (5) 17/F PN t(1 1;22)(q24;qI2) Type 2 LAP-35 (23) 12/F PNET of bone t(11;22)(q24;q12) Type 2 SK-PN-LI (24) 3/M PNET of bone t(21;22)(q22;qI2) Type 9e SK-PN-DW (24) 17/M PNET of soft parts -11;t(11;22)(q24;q12) Type 1 SK-N-MC (5) 14/F AT ofchest wall t(2;11;22;21)(q32;q24;q12;pl1) Type 1 U20S (25) 15/F Osteosarcoma Complex abnormalities None SK-N-SH 4/F Neuroblastoma der(22)t(22;?)(qI3;?) None Tumor

T89-135 17/M SRCTconsistent with ES -22 Type le

T91-124 hI/M PNET der(21)t(21;?)(q22;?) Type le

?der(22)t(22;?)(ql2;2)

T92-60 11/F ES ofbone der(22)t(22;?)(ql2;?) Type 3e

T93-98 12/F SRCTconsistent with -22 None alveolarrhabdomyosarcoma der(l6)t(16;22)(q?23;ql1-12)

der(22)t(22;?)(ql 1-12;?)

T93-101 14/F ES t(l1;22)(q24;ql2) Type 2 T93-113 13/M SRCTconsistent with PNET t(l2;22;22)(q14;pl;q12) Type 8

Variants ofspecific chromosomal translocations in multiple leukemia subtypes have been reported. The cloning of some of theserarevarianttranslocations has led to the discovery of new genes that share sequence homology and/or common motifs with the gene disrupted by the "typical" translocation. A nota-ble example is the group of translocations that fuse the MLL gene on llq23 (19-21) to genes from a variety of different chromosomes (22). These genes share extensive sequence ho-mology and presumably confer similar biological activities to thechimeric protein. Cloning of the genes involved in variant translocations in solidtumorsshould also result in anincreased understanding of the mechanisms involved in the development of thesemalignancies.

Inthis study,wehaveinvestigated the frequency and

speci-ficity of EWS alterations in a series of cell lines and tumors

obtainedfrompatients with SRCT. Cell lines and tumor

speci-mens with typical t( 1;22) translocations and with

re-arrangements of chromosome 22 in the presence of normal chromosomes11 werecharacterizedcytogeneticallyandstudied with molecular techniques. Novel transcripts involving EWS andthe ETS family-related erg orFlil genes wereobserved, expanding the repertoire of possible gene rearrangements

in-volved in thepathogenesisofESandPNET.

Methods

Cell lines and patientsamples. 11 tumorcell lines wereanalyzed in thisstudy.Their clinicalfeaturesatdiagnosisandpartial karyotypesare

listed in Table I(23-25).Cell linesRD-ES, SK-ES, SK-N-MC,

SK-N-SH,andU20SwereobtainedfromtheAmericanType Culture Collec-tion(Rockville, MD).SixSRCTcases werecytogenetically character-izedatTheChildren'sHospitalofPhiladelphia, accordingtostandard

methods(26).Cytogeneticabnormalities of the relevant chromosomes

arelisted in Table I. 17patient samplesofcytologically, histologically,

andimmunohistochemically confirmedSRCTswere not characterized

cytogeneticallyand wereobtained from the Istituti Ortopedici Rizzoli,

Bologna,Italy.Surgicaltumorsampleswerecryopreserved beforetheir

usein these studies.Informedconsentwasobtainedaccording to institu-tionalguidelines.

Reverse transcription PCR (RT-PCR) analysis of EWS-Flil and EWS-erg fusions. Total RNA was isolated from frozen tumorfragments

and from celllinesin culture as described(27). 1

,jg

of totalRNA was reverse transcribed with an oligo-d(T)16 primer using the GeneAmp RNA-PCR kit(Perkin-ElmerCorp.,Norwalk,CT). PCRamplification

was performed with 2.5 U of Taq DNA polymerase, 2 mM MgCl2,

lxPCRbufferH(Perkin-Elmer Corp.), 200

pM

deoxynucleotide tri-phosphate, and 0.15

jtM

each5' and3'primers. Oligonucleotide 22.1 (7)or22.3(6)wereused as5' (forward)EWSprimers.Primer 11.3

(6)wasthereverseprimeron exon9 of the human Fli1gene(Zucman, J., T. Melot, B. Plougastel,L.Selleri, M.Giovannini, G. A. Evans,0.

Delattre, and G. Thomas, manuscript submitted forpublication). The reverseprimerfor the human erg gene (28) 21.9, 5'-ATGAGAAGG-CATATGGCTGG-3', wasused to specifically amplify erg moieties.

The amplification profile consisted ofdenaturation at 94°C for 30 s,

primer annealingat65°Cfor 60 s, and extensionat72°Cfor 2 min, for

atotalof30cyclesonanautomatedheat block(GeneAmpPCR system

9600; Perkin Elmer-Cetus Instruments, Norwalk, CT). Positive and

negative controlswerecarried throughall steps. Asapositivecontrol for theintegrityof RNA isolated from eachtumorandcellline,PCR

amplificationof the/3-actingenewasperformed usingforwardprimer

f3Al

(nucleotides 2563-2582 of GenBank M10277) in combination with reverse primer /3A2 (nucleotides 2970-2989 of GenBank M10277),whichproducea314-bp product.

Directsequencing ofPCRproducts. PCRproducts werepurified

and concentratedon aMicrocon 100(Amicon, Beverly,MA).Primer 22.3 (6) was used for sequencing ofEWS-Flil and EWS-erg PCR

products usingaTaq DyeDeoxyterminatorcycle sequencingkit

(Ap-pliedBiosystems,Inc.,FosterCity, CA).Reactionswererunand

ana-lyzed on a fluorescent DNA sequencer (373A; Applied Biosystems,

Inc.).

Multicolorfluorescentin situhybridizationwith erg yeastartificial

H

l lto lo

Nr rw Ad _

M:

z l bd

XD N M

Type

_ 2~~-<--< ₁

-se

-314

00 0)iI

E-4

N M

T

(22.

UI(E2.

C

H L W >a0ich°H cN m li) _{N M}

500- 413- 311- 249- 200-

151-P-ACti

Figure 1.RT-PCR analysis of EWS-Flil and EWS-erg chimeric transcript expression in SRCT. (A) Cell lines. PCR analysis was per-formed onoligo-dT reverse-transcribed total RNAsamples (1 jug) with primers 22.1 (EWS exon 7) and 11.3 (Flil exon9). Am-plification products were separated by elec-trophoresis on 2% agarose gels and visual-ized by ethidium bromide staining. RNAs de-rived from t(11;22)-negative U20S and SK-N-SH were used asnegative controls. N,no template; M, molecular weight standards

bp

(OX

174/HinfIll).The fusion types are indi-cated on theright side, and their sizes with primers 22.1 and 11.3 or 21.3 are: type 1, 355 bp; type 2, 421 bp; and type 9e, 181 bp. The 314-bpfragment shown below is ampli-fied using b-actin primers for quality control of the RNAs. (B) Cytogenetically character-ized patient samples. Samples were amplified using primers 22.3 and 11.3. EWS-erg type 1 fusions isolated from patients T91-124 and T89-135 (lanes 3 and 5) were amplified

us-typ

e ing primer 21.9 that is specific for the erg

-1 e gene and does not amplify EWS-Fli I fusions.

3+21.9) The sizesof the fusion types indicated on the right are: type le, 328 bp (primers 22.3 and

2 11.3) and 735 bp (primers 22.3 and 21.9);

1e type2,394bp (primers 22.3 and11.3); type

3+11.3) 3e,580bp (primers22.3 and11.3);and type

8,202bp (primers22.3 and11.3).N, no

8 template; M, molecular weight standards

3 14 bp

(+X

174/HinfIll).

(C)

Cytogenetically

un-characterizedsamples. PCR analysis was performed on 17 cytologically,

histologi-cally,andimmunohistochemicallyconfirmed SRCTs. IncludedwereeightESs(lanes 1-5 and10-12), three metastatic ESs (lanes

6-8),onePNET(lane 9),twosmall cell

Type osteosarcomas (lanes 13 and 14), and three 12

non-Hodgkin lymphomas

of bone(lanes

15-|1 17).Samples were amplified using primers 22.3 and 11.3,and the sizesof the fusion

<-9e typesareindicated in B.N, notemplate; M,

1-3 14 bp molecular weight standards

(OX

174/Hin-fill).

prepared as described previously (25). Overlapping YAC clones

A125B12andB19C12from theWashington Universityhumangenomic YAClibrary wereshownpreviously tobe colinear andtocontainthe humanerggene(29).To confirm that the YAC strains A125B12and B19C12 contained the desired human insert, PCR analysis with two sets ofprimers corresponding tothe 5' and 3' regionsofthehuman erg gene was performed. Primers for the erg 5' region were erg5S (5'-CCTCTCGGTTATTCCAGGATC-3') (forward) and erg5A (5'-GTCCGGGACAGTCTGAATCAT-3') (reverse). Primers for the erg 3'regionwereerg3S(5'-TTCAAGATGACGGATCCCGAC-3') (for-ward) and primer 21.9 (reverse).Fluorescent in situsuppression hybrid-ization(FISSH) analysis using YAC clones A125B12 and B19C12was carried out as reported (30). For two-color FISSH, YACA125B12 DNAwaslabeled withbiotin-1l-dUTP (ENZO Diagnostics, Syosset, NY),andYAC B19C12 DNAwaslabeledwithdigoxigenin-l1-dUTP (BoehringerMannheim Corp., Indianapolis, IN) by random priming. Hybridization andposthybridization washes of the slideswerecarried

outasdescribed(25).Afterposthybridization washes,slideshybridized only to biotin-labeled probes were incubated in avidin-fluorescein as

described (25). Slideshybridized to both biotin- and digoxigenin-la-beledprobes wereamplifiedand detectedasdescribed(31).

Results

Identification ofdistinct EWS-Flil and EWS-ergfusion isoforms

in ES and PNET withoutt(11;22) (q24;q12)translocation. To

define the chromosomal translocations and/orgene fusions in

cases with variant rearrangements, we performed molecular

analysis on avariety ofcell lines and tumor material. RD-ES and SK-PN-LI cell lines (Table I)wereanalyzed byRT-PCR. The PCR amplification product generated using

reverse-tran-scribed RNA from cell lineRD-ES as atemplateandprimers

A

4IZ; I U) q r- Pa z X m I

I I I I I X

E t w C: Es a 50

413

311-24

200

15

M-ACtin R |ll

B

M

H H

HHI

o

Oi

HH

rn

I

I

I

I

I

I

I

m m w w am m m

H

H

p E

E

Ep

726

413

311

249

200-4

f3-Actinl

A

EWS-Flil

Type

8

P T 8 Y P P Q T a 8 Y S Q A P S Q Y S Q _tCCTAZZ=CW=QLAAaCTGTCCTXCAGCCALGCCA&GTCATATJGCCAA

22.1

Q8 8 8 Y QQ P Y Q I L G P T S S R

CAQLOCAGCTACOGGCGCAGl bTCCGTATCAGATCCTGGGCCCGACCAGCAGTCGC

L A N P C S G Q I Q L W Q F L L E L L S

CTAGCCAACCCTGGAAGCGGGCAGATCCAGCTGTGGCAATTCCTCCTGGAGCTGCTCTCC

D S A N A S C I T W E G T N G E

GACAGCOCCAACGCCAGCTGTATCACCTGGAGcG ,cAcGGGAGT

11.3

P T 8 Y P P Q T a 8 Y S Q A P 8 Q Y 8 Q

__-t"gTc~~j~ ~ WZATATAOCCAA_rlq

22.1 _(yele) 223

Q 8 S Y G Q Q L P Y E P R R S A W

CAGQCAOCTACQhOOQCA l TTTACCATATGAGCCCCCCAGGAGATCAGCCTGG

T G H G H P T P Q S K A A Q P S P S T V ACCGGTCACGGCCACCCCACGCCCCAGTCGAAAGCTGCTCAACCATCTCCTTCCACAGTG

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

CCCAAAACTGAAGACCAGCGTCCTCAGTTAGATCCTTATCAGATTCTTGGACCAACAAGT

s(Type 9e)

S R L A N P S G Q I Q L W Q F L L E L

AGCCGCCTTGCAAATCCAG CAGTGGCCAGATCCAGCTTTGGCAGTTCCTCCTGGAGCTC

L S D S S N S S C I T W E G T N G E F K

CTGTCGGACAGCTCCAACrCCAGCTGCATCACCTGGAAGGCACCAACcGGGAGTrCAAG

M T D P D E V A R R W G E R K S K P N M ATGACGGATCCCGACGAGGTGGCCCGGCGCTGGGGAGAGCGGAAGAGCAAACCCAACATG

N Y D K L S R A L R Y Y Y D K N I M T K AACTACGATAAGCTCAGCCGCGCCCTCCGTTACTACTATGACAAGAACATCATGACCAAG

V H G K R Y A Y K F D F H G I A Q A L Q GTCCATGGGAAGCGCTACGCCTACAAGTTCGACTTCCACGGGATCGCCCAGGCCCTCCAG

P H P P E S S L Y K Y P S D L P Y M G S CCCCACCCCCCGGAGTCATCTCTGTACAAGTACQCCTCAGACCTCCCGTACATGGGCTCC

Y H A H P Q K M N F V A P H P P A L P V TATCACGCCCACCCACAGAAGATGAACTTTGTGGCGCCCCACCCTCCAGCCCTCCCCGTG

T S S S F F A A P N P Y W N S P T G G I

ACATCTTCCAGTTTTTTTGCTGCCCCAAACCCATACTGGAATTCACCAACTGGGGGTATA

Y P N T R L P T S H M P S H TACCCCAACACTAGGCTCCC _{CAGCCATWGCCTTCTCAT}

N D 5 a P D L D L L P Y E P P R R ...ATGOUTAAGGACCAGATCT TTTACCATATGAGCCCCCCAGGAGA...

Figure2.Molecularanalysis ofEWS-erg

andEWS-Flilchimeric mRNAs.(A) Nucle-otidesequenceandpredictedamino acid

se-quenceof EWS-Flil type8andEWS-erg typele,3e,and 9e fusiontranscriptsobtained

by RT-PCR. Sequences underlined

corre-spondtotheprimers usedfor PCR

amplifi-cation and their names areindicated.Vertical bars indicate the differentjunctions,and the

contributionofthe EWS sequenceis high-lightedin boldcharacters. (B)Schematic

representationofEWS, Flil, and erg func-tionaldomains and their locationsonthe

non-rearrangedproteinsand thefusion proteins

translated from the cDNAsrepresentedin

(A). TheRNA-binding domain of EWS (RNA BD) is replaced by theETSdomains

(ETS D) of Flil orerg, dependingonthe

fusion type. Fusion proteins type 8andtype

9e arefully represented, whiletype le and type3ecanbededuced fromtype9e by the insertion of 59 additionalerg aminoacids. Intype3e, 84additional amino acidsare

derived from the EWS sequence. Interrupted

codons are indicated.

22.1 and 11.3 was 421 bp in size (Fig. 1 A). Nucleotide

se-quenceanalysis of thehybrid transcriptrevealedin-frame

junc-tions betweenEWS codon 265 and Flil codon 197,which is identicaltotype 2 fusion describedpreviously (6).

Amplifica-tion of SK-PN-LI cDNAs resulted in aproduct of 181 bp de-rived from the fusion of EWS with the human erg gene, located

onchromosome 21(Fig. 1 A).Althoughnotreportedpreviously (24), a t(21;22)(q22;ql2) translocation was observed in

karyotypesprepared from the SK-PN-LI cell line. Basic local

alignmentsearch tool (32) analysis (BLAST)of the GenBank database withprimer 11.3 nucleotide sequence revealed 90%

homology with the region extending from nucleotide 842 to

861of the published erg sequence(28).Therefore,primer 11.3

can amplify bothEWS-Flil and EWS-erg fusion transcripts.

Nucleotide sequence analysis of the fragment revealed an

in-framefusionoccurringbetween the first base(A)of codon 265

of EWS (Ser) and the G in the secondpositionofcodon 192 oferg(Gly), creating a newAGC codonfor aserineresidue (Fig. 2, A and B). This region of the erg gene shows 98% homologywith the exon 9regionof Fli1, and we willtherefore

refer to this fusion astype 9e.Sixdifferent erg isoformshave been described, generated by alternative splicing events (28, 33, 34). The ergmoieties clonedin thisstudyare in aregion

common to all the described erg isoforms. The erg-i cDNA

sequencewastherefore used for nucleotide and codon numera-tion (GenBank database accession number M21535). Oligo-dT-primed cDNAs from five SRCT patients that cytogeneti-cally donotcontain the typical t(11;22)(q24;q12)

transloca-tion(Table I)wereamplified usingthe combination ofprimers

22.3 and 11.3 (Fig. 1 B). Tumors from patients T91-124 and T89-135 showed a328-bpamplification productconsistent with an EWS-Flil type 1 fusion. Surprisingly, however, sequence

EWS-erg

Type

le

&

9e

EWS-erg

NH2

1 452

!11111

_m

IET,OoH

261

NH2 ETS D OOH

1 1265

RNA BD

265 349 4 Type 3e

NH2 j-

_jO~OHl

Type le

&

3eK/

133 192

NH2 TS OH

462 Figure2.Continued

analysis revealed anovel in-frame EWS-erg fusion occurring between the firstbase(A)of codon265 of EWS(Ser)andthe A in the secondposition of codon 133 oferg (Asp),creating a newAAT codonforanasparagine residue (Fig. 2,A andB). Because of thissimilarityin size withEWS-Flil fusiontype1, we define this fusion as type le. To improve the quality and

specificityof thePCRamplificationofEWS-ergfusions,a new reverseprimer, 21.9,wasdesigned. Primer 21.9 isintheregion immediately downstreamof the erg ETS domain(nucleotides

1249-1268 of GenBank M21535) that doesnotshow homology

withFlil.Asexpected, amplification of T91-124 and T89-135

cDNAsusing primers22.3 and21.9resulted insingle

amplifi-cationproductsof 735bp (Fig. 1 B, lanes 3 and5).Thesame combination ofprimersfailedtodetectthe EWS-Fliltranscripts

presentinSK-PN-DWandRD-EScelllines(datanotshown), confirming thepredicted specificity ofprimer21.9 for the hu-man erggene.Reexamination ofkaryotypesfromcaseT9 1-124 demonstrated a translocation involving 21q22 and a possible translocationinvolving 22ql2. CaseT89-135 wasmissing one chromosome 22, and no obvious rearrangements of chromo-some 21 were seen. To confirm that therearranged erginfact resulted in disruptionofthegene onchromosome 21,we per-formedinterphaseFISSHanalysis with twooverlapping YAC clonesencompassingtheerggene(29).PCRanalysis demon-strated that YAC clone A125B12 contains the 3'regionoferg andnotthe 5' and, conversely, YACB19C12 contains the5'

andnotthe 3'region (datanotshown,seeMethods fordetails onprimersequences). TogethertheseYACsspan - 430 kb of

genomic DNA on human chromosome 21q22.2 (29). After

FISSH, 46% of the nuclei fromtumor sample T89-135 con-tained threehybridization signals (Fig. 3), one yellow signal

duetotheoverlapof thetwoYACsonthe normal chromosome

21, onegreen signal corresponding toYAC B19C12 (5' erg

region),andoneredsignal correspondingtothe translocated3' ergregion contained in YAC A125B12. Theremaining nuclei

displayedtwoyellow hybridization signals indicatingtwo nor-mal erg genes andprobably representnormal diploid stromal

cellsinfiltratingthetumorbiopsy.Athird,distincttypeof

EWS-erg fusion was found in tumor T92-60. This tumor

demon-strated what appeared to be the derivative 22 from a

t( 1;22)(q24;q 1-2) translocation. Asingle fragmentof580bp

wasdetectedafter30 cyclesofamplification with primers 22.3 and 11.3 (Fig. 1 B). Sequence analysis of the region

encom-passing the fusion breakpoint revealed an in-frame junction between thefirst base(G)of codon349of EWS (Gly)and the A in the secondpositionof codon 133 oferg (Asp), creating a newGATcodon foranasparagine residue (Fig. 2,AandB).

EWScodon349wasreported tobeinterrupted byfusionwith Flil codon219. This was classifiedastype3 (6). Becauseof thestrikingsimilarities of thetwofusionmechanisms, werefer tothis newEWS-erg fusionas type 3e. AnEWS-Flil fusion

isoform was isolated in tumor T93-113. Cytogenetic studies from thistumordemonstratedavariantt( 12; 22; 22) transloca-tion in thepresenceoftwoapparentlynormal chromosomes 11. Arepresentative karyotype is showninFig. 4. One der(22)is similar to the der(22) seen as a result of the typical

t(11;22)(q24;ql2) translocation. RT-PCR amplification with

primers 22.3 and 11.3 (Fig. 1 B) produceda202-bp fragment

resultingfromanin-framejunctionbetween the A inposition

oneof EWS codon 265 (Ser) andthe A inposition 2 of Fill codon 261 (D), the first codon of Fill exon 8 (Fig. 2,A and

B).Acryptic translocationinvolvingchromosomes 11 and 22

wasthuspresentinthistumor.Wetermed thisEWS-Flilfusion as type 8. Despite amplification of the

_f3-actin

gene (Fig. 1 B), tumorT93-98 didnotdemonstrate EWS-Flil orEWS-erg fusionsbyRT-PCRanalysis.Thediagnosisfor thispatientwas undifferentiated sarcoma consistent with alveolar rhabdomyo-sarcoma. Cytogenetic studies demonstrated translocations

in-volvingbothchromosomes 22, althoughneitherresembled the

der(22) ofat(ll;22)(q24;q12) translocation. Thepresumed 22ql1-12 breakpoints are proximal to EWS and suggest the

presenceof another gene on chromosome 22 involved in the

developmentof solidtumors.

Incidence ofthe variousfusion transcripts in ES/PNET

B

Flil

EWS/ Fl ii

Type 8

EWS

656 k~~bOOH

EWS/erg

Type 9e

erg

__-m_

---1

Figure 3. FISSH with erg YAC clones. In-terphase nucleus from ES tumor T89-135 after two-colorFISSHwith YACsA125B12

and Bl9C12,biotin- and digoxigenin-la-beled, respectively. Hybridization signals were detected using Texas red avidin and fluorescein-anti-digoxigenin. Data were col-lected, and images were reconstructed using a confocalmicroscope(MRC-600;Bio-Rad, Cambridge, MA). Separate images of each

fluorochromewerecollected anddigitally

su-perimposed (imageone:red andgreen). De-lineation of the nucleus was obtained by blue pseudocoloring of a third image collected with the610-nmfilter to visualize the nucleus stained by diluted propidium iodide (image 2:blue). The final image was obtained by double-exposure photography of images 1 and 2onthe computer screen. The yellow

signal(thick arrow) corresponds to the

nor-mal, nontranslocatedergallele and is gener-atedby the proximity of green/red hybridiza-tionsignalsof the twooverlapping YAC clones.Disruptionof the erg gene with the translocation isvisualizedby separation of the red (left) and green(right)signals(thin arrows)correspondingtoYACA125B12

(erg3' region: ETSdomain) and B19C12 (erg 5' region),respectively.

carrying thetypicalt(JJ;22) translocation andin cytogeneti-cally uncharacterized tumors. To study the incidence of the

differentfusiontranscripts and their occurrence intumors with different phenotypes and cytogenetic abnormalities, we ex-tendedourRT-PCRanalysisto6 cell lines and1 patient sample (T93-101) with typical t(11;22)(q24;ql2) translocations, 1

cell line with a complex t(2;11;22;21) translocation, and 17

:*

#

I

& 'I

,4

!..

A

Isw:

6

ai

13

19

'I

2

7

14

..5

20

CN

3

i

3

..J

I

8

1.a

5

Vt

9 10

4.4

16

21

tumor samples withcytologically, histologically, and immuno-histochemically confirmed SRCT for which cytogenetic data were notavailable. AnEWS-Flil type1chimeric transcriptwas found ineight unkaryotyped Ewing'stumors(Fig. 1 C) and in

three cell lines (Fig. 1A). Of the cell lines, one wasderived from ES (TC-71), one from a PNET (SK-PN-DW), and one from an AT (SK-N-MC) carrying a complex t(2;l1;22;21)

4

45

as

r

Figure 4. Representative karyotypefrom

caseT93-113.Arrowspointtotheder(12)

and thetwoderivative chromosomes 22. Thechromosome 22ontheleft is similar

totheder(22) seenina

typical

8

t(11;22)(q24;ql2)

translocation. The

der(22)ontherightcontainsthelongarm

ofchromosome 12(q14-+qter)translocated

to22p.Theder(12)contains thelongarm

( I *, ofchromosome 22 (ql2-+qter).The

chro-mosomes _{11 appear}tobe normal. Loss of

2 ' _{X Y}

achromosome 14 and 21 arerandom, and

translocation. Karyotypes prepared from the SK-PN-DW cell

line demonstrated a typical t(11;22)(q24;ql2) translocation,

aswellasthe monosomy11whichhad beenreported previously (24). AnEWS-Flil Type2transcript was found in 3 of the 17 tumors (Fig. 1 C): 2 Ewing's Sarcomas and 1 PNET ofthe

bone,in 4cell lines(Fig. 1A):1 wasderived froma PN(TC32),

1 from a PNET of bone (LAP-35), 2 from ES (SK-ES; 6647), and in ESpatient T93-101 (Fig. 1 B). 1 additional ES among the 17 SRCT demonstrated a type 9e EWS-erg fusion (Fig. 1

C). Our RT-PCR assay didnotreveal the presence of the type 4 fusion in PNcell line TC-32, which had been described pre-viously (14).Despiteamplification of the

P-actin

gene (Fig. 1

C). RNA from fiveunkaryotypedtumorsamplesdid not result

inamplificationof fusion transcripts. On the basis ofcytologic,

histologic, and immunohistochemical analyses, three tumors

hadbeendiagnosed asnon-Hodgkin's lymphoma of bone and

two as small cellosteogenic sarcoma.

Discussion

Molecular studies ofprimary tumor specimens from ES and

PNETpatientsas well asestablishedcelllines demonstratethat the majority oftumors contain a typical t(ll;22)(q24;ql2)

translocation, resulting inatype 1 ortype2 EWS-Flil fusion transcript (6, 17).Atpresent, there doesnotappeartobe any correlation with tumorphenotype, and the relationship of the molecular findings to clinicalpatient outcomeis unknown. In

this report, wedescribe fivetumorspecimens which were

se-lected forstudybased on the cytogenetic finding of arearranged

ormissing chromosome 22, in the presence of normal chromo-somes 11.Inonecasewithavariantt(12;22;22)(q14;pl;q12)

translocation, we identifiedanovelEWS-Flil fusion (type 8). Thistumortherefore had acryptic ormasked translocation in-volving chromosomes 11 and 22and issimilartowhat has been

seen inchronicmyeloid leukemia (35). Three of these tumors,

as well as one cell line, and an additional SRCT in which cytogenetic analysis was notperformed donothave an EWS-Flil fusion, but containoneof three EWS- erg fusion transcripts.

Ineach case, theNH2-terminal domain ofEWSisfusedtothe

ETS DNA-bindingdomainof the erg gene. ERG is amember of the ETS family of genes which share highest homology with Flil(11, 12) andwasfound to be rearrangedinhumanmyeloid leukemia witht(16;21) translocation (36).

In recent studies (Zucman, J., T. Melot, B. Plougastel, L. Selleri, M. Giovannini, G. A. Evans, 0. Delattre, and G. Thomas,manuscript submitted for publication), we determined the genomic organization and exon structure of the Flil gene

and showed that it spans 120 kb of genomic DNA and is en-coded by nineexons. Flil exon9 encodesthe ETS domain and begins withaconserved glycine codon that is split by the8th intron/9th exon junction between nucleotides in position one and two (G/GA). Although the genomic structure and exon organization ofthehuman erg gene have not beendetermined yet, the striking amino acid sequence identities and the high degree of conservation of intron/exon boundaries found for the othermembers ofETS family for which the genomic structure is known (Flil, ETS1 and ETS2) imply that erg has an exon

structure similar to that of Flil. Furthermore, analysis of the

EWS-ergfusions we have isolated indicates two potential exon/

intronboundaries. Flil codon219 (asparagine), which issplit in EWS-Flil type 1 and 3 fusions (6), is interrupted by an intron/exon junction. In theEWS-erg fusion types le and 3e,

wefound erg codon 133(asparagine) to be split between the G

in the first position and theAin the second position, indicatinga

potential intron/exon junction. Another intron/exon junction could interrupt codon 192 oferg (glycine) between the G in

the firstposition and the G in the second position which was

found to be fused to EWS in EWS-erg fusion type 9e. This

fusion isoform, connecting theNH2-terminal domain ofEWS directlytotheergETSdomain,has not been describedforFlil and may indicate that the ETS domain itself is sufficient to

induce transformation. In fact, similar studies (37-39) have

demonstrated that theEWS/Flil chimericproteinis a

transcrip-tional activator, suggesting that itderegulates thetargetgenes

ofFlil.

Themapping of marker D21S60tothe ergYAC A125B 12

(29) gives us additional information regarding the genomic organization of erg. In anintegration mapof human chromo-some21, D21S60is located centromeric to theerg gene (40).

In addition to our PCRresults, this evidence stronglysuggests that the erg gene is transcribed in a telomere to centromere

direction. Since a simple balanced translocation would juxta-pose the 5'region ofEWS to the 5' end oferg and not the 3'

end, this putative erg orientation suggests that a productive EWS- erg fusion requires a complex chromosomal

re-arrangement. Additional FISSH studies with chromosome-spe-cificpainting probes andprobes encompassingorflankingthe

translocation breakpoints will be necessary toprecisely define

thedynamics of these chromosomal rearrangements.

Anotherrationale for this studywas toanalyze the incidence of the different fusion types inaspectrum oftumorswithknown

phenotype. The breakpoints resulting in EWS/Flil type 1 and type2 fusions appeartobe, byfar, the mostcommon, and the

occurrence of either type1 or 2doesnot seem tobe correlated

to whether the tumors are considered to be ESs or PNETs.

Although we have analyzed a significant number of tumors,

analysisofalargerseries is necessarytodetermine whether the

occurrenceof distinct fusion types isarandomevent. Consider-ing the number of novel fusion transcripts wehave identified inarelatively smallnumberof cases,wepredict that additional studies will probably reveal other fusion isoforms occurring

between EWS-Flil, EWS-erg, and fusion of EWS with genes

onotherchromosomes. Furtherbiologicalstudiesareneededto

determine the roleof the fusion transcripts in the development of thesetumors.At present, RT-PCR and FISSHclearly provide

a sensitive means by which to identify the molecular events

involved in the standard, variant, and masked translocations in ES andPNET.

Noteaddedinproof. Since submission of this article two independent groups(Zucman, J., T. Melot, C. Desmaze, J. Ghysdael, B. Plougastel, M.Peter, J. M. Zucker, T. J. Triche, D. Sheer, C. Turc-Carel, et al. 1993. EMBO [Eur.Moi.Bio. Organ.] J. 12:4481-4487) (Sorensen, P. H. B., S.-L. Lessnick, D. Lopez-Terrada,X. F. Liu, T. J. Triche, and C. T.Denny. 1994. Nat. Genet.6:146-151)have reported similar EWS-erg fusiontranscripts.

Acknowledgments

WeareindebtedtoDr.0. Delattreand Dr. G. Thomas, Laboratory of TumorGenetics,InstitutCurie (Paris, France), for invaluable discussion and for makingtheir unpublishedresults available. We thank Dr. T.

Triche,Children'sHospitalof Los Angeles (Los Angeles, CA) for the generousgiftof celllinesTC-71, TC-32, and 6647; and Dr. L. Helson and Dr.C.Helsen,New YorkMedicalCollege(Valhalla, NY) for cell lines SK-PN-DW and SK-PN-LI. We wish to thank Dr. P. Close,

Baker, Princess Margaret Hospital, Perth, Western Australia; Dr. C. Rubin, The University of Chicago Medical Center; and Dr. K. Leung, Kaiser Permanente, for contribution of patient samples.

M.Giovannini was a recipient of an Italian Research Council (CNR) fellowship. This work was supported by grantHG00202 from the De-partment of Energy, grants from the G. Harold and Leyla Y. Mathers Foundation (G. A. Evans), and grants CA 47983 (J. A. Biegel) and CA39926 (B. S. Emanuel) from the National Cancer Institute.

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