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`HOSPIRA EX. 1133
`Page 1
`
`

`

`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`• CIENCE ISSN 0036-8075
`
`9 JANUARY 1987
`VOLUME 235
`NUMBER4785
`
`139
`MDtiritiHM- 141
`M t i l i i i - 144
`
`This Week in Science
`
`Fraud in Science
`
`Misuse of the Freedom of Information Act: J. R. WILLIAMS• Riidenberg's
`Patents: J. L. HUMMER • Quality of Biomedical Literature: R. G . MARTIN •
`Quantitative Risk Aspects of the ''Woburn Case": M . T . SMITH • Moonlight and
`Circadian Rhytluns: R. M. SINCLAIR; C. A. CzEISLER AND J. S. ALLAN •
`Research Practices: W. W. STEWART AND N . FEDER
`
`•~1wi1~••.1m111111- 151
`153
`154
`155
`156
`158
`159
`
`Science Budget: More of the Same • R&D and the Deficit
`Peer Review-'oops-Merit Review in for Some Changes at NSF
`Cancer M.D.'s Clash over Interleukin Therapy
`Landsat Commercialization Stumbles Again
`Hazardous Waste: Where to Put It?
`EEC Research Program in Jeopardy
`Court Rejects Rifkin in Biotech Cases
`Math Papers Called Inaccessible
`Briefing: Comings and Goings
`
`Research News
`
`·\rticles
`
`Re::-earch Articles
`
`160
`161
`163
`165
`
`167
`
`172
`
`177
`
`182
`
`•
`
`•
`
`Oncogenes Give Breast Cancer Prognosis
`Materials Scientists Seek a Unified Voice
`Diabetics Should Lose Weight, Avoid Diet Fads • High-Carb Diets Questioned
`Delving into Faults and Earthquake Behavior: How to Stop a Quake by Jogging
`• Earthquakes Are Giving Little Warning • Coastal Ups and Downs Point to a
`Big Quake • Cutting the Gordian Knot of the San Andreas
`
`Community Diversity: Relative Roles of Local and Regional Processes:
`R. E. RICKLEFS
`Band-Gap Engineering: From Physics and Materials to New Semiconductor
`Devices: F. CAPASSO
`Human Breast Cancer: Correlation of Relapse and Survival with Amplification of
`the HER-2/neu Oncogene: D. J. SLAMON, G. M. CLARK, S. G. WONG,
`W. J. LEVIN, A. ULLRICH, W. L. McGUIRE
`The Atomic Structure of Mengo Virus at 3.0 A Resolution : M. Luo,
`G. VRIEND, G. KAMER, I. MINOR, E. ARNOLD, M. G. ROSSMANN, u. BOEGE,
`D. G. SCRABA, G. M. DUKE, A. c. PALMENBERG
`
`SCIENCE la published -ldy on Friday, except the last week In December, •nd with an extra luue In February
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`SCIENCE, VOL. 235
`
`HOSPIRA EX. 1133
`Page 2
`
`

`

`Lava fl.ow after eruption of Mauna Loa Volcano, March 1984. Lava
`COVER
`flows advanced nearly 20 kilometers in about 5 days toward the city of Hilo,
`largest city on the island of Hawaii. The fl.ow slowed and stopped about 8
`kilometers from nearest buildings on city's outskirts. All eruptive activity ceased
`by early April 1984. See page 196. [Scott Lopez, National Park Service]
`
`195
`
`202
`
`205
`
`193 Outward-Dipping Ring-Fault Structure at Rabaul Caldera as Shown by
`Earthquake Locations: J. MoRI AND C. McKEE
`Sensory Tuning of Lateral Line Receptors in Antarctic Fish to the Movements of
`Planktonic Prey: J.C. MONTGOMERY AND J. A. MACDONALD
`196 Disruption of the Mauna Loa Magma System by the 1868 Hawaiian Earthquake:
`Geochemical Evidence: R. I. TILLING, J.M. RHODES, J. W. SPARKS,
`J. P. LOCKWOOD, P. w. LIPMAN
`199 Oxygen Supersaturation in the Ocean: Biological Versus Physical Contributions:
`H. CRAIG AND T. HAYWARD
`Identification of a Juvenile Hormone-Like Compound in a Crustacean:
`H. LAUFER, D. BoRSr, F. C. BAKER, C. CARRAsco, M. SINKUS, C. C. REUTER,
`L. w. TSAI, D. A. SCHOOLEY
`Evolution of Male Pheromones in Moths: Reproductive Isolation Through Sexual
`Selection?: P. L. PHELAN AND T. C. BAKER
`207 Direct Activation of Mammalian Atrial Muscarinic Potassium Channels by GTP
`Regulatory Protein Gk: A. YATANI, J. CoDINA, A. M. BROWN, L. BIRNBAUMER
`211 Metalloregulatory DNA-Binding Protein Encoded by the merR Gene: Isolation
`and Characterization: T. O'HALWRAN AND C. WALSH
`214 Nerve Growth Factor Treatment After Brain Injury Prevents Neuronal Death:
`L. F. KROMER
`Lung Cancer and Indoor Air Pollution in Xuan Wei, China: J. L. MUMFORD,
`X. Z. HE, R. S. CHAPMAN, S. R. CAo, D. B. HARRis, X. M. LI, Y. L. XIAN,
`W. Z. JIANG, C. W. Xu, J. C. CHUANG, W. E. WILSON, M . CooKE
`Preferred Microtubules for Vesicle Transport in Lobster Axons: R . H . MILLER,
`R. J. LA.SEK, M. J. KATZ
`
`217
`
`220
`
`MGlllilHDIW§- 224
`
`MMiiiliiii&tlL1MIQIM¢11 229
`
`The Darwinian Heritage, reviewed by A. DESMOND • Confronting Nature,
`Y. GINGRAS • Are Australian Ecosystems Different?, G. H. ORIANs • Under the
`Cloud and Justice Downwind, R. A. DIVINE• Books Received
`
`Portable Macroscope • Diacylglycerol Kinase • Polyacrylarnide Gels for
`Electrophoresis • FORTRAN Language Subroutines • PVC Tubing • Miniature
`Centrifuge• Teflon-Coated Slides• Literature
`
`Board of Directors
`Gerard Piel
`Retiring President,
`Chairman
`Lawrence Bogorad
`President
`Sheila E. Widnall
`President-elect
`
`Robert McC. Adams
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`Floyd E. Bloom
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`William D. Carey
`Executive Officer
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`Editorial Boalrd
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`Walter Massey
`Oliver E. Nelson
`David V. Ragone
`David M. Raup
`Vera C. Rubin
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`Solomon H. Snyder
`Robert M. Solow
`James D. Watson
`
`Boalrd of Reviewing
`Editors
`John Abelson
`Qais Al-Awqati
`James P. Allison
`Don L. Anderson
`Elizabeth H. Blackbum
`Floyd E. Bloom
`Charles R. Cantor
`James H. Clark
`Bruce F. Eldridge
`Stanley Falkow
`Theodore H. Geballe
`Roger I. M. Glass
`Stephen P. Go«
`Robert B. Goldberg
`
`Corey S. Goodman
`Richard M. Held
`Gloria Heppner
`Eric F. Johnson
`Konrad B. Krauskopf
`I. Robert Lehman
`Karl L. Magleby
`Joseph B. Martin
`John C. McGif
`Alton Meister
`Mortimer Mishkin
`Peter Olson
`Gordon H. Orians
`John S. Pearse
`
`Y eshayau Packer
`Jean Paul Revel
`James E. Rothman
`Thomas C. Schelling
`Ronald H. Schwartz
`Stephen M. Schwartz
`Otto T. Solbrig
`Robert T. N. Tjian
`Virginia Trimble
`Geerat J. Vermeij
`Martin G. Weigert
`Harold Weintraub
`Irving L. Weissman
`George M. Whitesides
`Owen N. Witte
`William B. Wood
`
`9 JANUARY 1987
`
`TABLE OF CONTENTS
`
`137
`
`HOSPIRA EX. 1133
`Page 3
`
`

`

`Human Breast Cancer: Correlation of
`Relapse and Survival with Amplification
`of the HER-2/neu Oncogene
`
`D ENNIS J. S LAMON,* GARY M. CLARK, STEVEN G. WONG, WENDY J. L EVIN,
`AxEL ULLRICH, WILLIAM L. McGUIRE
`
`The HER-2/neu oncogene is a member of the erbB-like
`oncogene family, and is related to, but distinct from, the
`epidermal growth factor receptor. This gene has been
`shown to be amplified in human breast cancer cell 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(cid:173)
`tion with several disease parameters was evaluated. Am(cid:173)
`plification of the HER- 2/neu gene was a significant pre(cid:173)
`dictor of both overall survival and time to relapse in
`patients with breast cancer. It retained its significance
`even when adjustments were made for other known
`prognostic factors. Moreover, HER-2/neu amplification
`had greater prognostic value than most currently used
`prognostic factors, including hormonal-receptor status,
`in 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 HE EVIDENCE LINKING PROTO-ONCOGENES TO THE INDUC(cid:173)
`
`tion or maintenance of human malignancies is largely cir(cid:173)
`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(cid:173)
`oncogenes and the viral oncogenes of transforming retroviruses that
`are known to be tumorigenic in some species (1, 2); (ii) transfection
`studies showing the transforming potential of proto-oncogenes 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 leukosis virus ( 6) . Data from
`human tumors include: (i) increased expression of specific proto(cid:173)
`oncogenes in some human malignancies (7, 8); (ii) localization of
`proto-oncogenes at or near the site of specific, tumor-associated
`chromosomal translocations (9); and (iii) amplification of proto(cid:173)
`oncogenes in some human tumors (10, 11).
`Additional data linking proto-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(cid:173)
`oncogenes, three are related to a growth factor or a growth factor
`receptor. These genes include c-sis, which is homologous to the
`
`transforming gene of the simian sarcoma virus and is the f3 chain of
`platelet-derived growth factor (PDGF) (16, 17); c-fms, which is
`homologous to the transforming gene of the feline sarcoma virus
`and is closely related to the macrophage colony-stimulating factor
`receptor (CSF-l R) (18); and c-erbB, which encodes the EGF
`receptor (EGFR) and is highly homologous to the transforming
`gene of the avian erythroblastosis virus (19) . The two receptor(cid:173)
`related proto-oncogenes, c-fms and c-erbB, are members of the
`tyrosine-specific protein kinase family to which many proto-onco(cid:173)
`genes belong.
`Recently, a novel transforming gene was identified as a result of
`transfection studies with DNA from chemically induced rat neu(cid:173)
`roglioblastomas (20) . This gene, called neu, was shown to be related
`to, but distinct from, the c-erbB proto-oncogene (21) . By means of
`v-erbB and human EGFR as probes to screen human genomic and
`complementary DNA (cDNA) libraries, two other groups indepen(cid:173)
`dently isolated human erbB-related genes that they called HER-2
`(22) and c-erbB-2 (23). Subsequent sequence analysis and chromo(cid:173)
`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(cid:173)
`erbB as a probe, identified the same gene in a mammary carcinoma
`cell line, MAC 117, where it was found to be amplified five- to ten(cid:173)
`fold (26).
`This gene, which we will call HER-2/neu, encodes a new member
`of the tyrosine kinase family; and is closely related to, but distinct
`from, the EGFR gene (22). HER-2/neu differs from EGFR in that it
`is folind 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, HE R -2/neu is more closely related to the
`EGFR gene than to other members of the tyrosine kinase family
`(22). Like the EGFR protein, HE R-2/neu has an extracellular
`domain, a transmembrane domain that includes two cysteine-rich
`repeat clusters, and an intracellular kinase domain (21), indicating
`
`D. ]. Slamon, S. G. Wong, and W. ]. Levin are in the Division of Hematology(cid:173)
`Oncology, Department or Medicine and Jonsson Comprehensive Cancer Center,
`UCLA School of Medicine, Los Angeles, CA 90024. G. M. Clark and W. L. McGuire
`are in the Division of Oncology, Department of Medicine, University of Texas Health
`Science Center at San Antonio, San Antonio, TX 78284. A. Ullrich is in the
`Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080.
`
`*To whom correspondence should be addressed.
`
`9 JANUARY 1987
`
`ARTICLES 177
`
`HOSPIRA EX. 1133
`Page 4
`
`

`

`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/neu in a human mammary carcinoma cell line, and as part of an
`ongoing survey in our laboratory of proto-oncogene abnormalities
`in human tumors, we evaluated alterations of the HER-2/neu gene
`in a large series of human primary breast cancers. Our results show
`that amplification of this gene occurs relatively frequently in breast
`cancer, and that it is associated with disease relapse and overall
`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, which was conducted in two parts,
`involved the evaluation of tissue from 189 separate breast malignan(cid:173)
`cies that were part of a breast cancer study ongoing at the University
`of Texas, San Antonio. This cohort of tumors was of interest
`because considerable information was available on the majority of
`the specimens including size of the primary tumor, estrogen recep(cid:173)
`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/neu gene. DNA from
`individual tumors was prepared as described (30), digested with
`Eco RI, and subjected to Southern blot analysis with a 32P-labeled
`. HER-2/neu-l probe, which is known to detect a 13-kb hybridizing
`band in human DNA (22). Examples of tumors from the initial
`survey are shown in Fig. 1. Of the 103 samples examined, 19 ( 18%)
`showed evidence of HER-2/neu gene amplification. The degree of
`amplification in individual cases was determined by dilution analysis
`(Fig. 2A), as well as soft laser densitometry scanning. To determine
`that the amount of DNA loaded in each lane was equivalent, all
`filters were washed and rehybridized with a 32P-labeled arginase
`gene probe (31 ). This probe identifies a 15-kb hybridizing band on
`Eco RI-digested human DNA, and was selected as a control
`because it more appropriately assesses the relative amount and
`
`Table 1. Association between HER-2/neu amplification and disease parame(cid:173)
`ters in 103 breast tumors.
`
`Factor*
`
`ER+
`ER-
`PgR+
`PgR-
`
`s2
`2-5
`> 5
`Unknown
`
`s50
`> 50
`Unknown
`
`0
`1-3
`> 3
`Unknown
`
`Number of tumors
`
`Single
`copy
`
`2 to 5
`copies
`
`5 to 20
`copies
`
`> 20
`copies
`
`Pt
`
`Total
`
`1
`4
`2
`3
`
`Hormonal receptor status
`2
`9
`2
`1
`2
`6
`1
`5
`Tumor size (centi~tm)
`1
`1
`1
`5
`1
`2
`0
`3
`Age at diagnosis (years)
`1
`2
`1
`4
`2
`7
`0
`0
`2
`Number of positive lymph nodes
`1
`0
`3
`1
`1
`0
`2
`2
`4
`1
`3
`1
`
`0
`1
`2
`2
`
`53
`31
`42
`42
`
`13
`34
`17
`20
`
`21
`52
`11
`
`30
`20
`17
`17
`
`0.99
`
`0.85
`
`0.82
`
`0.83
`
`0.11
`
`65
`38
`52
`51
`
`15
`41
`22
`25
`
`25
`65
`13
`
`34
`22
`25
`22
`
`*Receptor status was analyzed as described (39). ER, estrogen receptor: + and - refer
`to the presence or absence of ;;.3 lino! of receptor per milligram of protein. PgR,
`progesterone receptor: + and - refers to th~resence or absence of ;;.5 lino! of receptor
`t Statistical an yses for correlation ofHER-2/neu amplifi-
`per milligram of protein.
`cation with disease parameters were performed by the x2 test. P values were computed
`after combining the cases with 5 to 20 and > 20 copies.
`
`transfer of high molecular weight species than a probe hybridizing
`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. 2B) . Individual
`tumors were assigned to groups containing a single copy, 2 to 5
`copies, 5 to 20 copies, and greater than 20 copies of the HER-2/neu
`gene (Fig. 1). Assignment of tumors to the various groups was done
`
`Fig. 1. Analysis of alterations of the HER-2/neu
`gene in human breast cancer. Shown arc 79 of the
`189 breast tumors used in this analysis. Tumors
`with a single copy of HER -2/neu: 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 o fHER-2/neu : 1, 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 -2/neu: 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/neu : 18, 30, 33,
`59, 68, and 78. Examples of tumors 77 to 79 have
`rearrangements in the HER-2/neu gene. DNA
`was extracted from tissues and digested with Eco
`Rl as described (30). A total of 12 µg of Eco Rl(cid:173)
`digcstcd DNA was loaded onto 0.8% agarosc
`gels, separated by electrophoresis, and transferred
`onto nylon filter papers (Biodync) (30). All filters
`were baked in a vacuum oven for 3 hours at 80°C,
`prehybridizcd in 5 X SSC (standard saline citrate)
`containing 50% formamide, 10% dcxtran sulfate,
`0.1 % SDS, denatured salmon sperm DNA ( 1 mg/
`ml), and 4 X Dcnhardts solution for 12 hours,
`then hybridized in the same solution containing
`32P-labeled nick-translated HER-2 probe (21 )
`specific activity of 1 x 108 cpm per microgram of
`DNA; 2 x 106 cprn/ml. H ybridization occurred
`at 42°C for 48 hours, followed by washing of
`filters under the following conditions in succes-
`
`1 2 3 4 5 • 6 7 8 9
`
`..
`
`• 10 11 12 13 14
`
`15 16 17 18 19 20
`
`21 22 23 24 25 26 27 28 29
`I
`
`41 42 43 44 45 46 47 48 49 50
`
`••
`
`•
`
`67 68 69 70 71 72 73 74 75 76
`
`n 1a 79
`
`kb
`·12
`
`- 4
`-3.1
`
`·18
`
`sion: 2 X SSC for 20 minutes at room tempera(cid:173)
`ture; two washes of 30 minutes each in 2 X SSC,
`0.1 % SDS at 65°C; one wash of 30 minutes in
`
`0.5 x SSC, 0.1 % SDS at 65°C . Filters were then
`exposed to XAR-5 x-ray film (Kodak) for autora(cid:173)
`diography.
`
`SCIENCE, VOL. 2 35
`
`HOSPIRA EX. 1133
`Page 5
`
`

`

`Fig. 2. (A) Example of dilutional analysis to assess degree of HER-2/neu
`gene amplification. Lanes a, g, k, and p were loaded with 12 µg of Eco Rl(cid:173)
`digested breast tumor DNA. Lane a is DNA from tumor 31(Fig. 1), which
`represents a tumor with a single copy of the HER/2-neu gene. Lane g is
`DNA from tumor 33, which represents a tumor with > 20 copies of the
`HER-2/neu gene. Lanes b to fare serial dilutions ( 1 : 100, 1 : 20, 1: 10, 1 : 5,
`and 1: 2, respectively) of the DNA sample in lane g. Lane k is DNA from
`rumor 35 (Fig. 1), which represents a tumor containing two to five copies of
`the HER-2/neu gene. Lanes h to j are serial dilutions ( 1 : 10, 1 : 5, and 1: 2,
`respectively) of the DNA sample in Jane k. Lane p is DNA from tumor 34
`(Fig. 1), which represents a tumor with 5 to 20 copies of the HER-2/neu
`gene. Lanes I to o are serial dilutions ( 1: 20, 1 : 10, 1: 5, and 1: 2, respective(cid:173)
`ly) of the DNA sample in lane p. The filter was prepared and hybridized with
`a 32P-labeled HER-2 probe as in Fig. 1. (B) Example of arginase probe
`hybridization to demonstrate that equivalent amounts of tumor DNA were
`loaded into each lane. Rehybridization of filter containing lanes 30 to 40
`(Fig. 1) . The filter was first stripped of label by washing in a buffer made up
`of 50% formarnide, 3 x 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 removal of all radioactive probe, then rehybridized as in Fig. 1 with a
`32P-labeled human arginase gene probe (31 ).
`
`in a blinded fashion, in that they were made without knowledge of
`disease parameters. Analysis of the data for association between gene
`amplification and a number of disease parameters was then per(cid:173)
`formed.
`Of 103 tumors evaluated in the initial survey, there was essentially
`no correlation between gene amplification and estrogen receptor
`status, progesterone receptor status, size of tumors, or age at
`diagnosis (Table 1). However, when analysis was performed for
`association between HER-2/neu amplification and number of posi(cid:173)
`tive lymph nodes, a trend was noted. This analysis showed that 4/34
`(11%) of patients with no involved nodes, 2/20 (10%) with 1to3
`involved nodes, and 8/25 (32%) 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 ofHER-2/neu gene amplifica(cid:173)
`tion in patients with > 3 axillary lymph nodes involved with disease.
`A multivariate regression analysis to correlate HER-2/neu amplifica(cid:173)
`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 indicated that it might be possible to discrimi(cid:173)
`nate among node-positive patients on the basis ofHER-2/neu gene
`
`1.0
`
`.~ 0.8
`~ 0.6
`.c e c.
`n; 0.4
`> ·;;
`:;
`"' 0.2
`
`0
`
`1.0
`E" 0.0
`ii
`"' .c 0.6
`e c.
`n; 0.4
`> ·;:
`:; 0.2
`"'
`
`A
`
`B
`
`Fig. 3. Actuarial curve for relapse in (A) node(cid:173)
`positive patients .with no amplification versus
`node-positive patients with any amplification (>2
`copies) of HER-2/neu and (C) node-positive pa(cid:173)
`tients with no amplification versus node-positive
`patients with greater than 5 copies ofHER-2/neu.
`Actuarial curve for overall survival in (B) node(cid:173)
`positive patients with no amplification versus
`node-positive patients with any amplification (>2
`copies) of HER-2/neu and (D) node-positive pa(cid:173)
`tients with no amplification versus node-positive
`patients with greater than 5 copies ofHER-2/neu.
`Actuarial curves for both relapse and overall sur(cid:173)
`vival were computed by the method of Kaplan
`and Meier (44 ) and compared by the log rank test
`(42--44 ).
`
`9 JANUARY 1987
`
`A
`a
`
`B
`30
`
`b
`
`c
`
`d
`
`e
`
`g
`
`h
`
`k
`
`m
`
`n
`
`o
`
`p
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`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/neu 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 analyzed 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 informa(cid:173)
`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) . Of these 100 samples, 86 yielded sufficient
`DNA for study. Amplification of the HER-2/neu gene was mea(cid:173)
`sured as in the initial survey, and examples of tumors from this study
`are shown (Fig. 1). Amplification was found in 34/86 (40% ) of
`these patients. For this larger sample of node-positive patients,
`several statistically significant or nearly significant relationships were
`observed. In agreement with the preliminary survey, there was an
`association between number of involved lymph nodes and HER-2/
`neu amplification (Table 2). In addition, the presence of gene
`amplification 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-2/neu amplifica(cid:173)
`tion with various disease parameters in the combined group is
`shown in Table 3.
`While these correlations were of interest, the strong relationship
`
`Amplified (n : 34)
`
`c
`
`Amplified (n=1 1)
`>5 copies
`
`Amplified (n =34)
`
`Amplified (n =11)
`>5 copies
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`D
`
`84
`0
`Ti me (months)
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`84
`
`ARTICLES 179
`
`HOSPIRA EX. 1133
`Page 6
`
`

`

`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 strong and highly statistically
`significant correlation was found 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 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 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 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 ofN-myc gene amplification in human
`neuroblastomas (32).
`To determine if amplification of HER-2/neu was independent of
`other known prognostic factors in predicting disease behavior,
`multivariate survival analyses were performed on the 86 node(cid:173)
`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 rehybridization of filter with human EGFR probe. Filters
`were stripped as in Fig. 2B, and hybridized with 32P-labeled human EGFR
`probe (28), as in Fig. 1. Shown are the lower molecular weight bands
`hybridized with 32P-labeled EGFR probe in filter-containing lanes 51 to 66
`(Fig. 1). The bands from top to bottom are 2.8, 2.2, and 1.8 kb, respectively.
`Lane 52 is an example of a tumor showing marked amplification (> SO
`copies) of the EGFR gene.
`
`patients, even when other prognostic factors were taken into
`account (Table 4).
`Rearrangement of the HER-2/neu gene was rare. Of the total 189
`tumors evaluated, three showed evidence of rearrangement, 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
`rearrangement as determined by Eco Rl digestion was too small to
`attempt statistical correlations.
`To determine whether the phenomenon of amplification ofHER-
`2/neu in breast cancer extended to related growth factor receptors,
`all filters were analyzed with the EGFR probe (Fig. 4). Amplifica(cid:173)
`tion of the EGFR gene was found in 4/189 (2%) of the cases, and
`rearrangement of the EGFR gene was found in one of those four
`cases. The incidence ofEGFR amplification and rearrangement was
`too small to attempt statistical correlation. Comparison ofHER-2/
`neu 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-oncogenes, abl and fas, in
`breast cancer did not show amplification of these genes (33).
`Alterations of non-tyrosine kinase-related proto-oncogenes in these
`
`Table 3. Association between HER-2/neu amplification and disease parame(cid:173)
`ters in combined surveys ( 189 patients).
`
`Table 2. Association between HER-2/neu amplification and disease parame-
`ters in 86 breast tumors from node-positive patients.
`
`Factor*
`
`Single
`copy
`
`2 to 5
`copies
`
`5 to 20
`copies
`
`>20
`copies
`
`Total
`
`Pt
`
`Factor*
`
`Single
`copy
`
`2 to 5
`copies
`
`5 to 20
`copies
`
`> 20
`copies
`
`Total
`
`Pt
`
`ER+
`ER-
`PgR+
`PgR-
`
`s2
`2-5
`> 5
`
`sSO
`> 50
`
`1-3
`> 3
`
`38
`14
`31
`21
`
`18
`28
`6
`
`16
`36
`
`31
`21
`
`1
`1
`1
`1
`
`H onnonal receptor status
`21
`5
`2
`4
`18
`4
`5
`5
`Tumor size (centimeters)
`8
`3
`2
`12
`3
`4
`Age at diagnosis (years)
`12
`1
`6
`11
`1
`3
`Number of positive lymph nodes
`7
`5
`0
`16
`4
`2
`
`0
`1
`1
`
`65
`21
`54
`32
`
`29
`43
`14
`
`35
`51
`
`43
`43
`
`0.05
`
`0.14
`
`0.09
`
`0.06
`
`0.06
`
`ER+
`ER-
`PgR+
`PgR-
`
`s2
`2-5
`> 5
`Unknown
`
`sSO
`>50
`Unknown
`
`0
`1-3
`> 3
`Unknown
`
`91
`45
`73
`63
`
`31
`62
`23
`20
`
`37
`88
`11
`
`30
`51
`38
`17
`
`2
`5
`3
`4
`
`Honnonal receptor status
`23
`14
`3
`6
`20
`10
`6
`10
`Tumor size (centimeters)
`9
`4
`13
`7
`4
`6
`0
`3
`Age at diagnosis (years)
`2
`13
`8
`13
`10
`5
`0
`2
`0
`Number of positive lymph nodes
`0
`3
`1
`7
`6
`1
`18
`8
`4
`1
`3
`1
`
`0
`2
`3
`2
`
`0.05
`
`0.06
`
`0.19
`
`0.11
`
`0.002
`
`130
`59
`106
`83
`
`44
`84
`36
`25
`
`60
`116
`13
`
`34
`65
`68
`22
`
`t Statistical analyses for correlation of
`*ER and PgR are as described in Table l.
`HER-2/neu amplification with various disease ~arameters were performed by the x2
`test. P values were computed after combining e 5 to 20 and > 20 cases, since therr
`were so few samples in t