Human Breast Cancer: Correlation of
`Relapse and Survival with Amplification
`of the HER-2/neu Oncogene
`
`DENNIS J. SLAMON,* GARY M. CLARK, STEVEN G. WONG, WENDY J. LEVIN,
`AxEL ULLRICH, WILLIAM L. McGUIRE
`
`The HER-2/neu on�cne is a member of the erbB-likc
`oncogene family, and ts related to, but distinct from, the
`epidermal growth factor .reccptoc. This gene has been
`shown to be amplified in human breast cancer cdl lines.
`In the current study, alterations of the gene in 189
`primary human breast cancers were investigated. HER-2/
`neu was found to be amplified from 2- to greater than 20-
`fold in 30% of the tumors. Correlation of gene amplifica­
`tion with several disease parameters was evaluated. Am­
`plification of the HER-2/IU'# gene was a significant pre­
`dictor of both overall survival and time to -�psc in
`patients with breast cancer. It retained its significance
`even when adjustments were made for other known
`prognostic factors. Moreover, HER-2/nn amplification
`had greater prognostic value than most currently used
`rn?SDostic factors, including hormonal-receptor status,
`m lymph node-positive disease. These data indicate that
`this gene may play a role in the biologic behavior and/or
`pathogenesis of human breast cancer.
`
`T HB BVIDENCE LINKING PROTO-ONCOGENES 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 transforming retroviruses that
`arc known to be tumorigcnic in some species (1, 2); (ii) ttansfcction
`studies showing the tranSforming potential of proro-onoogcnes in
`NIH 3T3 cells and primary embryo fibroblasts (3-5); and (iii) the
`central role of certain proto-oncogenes in tumorigenesis by chronic
`transforming retroviruses such as avian lcuk�is virus (6). Data from
`human tumors include: (i) increased expression of specific proto­
`oncogcnes in some human malignancies (7, 8); (ii) localization of
`proto-oncogcnes at or near the site of specific, tumor-associated
`chromosomal ttanslocations (9); and (iii) amplification of proto­
`oncogcnes in some human tumors (10, 11).
`Additional data linking proto-oncogcnes 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 arc related to a growth factor or a growth factor
`receptor. These genes include c-sis, which is homologous to the
`
`9 JANUARY 1987
`
`transforming gene of the simian sarcoma virus and is the � chain of
`platelet-derived growth factor (PDGF) (16, 17); c-ftn.s, which is
`homologous to the transfurming gene of the feline sarcoma virus
`and is cl�y related to the macrophage colony-stimulating factor
`receptor (CSF-lR) (18); and c-erbB, which encodes the EGF
`receptor (EGFR) and is highly homologous to the transforming
`gene of the avian crythroblastosis virus (19). The two rcccptor­
`related proto-oncogcnes, c-ftn.s and c-erbB, arc members of the
`tyrosine-specific protein kinase f;unily to which many proto-onco­
`gcnes belong.
`Recently, a novel transforming gene was identified as a result of
`transfcction studies with DNA from chemically induced rat ncu­
`roglioblastomas (20). This gene, called neu, was shown to be related
`to, but distinct from, the c-erbB proto-oncogcnc (21). By means of
`v-erbB and human EGFR as probes to screen human genomic and
`complementary DNA (cDNA) libraries, two other groups indepen­
`dently isolated human erbB-related genes that they cal.led HER-2
`(22) and c-erbB-2 (23). Subsequent sequence analysis and chromo­
`somal mapping studies revealed all three genes (neu, c-erbB-2, and
`HER-2) to be the same (22, 24, 25). A fourth group, also using v­
`erbB as a prob;c, identified the same gene in a mammary carcinoma
`cell line, MAC 117, where it was found to be amplified five- to ten­
`fold (26).
`This gene, which we will call HER-2/neu, encodes a new member
`of the tyrosine kinase family; and is cl�y related to, but distinct
`from, the EGFR gene (22). HER-2/neu differs from EGFR in that it
`is found on band q21 of chromosome 17 (22, 24, 25), as compared
`to band pll-pl3 of chromosome 7, where the EGFR gene is
`located (27). Also, the HER-2/neu gene generates a messenger
`RNA (mRNA) of 4.8 kb (22), which differs from the 5.8- and 10-
`kb transcripts for the EGFR gene (28). Finally, the protein encoded
`by the HER-2/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-2/neu is more closely related to the
`EGFR gene than to other members of the tyrosine kinase family
`(22). Like the EGFR protein, HER-2/neu has an extracellular
`domain, a ttansmembranc domain that includes two cystcine-rich
`repeat clusters, and an intracellular kinase domain (21), indicating
`
`D. J. Slamon, S. G. Wong, and W. J. Levin arc in the Division of Hcmatology­
`Onroloitv. Dcpartmc:nt or Medicine and Jonsson Compf'Cbcraive Cancer Center,
`UCLA� Of Medicine, Loi Angcb, CA 90024. G. M. C1arlt and W. L. Mc<JuiR:
`arc in the Division of Oncology, DCparuncm of Medicine, University of Texas Health
`Science Center at San Antonio, San Antonio, TX 78284. A Ullrich is in the
`Department of Molecular Biology, GcnCntcch, Inc., South San Francisco, CA �O.
`
`*To whom cor=pondcnce should be addtascd.
`
`AllTICLES 177
`
`1 of 6
`
`BI Exhibit 1045
`
`

`

`Tlble 1. Allociation bctwc:cn HER-2/11n1 amplification and disease paramc·
`ttn in 103 breast tumors.
`
`pt
`
`Toul
`
`Number of rumors
`Factor* Single
`>20
`5 to 20
`2 to 5
`copy copies copies copies
`1{-"' � stMW l
`
`53
`31
`42
`42
`
`4
`2
`3
`
`65
`38
`52
`51
`
`15
`41
`22
`25
`
`25
`65
`13
`
`0.99
`0.85
`
`0.82
`
`0.83
`
`0.11
`
`Ell+
`Ell-
`Pgll+
`Pgll-
`S2
`2-5
`>5
`Unknown
`
`13
`34
`17
`20
`
`21
`52
`11
`
`0
`l
`2
`2
`
`2
`l
`6
`2
`5
`1
`r-nu (mritutmJ
`l
`l
`l
`5
`2
`1
`3
`0
`"'81 "'� (pn)
`1
`2
`:sso
`l
`7
`4
`2
`>SO
`0
`2
`0
`Unknown
`N """-of positiPe lyrriph notlts
`34
`l
`3
`30
`0
`0
`l
`l
`20
`0
`1-3
`22
`25
`2
`2
`17
`4
`>3
`22
`1
`17
`1
`3
`Unknown
`*� ltaNI WU analyzed 11 dac:ribedJ39). ER, csttoga_I reccpax; + aDd - �
`plopst_JC:iODC rea:ptor: + and -rc:fi:n ID� QC abeaJa: of"'S fiDol of RlCCplX>r
`ID lbc pracnce QC abecoCc: o( ;i.3 final recepllCX ptt inillignril of protein. Pgll,
`pa� of prottin.
`n axrdllion ofHER·ll-amplili·
`tScatiltical
`abari with dilcatc pmmclen -�focmCd by the r tat. p va1uca -compWd
`aftrr mmbining lbc c:uca with 5 ID md > 20 aipics.
`
`that it too is likely to be a cellular receptor for an as yet unidentified
`ligand.
`As a result of the published data showing amplification ofHER-
`2/'le# in .a human mammary carcinoma cell line, and as part of an
`ongoing survey in our laboratory of proto-oncogcnc abnormalities
`in human tumors, we evaluated altcratiom of the HER-2/nn1 gene
`in a large series of human primary breast cancers. Our results show
`that amplification of this gene occurs rclativcly frequently in breast
`cancer, and that it is associated with disease relapse and overall
`patient survival.
`Factors that arc 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 (�itivc
`nodes) (29). The current study, which was conducted in two parts,
`involved the evaluation of tissue from 189 separate breast malignan·
`cies that were part of a breast cancer study ongoing at the University
`of Texas, San Antonio. This cohort of tumors was of inttrcst
`because considerable infOrmation was available on the majority of
`the specimens including size of the primary tumor, estrogen recep­
`tor status, progesterone receptor status, age of patient, disease stage,
`and status of the axillary lymph nodes.
`In the initial survey, tissue from 103 primary breast cancers was
`evaluated for alterations in the HER-2/nn1 gene. DNA froni
`individual tumors was prepared as dcacribcd (30), digested with
`Eco RI, and subjected to Southern blot analysis with a 32P-labcled
`HER-2/11eU-l probe, which is known to detect a 13-kb hybridizing
`band in human DNA (22). Examples of tumors from the initial
`survey arc shown in Fig. 1. Of the 103 samples c:xamincd, 19 (18'6)
`showed evidence of HER-2/nn1 gene amplification. The dcgrcc of
`amplification in individual cases was dctcnnincd by dilution analysis
`(Fig. 2A), as well as soft laser densitometry scanning. To dctcnn.inc
`that the amount of DNA loaded in each Jane was equivalent, all
`filters were washed and rchybridizcd with a "P-labclcd argin.asc
`gene probe (31). This probe identifies a 15-kb hybridWng band on
`Eco RI-digested human DNA, and was selected as a control
`because it more appropriately assesses the rclative amount and
`
`transfer of high molccuJar weight species than a probe hybridizing
`with low molccuJar weight species, which transfer more readily on
`Southern blotting. All lanes were shown to contain equivalent
`amounts of high molecular weight DNA (Fig. 2B). Individual
`tumors were assigned to groups oontaining a single copy, 2 to S
`copies, S to 20 copies, and greater than 20 copies of the HER-2/MN
`gene (Fig. 1 ). Assignment of nunors to the various groups was done
`
`Fig. 1. Analysis of alterations of the HER-2/­
`gcnc in human breast cancer. Shown arc 79 of the
`189 breast tumors used in this analysis. Twnors
`with a single copy ofHER·l/-: 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 ofHER-2/-: 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 ofHER.·21-: 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-2/-: 18, 30, 33,
`59, 68, and 78. Examplcsoftum0rs 77to 79 have
`rcarnngcments in the HER-2/11n1 gene. DNA
`was cnraacd from tissues and diRestcd with Eco
`R.l u described (30). A total of 12 .,.g of Eco R.l­
`digcsted DNA wu loaded onto 0.8'6 agaroec
`gels, separated by clcctrophorcsis and trmsttrmi
`omo nylon filttt papers (hiodync) (30). All filtas
`wae baked in a vacuum oven fur 3 hows at 80°C,
`pttbybridizcd in 5 x SSC (standard saline citrate)
`a>ll . .
`50'6 fonnamidc, 10'6 dc:nran sul&tc,
`0.1 :·��denatured salmons� DNA (l rDfl
`ml), and 4x Dcnhardts solunoo fur 12 hours,
`then hybridiud in the same solution c:ootaining
`»p. Jabclcd nick-tra.n.slatcd HER-2 probe (21)
`specific activity of l x 10' cpm per nucrogram of
`DNA; 2 x 106 cpmlml. Hybridization occum:d
`at 42°C foe 48 hours, followed by washing of
`filters under the following conditions in succcs-
`178
`
`12 345 . 8 789 · 1011121314
`..
`
`15 Ml 1718 19 20 • 212223�2526 2728 29
`.._
`....
`.
`
`30 31 32 33 34 35 38 :J1 38 39 40
`
`•
`
`51 52 53 54 55 58 SI 58 ee eo ei m 83 M es ee
`..
`
`87 ee ea 10 11 72 7J 111 75 16 n 78 79
`-·
`
`kb
`•12
`• 4
`·3.1
`
`•
`
`sioo: 2x SSC fur 20 minutes at room tempera·
`turc; two washes of 30 minutes each in 2 x SSC,
`0.1'6 SDS at 65"C; one wash of 30 minutes in
`
`O.Sx SSC, 0.1'6 SDS at 65"C. Filters were then
`c:xpoecd to XAR-5 x·ray film (Kodak) for autora­
`diography.
`
`SCIBNCB, VOL. 235
`
`2 of 6
`
`BI Exhibit 1045
`
`

`

`Fig. 2. (A) Example of dilutional analysis to :wcss degree of HER.-211.,,,
`gene amplification. Lanes a, g, k, and p were loaded with 12 p.� of Eco lU­
`digcstcd breast nunor DNA. Lane a is DNA from tumor 31(Fig.1), which
`rcprcscnts a tumor with a single copy of the HER/2-lUN gene. Lane g is
`DNA from rumor 33, which rcprcscrus a rumor with >20 a:>pia of the
`HER-2/nn gene. Lanes b to f arc serial dilutions ( 1: 100, 1: 20, 1: 10, 1: 5,
`and 1:2, respectively) ofthe DNA sample in lane g. Lane k is DNA from
`rumor 35 (Fig. 1 ), which rcprcscms a tumor containing two to five copies of
`the HER-2/nn gene. Lanes h to j arc serial dilutions (1:10, 1:5, and 1:2,
`respectively) of the DNA sample in lane k. Lane p is DNA from tumor 34
`(Fig. l), which represents a rumor with 5 to 20 copies of the HER-2/nn
`gmc. Lanes I too arc serial dilutions (1:20, 1:10, 1:5, and 1:2, rcspccrivc­
`lylpf the DNA sample in lane p. The filter was prepared and bybridiud with
`a 32P-labclcd HER-2 probe as in Fig. 1. (B) Example of arginasc probe
`hybridization to demonstrate that equivalent amounts of tumor DNA were
`loaded into each lane. Rchybrid.ization of filter a:>ntaining lanes 30 t0 .fO
`(Fig. 1). The filter was first snipped of label by washing in a buffer made up
`of 50% Wnnamidc, 3x SSC, and 0.1% SDS at 65"°C for 20 minutes,
`following by three successive washes of 5 minutes each in 0.1 x SSC at room
`temperature. Filters were exposed overnight on XAR-5 film (Kodak) to
`ensure rcmov;d of all radioactive probe, then rchybridiud as in Fig. 1 with a
`32P-labclcd human arginasc gene probe (31).
`
`in a blinded fashion, in that they were made without knowledge of
`disease parameters. Analysis of the data for association bctwccn gene
`amplification and a number of disease parameters was then per­
`formed.
`Of 103 tumors evaluated in the initial survey, there was essentially
`no correlation between gene amplification and estrogen receptor
`status, progesterone rcccptor status, size of tumors, or age at
`diagno,,is (Table 1). However, when analysis was pcrfonned for
`asscxiation between HER-2/nn amplification and number of posi·
`rive lymph nodes, a trend was noted. 'This analysis showed that 4/34
`(1196) of patients with no involved nodes, 1/20 (10%) with l to 3
`involved nodes, and 8125 (3296) with >3 involved nodes had gene
`amplification (P = 0.11). If these data were examined by comparing
`0 to 3 positive nodes versus > 3 positive nodes, the correlation with
`gene amplification became more significant (P < 0.05). Thus, there
`was a significant increase in incidence of HER-2/nn gene amplifica­
`tion in patients with > 3 axillary lymph nodes involved with disease.
`A multivariate regression analysis to correlate HER-2/nn amplifica­
`tion with various disease parameters identified the number of
`positive nodes as the only significant factor, either alone or in
`combination, to correlate with amplification.
`This initial study indieatcd that it might be possible to discrimi­
`nate among node-positive patients on the basis ofHER-2/nn gene
`
`1.0
`
`� 0.8
`·= •-"'
`ii o.s
`:a.
`:1 0.4
`Ci'l
`il 0.2
`A
`
`0
`
`1.0
`
`� 0.8
`
`:ii • ;i 0.6
`> _ o! 0.4
`$ • 0.2
`B
`
`Fig. 3. Acnwial curve for relapse in (A) nodc­
`positive F.ticnts with no amplification versus
`node-positive patients with any amplification (>2
`copies) ofHER-2/nn and (C) node-positive ,ea­
`ticnts with no amplification versus nodc-posiovc
`patients with greater than 5 copies ofHER-2/nn.
`Actuarial curve for overall survival in (B) nodc­
`positive rmcnts with no amplification versus
`node-positive patients with any amplification (>2
`copies) of HER-2/nn and (D) node-positive �­
`ticnts with no amplification versus node-posinve
`patients with greater than 5 copies ofHER.-2/11n1.
`Actuarial curves for both relapse and overall sur­
`vival were computed by the method of Kaplan
`and Meier (44) and compared by the log rank test
`(42-44).
`9 JANUARY 1987
`
`A
`• b c d •
`
`g
`
`h
`
`k
`
`m n o p
`
`B
`30
`
`37
`
`38
`
`39
`
`40
`
`amplification. It is well known that the number of positive nodes is
`the best prognostic factor for disease recurrence and survival in
`patients with breast cancer (29). Given .the correlation between
`number of nodes positive and HER· 2/nn amplification, one might
`predict that amplification of this gene might also have some
`prognostic value. No long-term follow-up data, however, were
`available on the 103 patients analy:zcd in the initial study. For this
`reason, a second study was conducted on 100 breast cancer samples
`from patients with positive axillary lymph nodes. All of the infonna.
`tion available for the first group of 103 patients was available for
`these patients. In addition, relapse and swvival information was
`available, since these cases had a median follow-up of 46 months
`(range 24 to 86 months). Of these l 00 samples, 86 yielded sufficient
`DNA for study. Amplification of the HER-2/neu gene was mea­
`sured as in the initial swvey, and examples of tumors from this study
`arc shown (Fig. 1). Amplification was found in 34186 (4096) of
`these patients. For this larger sample of node-positive patients,
`several statistically significant or nearly significant relationships were
`observed. In agrccmcnt with the preliminary swvey, there was an
`association between number of involved lymph nodes and HER· 21
`nn amplification (Table 2). In addition, the presence of gene
`amplification was corrclatcd with estrogen receptor status and siz.c
`of primary tumor (Table 2). Together, these two swvcys yielded
`data on 189 patients and the association of HER·2/nn amplifica­
`tion with various disease parameters in the combined group is
`shown in Table 3.
`While these correlations were of interest, the strong relationship
`
`Not emplttled (n =52)
`
`Amplified (na34)
`
`(n :34)
`Amplllled
`
`c
`
`D
`
`Amplified (11:11)
`
`>5 copies
`
`Ampllfled (II :11)
`
`>5 copies
`
`0
`0 12 24
`
`3S
`
`46
`
`12 24 36
`60 72 64
`0
`Time (monlhs)
`
`60 72
`
`ARTICLES 179
`
`3 of 6
`
`BI Exhibit 1045
`
`

`

`between HER-2/neu 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 (83%) 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 sttong and highly statistically
`significant correlation was round between the dcgrcc 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 ofHER-2/neu
`was found to be superior to all other prognostic factors, with the
`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-2/neu 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-2/neu gene in their tumors
`(Fig. 3, A and B), the greatest diffcrcnccs 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 ofHER-2/neu 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-myc gene amplification in human
`neuroblastomas (32).
`To determine if amplification ofHER-2/neu was independent of
`other known prognostic factors in pttdicting disease behavior,
`multivariate survival analyses were pcrfonncd on the 86 node­
`positive cases. Amplification of the gene continued to be a strong
`prognostic factor, providing additional and independent predictive
`information on both time to relapse and overall survival in these
`
`51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
`
`Fig. 4. Example of rchybridization of filter with human EGFR probe. Fitten
`were Stripped as in Fig. 2B, and hybridiud with 32P-labclcd human EGFR
`probe (28), as in Fig. 1. Shown arc the lower molc:cular weight bands
`(Fig. 1 ). The bands from top ro bottom arc 2.8, 2.2, �,respectively.
`hybridiud with 32P-labclcd EGFR probe in filter-con . . lanes SI to 66
`Lane S2 is m example of a tumor showing marked amplification (>SO
`copies) of the EGFR gene.
`
`patients, even when other prognostic factors were ta.ken into
`account (Table 4).
`Rearrangement of the HER-2/neu gene was rare. Of the total 189
`tumors evaluated, three showed evidence of rcarrangcincnt, and in
`two of the three cases, the rearrangement was identical (Fig. 1, cases
`77 to 79). Also, two of the rearranged HER-2/neu loci were
`amplified (Fig. 1, cases 78 and 79). The incidence of HER-2/neu
`rcarrangcmcnt as determined by Eco RI digestion was too small to
`attempt statistical correlations.
`To determine whether the phenomenon of amplification ofHER-
`2/neu in breast cane.er extended to related growth factor receptors,
`all filters were analyud with the EGFR probe (Fig. 4). Amplifica­
`tion of the EGFR gene was found in 41189 (2%) of the cases, and
`rearrangement of the EGFR gene was found in one of those four
`cases. The incidence of EGFR amplification and rearrangement was
`too small to attempt statistical correlation. Comparison of HER-2/
`MN 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 proto-oncogcnes, 1111' and fas, in
`breast cancer did not show amplification of these genes (33).
`Alterations of non-tyrosine kin�lated proto-oncogcnes in these
`
`Factor*
`
`ER+
`ER-
`PgR+
`6S 0.05
`PgR-
`21
`54
`32
`
`0.14
`:s2
`2-S
`>S
`Unknown
`29 0.09
`43
`14
`
`sso
`>SO
`Unknown
`3S 0.06
`SI
`
`2
`s
`3
`4
`
`130 o.os
`S9
`106 0.06
`83
`
`0.19
`
`0 44
`2
`84
`3
`36
`2
`2S
`
`0.11
`
`60
`116
`13
`
`0.002
`
`34
`6S
`68
`22
`
`Tllble 2. AssQciation between HER-2/-amplification and disease paramc-
`ters in 86 breast tumon from node-positive patients.
`2 to S S ro20 >20 Total pt
`Single
`copy copies copies copies
`H--1 reuptor stlll1IS
`s
`21
`2
`4
`18
`4
`s
`s
`r-nu (cmritfutmJ
`3
`8
`2
`12
`3
`4
`Aae Ill� (yetm)
`12
`6
`11
`3
`1
`Nut#ber <f ponme lymph -US
`7
`0
`s
`4
`16
`2
`
`Factor*
`
`ER+
`ER-
`PgR+
`PgR-
`
`s2
`2-S
`>S
`
`sSO
`>SO
`
`1-3
`>3
`
`38
`14
`31
`21
`
`18
`28
`6
`
`16
`36
`
`31
`21
`
`1
`1
`1
`1
`
`0
`1
`1
`
`tSwisticaJ analyses IOr rorrdarion of
`*ER and Pgll uc as described in Table 1.
`H:ER.-21- amplification with various discate � wae pcrfuancd by the x2
`S co 20 and > 20 cases, since thcrfo •
`-· P wlucs were computed after combining
`were to few samples in ihc > 20 group.
`180
`
`Table 3. Association between HER-2/11111 amplification and disease paramc-
`tcn in combined surveys (189 patients).
`.
`2 to s
`Single
`s to 20
`>20 Total pt
`copy copies copies copies
`H--1 reuptor st"'1IS
`14
`91 23
`6
`4S
`3
`73 20
`10
`6
`10
`63
`T11rfWI' siu (tmtinutm)
`31
`9
`4
`7
`62 13
`4
`6
`23
`3
`20
`0
`Aae tit� (yetm)
`37 13
`8
`2
`s
`88 13
`10
`11
`0
`0
`2
`N##lber <f ponme lymph -US
`3
`0
`30
`l
`7
`6
`SI
`1
`38 18
`8
`4
`1
`17
`3
`1
`
`0
`1-3
`43 0.06
`>3
`43
`Unknown
`tSwistical analyses foe corrcbtion of
`*ER and Pgll uc as described in Table 1.
`H:ER-2/""' amplification with various di.scasc paramctcn were pcrfunned by the x2
`tat. P wlucs were computed after combining die caKS with 5 IO 20 and > 20 copies.
`SCIBNCB, VOL. 23S
`
`4 of 6
`
`BI Exhibit 1045
`
`

`

`Factor
`
`Survival
`
`0.0003 (0.0938 ± 0.0256)
`0.02 (0.0872 ± 0.0388)
`
`Relapse
`0.001 (0.0849 ± O.Q266)
`0.001 (O.la78 ± 0.0425)
`
`Table 4. Univariate and multivariate analyses comparing disease-free survival (relapse) and overall survival to prognostic factors in node-positive patients.
`Univariate (P)
`Multivariate*
`Survival Relapse
`Number of positive nodes
`0.0002
`0.0001
`0.0011
`<0.0001
`HER-2/Mtl
`0.05
`0.05
`Log (PgR)
`TumorsU.c
`0.06
`0.06
`0.15
`0.10
`Log (ER)
`0.22
`0.61
`Age
`"Cox's partially nonparamaric rcgrcaion model was ulCd ro evaluarc the prcdictivc power of varioua oombinations and imuaaions of prognostic &cton in a multivariate manner
`(42-H'j. llaula arc shown as P (regression CXldlicicnt :I: SE).
`
`0.03 (-0.5158 ± O.UH)
`
`rumors 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 of disease, hormonal receptor status, histopathologic grade, or
`axillary node mcwta.ses showed no association. There was a statisti­
`cally significant association between c-mye amplification and age at
`diagnosis >50 years in a group of95 of these patients (34). Data on
`relapse and survival were not presented in this study; however, there
`was no correlation between c-myc amplification and nodal status to
`indicate an association with disease behavior.
`The exact role of various proto-oncogcncs in the pathogenesis of
`human malignancies remains unclear. One line of evidcacc implicat­
`ing abnormalities of these genes in human disca.sc is association of
`their amplification with nunor progression in specific cancers. The
`N-"'J' gene is frcquendy amplified in human ncuroblastomas and
`ncuroblastoma cell lines (35, 36). Srudies on the N-mye proto­
`oncogcnc were the first to show a direct association between
`abnormalities in a proto-onc:ogcnc and clinical behavior of a human
`twn0r. N·myc amplitication and aprcssioo rorrelatc both with stage
`of disease and overall survival in patients with ncuroblastoma (IO,
`32, 37). Moreover the greater the N-"'J' gene copy number, the
`worse the patient prognosis for all stages of the disease (32). Taken
`together, these data indicate a role for the N-myc gene in the
`pathogenesis of ncuroblastoma (32).
`Ncuroblastoma is a relatively rare disease with an incidence of one
`per 125,000 children. Carcinoma of the breast, however, is a
`common malignancy affecting one of every 13 women in the United
`States. There arc 119,000 new cases per year, and approximately
`40,000 women will die of the disease in 1986 (38). Current
`trcaancnt decisions for individual patients arc frcquendy based on
`specific prognostic parameters. The major prognostic factors for
`breast cancer include presence· or absence of tumor in the axillaiy
`nodes, size of the primary rumor, and prcscncc or absence of
`hormonal receptors (29). The current study indicates that amplifica­
`tion of the HER· 2/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 adjusancnts arc made for other
`known prognostic factors. Moreover, amplification of HER-2/nn1
`has greater prognostic value than most currcndy used prognostic
`factors, including progesterone and estrogen receptors, and is
`equivalent to and independent of the best known prognosticator­
`number of positive lymph nodes. Finally, the dcgrcc ofHER-2/nn1
`amplification appears to have an dfcct 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-"'J' gene amplifica­
`tion in human neuroblastoma (32).
`The potential role of HER-2/nn1 in the pathogenesis of breast
`cancer is wllmown. Like N-myc, the correlation of HER-2/neu
`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 transmcmbrane
`domain of the protein encoded by the rat neu oncogene appears to
`be all that is ncccssary 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 dctcnnining if HER-2/neu
`amplification results in an cxpcctcd increased gene expression. The
`question of amplification of HER-2/neu in metastatic as compared
`to primary lesions in a given patient is important. The current study
`utiliu:d only primary breast tumors for analyses. It would be of
`interest to determine if HER-2/neu copy number is altered as the
`twnor mcwwizcs. A recent study evaluating N-myc copy nwnber
`in human small cell carcinoma of the lung showed no di.ffcrcncc
`between primary and metastatic lesions (11).
`1bc initial survey from the current study showed that 15% of
`breast cancer patients with stage I disease (node-negative) have
`HER-2/neu amplification. Unfortunately, no long-tenn follow-up
`data were available for these patients. This stage I setting may be an
`additional group in which HER-2/neu mcasurcrnents will have an
`impact in predicting biologic behavior of the tumor, and as a result,
`in design of tteaancnt 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.
`
`lU!PBlU!NCES AND NOTES
`1. J.M. Bishop,A-. R.n. Biod#rrl. Sl, 301 (1983).
`2. H. E. Vannw,A-. R.n. c-t. 18, 553 (19114).
`3. H. Land, L. F. Panda, It A. Wc:inbcrg, Nlllfln (1.AttMM) 3M, 596 (1983).
`4. H. £. Ruley, illill., p. 602.
`5. M. Schwab, H. E. Vumus, J.M. B�, ilNt.. 316, 160 (1985).
`6. W. S. Hayward, B. G. Neel, S. M. Astrin, ilNt.. 290, 475 (1981).
`7. D. ]. Slamon a Ill., SMta lU, 256 (1984).
`8. M. D. Erisman a 111.,Mlll. QJJ. Bitrl. S, 1969 (1985).
`9. C. M. Ctooc and G. Klein, Sci. A"'. 252, S4 (Mm:h 1985).
`10. G. M. Brodeur a Ill., SMta lU, 1121 (1984).
`11. A. J. Woog a Ill., ilNt.. 233, 461 (1986).
`U. J. Campisi a Ill., Qll 33, 357 (1983).
`13. M. E. Grccnbcrg and E. B. Zif, Nlllfln (1.AttMM) 311, 433 (1984).
`14. It Mulkr a Ill., iftl. 299, 640 (1982).
`15. D. J. Slamon and M. J. Cline, Prw.. Nllll. A* Sci. U.S.A. 81, 7141 (1984).
`16. M. D. Wacafidd a Ill., Nlllfln (LoNioll) 3M, 35 (1983).
`17. It F. Doolittle, M. W. Hwlkapillcr, L. E. Hood, 5'""" 121, 275 (1983).
`18. C. J. Sherr a Ill., QJJ 41, 665 (1985).
`19. J. Downward a Ill., Nlllfln (1.AttMM) 307, 521 (1984).
`20. C. Shih, L. Padhy, M. Murray, It A. Weinberg, ilNI. 290, 261 (1981).
`21. A. L. Schech= a Ill., ilNt.. 3ll, 513 (1984).
`22. L. c.ousscns a Ill., 5'""" 230, 1132 (1985). ·
`23. K. Scmba, N. Kama12, K. Toyoshima, T. Yamamoto, hoe. Nllll.AcM. Sci. U.S.A.
`Bl, 6f97 (1985).
`
`·
`
`ARTICLES 181
`
`5 of 6
`
`BI Exhibit 1045
`
`

`

`24. A. L. Schcchtcr a Ill., Scima 229, 976 (1985).
`25. S. I. Fukushiac a lll.,.Mol. CllJ. Biol. 6, 955 (1985).
`26. C. R. �. �- H. Kraus, S. A. Aaromon, Scima 229, 97f (1985).
`27. D. Mccrillan and M. Smith, Cytqf-. Oil. Gma. 37, 71 (19M).
`28. A. Ullrich a Ill., N11t1m {LotNltii) 309, fl8 (l�).
`29. G. M. Chrk.etlll.,N.E"81.J.MM.. 309, la.3 (1983).
`30. T. Maniatis, E. F. Fricldi, J. Sambrook,� a-iltlr: A� MMlul,
`Cc.old Spring Harbor Laboratocy, c.o&d Spring Huboc, NY, 1982),
`pp. 282-285.
`31. G. J. DC7.W:S, W.W. Grody, R. M. � S. D. Ccdarbaum, Biod#lll. B�. b.
`c-., in pt'C$S .
`32. R. C. S«gcr et lll.,N. E¥l.J . Miil. 313, llll (1985).
`33. J. Yokoc2 a Ill., Sdma 231, 261 (1986).
`M. C. Escot a Ill., hoe. NMl. AIU. S&i. U.S.A. 83, .SM (1986).
`35. M. Schwab a Iii., N1111m {Ltnulon) 305, 245 (1983).
`
`36. N. E. Kohl a Ill., Qll 35, 359 (19M).
`37. R. C. Sccgcr and D. Slamon, unpublished data.
`38. E. Silverberg and J. Lubcra, ct 0..-J. C/in. 36, 9 (1986).
`39. K. B. Horwitz, W. L. McGuire, 0. H. Pcanon, A. ScgalolF, Scima 189, 726
`(1975).
`.0. W. L. McGuire, S-0.. Ortt:ol. 5, GS (1978).
`fl. C. I. Bargmann, M. C. Hung, R. A. Weinberg, CdJ 45, 6"9 (1986).