`
`Human Breast Cancer: Correlation of
`Relapse and Survival with Amplification
`of the HER-Z/neu Oncogene
`
`DENNIS I. SLAMON,* GARY M. CLARK, STEVEN G. WONG, WENDY I. LEVIN,
`AXEL ULLRICH, WILLIAM L. MCGUIRE
`
`The HER-Zlnm oncogene is a member of the erbB—like
`oncogene family, and is related to, but distinct from, the
`idermal growth factor receptor. This gene has been
`s own to be amplified in human breast cancer cell lines.
`In the current study, alterations of the gene in 189
`primary human breast eaneers were investigated. HER-Z/
`m was found to be amplified from 2- to
`ter than 20-
`fold in 30% ofthe tumors. Correlation 03:1: am lifiea-
`tion with several disease
`eters was evalua
`Am-
`
`plification of the HER-2km; gene was a signifieant pre-
`dictor of both overall survival and time to rela se in
`
`' cance
`patients with breast cancer. It retained its 3'
`even when adjustments were made for other known
`prognostic factors. Moreover, HER-2km; amplifieation
`ad greater prognostic value than most currently used
`prognostic factors, including hormonal-receptor status,
`in
`ph node-
`'tive disease. These data indicate that
`this gene may
`y a role in the biologic behavior and/or
`pathogenesis 0 human breast cancer.
`
`HE EVIDENCE LINKING PROTO-ONOOGENES TO THE INDUC-
`
`tion or maintenance of human malignancies is largely cir-
`cumstantial, but has become increasingly compelling. This
`circumstantial evidence is derived from studies of animal models,
`tumor cell lines, and actual human tumors. Data from animal models
`and cell lines include: (i) sequence homology between human proto-
`oncogenes and the viral oncogenes of transfomiing retroviruses that
`are known to be tumorigenic in some species (1, 2); (ii) transfection
`studies showing the transforming potential of prom-oncogenes in
`NIH 3T3 cells and primary embryo fibroblasts (3—5); and (iii) the
`central role of certain prom-oncogenes in tumorigenesis by chronic
`transforming retroviruses such as avian leukosis virus (6). Data fi'om
`human tumors include: (i) increased expression of specific proto-
`oncogenes in some human malignancies (7, 8); (ii) localization of
`prom-oncogenes at or near the site of specific, tumor-associated
`chromosomal translocations (9); and (iii) amplification of proto-
`oncogenes in some human tumors (10, 11).
`Additional data linking prom-oncogenes to cell growth is their
`expression in response to certain proliferation signals (12, 13) and
`their expression during embryonic development (14, 15). More
`direct evidence comes from the fact that, of the 20 known proto—
`oncogenes, three are related to a growth factor or a growth factor
`receptor. These genes include c-nk, which is homologous to the
`9 JANUARY 1987
`
`transforming gene of the simian sarcoma virus and is the B chain of
`platelet-derived growth factor (PDGF) (16, 17); c-fim‘, which is
`homologous to the transforming gene of the feline sarcoma virus
`and is closely related to the macrophage colony-stimulating factor
`receptor (CSF-lR)
`(18); and c-crbB, which encodes the EGF
`receptor (EGFR) and is highly homologous to the transforming
`gene of the avian erythroblastosis virus (19). The two receptor-
`related prom-oncogenes, c-fim‘ and c-crbB, are members of the
`tyrosine-specific protein kinase family to which many proto-onco-
`genes belong.
`Recently, a novel transforming gene was identified as a result of
`transfection studies with DNA firom chemically induced rat neu-
`roglioblastomas (20). This gene, called mu, was shown to be related
`to, but distinct from, the c-rrbB prom-oncogene (21). By means of
`v-crbB and human EGFR as probes to screen human genomic and
`complementary DNA (cDNA) libraries, two other groups indepen-
`dently isolated human crbB—related genes that they called HER-2
`(22) and c-erbB-Z (23). Subsequent sequence analysis and chromo-
`somal mapping studies revealed all three genes (neu, c-crbB-Z, and
`HER-2) to be the same (22, 24, 25). A fourth group, also using v-
`erbB as a probe, identified the same gene in a mammary carcinoma
`cell line, MAC 1 17, where it was found to be amplified five- to ten-
`fold (26).
`This gene, which we will call HER-Z/mu, encodes a new member
`of the tyrosine kinase family; and is closely related to, but distinct
`from, the EGFR gene (22). HER-Zlneu diifers from EGFR in that it
`is found on band q21 of chromosome 17 (22, 24, 25), as compared
`to band pll—p13 of chromosome 7, where the EGFR gene is
`located (27). Also, the HER-Zlm gene generates a messenger
`RNA (mRNA) of 4.8 kb (22), which dilfers from the 5.8- and 10-
`kb transcripts for the EGFR gene (28). Finally, the protein encoded
`by the HER-Z/neu gene is 185,000 daltons (21), as compared to the
`170,000-dalton protein encoded by the EGFR gene. Conversely, on
`the basis of sequence data, HER-Zlnm is more closely related to the
`EGFR gene than to other members of the tyrosine kinase family
`(22). Like the EGFR protein, HER-Zlneu has an extracellular
`domain, a transmembrane domain that includes two cysteine-rich
`repeat clusters, and an intracellular kinase domain (21), indicating
`
`and W. I. Levin are in the Division of Hematology-
`D. I. Slamon, S. G. Won
`Medicine and Ionmn Com
`ive Cancer Center,
`Onco
`, Department
`UCLA
`1 of Medicine, Los Angdes, CA 90024. G.
`. Clark and W. L. McGuire
`Science Career at San Antonio,
`an Antonio, TX 78284. A.
`lln'ch is in the
`are in the Division ofOncology, Department ofMedicine, Univets‘ ofTexas Health
`Department of Molecular Biology, Genentech, Inc, South San Francisco, CA 94080.
`
`“To whom correspondence should be addressed.
`
`ARTICLES I77
`
`(cid:43)(cid:82)(cid:86)(cid:83)(cid:76)(cid:85)(cid:68)(cid:3)(cid:89)(cid:17)(cid:3)(cid:42)(cid:72)(cid:81)(cid:72)(cid:81)(cid:87)(cid:72)(cid:70)(cid:75)(cid:3)
`Hospira v. Genentech
`(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:26)(cid:16)(cid:19)(cid:19)(cid:27)(cid:19)(cid:24)(cid:3)
`IPR2017-00805
`(cid:42)(cid:72)(cid:81)(cid:72)(cid:81)(cid:87)(cid:72)(cid:70)(cid:75)(cid:3)(cid:40)(cid:91)(cid:75)(cid:76)(cid:69)(cid:76)(cid:87)(cid:3)(cid:21)(cid:19)(cid:23)(cid:22)
`Genentech Exhibit 2043
`
`

`

`Title 1. Association betwwn HER-2km: amplification and disease parame-
`ten in 103 breast tumors.
`
`Number of tumors
`
`
`Fm"
`Single
`2 to 5
`5 to 20
`>2_o
`Tm,
`PT
`
`copy
`copies
`copies
`copies
`Hormonal m
`m?”
`
`1511+
`ER-
`
`53
`31
`
`1
`
`l
`4
`
`65
`38
`
`0.99
`
`ng+
`PgR-
`
`52
`2—5
`>5
`Unknown
`
`$50
`>50
`Unknown
`
`42
`42
`
`13
`34
`l7
`20
`
`21
`52
`11
`
`6
`2
`5
`1
`Twin (Wm)
`l
`1
`l
`5
`1
`2
`0
`3
`45: atW (was)
`1
`2
`2
`7
`0
`2
`Number {positive lynql: m
`34
`1
`0
`3
`30
`0
`22
`l
`0
`l
`20
`1—3
`25
`2
`2
`4
`17
`>3
`Unknown
`17
`1
`3
`1
`22
`
`
`2
`3
`
`0
`1
`2
`2
`
`l
`4
`0
`
`52
`51
`
`15
`41
`22
`25
`
`25
`65
`13
`
`0.85
`
`0.82
`
`0.83
`
`0.11
`
`thatittooislikelyto be acellularreceptorforanasyetunidentified
`ligand.
`As a result of the published data showing amplification of HER-
`2/neuinahumanmammarycarcinomacellline,andaspaitofan
`ongoing survey in our laboratory of pinto-oncogene abnortnalities
`in human tumors, we evaluamd alterations of the PIER-2km: gene
`inalargesetiesofhumanprimarybreastcancers.0urmultsshow
`that amplification of this gene occurs relatively frequently in breast
`cancer,anddiatitisassociatedwithdiseaserelapseandoverall
`patient survival.
`Factors that are known to be important in the prognosis of breast
`malignancies in individual patients include: size of the primary
`tumor, stage of disease at diagnosis, hormonal receptor status, and
`number of axillary lymph nodes involved with disease (positive
`nodes) (29). The current study, whidi was conductedm two parts,
`involved the evaluation of tissue from 189 separate breast malignan-
`ciesthatwerepartofa breastcancerstudyongoingattthniversity
`of Texas, San Antonio. This cohort of tumors was of interest
`because considerable information was available on the majority of
`the specimens including size ofdie primary tumor, estrogen recep-
`tor status, progesterone receptor status, age of patient, disease stage,
`andstatusoftheaxillarylymphnodes.
`Indieinitialsurvey,dssuefrom 103primarybreastcancerswas
`evaluated for alterations in the HER-2km: gene. DNA from
`individual tumors was prepared as described (30), digested with
`EcoRI, andsubjectedtoSouthern blotanalysiswithanP-labeled
`HER-Zlml probe, whichis known to detect a 13-kb hybridizing
`band in human DNA (22). Examples of tumors from the initial
`survey areshown in Fig. 1. Ofthe 103 samplesexamined, 19(1896)
`showed evidence of HER-2km; gene amplification. The degree of
`amplificationin individual cases was determined by dilution analysis
`(Fig. 2A), as well as soft laser densitometry scanning. To determine
`thatthe amountofDNAloadedineachlanewasequivalerit,all
`filters were washed and rehybridized with a 32P—labeled arginase
`gene probe (31). This probe identifies a 15-ltb hybridizing band on
`Eco Ill—digested human DNA, and was selected as a control
`because it more appropriately assesses the relative amount and
`
`
`
`Receptor mus was analysed as described 39). ER, estrogenreceptorreeepton+ ad -
`
`fixeorrelationofl-IER-Z/manylifi'-
`TStatistiml
`nulligram" ofproeein.’
`bydiex’tesLPvtluawereeomputed
`gmwididiseasepanmetenwere
`aficcombiningthecasetwidiSto
`and>20copies.
`
`with low molecular weight species, which transfer more readily on
`Southern blotting. All
`lanes were shown to contain equivalent
`amounts of high molecular weight DNA (Fig. 28). Individual
`tumorswere assigriedeogroupscontainingasinglecopy,2eo5
`copies, 5 to 20 copies, and greater than 20 copies of the HER-2km;
`gene (Fig. 1). Assignment oftumors to the various groups was done
`
`Fig.1.Analysisofalteiationsofthe1-1Ek-2/m
`inhurnanbreastcancer. Sbownare790fthe
`89breasttiunorsusediuthis.'1‘uriiots
`with a single
`of HER-Zines: 3, 4, 10 to 15,
`20, 23 to 25, 27 to 29, 31, 38, 42 to 46, 48, 49,
`52, 55, 61, 65, 66, 71, 72, and 74. Tumors with
`two to five copies of HER-2km: l, 2, 5, 7, 9, 16,
`17, 19, 21, 22, 32, 35, 36, 47, 50, 54, 56 to 58,
`60, 62, 70, and 75 to 77. Tumors with 5 to 20
`copies of HER-Wm: 6, 8, 26, 34, 37, 39 to 41,
`51, 53, 63, 64, 67, 69, 73, and 79. Tumors with
`more than 20 copies of HER-Zlm: 18, 30, 33,
`59,68,and78.
`oftumors77to79have
`rearrangements in the HER-2km: gene. DNA
`was attracted from tissues and ‘
`with Eco
`Rlasdescribed(30).Atotalof1ugofEcoRI—
`digestedDNAwasbadedontoO.agaroee8%
`WWW
`andtransferred
`onto
`filter
`iodyne 30). Allfilters
`werenywninavmbvenfor3hoursat80°q
`prehybridizedin 5X SSC (standard saline citrate)
`containin 50% formamide, 1096 dent-an sulfate,
`0.1% SD ,denaturedsalmonspetm DNA(1 mg!
`ml), and 4X Denhardts solution for 12 hours,
`then hybridizedin the same solution containing
`321’-labeled nick-translated HER-2 probe (21)
`specificactivityoflxlO’cp
`microgramof
`DNA; 2 x 10‘ m/ml. Hybcpnrfiznon occurred
`at42°Cfor48 cumbflowedbywashingof
`filters under the following conditionsin succes-
`
`178
`
`‘. 2 3‘ 5 '
`”a...
`g,
`1...
`
`6780'
`
`011121314
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`.v
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`, y.
`it» fi”~'
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`
`
`151011131920 2122232425$278291
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`--.flu I
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`
`
`4142644454647484950
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`”qfiswum
`
`51 52535456569390“ QBMGGGG
`
`
`
`-
`sion: 2xSSCfo‘r20minu‘eesatiroom
`ture;twowashesof30minuteseachin2x SC,
`0.1% SDS at 65°C; one wash of30 minutes in
`
`075x 880, 0.1% SDS at 65°C. Filters were then
`exposed to XAR-S x-ray film (Kodak) for aurora-
`Why-
`
`SCIENCE, VOL. 235
`
`

`

` Bs
`
`oatsaasasassssvssssao
`
`amplification. It is well known that the number of positive nodes is
`diebestprognosticlactorfordiseaserecurrenceandsunrivalin
`patients with breast cancer (29). Given the correlation between
`number of nodes positive and HER-Zlm amplification, one might
`predict
`that amplification of this gene might also have some
`prognostic vahte. No long-term follow-up data, however, were
`available on the 103 patients analyzed in the initial study. For this
`reason, a second study was conducted on 100 breast eancer samples
`from patients with positive axillary lymph nodes. All of the informa-
`tion available for the first group of 103 patients was available for
`these patients. In addition, relapse and survival information was
`available, since these cases had a median follow-up of 46 months
`(range 24 to 86 months). Ofthese 100 samples, 86 yielded suficient
`DNA for study. Amplification of the HER-2km: gene was mea-
`sured as in the initial survey, and examples oftumors from this study
`are shown (Fig. 1). Amplifiean'on was found in 34/86 (40%) of
`these patients. For this larger sample of node-positive patients,
`several statistically signifieant or nearly significant relationships were
`observed. In agreement with the preliminary survey, there was an
`association between number of involved lymph nodes and HER-2i
`m amplification (Table 2). In addition,
`the presence of gene
`amplifieation was correlated with estrogen receptor status and size
`of primary tumor (Table 2). Together, these two surveys yielded
`data on 189 patients and the association of HER-2km: amplifica-
`tion with various disease parameters in the combined group is
`shown in Table 3.
`
`While these correlations were of interest, the strong relationship
`
`ofHER-Z/m
`Fig.2.(A)Exampleofdilutionalanalysisto assess
`l2ugofEcoRI-
`geneamplifieation.1.anesa,s,k,andpwereloaded
`digestedbreastmniorDNAlaneaisDNAfromnmflms. l),which
`tsatumorwithasinslecopyofthel-IER/Z-mgene. Lanesis
`D Afromtumor33, whidtrepresentsatumorwith>20¢opieeofrhe
`HER-Zlmgene. Lanesbtofareserialdilutions(l:100, 1:20, 1:10, 1:5,
`andl.2, respectively)oftthNAsampleinlanes. LanekisDNAfroni
`tumor35(Fig. l),whichreptesentsatumorcontainingtwotofivecopiesof
`theHER-Z/mgzie. Laneshtoj areserialdilutions(l:10, 1:5,and122,
`DNAsantpleinlaneLIanepisDNAfromnimoru
`(Fig.l),whichtepresentsatumorwid15to20copiesofthel-IER-2lm
`.I.aneslmoareserialdilutions(1:20, 1:10, 1:5, and122,
`with
`)oftheDNA
`inlanep Thefilnetwas
`and
`be
`32P-Iabeledl-lER—Zprobeasinli
`1. (B)Exampleofarsinase
`hybridizationtodemonsu-ate that «.13th amounts oftumor DNAP'w‘Le
`loadcdintoeachlane.Reh
`offiltercontaininslanes30to40
`(Fig. l). 'I‘hefilserwasfirststrippedoflabelby
`inabufi‘ermadeup
`ofSO'isfm-namide, 3x SSC,and0.l%SDSat6°Clbr20minutet,
`followingbydtmenicmssivewashaofSntinuteseedflnOJx SSCatroom
`
`temperature. FilterswereexposedovemighovernishronXAR-Sfilm(Kodak)eo
`erumeranavalofaflradioamvepmbethen
`asinFis. lwitlia
`nPlabeled human arsinase gene probe (31).
`
`inablindedfashion,indiattheyweremadewithoutknowledgeof
`diseascparameters. Analysisofthedataforassociationbetweengene
`amplificationandanumberofdiseaseparameterswasthenper-
`formed.
`Of103 tumors evaluated inthe initial survey, therewasessentially
`nocorrelanonbetweengeneamplifieationandesn'ogenreceptor
`starus,prosesteronerecepcorstatus,sizeoftumots,orageat
`diagnosis (Table 1). However, when analysis was performed for
`association between HER-2km: amplification and number of posi-
`tive lymphnodes, atreridwasnoted.'I‘hisanalysisshowedthat4/34
`(11%) ofpatients with no involved nodes, 2/20 (10%) with l to 3
`involved nodes, and 8/25 (32%) with >3 involved nodes had gene
`amplification (P= 0.11). Ifthese data wereeramined bycornparing
`0 to 3 positive nodes versus >3 positive nodes, the correlation with
`gene amplification bceame more significant (P < 0.05). Thus, there
`was a significant increase in incidence of HER-2km: gene amplifies-
`tion in patients with >3 axillary lymph nodes involved with disease.
`A multivariate regression analysis to correlate HER-2km: amplifies-
`tion with various disease parameters identified the number of
`positive nodes as the only significant factor, either alone or iii
`combination, to correlate with amplification.
`This initial study indicated thatit might be possible to discrimi-
`nate among node-positive patients on the basis of HER-2/m gene
`
`1.0
`
`Disease-tree
`
`OVOVIII
`
`Fig. 3.Actuarialcurveforrelapsein(A)node-
`positive patients with no
`tion versus
`tients with any amplification (>2
`copies))oi'HE-Eflm and (C) node--positive pa-
`tients with no amp
`vermsof
`positive
`tients with greater than 5 copies HER-2mm.
`Ramada] curve for overall survivalin (B) node-
`positive patients with no
`versus
`positive
`dents with anyamamlifieation (>2
`copies))of HIE-g:2/m and (D)
`positive pa-
`tientswithnoamplificetionversusnodc-positrve
`patients with greater than 5 copies ofHERZlm.
`Actuarialcurvesforbothrelapseandoverallsur—
`vivalwerecomputcdbythemethodofKaplan
`andMeier(44)andcomparedbythelosranktest
`(42—44).
`
`9 JANUARY 1987
`
`
`
`survivalprobability 0 0
`
`9.
`
`.°to
`
`as O
`
`Pa
`
`
`
`survivalmuseum 9oOQ
`
`9n
`
`O
`
`
`
`Not ampmloa (n :52)
`
`
`
`Ampum (mat)
`
`
`
`
`
`Not emplliled (n .52)
`
` Not amplilled (n :52)
`
`
`Amplified (n=11)
`>5 copies
`
`
`
`Not amplllied (n =52)
`
`
`
`
`’5 copies
`
`
`Amplified 0! =34)
`
`Amplified (n :11)
`
`
`
`o
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`o
`84
`Time (months)
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`84
`
`ARTICLES
`
`179
`
`

`

`between HER-Zlm amplification and nodal status (P = 0.002)
`indicated that
`information on amplification of this gene may
`correlate with disease behavior; that is, recurrences and survival. To
`test this, univariate survival analyses were performed in which
`amplification was compared to relapse and survival in this patient
`group. A total of 35 patients had a recurrence of the disease, and 29
`had died at the time of the analyses. Median times to relapse and
`death were 62 months and 69 months, respectively. The median
`follow-up time for patients still alive was 47 months, ranging from
`24 to 86 months. A total of 71 of the 86 patients (8396) received
`some form of therapy after mastectomy: adjuvant systemic therapy
`alone, 47%; adjuvant systemic therapy plus local radiation, 19%;
`and local radiation alone, 17%. A strong and highly statistically
`significant correlation was fbund between the degree of gene
`amplification and both time to disease relapse (P = <0.0001) and
`survival (P = 0.0011) (Table 4). Moreover, when compared in
`univariate analyses to other parameters, amplification of HER-2km:
`was foundtobesuperiortoallotherprognosticfacuors,withdie
`exception of the number of positive nodes (which it equaled) in
`predicting time to relapse and overall survival in human breast
`cancer (Table 4). The association between HER-Wm amplification
`and relapse and survival can be illustrated graphically in actuarial
`survival curves (Fig. 3, A to D). While there was a somewhat
`shortened time to relapse and shorter overall survival in patients
`having any amplification of the HER-2km: gene in their tumors
`(Fig. 3, A and B),
`the greatest differences were found when
`comparing patients with >5 copies of the gene to those without
`amplification (single copy) (Fig. 3, C and D). Patients with greater
`than five copies of HER-2km: had even shorter disease-free survival
`times (P= 0.015) and overall survival
`times (P= 0.06) when
`compared to patients with no amplification. The phenomenon of
`greater gene copy number correlating with a worse prognosis has
`also been seen in evaluations of N-nqyc gene amplification in human
`neuroblastomas (32).
`To determine if amplification of HER-Zlm was independent of
`other known prognostic factors in predicting disease behavior,
`multivariate survival analyses were perfomied on the 86 node-
`positive cases. Amplification of the gene continued 00 be a strong
`prognostic factor, providing additional and independent predictive
`information on both time to relapse and overall survival in these
`
`Tablaz. Association between HER-21m amplificationanddiseaseparame-
`tersin86breasttumrsfromnode—positivepatients.
`2t05
`5to20
`>20
`copies
`copies
`00135“
`
`Factofl
`
`Sinsk
`copy
`
`Total
`
`PT
`
`ER+
`ER-
`
`PgR+
`PgR-
`
`52
`2—5
`>5
`
`550
`>50
`
`38
`14
`
`31
`21
`
`18
`28
`6
`
`16
`36
`
`Hormonal may”m
`21
`5
`2
`4
`
`4
`18
`5
`5
`Tumor size (M)
`8
`3
`12
`2
`3
`4
`
`A5: atW (years)
`12
`6
`11
`3
`
`l
`l
`
`1
`l
`
`0
`1
`1
`
`1
`l
`
`65
`21
`
`54
`32
`
`29
`43
`14
`
`35
`51
`
`0.05
`
`0.14
`
`0.09
`
`0.06
`
`43
`0
`5
`7
`31
`1-3
`>3
`21
`16
`4
`2
`43
`
`
`0.06
`
`'ERandPgRareasdeaaibediiiTablel.
`liER-flmamplificationwidivariousdisease
`thvalueswere
`aftereombining
`weresofewsamplesinthe >20group.
`180
`
`TStanstiealanalysesforeorrelationof
`wereperformed
`thex’
`5m20and>20cases,sineediere
`
`515253545558575859606162 83648566
`
`
`
`leofreh
`
`Fig.4.Exam
`werestri
`..(
`
`'
`ybridization offilter with human EGFR probe. Filters
`asinFig.ZB, andhybridized with nP-labeled human EGFR
`,asiiiFingShownarethekswermolecularweightbands
`widi’zP-labeledEGFRprobeinfilter
`lanesSlto66
`(Fig. l). Thebandsfi'om
`tobottomare28 22 andl8
`respectiv
`Lane52isanexample
`animorshowingmarkedamplificaiion (>50
`copies) oftheEGFRgene
`
`patients, even when other prognostic factors were taken into
`account (Table 4).
`Rearrangement ofthe HER-2km: gene was rare. Ofthe total 189
`tumors evaluated, three showed evidence of rearrangement, and in
`two ofthe three cases, the rearrangement was identical (Fig. 1, cases
`77 to 79). Also, two of the rearranged HER-2/m loci were
`amplified (Fig. 1, cases 78 and 79). The incidence of HER-2km:
`reamngementasdeterminedbyEcoRIdigestionwasmosmallto
`attempt statistical correlations.
`To determine whether the phenomenon of amplification ofHER-
`2/0» in breast cancer extended to related growth factor receptors,
`all filters were analyzed with the EGFR probe (Fig. 4). Amplifica-
`tion ofthe EGFR gene was found in 4/189 (2%) ofthe cases, and
`rearrangementoftheEGFRgenewasfoundinoneoftl'iosefour
`cases. The incidence of EGFR amplification and rearrangement was
`too small to attempt statistical correlation. Comparison of HER-ZI
`an amplification (53/189 or 28%) with that of the EGFR gene
`reveals the incidence of the former to be 14 times greater than that
`of the latter, indicating that the phenomenon of gene amplification
`is not a general one for a related tyrosine kinase—specific receptor in
`human breast cancer. Moreover, studies examining alterations of
`two other tyrosine kinase—specific prom-oncogenes, ab! and fogin
`breast cancer did not show amplification of these genes (33).
`Alterations ofnon—tyrosine kinase—related prom-oncogenes in these
`
`Tabb 3. Association between HER21m amplification and disease parame
`ters in combined surveys (189 patients).
`
`2t05
`5co20
`>20
`.
`copies
`copies
`copies
`
`Total
`
`H
`
`Factor'
`
`Single
`copy
`
`ER+
`ER-
`
`PgR+
`PgR—
`
`52
`2—5
`>5
`Unknown
`
`550
`>50
`Unknown
`
`91
`45
`
`73
`63
`
`31
`62
`23
`20
`
`37
`88
`1 1
`
`Home!W static
`23
`14
`3
`6
`
`10
`20
`10
`6
`Tumor size (W)
`9
`4
`13
`7
`4
`6
`0
`3
`
`43: a: liquid: (years)
`l
`13
`0
`
`10
`2
`
`2
`5
`
`3
`4
`
`0
`2
`3
`2
`
`2
`5
`0
`
`0.05
`
`0.06
`
`0.19
`
`0.11
`
`130
`59
`
`106
`83
`
`44
`84
`36
`25
`
`60
`1 16
`13
`
`Number d'pan'ts've W trader
`34
`0
`3
`1
`30
`0
`65
`7
`6
`l
`51
`1—3
`68
`18
`8
`4
`38
`>3
`Unknown
`17
`1
`3
`1
`22
`
`
`0.002
`
`TStatisticalamlysesforcot-relaiionof
`‘ERandPgRareasdescr-ibedinTablel.
`- HER-Zimampfificationwidivanmndiseuepanmewswueperfonnedbydiex’
`mat. PvalueswaemmputedafsacombiningdreaseswhhStoZOmd>20copiet
`
`SCIENCE, VOL. 235
`
`

`

`Table 4. Univariate and muln'variate analyses comparing disease-free survival (relapse) and overall survival to prognostic factors in node—positive patients.
`
`Multivariate“
`Univariate (P)
`
`Factor
`
`Number of positive nodes
`HER-2/neu
`
`Survival
`
`0.0001
`0.0011
`
`Relapse
`
`0.0002
`<0.0001
`
`Survival
`
`Relapse
`
`0.0003
`0.02
`
`(0.0938 t 0.0256)
`(0.0872 1 0.0388)
`
`0.001 (0.0849 t 0.0266)
`0.001 (0.1378 1 0.0425)
`
`0.05
`0.05
`Log (PgR)
`0.06
`0.06
`Tumor size
`0.10
`0.15
`Log (ER)
`0.61
`0.22
`Age
`
`parametric regression model was used to evaluate the predictive power ofvarious combinations andinteractions ofprognostic factorsin a multivariate manner
`(42-44)!)arucaslluymnonare shown as P (regression coeflicient : SE).
`
`(—0.5158 x 0.2414)
`
`0.03
`
`tumors have been examined. In a survey of 121 primary breast
`malignancies, amplification of the c-myc gene was found in 38
`(32%) (34). Attempts to correlate c—myc gene amplification with
`stage ofdisease, hormonal receptorvstatus, histopathologic grade, or
`axillary node metastases showed no association. There was a statisti-
`cally significant association between c-myc amplification and age at
`diagnosis >50 years in a group of95 ofthese patients (34). Data on
`relapse and survival were not presented in this study; however, there
`was no correlation between c-mye amplification and nodal status to
`indicate an association with disease behavior.
`
`The exact role of various prom-oncogenes in the pathogenesis of
`human malignancies remains unclear. One line of evidence implicat-
`ing abnormalities of these genes in human disease is association of
`their amplification with tumor progression in specific cancers. The
`N-myc gene is fiequently amplified in human neuroblastomas and
`neuroblastoma cell lines (35, 36). Studies on the N-mye proto-
`oncogene were the first to show a direct association between
`abnormalities in a prom-oncogene and clinical behavior of a human
`tumor. N-myc amplification and expression correlate both with stage
`of disease and overall survival in patients with neuroblastoma (10,
`32, 37). Moreover the greater the N-myc gene copy number, the
`worse the patient prognosis for all stages of the disease (32). Taken
`together, these data indicate a role for the Nmy: gene in the
`pathogenesis of neuroblastoma (32).
`Neuroblastoma is a relatively rare disease with an incidence ofone
`per 125,000 children. Carcinoma of the breast, however,
`is a
`common malignancy affecting one ofevery 13 women in the United
`States. There are 119,000 new cases per year, and approximately
`40,000 women will die of the disease in 1986 (38). Current
`treatment decisions for individual patients are frequently based on
`specific prognostic parameters. The major prognostic factors for
`breast cancer include presence or absence of tumor in the axillary
`nodes, size of the primary tumor, and presence or absence of
`hormonal receptors (29). The current study indicates that amplifica-
`tion of the HER-Z/neu gene is a significant predictor of both overall
`survival and time to relapse in node-positive patients with breast
`cancer. Amplification of the gene retains its prognostic significance
`in multivariate analysis, even when adjustments are made for other
`known prognostic factors. Moreover, amplification of HER-Zlneu
`has greater prognostic value than most currently used prognostic
`fa'ctors,
`including progesterone and estrogen receptors, and is
`equivalent to and independent of the best known prognosticator—
`number of positive lymph nodes. Finally, the degree of HER-2/neu
`amplification appears to have an effect on survival, with greater copy
`number being associated with a worse prognosis (Fig. 3, C and D).
`A similar phenomenon has been observed for N-myc gene amplifica-
`tion in human neuroblastoma (32).
`The potential role of HER-Zines; in the pathogenesis of breast
`cancer is unknown. Like N-myo, the correlation of HER-Zlneu
`amplification with disease progression indicates it may be an
`important gene in the disease process. The role of other cell
`
`9 JANUARY 1987
`
`receptors in the biology of breast cancer is well established (29, 39,
`40). It is easy to speculate that a gene encoding a putative growth
`factor receptor, when expressed in inappropriate amounts, may give
`a growth advantage to the cells expressing it. Alternatively, alter-
`ation in the gene product itself may lead to a critical change in the
`receptor protein. A single point mutation in the transmembrane
`domain of the protein encoded by the rat neu oncogene appears to
`be all that is necessary for the gene to gain transforming ability (41).
`Whether this or a similar alteration is found in the amplified HER-
`2/neu gene in human breast cancer will require sequence analysis of
`the homologous region in the amplified human gene. In addition,
`studies evaluating the expression of this gene at the RNA and/or
`protein level will prove important in determining if HER-Zlneu
`amplification results in an expected increased gene expression. The
`question of amplification of HER-2/neu in metastatic as compared
`to primary lesionsin a given patient is important. The current study
`utilized only primary breast tumors for analyses. It would be of
`interest to determine if HER-Zlneu copy number18 altered as the
`tumor metastasizes. A recent study evaluating N-myc copy number
`in human small cell carcinoma of the lung showed no difference
`between primary and metastatic lesions (11).
`The initial survey from the current study showed that 15% of
`breast cancer patients with stage I disease (node-negative) have
`HER-Zlneu amplification. Unfortunately, no long-term follow-up
`data were available for these patients. This stage I setting may be an
`additional group in which HER-Zlneu measurements will have an
`impact in predicting biologic behavior of the tumor, and as a result,
`in design of treatment strategy. Finally, if the HER-2/neu gene
`product functions as a growth factor receptor that plays a role in the
`pathogenesis of breast cancer,
`identification of its ligand and
`development of specific antagonists could have important therapeu-
`tic implications.
`
`REFERENCES AND NOTES
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`t from riton Bioscienees, Inc. We thank D. Keith, L. Gordon, and W. Aft for
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`assisuneeandDr. S. Cedarbaumforpmvidingthehumanarginase
`
`probe
`
` R meurrh \I'lit‘l(‘.~
`
`The Atomic Structure of Mengo Virus
`at 3.0 A Resolution
`
`MING LUO, GERRIT VRIEND*, GREG KAMER, IWONA MINOR, EDWARD ARNOLD,
`MICHAEL G. ROSSMANN, ULRIKE BOEGE, DOUGLAS G. SCRABA, GREG M. DUKE,
`ANN C. PALMENBERG
`
`'IhestrucmreofMengovirus,arepresentativememberof
`d1eeardiopieornavimses,issubstantiallydifi‘erentfi~omthe
`structures of rhino- and polioviruses. The structure of
`Mengoviruswassolvedwiththeuseofhumanrhinovirus
`l4asan8Aresolutionsu'ucmralapproximation.Phase
`infommdonwasumexmdfismsllwmambyuscof
`
`without the use of the isomorphous replacement tech-
`nique. Although the organization of the major eapsid
`proteins VPl, VP2, and VP3 ofMengo virus is essentially
`the same as in rhino- and polioviruses, large insertions
`
`and deletions, mostly in WI, radically alter the surface
`features. In particular, the putative receptor binding
`“canyon” ofhuman rhinovirus 14 becomes a deep “pit” in
`Mengo virus beeause of polypeptide insertions in WI
`that fill part of the eanyon. The minor eapsid peptide,
`VP4,
`is completely internal in Mengo virus, but its
`association with the other eapsid proteins is substantially
`difi'erent from that in rhino- or poliovirus. However, its
`carboxyl terminus is loeated at a position similar to that in
`human rhinovirus 14 and
`liovxrus, suggesting the same
`autoeatalytic cleavage of
`0 to VP4 and VP2 takes place
`during assembly in all these pieornaviruses.
`
`ICORNAVIRUSEs (1 ) COMPRISB A LARGE FAMILY OF SINGLE-
`
`P stranded RNA-containing animal viruses. They have been
`
`classified into four genera on the basis of physical properties
`of the vin'ons: (i) cardioviruses, such as encephalomyocarditis virus
`(EMCV), Theiler’s murine encephalitis virus, and Mengo virus; (ii)
`enteroviruses, such as poliovirus, hepatitis A virus, and Coxsackie
`virus; (iii) aphthoviruses, such as foot—and-mouth disease viruses
`(FMDV); and (iv) rhinoviruses, of which there are about 100
`serotypes. The physiological consequences of picomavir