[CANCERRESEARCH56, 3886-3890, September 1, 1996]

Advances in Brief

Two Distinct Amplified Regions at 17q11-.q21 Involved in Human PrimaryBreast Cancer'

Ivan Bièche, Catherine Tomasetto, Catherine H. Régnier,Christel Moog-Lutz, Marie-Christine Rio, andRosette Lidereau2

Laboratoire d'OncogénEtique, Centre RenéHuguenin, 35 rue Dailly, F-92211 St-Cloud [I. B., R. LI, and Institut de Génétiqueet de Biologie Moléculaireet Cellulaire, CentreNational de la Recherche Scientifique/Institut National de Ia Sante et de Ia Recherche Médicale/UniversitéLouis Pasteur, B P 163, 67404 Illkirch Cedex, C. U. de Strasbourg[C. T., C. H. R., C. M-L, M-C. R.), France

Abstract

Chromosomal segment 17q11—q21is a commonly amplified region inhuman breast carcinomas. Several lines ofevidence suggest that ERBB2 is

the gene responsible for the emergence of this amplicon, but four novelgenes (called MLNSO, MLN 51, MLN 62, and MLN 64) in 17q11—q21haverecently been found to be amplified and overexpressed in breast cancercell lines. We investigated 98 primary breast tumors for amplification ofthese five loci. Twenty-five tumors (25.5%) showed amplification of atleast one of these markers, but most amplifications did not encompass allof the testedloci. The genesmostfrequentlyamplifiedwereERBB2andMLN 64 (22 of 25 amplified cases). MLN 64 was always coampilfied with

ERBB2, and to a similar leveL Amplification of these five genes alwaysleads to overexpression of their mRNA; we observed no cases of overexpression without amplification in any of these genes. Our results suggestthat: (a) an independent, amplified region defined by MLN 62 (also calledCARTJ or TRAFJ) is located in 17q11-q12; (b) in addition to ERBB2,MLN 64 is a major target for the 17q12.-q21 amplicon; and (c) these MLN

genes could be of pathogeaetic significance in breast cancer.

Introduction

Gene amplification plays an important part in the pathogenesis andprognosis of various solid tumors, including breast cancer, probablybecause overexpression of the amplified target gene confers a selective advantage. The first technique used to detect gene amplificationwas cytogenetic analysis. Amplification of several chromosomal regions, visualized as either extrachromosomal double minutes or integrated homogeneously staining regions, are among the major visiblecytogenetic abnormalities found in breast tumors (1, 2). Other techniques, such as CGH3 and a novel strategy based upon chromosomemicrodissection and fluorescence in situ hybridization, have also beenused in broad searches for regions of increased DNA copy number intumor cells (3, 4). These different techniques have revealed some 20amplified chromosomal regions in breast tumors. The amplified regions result in 5- to 100-fold amplification of a small number ofgenes, few of which are thought to contribute in a dominant mannerto the malignant phenotype. Positional cloning efforts have beenstarted to identify the critical gene(s) in each amplified region. To

Received 6/3/96; accepted 7/17/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Ligue Nationale de Lutte Contre le Cancer and the

ComitésRégionauxdu Haut-Rhin, des Hauls de Seine, du Val d'Oise et des Yvelines, theInstitut National de [a Sante et de [a Recherche Médicale,the Centre National de IaRecherche Scientifique, the Centre Hospitalier Universitaire Regional, the MutuelleGénéralede l'Education Nationale, the Groupe de Recherches et d'Etudes sur lesGénomes(Grant 94/50), the Association pour la Recherche sur le Cancer, the Fondationpourla RechercheMédicaleFrançaise,andtheFondationde France.R. L. is a researchdirector with the Institut National de Ia Sante et de Ia Recherche Médicale.

2 To whom requests for reprints should be addressed. Phone: 47-11-15-66; Fax:

47-11-16-96.3 The abbreviations used are: CGH, comparative genomic hybridization; TRAF, tumor

necrosis factor receptor-associated factor.

date, genes known to be amplified in breast cancers include FGFRJ(8pl2); MYC (8q24); FGFR2 (10q26); CCNDJ, GSTPJ, and EMS](11q13),IGFR and FES (15q24—q25),and ERBB2 (17q12—q21)

(reviewed in Ref. 5).Segment qi 1—q21of chromosome 17 seems to be one of the most

commonly amplified DNA regions in human breast carcinomas. Fluorescence in situ hybridization, CGH, and chromosome microdissection have shown a major increase in DNA sequence copy number inthis region (3, 4, 6). Amplification of 17q12—q21 was originallydiscovered in breast carcinoma by using a probe to the ERBB2 gene(7). Other tumor types followed rapidly, including cancers of theovary, stomach, and bladder, and less frequently, lung and coloncarcinomas. Interestingly, amplification at 17q12—q21 has clinicalrelevance in breast cancer because independent studies have shownassociation with an increased risk of relapse (7, 8). To date, only onegene, ERBB2, has been implicated in the emergence of this amplicon.The ERBB2 proto-oncogene belongs to the ERBB family, the firstidentified member of which (ERBBJ) encodes the epidermal growthfactor receptor (9). ERBB2 amplification is associated with overexpression of its product. This gene is a good candidate for a role inbreast cancer because of its transforming potency (10) and the factthat transgemc mice carrying the ERBB2 gene show altered mammarycell proliferation and a high incidence of mammary adenocarcinomas(11).

All of these initial reports point to a potential role for the ERBB2proto-oncogene at 17q12—q21in human breast carcinomas. However,four novel genes (called MLN 50, MLN 51, MLN 62, and MLN 64) inthis chromosomal region have recently been identified by differentialscreening of a cDNA library established from breast cancer-derivedmetastatic axillary lymph nodes (12). The MLN 51 and MLN 64 genesshow no homology with other genes already described. MLN 62 (alsoknown as CART] or TRAF4) is a novel member of the TRAF family(13), whereas MLN 50 (also named Lasp-1) defines a new LIMprotein subfamily characterized by the association of a LIM motif anda domain of region 3 Src homology (SH3) at the amino- and carboxylterminal parts of the protein, respectively (14).

These four genes have been found to be amplified and overexpressed in breast cancer cell lines. Amplification of l7ql l—q21DNAsequences may thus be more complex than first suspected, and thenumber and identity of the target gene(s) are unknown.

We tested a large series of primary breast tumors for amplificationof ERBB2 and the four novel genes. We report that 25.5% of thebreast tumors show amplification of one or more of these genes.Preliminary mapping of the amplicons suggests the involvement oftwo distinct amplified regions at 17q11—q21in human primary breastcancer. Moreover, we suggest three genes (MLN 62/CARTJ,TRAF4,ERBB2, and MLN 64) as likely targets of the amplification event atthese two chromosomal regions.

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amplification of all genes and to similar amplification levels; B,amplification of all genes but to various amplification levels; and C,amplification of some genes only. Fig. 1 shows examples of the most

common patterns of genetic changes; Fig. 2 summarizes data in theform of amplification maps.

Group A (five cases) corresponded to the existence of a single butlarge amplicon at l7ql l—q2l. Amplification levels were always low

(2X-5X) in the five tumors, suggesting polysomies of the entire longarm of chromosome 17. This group is not of great interest to identifythe candidate genes responsible for the emergence of amplicons.

The two other groups (groups B and C; 2 and I 8 cases, respectively) showed that the size and the amplification level varied fromtumor to tumor. Tumors T0084, T0284, and TI 191 had the smallestamplicon, involving only MLN 62. With the exception of these threetumors, the amplicons in the remaining 17 tumors included ERBB2and MLN 64. Interestingly, ERBB2 and MLN 64 were always coamplified to similar levels. In three cases (T0l09, T1273, and T15l2),these were the only genes amplified at l7ql l—q21. In five othertumors (T0391, T0l83, T0309, T0559, and T0588), the ampliconswere discontinuous between MLN 62 and loci ERBB2 and MLN 64. Inthese tumors, there was no evidence of MLN 50 amplification.

These findings pointed to the existence of two distinct amplifiedregions at l7ql l—q12 and l7q12—q21 in human primary breast can

cer, one including the MLN 62 locus and the other including ERBB2and MLN 64 loci, respectively.

B MLN62

Patients and Methods

Tumor and Blood Samples. Ninety-eight primary breast tumor sampleswere obtained at the Centre RenéHuguenin (St-Cloud), and 8 samples of

normal breast tissue were obtained at the HOpital St. Louis (Paris, France).None of the 98 patients with primary breast cancer had undergone radiation

therapy or chemotherapy. Immediately following surgery, the tissue sampleswere placed in liquid nitrogen until extraction of high molecular weight DNAand RNA. A blood sample was also taken from each patient.

DNA Probes. A pMACI 17 probe (a 0.8-kb AccI DNA fragment from agenomic clone of ERBB2) was used to detect ERBB2 (ATCC No. 53408). The

four novel clones (MLN 50, MLN 51, MLN 62, and MLN 64) have been

described in detail by Tomasetto et a!. (1 2). These five probes have been

positioned and ordered by means of in situ hybridization ( 12). The control

probes for Southern blot analysis were the human @3-globin gene ( 15) and the

MOS proto-oncogene (ATCC No. 41004). The control probe for Northern blotanalysis was a 0.7-kb Psi'! fragment of the 36B4 cDNA, described by Masia

kowski et a!. (16).DNA Analysis. DNA was extracted from tumor tissue and blood leuko

cytes according to standard methods ( 17). Ten p@gof TaqI-restricted DNA were

separated by electrophoresis in agarose gel (leukocyte and tumor DNA sampies from each patient were run in adjacent lanes) and blotted onto nylon

membrane filters (Hybond Nv; Amersham Corp.) according to standard tech

niques. The membrane filters were hybridized with nick-translated 32P-labeledprobes, washed, and autoradiographed at —80'C for an appropriate period.

Detection of DNA Amplification. Restriction enzyme-digested tumorDNAs were compared with matching leukocyte DNA in the same agarose gels.

Blots of these gels were first hybridized with ERBB2 and the four MLN probes.

Rehybridization of the same blots with the MOS and f3-globin probes provided

a control for the amount of DNA transferred to the membranes. The proto

oncogene and control gene autoradiographs were first scored by visual inspec

tion and then by densitometry. Gene amplification was defined as two or more

gene copies per haploid genome. Amplification was quantified by serialdilutions of tumor DNA to obtain a Southern hybridization signal similar to

that obtained with the relevant leukocyte DNA.

RNA Analysis. RNA was extracted from normal and tumoral breast tissueby using the LiCI/urea method (18). Ten @.tgof mRNA were fractionated by

electrophoresis on 1.2% agarose gels containing 6% formaldehyde and ana

lyzed by blot hybridization after transfer onto nylon membrane filters (Hybond

N; Amersham). The same filters were first hybridized with ERBB2, and the

four MLN nick-translated 32P-labeledprobes were hybridized in 50% formamide at 42°C.Membranes were washed in stringent conditions in 0. 1X SSPEand 0.1% SDS at 50°Cand subjected to autoradiography for various periods at—80°C.Membranes were also rehybridized with a 36B4 eDNA probe corresponding to a ubiquitous RNA. The signal obtained was used to check the

amount of mRNA loaded on the gel in each experiment. The 36B4 signal also

showed that the mRNA samples were not extensively degraded.

Evaluation of RNA Overexpression. The relative intensity of the mRNAbands was assessed by visual examination and confirmed by means of densi

tometry, taking the ubiquitous 36B4 bands into account. An increase in

expression of at least 2-fold relative normal breast tissue was scored as

positive. Overexpression was quantified by serial dilution of tumor mRNA to

obtain a Northern hybridization signal similar to that obtained with normalbreast tissue.

Results

Normal DNA (peripheral blood leukocytes) and autologous tumorDNA from 98 breast cancer patients were screened on Southern blotsfor amplification of five genes (ERBB2, MLN 50, MLN 51, MLN 62,and MLN 64) located at l7ql l—q2l. Amplification was detected at atleast one locus in 25 of the 98 tumors (25.5%). Densitometric analysis

revealed that amplification levels varied not only from case to casebut, in some tumors, from gene to gene also. Amplification rangedfrom 2-fold to more than 30-fold.

17q11—q21Amplicon Maps in Human Breast Carcinomas. The25 amplified tumors were subdivided into three groups on the basis ofthe pattern and level of amplification as follows: A, tumors with

309 11911512LT

LT LT

.MLN5O

.ERBB2

.MLN64 ‘@

., .,*

.MLN51

Fig. I . Southern blot analysis of three representative breast cancer tumor DNAs withamplifications ofchromosomal region l7qll—q21. L and T, matched Taql-digested DNAsamples isolated from peripheral leukocytes and tumor tissue, respectively. Hybridizationswere carried out successively with probes MLN 50. MLIV 51. MLN 62. MLN 64. andERBB2. Case 309 shows amplifications for MLN 62. ERBB2, and MLN 64. Case I I91shows amplification for only MLN 62. Case 1512 shows amplifications for ERBB2 andMLN 64.

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TWO I7q1l-q21 AMPLIFIED REGIONS IN BREAST CANCER

MLN 62 MLN 50 ERBB2 MLN 64 MLN 51

F ______________________________@ 11 ]I@ II IF I

F 11 @I IF 11 1@ 1@ 11 II 1@ I@@@@ F:::1

T0038

10179

GroupA 10379

11549

11628

I I1:::;:::::.:::.::::i@ I:;.@;:..i.@:.:.:@:1F:.::.:..:..:.::.:.:..II I@ II I I II III II I F II II II IF II II II I I II II I I I

@ I IF II I

@ II II. II II II I I II I I I

@ II II II II I

F II II II II II I I II II II II II II I

10523

10540

T1373

10212

10402

10109

11273

11512

GroupC 11290

11767

10391

10183

10309

10559

10588

10084

10284

11191

Fig. 2. 17q1 1—q21amplicon maps in human breast cancer. Lines correspond to each tumor sample; columns correspond to each marker. The densitometrically determinedgene dosages (amplification levels) were subdivided intofour categories. White boxes, a normal copy number; shadedboxes, 2—5times amplification; dark shaded boxes, 6—10times amplification; black boxes, >10 times amplification.The loci from 17q11—q21are orderedaccordingto theirchromosomal location, from the most centromeric locus(MLN 62) to the most telomeric locus (MLW 51; Ref. 12).

I I<2X2X-5X 5X-1OX >1OX

Expression of ERBB2 and the Four MLN Genes in HumanBreast Carcinomas. To determine whether the amplification ofERBB2 and the four MLN genes contributed to elevated expression,we compared RNA expression with DNA amplification. This wasdone on a total of 20 tumor samples for which total RNA wasavailable; 10 samples among the 25 tumors amplified at least onelocus and 10 un-amplified tumors.

Fig. 3 shows examples of overexpressed tumors evaluated byNorthern blot analysis. No gross alteration in the size of the mRNAwas detected in any of the samples. We also observed perfect overlapbetween RNA overexpression and DNA amplification. The amplifiedtumors were always overexpressed for amplified genes, and the fivegenes were never overexpressed in the 10 un-amplified tumor DNAspecimens. Despite the technical difficulty of obtaining quantitativedata from Northern blots, there was an apparent correlation betweenRNA levels and the degree of DNA amplification. Tumors with highdegrees of amplification showed higher mRNA levels, irrespective ofthe analyzed gene.

Discussion

There are various approaches to detecting genes whose amplification may be responsible for tumorigenesis. Cytogenetic analysis,CGH, and chromosome microdissection have led to the identificationof distinct amplified chromosomal regions that might harbor genescontributing to tumorigenesis. Studies using pulsed-field electrophoresis have shown that amplicons in human tumor cells usuallycomprise large regions of genomic DNA, which can be up to severalmegabases in length and contain several genes (19). Fine-scale molecular mapping of amplified regions is needed to locate such genesprecisely. Coamplification of genes located in a limited chromosomalregion has been described in human tumors. Examples include thecomplex coamplification of multiple genes from I 1q13 in humanbreast cancer (20) and from 12q13—q14in human malignant gliomas(21).

Several authors have observed amplification of the ERBB2 genefrom 17q11—q21 in human breast cancer (7, 22—24).Because fournovel genes from this chromosomal segment have recently been

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11318 F I

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Two 17q1l-q2l AMPLIFIEDREGIONSINBREASTCANCER

mechanisms distinct from DNA amplification (e.g., alteration of theregulatory sequence of the genes).

In the majority of the altered tumors, the amplification did notencompass all of the tested loci. The two genes most frequentlyamplified on 17q1 l—q21 in our series were ERBB2 and MLN 64

@ 2 2 (22.5%),whichwerealwayscoamplifiedandoverexpressedtosimilar. levels. The invariable coamplification of ERBB2 and MLN 64 seen in

our study indicates that the two genes are likely to be located in closeproximity to each other at 17q12—q2l, as proposed previously (12). Inconsequence, the amplification and consequent overexpression of

@ 4.0 MLN64andERBB2couldbeofpathogeneticsignificanceforneoplastic breast tissue growth.

A third gene, MLN 62, is a possible target for a second amplicon.It is located centromeric to MLN 64 and ERBB2 at 17q1 1-12 (12).Although MLN 62 was less frequently amplified (17.5%) than MLN64 and ERBB2, it was strongly amplified in most tumors, showing twodistinct amplified regions at 17q11—q21,and was the only amplifiedand overexpressed gene in three tumors (T0084, T0284, and Tll9l).These findings suggest that, in some tumors, amplification of MLN 62

2.2 mayprovideaselectivegrowthadvantage.Evenif theampliconsobserved in our breast tumor series frequently contained MLN 50 andMLN 51, the amplification maps suggest that these two genes are notthe target genes of the amplification, because they were always

j:.: @.. : • 4 Q coamplified with MLN 64 and ERBB2 and never had the highest

S amplification level in individual tumors. Four other genes close to

ERBB2 and found to be coamplified with ERBB2 in 10 to 50% ofERBB2-amplified tumors are THRAJ (25), RARA (26), GRB-7 (27),and TOP2A (28). These four genes were never amplified in theabsence of ERBB2 amplification. Our data, together with these other

)@. I .5 results, suggest that MLN 50 and MLN 51, as well as THRAJ, RARet,

GRB-7,and TOP2A,arejust incidentally included in some l7q12—q21 amplicons.

Little is known about the physiological and pathological functionsof MLN 62 and MLN 64. MLN 64 shows no clear homology withknown genes, whereas MLN62/CARTIITRAF4 encodes a protein withthree domains also observed in the CD4O-binding protein and TRAF2,both of which are involved in signal transduction mediated by thetumor necrosis factor receptor family. The MLN 62/CARTJII'RAF4gene may thus be involved in tumor necrosis factor-related cytokinesignal transduction in breast carcinoma.

In conclusion, this study shows that DNA amplification is frequently observed in two different regions at l7qll—q21 in humanbreast cancer. This suggests that several genes in these two regions areinvolved in the initiation and/or progression of human breast cancer.Our preliminary mapping of these l7ql l—q21amplicons in 25 amplified breast tumors shows that they consistenfly include either MLN62/CARTJIfRAF4 (17q11—q12)or MLN 64 and ERBB2 (17q12—

q21). The two new genes are good candidates for a role in breastcancer because, like ERBB2, their amplification leads to high overexpression. The main conclusion to be drawn from our data is that,although ERBB2 remains a good candidate as one of the genes underselection in 17q1 l—q21amplicons, two novel candidate genes havebeen identified as driver genes of these amplicons. Elucidation of thephysiological and pathological significance of MLN 62/CART]!TRAF4 and MIX 64 should confirm the involvement of these twogenes in breast carcinogenesis.

Acknowledgments

We are indebted to Dr. P. Chambon for kindly providing probe 36B4 andDr. J. T. Wilson for probe human (3-globin.We thank Prof. F. Calvo for hiscollaboration.

.@@ .,@

Fig. 3. Northern blot analysis of MLN 50. MLN 51, MU'/ 62, MLN 64, and ERBB2 innormal and tumoral breast tissues. NJ and N2, normal breast tissues; T309, 11191, andT1512. breast tumor tissues. Hybridizations were carried out successively with probesMLN 50, MU'I 51, MLN 62, MLN 64, and ERBB2. Control hybridizations with the 36B4probe showed that similar amounts of mRNA were loaded in each case. Right, approximate sizes of the mRNAs are indicated in kilobases. Case 309 shows overexpressions forMLN 62, ERBB2, and MLN 64, compared with normal breast tissues. Case 1191 showsoverexpression for only MLN62. Case 1512 shows overexpressions for ERBB2and MLN64.

identified and found to be amplified and overexpressed in breastcancer cell lines (12), we decided to further characterize the l7ql 1—q21 region in breast cancer biopsies by studying amplification of thesefour genes, in addition to the ERBB2 gene, in a large series of tumorDNAs. The aim was to identify the genes within the amplicon,together with their frequency and their level of amplification, andthereby to more precisely define the actual driver gene(s) in thisamplicon(s).

Twenty-five (25.5%) of the 98 tumors showed amplification of atleast one of the five genes. In addition, amplification of these fivegenes was always accompanied by mRNA overexpression. However,it is also known that some tumors with single-copy oncogenes mayoverexpress the corresponding mRNA. We also examined the mRNAexpression of ERBB2 and the four MLN genes in 10 breast tumors thatdid not show amplification. We observed no mRNA overexpression inun-amplified tumors. It thus seems that the four MLN genes, likeERBB2, are mainly activated by gene amplification in breast carcinoma. However, because we examined the mRNA expression in only10 un-amplified tumors, we cannot rule out the possibility that theRNA overexpression of these genes can also be accomplished by

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1996;56:3886-3890. Cancer Res   Ivan Bièche, Catherine Tomasetto, Catherine H. Régnier, et al.   Human Primary Breast Cancer

q21 Involved in−Two Distinct Amplified Regions at 17q11

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